Dissertations / Theses on the topic 'Autophagy'
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1
Vigié, Pierre. "Mitochondrial quality control : roles of autophagy, mitophagy and the proteasome." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0202/document.
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La mitophagie, la dégradation sélective des mitochondries par autophagie, est impliquée dans l’élimination des mitochondries endommagées ou superflues et requiert des régulateurs et protéines spécifiques. Chez la levure, Atg32, localisée dans la membrane externe mitochondriale, interagit avec Atg8, et permet le recrutement des mitochondries et leur séquestration à l’intérieur des autophagosomes. Atg8 est conjuguée à de la phosphatidyléthanolamine et est ainsi ancrée aux membranes du phagophore et des autophagosomes. Chez la levure, plusieurs voies de synthèse de PE existent mais leur contribution dans l’autophagie et la mitophagie est inconnue. Dans le premier chapitre, nous avons étudié la contribution des différentes enzymes de synthèse de PE, dans l’induction de l’autophagie et la mitophagie et nous avons démontré que Psd1, la phosphatidylsérine décarboxylase mitochondriale, est impliquée dans la mitophagie seulement en condition de carence azotée alors que Psd2, localisée dans les membranes vacuolaires, endosomales et de l’appareil de Golgi, est nécessaire en phase stationnaire de croissance. Dans le second chapitre, la relation entre Atg32, la mitophagie et le protéasome a été étudiée. Nous avons démontré que l’activité du promoteur d’ATG32 et la quantité de protéine Atg32 exprimée sont inversement régulées. En phase stationnaire de croissance, l’inhibition du protéasome empêche la diminution de l’expression d’Atg32 et la mitophagie est stimulée. Nos données montrent ainsi que la quantité d’Atg32 est reliée à l’activité du protéasome et que cette protéine pourrait être ubiquitinylée. Dans le troisième chapitre, nous nous sommes intéressés au rôle potentiel de Dep1, un composant du complexe nucléaire Rpd3 d’histones déacétylases, dans la mitophagie. Dans nos conditions, Dep1 semble être mitochondriale et elle est impliquée dans la régulation de la mitophagie. BRMS1L (Breast Cancer Metastasis suppressor 1-like) est l’homologue de Dep1 chez les mammifères. Cette protéine possède un rôle anti-métastatique dans des lignées de cancer du sein. Nous avons trouvé que l’expression de BRMS1L augmente en présence de stimuli pro-mitophagieMitophagy, the selective degradation of mitochondria by autophagy, is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with Atg8, promoting mitochondria recruitment to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to phosphatidylethanolamine (PE). In yeast, several PE synthesis pathways have been characterized, but their contribution to autophagy and mitophagy is unknown. In the first chapter, we investigated the contribution of the different enzymes responsible for PE synthesis in autophagy and mitophagy and we demonstrated that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only in nitrogen starvation, whereas Psd2, located in vacuole/Golgi apparatus/endosome membranes, is required preferentially for mitophagy induction in stationary phase of growth. In the second chapter, we were interested in the relationship between Atg32, mitophagy and the proteasome. We demonstrated that ATG32 promoter activity and protein expression are inversely regulated. During stationary phase of growth, proteasome inhibition abolishes the decrease in Atg32 expression and mitophagy is enhanced. Our data indicate that Atg32 protein is regulated by the proteasome activity and could be ubiquitinated. In the third chapter, we investigated the involvement of Dep1, a member of the nuclear Rpd3L histone deacetylase complex, in mitophagy. In our conditions, Dep1 seems to be located in mitochondria and is a novel effector of mitophagy both in nitrogen starvation and stationary phase of growth. BRMS1L (Breast Cancer Metastasis suppressor 1-like) is the mammalian homolog of Dep1 and has been described in breast cancer metastasis suppression. We found that BRMS1L protein expression increases upon pro-mitophagy stimuli
2
Akinduro, Olufolake A. E. "Autophagy in epidermis." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8703.
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Organ‐transplant recipients (OTRs) on a new class of immunosuppressants, rapamycin and its analogues, have reduced cutaneous Squamous Cell Carcinomas (cSCCs). Rapamycin, an mTORC1 inhibitor, is also a known autophagy inducer in experimental models. Autophagy, which literally means self‐eating, is a cell survival mechanism but can also lead to cell death. Therefore, the main hypothesis behind this work is that rapamycin prevents epidermal tumourigenesis by either affecting epidermal mTOR regulation of autophagy and/or selectively affecting epidermal AKT isoform activity. Epidermal keratinocytes move from the proliferating basal layer upwards to the granular layers where they terminally differentiate, forming a layer of flattened, anucleate cells or squames of the cornified layer which provides an essential environmental barrier. However, epidermal terminal differentiation, a specialised form of cell death involving organelle degradation, is poorly understood. The work presented in this thesis shows that analysis of the autophagy marker expression profile during foetal epidermal development, indicates autophagy is constitutively active in the terminally differentiating granular layer of epidermis. Therefore, I hypothesize that autophagy is a mechanism of organelle degradation during terminal differentiation of granular layer keratinocytes. In monolayer keratinocytes, activation of terminal differentiation is accompanied by autophagic degradation of nuclear material, nucleophagy. This suggests that constitutive autophagy is a pro‐death mechanism required for terminal differentiation. In cultured keratinocytes and in epidermal cultures, rapamycinmediated mTORC1 inhibition strongly increases AKT1 activity as well as up‐regulates constitutive granular layer autophagy promoting terminal differentiation. Therefore, autophagy is an important fundamental process in keratinocytes which may be the mechanism by which terminally differentiating keratinocytes of the epidermal granular layer degrade their organelles required for barrier formation. This may have implications for the treatment of patients with barrier defects like psoriasis. In immunosuppressed OTRs, rapamycin may promote epidermal autophagy and AKT1 activity adding to its anti‐tumourigenic properties.
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Malik, Shoaib Ahmad. "Crosstalk Between Apoptosis and Autophagy : BH3 Mimetics Activate Multiple Pro-Autophagic Pathways." Thesis, Paris 11, 2012. http://www.theses.fr/2012PA11T044/document.
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La macro-autophagie est une voie catabolique conservée dans l’évolution permettant la dégradation des organites endommagés ou vieux, des protéines à longue durée de vie ou agrégées et des portions du cytosol pour le recyclage métabolique afin de maintenir l'homéostasie cellulaire. L'absence d'autophagie est fréquemment observée dans de nombreuses pathologies incluant les cancers et les maladies neurodégénératives. Beclin 1, un suppresseur de tumeur,est une protéine clé dans la régulation de l’autophagie et participe à la nucléation de l’autophagosome. Beclin 1 est une protéine “BH3-only” pouvant interagir avec le site de fixation au domaine BH3 présent dans la protéine Bcl-2 et ses homologues. Cette interaction inhibe l’autophagie. Certains agents pharmacologiques tels qu’ABT737, appelés«BH3 mimetics», occupent le site de fixation du domaine BH3 de façon compétitive pour perturber l'interaction inhibitrice entre Beclin 1 et Bcl-2/Bcl-XL. Ceci permet à Beclin 1 de maintenir l’activité classe IIIphosphatidylinositol-3-kinase de Vps34 pour la formation du phagophore. L'autophagie est un processus finement régulé par de nombreux complexes protéiques. Les senseurs de la charge énergétique comme l’AMP-dependant kinase(AMPK), la cible mammalienne de la rapamycine (mTOR), la Sirtuin1 (SIRT1) ou les voies d’intégration du stress telles que celles impliquant l'inhibiteur des kinases NF-κB (IκBα) (IKK) et le suppresseur de tumeur p53, ont tous un impact majeur dans la régulation de l'autophagie. Dans de nombreux paradigmes de stimulation autophagique, ils semblent tous agir en amont de la dissociation Beclin 1-Bcl-2. Nos résultats révèlent qu’ABT737 stimule plusieurs voies pro-autophagiques pour obtenir une efficacité optimale. Ces résultats placent la SIRT1, AMPK / mTOR, HDM2et IKK en aval de la dissociation du complexe Beclin 1-Bcl-2. Cette étude démontre que les BH3-mimetics activent des voies multiples de stimulation de l’autophagie, peut-être en raison du degré élevé de connectivité qui existe entre les complexes protéiques de régulation de l’autophagie. Cela signifie qu’un effet spécifique sur l’interactome de Beclin 1 peut affecter d'autres voies dans le réseau du contrôle autophagique. Ces voies ne semblent pas suivre une hiérarchie linéaire, mais doivent être plutôt interconnectées dans un circuit complexe dans lequel la stimulation de l'autophagie par des déclencheurs physiologiques (tels que la carence en nutriments ou le stress des organites) induit un ensemble de changements intimement liés et impliqués dans une boucle de régulation positive qui constituerait un ensemble indissociable composant l’«autophagy switch»Macro-autophagy is a conserved catabolic pathway that culminates in the degradation of old/damaged organelles,long-lived/aggregated proteins and portions of the cytosol for metabolic recycling to maintain cellular homeostasis.The absence of autophagy is frequently observed in many pathologies including cancers and neurodegenerative diseases. Beclin 1, a bona fide tumour suppressor, is the key autophagy regulatory protein that participates in autophagosome nucleation. Infect, Beclin 1 is a BH3-only protein that can interacts with the BH3 receptor domain contained within Bcl-2 and its homologues. This interaction functions as a inhibitory check on autophagy. Some pharmacological agents such as ABT737, referred to as ‘BH3 mimetics’, occupy the BH3-binding grooves to competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-XL allowing Beclin 1 to maintain the class III phosphatidylinositol-3-kinase activity of Vps34 for the phagophore formation. Autophagy is a complex process that is regulated by multiple protein complexes beyond that organized around Beclin 1. The energy sensors including AMP-dependent kinase (AMPK), mammalian target of rapamycin (mTOR), Sirtuin1 (SIRT1) as well as stress-integrating pathways such as those involving the inhibitor of NF-κB (IκB) kinases (IKK) and the tumour suppressor protein p53, all have a major impact on the regulation of autophagy. In many paradigms of autophagic stimulation, they all seem to act upstream of the dissociation of Beclin 1-Bcl-2. Our results reveal that ABT737stimulate multiple pro-autophagic pathways to be optimally efficient. These results place SIRT1, AMPK/mTOR,HDM2 and IKK downstream of the dissociation of the Beclin 1-Bcl-2 complex. This study advocates that BH3mimetics trigger multiple autophagy-stimulatory pathways maybe due to the high degree of connectivity that exists among autophagy-regulatory protein complexes meaning that a specific effect on the Beclin 1-interactome might affect other nodes in the autophagy-controlling network. These pathways cannot follow a linear hierarchy and rather must be interconnected in a complex circuitry, in which stimulation of autophagy by physiological triggers (such as starvation or organelle stress) induce an ensemble of intimately linked changes that are coupled to each other in positive feed forward loops constituting an indissociable ensemble that composes the “autophagic switch”
4
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 répliquerMacroautophagy 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
5
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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Scrivo, Aurora. "Régulation de la voie autophagique par la Gigaxonine E3-ligase, et implication dans les maladies neurodégénératives." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT090.
