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Da, Silva Alison. "Étude de la reconnaissance des Escherichia coli adhérents et invasifs (AIEC) associés à la maladie de Crohn par l'autophagie : identification des récepteurs autophagiques et des facteurs de virulence." Electronic Thesis or Diss., Université Clermont Auvergne (2021-...), 2023. http://www.theses.fr/2023UCFA0117.
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La maladie de Crohn (MC) est une maladie inflammatoire chronique de l'intestin, dont l'étiologie est multifactorielle. Elle résulte de l'interaction complexe entre des prédispositions génétiques, des facteurs environnementaux et des altérations de la composition du microbiote intestinal, induisant une dérégulation du système immunitaire intestinal. À ce jour, la MC est incurable, seuls des traitements visant à soulager les symptômes et à prévenir les récidives et complications sont disponibles. Chez les patients atteints de la MC, une augmentation de la prévalence de souches particulières d'Escherichia coli, appelées les AIEC (adherent-invasive E. coli), a été rapportée. Les AIEC sont des pathobiontes capables d'adhérer et d'envahir les cellules épithéliales intestinales ainsi que de se répliquer en macrophages sans induire la mort cellulaire, entraînant une réponse immunitaire dérégulée. Par ailleurs, des études ont montré que plusieurs polymorphismes dans les gènes de l'autophagie (ATG16L1, IRGM, ULK1, etc.) sont associés à un risque augmenté de développer la MC. L'autophagie est un processus essentiel au maintien de l'homéostasie cellulaire permettant la dégradation et le recyclage de composants cytoplasmiques et de pathogènes via le lysosome. Toutefois, certains pathogènes intracellulaires développent diverses stratégies pour échapper à la dégradation par l'autophagie. Dans ce contexte, l'objectif de mes travaux de thèse était d'identifier les récepteurs autophagiques responsables de la reconnaissance des AIEC, ainsi que les gènes nécessaires aux AIEC pour échapper à l'autophagie.Le 1er axe de mes travaux de thèse a montré que la déficience de p62 ou NDP52 dans les cellules HeLa entraîne une augmentation de la réplication intracellulaire de la souche de référence AIEC LF82 et de la production de cytokines pro-inflammatoires. L'analyse par microscopie confocale a révélé la colocalisation de p62 ou NDP52 avec la bactérie AIEC LF82 et la protéine LC3, un marqueur de l'autophagie. Ainsi, nos résultats suggèrent que p62 et NDP52 agiraient comme des récepteurs autophagiques pour contrôler la réplication intracellulaire des AIEC. De plus, nous avons étudié l'impact d'un polymorphisme du gène NDP52 associé à une susceptibilité augmentée de développer la MC, appelé NDP52Val248Ala, sur le contrôle des AIEC. Aucune différence n'a été observée dans le nombre de bactéries AIEC LF82 intracellulaires entre les cellules HeLa exprimant le variant à risque NDP52Val248Ala et celles exprimant l'allèle sauvage, suggérant un autre rôle de ce variant, probablement dans le contrôle de l'inflammation.Le 2ème axe de mes travaux de thèse s'est concentré sur l'identification de gènes nécessaires aux AIEC pour échapper au contrôle par l'autophagie en utilisant la technique Transposon Sequencing (Tn-Seq). Brièvement, une banque de mutants de la souche AIEC LF82 a été créée de manière « saturée », c'est-à-dire que chacun des gènes du génome bactérien a été interrompu par au moins un transposon, conduisant à son invalidation. Cette banque de mutants a été utilisée pour infecter des cellules HeLa contrôles et déficientes pour l'autophagie. À 24h post-infection, l'ADN des mutants a été extrait et les sites d'insertion du transposon déterminés par séquençage ont permis d'identifier 68 gènes différentiellement représentés entre nos deux conditions. Les gènes sur-représentés dans les cellules HeLa déficientes pour l'autophagie par rapport aux cellules contrôles, sont les gènes potentiellement nécessaires aux AIEC pour échapper à l'autophagie. Ainsi, cette étude permettrait d'identifier de nouvelles cibles pour limiter la virulence des AIEC.En conclusion, ces travaux contribuent à la compréhension des divers aspects de l'interaction entre les cellules hôtes et les bactéries AIEC associées à la MC et permettront, à l'avenir, de mieux caractériser la pathogenèse de cette maladie<br>Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology is multifactorial. It results from the complex interaction between genetic predispositions, environmental factors and alterations in the intestinal microbiota composition, inducing a deregulation of the intestinal immune system. To date, CD is incurable, only treatments aimed at alleviating symptoms and preventing recurrences and complications are available. In CD patients, an increase in the prevalence of particular strains of Escherichia coli, called AIEC (adherent-invasive E. coli) strains, has been reported. AIEC are the pathobionts able to adhere to and to invade intestinal epithelial cells as well as replicate inside macrophages without inducing cell death, leading to a dysregulated immune response. Furthermore, it has been shown that several polymorphisms in autophagy-related genes (ATG16L1, IRGM, ULK1, etc.) are associated with an increased risk to develop CD. Autophagy is an essential process for maintaining cellular homeostasis, which allows the degradation and recycling of cytoplasmic components and pathogens via the lysosome. However, some intracellular pathogens develop various strategies to escape autophagy degradation. In this context, the aim of my thesis was to identify the autophagic receptors responsible for AIEC recognition, as well as the genes necessary for AIEC to escape autophagy.The first part of my thesis showed that the depletion of p62 or NDP52 in HeLa cells leads to an increase in the intracellular replication of the AIEC LF82 reference strain and the production of pro-inflammatory cytokines. Confocal microscopy analysis revealed the colocalization of p62 or NDP52 with AIEC LF82 bacteria and LC3 protein, a marker of autophagy. Thus, our results suggest that p62 and NDP52 could act as autophagic receptors to control AIEC intracellular replication. Additionally, we investigated the impact of a polymorphism in the NDP52 gene associated with increased susceptibility to develop CD, called NDP52Val248Ala, on the control of AIEC. No difference was observed in the AIEC LF82 intracellular number between HeLa cells expressing the NDP52Val248Ala risk variant and those expressing the wild-type allele, suggesting another role for this variant, probably in the control of inflammation.The second part of my thesis focused on the identification of the genes necessary for AIEC to escape from autophagy control by the Transposon Sequencing (Tn-Seq) technique. Briefly, a mutant library of the AIEC LF82 strain was created in a “saturated” manner, meaning that each gene of the bacterial genome was inserted by at least one transposon, leading to its invalidation. This mutant library was used to infect control and autophagy-deficient HeLa cells. At 24 hours post-infection, the mutants DNA was extracted and transposon insertion sites determined by sequencing allowed the identification of 68 genes differentially represented between our two conditions. The genes over-represented in autophagy-deficient HeLa cells compared to control cells, are potential genes necessary for AIEC to escape from autophagy control. Thus, this study could allow the identification of new targets to limit the virulence of AIEC.In conclusion, this work contributes to the understanding of various aspects of the interaction between host cells and CD-associated AIEC bacteria and, in the future, will aide to better characterize the etiopathogenesis of this disease
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Verlhac, Pauline. "Rôle des récepteurs autophagiques dans la maturation des autophagosomes." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1138/document.
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La xénophagie est une forme d'autophagie sélective permettant de capture des pathogènes dans les autophagosomes et de les dégrader dans les autolysosomes. Cette sélectivité est assurée par une famille de protéines ; les récepteurs autophagiques qui reconnaissent des substrats cytosoliques d'un côté et les membres de la famille LC3 ancrés dans la membrane de l'autophagosome de l'autre. Parmi ces récepteurs, NDP52 cible la bactérie Salmonella Typhimurium vers l'autophagie.Nous décrivons un rôle nouveau et inattendu pour NDP52 ; assurer la maturation d'autophagosomes durant l'infection par Salmonella mais aussi durant l'autophagie basale. De manière intéressante, ce rôle de NDP52 dans la maturation est indépendant de son rôle dans le ciblage de la bactérie puisque ces fonctions nécessitent des domaines et des partenaires moléculaires de NDP52 distincts. Nous montrons aussi que d'autres récepteurs peuvent participer à la maturation comme Optineurine. Ce travail montre donc que NDP52 assure deux rôles durant la xénophagie en ciblant les bactéries vers les autophagosomes en formation puis en promouvant la maturation de l'autophagosome. De plus, nous proposons aussi un possible mécanisme de régulation de ces deux fonctions par des modifications post-traductionnelles des récepteurs autophagiques.Ce travail démontre que les récepteurs autophagiques jouent des rôles au-delà du ciblage des pathogènes qui sont aussi cruciaux pour une xénophagie efficace. De plus, les récepteurs autophagiques sont aussi nécessaires pour le déroulement de l'autophagie basale. Ces travaux offrent une nouvelle compréhension de la régulation moléculaire de l'autophagie et de la xénophagie<br>Xenophagy relies on the ability of the autophagy process to selectively entrap intracellular pathogens within autophagosomes to degrade them into autolysosomes. The selectivity of the process relies on proteins named autophagy receptors that share the ability to recognise cytosolic cargos on one hand and autophagosome-bound members of the ATG8 family on the other. Among autophagy receptors NDP52 has been described to target Salmonella Typhimurium to the growing autophagosome. We describe a new unexpected role for NDP52, as this receptor also regulates the maturation of Salmonella-containing autophagosomes and during ongoing autophagy. Interestingly, the role of NDP52 in maturation is independent from its role in targeting as they rely on different binding domains and protein partners. We also show that other autophagy receptors also mediate autophagosome maturation such as Optineurin. Therefore, our work shows that NDP52 plays a dual function during xenophagy first by targeting bacteria to growing autophagosomes and then by assuring autophagosome maturation. Moreover, we also provide insights as to how these dual roles are regulated by post-translational modifications of autophagy receptors.This work demonstrates that autophagy receptors have other roles beyond pathogen targeting that are also crucial for an efficient xenophagy. Moreover, autophagy receptors are also necessary for autophagy completion in uninfected cells. These results strengthen our understanding of both ongoing autophagy and xenophagy molecular mechanisms
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Petkova, Denitsa. "Étude du rôle de récepteurs autophagiques lors de l'infection par le virus de la rougeole." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10311/document.
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La macroautophagie assure l'homéostasie cellulaire en recyclant du matériel cytosolique obsolète ou délétère et sa dérégulation est associée à plusieurs pathologies. Elle constitue aussi un mécanisme de défense car elle peut éliminer des pathogènes intracellulaires. L'étape cruciale de l'autophagie est la maturation lors de laquelle la vésicule renfermant des substrats cytosoliques, l'autophagosome, fusionne avec des lysosomes et la dégradation a lieu. Nous nous intéressons à la régulation de l'autophagie et aux conséquences de sa perturbation lors des infections, notamment par le virus de la rougeole (VR). Les données de l'équipe montrent qu'il induit et utilise toutes les étapes de l'autophagie, afin de se répliquer efficacement. Mes travaux montrent que des protéines du virus peuvent interagir avec au moins deux protéines cellulaires NDP52 et T6BP qui sont des récepteurs autophagiques (protéines cytosoliques ayant un domaine de liaison aux autophagosomes et un domaine de liaison au substrat à dégrader, par exemple des pathogènes). J'ai alors étudié le rôle des récepteurs autophagiques T6BP, NDP52 et Optineurine dans la réplication virale. J'ai aussi participé à une étude décrivant que NDP52 et Optineurine régulent en plus la maturation. Mes travaux de thèse démontrent un tel double rôle pour T6BP. Cependant, seuls T6BP et NDP52 sont nécessaires à la réplication du VR bien qu'elle requiert la maturation autophagique. Ainsi mes résultats suggèrent d'une part que les trois récepteurs puissent réguler la maturation d'autophagosomes distincts.D'autre part, le VR pourrait exploiter individuellement les autophagosomes dont la maturation dépend de T6BP et NDP52 pour se répliquer<br>Macroautophagy ensures cell homeostasis through the recycling of obsolete or deleterious cytosolic components and its deregulation is associated with several pathologies. It is also a defense mechanism as it allows the elimination of intracellular pathogens. The most important autophagic step is maturation, during which the cytosolic substrate-containing vesicle, the autophagosome, fuses with lysosomes and the degradation occurs. We study autophagy regulation and the consequences of its disruption during infections and in particular by measles virus (MeV). Our team has shown that MeV induces and exploits all steps of autophagy, to replicate more efficiently. My results indicate that viral proteins can interact with at least two cellular proteins, NDP52 and T6BP, which are autophagy receptors (cytosolic proteins that carry an autophagosome-binding domain and a domain binding substrates that would be degraded, such as intracellular pathogens). I then studied the role of autophagic receptors T6BP, NDP52 and OPTINEURIN in viral replication. I also took part in a study describing NDP52 and OPTINEURIN as autophagosome maturation regulators. My work depicts the same dual role for T6BP. However, only T6BP and NDP52 are necessary for MeV replication even though it requires autophagosome maturation. Thus, my results suggest that the three autophagy receptors might regulate distinct autophagosome maturation on one hand. On the other, MeV could individually exploit autophagosomes, the maturation of which is regulated by T6BP or NDP2 to replicate efficiently
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Negulescu, Ana-Maria. "Caractérisation des récepteurs à dépendance Notch3 et Kremen1 dans les cancers." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1265.
