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CONTRIBUTION OF THE UNFOLDED PROTEIN RESPONSE (UPR) TO ADIPOGENESIS AND WHOLE BODY ENERGY HOMEOSTASISBasseri, Sana 04 1900 (has links)
<p>The endoplasmic reticulum (ER) is a specialized organelle that facilitates correct protein folding and maturation. Disruptions in ER homeostasis lead to ER stress and activation of a series of signal transduction cascades known as the unfolded protein response (UPR), which acts to restore ER homeostasis. In recent years, ER stress and UPR dysfunction have been linked to obesity, fatty liver and insulin resistance. Lipid-laden adipocytes, the main cellular component of white adipose tissue (WAT), play a critical role in whole body energy homeostasis as well as lipid and carbohydrate metabolism. Mature adipocytes, which are metabolically active endocrine cells, differentiate from precursor fibroblast-like preadipocytes, through a process called adipogenesis, leading to formation of cells capable of secreting numerous proteins, cytokines and hormones. ER homeostasis and UPR activation are essential to the function/differentiation of highly secretory cells, however, the role of ER stress/UPR activation in adipogenesis had previously not been examined. We hypothesized that<em> adipogenesis may rely on physiological UPR activation to accommodate the demand on the ER for increased folding and secretion of proteins.</em></p> <p>Initial experiments examining UPR activation during 3T3-L1 adipogenesis identified that expression of ER stress/UPR markers was modulated during adipocyte differentiation. Furthermore, inhibition of ER stress/UPR activation by the chemical chaperone, 4-phenyl butyric acid (4-PBA), inhibited adipogenesis and blunted high fat-diet induced weight gain in 4-PBA supplemented mice. These findings suggested that UPR activation modulates adipogenesis and adipose tissue metabolism.</p> <p>Subsequently, we sought to identify novel candidate ER stress/UPR responsive genes that may be involved in adipogenesis and WAT metabolism. The expression of a recently recognized ER stress-responsive gene, T-cell death associated gene 51 (TDAG51) was identified to be differentially regulated during adipogenesis. However, the function of TDAG51 in adipogenesis or energy regulation was not known. Studies from this thesis showed that TDAG51 protein expression is attenuated by ER stress/UPR activation in preadipocytes and declines during adipogenesis. Based on these results, and given the importance of adipogenesis in WAT function and whole body energy metabolism, it was<em> </em>hypothesized that<em> TDAG51 may be a novel regulator of adipogenesis and energy homeostasis.</em> Indeed, as reported here, knock-down or absence of TDAG51 (<em>TDAG51<sup>-/-</sup></em>) in pre-adipocytes increased lipogenesis and lead to earlier and more potent expression of adipogenic markers.</p> <p>Finally, we investigated whether absence of TDAG51 in mice affected adiposity and metabolic outcomes. Consistent with the <em>in vitro </em>results, we found that <em>TDAG51<sup>-/-</sup></em><sup> </sup>mice fed a standard chow diet, exhibited an age-associated increase in WAT, developed fatty liver, and exhibited insulin resistance as compared to wild-type mice.</p> <p>Taken together, the findings in this thesis indicate that physiological UPR activation and the UPR-responsive gene TDAG51 play important roles in regulating adipogenesis, lipogenesis and whole-body energy metabolism. Thus, therapeutic approaches aimed at modulating ER folding capacity, UPR activation and/or TDAG51 expression may have great potential in the treatment of obesity and its co-morbidities.</p> / Doctor of Philosophy (PhD)
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Développement de modèles C. elegans de Sclérose Latérale AmyotrophiqueVaccaro, Alexandra 12 1900 (has links)
Les gènes TDP-43 (TAR DNA Binding Protein 43) et FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma) sont actuellement à l’étude quant à leurs rôles biologiques dans le développement de diverses neuropathies telles que la Sclérose Latérale Amyotrophique (SLA). Étant donné que TDP-43 et FUS sont conservés au cours de l’évolution, nous avons utilisé l’organisme modèle C. elegans afin d’étudier leurs fonctions biologiques. Dans ce mémoire, nous démontrons que TDP-1 fonctionne dans la voie de signalisation Insuline/IGF pour réguler la longévité et la réponse au stress oxydatif. Nous avons développé des lignées C. elegans transgéniques mutantes TDP-43 et FUS qui présentent certains aspects de la SLA tels que la dégénérescence des motoneurones et la paralysie adulte. La protéotoxicité causée par ces mutations de TDP- 43 et FUS associées à la SLA, induit l’expression de TDP-1. À l’inverse, la délétion de tdp-1 endogène protège contre la protéotoxicité des mutants TDP-43 et FUS chez C. elegans. Ces résultats suggèrent qu’une induction chronique de TDP-1/TDP-43 sauvage propagerait la protéotoxicité liée à la protéine mutante. Nous avons aussi entrepris un criblage moléculaire pilote afin d’isoler des suppresseurs de toxicité neuronale des modèles transgéniques mutants TDP-43 et FUS. Nous avons ainsi identifié le bleu de méthylène et le salubrinal comme suppresseurs potentiels de toxicité liée à TDP-43 et FUS via réduction de la réponse au stress du réticulum endoplasmique (RE). Nos résultats indiquent que l’homéostasie de repliement des protéines dans le RE représente une cible pour le développement de thérapies pour les maladies neurodégénératives. / Two recently discovered causative genes for ALS, TDP-43 (TAR DNA Binding Protein 43) and FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma) are under further investigation regarding their biological roles in neuropathies such as Amyotrophic Lateral Sclerosis (ALS). Since TDP-43 and FUS are evolutionarily conserved we turned to the model organism C. elegans to learn more about their biological functions. Here we report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response. We have generated mutant TDP-43 and FUS transgenic lines in C. elegans that recapitulate certain aspects of ALS including motor neuron degeneration and adult-onset paralysis. Proteotoxicity caused by ALS- associated mutations in TDP-43 or FUS also induce TDP-1 expression and consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild type TDP-1/TDP-43 by proteotoxicity may actively promote neurodegeneration. We also screened for small- molecule suppressors of mutant TDP-43 and FUS neuronal toxicity in transgenic C. elegans and identified methylene blue and salubrinal as potent suppressors of TDP-43 and FUS toxicity in our models through induction of the endoplasmic reticulum (ER) stress response. Our results indicate that protein folding homeostasis in the ER may be an important target for therapeutic development in neurodegenerative diseases.