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L'autophagie est l'une des voies de signalisation qui maintiennent l'homéostasie cellulaire en condition basale, mais aussi en réponse à un stress. Son rôle est essentiel pour assurer plusieurs fonctions physiologiques, et son altération est associée à de nombreuses maladies, parmi lesquelles le cancer, les maladies immunitaires et les maladies neurodégénératives. Un nombre croissant d'études a établi que la voie autophagique est finement contrôlée. Cependant, très peu est connu sur les mécanismes moléculaires assurant sa régulation mais la famille des E3-ligases joue un rôle primordiale. La Gigaxonine est un adaptateur de la famille des E3 ligases CUL3, qui spécifie les substrats pour leur ubiquitination et leur successive dégradation. Des mutations «perte de fonction» de la Gigaxonine causent la Neuropathie à Axones Géants (NAG), une maladie neurodégénérative sévère et fatale, qui impacte tout le système nerveux et provoque une agrégation anormale des Filaments Intermédiaires (FI) dans l'organisme entier. Grâce à la modélisation de la pathologie dans les cellules de patients et chez la souris, le laboratoire a pu mettre en avant le rôle crucial de la Gigaxonine dans la dégradation de la famille des FIs, à travers son activité d'ubiquitination.Au cours de ma thèse, j'ai étudié les mécanismes de neurodégénerescence de la NAG, et la possible altération de la voie autophagique.Pour cela, j'ai développé un nouveau modèle neuronal de la maladie, à partir de notre modèle murin NAG, qui reproduit la mort neuronale et l'agrégation des FIs retrouvées chez les patients. Pour étudier l'implication de l'autophagie dans la neurodégénérescence, j'ai évalué l'effet de la déplétion de la Gigaxonine sur la formation des autophagosomes, le flux autophagique, la fusion avec le lysosome et la dégradation. J’ai ainsi révélé un défaut dans la dynamique autophagique dans les neurones NAG -/-. Pour déchiffrer les mécanismes moléculaires sous-jacents, j'ai étudié l'effet de l'absence de la Gigaxonine sur différentes régulateurs de la voie. En utilisant des techniques complémentaires, j'ai montré que la Gigaxonine est essentielle pour le turn-over d’un interrupteur autophagique, à travers son activité d’E3-ligase.En conclusion, nous avons identifié un nouveau mécanisme moléculaire impliqué dans le contrôle des premières phases de l'autophagie. Non seulement ces résultats présentent une avancée significative dans le domaine de l'autophagie, ils contribuent également à la compréhension de son dysfonctionnement dans les maladies neurodégénératives, et pourraient générer une nouvelle cible pour une intervention thérapeutique chez l'hommeThe autophagic route is one of the signaling pathways that sustain cellular homeostasis in basal condition, but also in response to stress. It has been shown to be crucial for several physiological functions and its impairment is associated with many diseases, including cancer, immune and neurodegenerative diseases. While an expanding number of studies have shown that autophagic route is finely controlled, little is known about the molecular mechanisms ensuring its function, but a fundamental role is sustained by the family of E3 ligases. Gigaxonin is an adaptor of a Cul3-E3 ligase, which specifies the substrates for their ubiquitination and their subsequent degradation. “Loss of function” mutations in Gigaxonin cause Giant Axonal Neuropathy (GAN), a severe and fatal neurodegenerative disorder that impacts broadly the nervous system and cause an abnormal aggregation of Intermediate Filaments (IFs) through the body. Modeling the disease in patient’s cells and in mouse, the laboratory has demonstrated the crucial role of Gigaxonin in degrading the entire family of IFs through its ubiquitination activity.During my PhD, I studied the neurodegenerative mechanisms in GAN disease, and the possible impairment of autophagy pathway.For that purpose, I developed a new neuronal model of the disease from our GAN mouse, which reproduced the neurodegeneration and the IF aggregation found in patients. To investigate the involvement of autophagy in neurodegeneration, I evaluated the effect of Gigaxonin depletion on autophagosome formation, autophagic flux, lysosome fusion and degradation, and I revealed a defect in autophagy dynamics. To decipher the molecular mechanism of autophagosome impairment, I investigated the effect of Gigaxonin depletion on different autophagy regulators. Using complementary techniques, I showed that Gigaxonin is essential for the turn-over of a specific molecular switch, through its E3 ligase activity.Altogether, we identified a new exciting molecular mechanism in the control of autophagy. Not only these findings present a significant advance in the comprehension of the fundamental field of autophagy, but it also contribute in the understanding of its dysfunction in neurodegenerative diseases, and may generate a new target for therapeutic intervention in humans
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Osman, Ayman. "Autophagy in Peripheral Neuropathy." Doctoral thesis, Linköpings universitet, Avdelning för neurobiologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-142125.
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Peripheral neuropathy includes a wide range of diseases affecting millions around the world, and many of these diseases have unknown etiology. Peripheral neuropathy in diabetes represents a large proportion of peripheral neuropathies. Nerve damage can also be caused by trauma. Peripheral neuropathies are a significant clinical problem and efficient treatments are largely lacking. In the case of a transected nerve, different methods have been used to repair or reconstruct the nerve, including the use of nerve conduits, but functional recovery is usually poor. Autophagy, a cellular mechanism that recycles damaged proteins, is impaired in the brain in many neurodegenerative diseases affecting animals and humans. No research, however, has investigated the presence of autophagy in the human peripheral nervous system. In this study, I present the first structural evidence of autophagy in human peripheral nerves. I also show that the density of autophagy structures is higher in peripheral nerves of patients with chronic idiopathic axonal polyneuropathy (CIAP) and inflammatory neuropathy than in controls. The density of these structures increases with the severity of the neuropathy. In animal model, using Goto-Kakizaki (GK) rats with diabetes resembling human type 2 diabetes, activation of autophagy by local administration of rapamycin incorporated in collagen conduits that were used for reconnection of the transected sciatic nerve led to an increase in autophagy proteins LC3 and a decrease in p62 suggesting that the autophagic flux was activated. In addition, immunoreactivity of neurofilaments, which are parts of the cytoskeleton of axons, was increased indicating increased axonal regeneration. I also show that many proteins involved in axonal regeneration and cell survival were up-regulated by rapamycin in the injured sciatic nerve of GK rats four weeks after injury. Taken together, these findings provide new knowledge about the involvement of autophagy in neuropathy and after peripheral nerve injury and reconstruction using collagen conduits.
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Yassine, Maya. "Calcium, Calcium-permeable channels and autophagy modulators in control of autophagy and cancer." Thesis, Lille 1, 2013. http://www.theses.fr/2013LIL10159/document.
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L'autophagie est une voie cellulaire strictement régulée dont le but principal est la dégradation lysosomale et le recyclage ultérieur du matériel cytoplasmique afin de maintenir l'homéostasie cellulaire normale. Des défauts dans l'autophagie sont liés à une variété d'états pathologiques, dont le cancer. Le cancer est une maladie associée aux modifications des processus cellulaires fondamentaux tels que l'apoptose et l'autophagie. Le calcium régule une série de processus physiologiques et pathologiques tels que le vieillissement, la neurodégénérescence et le cancer. Si le rôle du calcium et des canaux calciques dans le cancer est bien établi, l'information sur la nature moléculaire des canaux régulant l’autophagie ainsi que les mécanismes de cette régulation reste encore limitée. Le rôle de l'autophagie dans le cancer est complexe. En effet, elle peut favoriser à la fois la prévention tumorale et la résistance aux traitements. Elle est souvent détectée dans les cellules cancéreuses en réponse aux expositions aux rayons et la chimiothérapie. Elle semble contribuer à la résistance thérapeutique de certains cancers. Il est maintenant bien établi que sa modulation peut potentiellement contribuer à la mise en œuvre des méthodes de traitement du cancer. Dans cette étude, nos travaux ont permis d’identifier le calcium intracellulaire, comme un régulateur important de l'autophagie. Ainsi, nous proposons un lien possible entre le calcium, les canaux calciques, l’autophagie et la progression du cancer. De plus, nous avons mis en évidence un nouveau modulateur de l’autophagie, le ML-9. Cet outil pourrait cibler l'autophagie et être utilise dans le traitement des cancersAutophagy is a tightly regulated cellular pathway the main purpose of which islysosomal degradation and subsequent recycling of cytoplasmic material to maintain normal cellular homeostasis. Defects in autophagy are linked to a variety of pathological states,including cancer. Cancer is the disease associated with abnormal tissue growth following an alteration in such fundamental cellular processes as apoptosis, proliferation, differentiation,migration and autophagy. Calcium is a ubiquitous secondary messenger which regulates plethora of physiological and pathological processes such as aging, neurodegeneration and cancer. The role of calcium and calcium-permeable channels in cancer is well-established, whereas theinformation about molecular nature of channels regulating autophagy and the mechanisms of this regulation is still limited. The role of autophagy in cancer is complex, as it can promoteboth tumor prevention and survival/treatment resistance. Elevated autophagy is often detected in cancer cells in response to radiation and chemotherapy. Furthermore, autophagy seems to contribute to the therapeutic resistance of some cancers. It's now clear that modulation of autophagy has a great potential in cancer diagnosis and treatment. Our findings identified intracellular calcium as an important regulator of autophagy. We propose a possible link between calcium, calcium permeable ion channels, autopohagy and cancer progression. Further, our results revealed a new autophagy modulator ML-9 as an attractive tool for targeting autophagy in cancer therapy
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McKnight, N. C. "A genome-wide screen for starvation-induced autophagy : identifies new modulators of autophagy." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302281/.
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Autophagy is a catabolic mechanism by which cytoplasmic components are sequestered and transported by a double-membrane vesicle called an autophagosome to the lysosome for degradation. This recycling of organelles and macromolecules provides the cell with amino acids in times of nutrient deprivation though we do not fully know how the process is triggered or controlled. It is a highly regulated process in mammalian cells and its deregulation has been shown to contribute to multiple diseases. In order to find new regulators of mammalian autophagy, I performed a genome-wide screen using the Dharmacon human siRNA library in a stable human cell line expressing GFP-LC3, a specific marker for autophagosomal membranes. First I incubated the cells with the siRNA pools then I starved the cells of amino acids. This initiated the formation of GFP-LC3-labelled autophagosomes that I quantified using the Cellomics VTiScan microscope and accompanying software. I measured the effect of specific siRNA-mediated knock-down on multiple parameters including spot count. Accounting for cell death and normalising the data, I generated a Z-score for each siRNA pool and retested the best 500 autophagy-increasing and 500 autophagydecreasing siRNAs as above. The 190 strongest siRNA pools were deconvoluted leaving 20 hits that reproduced the phenotype with three or four out of four duplexes. These 20 hits were then assayed for endogenous LC3 lipidation in a different cell line and the ability of their siRNA to reduce mRNA levels was determined. Four increasers of GFP-LC3 spots increased endogenous LC3 lipidation, suggesting that these proteins are either negative regulators of autophagy or inhibit the maturation or degradation of autophagosomes. Five decreasers of GFP-LC3 spots also inhibited endogenous LC3 lipidation and I have characterised two of these proteins required for autophagy. SCOC colocalises with early autophagy markers and may be providing a scaffold for autophagy machinery. WAC, through its reported binding partners, may be playing a role in both the autophagic and ubiquitin/proteasome pathways.
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Otten, Elsje Gesina. "Molecular mechanisms of autophagy and the effect of autophagy dysfunction on mitochondrial function." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3953.
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Long lifespan of evolutionary higher organisms including humans is associated with the challenge to maintain viability of post mitotic cells, such as neurons, for decades. Autophagy is increasingly recognized as an important prosurvival pathway in oxidative and proteotoxic stress conditions. Autophagy degrades cytosolic macromolecules in response to starvation and is involved in the selective degradation of damaged/toxic organelles, such as mitochondria. With age autophagy function declines, and is also compromised in several neurodegenerative diseases. We identified a novel role for autophagy in the maintenance of mitochondrial health, specifically respiratory complex I. Intriguingly, galactose-induced cell death of autophagy deficient cells was rescued by preventing ROS production at complex I or bypassing complex I-linked respiration. We propose that aberrant ROS production via complex I in response to autophagy deficiency could be pathogenic and result in neurodegeneration and preventing this could be an interesting therapeutic target. Furthermore, we found that vertebrates have evolved mechanisms to induce autophagy in response to oxidative stress. This involves the oxidation of the autophagy receptor p62, which promotes autophagy flux and the clearance of autophagy cargo, resulting in increased stress resistance in mammalian cells and survival under stress in flies. In addition, we obtained data revealing an important role for redox-regulated cysteines in NDP52 for the degradation of mitochondria via mitophagy and tools were created to study the role of other autophagy receptors in autophagy initiation and selective autophagy.
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Motta, Isabelle. "Interactions reciproques de la proteine de l'autophagie Gabarap et de membranes modèles." Thesis, Paris, Ecole normale supérieure, 2015. http://www.theses.fr/2015ENSU0009/document.