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Les récepteurs membranaires sont des acteurs majeurs des interactions entre la cellule et son environnement. Ils peuvent être à l'origine des signaux de survie, de différentiation, de migration ou bien de mort cellulaire. Les travaux de ce manuscrit ont été faits sur une famille de récepteurs nommés "récepteurs à dépendance". Ils sont caractérisés par leur fonctionnement dans la cellule plutôt que par leur structure: en présence de leurs ligands ces récepteurs induisent un signal de survie et en l'absence de ces mêmes ligands ils induisent un signal actif de mort cellulaire. Deux nouveaux récepteurs à dépendance ont été étudiés: Notch3 et Kremen1 dans le contexte du contrôle de l'homéostasie et plus particulièrement dans le contrôle de la tumorigenèse du cancer du sein. Nous montrons que le récepteur à dépendance Notch3 est perdu dans le cancer du sein, dû à un gain significatif de méthylation, entre le tissu normal et le tissu tumoral dans les patients. Notch3 a également un rôle pro-apoptotique dans les cellules endothéliales dans le cancer du poumon. Des études effectuées sur des cohortes de cancer nous ont permis de voir que le ligand Dickkopf1 (Dkk1), qui lie le récepteur Kremen1, est sur-exprimé dans plusieurs cancers tandis que le récepteur il est perdu dans les cancers. Rétablir l'expression de Kremen1 ou invalider Dkk1 dans la lignée de cancer du sein de type basal MDA-MB 231 conduit à une forte mort cellulaire de type autophagique. En ce qui concerne les enjeux thérapeutiques de ces travaux, nous avons pu sélectionner plusieurs anticorps dirigés contre le domaine extracellulaire de Kremen1, qui induisent la mort des cellules cancéreuses<br>Membrane receptors are major actors of the interaction between a cell and its environment. They are able to trigger different types of signals such as survival, differentiation, migration or cell death. The work presented in this manuscript has been done on a particular family of receptors called dependence receptors. They are characterized by their function rather than by their structure. In the presence of their ligand they induce a survival signal whereas in the absence of the ligand they induce an active signal of cell death. Two new dependence receptors have been studied: Notch3 and Kremen1, in the context of homeostasis control, and more particularly in the control of breast cancer tumorigenesis. We show that Notch3 dependence receptor is lost in breast cancer, because of a significant gain of methylation observed between the normal tissu and the tumoral tissue within the same patient. Notch3 plays also a pro-apoptotic role in endothelial cells of lung cancer. Experiences carried on cancer cohorts have allowed us to notice that the Dickkopf (Dkk1) ligand, which links the Kremen1 receptor, is over-expressed in several cancers whereas the receptor is lost in different cancers. Restoring Kremen1 expression or disabling Dkk1 in breast cancer basal type MDA-MB 231 cells, leads to large autophagic cell death. Concerning therapeutic approaches, we selected several antibodies against Kremen1 extra-cellular domain, which induce the death of cancer cells
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Runwal, Gautam. "The study of two transmembrane autophagy proteins and the autophagy receptor, p62." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290149.
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Autophagy is an evolutionarily conserved process across eukaryotes that is responsible for degradation of cargo such as aggregate-prone proteins, pathogens, damaged organelles, macromolecules etc. via its delivery to lysosomes. The process is known to involve the formation of a double-membraned structure, called autophagosome, that engulfs the cargo destined for degradation and delivers its contents by fusing with lysosomes. This process involves several proteins at its core which include two transmembrane proteins, ATG9 and VMP1. While ATG9 and VMP1 has been discovered for about a decade and half, the trafficking and function of these proteins remain relatively unclear. My work in this thesis identifies and characterises a novel trafficking route for ATG9 and VMP1 and shows that both these proteins traffic via the dynamin-independent ARF6-associated pathway. Moreover, I also show that these proteins physically interact with each other. In addition, the tools developed during these studies helped me identify a new role for the most common autophagy receptor protein, p62. I show that p62 can specifically associate with and sequester LC3-I in autophagy-impaired cells (ATG9 and ATG16 null cells) leading to formation of LC3-positive structures that can be misinterpreted as mature autophagosomes. Perturbations in the levels of p62 were seen to affect the formation of these LC3-positive structures in cells. This observation, therefore, questions the reliability of LC3-immunofluorescence assays in autophagy-impaired cells as method of assessing autophagy and points towards the homeostatic function played by p62 in autophagy-impaired cells.
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Coly, Pierre-Michaël. "Régulation de l'activité autophagique par les récepteurs chimiotactiques couplés aux protéines G : rôle essentiel dans la migration directionnelle." Thesis, Normandie, 2017. http://www.theses.fr/2017NORMR004/document.
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L’autophagie est un processus catabolique par lequel certaines protéines cytosoliquessont dirigées vers le compartiment lysosomial, afin d’y être dégradées. Ce processus débutepar la séquestration de constituants cytoplasmiques par une structure multimembranaireappelée phagophore. La fermeture du phagophore donne naissance à une vésicule à doublemembrane nommée autophagosome, qui fusionne avec les lysosomes, ce qui conduit à ladégradation du contenu de sa lumière. Ainsi, la modulation de l’autophagie permet unremodelage dynamique du protéome cellulaire. Bien que des données récentes ont permis dedémontrer la dégradation autophagique de protéines impliquées dans la migration cellulaire,telles que des intégrines, ou encore les protéines RhoA et Src, l'impact fonctionnel del'autophagie sur la migration cellulaire demeure sujet à controverse. Alors que l'autophagie estdécrite comme un processus pro-migratoire et pro-invasif dans certaines études, d'autrestravaux indiquent que l'inactivation des protéines pro-autophagiques stimule l'invasion descellules cancéreuses. De plus, l'effet fonctionnel des RCPG chimiotactiques sur l’activitéautophagique reste totalement inexploré. Sur la base de ces données, les objectifs de mon travail de thèse ont été i) d’évaluer les effets des RCPG chimiotactiques, le CXCR4 et l’UT,sur le processus autophagique et ii) d’étudier l’impact de cette modulation sur la migrationcellulaire. Pour ce faire, nous avons utilisé des cellules HEK-293, transfectées à l’aide deconstruits permettant l’expression des RCPG CXCR4 et UT, ainsi que la lignée deglioblastome humain U87, exprimant ces deux récepteurs de manière endogène.Nous avons dans un premier temps évalué l’activité autophagique à l’aide de laprotéine de fusion EGFP-LC3, marqueur des autophagosomes. Nous avons ainsi démontréque l’activation du CXCR4 et de l’UT provoque une diminution significative de la biogénèsedes autophagosomes. Une étape essentielle de cette biogenèse est le recrutement des protéinesAtg16L1 et Atg5 à la membrane plasmique, conduisant à la formation d'endosomes Atg16L1-Atg5-positifs, appelés « endosomes pré-autophagiques ». Cette population d’endosomesconstitue une source importante de phospholipides nécessaire à l’expansion du phagophore etla formation d’un autophagosome mature. Afin d’évaluer l’impact des RCPG chimiotactiquessur le recrutement de la protéine Atg16L1 à la membrane plasmique, nous avons bloqué leprocessus d’endocytose par l’utilisation d’un inhibiteur de la dynamine, le Dynasore. Cettemolécule provoque une accumulation marquée de la protéine Atg16L1 dans les endosomespré-autophagiques en formation, retenus à la membrane plasmique<br>Autophagy is a catabolic process by which certain cytosolic proteins are directed to thelysosomal compartment to be degraded. This process begins with the sequestration ofcytoplasmic components, by a multimembrane structure called the phagophore. The closure ofthe phagophore gives rise to a double membrane vesicle called autophagosome, which thenmerges with lysosomes in order to degrade its luminal content. Autophagy modulation allowsa dynamic remodeling of the cellular proteome. Although recent evidence has demonstratedautophagic degradation of key proteins involved in cell migration, such as integrins, RhoAand the Src kinase, the functional impact of autophagy on cell migration remainscontroversial. While autophagy is described as a pro-migratory and pro-invasive process insome studies, others indicate that the inactivation of pro-autophagic proteins stimulates thecancer cell invasion. In addition, the functional effect of chemotactic GPCR on autophagicactivity remains unexplored. On the basis of these data, the objectives of my thesis were i) toevaluate the effects of the chemotactic GPCRs for SDF-1 (CXCR4) and for the vasoactivepeptide urotensin II (UT), on the autophagic process and ii) to study the impact of thismodulation on cell migration. In order to do this, we used HEK-293 cells, transfected with constructs allowing the expression of CXCR4 and UT, as well as the human glioblastomaline, U87, which endogenously expresses these two receptors. Previous studies have demonstrated a direct interaction of Atg5 with membranes,suggesting that recruitment of Atg16L1 to the plasma membrane may depend on Atg5. This prompted us to evaluate the formation of Atg16L1-positive pre-autophagic endosomes,following depletion of Atg5 levels. Several interfering RNAs, targeting the transcriptencoding Atg5, have been tested and, as expected, these interfering RNAs completely blockedthe recruitment of Atg16L1 to forming pre-autophagic endosomes. We then tested the effectsof chemotactic GPCRs on the subcellular localization of the Atg5 protein. By confocalmicroscopy, we found that a significant fraction of Atg5 localized to the plasma membraneunder basal conditions. The activation of CXCR4 or UT is accompanied by a marked decreaseof the Atg5 pool localized at the plasma membrane. Furthermore, we have demonstrated thatthe anti-autophagic effects of chemotactic GPCRs are completely abrogated byoverexpression of a recombinant Atg5 protein, suggesting that chemotactic GPCRs exert theiranti-autophagic effects by reducing the membrane pool of Atg5, necessary for the productionof pre-autophagic endosomes, and the expansion of the phagophore
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Bigford, Gregory E. "Activation of NR2B and Autophagy Signaling Pathways Following Traumatic Brain Injury." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/204.
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Hyper-activation of N-methyl-D-aspartate receptors (NRs) is associated with excitotoxic cell death during secondary injury following traumatic brain injury (TBI). The efficiency of the NR is dependent on the location of receptors in membrane raft microdomains that provide a platform for coupling of NRs and effector proteins. In many neurodegenerative diseases, activation of the autophagy pathway has been suggested to contribute to glutamate excitotoxicity, but whether increased autophagy signaling contributes to pathology after TBI has not been defined. In these studies, I investigate whether membrane rafts mediate NR signaling and autophagy in cortices of adult male rats subjected to moderate TBI and in sham-operated controls. These studies demonstrate that membrane rafts of the normal rat cortex contain a novel multi-protein signaling complex that links the NR2B glutamate receptor and the autophagic protein Beclin 1. TBI caused a rapid disruption of this complex in which NR2B and pCaMKII were recruited to membrane microdomains. Alteration in NR2B-Beclin 1 association in membrane rafts resulted in activation of autophagy as demonstrated by increased expression of key autophagic proteins Beclin 1, ATG 5 and ATG 7, and significant increases in autophagic vacuoles in neurons of traumatized brains. Administration of the NR2B antagonist RO 25-6981 significantly blocked TBI-induced redistribution of NR2B signaling intermediates and Beclin 1 and delayed the increase in autophagy protein expression in traumatized cortices. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin 1 in membrane rafts after TBI.
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Vicencio, Bustamante José Miguel. "The inositol-1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1." Paris 11, 2009. http://www.theses.fr/2009PA11T045.
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Manni, Diego. "Oxidation-dependent regulation of the selective autophagy receptor SQSTM1/p62." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3675.
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Oxidative stress and impairment of autophagy can lead to the accumulation and aggregation of damaged proteins, a common feature of most age-related neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. SQSTM1/p62, a receptor and a substrate of selective autophagy, is implicated in the degradation of damaged and polyubiquitinated substrates. Importantly, p62 has been detected in many types of protein inclusions found in neurodegenerative diseases, together with other disease-related proteins. However, the mechanisms allowing p62 to selectively recruit and degrade autophagic substrates in conditions of oxidative stress remain unknown. The aim of this thesis work is to understand the mechanisms underlying the oligomerisation and the aggregation of p62 during oxidation, looking at post-translational modifications that can lead to the formation of protein aggregates. We found that p62 senses and is regulated by oxidative stress. In response to oxidation, two cysteine (Cys) residues C105 and C113 of p62 mediate the formation of disulphide-linked conjugates (DLC). The formation of p62 DLC was reduced upon antioxidants addition, while inhibitors of the antioxidant system enhanced their development. This feature was critical for the function of p62 as an autophagy receptor as well as for the accumulation of polyubiquitinated aggregates. Indeed, the accumulation and degradation of p62 and its substrates was impaired following mutation of the Cys residues implicated in DLC formation, while the interaction between p62 and polyubiquitinated substrates was not affected. Oxidation of p62 was also required for cell survival in conditions of oxidative stress, indicating the physiological importance of the correct function of p62 in selective autophagy. In addition, formation of p62 DLC was increased in ageing and age-related neurodegenerative diseases, possibly as a compensatory mechanism to protect cells in increased oxidative conditions. In conclusion, we reveal a new mechanism of p62 oligomerisation aiding the selective autophagy of dysfunctional proteins under oxidative stress conditions.
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Singh, Madhu [Verfasser]. "Autophagy and Listeria monocytogenes : the role(s) of cargo receptors / Madhu Singh." Gießen : Universitätsbibliothek, 2014. http://d-nb.info/1068773235/34.
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Walinda, Erik. "Structural Study of Proteins Involved in Autophagy." 京都大学 (Kyoto University), 2015. http://hdl.handle.net/2433/202720.
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BRILLANTE, SIMONA. "THE OFD1 PROTEIN CONTROLS AUTOPHAGOSOME BIOGENESIS THROUGH SELECTIVE AUTOPHAGY." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/793410.
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Cilia are cellular projections that serve a wide variety of essential functions in mammals. Defects in cilia structure or function have emerged as etiological mechanisms underpinning human diseases called ciliopathies. The OFD1 gene, defective in a rare developmental ciliopathy known as Oral facial digital syndrome type I, encodes for a centrosomal/basal body protein required for cilia formation. Recent data link ciliary structures to autophagy, the major intracellular degradation system, although the mechanisms and the main players underlying this connection are still to be determined.