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Análise da expressão de genes ligados ao estresse de retículo endoplasmático no adenoma de paratireoide / Gene expression of endoplasmic reticulum stress in the parathyroid adenomaIwakura, Ricardo 29 October 2018 (has links)
Introdução: O hiperparatireoidismo primário (HP) é a terceira maior causa de doenças endocrinológicas na população, sendo a principal causa de hipercalcemia. Caracteriza-se por hipersecreção do paratormônio (PTH) pelas células principais da paratireoide, levando a um distúrbio da homeostase do cálcio (Ca). O adenoma de paratireoide é a principal causa do HP, com 80-85% dos casos, sendo o objeto deste estudo. Mutações genéticas podem corresponder a mais de 11% da origem deste tumor benígno hipersecretor. Apesar dos avanços dos métodos de diagnóstico, o tratamento é essencialmente cirúrgico, havendo carência de tratamentos alternativos eficientes, que podem ser aprimorados com maior conhecimento fisiopatológico. Como as células do adenoma são hipersecretoras de proteínas, possuem abundante quantidade de retículo endoplasmático (RE), onde as proteínas sofrem dobramento, adquirindo sua conformação nativa. Em células hipersecretoras é comum o aumento da atividade da maquinaria de tratamento protéico, gerando sinais de sobrecarga no RE e citoplasma, sendo denominado de estresse do retículo endoplasmático (ERE). O ERE e as suas vias efetoras, a UPR (resposta a proteínas não-dobradas) e o ERAD (degradação associada ao retículo endoplasmático), estão presentes na fisiopatologia de diversas doenças ou de células hipersecretoras, servindo como importante alvo terapêutico. Objetivos: Analisar a atividade do ERE nas células do adenoma de paratireoide. Casuística e Métodos: Análise da expressão dos principais genes do ERE por RealTime-PCR, em 14 pacientes portadores de adenoma de paratireoide operados no Serviço de Cirurgia de Cabeça e Pesoco do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, comparando-se a expressão do adenoma em relação ao controle (tecido normal). Resultados: Houve aumento da expressão de vários genes relacionados ao ERE, ERAD e à UPR, com significância estatística, principalmente da via de PERK (Pancreatic endoplasmic reticulum kina), do ERAD e da autofagia, evidenciando um quadro de cronicidade e eficência da maquinaria do ERE, com finalidade antiapoptótica. O resultado foi compatível com a manifestação clínica do adenoma de paratireoide, que cursa com raros casos de remissão espontânea e necrose central. Conclusão: Verificou-se que há uma atividade do ERE na fisiopatologia do adenoma de paratireoide, com efetividade na síntese e seleção protéica do PTH, trazendo longevidade às células do adenoma por meio da prevalência dos mecanismos de citoproteção, em detrimento da via da apoptose. O estudo traz importantes informações que podem ser úteis na elaboração de novos tratamentos medicamentosos, fazendo com que o ERE seja um futuro alvo terapêutico também no adenoma de paratireoide, assim como já é a realidade em outras patologias. / Background: Primary Hyperparathyroidism (PHP) is the third most common cause of endocrinologic disorders in the world, and the main cause of hypercalcemia. It is manifested by PTH (parathormone) hypersecretion by the parathyroid chief cells and consecutive calcium (Ca) homeostasis disturbance. Parathyroid adenoma (PA) is present in 80-85% of patients and, for this reason, is the aim of this study. Gene mutations is associated with at least 11% of this hypersecretory benign tumor. Even though the clinical presentation is much better than the past few decades, therapeutic options beside surgery do not advance along increasing efficiency in diagnostic tools. This is partly because there are many lacks in the pathophysiology of Pas, that would give new possibilities of medical treatment. PA is composed of hypersecretory cells and rich endoplasmic reticulum cytoplasm, where the main protein treatment and selection factors are situated. Considering that PA cells have protein hypersecretory activities, it is expected an abundant ER mass, that provides the machinery to treat and fold the great amount of nascent protein, to turn it to native form. As protein traduction increases, more energy is needed to keep ER function and this may result in the endoplasmic reticulum stress (ERE) in the cells. ERE and the downstream effectors, UPR (unfolded response protein) and ERAD (endoplasmic reticulum associated degradation), are acting in the physiology of several diseases and others hypersecretory cell types, providing important treatment targets. Objectives: To analyze ERE activity in PA cells. Casuistic and Methods: Evaluation of the main ERE genes expressed with Real Time-PCR analysis, in 14 patients with PA PHP and that were treated with conventional surgery, with further comparison between PA and control groups. Results: There were significant expression elevation of ERE, UPR and ERAD related genes, with statistical significance, specially of PERK downstream, ERAD and autophagy induction, suggesting efficient, though chronic, ERE activity level, with stimulated anti-apoptosis pathway. The final results confirmed our hypothesis that there is lower pro-apoptosis activity than expected by some authors, but this is compatible with low incidence of spontaneous remission or PA necrosis. Conclusion: There is contundent ERE activity in the PA pathophysiology, with great protein metabolism effectiveness expressed by PTH bioactivity, increasing cell longevity by stimulating cytoprotection pathways, instead of pro-apoptosis one. We believe this outcome will influence others research on this challenging and gratifying field of investigation, that will certainly provide new treatment options to PA, as ERE has been playing a significant role as therapeutic target with others hypersecretory diseases.