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La macro-autophagie est un processus de dégradation intracellulaire qui impliquela formation d'une vésicule à double membranes, l'autophagosome, permettantla séquestration des molécules à dégrader. La formation de l'autophagosome dé-bute par la nucléation de membranes isolées dans le cytosol. Elle se poursuit parune phase de croissance de la membrane en forme de coupe. Dans l'étape _nale,l'autophagosome se referme en fusionnant par ses bords pour former la vésicule.La morphologie de l'autophagosome évolue donc durant sa formation. Certainesétudes montrent que cela est dû à l'implication de machineries protéiques. Indé-pendamment, d'autres recherches mettent en avant les rôles joués par les propriétésphysiques de la membrane.Dans mon travail de thèse, j'ai cherché à montrer un couplage entre l'activité d'uneprotéine impliquée dans l'autophagie et les caractéristiques physiques de la membrane.Cette protéine, nommée GABARAP, est considérée comme le marqueurde l'autophagosome car elle est la seule protéine, avec ses homologues, à s'ancrerspécifiquement sur sa membrane lors de sa formation. En incorporant GABARAPdans des vésicules géantes unilamellaires micromanipulées, j'ai pu montrer unein_uence de la composition et de la courbure de la membrane sur la distribution,l'oligomérisation et la dynamique de la protéine. Ensuite, j'ai mesuré une diminutiondu module de courbure de la membrane lorsque la protéine y était ancrée.Cette dernière étude m'a amenée à développer un modèle permettant de prédire ladistribution de la protéine sur une membrane possédant deux régions de courburesdiérentes. Enfin, j'ai déterminé la nature de l'interaction en trans de GABARAPMacro-autophagy is an intracellular degradation process that involves a doublemembrane vesicle, the autophagosome, to engulf a cargo. Its formation starts withthe nucleation of isolated membrane in the cytosol. Then the membrane growsas a cup-shape around the cargo to finally fuse at its edge and enclose the molecules to be degraded. Thus, the autophagosome morphology evolves during itsformation. Studies show that protein machineries support such shape changes. Independently, other researches point membrane physical properties roles during itsrearrangement.During my PhD, I investigated the coupling between the activity of one autophagyprotein and membranes physical characteristics. This protein, GABARAP,is considered as the autophagosome marker, because, with its homologs, it is theonly protein to be specifically anchored to its membrane during all its formation.The reconstitution of GABARAP in micromanipulated giant unilamellar vesicles(GUVs) allowed me to study the interplay between membrane characteristics andprotein behaviours. In a first part, I showed that membrane composition and curvature trigger specific distribution, oligomerization and dynamic of GABARAP.Then I measured a decrease of the membrane bending modulus when the proteinwas anchored. This last result led me to propose a model that predicts proteinsdistribution on membranes with two regions of diferent curvatures. Finally, I determined the nature of GABARAP / GABARAP trans interaction
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Nowosad, Ada. "Rôle de p27/Kip1 dans l'autophagie induite par le stress métabolique." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30194.
Full textAbstract:
Les cancers sont caractérisés par une prolifération anarchique des cellules causée par une dérégulation des mécanismes de contrôle du cycle cellulaire, comme la protéine p27Kip1 (p27). Dans le noyau, p27 inhibe les complexes cycline-CDK, bloquant ainsi la progression du cycle de division cellulaire, et par conséquent, la prolifération cellulaire. Cette propriété confère à p27 un rôle de suppresseur de tumeurs. Toutefois, dans certains cancers, p27 est relocalisé dans le cytoplasme où il exerce un rôle oncogénique, cependant les mécanismes moléculaires par lesquels p27 agit comme un oncogène restent largement inconnus. Des études récentes montrent que sa localisation cytoplasmique permet à p27 d'activer l'autophagie, un processus de recyclage des constituants intracellulaires dans les cellules carencées en nutriments. Dans les cellules cancéreuses, l'autophagie est un des mécanismes d'adaptation au microenvironnement tumoral et leur permet de survivre malgré des conditions défavorables. En outre, l'autophagie peut être induite par le stress causé par les traitements anti-cancéreux, ce qui retarde l'apoptose en permettant aux cellules d'échapper aux traitements. L'autophagie induite par la localisation cytoplasmique de p27 pourrait ainsi compromettre l'efficacité des thérapeutiques anti-tumorales. Le but de mon projet de thèse était de déterminer par quels mécanismes p27 contrôle l'autophagie et participe ainsi à la survie des cellules dans des conditions de stress métabolique. Durant ma thèse, j'ai pu mettre en évidence un rôle essentiel de p27 dans la régulation de l'autophagie et de la mort cellulaire. Mes travaux indiquent que le statut de p27 cytoplasmique ou nucléaire détermine à la fois le degré d'autophagie et la susceptibilité des cellules à l'apoptose induite par la privation nutritionnelle. En utilisant des méthodes de biologie cellulaire et moléculaire, j'ai disséqué les voies de signalisation et le mécanisme moléculaire expliquant le rôle pro-autophagique de p27. De manière surprenante, il apparait que p27 régule l'autophagie par différents mécanismes en fonction de la carence infligée aux cellules. [...]P27 controls cell cycle progression via its ability to block cyclin-CDK activity. Thus, it acts as a tumor suppressor in the nucleus. However, in certain cancers, p27 relocalizes in the cytoplasm where it may promote tumorigenesis by still largely unknown mechanisms. Recent studies have shown that the cytoplasmic localization of p27 induces autophagy, a catabolic process whereby intracellular constituents are recycled in response to nutrient depletion. In cancer cells, autophagy acts as as an adaptive response to metabolic stress in tumor tissues. Furthermore, autophagy may be induced by various cancer therapies, leading to chemotherapeutic resistance and promoting cancer cell survival. The aim of my PhD project was to determine by which mechanisms p27 controls autophagy and cell survival upon metabolic stress conditions. My results indicate that p27 plays a prominent role in the regulation of autophagy and cell death during nutrient deprivation. The status of p27 determines the rate of autophagy and the susceptibility of cells to apoptosis. Importantly, the mechanisms underlying the role of p27 in autophagy appears to be different in function of the nature of the metabolic stress. Amino acid deprivation leads to translocation of p27 to lysosomes where it participates in the inhibition of mTOR, a kinase that acts as a master regulator of cellular metabolism and autophagy. In contrast, the effect of p27 in glucose starved cells depends mostly on its role in the regulation of microtubule dynamics, which controls intracellular vesicle trafficking. Thus, in glucose starved cells, p27 promotes the fusion of autophagic vesicles and degradation of autophagy cargo. To conclude, my results show that p27 is a critical modulator of starvation-induced autophagy and its status determines the response of cells to metabolic stress. Therefore, p27 may serve as a predictive marker for treatment response targeting specific metabolic pathways and may constitute a promising target for anticancer treatment affecting these pathways
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Dai, Yun. "Targeting Autophagy in Multiple Myeloma." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3933.
Full textAbstract:
Apoptosis (Type I) and autophagy (Type II) represent two major forms of programmed cell death. Numerous anticancer agents employed in standard chemotherapy or novel targeted therapy induce both apoptosis and autophagy. Of note, a cytoprotective autophagic response often counteracts apoptosis triggered by such agents, potentially contributing to drug-resistance. Mechanistically, autophagy and apoptosis share molecular regulatory mechanisms primarily governed by the Bcl-2 family proteins. However, since autophagy acts as the double-edge sword in cancer, whether autophagy should be inhibited or activated in cancer treatment remains the subject of debate. Here we report a) a novel autophagy-targeted strategy that targeting the adaptor SQSTM1/p62 induces “inefficient” autophagy due to cargo-loading failure and converts cytoprotective autophagic response to apoptosis via the BH3-only protein NBK/Bik (Part 1); and b) a new mechanism for acquired drug-resistance in which the BH3-only protein Bim acts as a dual-agent regulating both autophagy and apoptosis (Part 2).
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Loska, Stefan. "Regulation of ULK1 in autophagy." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/regulation-of-ulk1-in-autophagy(2cc61e06-ee38-451b-b7ca-83b3a96e181c).html.
Full textAbstract:
ULK1 (UNC-51 like kinase 1) is a serine/threonine protein kinase that has been shown to play a crucial role in autophagy, a process of self digestion implicated in maintaining cellular homeostasis and in mediating type II programmed cell death. However, the exact mechanism by which ULK1 controls autophagy remains elusive, mostly because none of the known ULK1 targets have been directly linked to autophagy. To address this issue, I have employed a protein microarray screening approach to identify novel ULK1 substrates. I found five putative targets: MERTK (proto-oncogene tyrosine-protein kinase MER), B-RAF (v-raf murine sarcoma viral oncogene homologue B1), NOL4 (nucleolar protein 4), TBC1D22B (TBC1 domain family member 22B) and ACVRL1 (activin A receptor type II-like 1). My preliminary experiments have not confirmed that MERTK or B-RAF can be phosphorylated by ULK1 in vitro. However, further investigation will be required to firmly rule out MERTK and B-RAF as downstream targets of ULK1 and to test the ability of ULK1 to phosphorylate the other candidates. In addition, I have identified by in-gel kinase assay a ULK1 kinase at 34-kDa whose ability to phosphorylate the kinase domain of ULK1 was increased upon starvation. Using the genome information, I predicted this upstream kinase to be Pim1 (Proto-oncogene serine/threonine-protein kinase pim-1). I confirmed that Pim1 phosphorylated ULK1 in vitro at S147 and S224. Results of site directed mutagenesis suggest that phosphorylation at S224 correlates with increased ULK1 activity. This is consistent with observation that Pim1 is capable of activating ULK1 in vitro. Furthermore, I present preliminary data suggesting that Pim1 promotes autophagy in HeLa cells.
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Clarke, Alexander James. "Autophagy in Systemic Lupus Erythematosus." Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/autophagy-in-systemic-lupus-erythematosus(1e5a4a5e-99f7-456e-bcb4-634a4e3fd986).html.
Full textAbstract:
Autophagy has emerged as a critical homeostatic mechanism in lymphocytes, influencing proliferation and differentiation. I sought to explore the role of autophagy in the pathogenesis of human and murine lupus, a disease in which B cells are critical effectors of pathology. Autophagy was assessed using multiple techniques in NZB/W and control mice, and in patients with SLE compared to healthy controls. I evaluated the phenotype of the B cell compartment in Vav-Atg7-/- mice in vivo, and examined human and murine plasmablast formation following inhibition of autophagy. I found activation of autophagy in early developmental stages of B cell development in a lupus mouse model even before disease-onset, and which progressively increased with age. In human disease, again autophagy was activated compared with healthy controls, principally in naïve B cells. B cells isolated from Vav-Atg7-/- mice failed to effectively differentiate into plasma cells following stimulation in vitro. Similarly, human B cells stimulated in the presence of autophagy inhibition did not differentiate into plasmablasts. My data suggest activation of autophagy is a mechanism for survival of autoreactive B cells, and also demonstrate that it is required for plasmablast differentiation, processes that induce significant cellular stress. The implication of autophagy in two major pathogenic pathways in SLE suggests the potential to use inhibition of autophagy as a novel treatment target.
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Ragimbeau, Romain. "Etude du rôle de la co-chaperonne BAG6 dans la régulation de la mitophagie." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT022.
Full textAbstract:
L'autophagie est un processus d'autodigestion du matériel intracellulaire qui repose sur la formation de vésicules à double membrane nommées autophagosomes qui séquestrent les organites et macromolécules puis fusionnent avec les lysosomes pour permettre leur dégradation.La dégradation sélective des mitochondries par autophagie se nomme mitophagie. En éliminant les mitochondries superflues ou endommagées à l'origine d'un stress oxydant toxique et mutagène, elle contribue au maintien de l'homéostasie cellulaire. Sa régulation met en jeu des voies de signalisations telles que la voie PINK1-PARKIN et des récepteurs spécifiques appelés récepteurs de la mitophagie.La protéine BAG6 est une co-chaperonne des HSP70 et possède des fonctions variées telles la régulation de l'apoptose, les modifications épigénétiques, le repliement des protéines, ou encore l’élimination de peptides par le système ubiquitine-protéasome. BAG6 est également essentielle pour l’autophagie en régulant la localisation intracellulaire de l’acétyltranférase EP300 responsables de l’acétylation des protéines ATGs.Dans ce travail de thèse, nous montrons que BAG6 se localise dans la mitochondrie. Plus précisément, BAG6 est située dans la matrice mitochondriale en conditions basales puis se délocalise dans la membrane externe lorsque les mitochondries sont dépolarisées. En parallèle, BAG6 promeut le recrutement du complexe de fission mitochondrial DRP1 et des protéines PINK1 et PARKIN impliquées dans la signalisation mitophagique ce qui lui confère un effet pro-mitophagique. Enfin, BAG6 interagit également avec LC3 par l’intermédiaire d’un domaine LIR (LC3 Interacting Region) et son effet sur la mitophagie dépend de cette interaction, faisant de BAG6 un potentiel récepteur de la mitophagieAutophagy is a process of self-digestion of intracellular material based on the formation of double membrane vesicles called autophagosomes that sequester organelles and macromolecules and then fuse with lysosomes to allow their degradation.The selective degradation of mitochondria by autophagy is named mitophagy. By eliminating superfluous or damaged mitochondria, that cause toxic and mutagenic oxidative stress, mitophagy contributes to the maintenance of cellular homeostasis. Its regulation involves signaling pathways such as the PINK1-PARKIN pathway and specific receptors called mitophagy receptors.The BAG6 protein is a co-chaperon of HSP70 and has a variety of functions such as regulation of apoptosis, epigenetic modifications, protein folding, and peptide removal by the ubiquitin-proteasome system. BAG6 is also essential for autophagy by regulating the intracellular localization of EP300 acetyltransferase responsible for ATGs protein acetylation.In this thesis, we showed that BAG6 is located in the mitochondria. More precisely, BAG6 is located in the mitochondrial matrix under basal conditions and then relocalizes in the outer membrane when the mitochondria are depolarized. In parallel, BAG6 promotes the recruitment of the mitochondrial fission complex DRP1 and the proteins PINK1 and PARKIN involved in mitophagic signalling, which gives it a pro-mitophagic effect. Finally, BAG6 also interacts with LC3 via a LIR domain (LC3 Interacting Region) and its effect on mitophagy depends on this interaction, making BAG6 a putative mitophagy receptor
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Mei, Yang. "Structural Studies of BECN1, A Key Autophagy Protein, and Intrinsically Disordered Regions in Autophagy Proteins." Diss., North Dakota State University, 2016. https://hdl.handle.net/10365/28030.