Autophagy is an evolutionarily conserved and strictly regulated lysosomal pathway which plays a wide variety of physiological and pathophysiological roles in cellular homeostasis. Either too little or too much autophagy may contribute to pathological conditions.
In the past decade a great deal of progress has been made in the molecular dissection of stimulatory autophagy inputs. On the other hand, our understanding of the mechanisms that restrain autophagy is only partial and far from being complete. Data obtained during my PhD program contribute to the description of a new negative feedback mechanism that inhibits autophagosome biogenesis through selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. I demonstrate that the ciliary OFD1 protein is involved in selective autophagy and acts as autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. Preliminary data also indicate that excessive autophagy may contribute to the disease pathogenesis. My results add a new tile to the puzzle of the cilia/autophagy interconnection and will help shedding light on these complex biological processes.
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Ellison, Cara Jane. "Sphingomyelin as a danger signal in cell-autonomous immunity." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267993.
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Individual cells employ mechanisms of cell-autonomous immunity to defend their cytosol against bacterial invasion. One such mechanism involves indirect detection of the pathogen through recognition of pathogen-induced disturbances causing the appearance of specific host molecules in an abnormal location. For example, glycans, which are located on the extracellular leaflet of the plasma membrane under homeostatic conditions, become hidden inside bacteria-containing vacuoles (BCVs) during bacterial entry into the cell. Upon BCV rupture, glycans become exposed to the cytosol where they act as a danger signal and are detected by the cytosolic danger receptor, Galectin 8. My research reveals that sphingomyelin, a host lipid predominantly located on the outer leaflet of the plasma membrane, is exposed to the cytosol on damaged BCVs. I visualised the appearance of intracellular sphingomyelin by utilising Lysenin - a sphingomyelin-specific toxin from earthworms – as a cytosolic sphingomyelin reporter. Lysenin is recruited to BCVs in a sphingomyelin-dependent manner upon cytosolic entry of both Gram-negative and Gram-positive bacteria. Lysenin co-localises with Galectin 8 on a proportion of BCVs, indicating that sphingomyelin exposure occurs upon membrane damage. Moreover, I elucidated that sphingomyelin exposure occurs before glycan exposure on damaged BCVs indicating that BCV rupture may proceed through two stages: ‘minor’ and ‘major’ damage. My investigations into possible causes of vacuole rupture are on going. To identify endogenous cellular receptors for cytosol-exposed sphingomyelin, I established and executed an assay to compare enrichment of mammalian cell lysate proteins on liposomes containing or lacking sphingomyelin. Following mass spectrometry analysis, 49 candidate proteins were tested for recruitment to Salmonella. Twelve candidates were recruited to BCVs upon infection. Of these twelve, I pursued five candidates in greater detail due to their recruitment to Salmonella being either entirely unknown, or known, but via a non-sphingomyelin mechanism. Further analysis of one candidate in particular, TECPR1, elucidated that TECPR1 is recruited to Salmonella in a sphingomyelin-dependent manner and possesses sphingomyelin-specific binding properties in vitro. Therefore, my thesis research identifies TECPR1 as an endogenous sphingomyelin-binding protein.
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Äijälä, M. (Meiju). "Studies about contribution of leptin receptor in cardiovascular risk." Doctoral thesis, Oulun yliopisto, 2013. http://urn.fi/urn:isbn:9789526203058.
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Abstract Leptin is a hormone secreted by adipose tissue. It is involved in the regulation of appetite and energy expenditure. Leptin binds to its receptor (LEPR) that is expressed in the central nervous system as well as in other tissues including adipocytes and endothelial cells. Plasma leptin level reflects the amount of adipose tissue and previously, it has been shown to be associated with the risk for coronary artery disease. Two LEPR polymorphisms, Lys109Arg and Gln223Arg, have been extensively studied and they have been associated with several risk factors of atherosclerosis. Earlier studies have also shown that the risk for developing atherosclerosis and various other diseases might already be determined during the fetal period or immediately after birth. It seems that intrauterine undernourishment might cause changes on epigenetic level and result in alterations in gene expression. It has been suggested that the impaired fetal growth could affect plasma leptin level and leptin messenger RNA expression from adipose tissue. Long-term fructose consumption has also been shown to result in leptin resistance. Recently, leptin has been observed to be associated with autophagy. Autophagy has been demonstrated to act in several interesting processes such as fat storage in adipocytes and liver. Autophagy and the leptin system might also regulate each another. The aim of this thesis was to investigate the association of LEPR polymorphisms with thickness of the wall of carotid artery as well as with fatal and nonfatal cardiovascular disease events. In addition, we aimed to clarify the effects of fetal undernourishment and fructose consumption on the leptin system and autophagy. We were also interested in studying the role of the leptin system and autophagy in elevated triglycerides and liver fat accumulation seen as a result of high-fructose diet. In our studies, we observed that LEPR polymorphisms, Lys109Arg and Gln223Arg, are associated with intima-media thickness of carotid artery. Moreover, 19-year follow-up study showed that 109Arg homozygotes display lower incidence of cardiovascular events and lower total mortality. In our animal experiments, we were able to detect that fructose diet affects both LEPR isoform and autophagy gene expression. It seems that these changes might partly explain the mechanism behind the rise in blood triglyceride levels and liver fat accumulation caused by fructose diet. In conclusion, the results of this study clarify the role of leptin receptor in cardiovascular diseases. In addition, they offer new information especially about the effects of fructose diet on the leptin system, the dysfunction of which might predispose to the development of diseases<br>Tiivistelmä Leptiini on rasvakudoksen tuottama hormoni. Se osallistuu ruokahalun ja energiankulutuksen säätelyyn. Leptiini sitoutuu reseptoriinsa (LEPR), joita on sekä keskushermostossa että muissakin kudoksissa, myös adiposyyteissä ja endoteelisoluissa. Plasman leptiinitaso heijastaa rasvakudoksen määrää ja sen on aiemmin osoitettu olevan yhteydessä sepelvaltimotaudin riskiin. Erityisesti kahta LEPR:n polymorfiaa, Lys109Arg ja Gln223Arg, on tutkittu aiemmin ja niiden on osoitettu olevan yhteydessä useisiin ateroskleroosin riskitekijöihin. Aiemmat tutkimukset ovat myös osoittaneet, että ateroskleroosiin ja useisiin muihin sairauksiin sairastumisen riski voi osittain määräytyä jo sikiöaikana tai varhain syntymänjälkeisen kehityksen aikana. Vaikuttaa siltä, että sikiöaikainen aliravitsemus voi aikaansaada muutoksia epigeneettisellä tasolla ja aiheuttaa näin muutoksia geeniekspressiossa. On ehdotettu, että sikiön heikentynyt kasvu vaikuttaisi plasman leptiinitasoon ja rasvakudoksen leptiinin lähetti-RNA:n ilmentymiseen. Pitkäaikaisen fruktoosinkulutuksen on myös osoitettu aiheuttavan leptiiniresistenssiä. Hiljattain leptiinin on havaittu olevan yhteydessä myös autofagiaan. Autofagian on osoitettu vaikuttavan useisiin kiinnostaviin prosesseihin, kuten rasvan varastoitumiseen adiposyytteihin sekä maksaan. Autofagia ja leptiinijärjestelmä mahdollisesti myös säätelevät toisiaan. Tämän väitöskirjan tavoitteena oli tutkia LEPR-polymorfioiden yhteyttä kaulavaltimon seinämän paksuuteen sekä sydän- ja verisuonitautitapahtumiin ja kuolleisuuteen. Pyrimme lisäksi selvittämään sikiöaikaisen aliravitsemuksen ja fruktoosin käytön vaikutusta leptiinijärjestelmään sekä autofagiaan ja olimme kiinnostuneita tutkimaan näiden osuutta fruktoosin kulutuksen seurauksena nähtävien metabolisten muutosten, kuten kohonneiden triglyseridien sekä maksan rasvoittumisen, synnyssä. Tutkimuksessamme havaittiin yhteys LEPR polymorfioiden Lys109Arg ja Gln223Arg sekä kaulavaltimon paksuuden välillä. Lisäksi 19-vuoden seurantatutkimus osoitti 109Arg-homotsygotian liittyvän pienentyneeseen sydän- ja verisuonitapahtumien ilmaantuvuuteen sekä matalampaan kokonaiskuolleisuuteen. Eläinmallissamme havaitsimme sekä LEPR-muotojen että autofagiageenien ilmentymisen muuttuneen fruktoosidieetin vaikutuksesta. Vaikuttaa siltä, että nämä muutokset voisivat osaltaan selittää esimerkiksi fruktoosiruokavalion aiheuttaman veren triglyseriditasojen nousun sekä maksan rasvoittumisen rotilla. Tutkimuksen tulokset selventävät leptiinireseptorin roolia sydän- ja verisuonitautien taustalla. Lisäksi ne tarjoavat uutta tietoa erityisesti fruktoosinkulutuksen vaikutuksesta leptiinijärjestelmään, jonka häiriöt altistavat sairauksien kehittymiselle
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LE, Thi Yen Loan. "Role of mineralocorticoid receptor regulation during experimental myocardial infarction." Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/10270.
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Ischaemic heart disease remains the leading cause of death worldwide. Following an ischaemic event, the primary strategy is to restore blood flow (reperfusion). However, this triggers release of reactive oxygen species, activation of stress-related gene transcription, autophagy and cell death processes leading to further injury (reperfusion injury). Elevated plasma aldosterone levels produce adverse cardiac effects, while mineralocorticoid receptor (MR) antagonists (spironolactone or eplerenone) reduce mortality, although mechanisms have not been defined. The aim of this thesis was to determine the role of MR regulation during experimental myocardial infarction (MI). This was achieved by using an ex-vivo isolated rat heart model and occluding a branch of the left coronary artery (30min) followed by reperfusion (150min). Increased levels of oxidative stress with activated autophagy and apoptosis confirmed our model of MI. Since there are sex differences in cardiac damage during MI, our studies show that androgens downregulate anti-apoptotic protein Bcl-xL, which shifts the balance towards apoptosis leading to aggravated cardiac damage in males compared to females. Expression levels of MR have been reported to be upregulated during MI in males, which could contribute to the aggravated damage, we did not find any significant change in MR expression between male and female rats and hence male rats were used for subsequent studies. Activation of MR by aldosterone (10 nM) increased cardiomyocyte apoptosis and aggravated infarct size during MI; prevented by low-dose MR antagonists. Low-dose (10 nM) spironolactone alone maintained redox balance, prevented activation of stress-related gene transcription and degradation of anti-apoptotic protein ARC, which prevented initiation of apoptosis. These studies provide direct evidence that MR activation aggravates cardiac damage during MI and provide mechanisms for the cardioprotective action of low-dose MR antagonists clinically.
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Blasi, Beriain Ignacio. "Porphyromonas gingivalis LPS stimulates autophagy using a TLR mediated pathway." Doctoral thesis, Universitat Internacional de Catalunya, 2017. http://hdl.handle.net/10803/461098.
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Resum:
Porphyromonas gingivalis often subverts host cell autophagic processes for its own survival. Our previous studies document the association of the cargo sorting protein, melanoregulin (MREG), with its binding partner, the autophagic protein, microtubule-associated protein 1 light chain 3 (LC3) in macrophages incubated with P. gingivalis (strain 33277). Differences in the lipid A moiety of lipopolysaccharide (LPS) affect the virulence of P. gingivalis; penta-acylated LPS1690 is a weak Toll-like receptor 4 agonist compared with Escherichia coli LPS, whereas tetra-acylated LPS1435/1449 acts as an LPS1690 antagonist. To determine how P. gingivalis LPS1690 affects autop- hagy we assessed LC3-dependent and MREG- dependent processes in green fluorescent protein (GFP)-LC3-expressing Saos-2 cells. LPS1690 stimu- lated the formation of very large LC3-positive vac- uoles and MREG puncta. This LPS1690-mediated LC3 lipidation decreased in the presence of LPS1435/1449. When Saos-2 cells were incubated with P. gingivalis the bacteria internalized but did not traffic to GFP-LC3-positive structures. Never- theless, increases in LC3 lipidation and MREG puncta were observed. Collectively, these results suggest that P. gingivalis internalization is not necessary for LC3 lipidation. Primary human gin- gival epithelial cells isolated from patients with periodontitis showed both LC3II and MREG puncta whereas cells from disease-free individu- als exhibited little co-localization of these two pro- teins. These results suggest that the prevalence of a particular LPS moiety may modulate the degradative capacity of host cells, so influencing bacterial survival.
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Rohatgi, Rasika. "Autophagy-Independent Role for Beclin 1 in the Regulation of Growth Factor Receptor Signaling: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/873.
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Beclin 1 is a haplo-insufficient tumor suppressor that is decreased in many human tumors. The function of Beclin 1 in cancer has been attributed primarily to its role in the degradative process of autophagy. However, the role of autophagy itself in tumorigenesis is context-dependent and can be both preventive and promoting. Due to its dual function in cancer a better understanding of this process is necessary to develop potential novel cancer therapies. To gain insight into the role of autophagy in breast carcinoma, I analyzed the autophagydependency of different subtypes of breast cancer. My results implicate that triple-negative breast carcinoma cells are more dependent on autophagy than luminal breast carcinoma cells. Chemical inhibition of autophagy decreased the tumorigenicity of triple-negative breast carcinoma cells with regard to proliferation and anchorage-independent growth. However, RNAi-mediated suppression of two autophagy genes, ATG5 and Beclin 1, revealed different outcomes. While suppression of ATG5 decreased glycolysis, Beclin 1 depletion did not affect the glycolytic rates. These results suggest autophagy-independent pro-tumorigenic effects of loss of Beclin 1 in cancer.