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Rôles de la méthionine sur le métabolisme hépatique de la truite arc-en-ciel (Oncorhynchus mykiss) : focus sur les mitochondries / Roles of methionine on hepatic metabolism of rainbow trout (Oncorhynchus mykiss) : focus on mitochondriaSéité, Sarah 14 May 2019 (has links)
L’impératif d’une aquaculture durable conduit à orienter l’alimentation des poissons vers la substitution de la farine de poisson par des produits végétaux renouvelables. Toutefois, ce remplacement est souvent limité par des niveaux trop faibles en méthionine dans les matières premières végétales. Ainsi la supplémentation en méthionine de ces nouveaux régimes à base de végétaux est essentielle, mais requière une bonne connaissance de son rôle pour adapter les apports aux conditions physiologiques des poissons tout en prenant en compte les contraintes économiques et environnementales de production. Dans ce contexte, cette thèse avait pour principal objectif de caractériser les effets induits par une carence en méthionine sur le métabolisme mitochondrial de la truite arc en ciel. Les résultats obtenus dans notre première étude montrent que l'alimentation de truites avec un régime déficient en méthionine entraîne une baisse des performances de croissance associée à une baisse de l’intégrité mitochondriale et une diminution du statut oxydatif dans le foie. Nous démontrons également que ces perturbations s’accompagnent de l’induction d’un processus de dégradation des mitochondries par autophagie (appelé mitophagie) ainsi que d’une augmentation du stress du Réticulum Endoplasmique (RE) et de l’apoptose. Ces données originales publiées dans le journal Scientific Reports mettent ainsi en évidence les liens étroits qui existent entre différentes fonctions cellulaires pour faire face à un déséquilibre nutritionnel en méthionine. Outre cet effet à court terme, nous démontrons également, dans un seconde étude, qu’une carence en méthionine alimentaire pendant une courte période (2 semaines) lors des premiers repas des alevins entraîne une induction à long terme de facteurs liés à la mitophagie. Ces résultats, soumis à publication dans Journal of Experimental Biology, démontrent ainsi pour la première fois la mise en place d’un processus de programmation de cette fonction cellulaire par une carence précoce en méthionine. L’enrichissement en H3K4me3 et H3K36me3 des foies des poissons issus d’alevins carencés en méthionine par rapport aux poissons témoins suggère une implication de mécanismes épigénétiques dans ces effets. Enfin, dans une troisième étude qui se détache de la thématique principale de la thèse et qui a fait l’objet d’une publication dans le journal Frontiers in Physiology, nous nous sommes attachés à préciser les interactions existante entre l’autophagie, l’homéostasie du RE et le métabolisme intermédiaire. Dans l’ensemble, ces données approfondissent notre compréhension du rôle de la méthionine alimentaire au niveau cellulaire et soulignent le potentiel de cet acide aminé en tant que levier pour appliquer de nouvelles stratégies alimentaires, comme la programmation nutritionnelle, afin d’optimiser la nutrition et la santé des poissons d'élevage. / The expansion of the aquaculture industry in combination with limited availability and high prices of fishmeal has prompted feed producers to include more plant proteins in the aquaculture feeds. However, replacement of fish meal with plant proteins is often limited by the level of methionine in the alternative plant protein sources. Understanding of the different roles of methionine in fish is therefore essential to develop new diets and/or feeding strategies that are in tune with optimal fish growth, environmental and economic constraints. In this context, the main objective of this thesis was to characterize the effects induced by methionine deficiency on the hepatic mitochondrial metabolism in rainbow trout. The results obtained in our first study show that feeding trout with a methionine deficient diet leads to a decrease in growth performance associated with a decrease in both mitochondrial integrity and oxidative stress in the liver. We also demonstrate that these defects are accompanied by the induction of an autophagy-dependent mitochondrial degradation process (called mitophagy) as well as an increase in Endoplasmic Reticulum (ER)-stress and apoptosis. These original data published in Scientific Reports thus highlight the existence of close interactions between different cellular functions to cope to a dietary methionine deficiency. In addition to this short-term effect, we also demonstrate in a second study (submitted for publication in the Journal of Experimental Biology), that early nutritional stimulus during two weeks with a methionine deficient diet resulted in a long term programming of mitophagy. The enrichment of H3K4me3 and H3K36me3 in the liver of fish from methionine-deficient fry compared to their control counterparts suggests that epigenetic mechanisms are involved in these effects. Finally, in a third and last study, recently accepted for publication in Frontiers in Physiology, we sought to clarify, in primary culture of trout hepatocytes, the existing interactions between autophagy, ER homeostasis and intermediate metabolism under amino acid deprived conditions. Together, the results obtained in the present thesis extended our understanding of the role of dietary methionine at cellular level and emphasize the potential of this amino acid to apply new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish.