Full textAbstract:
Autophagy, a conserved catabolic process required for cellular homeostasis in
eukaryotes, is regulated by many proteins. The central goal of my doctoral research is to
investigate conformational flexibility of autophagy proteins, with a special focus on BECN1, a
core component of the class III phosphatidylinositol-3 kinase autophagosome nucleation
complex that may serve as an autophagy interaction hub.
Our rigorous bioinformatics analysis predicts that 57% of 59 key human autophagy
proteins contain intrinsically disordered regions (IDRs), which lack stable secondary and tertiary
structure. The prevalence of IDRs suggests that IDRs play an important, yet hitherto
uninvestigated, role in autophagy. We confirm disorder of selected IDRs via biophysical
methods, and use additional bioinformatics tools to predict protein-protein interaction and
phosphorylation sites within IDRs, identifying potential biological functions.
We experimentally investigate four distinct BECN1 domains: (i) The IDR, which
includes a functional BCL2 homology 3 domain (BH3D) that binds BCL2 proteins, undergoing a
binding-associated disorder-to-helix transition and enabling BCL2s to inhibit autophagy. (ii) The
flexible helical domain (FHD) which has an unstructured N-terminal half and structured Cterminal
half forming a 2.5-turn helix in our 2.0 ? X-ray crystal structure. Our molecular
dynamics simulations and circular dichroism spectroscopy analyses indicate the FHD transiently
samples more helical conformations and likely undergoes a binding-associated disorder-to-helix
transition. We also show that the FHD bears conserved residues critical for AMBRA1 interaction
and for starvation-induced autophagy. (iii) A coiled-coil domain (CCD) which forms an antiparallel
homodimer in our 1.46 ? X-ray crystal structure. We have also built a atomistic model
of an optimally packed, parallel BECN1:ATG14 CCD heterodimer that agrees with our experimental SAXS data. Further, we show that BECN1:ATG14 heterodimer interface residues
identified from this model are important for heterodimer formation and starvation-induced
autophagy. (iv) A ?-? repeated autophagy-specific domain which bears invariant residues that
we show are important for starvation-induced autophagy. Thus, we demonstrate that
conformational flexibility is a key BECN1 feature.
Lastly, we show that multi-domain BECN1 constructs have extended conformations with
no intra-domain interactions that impact structure of other domains, suggesting that BECN1
structure and conformational flexibility enable its function as an autophagy interaction hub.
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Siddiqui, Mohammad Adnan. "Role of RNase L in Inducing Autophagy and Regulating the Crosstalk from Autophagy to Apoptosis." University of Toledo / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1438904772.
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Emond-Boisjoly, Marc-Alexandre. "Rôle de la protéine DUSP5 dans l’autophagie des cardiomyocytes." Mémoire, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8908.
Full textAbstract:
Résumé: L’autophagie est un processus essentiel au maintien de l’homéostasie cellulaire. Elle permet de dégrader et recycler aussi bien des organelles entières que des composants cytoplasmiques non fonctionnels. De plus, l’augmentation d’autophagie en condition de stress constitue une réponse adaptative favorisant la survie cellulaire. Chez les cardiomyocytes, l’autophagie en condition basale est indispensable au renouvellement, entre autres, des mitochondries et des protéines formant les sarcomères. De plus, les stress tels l’ischémie cardiaque ou la carence en nutriments induisent une augmentation de l’autophagie protectrice. Dans certaines conditions extrêmes, il a été suggéré qu’un surcroît d’autophagie puisse toutefois exacerber la pathologie cardiaque en provoquant la mort des cardiomyocytes. Considérant l’importance de ce processus dans la physiopathologie cardiaque, l’identification des mécanismes signalétiques régulant l’autophagie chez les cardiomyocytes a été le sujet de recherches intenses. À cet effet, l’activation des Mitogen-Activated Protein Kinase (MAPK) a été démontrée pour réguler, avec d’autres voies signalétiques, l’autophagie et l’apoptose des cardiomyocytes. Il est donc probable que les Dual-Specificity Phosphatase (DUSP), enzymes clés contrôlant l’activité des MAPK, participent aussi à la régulation de l’autophagie. Afin de vérifier cette hypothèse, nous avons induit l’autophagie chez des cardiomyocytes isolés de rats nouveau-nés en culture. L’analyse de marqueurs d’autophagie par immunobuvardage démontre que l’activation des MAPK ERK1/2 et p38 corrèle avec l’activité autophagique chez les cardiomyocytes. Dans ces conditions, la diminution d’expression de la majorité des ARNm encodant les différentes DUSP retrouvées chez les cardiomyocytes contraste de façon marquée avec l’augmentation d’expression de l’ARNm Dusp5. De plus, nous avons démontré par une étude de gain de fonction que l’activation soutenue de p38 par surexpression d’un mutant MKK6 constitutivement actif stimule l’autophagie chez les cardiomyocytes. De façon surprenante, la perte de fonction de p38 obtenue par surexpression d’un mutant p38 dominant négatif n’altère en rien la réponse autophagique initiatrice dans notre modèle in vitro. Nos résultats suggèrent que les DUSP puissent réguler, via leurs actions sur les MAPK, d’importantes étapes du processus autophagique chez les cardiomyocytes.Abstract: Autophagy is a process essential to the maintenance of cellular homeostasis. It helps degrade and recycle whole organelles and nonfunctional cytoplasmic components. In addition, the adaptative up regulation of autophagy in stress condition promotes cell survival. In cardiomyocytes basal autophagy is essential to the renewal of, among others, mitochondria and proteins forming sarcomeres. In addition, stresses such as ischemic heart or nutrient deficiency induce an increase in protective autophagy. In extreme conditions, it has been suggested that autophagy may exacerbate cardiac disease causing the death of cardiomyocytes. Considering the importance of this process in cardiac pathophysiology, identify ing safety mechanisms regulating autophagy in cardiomyocytes has been the subject of intense research. To this end, activation of mitogen-activated protein kinase (MAPK) has been demonstrated to regulate, with other signaling pathways, autophagy and cardiomyocyte apoptosis. It is therefore likely that Dual-Specificity Phosphatases (DUSPs), key enzymes that control the activity of MAPKs, also participate in the regulation of autophagy. To test this hypothesis, we have induced autophagy in isolated cardiomyocytes of newborn rats in culture. Analysis of autophagy markers by immunoblotting demonstrated that the activation of MAPKs ERK1/2 and p38 correlates with autophagic activity in cardiomyocytes. Under these conditions, the decrease in expression of the majority of mRNAs encoding different DUSPs found in cardiomyocytes contrast sharply with the increase mRNA expression of Dusp5. Furthermore, we demonstrated by again of function study that sustained activation of p38 by overexpression of a constitutively active MKK6 mutant stimulates autophagy in cardiomyocytes. Surprisingly, the loss of p38 function obtained by overexpression of a dominant negative p38 mutant does not affect the autophagic response in our in vitro model, but increases the lipidation of autophagosomes marker LC3. Our results suggest that DUSPs can regulate, through their actions on MAPKs, important stages of autophagy in cardiomyocytes.
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El, Kebriti Leïla. "BAG6, un nouveau régulateur de la mitophagie." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT053.
Full textAbstract:
L’autophagie est un processus d’autodigestion qui se produit dans toutes les cellules eucaryotes et conduit à la dégradation d’éléments du cytoplasme (organites, macromolécules) par le lysosome. Elle peut se produire au hasard dans le cytoplasme où elle peut être sélective, par exemple d’un organite intracellulaire. Lorsque les mitochondries sont sélectivement dégradées par autophagie, on parle de mitophagie. L’autophagie et la mitophagie sont impliquées dans diverses pathologies comme les maladies neurodégénératives et le cancer car leur dérégulation peut grandement perturber l’homéostasie cellulaire.Mon projet de thèse porte sur le rôle de la protéine co-chaperonne BAG6 dans la régulation de la mitophagie.BAG6 est une protéine de 150 kDa, également appelée BAT3 ou Scythe, dont la fonction majeure réside dans le contrôle qualité du cytoplasme mais BAG6 est également impliquée dans l’immunité, l’apoptose ou l’autophagie. Nous montrons que son mécanisme d’action passe, tout d’abord, par la régulation de la morphologie mitochondriale en induisant la fission des mitochondries. Ensuite, la protéine BAG6 induit la mitophagie : les protéines impliquées dans la mitophagie (PINK1 et PARKIN) s’accumulent à la mitochondrie alors que les protéines de la mitochondrie (TOM20, TFAM et TIM23) voient leur expression diminuée. BAG6 diminue également la masse mitochondriale par un mécanisme dépendant de l’autophagie. L’analyse de la séquence de BAG6 montre qu’elle est composée de nombreux domaines protéiques incluant les domaines UBL et deux domaines LIR (LC3-Interacting Region) et nous avons montré que BAG6 interagit avec LC3 grâce à son domaine LIR2. Ces caractéristiques identifient la protéine BAG6 comme un nouveau récepteur potentiel de la mitophagieAutophagy, literally meaning self-eating, is a highly evolutionary conserved process in eukaryotes where elements of the cytoplasm (organelles, macromolecules) are degraded by lysosomes. Autophagy can occur randomly in the cytoplasm or can be selective of a specific organelle. Among other, the specific degradation of mitochondria is called mitophagy. Autophagy and mitophagy have been implicated in several physiopathologies such as neurodegenerative diseases or cancer. Deregulations of autophagy/mitophagy may profoundly affect homeostasis.The aim of my thesis is to characterize the role of the co-chaperonne protein BAG6 in the regulation of mitophagy.BAG6 is a 150kDa protein, also known as BAT3 or Scythe, which functions in the quality control of the cytoplasm. Moreover BAG6 is also involved in immunity, apoptosis or autophagy. Our work showed that it is implicated in the regulation of mitochondrial morphology by inducing mitochondrial fission. Also, BAG6 induces mitophagy: in presence of BAG6, mitophagy markers such as PINK1 and PARKIN are more localized at the mitochondria whereas the expression of mitochondrial specific protein’s (TOM20, TFAM and TIM23) decreases. After its sequence analysis, we discovered that BAG6 is composed of many domains such as the UBL domains and two LIR domains (LC3- Interacting Region) and that BAG6 interacts with LC3 through its LIR2 domain. These features lead to identify BAG6 as a new potential receptor of mitophagy
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Sukkurwala, Abdul Qader. "Autophagy : A New Modulator of Immunogenic Cell Death for Cancer Therapy." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T031.