Beclin 1 is a core component of the Vps34/Class III PI3K (PI3KC3) and Vps15/p150 complex that regulates multiple membrane trafficking events. I describe a novel mechanism of action for Beclin 1 in breast cancer involving its control of growth factor receptor signaling. I identify a specific stage of early endosome maturation that is regulated by Beclin 1, the transition of APPL1- containing phosphatidyIinositol 3-phosphate-negative (PI3P-) endosomes to PI3P+ endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P-/APPL+ signaling competent compartment. As a result, suppression of BECN1 sustains growth factor stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, Beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Taken together my data identify a novel role for Beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of Beclin 1 expression would enhance breast cancer progression independent of its impact on autophagy.
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Lin, Ching-Yu. "LAMTOR2/LAMTOR1 complex is required for TAX1BP1-mediated xenophagy." Kyoto University, 2020. http://hdl.handle.net/2433/253144.
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Chen, Jinyun. "REGULATION OF INTRACELLULAR ARYL HYDROCARBON RECEPTOR PROTEIN LEVELS." Scholarly Commons, 2020. https://scholarlycommons.pacific.edu/uop_etds/3675.
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The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which controls tumor growth and metastasis, T cell differentiation, and liver development. Expression levels of this receptor protein are sensitive to the cellular p23 protein levels in immortalized cancer cell lines. As little as 30% reduction of the p23 cellular content can suppress the AHR function. Here we reported that down-regulation of the p23 protein content in normal, untransformed human bronchial/tracheal epithelial cells to 48% of its content also suppresses the AHR protein levels to 54% of its content. This p23-mediated suppression of AHR is responsible for the repression of (1) the ligand-dependent induction of the cyp1a1 gene transcription; (2) the benzo[a]pyrene- or cigarette smoke condensate-induced CYP1A1 enzyme activity, and (3) the benzo[a]pyrene and cigarette smoke condensate-mediated production of reactive oxygen species. Reduction of the p23 content does not alter expression of oxidative stress genes or production of PGE2. Down-regulation of p23 suppresses the AHR protein levels in two other untransformed cell types, namely human breast MCF-10A and mouse immune regulatory Tr1 cells. Collectively, down-regulation of p23 suppresses the AHR protein levels in normal and untransformed cells and can in principle protect our lung epithelial cells from AHR-dependent oxidative damage caused by exposure to agents from environment and cigarette smoking.
The AHR is expressed in triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with the estrogen receptor signaling. Normally the AHR is degraded shortly after ligand activation via the action of 26S proteasome. Here we report that the piperazinylpyrimidine compound Q18 triggers AHR protein degradation which is mediated through chaperone-mediated autophagy in triple-negative breast cancer cells (MDA-MB-468 and MDA-MB-231). This lysosomal degradation of AHR exhibits the following characteristics: (1) not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (2) inhibited by progesterone receptor B but not estrogen receptor alpha; (3) reversed by chloroquine but not MG132; (4) required LAMP2A; (5) triggered by 6 amino-nicotinamide and starvation and (6) involved AHR-LAMP2A interaction mediated by 6 amino-nicotinamide and starvation. The NEKFF sequence localized at amino acid 558 of human AHR is a KFERQ-like motif of chaperone-mediated autophagy, essential for the LAMP2A-mediated AHR protein degradation.
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Ruddy, Samantha. "Preferential Estrogen Receptor β Ligands Inhibit Proliferation and Reduce Bcl-2 Expression in Fulvestrant-resistant Breast Cancer Cells". Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23669.
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Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2.
Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland.
Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.
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Lopez, Corcino Yalitza Z. "Inhibition of Epidermal Growth Factor Receptor (EGFR) Leads to Autophagy-mediated Killing of Toxoplasma gondii and Control of Disease." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1560350001767936.
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Gao, Jianqun. "TLR2 and α-synuclein mediated pathology in human neuronal cell models". Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/20502.
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Parkinson’s disease (PD) is a progressively debilitating neurodegenerative disorder with the formation and development of Lewy bodies (LBs) and Lewy neurites (LNs) as its pathological characteristics. The most prominent and well-studied component of LBs and LNs is α-synuclein, which is thought to propagate through PD brain and contribute to neural dysfunction and clinical symptoms. The α-synuclein protein invades vulnerable neurons in the PD brain in a predictable, staged pattern. Recent evidence suggests that this propagation has some characteristics similar to the propagation of the prion protein, with distinct toxic α-synuclein species triggering the pathological conversion of normal endogenous α-synuclein in neighboring cells. However, the mechanism by which α-synuclein is taken up, accumulated and transferred within neurons remains to be fully defined. In this study I have used two different models of PD pathology, α-synuclein pre-formed fibrils (PFFs) and TLR2 activation with Pam3CSK4, to induce α-synuclein accumulation in differentiated SH-SY5Y cells and primary induced pluripotent stem cells (IPSCs)-derived neurons. Both cell models resulted in the accumulation of endogenous α-synuclein which peaked at 4-6 days post treatment. Such accumulation was absent in α-synuclein knockout SH-SY5Y cells, indicating the essentiality of endogenous α-synuclein for PD pathology. The PFF- or Pam3CSK4- triggered α-synuclein aggregation was associated with a temporal block in autophagy, as indicated by an increase in the selective autophagy markers p62/SQSTM1, LC3 and LAMP2, suggesting α-synuclein may be increased due to impaired macroautophagy / chaperone-mediated autophagy (CMA) clearance. Moreover, the accumulation of α-synuclein could be ameliorated by promoting autophagy with rapamycin or activators of the 5’ AMP-activated protein kinase (AMPK). TLR2 has recently been suggested as a receptor for endocytosis of α-synuclein. I generated TLR2 KO cells and observed an attenuation of α-synuclein accumulation as well as a suppression of inflammatory responses post Pam3CSK4 treatment in SH-SY5Y cells. Finally, a number of small molecule inhibitors targeting the TLR2 pathway were identified to ameliorate the accumulation of α-synuclein in neural cells, albiet less significantly than the AMPK agonists. These observations increase understanding of the mechanisms involved in PD pathology and provide a convenient model for the screening of potential therapeutics to prevent α-synuclein accumulation. These results suggested that targeting the AKT-mTORC1 and/or TLR2 signaling pathways might be potential therapeutic options for preventing α-synuclein accumulation in PD.
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Giorgetti, E. "DIFFERENT APPROACHES TO UNDERSTAND AND COUNTERACT SPINAL AND BULBAR MUSCOLAR ATROPHY (SBMA)." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/246561.
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Spinal and Bulbar Muscular Atrophy (SBMA), or Kennedy’s disease, is a hereditary neuromuscular disorder that affect only men and is characterized by slowly progressive weakness and atrophy of bulbar, facial, and limb muscles, which are attributable to degeneration of lower motor neurons in the spinal cord and brainstem. The disease is associated with an abnormally expanded CAG repeat in the androgen receptor (AR) gene which results in a longer polyglutamine tract (polyQ) at the N-terminus of the protein. PolyQ tract triggers AR protein misfolding and aggregation and leads to nuclear toxicity and cell death. Many efforts have been done to examine in depth disease pathogenesis and to find strategies to counteract polyQ AR toxicity but many aspects remain incompletely understood. Here we propose three different approaches aimed to contrast the disease.
In the first chapter, the anti-androgen Bicalutamide is used in combination with the autophagy activator trehalose, in order to slowdown polyQ AR nuclear translocation and promote its clearance through the cytoplasmic autophagic machinery. The combined treatment strongly reduces polyQ AR protein levels more than the single treatment, thus suggesting an autophagy-mediated clearance as a potential therapeutic strategy.
The second chapter focuses on polyQ AR degradation via the ubiquitin-proteasome system (UPS), another major degradation process within the cell. We stabilized Hsp70 in its more active ADP-bound state with the small molecule JG98 and promoted a selective CHIP-mediated ubiquitination and proteasomal degradation of polyQ AR. This confirms the involvement of UPS in SBMA pathogenesis and proposes UPS-mediated degradation of polyQ AR as a possible approach for the treatment of the disease.
The last chapter proposes exercise as a non-pharmacological intervention for the treatment of SBMA. Wild type (wt) and AR113Q knock-in mice followed a mild exercise regimen for six weeks, then mice were run to exhaustion and sacrificed. Muscle tissues analysis revealed a significant decrease in type I and II fiber size in the exercised cohort of AR113Q mice compared to control rest mice. These data suggested the existence of muscle abnormalities, possibly exacerbated by exercise, in AR113Q mice.
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Janota, Danielle Marie. "Alpha1-Adrenergic Receptor Activation Mimics Ischemic Postconditioning in Cardiac Myocytes." Kent State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=kent1406562863.
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Wang, Qian, and 王倩. "Mechanistic study of the transient receptor potential melastain 2 (TRPM2)-Ca²⁺ signaling in ROS induced switch between apoptosis and autophagy." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/206750.
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Autophagy is a major catabolic pathway for maintaining cell homeostasis through degradation and recycle of macromolecules and organelles. Autophagy can be activated under environmental stress conditions, including reactive oxygen species (ROS). TRPM2, a non-selective trans-membrane calcium channel, can be activated by ROS that, in turn, leads to intracellular 〖Ca〗^(2+) increase through 〖Ca〗^(2+) influx. It is well known that ROS regulates autophagy, and vice versa. Yet, the molecular mechanisms underlying the interplay between ROS and autophagy remain elusive. Here we studied the role of TRPM2-mediated 〖Ca〗^(2+) influx in interplay between ROS and autophagy.
From our study, we found that ROS activated TRPM2 for 〖Ca〗^(2+) influx via ADPR to inhibit early autophagy induction, which ultimately led to apoptosis in TRPM2 expressing cancer cell lines. On the other hand, ROS induced autophagy, not apoptosis, for cell survival in cancer cell lines which do not express TRPM2, and autophagy inhibition, either by ATG5 knockdown or by treating cells with bafilomycin A1 (an autophagy inhibitor), converted cells to apoptosis upon ROS treatment. In addition, ROS dramatically changed mitochondrial morphology, increased mitochondrial 〖Ca〗^(2+) content, and abolished mitochondrial membrane potential in TRPM2 expressing cells. Moreover, we found that ROS-induced Ca2+ influx via TRPM2 actually activated calmodulin-dependent protein kinase II (CaMKII) to phosphorylate Ser295 on Beclin1. Phosphorylated Beclin1, in turn, decreased the association between Beclin1 and VPS34, but induced the binding between Beclin1 and BCL-2. In summary, our data demonstrated that the TRPM2/〖Ca〗^(2+)/CaMKII/ Beclin1 cascade is the molecular switch between autophagy and apoptosis in response to ROS. Since dysregulation of ROS and autophagy has been associated with a variety of human diseases, e.g. cancer, neurological disorders, heart diseases, and liver diseases, manipulating the TRPM2/〖Ca〗^(2+)/CaMKII/ Beclin1 cascade should provide novel treatment option for these diseases.<br>published_or_final_version<br>Physiology<br>Doctoral<br>Doctor of Philosophy
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Nguyen, The Duy [Verfasser], Rolf [Gutachter] Marschalek, and Christian [Gutachter] Brandts. "The role of the selective autophagy receptor p62 in acute myeloid leukemia / The Duy Nguyen ; Gutachter: Rolf Marschalek, Christian Brandts." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2018. http://d-nb.info/1157097979/34.
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Lajoie, Patrick. "Regulation of receptor signaling and membrane trafficking by beta1,6-branched n-glycans and caveolin-1/cholesterol membrane domain organization." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/336.
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Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis.
Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor.
I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis
Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions.
In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases.
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Schuh, Mélanie. "Caractérisation des voies de signalisation contrôlées par les androgènes dans le muscle strié chez la souris." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ106/document.
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Les muscles permettent de générer force et mouvements et ont des fonctions métaboliques importantes. Mon travail a consisté à caractériser le rôle et les mécanismes d’actions des androgènes dans le muscle strié. Nous avons montré que l’ablation du récepteur des androgènes dans les myofibres n’affecte pas la masse musculaire car à la fois les voies anaboliques (IGF1) et cataboliques (myostatine) sont dérégulées. Cependant, l’absence du récepteur dans les myofibres diminue l’hypertrophie musculaire induite par une surcharge mécanique et limite l’atrophie induite par les glucocorticoïdes. Son ablation augmente également l’autophagie, entrainant une déstructuration des sarcomères, conduisant à une diminution de la force musculaire. De plus, sa délétion diminue la vitesse d’absorption du glucose lors d’une surcharge glucidique. Le récepteur des androgènes dans les myofibres régule donc la masse et la force musculaire, ainsi que l’import du glucose<br>Muscles generate strength and movement, and have important metabolic functions. The aim of my work was to characterize the role and mechanisms of action of androgen receptor in skeletal muscle. We show that ablation of the androgen receptor in skeletal muscle myofibers does not affect muscle mass as both anabolic (IGF1) and catabolic pathways (myostatin) are deregulated. However, the absence of this receptor in myofibers decreases muscle hypertrophy induced by mechanical overload and limits glucocorticoids-induced muscle atrophy. Its ablation also increases autophagy, leading to sacromeres destructuration, resulting in decreased muscle strength. Moreover, its deletion reduced the rate of glucose absorption during a glucidic overload. Thus, myofibres androgen receptor regulates muscle mass and strength, as well as glucose import
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Yang, Yujie. "POST-TRANSLATIONAL MODIFICATION AND DEGRADATION MECHANISMS OF THE ARYL HYDROCARBON RECEPTOR." Scholarly Commons, 2021. https://scholarlycommons.pacific.edu/uop_etds/3753.