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L’angiogénine : un nouveau médiateur de la réponse au stress du Réticulum Endoplasmique / Angiogenin : a novel mediator of the Endoplasmic Reticulum stress responseMami, Iadh 28 October 2015 (has links)
Le stress du Réticulum Endoplasmique (RE) est impliqué dans la physiopathologie des maladies rénales, et la réponse UPR (Unfolded Protein Response), qui est activée en réponse à ce stress, joue un rôle important dans l'homéostasie des cellules tubulaires rénales et des podocytes. L’étude des mécanismes moléculaires et des conséquences de l'activation de cette voie est donc importante dans la compréhension de la physiopathologie des maladies rénales et dans la caractérisation de biomarqueurs de lésions évolutives. L’Angiogénine (ANG, appelée également RNase 5) est une ribonucléase secrétée, qui est impliquée dans la réponse à certains stress cellulaires, et permet une adaptation cellulaire et tissulaire.
L'objectif de ce travail a été de mettre en évidence les mécanismes de régulation et les fonctions biologiques de l'ANG en réponse au stress du RE. A partir d'un modèle de cellules tubulaires rénales humaines en culture, nous avons montré que le stress du RE induisait l’expression de l’Angiogénine ainsi que sa sécrétion. Cette observation a été également faite sur différents modèles murins de lésions rénales. Le facteur transcriptionel sXBP1, activé par le transducteur de la réponse UPR, IRE1a, est directement impliqué dans la régulation de l'expression de l'Angiogénine.
Nous avons mis en évidence que l'Angiogénine participait à l’inhibition de la traduction protéique en réponse au stress du RE en produisant des fragments d'ARN de transfert appelés tiRNAs (stress-induced tRNA fragments) qui répriment la traduction des protéines en interférant avec le complexe initiateur de la traduction. L'Angiogénine favorise la survie cellulaire en réduisant l'apoptose induite par le stress du RE, et des souris invalidées pour le gène codant l'Angiogénine sont plus sensibles aux lésions de nécrose tubulaire aigues induites par la Tunicamycine. Outre les propriétés cellulaires "intrinsèques" de l'Angiogénine, nous avons également caractérisé les mécanismes de sécrétion de l'Angiogénine par l'épithélium rénal en situation de stress du RE. La sécrétion épithéliale de l'Angiogénine est sous le contrôle des facteurs transcriptionnels NF-κB et sXBP1, et se produit sous un mode conventionnel, c’est-à-dire dépendant du transit par l'appareil de Golgi. A ce titre, la régulation de l'Angiogénine est similaire à celle de l'Interleukine 6. L'Angiogénine induit une polarisation des macrophages vers un phénotype pro-inflammatoire. Enfin, considérant que l'Angiogénine est secrétée par l'épithélium rénal en situation de stress, nous avons montré que l’Angiogénine peut être un marqueur non invasif de souffrance rénale. L'Angiogénine peut être quantifiée dans les urines de patients porteurs de maladies rénales, et sa concentration est corrélée à la concentration urinaire de Retinol Binding Protein (une protéine de petit poids moléculaire, marqueur de dysfonction tubulaire), mais pas avec celle de l'Albumine. En outre, la concentration urinaire d'Angiogénine est significativement plus élevée dans les urines de patients transplantés rénaux dont la biopsie rénale met en évidence des lésions de tubulite (rejet aigu cellulaire et néphropathie associée au BK virus) que dans les urines de patients indemnes de lésions tubulaires (rejet humoral, ou absence de lésions histologiques). Nous avons mis en évidence par immuno-histochimie un marquage nucléaire du facteur transcriptionnel sXBP1 dans les tubules de reins porteurs de lésions de tubulite, suggérant un lien potentiel entre sécrétion d'Angiogénine et activation du facteur transcriptionnel sXBP1 dans un environnement inflammatoire. En conclusion, nous avons intégré la régulation l'Angiogénine dans la réponse épithéliale rénale au stress du RE, et caractérisé ses fonctions biologiques intracellulaires et paracrines. Notre travail a identifié l'Angiogénine urinaire en étant que potentiel marqueur de lésions rénales tubulaires. / The Endoplasmic Reticulum (ER) stress is involved in the pathophysiology of renal diseases ; the UPR (Unfolded Protein Response), which is activated in response to that stress plays an important role in renal tubular cells and podocytes homeostasis and consequently in tissu homeostasis. Understanding the molecular mechanisms and the consequences of the activation of this pathway is important to characterize the pathophysiology of renal diseases and identification of biomarkers of ongoing lesions. Angiogenin (ANG, also known as RNase 5) is a secreted ribonuclease, which is involved in the cellular stress response, it allows cell and tissue adaptation. The goal of this work was to clarify and identify the mechanisms regulating Angiogenin’s expression and its biological functions during ER stress. Using a human renal tubular cell line, we have shown that ER stress induces the expression of angiogenin and its secretion. This observation was also made on several murine models of renal injury. The transcriptional factor sXBP1 activated by the UPR transducer, IRE1α, is directly involved in regulating the expression of angiogenin. We have shown that angiogenin participates in the inhibition of protein translation in response to ER stress by cleaving transfer RNA and generating tiRNAs (stress-induced tRNA fragments) that