Les inhibiteurs de la protéase virale (IPs) sont utilisés avec succès dans le cadre d'une thérapie anti-VIH-1. L'efficacité de ce traitement est incontestable notamment en terme de réduction de la charge virale et de maintient du taux de lymphocytes CD4 circulants. Depuis l'incorporation des IPs dans la prise en charge thérapeutique, on note une réduction significative de la morbidité et de la mortalité ainsi qu'un allongement de la durée de vie des patients. Cependant, la prise de ces molécules anti-rétrovirales s'accompagne de nombreux effets secondaires. Les plus préoccupants d'entre eux sont : une lipodystrophie partielle, une hyperlipidémie, une insulino-résistance, une athérosclérose prématurée, ainsi que des infarctus du myocarde. Les patients sont également confrontés à l'apparition de maladies généralement associées à l'âge telles que la neurodégenérescence, l'ostéopénie et le développement de tumeurs malignes. Les mécanismes cellulaires et moléculaires impliqués dans ces altérations métaboliques n'ont pas encore été élucidés. L'objectif de cette étude était d'étudier les effets cellulaires de quatre IPs (Atazanavir, Lopinavir, Ritonavir et Saquinavir) sur la cellule musculaire striée squelettique humaine. Ces travaux ont permis d'identifier une augmentation de la production d'espèces oxygénées réactives (ERO), une altération morphologique du réticulum sarco/endoplasmique (RS/RE) ainsi qu'une augmentation de l'expression de CHOP, un marqueur du stress de ce compartiment et une diminution de l'expression et de la localisation dans les microdomaines membranaires (lipid rafts) de la cavéoline 3 et de la flotilline 1. Enfin, l'utilisation d'un antioxydant, le Resvératrol protége le myotube primaire humain de ces différentes altérations engendrées par les IPs. Ces données suggèrent un rôle central de la surproduction d'ERO dans le développement du stress du RE/RS et de la perte de localisation des protéines résidantes des microdomaines membranaires. De plus , en l'absence de persceptive vaccinale concrète, le Resvératrol, au travers de ces effets protecteurs, pourrait se révéler un atout de choix dans l'atténuation des effets secondaires des IPs en contribuant ainsi à l'amélioration de la prise en charge du patient séropositif. / HIV protease inhibitors (PI) have been successfully used in highly active antiretroviral therapy (HAART) of HIV-1 infection, the most effective treatment currently available. Incorporation of protease inhibitors in HAART has significantly reduced the morbidity and mortality and prolonged the lifespan of patients with HIV infection. Protease inhibitor benefits are unfortunately compromised by a number of clinically important adverse side-effects. Most patients on HAART develop a metabolic syndrome associated with partial lipodystrophy, hyperlipidemia, insulin resistance, premature atherosclerosis and myocardial infarction. Moreover, these patients face a growing number of other age-related comorbidities, such as neurodegeneration, osteopenia and malignancies. The cellular and molecular mechanisms underlying protease inhibitor-associated metabolic abnormalities remain elusive, but they seem to be related to overproduction of reactive oxygen speci es (ROS), induction of endoplasmic reticulum stress and activation of the unfolded protein response (UPR). The objective of this thesis was to determine the cell effects of four PIs (Atazanavir, Lopinavir, Ritonavir and Saquinavir) in cultures of primary human skeletal myotubes. This study showed that PIs increased ROS production, altered sarco/endoplasmic reticulum (SR/ER) morphology, increased expression of C/EBP homologous protein, a SR/ER stress marker, and decreased expression and localization at lipid rafts of Caveolin 3 and Flotillin 1. In addition, we showed that the antioxidant Resveratrol protected the human primary myotube of these iatrogenic effects. These data suggest a central role of the overproduction of ERO in the development of SR/ER stress and mislocalization of lipid raft proteins induced by PIs. Besides, in absence of vaccine, Resveratrol may be used by a potentiel therapeutic agent to attenuate PI-induced side effects

Iron is an essential trace element for almost all living organisms. Iron overload in cells and tissues, however, leads to their disruption. Most oftenly damaged are parenchymatic organs such as the liver, pancreas and heart. The aim of this thesis was to create cellular in vitro models for the investigation of effects of excess iron on hepatocytes and pancreatic beta cells and on these models to investigate cellular processes which lead to cellular damage during iron overload. We focused on examining the presence of oxidative and endoplasmic reticulum stress and the activation of apoptotic cell death. For our experiments, we used HEP-G2 cell line which represents human hepatocytes and NES2Y cell line which represents human pancreatic beta cells. To study the mechanisms of cellular damage during iron overload, we used two approaches by which we observed both acute and long-term effects of high levels of iron on damage of the tested cell lines. When studying the acute effect of excess iron on the cells, we applied high doses of iron (using 15 mM ferric citrate in medium) that led to the activation of cell death in hours. Long-term effects of iron overload were tested on cells regularly cultivated in the presence of 50 μM and 100 μM ferric citrate over a period of several months. Iron concentrations...