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The aryl hydrocarbon receptor (AHR) is a transcription factor first discovered to be activated by exogenous ligands, such as dioxins, and helps promote downstream gene (e.g. CYP1A1) transcription to metabolize the toxicants. With the reports of various AHR targets genes, the expression levels and activities of AHR have been implicated in many physiological and pathological situations. Understanding how AHR protein level is regulated would provide more information to target AHR. AHR stays in the cytosol in the absence of ligand in a complex with HSP90, p23 and XAP2. After ligand activation, AHR translocates into the nucleus, fulfilling its transactivation function and then is finally degraded by proteasomes. Here, we discovered a new mechanism that controls basal AHR protein level: the selective autophagy. Loss of AHR co-chaperone p23 leads to increased protein degradation of AHR through autophagy in HeLa cells. Inhibition of autophagy using several inhibitors (chloroquine, bafilomycin A1 or 3-methyladenine) increased AHR protein levels. Knocking down of key macroautophagy protein LC3B increases AHR protein levels and decreases the responsiveness of AHR to CQ treatment. The interaction between AHR and LC3B as well as AHR and autophagy receptor p62 were confirmed in vitro and in situ. AHR is found to be lysine (K) 63-ubiquitinated in HeLa cells, which is a common signal for the autophagy-lysosomal degradation.6 We also discovered that AHR is controlled by glycogen synthase kinase 3β (GSK3β) phosphorylation. Inhibition of GSK3β activity or its expression level increased AHR protein levels while expression of HA tagged-GSK3β lowers AHR protein levels. AHR protein level is regulated through autophagy. We confirmed the GSK3β-mediated phosphorylation of AHR by phos-tag gel electrophoresis couples with Western blot analysis and identified three putative phosphorylation sites of AHR in the C-terminal half of AHR sequence. Moreover, phosphorylated AHR constitutes the active pool for transactivation and phosphorylation tagged AHR for the autophagy-lysosomal degradation, which may act as way to limit its function.
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Eimer, Sandrine. "Etude des réponses induites par l’erlotinib dans des cellules de lignées de glioblastome." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21822/document.
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Le glioblastome (GBM), tumeur de plus haut grade du système nerveux central (OMS grade 4) a un pronostic très sombre, quelque soit le traitement, lié à une résistance à l’apoptose. L’erlotinib (Tarceva®, OSI 774) est un inhibiteur de la tyrosine kinase du récepteur au facteur de croissance épithélial (EGFR). L’hyper-expression et l’amplification du gène de l’EGFR dans 40 à 60% des GBM, fourni un rationnel pour utiliser l’erlotinib. Nous avons montré sur U87-MG et DBTRG-05MG, deux lignées de GBM, l’absence d’apoptose avec l’erlotinib, liée soit à un déficit en pro-caspase 3, soit à une accumulation d’αB-crystalline bloquant l’activation de la caspase-3. L’absence d’apoptose dévie alors la cellule vers l’autophagie. L’inhibition de l’autophagie par ARN interférents ou par la chloroquine permet d’obtenir une synergie avec l’erlotinib en induisant la mort des cellules tumorales à des doses acceptables.Les GBM ont composition cellulaire hétérogène, avec un petit nombre d’éléments appelés cellules souches cancéreuses (CSC). Douées d’auto-renouvellement, elles participent à la propagation tumorale et à la résistance aux traitements. Nous avons testé l’erlotinib sur trois lignées issues de GBM humains, ayant deux modes de croissance distincts selon les conditions de milieu: en neurosphères (NS) et de type adhérent. Erlotinib a un effet inhibiteur minime sur les trois lignées adhérentes, alors que l’effet est significatif sur les lignées NS, traduisant l’importance de la voie d’EGFR pour les NS. Dans les lignées en NS, l’erlotinib est efficace sur les cellules progénitrices, mais n’a pas d’action ni sur les cellules initiatrices de NS, ni sur les cellules différenciées. L’auto-renouvellement des NS n’est pas non plus altéré. L’association cyclopamine, inhibiteur pharmacologique de la voie de Hedgehog, -erlotinib est synergique en bloquant la croissance et l’initiation des NS, laissant présager une efficacité sur les CSC. Les résultats obtenus sur ces différents modèles permettent d’une part de préciser certains mécanismes de résistance des cellules de GBM, et aussi d’orienter les indications et le choix des traitements susceptibles d’être les plus efficaces<br>Glioblastoma (GBM) is the most common primary central nervous system tumor in adults and the prognosis remains dismal, any treatment used. Epidermal Growth Factor Receptor (EGFR) is amplified, overexpressed, and/or mutated in GBM, making it a rational for therapy. Erlotinib, an EGFR kinase inhibitor is strongly associated with clinical response in several cancers. We showed for U87-MG and DBTRG-05MG, two human GBM cell lines, that erlotinib can’t trigger apoptosis, related either to accumulation of αB-crystallin capable to impair caspase 3 cleavage, or to constitutive deficit for procaspase 3 in DBTRG-05MG. Apoptosis deficit switches the cell to autophagic process. Inhibition of autophagy with RNA interference or chloroquine resulted in sensitization of U87 and allowed a synergistic effect with erlotinib at therapeutic doses.Moreover, GBM showed a heterogeneous cell composition with cancer stem cells, progenitors and more differentiated cells. In this study, we test erlotinib in vitro on other GBM models: three cell lines established from surgically resected GBM specimens, grown along two features adherent and neurospheres. On the three differentiated adhering cell lines, erlotinib had only a moderate activity. Conversely, on neurosphere forming cell lines, erlotinib induced a strong inhibition of cell growth related to the EGFR amplification and EGFR expression. A short erlotinib exposure induced cell death primarily in nestin-positive cells; however it was found without effect on neurosphere initiating activity and self renewal. These results suggest that EGFR pathway activation is essential for the proliferation of GBM progenitor cells but dispensable for stem-like cancer cells self–renewal. As Hedgehog pathway is known to be activated in neural stem cells, we assayed the Hedgehog pathway inhibitor cyclopamine in association with erlotinib. While each drug separately was without effect on sphere initiation, their combination led to a 25 fold decrease in the sphere number (p=0.0004).These in vitro models are convenient to investigate resistance mechanisms in GBM. Furthermore, they focus on the necessity to exploit drug combinations for greatest efficiency
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Ségala, Grégory. "Caractérisation des mécanismes moléculaires impliqués dans l'activité anti-cancéreuse du Tamoxifène et de la Dendrogénine A." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1694/.
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Le tamoxifène (Tam) est l'un principaux médicament utilisé pour le traitement des cancers du sein exprimant les récepteurs des œstrogènes (ER). Des résistances au Tam limitent son utilisation thérapeutique et l'identification des mécanismes responsables de ces résistances nécessite une connaissance approfondie de la pharmacologie du Tam. L'ER est la cible la mieux connue du Tam mais d'autres cibles existent parmi lesquelles le site de liaison des anti-oestrogènes (AEBS : AntiEstrogen Binding Site). L'équipe de Marc Poirot a montré qu'AEBS est impliqué dans les effets anti-cancéreux du Tam par un mécanisme qui fait intervenir le métabolisme des stérols. Au cours de ma thèse, nous avons découvert qu'AEBS porte l'activité enzymatique cholestérol époxyde hydrolase (ChEH) qui catalyse la transformation des cholestérol-5,6-époxydes (5,6-CE) en cholestane-3,5,6-triol. Nous avons montré que le Tam induit une production de 5,6-CE dans les cellules cancéreuses mammaires et stimule leur accumulation en inhibant la ChEH. Les métabolites de 5,6-CE sont des modulateurs du récepteur nucléaire des oxystérol LXRß. Nous avons caractérisé l'implication de LXRß dans les effets différenciants et cytotoxiques du Tam et montré une dérégulation de cette voie dans une lignée cellulaire résistante au Tam. En parallèle, nous avons découvert que le 5,6-CE alpha est métabolisé dans les tissus sains en Dendrogénine A (DDA), qui est le premier alkaloïde stéroïdien découvert chez les mammifères, et qui est absente dans les tissus tumoraux ce qui suggère un lien entre le métabolisme de la DDA et l'oncogenèse. Nous avons observé que la DDA a une forte activité anti-tumorale sur des cancers du sein et sur des mélanomes métastatiques en provoquant la différenciation et la mort cellulaire, ce qui a motivé son développement pour une utilisation thérapeutique. Nous avons identifié que LXRß est une cible directe de la DDA et nous avons établi que la cytotoxicité de la DDA dépend de LXRß et fait intervenir une apoptose ainsi qu'une autophagie cytotoxique. La caractérisation des mécanismes d'action du Tam et de la DDA pourront permettre une utilisation thérapeutique optimale de ces deux molécules ainsi que le développement de nouvelles thérapies anti-cancéreuses personnalisées<br>Tamoxifen (Tam) is one the leading drug used for the treatment of estrogen receptor (ER)-positive breast cancers. Resistances to Tam limit its therapeutic use and the identification of mechanisms involved in these resistances requires an accurate knowledge of its pharmacology. ER is the best-known target of Tam but other targets exist such as the Antiestrogen Binding Site (AEBS). Marc Poirot's team showed that AEBS is involved in the anti-cancerous effects of Tam by a mechanism that induced a perturbation in sterol metabolism. During my thesis, we discovered that the AEBS carried out the cholesterol-5,6-epoxide hydrolase (ChEH) activity which catalyzes the transformation of 5,6-epoxy-cholesterol (5,6-EC) into cholestane-3,5,6-triol. We showed that Tam induced the production of 5,6-EC in tumor cells and stimulated their accumulation through ChEH inhibition. We found that some 5,6-EC metabolites were modulators of the oxysterols nuclear receptor LXRß. We characterized the involvement of LXRß in the anticancer action of Tam and showed a deregulation of this oxysterol signaling pathway in a Tam-resistant cell line. At the same time, we discovered that one 5,6-EC was metabolized in normal tissue into Dendrogenin A (DDA), the first steroidal alkaloid discovered in mammals, but DDA was found absent in neoplastic tissues, suggesting a link between DDA metabolism and oncogenesis. We found that DDA has a strong anti-tumor potency on breast cancers and metastatic melanoma through the induction of tumor cell differentiation and death, which prompted its development for clinical applications. We demonstrated that LXRß is a direct target of DDA and established that the cytotoxicity of DDA was LXRß-dependent and involved apoptosis and cytotoxic autophagy. The characterization of the mechanisms of action of Tam and DDA will allow an optimal therapeutic use of these two molecules and also the development of new personalized anti-cancer therapies
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Peng, Luo-Gen [Verfasser]. "Urokinase-type plasminogen activator receptor contributes to chemosensitivity and epithelial-to-mesenchymal transition in PDAC : uPAR and p38 regulate autophagy dependent gemcitabine resistance in AsPC1: autophagy inhibitors and gemcitabine as a potential combined therapy for a subgroup of pancreastic cancers / Luogen Peng." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1221802313/34.
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Peng, Luogen [Verfasser]. "Urokinase-type plasminogen activator receptor contributes to chemosensitivity and epithelial-to-mesenchymal transition in PDAC : uPAR and p38 regulate autophagy dependent gemcitabine resistance in AsPC1: autophagy inhibitors and gemcitabine as a potential combined therapy for a subgroup of pancreastic cancers / Luogen Peng." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1221802313/34.
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Berleth, Niklas [Verfasser], Björn [Akademischer Betreuer] Stork, and Thomas [Gutachter] Klein. "NRBF2, a novel component of the class III PtdIns3K complex, regulates starvation-induced autophagy and nuclear receptor-mediated gene expression / Niklas Berleth ; Gutachter: Thomas Klein ; Betreuer: Björn Stork." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1172968012/34.
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Laurent, Anne-Coline. "Rôles et mécanismes d’action de la protéine Epac dans l’hypertrophie cardiaque." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T044/document.
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Les catécholamines induisent la synthèse d’AMPc par une stimulation des récepteurs β-adrénergiques et contrôlent ainsi la fonction cardiaque en activant une pléiade de voies de signalisation intracellulaires. Les protéines Epac sont des facteurs d’échange pour les petites protéines G et sont directement activés par l’AMPc. Devant l’importance de la voie β-adrénergique dans la physiopathologie cardiaque et dans le but de mieux comprendre la régulation des processus cellulaires dépendants de l’AMPc dans le cœur, il apparaît essentiel de caractériser le rôle des facteurs d’échange Epac dans le myocarde. Dans une première partie, cette étude démontre que les effets de Epac sur l’hypertrophie des cardiomyocytes ventriculaires de rats nouveaux nés requièrent les GTPases H-Ras et Rap2B. Epac active la voie PLC/IP3/Ca2+ qui est nécessaire pour l’activation de H-Ras. Au niveau transcriptionnel, Epac induit l’export nucléaire de HDAC4 permettant l’activation d’un programme génique d’hypertrophie. Dans une deuxième partie, cette étude révèle l’implication de Epac1 dans l’hypertrophie des cardiomyocytes in vivo, chez la souris. La délétion de Epac1 protège du remodelage cardiaque induit par l’activation prolongée des récepteurs β-adrénergiques et améliore la fonction cardiaque. La surexpression de Epac1 spécifiquement dans le myocarde entraîne une hypertrophie des cardiomyocytes. Par ailleurs, la voie β-AR/Epac1 induit l’accumulation de protéines ubiquitinylées et provoque l’activation du processus d’autophagie in vitro et in vivo. L’autophagie protège des effets délétères de la voie β-adrénergique/Epac en participant à l’élimination des agrégats protéiques et en contrant les effets hypertrophiques de Epac1. Ces résultats ouvrent de nouvelles perspectives pour le traitement de l’hypertrophie et de l’insuffisance cardiaque<br>Catecholamines regulate cardiac function by stimulating β-adrenergic receptors (β-AR), leading to cAMP production and activation of a multiplicity of signaling pathways. Epac proteins are exchange factors for small G proteins which are directly activated by cAMP. Given the importance of the β-adrenergic pathway in cardiac physiopathology, it becomes essential to characterize functions of Epac protein in myocardium. In a first part, this study shows that H-Ras and Rap2B GTPases are involved in Epac-induced neonatal rat cardiac myocytes hypertrophy. Epac induces activation of the PLC/IP3/Ca2+ pathway which is necessary for H-Ras activation. At the transcriptional level, Epac causes HDAC4 nuclear export leading to activation of a hypertrophic gene program. In a second part, this study reveals implication of Epac1 in cardiac hypertrophy in vivo. Deletion of Epac1 in mice protects from cardiac remodeling induced by chronic isoproterenol infusion and enhances cardiac function. Cardiac specific overexpression of Epac1 in mice induces cardiac myocytes hypertrophy. Interestingly, β-AR/Epac1 pathway triggers ubiquitinated proteins accumulation and activation of autophagy both in vitro and in vivo. By eliminating aggregates and by counteracting hypertrophic effects of Epac, autophagy protects from deleterious effects of the β-AR/Epac pathway. These results open news insights into the treatment of cardiac hypertrophy and heart failure
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Sahin, Katherine B. "Evaluation of cell division cycle associated protein 3 (CDCA3) as a novel prognostic/therapeutic target for EGFR-mutant non-small cell lung cancer." Thesis, Queensland University of Technology, 2022. https://eprints.qut.edu.au/231468/1/Katherine_Sahin_Thesis.pdf.