Full textAbstract:
Certains agents chimiothérapeutiques tels que les anthracyclines ou l'oxaliplatine induisent une mort cellulaire immunogène, ce qui implique que les cellules mourrantes du patient servent de vaccin thérapeutique en stimulant une réponse immunitaire antitumorale. La mort cellulaire immunogène est caractérisée par la libération de signaux d'alarme par la cellule tumorale mourante qui permettent l’activation du système immunitaire. En premier lieu, l'exposition de la calréticuline à la surface de la cellule tumorale mourante va agir comme un signal de type «eat-me» pour les cellules dendritiques. Une fois relâchée, la protéine nucléaire HMGB1 se lie au récepteur TLR4 afin de faciliter la présentation antigénique. Les cellules mourantes vont également libérer de l'ATP qui agit sur les récepteurs P2X7 et active l’inflammasomme NLRP3, conduisant à la libération d'IL-1β et ainsi à l’activation des cellules T CD8+ productrices d’IFN-γ. L’autophagie est un mécanisme cellulaire qui est activé en réponse à la chimiothérapie. L'autophagie signifie «self-ating», il s'agit d'un processus cellulaire activé par diverses conditions de stress, par lequel les cellules peuvent dégrader les protéines et les organites. Il peut aussi être induit par un stress du réticulum endoplasmique. Ce dernier étant également impliqué dans l'exposition de la calréticuline pendant la mort cellulaire immunogène, nous avons au cours de cette étude cherché à déterminer le rôle de l'autophagie dans la mort cellulaire immunogène. Nous avons constaté que l'autophagie est nécessaire pour la libération de l'ATP après un traitement par des chimiothérapies immunogènes, en observant que le nockdown de gènes essentiels de l'autophagie limitait la sécrétion d'ATP. Nous avons également observé que des cellules déficientes pour l'autophagie traitées par une chimiothérapie immunogène sont incapables d’immuniser des souris contre une injection de cellules vivantes. En outre, les tumeurs déficientes pour l’autophagie ne répondent pas à un traitement systémique immunogène dans des souris immunocompétentes et continuent à proliférer en comparaison à des tumeurs “wild-type”. De plus, nous avons montré que les cellules déficientes pour l'autophagie ne sont pas en mesure de recruter des cellules dendritiques dans le lit tumoral ou d'induire l’activation des cellules T CD8+. A l'inverse, l'inhibition des enzymes de dégradation de l’ATP extracellulaire accroit les concentrations d'ATP dans les tumeurs déficientes pour l'autophagie, ce qui rétablit le recrutement des cellules immunitaires dans le lit tumoral et restaure la réponse chimiothérapeutique des cancers déficients pour l'autophagie. Ainsi, cette étude a montré l'importance de l'autophagie dans la réponse anti-tumorale spécifique, après traitement par des chimiothérapies immunogènes. Ces résultats ouvrent de nouvelles perspectives dans le concept de la mort cellulaire immunogèneIn recent years it has been demonstrated that some chemotherapeutic agents such as anthracyclines or oxaliplatin can induce a type of tumor cell death that is immunogenic, implying that the patient’s dying cancer cells serve as a therapeuticvaccine that stimulates an antitumor immune response, which in turn can control or eradicate residual cancer cells. Immunogenic cell death is characterized by the emission of danger signals from the dying tumor cell, which activate the immune system. At first the exposure of calreticulin, acts as an «eat-me» signal for dendritic cells (DCs). Once released, the nuclear protein HMGB1 binds to TLR4 on DCs, facilitating antigen processing and presentation. The dying tumor cells also releases ATP, which acts on P2X7 receptors on DCs and activates the NLRP3 inflammasome, leading to IL-1β release, necessary for IFN-γ-producing CD8+ T cell activation. Autophagy literally ‘self-eating’ is a cellular process activated in response to various conditions of cellular stress, whereby cells can liberate energy resources via the degradation of proteins and organelles. Recently autophagy has been found activated in response to chemotherapy and in this project we aimed to determine the potential role of autophagy in immunogenic cell death. We found that autophagy isrequired for the release of ATP in response to immunochemotherapeutic treatment, as we observed that the knockdown of essential autophagy-related genes abolished its secretion. We observed that autophagy deficient cells treated with immunogenic cell death inducers failed to immunize mice against a re-challenge with living cells. Furthermore, autophagy deficient tumors growing on immunocompetent mice did not respond to systemic immunogenic treatment and continued proliferating in contrast to autophagy proficient tumors. We showed that autophagy deficient cells were neither able to recruit DCs into the tumor bed nor to activate CD8+ T cells. Conversely, the inhibition of extracellular ATP degrading enzymes increased extracellular ATP concentrations in autophagy deficient tumors, which reestablished the recruitment of immune cells into the tumor bed, and restored chemotherapeutic responses in autophagy-deficient cancers. Altogether, this study showed the importance of autophagy in tumor-specific immune response after treatment with chemotherapy, thus giving new insights into the concept of immunogenic cell death
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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 xénophagieXenophagy 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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Castoldi, Francesca. "L'aspirine récapitule les caractéristiques de la restriction calorique." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS440.
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L'autophagie est un processus d'auto-digestion durant lequel les cellule dégradent leurs propres composants afin de maintenir l’homéostasie en conditions basales. L'autophagie est donc nécessaire à l’échelle de la cellule et de l’organisme car elle joue un rôle dans l’élimination des organites endommagés et des agrégats de protéines potentiellement nocifs et a la capacité de mobiliser les métabolites essentiels des réserves énergétiques en conditions de stressLa détérioration des fonctions cellulaires et au niveau de l’organisme liée à l'âge est associée à une dérégulation des voies de détection des nutriments ainsi qu’à une autophagie déficiente. La réactivation du flux autophagique peut prévenir ou améliorer ces dysfonctionnements métaboliques liés à l'âge. Les composés non toxiques capables de réduire les taux globaux d'acétylation des protéines et d'induire l'autophagie ont été classés dans la catégorie des agents mimétiques de restriction calorique (CRMs, de l’anglais « caloric restriction mimetic »). Nous montrons ici que l'aspirine et son métabolite actif, le salicylate, induisent une autophagie en raison de leur capacité à inhiber l'activité acétyltransférase de EP300. Alors que le salicylate stimule le flux autophagique dans les cellules « Wild Type », il ne permet pas d’augmenter le niveau d'autophagie dans les cellules déficientes en EP300, ni dans les cellules dans lesquelles EP300 endogène a été remplacé par les mutants EP300 résistants au salicylate. En conséquence, l'activité pro-autophagique de l'aspirine et du salicylate sur le nématode Caenorhabditis elegans est perdue lorsque l'expression de l'orthologue EP300 cpb-1 est réduite. Ces résultats permettent de conclure que l'aspirine est un CRM dont le mécanisme est conservé au cours de l’évolutionAutophagy is a self-digestion process in which cell degrades its own components in order to maintain homeostasis in basal conditions; autophagy is required for the maintenance of cellular and organismal fitness due to its role in eliminating damaged organelles and potentially harmful protein aggregates, as well as its unique capacity to mobilize essential metabolites from complex energy stores in conditions of stress.The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300. While salicylate readily stimulates autophagic flux in control cells, it fails to further increase autophagy levels in EP300-deficient cells, as well as in cells in which endogenous EP300 has been replaced by salicylate-resistant EP300 mutants. Accordingly, the pro-autophagic activity of aspirin and salicylate on the nematode Caenorhabditis elegans is lost when the expression of the EP300 ortholog cpb-1 is reduced. Altogether, these findings identify aspirin as an evolutionary conserved CRM
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Zhang, Lingzhi. "Synthesis of autophagy inhibiting virantmycin analogs." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/54843.
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(–)-Virantmycin (1.12), first isolated from Streptomyces nitrosporeus in 1981, was found to be a potent inhibitor of autophagy with an IC₅₀ of 0.5 μM against rapamycin-induced autophagy in MCF-7 cells. Recent studies showed that autophagy inhibition considerably reduced the growth of pancreatic ductal adenocarcinoma (PDAC) in mouse models. Therefore, virantmycin’s sub- μM potency as an early stage autophagy inhibitor makes it an interesting “lead compound” for the development of a treatment for PDAC. Previous attempts failed to make the benzoic acid from aryl iodide, using Kogen’s method. The current method for synthetic access to virantmycin analogs employs microwave irradiation to generate aryl nitriles, such as 2.144, for further installation of the carboxyl group at the aryl ring. Analogs 2.108 and 2.152 show the most potent autophagy inhibiting activity among the synthetic analogues prepared to date. The construction of simplified pharmacophore analogs 2.108 and 2.152 allows for scalable synthesis to provide quantities for animal testing.Science, Faculty of
Chemistry, Department of
Graduate
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Bortnik, Svetlana. "Investigating autophagy modulation in breast cancer." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62396.
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Woods, Kerry Louise. "Regulators of autophagy in Leishmania major." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/1211/.
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Autophagy is a conserved lysosomal degradation pathway for recycling long-lived proteins and organelles that is thought to be required for life cycle progression and virulence of Leishmania. ATG8 is a ubiquitin like protein that is required for the formation of autophagosomes, and Leishmania uniquely possesses a set of ATG8-like proteins in addition to ATG8, that are distributed in three multi-gene families called ATG8A, ATG8B and ATG8C. The localisation and expression of ATG8A, ATG8B and ATG8C were analysed using GFP fusion proteins and affinity purified antibodies. ATG8A exhibited a dramatic relocalisation to punctate structures under starvation conditions, suggestive of a specific role for ATG8A in starvation induced autophagy. Although ATG8 and ATG8A both participate in a response to starvation, they differed in their sensitivity to the PI(3) kinase inhibitor wortmannin, responded differently to the presence of energy sources, and labelled distinct subsets of vesicles. When the data generated in this thesis was considered together with recent analyses of the functions of the cysteine peptidases ATG4.1 and ATG4.2, evidence for distinct roles of ATG8 and ATG8A emerged. ATG8B and ATG8C localised to single punctate structures close to the flagellar pocket in a small proportion of promastigotes grown under nutrient rich conditions. The distribution of ATG8B and ATG8C labelled structures did not change during differentiation or starvation, suggestive of a role distinct from autophagy. ATG8B labelled structures appeared to be duplicated during cell division, and might be derived from endosomal membranes. ATG8A, ATG8B and ATG8C expression was shown to be developmentally regulated with all expressed at high levels in stationary phase promastigotes. Conjugation of ATG8 to phosphatidylethanolamine (PE) is required for the association of ATG8 with autophagosome membranes, and while ATG8 was shown to be conjugated to a phospholipid, no evidence was obtained to suggest that ATG8 paralogues are modified by a lipid. High molecular weight proteins were detected by western blot with anti-ATG8, ATG8A, ATG8B and ATG8C antibodies, perhaps indicating associations with other proteins in complexes. Two ubiquitin fusion proteins and a putative SNARE were identified in co-immunoprecipitation experiments performed with anti-ATG8B antibodies, although further experiments are required to determine the validity of these interactions. To analyse the role of a predicted presenilin-1 (PS1) homologue in L. major, Δps1 null mutants were generated. These mutants were not defective in their ability to differentiate into infective metacyclic promastigotes, and could establish infections in vivo and in vitro, demonstrating that PS1 is not essential and is not a good target for drug development. Large autophagosomes accumulated in Δps1 mutants suggesting that PS1 might be involved in the regulation of autophagy, although it seemed that the parasites could compensate for this, as autophagy was restored to normal levels in Δps1 mutants that had undergone differentiation into amastigotes. Antibodies were raised against a PS1 peptide that recognised L. major PS1 only when over-expressed, suggesting that endogenous PS1 is expressed at a low level. PS1-HA that was stably integrated into the genome localised to a structure close to the flagellar pocket, although a different localisation was observed when PS1-GFP was over-expressed, and investigation is required to clarify the subcellular localisation. In summary, the regulation of autophagy in L. major has been investigated from two different angles, leading to the characterisation of a unique family of ATG8-like proteins and an aspartic peptidase, presenilin-1.
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Tomlins, Andrew Michael. "Autophagy in Plasmodium falciparum intraerythrocytic stages." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/4553/.
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Tasdemir, Ezgi. "Regulation of autophagy by cytoplasmic p53." Paris 11, 2009. http://www.theses.fr/2009PA11T008.
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PARISI, BARBARA. "Role of the novel neuronal protein APache in autophagy." Doctoral thesis, Università degli studi di Genova, 2022. http://hdl.handle.net/11567/1090471.
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The central event driving neuronal activity is represented by synaptic transmission, a process that relies on regulated cycles of synaptic vesicle (SV) exocytosis and endocytosis at presynaptic terminal level. Neurons, polarized and perennial cells, to guarantee an efficient neurotransmitter release, to maintain cellular homeostasis and promote neuronal survival, are particularly dependent on efficient quality control pathways to continuously remove dysfunctional presynaptic proteins and organelles. The main mechanisms used by neurons to achieve these goals are endosomal sorting and autophagy, a highly conserved endo-lysosomal degradation pathway required to recycle basic nutrients by the clearance of damaged or aged proteins and organelles.
Several presynaptic endocytic proteins have been shown to regulate both SV recycling and autophagy and defects in both pathways have been linked to neurodevelopmental abnormalities and neurodegeneration in mouse and humans.