suppress protein translation by interfering with the translation initiation complex. Angiogenin promotes cell survival by reducing ER stress-induced apoptosis, ANG knockout mice are more sensitive to acute tubular necrotic lesions induced by tunicamycin. In addition to the cell-autonomous effects of angiogenin, we also characterized the mechanisms by which Angiogenin is secreted by the renal epithelium under ER stress. Angiogenin is secreted in a conventional manner under the control of the transcriptional factors NF-kB and sXBP1. As such, the regulation of angiogenin is similar to Interleukin-6. We also demonstrated that Angiogenin induces macrophage polarization to a pro-inflammatory phenotype. Finally, considering that angiogenin is secreted by the renal epithelium under stress, we have shown that angiogenin may be a noninvasive marker of kidney injury. Angiogenin can be quantified in the urine of patients with kidney disease, its urinary concentration is correlated to the urinary concentration of Retinol Binding Protein (a low molecular weight protein marker of tubular dysfunction), but not with that of Albumin . In addition, the urinary concentration of angiogenin is significantly higher in the urine of renal transplant patients whose renal biopsy highlights tubulitis lesions (cell acute rejection and BK virus associated nephropathy) than in the urine of patients without histological tubular damage (antibody-mediated rejection, or no visible histological lesions). We have demonstrated by immuno-histochemistry a tubular nuclear localization of the activated transcriptional factor sXBP1 in the biopsies of patients with high tubulitis score, suggesting a potential relationship between the secretion of Angiogenin and the activation of transcriptional factor sXBP1 within an inflammatory environment. To conclude, we have described Angiogenin as a new mediator of the integrated ER stress response, and characterized its cell- and non-cell-autonomous biological functions. Our study have identified urinary angiogenin as a potential marker of ongoing kidney tubular injuries.
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Inibição do estresse do retículo endoplasmático restaura o conteúdo de ABCA-1 e o efluxo de colesterol em macrófagos tratados com albumina modificada por glicação avançada / Inhibition of endoplasmic reticulum stress restores the ABCA-1 protein level and cholesterol efflux in advanced glycated albumin-treated macrophagesCastilho, Gabriela 14 August 2012 (has links)
Produtos de glicação avançada (AGE) prejudicam o metabolismo de lipoproteínas e o transporte reverso de colesterol, o que contribui para a aterosclerose no diabete melito (DM). Em particular, a albumina modificada por AGE (albumina-AGE) reduz a remoção de colesterol por diminuir o conteúdo do receptor ABCA-1 em macrófagos. Isto se vincula ao insulto oxidativo e inflamatório, os quais são indutores do estresse do retículo endoplasmático (RE). O objetivo do presente estudo foi avaliar, em macrófagos, os efeitos do tratamento com albumina-AGE sobre o estresse do RE e suas vias adaptativas (UPR), relacionando-os com o prejuízo na expressão do ABCA-1 e efluxo de colesterol celular. Albumina-AGE foi produzida pela incubação de albumina isenta em ácidos graxos com glicolaldeído 10 mM e, albumina controle (albumina-C) com PBS apenas. Albumina foi isolada do soro de pacientes portadores de DM com controle glicêmico inadequado (albumina-DM) ou indivíduos controles (albumina não- DM) por cromatografia para separação rápida de proteínas seguida por purificação alcoólica. Macrófagos de peritônio de camundongos ou macrófagos da linhagem J774 foram tratados com os diferentes tipos de albumina na presença ou ausência de ácido fenil butírico (PBA; chaperona química que alivia o estresse do RE) ou MG-132 (inibidor do sistema proteasomal) por diferentes intervalos de tempo. A expressão de marcadores do estresse do RE, UPR, proteína dissulfeto isomerase (PDI), calreticulina e ubiquitina foi determinada por imunoblot e o conteúdo de ABCA-1, por citometria de fluxo e imunocitoquímica. O efluxo de 14Ccolesterol foi avaliado, utilizando-se apoA-I como aceptora de colesterol. A albumina-AGE induziu aumento tempo-dependente na expressão das chaperonas marcadoras do estresse do RE - Gr78 e Grp94 - e de proteínas da UPR (ATF6 e eIF2-P) em comparação à albumina-C. O conteúdo de ABCA-1 e o efluxo de colesterol foram reduzidos em, respectivamente, 33% e 47% e ambos foram restaurados pelo tratamento com PBA, o qual também reduziu o estresse do RE. A associação entre estresse de RE e redução de ABCA-1 foi confirmada pelo uso da tunicamicina (indutor clássico de estresse do RE), que diminuiu em 61% o conteúdo de ABCA-1, prejudicando em 82% o efluxo de colesterol. A albumina-AGE aumentou o conteúdo total de ubiquitina. A inibição do sistema proteasomal não foi capaz de restaurar o conteúdo de ABCA-1 em células tratadas com albumina-AGE. Em macrófagos expostos à albumina-DM evidenciou-se maior expressão da PDI e calreticulina, com tendência à maior expressão da Grp94. A albumina-AGE (produzida in vitro ou isolada de portadores de DM) induz estresse de RE, o qual se vincula à redução no conteúdo de ABCA-1 e efluxo de colesterol. Estes eventos podem contribuir para a aterosclerose no DM. Chaperonas químicas, que aliviam o estresse do RE, podem ser ferramentas úteis na prevenção e tratamento da aterosclerose / Advanced glycation end products (AGE) disturb lipoprotein