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This thesis defined a unique role for the protein cell division cycle associated protein-3 (CDCA3) in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). This thesis has established an association between the levels of CDCA3 expression and the tumour response to tyrosine kinase inhibitors (TKI), which are the front-line therapy for EGFR-mutant NSCLC. In this disease, CDCA3 functions to modulate cellular growth pathways to impact sensitivity towards TKI therapy. Future work might enable development of a clinical stratification tool to discern TKI responsive from non-responsive EGFR-mutant NSCLC tumours.
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Weng, Shu-Chuan. "Preclinical exploration of novel small molecules as anticancer agents in triple-negative and HER2/neu-positive breast cancers." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1227727553.
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Morelli, E. "NOVEL FUNCTIONS OF THE SNARE PROTEIN SNAP29IN MEMBRANE TRAFFICKING AND CELL DIVISION." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/265475.
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Vesicular trafficking within cells is an important process for tissue development and homeostasis. A key step of vesicular trafficking is the fusion between two membranes, a process in which SNARE (Soluble NSF Attachment Protein Receptors) proteins play a fundamental role. SNAP29 (SyNaptosomal Associated Proteins 29) is a ubiquitous SNARE, regulating membrane fusion in different trafficking compartments and in different contexts in non dividing cells. We isolated a loss of function mutant in usnp, the gene encoding the Drosophila homolog of the human protein SNAP29 (Snap29 hereafter), that, when made homozygous in developing epithelial organs, disrupts epithelial architecture. In vivo, we find that Snap29 interacts with multiple SNARE proteins, localizes to a number of trafficking organelles, and is required for proper Golgi Apparatus morphology. In addition, we show that Snap29 is required for fusion of autophagosomes with lysosomes together with Syx17 and Vamp7, and that lack of Snap29 results in excess secretion, suggesting that Snap29 might act negatively in regulation of vesicle fusion at the plasma membrane.
Interestingly, at the onset of mitosis, when trafficking compartments re-shape to allow the formation of the mitotic spindle, Snap29 is found at the outer KT in Drosophila S2 cells and localizes at spindle microtubules and centrosomes in mammalian cells. Depletion of Snap29 in Drosophila and mammalian cells leads to spindle assembly defects, associated to pro-metaphase delay in mammalian cells, and to the formation of daughter cells containing mininuclei. Mechanistically, lack of SNAP29 correlates with absence at KT of ZWINT-1 and ZWILCH, a component of RZZ complex, and with weak KTs-MTs attachments. In addition, we find that SPINDLY, the adaptor for recruitment to KTs of dynein/dynactin and MAD1, a component of the Spindle Assembly Checkpoint machinery, fail to be removed from KTs at the end of metaphase in SNAP29 depleted mammalian cells forced to reassemble the spindle after treatment with microtubules depolymerization drug.
Finally, we show that cell division is impaired in Snap29 mutant tissues in vivo, that autophagy defects are not the cause of the altered epithelial tissues architecture in Snap29 mutants and that the trafficking and cell division function of Snap29 are molecularly distinct.
All together our findings support a role of Snap29 at key steps of membrane trafficking and in cell division. Our study contribute to shed light on the pathogenesis of CEDNIK, a human congenital syndrome caused by SNAP29 inactivation. In addition to this, we propose that the function of SNAP29 in cell division might be evolutionarly related to that of complexes tethering MTs to vesicular organelles in interphase. We surmise that such function could be potentially relevant to development of aneuploidy in tumor-like masses.
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"Expression patterns of estrogen receptor isoforms in thyroid cancer and the role of estrogen receptor alpha in autophagy of thyroid cancer cells." 2013. http://library.cuhk.edu.hk/record=b5884401.
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Fan, Dahua.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.<br>Includes bibliographical references (leaves 117-155).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts also in Chinese.
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Rocha, Mariana Botelho da. "Autophagy in the hypothalamus: role of Neuropeptide Y and impact on Synaptic Plasticity." Doctoral thesis, 2016. http://hdl.handle.net/10316/29288.
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Tese de doutoramento em Ciências Farmacêuticas, na especialidade Farmacologia e Farmacoterapia, apresentada à Faculdade de Farmácia da Universidade de Coimbra<br>O hipotálamo é uma região do cérebro que regula o desenvolvimento, o crescimento e o metabolismo. Recentemente, foi também demonstrado que o hipotálamo desempenha um papel chave no desenvolvimento generalizado do envelhecimento. A autofagia é um processo intracelular envolvido na reciclagem dos constituintes da célula e na manutenção da homeostase celular. Durante o envelhecimento e em doenças associadas ao envelhecimento ocorre diminuição da autofagia.
Por outro lado, em diversas espécies de animais, a restrição calórica (RC) é uma robusta intervenção anti-envelhecimento, aumentando o tempo de vida e diminuindo a incidência de doenças associadas à idade. A RC estimula a autofagia e também aumenta os níveis do neuropeptídeo Y (NPY) no hipotálamo. Diversos trabalhos mostram que o NPY tem um papel neuroprotector, aumenta a resistência ao stress. Contudo, o papel do NPY na autofagia nunca foi investigado.
Desta forma, o primeiro objectivo deste trabalho foi estudar o papel do NPY na autofagia em neurónios do hipotálamo. Os resultados mostraram que o NPY estimula a autofagia numa linha de neurónios hipotalâmicos de murganho (mHypo-N42) e também em culturas primárias de células hipotalâmicas neurais diferenciadas de rato. O NPY aumentou o fluxo autofágico em neurónios do hipotálamo através da activação dos receptores Y1 ou Y5, e que activam as vias de sinalização intracelular PI3K, ERK e PKA. O efeito do NPY na autofagia num modelo in vivo também foi avaliado, através da sobre-expressão do NPY no núcleo arqueado do hipotálamo de murganhos C57BL/6, pela tecnologia de transferência génica usando vírus adenoassociados. Os resultados mostraram que o NPY também estimula a autofagia no hipotálamo in vivo.
O núcleo arqueado do hipotálamo, responsável pela homeostase energética, é composto por duas populações neuronais distintas – neurónios que expressam POMC e CART, e neurónios que expressam NPY e AgRP. Estas duas populações regulam o anabolismo e catabolismo, recebendo e integrando sinais nutricionais e hormonais da periferia. Estudos recentes sugerem que a plasticidade sináptica dos circuitos hipotalâmicos envolvidos na ingestão alimentar também tem um papel na regulação da homeostase energética. Contudo, o papel da autofagia na plasticidade dos circuitos hipotalâmicos nunca foi investigado.
Desta forma, o segundo objectivo deste trabalho foi investigar o papel da inibição específica de uma proteína fundamental do processo de autofagia, a Atg7, na organização sináptica, com uma dieta normal e em privação de alimentos. Os murganhos com inibição específica da proteína Atg7 nos neurónios POMC (POMC-Cre; Atg7loxP/loxP) foram usados como modelo animal de estudo e os murganhos com expressão inalterada de Atg7 (Atg7loxP/loxP mice) como controlos. Nestes animais avaliou-se a organização sináptica dos neurónios POMC. Os animais foram mantidos durante cerca de 10 semanas com acesso livre a uma dieta padrão ou sem acesso a comida durante uma noite. O núcleo arqueado do hipotálamo destes animais foi analisado por microscopia electrónica, microscopia de fluorescência e por microscopia óptica de luz visível.
Os neurónios hipotalâmicos POMC dos murganhos POMC-Cre; Atg7loxP/loxP, com ausência de Atg7 nos neurónios hipotalâmicos POMC, apresentaram um aumento da área e do perímetro desses neurónios, e apresentaram acumulação de nematossomas. Além disso, os neurónios hipotalâmicos POMC dos murganhos POMC-Cre; Atg7loxP/loxP apresentaram mais contactos sinápticos, que se traduzem num aumento dos contactos simétricos inibitórios. Depois de uma noite sem acesso a comida, os neurónios do núcleo arqueado do hipotálamo dos murganhos POMC-Cre; Atg7loxP/loxP apresentaram menor imunorreactividade para c-Fos, que sugere menor activação neuronal.
Em conclusão, os resultados desta tese mostram que o NPY induz o fluxo autofágico em neurónios do hipotálamo, e que a autofagia desempenha um papel na regulação da plasticidade sináptica dos neurónios POMC. Uma vez que a autofagia no hipotálamo e os níveis do NPY diminuem com o envelhecimento, a modulação do NPY pode ser um mecanismo protector contra a disfunção hipotalâmica associada ao aumento da idade. Por outro lado, a modulação da autofagia, através de um mecanismo sináptico subjacente, pode oferecer estratégias para a regulação do peso corporal.<br>The hypothalamus is the brain region that regulates development, growth and metabolism, and has gained increased attention for its key role in the progression of whole body aging. Additionally, autophagy, a highly regulated intracellular process involved in the turnover of most cellular constituents and in the maintenance of cellular homeostasis, is impaired in aging, contributing to the aging phenotype and to the aggravation of age-related diseases.
On the other hand, caloric restriction (CR) is a robust anti-aging intervention, increasing lifespan and decreasing the incidence of age-related diseases. CR increases autophagy in different brain areas and increases neuropeptide Y (NPY) levels in the hypothalamus. Moroever, NPY has neuroprotective effects and increases resistance to stress and mean lifespan. However, the role of NPY on autophagy has never investigated before.
Therefore, the first aim of this study was to investigate the role of NPY on autophagy in hypothalamic neurons. The results show that NPY stimulated autophagy in mouse hypothalamic cell line N42 (mHypo-N42) and also in rat differentiated hypothalamic neural cell cultures. Moreover, NPY stimulated the autophagic flux in hypothalamic neurons by activating NPY Y1 or Y5 receptors, through PI3K, ERK and PKA intracellular signaling pathways. We also evaluated the role of NPY on autophagy in vivo, by overexpressing NPY in the arcute nucleus (ARC) of hypothalamus of C57BL/6 mice, using adenoassociated viral (AAV) gene transfer technology. The results show that NPY also stimulated autophagy in hypothalamus in vivo.
The hypothalamic ARC, responsible for energy homeostasis, is composed by two major neuronal populations – cocaine- and amphetamine-regulated transcript (CART)/Pro-opiomelanocortin (POMC) expressing neurons and agouti-related peptide (AgRP)/ neuropeptide Y (NPY) expressing neurons. These two neuronal populations regulate anabolic and catabolic state, receiving and integrating peripheral nutritional and hormonal signals. Recent observations suggest that synaptic plasticity in the hypothalamic feeding circuits has also a critical role in regulation of energy homeostasis, since the neuronal synaptic input organization in the hypothalamus is able to adapt and rearrange rapidly in response to metabolic hormones. In addition, autophagy in the hypothalamus was identified as a player in metabolic regulation. However, a role for autophagy in plasticity of hypothalamic feeding circuits has not been explored.
Therefore, the second aim of this study was to investigate the role of Atg7 deletion in POMC neurons in the synaptic organization in mice under standard diet and food deprivation. POMC-specific Atg7 knockout mice (POMC-Cre; Atg7loxP/loxP) were used as animal model and Cre-negative Atg7loxP/loxP mice as controls, to evaluate the synaptic organization of the hypothalamic POMC neurons and neuronal activation in hypothalamic ARC. Animals were maintained during 10 weeks with standard diet or overnight fasting, and then brains slices containing arcuate nucleus of the hypothalamus were stained for electron microscopy and for fluorescence and light microscopy.
The specific Atg7 deletion in POMC neurons resulted in an increased cell area and perimeter, and nematosomes accumulation. Moreover, we observed that POMC-Cre; Atg7loxP/loxP neurons have more synaptic inputs and more symmetric, putatively inhibitory inputs. After an overnight fasting, POMC-specific Atg7 knockout mice show no normal adaptation to food deprivation, with an impaired neuronal activation in hypothalamic ARC.
Overall, these results show that NPY induces autophagic flux in hypothalamic neurons, and that autophagy has a role in the control of synaptic plasticity of POMC neurons. Since both hypothalamic autophagy and NPY levels decrease with age, modulation of NPY may act as a protective mechanism against impaired hypothalamic dysfunction associated with age. Moreover, autophagy modulation, through underlying synaptic mechanism, might offer strategies to the body weight regulation.<br>FCT - SFRH/BD/73004/2010
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Lin, Yi-Sheng, and 林易陞. "Mechanism study on autophagy cargo receptor Joka2-involved chloroplast degradation." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/68929480746577128696.