Le myélome multiple (MM) est une hémopathie dont la croissance et la prolifération sont liés à une variété de facteurs de croissance, y compris « insulin-like growth factor type 1 » (IGF-1). Bortézomib est le premier inhibiteur protéasome ayant une activité anti-tumorale significative dans le myélome multiple. Nous avons analysé l'impact de l'IGF-1 recombinant associé à l'inhibiteur du protéasome bortezomib sur des lignées humaines de MM, in vivo et sur des cellules de myélome frais humaines ex vivo. Nous avons montré que l'IGF-1 améliore l'activité cytotoxique du bortezomib in vitro, in vivo et ex vivo. Nous avons montré que l'accroissement de la toxicité peut être inhibé par la présence d'un anticorps monoclonal dirigé contre le récepteur de l'IGF-1 (IGF1-R). IGF-1 renforce l'activité cytotoxique des autres inhibiteurs de protéasome, y compris MG115, MG132, PSI et epoxomicin. Nos résultats confirment le fait que l'IGF-1sensibilise des cellules de myélome à l'activité cytotoxique des inhibiteurs du protéasome tels que le bortezomib, en raison du niveau accru du stress de réticulum endoplasmique et l'induction de la une réponse protéine dépliée (UPR) / Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib in MM cell lines, in vivo and on fresh human myeloma cells ex vivo. We found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro, in vivo and ex vivo. We showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 enhances the cytotoxic activity of other proteasome inhibitors, including MG115, MG132, PSI and epoxomicin. Our results support the fact that IGF-1sensitize myeloma cells to the cytotoxic activity of proteasome inhibitors such as bortezomib, as a consequence of enhanced level of endoplasmic reticulum stress and the induction of an unfolded protein response (UPR)

Les cellules épithéliales nasales humaines (CENH) sont des acteurs importants de la physiopathologie de la polypose naso-sinusienne (PNS), le plus souvent la PNS est primitive (étiologie inconnue), plus rarement, elle est secondaire à la mucoviscidose (PNS CF) ou la dyskinésie ciliaire primitive (PNS DCP). L'objectif de cette thèse est d'identifier des protéines différentiellement exprimées dans les PNS primitives et secondaires par approche protéomique puis de les caractériser par des techniques classiques. Dans cette étude, nous avons utilisé des cultures primaires (interface air/liquide) de CENH, dérivant de déchet per-opératoire (PNS) ou de brossages (muqueuse saine). Le marquage iTRAQ suivie d'une analyse par nano-LC-MALDI-TOF-TOF à en particulier montré : 1) une augmentation de six marqueurs du stress du réticulum endoplasmique (RE) dans la PNS primitive et la PNS CF, 2) une altération du métabolisme du glucose dans la PNS CF (sous-expression de 6 protéines dont la pyruvate kinase, enzyme clef de la glycolyse) et 3) une sous expression de 12 protéines des filaments acto-myosines dans la PNS DCP (dont 3 tropomyosines). Nous avons caractérisé le stress du RE dans la PNS primitive, montrant qu'il est induit par le biais d'une sensibilité des CENH à un stress oxydatif probablement d'origine mitochondrial et qu'il participe activement à l'inflammation (sécrétion d'IL-8 et de LTB4). Ces observations nous permettent de proposer que, dans la PNS primitive, il existe un cercle vicieux entre stress du RE, stress oxydatif et inflammation et que la sensibilité au stress oxydatif observée apparaît comme une nouvelle cible thérapeutique dans le traitement de la PNS. / Human nasal epithelial cells (HNEC) play an important role in nasal polyposis(NP). NP have generally an unknown etiology, less frequently, NP is secondary to cystic fibrosis (CF NP) or primary ciliary dyskinesia (PCD NP). The aim of this thesis is to identify differentially expressed proteins in NP, CF NP and PCD NP using proteomic approach then to characterize them by classical methodes. In this study, we used primary culture (air/liquid)of HNEC derived from NP patients (surgery