metabolism and reverse cholesterol transport, contributing to atherosclerosis in diabetes mellitus (DM). Particularly, advanced glycated albumin (AGE-albumin) reduces cell cholesterol removal by impairing the expression of ABCA-1 in macrophages. This is ascribed to the oxidative and inflammatory stress, conditions that elicit endoplasmic reticulum (ER) stress. In this study it was investigated the effect of AGE-albumin on ER stress and adaptative pathways (UPR) development in macrophages, and its relationship to the reduction in ABCA-1 expression and cholesterol efflux. AGE-albumin was prepared by incubating fatty acid free albumin with 10 mM glycolaldehyde and control albumin (C-albumin) with PBS only. Albumin was isolated from poorly controlled DM patients (DM-albumin) and control individuals (nonDMalbumin) by fast liquid chromatography and purified by alchoolic extraction. Mouse peritoneal macrophages or J774 cells were treated along time with the different types of albumin in the absence or presence of phenyl butiric acic (PBA; a chaperone that aleviates ER stress) or MG132 (a proteasomal inhibitor). The expression of ER stress and UPR markers, protein disulfide isomerase (PDI), calreticulin and ubiquitin was determined by immunoblot and ABCA-1 protein level, by flow cytometry and imunocytochemistry. 14Ccholesterol efflux was evaluated utilizing apo A-I as cholesterol acceptor. AGE-albumin induced a time-dependent increase in the expression of ER stress chaperone markers - Gr78 and Grp94 - and UPR proteins (ATF6 and eIF2-P) in comparison to C-albumin. ABCA-1 content and cholesterol efflux were diminished by, respectively, 33% and 47% and both were recovered by the treatment with PBA. The association between ER stress and ABCA-1 reduction was confirmed by the reduction, induced by tunicanycin (a classical ER stress inductior) in ABCA-1 protein level (61%) and cholesterol efflux (82%). AGE-albumin increased the amount of cellular total ubiquitin. The inhibiton of proteasomal system was unable to restore ABCA-1 protein level in cells treated with AGE-albumin. In macrophages exposed to DM-albumin a higher expression of PDI and calreticulin was observed together with a trend of enhanced Grp94 expression. In conclusion, AGE-albumin (produced in vitro or isolated from DM patients) induces ER stress which is related to the reduction in ABCA-1 level and cholesterol efflux in macrophages. These events can contribute to atherosclerosis in DM. Chemical chaperones that alleviate ER stress may be useful in the prevention and treatment of atherosclerosis
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Implication du métabolisme des phospholipides dans la progression et la résistance des cancers digestifs / Study of the involvement of phospholipid metabolism in the progression and the resistance of digestive cancersCotte, Alexia 03 May 2017 (has links)
Le métabolisme des lipides joue un rôle prépondérant dans le cancer. Ce métabolisme a pour effet, particulièrement grâce à la production de phospholipides (PLs), de supporter le niveau accru de prolifération mais aussi de réguler finement des mécanismes intra-cellulaires et extra-cellulaires qui promeuvent le maintien et la progression des cellules cancéreuses. Parmi tous ces acteurs, les gouttelettes lipidiques (GLs), connues pour leur fonction de réservoir, commencent à dévoiler leurs côtés sombres. Notre premier projet nous a permis de mettre en avant l’accumulation de GLs par des cellules de cancer colorectal (CCR) chimiorésistantes. La formation de GLs est régie par l’expression de l’enzyme lysophosphatidylcholine acyltransférase 2 (LPCAT2), permettant la production de phosphatidylcholine. Elle a pour effet de protéger le réticulum endoplasmique (RE) de l’induction d’un stress prévenant l’activation d’une mort cellulaire immunogène. Ces modulations lipidiques peuvent également se retrouver dans le plasma, où elles font l’objet de l’identification de biomarqueurs. Dans ce contexte, nous avons montré dans un second projet, que certains PLs pouvaient diagnostiquer la présence d’un carcinome hépatocellulaire (CHC) sur un foie cirrhotique. Ces deux aspects soulignent l’importance du métabolisme des PLs dans les cancers digestifs. / Among all altered cancer metabolic pathways, lipid metabolism has a preponderant role in cancer development. This metabolism, especially through the production of phospholipids, supports high level of proliferation and carefully regulates intra-cellular and extra-cellular mechanisms promoting maintenance and progression of cancer cells. Among all metabolic players, lipid droplets (LD), known for their storage function, begin to reveal dark sides. Our first project led us to highlight LD involvement in the chemoresistance of colorectal cancer (CRC) cells. This resistance carries out thanks to LD accumulation during chemotherapy treatment. Their accumulation is regulated by the expression of lysophosphatidylcholine acyltransferase 2 (LPCAT2), leading to the production of phosphatidylcholine. It causes the protection of the endoplasmic reticulum (ER) stress induction preventing the activation of immunogenic cell death. These lipid modulations can also be found in plasma where they can be identified as biomarkers. In this context, we have shown that some phospholipids could prognosticate hepatocellular carcinoma (HCC) upon cirrhotic liver. These two aspects highlight the significance of phospholipid metabolism in digestive cancers.