In 2017 we characterized the previously unknown protein APache (KIAA1107) as a neuronal-specific protein, novel interactor of the adaptor protein AP-2 essential in the regulation of neuronal development and SV cycle in vitro and in vivo.
In this work, we intended to define APache functional role in neuronal autophagy by combining electron microscopy, immunofluorescence, live-cell imaging microscopy and biochemistry. We observed that APache is actually involved in autophagy: the induction of the process increases APache levels in mature neurons and, conversely, APache silencing leads to a severe accumulation of late-stage autophagosomes in neurons, also at synaptic level, due to autophagic blockade. Interestingly, APache expression is significantly reduced in the brain of sporadic Alzheimer’s disease patients.
These data point to APache as a novel key regulator of neuronal autophagy. Its altered levels, resulting in defective autophagy, may contribute to the precocious cellular alterations and synaptic dysfunctions observed in neurodegenerative diseases. The further elucidation of its functional role in neurons and of its precise molecular mechanism will help our understanding of the physiology and pathology of synaptic function.
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Lescat, Laury. "Caractérisation et étude du rôle de lamp2a chez les poissons." Thesis, Pau, 2019. http://www.theses.fr/2019PAUU3014.
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L’Autophagie médiée par les protéines chaperonnes (ou CMA pour Chaperone-Mediated Autophagy) est une voie majeure du catabolisme lysosomal considérée aujourd’hui comme un acteur central de contrôle de nombreuses fonctions cellulaires, et dont les défauts sont associés à plusieurs pathologies humaines, dont des maladies neurodégénératives, des cancers et des troubles du système immunitaire. Selon l’idée actuellement admise, cette fonction cellulaire n’existerait que chez les mammifères ou les oiseaux, qui seraient les seuls à exprimer la protéine LAMP2A, une protéine nécessaire au fonctionnement de la CMA. Or, récemment, nous avons pu mettre en évidence l’existence de séquences exprimées présentant une forte homologie avec LAMP2A de mammifères chez plusieurs espèces de poissons, remettant ainsi en question ce point de vue et suggérant que la CMA soit apparue beaucoup plus tôt au cours de l'évolution qu'on ne l'avait initialement cru. Dans cette thèse, nous retraçons l’histoire évolutive du gène LAMP2 chez les vertébrés. Nous démontrons que ce gène est apparu après la seconde duplication complète du génome survenue chez l'ancêtre commun des vertébrés il y a environ 500 millions d'années. En outre, en adaptant une méthode récemment décrite pour mesurer l’activité de la CMA dans des cellules de mammifères à une lignée de fibroblastes de medaka (Oryzias latipes), nous apportons la preuve de l’existence de cette fonction cellulaire chez cette espèce de poisson. Enfin, afin de caractériser le rôle physiologique de cette fonction chez les poissons, nous avons procédé à l’invalidation par crispR-cas9 de lamp2a chez le medaka. Les poissons générés présentaient de sévères perturbations du métabolisme intermédiaire, comme précédemment décrit chez des souris dont LAMP2A a été invalidée dans le foie. Dans l’ensemble, ces résultats démontrent clairement, et pour la toute première fois, qu’il existe bien une activité CMA fonctionnelle chez les poissons, et apportent ainsi de nouvelles perspectives dans ce domaine de recherche, notamment en autorisant l'utilisation de modèles génétiques complémentaires, tels que le poisson zèbre ou le medaka, pour faire avancer nos connaissances sur les mécanismes régissant cette fonction cellulaireChaperone-Mediated Autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player in the control of numerous cellular functions, and whose defects have been associated to several human pathologies, including neurodegenerative diseases, cancers and immune disorders. To date, this cellular function was presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in non-tetrapod species. However, we recently identified the existence of expressed sequences displaying high homology with the mammalian LAMP2A in several fish species, challenging that view and suggesting that CMA appeared much earlier during evolution than initially thought. In the present thesis, we first present new evidences about the evolutionary history of the gene LAMP2 in vertebrates. We demonstrate that LAMP2 appeared after the second whole genome duplication that occurred at the root of the vertebrate lineage approximately 500 million years ago. By using a fluorescent reporter previously used to track CMA in mammalian cells, we then revealed the existence of a CMA-like pathway in a fibroblast cell line of the fish medaka (Oryzias latipes). Finally, to address the physiological role of Lamp2a in fish, we generated, medaka knockout for the splice variant lamp2a, and found severe alterations in the intermediary metabolism, as previously demonstrated in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective
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Liang, Ning. "Regulation of YAP by mTOR and autophagy reveals a therapeutic target of Tuberous Sclerosis Complex." Thesis, Paris 5, 2014. http://www.theses.fr/2014PA05T055/document.
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La sclérose tubéreuse complexe (TSC) est une maladie génétique caractérisée par une croissance des hamartomes dans différents organes y compris le cerveau, les reins, les poumons, la peau et le cœur. Ces lésions sont des sources de morbidité et de mortalité chez les patients TSC, car ils peuvent provoquerl’ épilepsie, l’autisme, le retard de développement et l’insuffisance rénale et pulmonaire. Les causes connues de TSC sont la perte de la fonction et les mutations des gènes TSC1 et TSC2. La majorité des lésions TSC contient plusieurs types cellulaires de la lignée mésenchymateuse, comme dans le cas des angiomyolipomes, l’lymphangioleiomyomatose et les angiofibromes. Un type unique de cellules épithélioïdes périvasculaires nommé (PEC) est constamment présent dans les lésions de TSC mésenchymateuses, comme angiomyolipomes et lymphangioleiomyomatose, basant sur les caractérisations morphologiques et l'expression des marqueurs communs mélanocytaires et myogéniques. Par conséquent, ces lésions sont officiellement classées, ainsi que d'autres tumeurs, comme PEComes. Leur origine cellulaire et les mécanismes moléculaires impliqués dans la pathologie restent à élucider. Ici, nous avons généré un modèle souris mosaïque TSC1 knockout qui développe des lésions rénales mésenchymateuses récapitulant périvasculaire épithélioïde cellules tumorales humaines (Pecoma) observés chez les patients TSC. Nous avons identifié YAP, le co-activateur transcriptionnel de la voie Hippo, a été régulée d'une manière mTOR-dépendante dans les lésions rénales de notre TSC1 knockout souris et les échantillons de l’angiomyolipome humaines. L'inhibition de YAP avec des outils génétiques ou pharmacologiques atténue considérablement la prolifération et la survie des cellules nulles TSC1 in vivo et in vitro. En outre, l’accumulation de YAP dans les cellules déficientes TSC1 / TSC2 pourra être dû à la dégradation de la protéine altéré par le système de l’autophagosome / lysosome. Ainsi, la régulation de YAP par mTOR et l'autophagie est un nouveau mécanisme de contrôle de la croissance, l'activité de YAP correspondant à la disponibilité des éléments nutritifs dans les conditions de croissance permissives. Il pourra servir comme une cible thérapeutique potentielle pour TSC et d'autres maladies avec une activité de mTOR déréguléeThe Tuberous Sclerosis Complex (TSC) is a genetic disease characterized by growth of hamartomas in different organs including brain, kidney, lung, skin, and heart. These lesions are sources of morbidity and mortality in patients with TSC, as they may cause intractable epilepsy, autism, developmental delay, renal and pulmonary failure. Known causes of TSC are loss of function mutations in TSC1 and TSC2 genes. The majority of TSC lesions contain multiple cell types of the mesenchymal lineage, as in the case of angiomyolipomas, lymphangioleiomyomatosis and angiofibromas. A unique cell type named perivascular epithelioid cell (PEC) is constantly present in mesenchymal TSC lesions, such as angiomyolipomas and lymphangioleiomyomatosis, basing on morphological features and the common expression of melanocytic and myogenic markers. Therefore, these lesions are officially classified, along with other tumors, as PEComas. Their cell of origin and the molecular mechanisms underlying their pathogenesis remain poorly defined. Here we generated a novel mosaic Tsc1 knockout mouse model which develop renal mesenchymal lesions recapitulating human Perivascular Epithelioid Cell tumor (PEComa) observed in TSC patients. We identified YAP, the transcriptional coactivator of Hippo pathway, was upregulated in both renal lesions of TSC mouse model and human angiomyolipoma samples in a mTOR-dependent manner. Inhibition of YAP with genetic or pharmacological tools greatly attenuates the proliferation and survival of Tsc1 null cells in vivo and in vitro. Futhermore, we found YAP accumulation in TSC1/TSC2 deficient cells is due to impaired degradation of the protein through the autophagosome/lysosome system. Thus the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth permissive conditions. It may serve as a potential therapeutical target for TSC and other diseases with dysregulated mTOR activity
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Heiseke, Andreas. "Prions and autophagy: Effect of lithium on prion infection and role of basal autophagy in primary prion infection." kostenfrei, 2010. https://mediatum2.ub.tum.de/node?id=818228.
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Lelogeais, Virginie. "Étude de l’interaction entre L. pneumophila et l’autophagie de la cellule hôte." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1168/document.
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L. pneumophila est l'agent responsable de la légionellose, une pneumonie sévère associée à 10% de mortalité. Cette bactérie intracellulaire a acquis la capacité de survivre et de se répliquer dans des cellules humaines. Notamment, L. pneumophila sécrète un grand nombre d'effecteurs par son système de sécrétion de type IV, qui interagissent avec différentes voies cellulaires, dont l'autophagie. L'autophagie est une voie de dégradation conservée qui permet aux cellules eucaryotes de réguler l'homéostasie cellulaire et d'éliminer les agents pathogènes intracellulaires. Néanmoins, nombre d'entre eux ont évolué pour manipuler cette voie à leur propre avantage. Même si l'interaction entre L. pneumophila et l'autophagie a été rapportée, aucun modèle clair n'est déterminé. Dans cette étude, nous montrons qu'une infection à L. pneumophila induit une stimulation globale de l'autophagie, mais que ce phénotype dépend des souches utilisées, et notamment de la présence de certains effecteurs. De plus, l'inhibition de l'autophagie est liée à un défaut de réplication intracellulaire suggérant que cette voie est bénéfique à la bactérie. Afin de rechercher les déterminants génétiques impliqués dans cette interaction, nous avons identifié des effecteurs communs sécrétés par le système de sécrétion de type IV entre L. pneumophila et Coxiella burnetii, une bactérie de l'ordre des Legionellales connue pour stimuler et détourner l'autophagie. La capacité des mutants de ces effecteurs à stimuler l'autophagie chez L. pneumophila a été analysée. Si aucun d'entre eux ne semble impliqué dans la modulation de l'autophagie, cette étude suggère d'autres fonctions pour ces effecteurs conservésLegionella pneumophila is responsible for the legionellosis disease, a severe pneumonia associated with 10% mortality rate. This intracellular bacterium has evolved the ability to survive and replicate within human cells. Notably, L. pneumophila secretes a high number of type IV secretion system effectors that interfere with many cellular pathways including autophagy. Autophagy, a highly conserved degradative pathway, allows eukaryotic cells to regulate cell homeostasis and fight intracellular pathogens. Nevertheless numerous microorganisms have evolved strategies to subvert this mechanism to their own advantage. The interaction between L. pneumophila and autophagy has been reported but remains unclear. In this study, we show that L. pneumophila infection induces a global stimulation of autophagy, but importantly this autophagy stimulation depends on the bacterial strain. Moreover, we also observed that inhibition of autophagy results in decreased intracellular bacterial proliferation suggesting that host cell autophagy is benificial for L. pneumophila. In order to decipher the molecular determinants involved in the interaction with autophagy, we identified common effectors secreted by the type IV secretion system between L. pneumophila and Coxiella burnetii, a bacterium from the order Legionellale responsible for Q fever and known to stimulate and hijack host cell autophagy. Mutant of these common effectors in L. pneumophila were analysed. While, none of them seems to be implicated in autophagy modulation, this study suggests other functions for these conserved effectors
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Liu, Dawei. "Target and small molecule discovery for therapeutic innovation in cardiovascular area." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS324.