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碩士<br>國立中興大學<br>生物化學研究所<br>105<br>Plant selective autophagy plays a significant role in stress responses, delay aging and nutrient shortage. The main function of selective autophagy is to recycle the specific elements like organelles, aggregated proteins or specific proteins. Previous studies indicated that phytoene desaturase (pds) silencing not only lead to the decrease of carotenoid and chlorophyll contents, but also cause an albino phenotype. Here, we characterized the relationship between pds silencing and leaf albino were characterized. We observed the number of chloroplasts was reduced in the albino leaves via tissue slices. Chloroplast degradation has been reported via Rubisco-Containing Bodies (RCBs), which was regarded as autophagy. However, it is still unknown how specific chloroplast substrates are recognized and obtained in RCBs. In this study, we found that the expression level of cargo receptor Joka2 was highly increased in albino leaves. Moreover, in Joka2 silencing plant, vesicle-like structures were observed and accumulated after Amitrole, a PDS inhibitor, treatment. Taking together, these results suggest that Joka2 is involved in chloroplast degradation.
It has been known that Xanthomonas can interfere to plant immunity by virulence effectors, which are secreted via type III secretion system. The overexpression of XopN(Xanthomonas outer protein N) and XopQ effectors in leaves could trigger severe hypersensitive response (HR) and cause cell death in tobacco leaves. In this study, we found that HR can also induce the expression of Joka2. Thus, these results suggest that Joka2 is also involved in HR triggered by Xanthominas type III effectors.
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Balounová, Jana. "Toll like receptory a myeloidní buňky ve vývoji a nemoci." Doctoral thesis, 2014. http://www.nusl.cz/ntk/nusl-342350.
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Toll like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to pathogens. Primarily they are responsible for induction and regulation of the innate and adaptive immune responses whereby the effector function is executed chiefly by differentiated myeloid cells. Somewhat unexpectedly, TLRs have been also shown to be involved in direct pathogen sensing by bone marrow-derived hematopoietic stem cells (HSCs) and hematopoietic progenitors when, under inflammatory conditions, the rapid generation of innate immune effector cells that effectively combat the infection is of utmost priority. While it has been recognized that the release of inflammatory cytokines from inflamed tissues along with the changes in proportions of differentiating cells in the bone marrow (BM) as well as the BM niche can nudge the differentiation of adult BM-derived cells towards myeloid cells and granulocytes, a direct role of TLRs expressed by HSCs in this process has been demonstrated only recently. However, whether a similar mechanism operates also during embryonic hematopoiesis is unknown. Here we show that TLRs and their adaptor proteins are functionally expressed during early stages of embryogenesis by short-lived maternally-transferred...
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Tung, Ying-Tsen, and 董盈岑. "The role of the autophagic cargo receptor p62 in the clearance of aggregation-prone proteins." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/80367896685135791504.
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博士<br>國立臺灣大學<br>動物學研究所<br>101<br>The accumulation of certain misfolded protein aggregates in the brain is a common feature in various neurodegenerative diseases, and is accepted as a major causative factor of neurodegeneration. Aggrephagy, the process by which protein aggregates are selectively degraded through macroautophagy, plays an essential role in protecting neurons from aggregate-induced neurotoxicity. Recent findings have identified p62/sequestosome1 as a cargo receptor that interacts with the autophagosomal membrane associated protein LC3, and recruits ubiquitin-positive protein aggregates into autophagosomes. The finding that p62 is co-localized with inclusion bodies in the brains of patients with Huntington’s disease (HD) and Alzheimer’s disease (AD) suggests a critical role for p62 in neurodegeneration.
Previous findings have identified residues in a yeast LC3 homologue, Atg8, that are essential for interaction of Atg8 with the cargo receptor Atg19 in selective autophagic processes. In the first part of my thesis, I describe our attempts to determine whether such interaction is evolutionally conserved from yeast to mammals. By using an amino acid replacement approach, we determined that three residues in LC3 corresponding to those in Atg8 were essential for p62 binding. Furthermore, while disruption of the LC3-p62 complex formation did not alter overall autophagic activity, it was sufficient to impede the autophagy-mediated clearance of aggregation–prone mutant Huntingtin (Htt), the cytotoxic protein which induces the pathological phenotypes of HD.
The protective role of p62 in the clearance of aggregation-prone proteins prompted us to investigate how p62 expression is regulated under pathological conditions. In the second part of my thesis, I describe our discovery that p62 expression is transcriptionally regulated by presenilin 1 (PS1), a protein which is mutated in the majority of patients with early-onset familial Alzheimer’s disease (FAD). The PI3K/Akt/AP-1 pathway was found to be required for PS1-mediated regulation of p62 expression. Moreover, down-regulation of p62 by either PS1 deficiency or over-expression of FAD-linked PS1 mutants compromised clearance of aggregation-prone Tau, which forms intracellular neurofibrillary tangles in the AD brain; these findings thus confirm the essential role of p62 in the clearance of neurotoxic protein aggregates.
Together, our studies emphasize the importance of the LC3-p62 interaction in selective autophagy, and the requirement of p62 for the removal of neurodegeneration-associated protein aggregates. Furthermore, the identification of PS1-dependent transcriptional regulation of p62 expression uncovers a novel PS1/p62-mediated molecular mechanism underlying the pathogenesis of AD and related neurodegenerative diseases.
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Pascoal, Jorge Filipe da Conceição. "Autophagy in hypothalamic cells: role of Neuropeptide Y." Master's thesis, 2011. http://hdl.handle.net/10400.1/2259.
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Dissertação de mest.Ciências Biomédicas. Departamento de Ciências Biomédicas e Medicina, Univ. do Algarve, 2011<br>A autofagia é um mecanismo celular, presente em todas as células eucariotas, responsável pela degradação e reciclagem de proteínas de longa vida e organelos danificados. É caracterizada pela formação de uma vesícula de membrana dupla, designada autofagossoma, que captura as proteínas ou os organelos a degradar e, posteriormente, se funde com lisossomas, levando à degradação dos substratos. Embora ocorra ao nível basal, a sua estimulação é normalmente provocada por sinais de privação de nutrientes ou energia, sendo por isso, um mecanismo de resposta ao stress, com o intuito de recuperar nutrientes ao nível celular e repor a homeostasia. No entanto, a sua importância ao nível do organismo vai muito mais longe, sendo que está envolvida na degradação de agregados proteicos, que podem levar a doenças neurodegenerativas, bem como na prevenção da tumorogénese. De facto, o seu mau funcionamento pode mesmo levar a estas ou outras condições patológicas. Retardando doenças normalmente associadas ao envelhecimento, está também envolvida no aumento da longevidade de organismos eucariotas. No entanto, a estimulação desregulada de autofagia pode também ser prejudicial, levando à morte celular por apoptose, razão pela qual a sua indução é, necessariamente, bem controlada. A principal via de sinalização responsável pela indução de autofagia passa pela inibição da cinase de serina/treonina mTOR (do inglês, mammalian target of rapamycin). Esta enzima é um dos principais “interruptores” metabólicos da célula, determinando se esta deve gastar energia e proliferar, ou, pelo contrário, produzir energia, reciclando conteúdo citoplasmático. Um dos seus principais substratos, a S6K1/p70, regula a expressão proteica, modulando a tradução ao nível dos ribossomas. Existem muitas proteínas envolvidas na regulação da autofagia, normalmente designadas Atg (do inglês autophagy-related genes). Especificamente, a MAP1LC-3 (do inglês microtubule associated protein 1 light chain 3), ou simplesmente LC-3, que participa na formação dos autofagossomas e é o principal marcador utilizado no estudo da indução de autofagia. Esta proteína pode assumir duas formas: forma lipidada, designada LC-3II, ou não lipidada, designada LC-3I. A LC-3I encontra-se normalmente dispersa no citoplasma celular, no entanto, ao haver a indução de autofagia, associa-se a uma fosfatidiletanolamina, convertendo-se em LC-3II e ligando-se às membranas do autofagossoma. Essa conversão, analisável por Western blotting, é o melhor marcador actualmente existente para a autofagia.
A restrição calórica (CR, do inglês calorie restriction) é um conhecido indutor de autofagia, tendo, no entanto, implicações muito mais variadas nos organismos, desde a alteração da secreção de hormonas, ao aumento da resistência ao stress oxidativo. Esse mecanismo de aumento da resistência a stresses ambientais é conhecido como hormese,
sendo a hipótese que melhor se adequa aos efeitos da CR. Foi demonstrado que a CR previne o surgimento de cancro e doenças neurodegenerativas, pelo que é um potente alvo terapêutico. A CR é, também, a mais eficaz terapia para o aumento da longevidade, havendo estudos que o comprovam em eucariotas inferiores e superiores, bem como indícios favoráveis em estudos a decorrer em primatas. Existem, assim, muitas semelhanças entre os efeitos da autofagia e da CR, mas mesmo sabendo que a CR induz autofagia, os mecanismos envolvidos não estão ainda totalmente esclarecidos, uma vez que a CR produz uma grande variedade de alterações fisiológicas.
Um dos principais efeitos neuroendócrinos da CR é a libertação de neuropéptido Y (NPY), o mais potente factor orexigénico endógeno conhecido. Este péptido é principalmente produzido e libertado no hipotálamo, o centro da fome e saciedade, mas produz efeitos ao nível de todo o organismo. Actua através de pelo menos 5 receptores, designados receptores NPY, acoplados a proteína G, levando à inibição da enzima adenilato ciclase e do consequente aumento de monofosfato de adenosina cíclico (cAMP). Adicionalmente, tem efeito neuroprotector em vários tipos celulares, prevenindo a apoptose por excitotoxicidade. Tendo um papel muito abrangente em resposta à privação de nutrientes, não seria estranho que estivesse envolvido na regulação da autofagia induzida por CR. No entanto este envolvimento nunca foi estudado.
Os objectivos deste estudo passaram, então, por verificar o efeito do NPY na indução de autofagia em culturas de células hipotalâmicas e, de seguida, verificar o seu papel na autofagia induzida por CR. Foi também pretendido verificar as vias de sinalização activas, aquando a possível indução de autofagia. Como modelos celulares, foram utilizadas a linha mHypoE-N42 (N42), de neurónios embrionários de murganho e culturas primárias de células hipotalâmicas neurais diferenciadas de rato, originadas a partir de neuroesferas hipotalâmicas embrionárias.
Este estudo demonstrou que o NPY induz autofagia em culturas in vitro de células hipotalâmicas. Analisando o rácio entre LC-3II e LC-3I, verificou-se que este aumentava após tratamento com NPY, de uma forma dependente do tempo de incubação, nos dois modelos in vitro de células hipotalâmicas. Utilizando um inibidor de degradação lisossomal – cloroquina – comprovou-se o aumento de fluxo autofágico em células N42, uma vez que se observou a acumulação da forma LC-3II. Verificou-se, também, que a utilização de antagonistas dos receptores Y1, Y2 e Y5 de NPY, aparentemente, preveniu esse efeito, em parte, o que fortaleceu a hipótese de que o NPY esteja, de facto a induzir autofagia. Com o objectivo de determinar se o NPY estaria a induzir autofagia através da via canónica, avaliou-se a fosforilação de mTOR e
um dos seus substratos, p70. No entanto, não se verificou qualquer alteração significativa na fosforilação do mTOR ou da p70, indicando que o NPY poderá estar a induzir autofagia por uma via independente de mTOR. Para estudar o eventual papel do NPY na autofagia induzida por CR, as culturas celulares foram submetidas a meios com baixo teor energético e incubadas com antagonistas de receptores de NPY. Verificou-se que os antagonistas aparentemente preveniram, em parte, a autofagia induzida por CR, em células N42, sendo que nas células hipotalâmicas neurais diferenciadas ainda é necessário um maior número de experiências. Foi ainda verificado que a privação de nutrientes levava a um aumento da expressão de NPY, indicando que este poderá, de facto, ter um importante papel na indução de autofagia, após CR.
Estes resultados são a base para um estudo mais aprofundado do papel do NPY na regulação do fluxo autofágico. A utilização de outros modelos experimentais contribuirá para a melhor compreensão destes fenómenos e da importância do NPY como resposta à privação de nutrientes.
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Peng, Kuan-Jen, and 彭冠蓁. "TIM-1 receptor-mediated dengue virus-induced autophagy facilitates virus production." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/zrx3xn.
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Liang, Jing-Zhang, and 梁晉彰. "Functional Studies of Rice Autophagy Cargo Receptor Under Abiotic Stress Conditions." Thesis, 2019. http://ndltd.ncl.edu.tw/cgi-bin/gs32/gsweb.cgi/login?o=dnclcdr&s=id=%22107NCHU5107008%22.&searchmode=basic.
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碩士<br>國立中興大學<br>生物化學研究所<br>107<br>Plants frequently encounter adverse environmental conditions, such as heat stress or salt stress. Autophagy not only plays an important role in nutrient recycling and utilization, but also can reduce energy consumption during stress conditions. Next to BRCA1 gene 1 (NBR1), a selective autophagy cargo receptor, is to recognize degraded protein.ACR1 is a homologous protein of NBR1.However, the function of ACR1 in rice is still unknown. In this study, the role of ACR1 in rice was explored. We found that the expression levels (protein and mRNA) of ACR1 were induced by salt, heat and chilling stresses. In addition, the T-DNA insertion mutant lines (ACR1 overexpression and acr1 knockout) were identified from the TRIM database. With RNA-seq analysis, 164 genes with significantly differential expression levels between ACR1 overexpression mutant and wild-type were identified; 65 genes with significantly differential expression levels between acr1 knockout mutant and wild-type were identified. Among them, genes involved in stress responds, such as heat stress and salt stress, were correlated with the expression of ACR1 to a certain extent. These results suggest that the selective autophagy cargo receptor, ACR1 might play an important role in stress response in rice.
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Lai, Yi-Ping, та 賴益平. "Regulatory Mechanisms of Estrogen Receptor β on Hypoxia-induced Autophagic and Apoptotic Pathways in Myocardial Cells". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/87922722926966670275.