waste) or healthy patients (brushing). The iTRAQ labeling folowing by nano-LC-MALDI-TOF-TOF analysis shown in particular :1)an increased expression of six endoplasmic reticulum (ER) stress markers in NP and CF NP, 2)an alteration of glucose metabolism (decreased expression of six proteins with in particular the pyruvate kinase, glycolysis key enzyme) and 3)an decreased expression of 12 acto-myosin filament proteins (in particular three tropomyosins). We caracterized the ER stress in NP showing that ER stress is activated by an oxydative stress susceptibility of NP HNEC probably of mitochondrial origin and that it directly participate to inflammation (IL-8 and LTB4 secretions). Altogether, our results underline a vicious circle that exists between oxidative stress, ER stress and inflammation.The oxydative stress susceptibility observed in NP may represent new therapeutic target in such a pathology.

As doenças cardiovasculares são a principal causa de morte no mundo, sendo a cardiomiopatia isquêmica a mais prevalente. Independente da sua etiologia, a via final comum da maioria das doenças cardiovasculares é a insuficiência cardíaca. Nos últimos anos, tem sido reportado que o acúmulo de proteínas mal enoveladas no retículo endoplasmático (estresse do RE) pode contribuir para redução da função cardíaca e instalação da insuficiência cardíaca. Apesar do mecanismo responsável pela disfunção contrátil induzida pelo estresse do RE ainda não ser conhecido, evidências sugerem que a inibição da via de sinalização PI3K/AKT pela proteína JNK pode estar envolvida nessa resposta. Na primeira parte desta tese, verificamos que a indução do estresse do RE em cardiomiócitos isolados ativou a JNK, mas não inibiu a via de sinalização PI3K/AKT. A inativação de JNK reverteu a disfunção contrátil e a redução da amplitude do transiente de Ca+2 de cardiomiócitos causados pelo estresse do RE. Pelo fato da via sinalização PI3K/AKT não estar envolvida na disfunção contrátil causada pelo estresse do RE, analisamos outro alvo de JNK, a proteína BNIP3, proteína pró-apoptótica e envolvida no controle de qualidade mitocondrial promovendo mitofagia quando ativada. O estresse do RE aumentou a expressão de BNIP3, a qual foi atenuada pela inibição de JNK. A depleção de BNIP3 impediu a disfunção contrátil dos cardiomiócitos e a redução da amplitude do transiente de Ca+2 induzidos pelo estresse do RE. Na segunda parte da tese, o objetivo foi avaliar se os efeitos observados em cardiomiócitos submetidos a estresse do RE poderiam ser observados em modelo experimental de doença cardiovascular. Nesse sentido, observamos que a disfunção cardíaca provocada pelo infarto do miocárdio em ratos foi acompanhada pelo quadro de estresse do RE e pela ativação da via de sinalização JNK/BNIP3. Entretanto, o treinamento físico aeróbico (TFA), uma das principais terapias não farmacológicas mais eficazes das doenças cardiovasculares, foi capaz de atenuar o estresse RE, a ativação da via de sinalização JNK/BNIP3 e a disfunção cardíaca de ratos infartados. Na terceira parte da tese, verificamos que o TFA aumentou a expressão proteica de DERLIN-1, uma proteína que atua retro-translocando proteínas mal enoveladas para o citosol, no miocárdio de ratos saudáveis. O aumento dos níveis proteicos de DERLIN-1 observado em ratos infartados foi atenuado pelo TFA. Apesar do aumento da proteína DERLIN-1, observamos que nos animais infartados as proteínas mal enoveladas acumulavam na forma de oligômeros e que o TFA atenuou essa resposta. Em conjunto, os resultados da presente tese sugerem que a ativação da via de sinalização JNK/BNIP3 pelo estresse do RE causa disfunção contrátil de cardiomiócitos e que o TFA é capaz de atenuar essa resposta no coração de ratos infartados, melhorando o controle de qualidade de proteína no músculo cardíaco / Cardiovascular diseases are currently the main cause of death worldwide, with the ischemic cardiomyopathy as the most prevalent ethiology. This is