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La production cyclique d'adénosine monophosphate (AMPc) régule certains aspects de la fonction mitochondriale des cardiomyocytes de rongeurs, tels que la production d'ATP, la consommation d'oxygène, les importations de calcium et la transition de perméabilité mitochondriale (MPT), mais le contrôle de ce pool d'AMPc n'est pas bien connu. Dans la première partie de cette thèse, nous avons étudié l'expression, la localisation et l'activité de plusieurs enzymes dégradant l'AMPc, les phosphodiestérases (PDEs), dans des mitochondries cardiaques isolées de rongeurs. L'expression de la PDE2 a été principalement détectée dans les mitochondries sous-sarcologiques, et l'activité de la PDE2 stimulée par le GMPc était plus importante que celle de la PDE3 et de la PDE4; leurs activités ont ensuite été confirmées dans les cardiomyocytes de rats nouveau-nés par analyse FRET en temps réel. De plus, l’inhibition pharmacologique ou la surexpression cardiaque spécifique de la respiration mitochondriale modulée par la PDE2, la perte de potentiel de la membrane mitochondriale, le MPT et l’importation de calcium. Ainsi, la dégradation de l'AMPc par les PDE représente un nouveau mécanisme de régulation de la fonction mitochondriale et devient une cible potentielle dans le traitement des maladies cardiovasculaires.En outre, l'amélioration récente du traitement anticancéreux entraîne une augmentation du nombre de patients survivants, mais un risque de cardiotoxicité à long terme. Ainsi, dans la deuxième partie de cette thèse, nous avons identifié des molécules cardioprotectrices à partir de bibliothèques chimiques en développant un test de criblage à haut débit. Nous avons identifié 6 molécules à effets puissants et spécifiqies et les avons validés dans 3 modèles cellulaires. Nous avons étudié les mécanismes d'action de chaque molécule en utilisant l’extinction par siARN, l'analyse par western blot, l'imagerie par fluorescence et les analyses métaboliques en temps réel. Trois molécules pourraient entrer rapidement dans les études précliniques et cliniques en association avec des agents de radiothérapie ou des agents chimiothérapeutiques pour le développement thérapeutique, tandis que les trois autres molécules pourraient nécessiter une optimisation chimique supplémentaireCyclic adenosine monophosphate (cAMP) production regulates certain aspects of mitochondria function in rodent cardiomyocytes, such as ATP production, oxygen consumption, calcium imports and mitochondrial permeability transition (MPT), but how this cAMP pool is controlled is not well known. In the first part of this thesis, we investigated the expression, localization and activity of several cAMP-degrading enzymes, phosphodiesterases (PDEs), in isolated rodent cardiac mitochondria. PDE2 expression was mainly detected in subsarcolemmal mitochondria, and cGMP-stimulated PDE2 activity was largest than PDE3 and PDE4, their activities were further confirmed in neonatal rat cardiomyocytes by real time FRET analysis. Moreover, the pharmacological inhibition or the cardiac-specific overexpression of PDE2 modulated mitochondrial respiration, mitochondrial membrane potential loss, MPT and calcium import. Thus, cAMP degradation by PDEs represents a new regulatory mechanism of mitochondrial function, and becomes a potential target in cardiovascular diseases therapy.In addition, the recent improvement of anticancer treatment results in an increase in surviving patients, but with a risk of long-term cardiotoxicity. Thus, in the second part of this thesis, we identified cardioprotective molecules from chemical libraries by developing a high throughput screening assay. We identified 6 potent and specific hits and validated them in 3 cellular models. We investigated the mechanisms of actions of each molecule and their cellular impact by using siRNA silencing, western-blot analysis, fluorescent imagery and real-time metabolic analyses. Three molecules could enter rapidly in preclinical and clinical studies in combination with radiation or chemotherapeutic agents for therapeutic development, while other three molecules may require further chemical optimization
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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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Nozawa, Atsuko. "Rab35 GTPase recruits NPD52 to autophagy targets." Kyoto University, 2018. http://hdl.handle.net/2433/230994.
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Zhang, Hanlin. "Translational control of autophagy rejuvenates immune responses." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:9950cef9-7592-41b4-973c-c906edad23c8.
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As our body's guardian, the immune system maintains systemic health through removal of pathogens, damage and cancer. Ageing of the immune system is associated with compromised immune responses as well as decreased tumour surveillance and is therefore a key risk factor for major diseases in the elderly. Adaptive immune responses are mediated by T and B lymphocytes, and failure in adaptive immunity is a particular hallmark of the ageing organism. Here we show that autophagy is impaired in aged murine B lymphocytes, and loss of autophagy causes severely reduced B cell responses. Our data demonstrate that B cell senescence can be reversed in an autophagy-dependent manner by spermidine, a naturally occurring polyamine metabolite. Mechanistically, our study reveals that the translation factor eIF5A, that requires spermidine for its activation, regulates the expression of the master autophagy/lysosomal transcription factor TFEB. Importantly, we show in humans that spermidine, eIF5A and TFEB levels decrease with age and may serve as ageing biomarkers. Taken together our results indicate that the translational control of autophagy by eIF5A is dysregulated with ageing, and identify a novel pathway with therapeutic implications.
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Winslow, Ashley Regan. "The role of impaired autophagy in neurodegeneration." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608894.
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Cull, Benjamin. "Autophagy and organelle turnover in Leishmania major." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/4396/.
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Paro, Simona. "RNA editing and autophagy in Drosophila melanogaster." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/8254.
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Post-transcriptional regulation of gene expression involves a diverse set of mechanisms such as RNA splicing, RNA localization, and RNA turn-over. Adenosine to Inosine (A-to-I) RNA editing is an additional post-transcriptional regulatory mechanism. Temporally, it occurs after transcription and before RNA splicing and has been shown in some instances to possibly modulate alternative splicing events. This is the case for example, with the pre-mRNA encoding the GluR- 2 subunit of AMPA receptor, a glutamate-activated ion channel. ADAR (Adenosine deaminase acting on RNA) proteins bind to double-stranded regions in pre-messenger RNAs. They deaminate specific adenosines, generating inosines; if the editing event occurs within the coding region, inosine is then interpreted as guanosine by the ribosomal translational machinery, changing codon meaning. These editing events can increase the repertoire of translated proteins, generating molecular diversity and modifying protein function. In mammals there are four ADAR genes: ADAR1, ADAR2, ADAR3 and TENR. ADAR3 and TENR are enzymatically inactive. All the proteins have two types of functional domains: (i) the catalytic deaminase domain at the carboxyl-terminus and (ii) the double stranded RNA binding domains, dsRBDs, at the amino terminus. ADAR1 and ADAR2 differ significantly at the amino terminus, by the number of the dsRNA binding domains (three and two dsRBDs for ADAR1 and ADAR2 protein, respectively). The differences observed between ADAR1 and ADAR2 are likely to reflect the different repertoires of substrates edited by these two enzymes. Data concerning the conservation of Adar genes throughout evolution suggest that Drosophila melanogaster has a unique Adar gene which is a true ortholog of human ADAR2 rather than an invertebrate gene ancestral for both vertebrate genes. Flies that are null mutants for Adar (Adar5G1 mutants) display profound behavioral and locomotive deficits. Impairment in motor activity of the mutants is succeeded by age-dependent neurodegeneration, characterized by swelling within the Adar-null mutant fly brain. The initial focus of my thesis was to elucidate what causes Adar mutant phenotypes or, whether it is possible, to suppress them. I took advantage of Drosophila genetics to establish a forward genetic screen for suppressors of reduced Adar5G1 viability which is approximately 20-30% in comparison to control flies at eclosion. The results from an interaction screen on Chromosome 2L were further confirmed using Exelixis P-element insertion lines. The screen revealed that decreasing Tor (Target of rapamycin) expression suppresses Adar mutant phenotypes. TOR plays a role in maintaining cellular homeostasis by balancing the metabolic processes. It controls anabolic events by phosphorylating eukaryotic translation initiation factor 4E-binding protein (4E-BP) and p70 S6 kinase (S6K) and inducing cap-mediated translation. However, different types of stress, signals or increased demand in catabolic processes, converge to reduce TOR enzymatic activity. This results in long-lived proteins and organelles being engulfed in double-membrane vesicles and degraded; this bulk degradation process is called (macro)autophagy. The second aim of my thesis was to clarify which pathway, downstream to TOR, was responsible for the suppression of Adar-null phenotypes. I mimicked the effect of reduced Tor expression by manipulating genetically the cap-dependent translation and the autophagy pathways. Interestingly, boosting the expression of Atg (autophagy specific genes) genes, such as, Atg1 and Atg5, thereby increasing the activation rate of the autophagy pathway, suppresses Adar5G1 phenotypes. Finally, I found that Adar5G1 mutant flies have an increased level of autophagy that is observable from the larval stage. I investigated possible stresses affecting our mutants; Adar-mutant larval fat cells show ER stress triggering an unfolded protein response as indicated by expression of XbpI-eGFP reporter. Thus, ER stress might induce increased autophagy and it can lead to locomotive impairments and neurodegeneration in Adar-null mutants. These results suggest a function for the Adar gene in regulating cellular stress.
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Watson, Alexander Scarth. "Autophagy in hematopoiesis and acute myeloid leukemia." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:2e66c5c3-4774-44d1-8345-d0dc827da16d.
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Acute myeloid leukemia (AML) develops following oncogenic alterations to hematopoietic stem (HSC) and progenitor cells (HSPCs) in the bone marrow, resulting in dysregulated proliferation of immature myeloid progenitors that interferes with normal hematopoiesis. Understanding the mechanisms of HSPC protection against damage and excessive division, and how these pathways are altered during leukemic progression, is vital for establishing effective therapies. Here, we show that autophagy, a lysosomal degradation pathway, is increased in HSPCs using a novel imaging flow cytometry autophagy assay. Loss of hematopoietic autophagy following deletion of key gene Atg5 resulted in increased HSC proliferation, leading to HSC exhaustion and bone marrow failure. Although erythrocyte and lymphocyte populations were negatively impacted by autophagy loss, myeloid cells showing immature characteristics were expanded. Deletion of Atg5 in an AML model resulted in increased proliferation under metabolic stress, dependent on the glycolytic pathway, and aberrant upstream mTOR signaling. Moreover, modulation of Atg5 altered leukemic response to culture with stromal cells. Finally, primary AML cells displayed multiple markers of decreased autophagy. These data suggest a role for autophagy in preserving HSC function, partially through suppression of HSPC proliferation, and indicate that decreased autophagy may benefit AML cells. We postulate that modulation of autophagy could help maintain stem cell function, for example during transplantation, and aid AML therapy in a setting-specific manner.
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Lin, Lin. "Complement-Related Regulates Autophagy in Neighboring Cells." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/911.
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Autophagy is a conserved process that cells use to degrade their own cytoplasmic components by delivery to lysosomes. Autophagy ensures intracellular quality control and is associated with diseases such as cancer and immune disorders. The process of autophagy is controlled by core autophagy (Atg) genes that are conserved from yeast to mammal. Most Atg proteins and their regulators were identified through pioneering studies of the single cell yeast Saccharomyces cerevisiae, and little is known about factors that systematically coordinate autophagy within the tissues of multicellular animals. The goal of this thesis is to identify new autophagy regulators and provide a better understanding of the regulatory mechanisms within multicellular animals. My research determined Macroglobulin complement-related (Mcr), a Drosophila complement orthologue, can activate autophagy during developmental cell death. Unlike most known autophagy regulators, Mcr functions in a cell non-autonomous manner to trigger autophagy in neighboring cells. To my knowledge, this is the first identified autophagy factor that cell non-autonomously activates autophagy. Additionally, I found that Mcr, a secreted protein, instructs the autophagy machinery through the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Lastly, Mcr is dispensable for both nutrient deprivation-induced autophagy in the fat body and developmentally programmed autophagy in the dying midgut of Drosophila. Therefore, this study unveils a mechanism in a multicellular organism by which autophagy is systematically controlled in distinct cell contexts.
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Liu, Elizabeth. "The Autophagy Pathway and Toxoplasma gondii Infection." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1428103561.
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Ballhaus, Florentine. "Investigating plant autophagy with new chemical modulators." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-428075.
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Autophagy is a major catabolic pathway in which cell components get sequestered in a double membrane vesicle, transported to the vacuole, degraded by vacuolar hydrolases and recycled. Through this process, cells ensure cell homeostasis and remobilise nutrients. The autophagic flux can be enhanced as an adaptive stress response, improving plants resistance against stress, reducing aging and ultimately increasing yield. However, autophagy regulation in plants remains poorly understood. Novel plant-specific modulators can be used in a chemical genetic approach for identification of proteins involved in the autophagy pathway. Furthermore, autophagy enhancers can find their application in agriculture for improved plant fitness. Known autophagy modulators have severe off-target effects, affecting plant growth and development. A recent screening identified two potential autophagy modulators. We developed a novel method for photoaffinity labelling and pulldown assay in Arabidopsis thaliana to identify potential interactors of the modulators. The identification of autophagy-related proteins will help to further elucidate the autophagic pathway in plants. The effect of the new autophagy enhancers on plant growth and development was analysed by automated growth assays. In comparison with a currently available autophagy enhancer, treated plants showed higher viability, indicating possible further applications for the new autophagy modulators in planta.