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Efeitos da suplementação do ácido alfa-linolênico no estresse do retículo endoplasmático em tecido adiposo subcutâneo abdominal de indivíduos com diabetes mellitus tipo 2 / Alpha-linolenic acid supplementation effect on endoplasmic reticulum stress in abdominal subcutaneous adipose tissue from type 2 diabetes mellitus patientsMiranda, Wallace Rodrigues de Holanda 24 June 2016 (has links)
Diabetes mellitus tipo 2 (DM2) está associado a um estado de inflamação crônica e ativação do estresse do retículo endoplasmático (ERE). Nesse contexto, são necessários estudos para encontrar alternativas que melhorem o quadro inflamatório, como os ácidos graxos poli-insaturados ômega 3 (n-3 PUFA), um conhecido agente anti-inflamatório. Esse estudo teve por objetivo avaliar o efeito da suplementação do ácido alfa-linolênico (ALA, um n-3 PUFA) no estresse do retículo endoplasmático e no estado inflamatório no tecido adiposo subcutâneo abdominal (TASC) em pacientes com DM2. Foi conduzido um estudo duplo-cego, prospectivo, placebo-controlado. Vinte pacientes com DM2 foram randomizados para suplementação com 3g/dia de ALA ou placebo durante 60 dias. O tecido adiposo foi coletado através de punção aspirativa por agulha fina do abdome antes e após a suplementação e os genes e proteínas foram avaliados através de PCR em tempo real e western blot. Foi encontrada, após suplementação, uma redução da expressão gênica do XBP1 (20%), sXBP1 (70%) e aumento da expressão gênica do GRP78 (150%), confirmado na expressão proteica. Além disso, foi encontrado aumento da expressão gênica da adiponectina (90%) e redução da expressão gênica do IL-6 (80%) e IRS-1 (60%), sem correlação com a expressão proteica, no tempo pós-suplementação com ALA. Portanto, foi demonstrado que o ALA pode modular o ERE através da via da IRE1/XBP, levando ao aumento das chaperonas (BIP/GRP78), além de um efeito adicional na expressão gênica da adiponectina, IL-6 e IRS-1, o que pode demonstrar um potencial terapêutico do ALA em pacientes com DM2. / Type 2 diabetes mellitus (T2DM) is a state of chronic inflammation and activation of endoplasmic reticulum stress (ERS). In this context, studies are necessaries to find new possibilities to improve this inflammation such as the n-3 polyunsaturated fatty acid (n-3 PUFA) acting as an anti-inflammatory agent. In this study, we aimed to evaluate the effect of n-3 PUFA alpha-linolenic acid (ALA, a n-3 PUFA) supplementation in T2DM patients on the molecular expression of ERS genes in abdominal subcutaneous adipose tissue (SAT). We performed a placebo-controlled study, in a double-blind design with 20 T2DM patients, receiving, randomly, 3g/day of ALA or placebo for 60 days. The adipose tissue was collected by fine-needle aspiration in abdomen before and after the supplementation and the genes and proteins were evaluated by real-time PCR and western blot. It was seen, after the supplementation, a reduction in XBP1 (20%), sXBP1 (70%) and an increase in Grp78 (150%) gene expression, likewise same results in protein concentration. Furthermore, it was observed an increase in adiponectina (90%) gene expression and reduction in IL-6 (80%) and IRS-1 (60%) gene expression, with no correlation to protein expression after supplementation of ALA. Therefore, we have provided evidence that ALA may modulate ERS by the IRE1/XBP pathway leading to an increase in chaperones (BIP/GRP78), additionally its effect on adiponectina, IL-6 and IRS-1 gene expression can demonstrate a therapeutic potential in T2DM.
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Développement de modèles C. elegans de Sclérose Latérale AmyotrophiqueVaccaro, Alexandra 12 1900 (has links)
Les gènes TDP-43 (TAR DNA Binding Protein 43) et FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma) sont actuellement à l’étude quant à leurs rôles biologiques dans le développement de diverses neuropathies telles que la Sclérose Latérale Amyotrophique (SLA). Étant donné que TDP-43 et FUS sont conservés au cours de l’évolution, nous avons utilisé l’organisme modèle C. elegans afin d’étudier leurs fonctions biologiques. Dans ce mémoire, nous démontrons que TDP-1 fonctionne dans la voie de signalisation Insuline/IGF pour réguler la longévité et la réponse au stress oxydatif. Nous avons développé des lignées C. elegans transgéniques mutantes TDP-43 et FUS qui présentent certains aspects de la SLA tels que la dégénérescence des motoneurones et la paralysie adulte. La protéotoxicité causée par ces mutations de TDP- 43 et FUS associées à la SLA, induit l’expression de TDP-1. À l’inverse, la délétion de tdp-1 endogène protège contre la protéotoxicité des mutants TDP-43 et FUS chez C. elegans. Ces résultats suggèrent qu’une induction chronique de TDP-1/TDP-43 sauvage propagerait la protéotoxicité liée à la protéine mutante. Nous avons aussi entrepris un criblage moléculaire pilote afin d’isoler des suppresseurs de toxicité neuronale des modèles transgéniques mutants TDP-43 et FUS. Nous avons ainsi identifié le bleu de méthylène et le salubrinal comme suppresseurs potentiels de toxicité liée à TDP-43 et FUS via réduction de la réponse au stress du réticulum endoplasmique (RE). Nos résultats indiquent que l’homéostasie de repliement des protéines dans le RE représente une cible pour le développement de thérapies pour les maladies neurodégénératives. / Two recently discovered causative genes for ALS, TDP-43 (TAR DNA Binding Protein 43) and FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma) are under further investigation regarding their biological roles in neuropathies such as Amyotrophic Lateral Sclerosis (ALS). Since TDP-43 and FUS are evolutionarily conserved we turned to the model organism C. elegans to learn more about their biological functions. Here we report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response. We have generated mutant TDP-43 and FUS transgenic lines in C. elegans that recapitulate certain aspects of ALS including motor neuron degeneration and adult-onset paralysis. Proteotoxicity caused by ALS- associated mutations in TDP-43 or FUS also induce TDP-1 expression and consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild type TDP-1/TDP-43 by proteotoxicity may actively promote neurodegeneration. We also screened for small- molecule suppressors of mutant TDP-43 and FUS neuronal toxicity in transgenic C. elegans and identified methylene blue and salubrinal as potent suppressors of TDP-43 and FUS toxicity in our models through induction of the endoplasmic reticulum (ER) stress response. Our results indicate that protein folding homeostasis in the ER may be an important target for therapeutic development in neurodegenerative diseases.