of particular interest, since ischemic cardiomyopathy advances to heart failure, a common endpoint of the most cardiovascular disease. In the last years, it has been showed that accumulation of unfolded protein in the endoplasmic reticulum (ER stress) may cause cardiac dysfunction and heart failure development. Despite the mechanisms behind this cardiac deterioration is still unknown, evidences suggest that ER stress-induced cardiomyocytes contractile dysfunction results from PI3K/AKT signaling pathway inhibition, which would be caused by JNK activation. In the first part of this thesis, we found that the ER stress activated JNK, but different from our hypothesis it was not accompanied by an inactivation of PI3K/AKT signaling pathway. The inhibition of JNK mitigated the reduction in cardiomyocytes shortening and amplitude of Ca+2 transient caused by ER stress. Once the PI3K/AKT signaling pathway was not involved in the ER stress-induced cardiomyocytes contractile dysfunction, we have analyzed protein expression of BNIP3, another JNK target involved in apoptosis and mitochondria quality control. We observed that the elevation in BNIP3 proteins levels after ER stress induction was prevented by inhibition of JNK. BNIP3 depletion attenuated the reduction in cardiomyocytes contractility and amplitude of Ca+2 transient induced by ER stress. In the second part of the thesis, we found that myocardial infarction-induced cardiac dysfunction in rats was accompanied by ER stress and activation of JNK/BNIP3 signaling pathway. However, the AET mitigated ER stress, activation of JNK/BNIP3 signaling pathway and cardiac dysfunction in infarcted rats. In third part of the thesis, we have identified that AET increased the protein expression of DERLIN-1 an ER membrane protein that retro-translocates unfolded proteins to cytosol in the myocardial of healthy rats. We observed that the increased DERLIN-1 protein levels in infarcted rats were mitigated by AET. Despite increased DERLIN-1 protein expression, we found high levels of oligomers in the myocardium of infarcted rat, which was reduced by AET. It suggests that unfolded protein degradation was reduced in infarcted hearts. Taken together, these results suggest that ER stress causes cardiomyocytes contractile dysfunction through JNK/BNIP3 signaling pathway activation and that AET mitigates the myocardial infarction-induced ER stress and activation of JNK/BNIP3 signaling pathway by restoring of ER-associated protein quality control in the cardiac muscle

de tumorigênese em melanomas, ainda não há tratamento eficaz para melanomas metastáticos. Esta ineficácia terapêutica pode estar relacionada com a adaptação e seleção de células de melanoma à indução de estresse de RE. Ultrapassar os níveis sustentados de estresse de RE, interferindo nas vias de adaptação a este estresse, foi o alvo deste estudo na tentativa de propor uma nova estratégia terapêutica para sensibilizar células de melanoma a morte induzida por cisplatina. Mostramos que GADD153, um dos componentes da via de UPR (Unfolded Protein Response) responsável por induzir apoptose em reposta ao estresse de RE, está excluída do núcleo em melanomas primários, metástases ganglionares e viscerais. Este dado sugere que a localização citoplasmática do fator de transcrição GADD153 possa estar envolvida na resposta adaptativa de melanomas ao estresse de RE, uma vez que se sabe que GADD153 se acumula no núcleo em resposta a este estresse. Investigamos se a indução de estresse de RE seria capaz de induzir a translocação de GADD153 para o núcleo e resultar na sensibilização de células de melanoma a morte induzida por cisplatina (CDDP). Realizamos o tratamento de células de melanoma (SbCl2, Mel85, SK-MEL- 29, SK-MEL-28 e SK-MEL-147) com tunicamicina (Tuni), indutor clássico de estresse de RE, previamente ao tratamento com CDDP. Demonstramos que em todas as linhagens exceto em SK-MEL-29, houve um aumento na porcentagem de células hipodiploides (>50%) no tratamento combinado (Tuni>CDDP) comparado ao tratamento com CDDP. As células SK-MEL-147 se mostraram mais sensíveis à indução de estresse de RE e as células SK-MEL-29 mais resistentes. Algumas diferenças entre estas linhagens como a expressão de GRP78 de superfície e presença de oligossacarídeos ?1-6 ligados de superfície podem estar relacionadas com esta resposta diferencial ao estresse de RE. Em todas as linhagens verificamos a acúmulo dos marcadores de UPR, GRP78 e GADD153, após o tratamento com tunicamicina. Além disso, GADD153 foi direcionada para o núcleo em reposta ao tratamento com tunicamicina. O acúmulo de vacúolos acídicos, da proteína autofágica LC3-II e de ROS após o tratamento com Tuni>CDDP, sugerem que tanto a autofagia quanto o estresse oxidativo parecem estar envolvidos na resposta de sensibilização. A inibição de autofagia com cloroquina aumentou a morte induzida por Tuni>CDDP, sugerindo que autofagia desempenha função protetora neste esquema terapêutico. Testamos um segundo agente genotóxico, temozolomida (TMZ), uma droga equivalente à dacarbazina, e a mesma capacidade de sensibilização foi observada pelo prévio tratamento com tunicamicina. A validação deste conceito in vivo foi dificultada pela acentuada toxicidade apresentada por tunicamicina. Avaliamos alguns candidatos a agentes estressores do RE que poderiam apresentar menor toxicidade celular, como swainsonina, atorvastatina, metformina e o composto de cobre [Cu2(apyhist)2(dpam)](ClO4)4. No entanto, não obtivemos resultados promissores com nenhum destes candidatos. Estes resultados mostram que as células tumorais podem ser pré-condicionadas à morte celular se expostas a um prévio estressor de RE, como Tuni, o que leva ao comprometimento da resposta adaptativa a indutores de morte celular como CDDP e TMZ. No entanto, ainda é necessário o estudo de agentes indutores de estresse de RE pouco tóxicos para que esta estratégia terapêutica possa ser utilizada em pacientes com melanoma / Melanoma is among the most aggressive malignancies with increasing worldwide incidence and there is no effective treatment for the metastatic disease. The absence of an effective therapy may be due to adaptation and selection of melanoma cells to endoplasmic reticulum (ER) stress. We showed that GADD153, one of the components of the ER stress-mediated apoptosis pathway, was mostly excluded from the nucleus of primary and metastatic melanoma cells compared to nevus cells. These data suggest that the unexpected GADD153 cellular localization could be involved in melanoma cell adaption to ER stress, since GADD153 accumulates in the nucleus during ER stress. Unfolded protein response (UPR) signaling induced in response to ER stress, is a dual process that induces a protective response to restore ER homeostasis or cell death if ER stress is severe or persistent. We investigated if induction of ER stress was a potential strategy to chemosensitize melanoma cells to a second insult by surpassing the adaptive levels to ER stress. We first treated human melanoma cells (SbCl2, SK-MEL-28, Mel85, SK-MEL-29 and SK-MEL-147) with tunicamycin (Tuni), an ER stress inducer, before cisplatin (CDDP) treatment. CDDP is a low cost chemotherapeutic drug currently used in Brazil as a second line for melanoma treatment, especially in youngsters. All cell lines, except SK-MEL-29, demonstrated an >50% increase in the percentage of hypodiploid cells with Tuni>CDDP treatment when compared to CDDP only. The same results were obtained with temozolomide (TMZ), equivalent drug to the active form of dacarbazine, the first line of cytotoxic treatment of melanomas. UPR markers, GRP78 and nuclear translocation of GADD153 were induced by Tuni. Differences between SK-MEL-29 and SK-MEL-147 as cell surface GRP78 and ?1-6 oligossacharides can be related with the differential ER stress sensitization observed in these cells. One of the cellular mechanisms that are regulated by ER stress is