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Simões, Ana Marisa Henriques Duarte. "Structural characterization of proteins associated to autophagy." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/9691.
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Mestrado em Biologia Molecular e CelularDOR (ou Tumor protein p53 inducible nuclear protein 2 - tp53inp2) é uma proteína bifuncional que atua no núcleo e no citosol. No núcleo DOR atua como um co-fator nuclear, liga-se e co-ativa no recetor da hormona da tiróide. No último, DOR desloca-se do núcleo para o citoplasma em situações de ativação da autofagia ou stress celular, localiza-se no autofagossoma e interage diretamente com as proteínas associadas à membrana do autofagossoma, LC3 e GATE16. A caraterização da interação entre a DOR e os seus parceiros e a relevância da DOR na autofagia é muito importante. A autofagia tem um papel importante no envelhecimento, morte celular, defesa contra agentes intracelulares patogénicos, doenças neurodegenerativas e tumorogenesis, o que demonstra a importância biológica e médica de estudar as proteínas envolvidas neste processo. A proteína de fusão NusA-DOR e os seus interatores, LC3 e GATE16, foram expressos em E.coli. Todas as proteínas foram purificadas por cromatografia de afinindade, seguida por cromatografia de exclusão molecular (DOR) ou por cromatografia de troca iónica (LC3 e GATE16). A estabilidade da DOR e a interação com os seus parceiros intracelulares foi analisada estruturalmente, através de ressonância plasmónica de superfície, circular dicroísmo e estabilidade térmica. Um péptido da DOR contendo o local de interação (região LIR) foi produzido para os ensaios de co-cristalização por difusão vapor. O péptido da DOR liga num sulco da LC3 numa conformação em gancho, dois importantes aminoácidos medeiam a interação com LC3, Trp35 e a Leu38. A conformação desta estrutura é diferente das outas estruturas conhecidas da LC3 com domínios LIR.
DOR (or Tumor protein p53 inducible nuclear protein 2 - tp53inp2) is a bifunctional protein that operates both in the nucleus and in the cytosol. In the nucleus, DOR acts as a nuclear co-factor, and binds to and co-activates the thyroid hormone receptor. In the later, DOR moves from the nucleus to the cytoplasm under conditions characterized by the activation of autophagy or cellular stress and can be localized to early autophagosome and interact directly with the autophagosome membrane associated protein LC3 and GATE16. Characterization of the interaction between DOR and its interacting partners is very important to understand the relevance of DOR in autophagy. Since autophagy plays a protective role in aging, cell death, defense against intracellular pathogens, neurodegenerative diseases and tumorogenesis, studying DOR might have a large biological and medical relevance. The fusion protein NusA-DOR and its interactors, LC3 and GATE16, were expressed in E.coli. All the proteins were purified by affinity chromatography, followed by size exclusion chromatography (DOR) or ion exchange chromatography, (LC3 and GATE16). The stability of DOR and the interaction with intracellular partners has been structurally analyzed, by surface plasmon resonance, circular dichroism and differential scanning fluorimetry. A DOR peptide containing the interaction site (LIR motif) has been produced for cocrystallization experimentss. The DOR peptide binds within LC3 groove in a hairpin conformation, two important amino acids, Trp35 and Leu38 mediated the insertion into pockets of LC3. This peptide displays a new conformation, when compared with the known three-dimensional strutures of LC3:LIR complexes.
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Karnes, Jonathan Burgess. "PI3K Class IIalpha Is Required for Autophagy." Thesis, Van Andel Research Institute, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10268645.
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Autophagy is a cellular recycling process in which cytoplasmic proteins and organelles are sequestered in a double membrane vesicle, delivered to the lysosome, and degraded following fusion of the two vesicles. A key part of the initiation signaling for autophagy is the generation of phosphoinositol 3-phosphate (P13P) by class III phosphoinositol 3-kinase also knows as Vps 34. In humans there are eight P13K isoforms divided into three classes, four class I enzymes, three class II enzymes, and a single class III enzyme. Of these eight enzymes, only the class III isoform is thought to participate directly in autophagic signaling. A quantitative microscopy based, loss-of-function survey of all eight P13K isoforms was used to determine their relative contribution to autophagic signaling, as measured by LC3 positive autophagic vesicles. As predicted, knockdown of P13K-class III reduced the number of autophagic vesicles in cells. Interestingly, knockdown of the P13K-class IIα isoform had an even more potent effect on reducing the number of autophagic vesicles than knockdown of P13K-class III. In follow up studies, knockdown of P13K-class IIα reduced endogenous LC3 conversion, caused the accumulation of p62 and lipid droplets, and colocalized with endosomal markers. These results suggest P13K-class IIα may act to promote autophagy through the shuttling of endosomal vesicles into the autophagic pathway and approaches to test this hypothesis will be discussed. The requirement of P13K-class IIα for autophagy is an important finding as it indicates a role for class II P13Ks in autophagy.
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Lin, Lin. "Complement-Related Regulates Autophagy in Neighboring Cells." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/911.
Full textAbstract:
Autophagy is a conserved process that cells use to degrade their own cytoplasmic components by delivery to lysosomes. Autophagy ensures intracellular quality control and is associated with diseases such as cancer and immune disorders. The process of autophagy is controlled by core autophagy (Atg) genes that are conserved from yeast to mammal. Most Atg proteins and their regulators were identified through pioneering studies of the single cell yeast Saccharomyces cerevisiae, and little is known about factors that systematically coordinate autophagy within the tissues of multicellular animals. The goal of this thesis is to identify new autophagy regulators and provide a better understanding of the regulatory mechanisms within multicellular animals. My research determined Macroglobulin complement-related (Mcr), a Drosophila complement orthologue, can activate autophagy during developmental cell death. Unlike most known autophagy regulators, Mcr functions in a cell non-autonomous manner to trigger autophagy in neighboring cells. To my knowledge, this is the first identified autophagy factor that cell non-autonomously activates autophagy. Additionally, I found that Mcr, a secreted protein, instructs the autophagy machinery through the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Lastly, Mcr is dispensable for both nutrient deprivation-induced autophagy in the fat body and developmentally programmed autophagy in the dying midgut of Drosophila. Therefore, this study unveils a mechanism in a multicellular organism by which autophagy is systematically controlled in distinct cell contexts.
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Kim, Insil Lemasters John J. "Mitochondrial degradation by autophagy mitophagy in hepatocytes." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1743.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell and Developmental Biology." Discipline: Cell and Developmental Biology; Department/School: Medicine.
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Chehab, Tala. "The role of calcium signalling in autophagy." Thesis, Open University, 2018. http://oro.open.ac.uk/55091/.
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Autophagy is a catabolic process that is important for degradation of cellular components, and for cell survival, and has also been associated with pathological disorders and tumour growth. Autophagy is a complex process; many factors and messengers converge to control steps along the autophagic pathway. Ca2+ has been proposed to regulate autophagy. However, Ca2+ has been proposed to be both pro- and anti-autophagic. To better understand how Ca2+ has these opposing effects, this study investigated in what ways particular sources of Ca2+, and the characteristics of Ca2+ signals impacted on autophagy. The fundamental need for Ca2+ in the activation of autophagy was demonstrated by loading cells with an exogenous Ca2+ buffer, which prevented various stimuli from triggering autophagy. Autophagy could be activated by inhibiting the transfer of Ca2+ from the endoplasmic reticulum to the mitochondrial matrix. This was achieved by expressing an enzyme that prevented Ca2+ release from inositol 1,4,5-trisphosphate receptors, inhibition of mitochondrial respiration, and knockdown of the mitochondrial Ca2+ uniporter. The triggering of autophagy under these conditions was due to reduced cellular ATP levels. These data suggest that Ca2+ signals arising from InsP3Rs suppress autophagy. Additional studies used a well-characterised Ca2+ transport pathway to generate cellular Ca2+ signals, and examined their ability to trigger autophagy. This pathway, known as ‘store-operated Ca2+ entry’ (SOCE), was activated by depleting endoplasmic reticulum Ca2+ stores using inhibitors of sarco/endoplasmic reticulum ATPases (SERCA). It was found that sustained cellular Ca2+ signals arising via chronic inhibition of SERCA were pro-autophagic. The activation of autophagy absolutely required the presence of extracellular Ca2+, and was not due to cellular stress. Using pharmacological inhibition of various Ca2+-sensitive kinases, it was found that at least part of the autophagy that occurred during SOCE was due to activation of Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β, also known as CaMKK-2).
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NINFOLE, ELISABETTA. "Involvement of autophagy in cholestatic liver diseases." Doctoral thesis, Università Politecnica delle Marche, 2022. https://hdl.handle.net/11566/298981.
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L'autofagia è un processo fisiologico di degradazione lisosomiale, essenziale per l'omeostasi cellulare e onnipresente in tutte le cellule eucariotiche. La deregolazione epatica dell'autofagia è stata descritta in diverse condizioni, dall'obesità al diabete e alle malattie colestatiche, mentre la stimolazione dell'autofagia sembra migliorare il danno epatico.
Il progetto è focalizzato sull'identificazione di vie molecolari che si attivano durante il processo autofagico in risposta al danno del dotto biliare e se l'autofagia svolge un ruolo nella regolazione dei processi di invecchiamento cellulare a livello dell'epitelio biliare.
I colangiociti normali di ratto (NRC), sono una linea cellulare murina del dotto biliare intraepatico, utilizzati per studiare il processo autofagico. Abbiamo analizzato il ruolo dell'autofagia nei colangiociti, il legame tra autofagia e senescenza e l'uso di inibitori e attivatori autofagici.
In vitro, l'autofagia viene attivata a causa dell'azione degli induttori con un concomitante declino dei marker di senescenza. Come ulteriore conferma, sono stati eseguiti esperimenti per modulare l'attività dell'autofagia stessa, utilizzando un inibitore. Il nostro gruppo di ricerca ha recentemente dimostrato che la proteina Twinfilin-1 (TWF1) modula la risposta al danno dei colangiociti all'invecchiamento. Successivamente, abbiamo studiato se Twf1 possa svolgere un ruolo nelle prime fasi del destino cellulare tra autofagia e senescenza.
L'uso di modulatori dell'autofagia (induttori/inibitori) combinati con agenti farmacologici sembra essere una strategia promettente per il trattamento di una varietà di condizioni colestatiche. In questo contesto, la modulazione Twf1 della biologia dei colangiociti può svolgere un ruolo rilevante nel decidere il destino cellulare tra autofagia e senescenza. Ulteriori studi forniranno con modelli sperimentali in animali da laboratorio potranno rafforzare i risultati ottenuti in vitro.Autophagy is a physiological lysosomal degradation process, essential for cellular homeostasis and ubiquitous in all eukaryotic cells. Dysregulation of hepatic autophagy has been described in several conditions, from obesity to diabetes and cholestatic disease, while stimulation of autophagy seems to ameliorate the liver damage. The project was focused on the identification of molecular pathways which are activated during autophagic process in response to damage of the bile duct and whether autophagy plays a role in regulating cellular aging processes at the level of the biliary epithelium. Normal rat cultured cholangiocytes (NRC), a murine intrahepatic bile duct cell line, were used to investigate the autophagic process. We analyzed the role of autophagy in cholangiocytes, the link between autophagy and senescence and the use of autophagic inhibitors and activators. In vitro, autophagy is activated due to the action of the inductors with a concomitant decline of senescence marker. As further confirmation, we perform experiments to modulate the activity of autophagy itself, by using inhibitor. Our research group has recently shown that the Twinfilin-1 protein (TWF1) modulates the response to damage of cholangiocytes to aging. Subsequently, we investigated if Twf1 may play a role in the early stages of cell fate between autophagy and senescence. The use of autophagy modulators (inductors / inhibitors) combined with pharmacological agents appears to be a promising strategy to treat a variety of cholestatic conditions. In this settings, Twf1 modulation of cholangiocyte biology may play a relevant role when deciding cell fate between autophagy and senescence. Further studies will provide the use of experimental models in laboratory animals to strengthen the results obtained in vitro.