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碩士<br>中國醫藥大學<br>基礎醫學研究所碩士班<br>100<br>Myocardial infarction (MI) is the common cause of cardiomyocyte death. Even hypoxia alone is sufficient to induce apoptosis of cardiomyocytes. In hearts, autophagy play important roles in hypoxia-mediated cardioprotection or myocardial injury effects. To date, the hypoxia-inducible factor-1α (HIF-1α) transcriptional factor and the BH-3 only protein, Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3), are known to play fundamental roles in adaptive or death process in response to hypoxia. In addition, hypoxia induces insulin-like growth factor binding protein 3 (IGFBP-3) to block the IGF1R/PI3K/Akt survival pathway. Therefore, we aim to investigate the molecular mechanisms and the correlation of HIF-1α, BNIP3 and IGFBP-3 in hypoxia-induced cardiomyocytes injuries. In the present study, heart-derived H9c2 cells and neonatal rat ventricular myocytes (NRVMs) were incubated in normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for up to 48 h. Results showed that hypoxia primarily highly increased HIF-1α expression, then activated downstream genes such as BNIP3 and IGFBP-3, and further triggered mitochondria-dependent apoptotic pathways. Moreover, IGF1R/PI3K/Akt signaling obviously attenuated by up-regulated expression of HIF-1α-dependent IGFBP-3 expression to enhance hypoxia-induced cell apoptosis. In addition, suppression of autophagy with 3-methyladenine (3MA) or siRNA of ATG5 or Beclin-1 significantly decreased the myocardial apoptosis under hypoxic conditions. The data also showed that the activation of autophagy during hypoxia was obviously induced by Forkhead box O3 (FoxO3a)-dependent BNIP3 expression. Importantly, knockdown of FoxO3a or BNIP3 significantly abrogated hypoxia-induced autophagy and mitochondria-dependent apoptosis effects. Taken together, our present results confirmed that autophagy is a pivotal regulator for hypoxia-induced cardiomyocyte apoptosis modulated by FoxO3a-dependent BNIP3 expression. Moreover, prolonged-hypoxia induced HIF-1α not only stimulated BNIP3 expression but also enhanced IGFBP-3 activation to inhibit IGF1R/PI3K/Akt survival pathway and mediate mitochondria-dependent cardiomyocyte apoptosis. We believe that HIF-1α and FoxO3a blockage are sufficient to annul the change of excessive hypoxia of hearts.
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Conlon, Donna Marie. "Role of Autophagy and Peroxisome Proliferator-Activated Receptor Gamma2 in Hepatic Lipid Homeostasis." Thesis, 2014. https://doi.org/10.7916/D81C1V2R.
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The liver maintains lipid homeostasis by regulating hepatic uptake of circulating fatty acids (FA) and triglycerides (TG), de novo lipogenesis (DNL), FA, and secretion of TG in very low density lipoproteins (VLDL). To investigate the effects of reduced VLDL secretion on hepatic lipid homeostasis, we examined the effects of knockdown of either apolipoproteinB (apoB) or microsomal triglyceride transfer protein (MTP) using antisense oligonucleotides (ASO) for 6 weeks in apobec-1 knockout mice. Despite a similar decrease in VLDL secretion in mice treated with either apoB ASO or MTP ASO, there was an increase in liver TG content only in the MTP ASO-treated mice. There were no differences in either FA uptake or secretion, or lipid synthesis from DNL. However, there was an increase in autophagosomes that co-localized with the endoplasmic reticulum (ER) in the apoB ASO-treated livers. We hypothesized that there is an accumulation of lipid in the ER due to the absence of apoB, the necessary protein for the formation and secretion of VLDL, and so the lipid becomes trapped inside the lumen of the ER. We provide evidence that the ER was engulfed by autophagosome and shuttled to the lysosome where the ER and its lipid content were degraded, leading to an increase in FA oxidation. This increase in autophagy of the ER prevented steatosis. We were surprised, however, that there was no evidence for ER stress after 6 weeks of knockdown of apoB and so we next examined the effect of only 3 weeks of ASO treatment and found that at this earlier time point, apoB ASO-treated mice had increased steatosis as compared to control ASO-treated mice and that the level of steatosis was similar to that caused by MTP ASO-treated mice. Furthermore, at 3 weeks of apoB ASO treatment, there was an increase in markers of ER stress in the apoB ASO-treated mice, but no evidence of an increase in the autophagy. After inhibition of autophagy, both ER stress and apoptosis were markedly increased in the livers of the apoB ASO-treated mice, indicating that autophagy protected the hepatocyte when apoB was knocked down. Thus, in this model of inhibition of apoB synthesis, with markedly reduced secretion of VLDL, TG that enters the ER gets trapped there and first induces ER stress. The ER stress response is unable to repair the defect, lipid accumulation in the ER continues to increase, and autophagy of the lipid-filled ER is induced, allowing the lysosome to act as an alternative pathway for oxidation of FA by the mitochondria. Our results suggest, therefore, that by stimulating autophagy, it may be possible to lower plasma TG levels by inhibiting VLDL secretion without causing hepatic steatosis.
PPARgamma2, which has been previously shown to contribute to increased lipid accumulation through decreased TG turnover of the lipid droplets and increased DNL, is aberrantly expressed in hepatic steatosis. However, the basis for increased expression of the PPARgamma2 specific isoform in the liver is unknown. We used hepatocytes and in vivo models to study the relative effects of hyperinsulinemia and/or increased FA delivery on hepatic PPARgamma2 and PPARgamma1 expression. Hepatic PPARgamma2 expression is not increased by increased fatty acid delivery in the absence of hyperinsulinemia but is regulated by changes in insulin signaling. Since hyperinsulinemia often occurs in the presence of excess nutrients, including glucose and FA, expression of PPARgamma2 in the liver, with subsequent effects on hepatic lipid droplet formation and stability, may be a means of protecting hepatocytes from lipotoxicity.
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Yueh, Yi-Mei, and 樂以梅. "Toll-like Receptor 7 Ligands Induce Autophagy and Their Effects on B cell Antigen Receptor Mediated Apoptosis in Ramos B cells." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/72030476377955515377.
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碩士<br>國立中興大學<br>生物醫學研究所<br>99<br>Autophagy is a highly conserved degradative process for cellular maintenance in all eukaryotic cells and its functional relationship with apoptosis is complex. Recent studies have shown that autophagy is important for the regulation of innate immunity and Toll-like receptor ( TLR ) ligands are potent autophagy inducers in macrophage. In addition, TLR ligands have been demonstrated to protect B cells from B cell antigen receptor ( BCR ) mediated apoptosis, an important mechanism to eliminate the autoreactive B cells. However, whether the TLR ligands could induce the autophagy in B cells and the biological significance of TLR induced autophagy in BCR mediated apoptosis is still unknown. In this study, we found that Imiquimod and Resiquimod, two synthetic TLR7 ligands, could induce autophagy in Ramos B cells. Moreover, Imiquimod and Resiquimod induced autophagy could rescue BCR mediated apoptosis via cross-linking with anti-IgM μ-chain antibodies in Ramos B cells and this protect effect was disrupted when autophagy was inhibited by 3-MA or Bafilomycin A1. These results not only presented a model for TLR-induced autophagy in BCR mediated apoptosis but also provided insight into the pathogenesis of autoimmune disease and a way of developing novel therapies.
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"CXCL10 and its receptor CXCR3 promote non-alcoholic steatohepatitis through mediating inflammatory cytokines and autophagy." 2014. http://library.cuhk.edu.hk/record=b6115759.
Full textAbstract:
研究背景及實驗目的: 非酒精性脂肪性肝炎(NASH)使得肥胖和2 型糖尿病變得複雜，肝臟炎症的持續產生是其主要的發病機理。CXCL10 是一種促進炎症的細胞因數，其在肥胖和2 型糖尿病中的表達顯著升高。CXCL10 以及其受體CXCR3 是否在NASH 的發生發展中起作用尚不清楚。在本研究中，我們探索了CXCL10 以及其受體CXCR3 在脂肪性肝炎中的功能， 並評估了CXCL10 在NASH 中的臨床價值。<br>實驗方法：CXCL10 基因敲除鼠，CXCR3 敲除鼠以及野生型C57BL/6 小鼠給予蛋氨酸膽鹼缺乏食(MCD)4 周或者8 周。CXCL10 的信號通路以及下游靶點通過細胞因數分析，cDNA array, 蛋白DNA 結合實驗，自噬溶酶體系統分析進行檢測。為了闡明CXCL10 抑制對NASH 的預防治療作用，我們給MCD 餵養的小鼠注射抗CXCL10 抗體。用不同濃度的CXCL10 抗體以及CXCR3 抑制劑NIBR2130 幹預MCD 培養的肝細胞株AML-12。臨床研究中，我們收集了147個非酒精性脂肪肝患者以及73 個健康對照的血清，用酶聯免疫吸附試驗檢測血清中CXCL10 的水準。<br>結果：野生型小鼠給予MCD 餵養後，CXCL10 以及CXCR3 的表達升高，並出現脂肪性肝炎的表現。然而，MCD 飼養的CXCL10 以及CXCR3 基因敲除鼠中，脂肪性肝炎明顯減輕。CXCL10 通過促炎細胞因數的產生以及NK-κB 信號通路促進MCD 飼養的小鼠NASH 的發生。CXCL10 通過促進脂質合成的基因SREBP-1c, ChREBP 和 SCD-1 引起脂肪變性，並通過CYP2E1 以及 C/EBPβ 的上調引起氧化應激。值得注意的是，自噬的損傷在CXCL10 以及CXCR3 導致的脂肪性肝炎的進展中起重要作用。 MCD 飼養的野生型小鼠中p62 以及LC3-II 表達明顯高於CXCL10 以及CXCR3 基因敲除鼠。通過抗CXCL10 抗體中和CXCL10 可以減輕MCD 食引起的小鼠脂肪性肝炎以及MCD 培養液引起的AML-12 細胞損傷。高選擇性的CXCR3 抑制劑NIBR2130 也可以抑制MCD 引起的肝細胞損傷。我們進一步研究了CXCL10 的臨床應用價值，發現NASH 患者血清以及肝臟中CXCL10 的水準明顯升高。更重要的是，血液中CXCL10 的水準與肝小葉炎症程度有關，是NASH 的獨立危險因素。<br>結論：我們的研究首次發現CXCL10 以及其受體CXCR3 通過促進炎症，脂質聚集，氧化應激以及自噬缺乏在NASH 的發病中起重要作用。抑制CXCL10 或者CXCR3 為NASH 患者的治療提供了新的方法。CXCL10 可作為NASH 患者非侵入性診斷的標誌物。<br>Background and aims: Non-alcoholic steatoheaptitis (NASH) complicates obesity and type 2 diabetes, while recruitment and perpetuation of liver inflammation is central to its pathogenesis. Expression of C-X-C motif chemokine 10 (CXCL10), a proinflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 and its receptor CXCR3 play a role in NASH is unknown. In this study, we investigated the functional significance of CXCL10 and its receptor CXCR3 in steatoheaptitis. Moreover, the clinical impact of CXCL10 in NASH was examined.<br>Methods: Gene-deleted CXCL10 (CXCL10-/-), CXCL10 receptor CXCR3 (CXCR3-/-) and C57BL/6 wildtype (WT) mice were fed methionine and choline-deficient (MCD) diet for 4 or 8 weeks. Cytokine profiling assay, cDNA array, protein-DNA binding activity assay and autophagosome-lysosome system analysis of CXCL10 signaling and downstream targets were performed. In other experiments, we injected neutralizing anti-CXCL10 monoclonal antibodies (mAb) into MCD diet-fed WT mice, while AML-12 cells were cultured in MCD medium in the presence of anti-CXCL10 mAb or CXCR3 inhibitor (NIBR2130) for 24 hours. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 controls. Circulating CXCL10 levels were determined by enzyme-linked immunosorbent assay.<br>Results: MCD-fed WT mice developed steatohepatitis with higher hepatic CXCL10 and CXCR3 expression. CXCL10-/- and CXCR3-/- mice were refractory to MCDinduced steatohepatitis. In WT mice with steatohepatitis, but not in CXCL10-/- mice, CXCL10 was associated with the induction of pro-inflammatory chemokines and cytokines, as well as activation of nuclear factor-κB (NF-κB) signaling. CXCL10 expression was linked to steatosis through lipogenic factors, including liver X receptors and its downstream targets (SREBP-1c, ChREBP and SCD-1), and also to oxidative stress (up-regulation of CYP2E1 and C/EBPβ). In particular, autophagy deficiency was involved in CXCL10- and CXCR3-induced steatohepatitis as indicated by p62 and LC3-I/II protein accumulation in MCD-fed WT mice than in CXCL10-/- and CXCR3-/- mice. Moreover, the impaired autophagic function was related to the reduction of lysosomal function in CXCL10- or CXCR3-induced NASH. Blockade of CXCL10 by anti-CXCL10 mAb protected against MCD-induced steatohepatitis in vivo and against MCD-mediated injury to AML-12 cells in vitro. The highly selective CXCR3 antagonist NIBR2130 also inhibited MCD-induced injury in AML-12 hepatocytes. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients.<br>Conclusions: We demonstrate for the first time that CXCL10 and its receptor CXCR3 plays a pivotal role in the pathogenesis of NASH by promoting inflammation, fatty acid accumulation, oxidative stress and autophagy deficiency. Blockade of CXCL10 or CXCR3 is a potential novel approach for NASH intervention. CXCL10 is a noninvasive biomarker for NASH patients.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Zhang, Xiang.<br>Thesis (Ph.D.) Chinese University of Hong Kong, 2014.<br>Includes bibliographical references (leaves 145-167).<br>Abstracts also in Chinese.