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Rôle de l'hypoxie intermittente dans la maladie ischémique cardiaque associée au Syndrome d'Apnées Obstructives du Sommeil / Role of intermittent hypoxia in ischemic disease associated with Obstructive Sleep Apnea SyndromeBourdier, Guillaume 18 December 2017 (has links)
Le syndrome d’apnées obstructives du sommeil (SAOS) est un problème de santé publique majeure affectant 6-13% de la population d’âge moyen. Des études épidémiologiques et l’accumulation de données cliniques ont montré que le SAOS joue un rôle important dans l’initiation et la progression des pathologies cardiovasculaires (CV) comme l’infarctus du myocarde (IM). Les patients hospitalisés post-IM présentent une prévalence pour le SAOS de l’ordre de 50%. De plus, le SAOS augmente la vulnérabilité du cœur à l’infarctus, ce qui se traduit par une taille d’IM plus grande, une ischémie myocardique prolongée, et une aggravation des évènements cardiovasculaires au long-terme, prédisposant les patients apnéiques à des infarctus surnuméraires, à l’insuffisance cardiaque (IC) et au décès. Il semble donc important de comprendre précisément les mécanismes impliqués dans cette susceptibilité accrue à l’ischémie myocardique afin de proposer de nouvelles cibles thérapeutiques et améliorer la prise en charge du risque CV chez les patients apnéiques. L’hypoxie intermittente chronique (HI) est le substrat physiopathologique majeur des complications CV du SAOS via l’activation de mécanismes physiopathologiques variés, tels que l’inflammation, le stress oxydant ou encore l’activation sympathique. Ce travail de thèse avait pour but de 1) caractériser la réponse aigue et chronique à l’IM chez des animaux exposés à l’HI, 2) de disséquer les mécanismes cellulaires impliqués dans la susceptibilité accrue à l’IM chez ces mêmes animaux.Nos travaux ont confirmé que l’HI induit une majoration de la taille d’infarctus suite à un évènement ischémique aigue et aggrave le remodelage cardiaque et la dysfonction contractile dans un modèle de cardiopathie ischémique chronique chez le rat. Nous avons également mis en évidence que l’HI induisait dans ce contexte une hyperactivation sympathique persistante, un stress du RE proapoptotique et l’activation du facteur de transcription HIF-1 contribuant à l’augmentation de la vulnérabilité du cœur à l’infarctus et l’aggravation post-IM des complications cardiaques au long-terme. Ces différents facteurs pourraient représenter des biomarqueurs intéressants pour prédire le risque CV chez les patients apnéiques sévères et pourraient être considérés comme des pistes thérapeutiques potentielles pour améliorer la prise en charge des patients SAOS à haut risque CV. / Obstructive sleep apnea syndrome (OSA) is a common disease that affects 6-13% of the middle-aged population. Epidemiological and clinical data support the notion that OSA has a role in the initiation or progression of several cardiovascular (CV) diseases, including myocardial infarction (MI). Indeed, patients hospitalized with acute MI present high prevalence for OSA. Furthermore, OSA is known to major infarct size in patients that persists over time and aggravates long-term adverse events post-MI, as reinfarction, heart failure (HF) and death. OSA is characterized by intermittent hypoxia (IH) which results in desaturation-reoxygenation sequences and appears to be the major consequence of OSA in term of cardiovascular alterations associated with apneas. However, the mechanisms remain unclear. Therefore, the understanding of pathophysiologic mechanisms involved in cardiac disorders is a research priority for OSA in order to develop new therapeutic targets and improve the management of CV risk in apneic patients. There are growing evidences suggesting a major role of endoplasmic reticulum (ER) stress and HIF-1 activation in the vulnerability to acute ischemic events and in long-term adverse complications associated with prolonged MI. Furthermore, the progression of ischemic cardiomyopathy following MI is also associated with activation of the sympathetic nervous system which substantially contributes to cardiac alterations. Furthermore, these are three mechanisms known to be activated with IH. This project aimed 1) to assess the IH-induced acute and chronic cardiac alterations following MI, 2) to study the implication of cellular mechanisms involved in the adverse ischemic events related to OSA.We have shown that IH increases infarct size following acute MI and aggravates cardiac remodeling and contractile dysfunction in a rat model of chronic ischemic cardiomyopathy. In these contexts, IH is associated with a sympathetic over activity, a proapoptotic ER stress and the activation of HIF-1, which substantially contribute to increased heart vulnerability to infarction and worsening of long-term heart complications post-MI. These different factors may represent interesting biomarkers for predicting CV risk in severe apneic patients and may be considered as potential therapeutic targets to improve the management of OSA patients with high CV risks.
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