autophagy. Accordingly, we observed an increase in the acidic vesicular organelles and accumulation of LC3II in response to Tuni>CDDP treatment. Autophagy inhibition with chloroquine increased Tuni>CDDP induced-cell death, suggesting that autophagy plays a protective role in this response. Oxidative stress can be involved in this scenario since we demonstrated an accumulation of reactive oxygen species in response to Tuni>CDDP. Tunicamycin was cytotoxic in vivo and we investigated alternatives to this antibiotic as swainsonine, atorvastatin, metformin and [Cu2(apyhist)2(dpam)](ClO4)4 but we did not observed ER stress induction. These results indicate that tumor cells could be preconditioned to cell death if exposed to a first ER stressor, such as Tuni, which would compromise an effective adaptive response to a cell death inducer, as CDDP and TMZ. This combined approach may be a promising strategy for melanoma therapy but further studies are necessary to find noncytotoxic alternatives to tunicamycin

A Síndrome de Marfan (SMF) é a enfermidade hereditária mais comum dentre as que afetam o sistema conjuntivo, causada por mutações da glicoproteína fibrilina-1, o principal componente estrutural das microfibrilas elásticas da matriz extracelular. As manifestações fenotípicas da SMF são sistêmicas e acometem tipicamente os sistemas ocular, esquelético e cardiovascular, este uma importante causa de morbi-mortalidade. Entretanto, não está claro como a mutação induz a doença. Estudos anteriores sugerem anomalias morfológicas do retículo endoplasmático (RE) ou retenção intracelular da fibrilina-1 nos estágios avançados da SMF. Entretanto, a contribuição do enovelamento da fibrilina-1 mutada e do estresse do RE na fisiopatologia celular da SMF não é conhecida. Proteínas mal-enoveladas podem levar à retenção intracelular e/ou aumento da degradação através da via de degradação associada ao RE (ERAD), além da indução da resposta a proteínas mal-enoveladas (UPR), ambas com potencial contribuição à fisiopatologia de doenças, incluindo a SMF. Assim, estudamos em fibroblastos embrionários isolados de camundongos (MEFs) com SMF se a fibrilina-1 mutada é reconhecida pelo controle de qualidade do RE pelo seu mal- enovelamento e induz estresse do RE por sua retenção intracelular. Demonstramos que a mutação na fibrilina-1 per se não promoveu chaperonas marcadoras de UPR ou geração de oxidantes. Além disso, não levou a uma maior sensibilização das células à indução exógena de estresse do RE, nem promoveu maior morte celular após inibição do proteassoma. Além disso, não foi observada retenção intracelular da fibrilina-1 nas células SMF, e mesmo após inibição da via secretora ou indução de estresse do RE, a inibição da secreção da fibrilina-1 foi similar nos MEFs SMF e wild-type (WT). A dissulfeto isomerase proteica (PDI), uma importante chaperona redox do RE, interage com fibrilina-1, e seu silenciamento levou a um aumento na secreção da fibrilina-1 pelos MEFs WT, mas não SMF. Além disso, o silenciamento da PDI promoveu a desorganização da matriz extracelular depositada de fibrilina-1 nos MEFs WT, enquanto nos MEFs SMF, a desorganização basal da matriz não foi adicionalmente alterada. Em paralelo, investigações in vivo mostraram que o estresse do RE não é induzido em camundongos SMF com 1 ou 3 meses de idade, apesar de manifestações fenotípicas evidentes. Entretanto, concomitante à progressão da doença, detectamos a ocorrência de estresse do RE nas aortas ascendentes dos camundongos aos 6 meses. Esta detecção foi exclusiva desta região da aorta e não ocorreu em outros órgãos afetados ou não afetados pela SMF. Assim, a manifestação do fenótipo clássico da SMF não requer uma perda da homeostase do RE diretamente induzida pela fibrilina-1 mutada. Ao contrário, esta é capaz de evadir mecanismos de controle de qualidade mediados pela PDI, sendo secretada normalmente. Assim, esta evasão do controle de qualidade pela PDI é uma condição permissiva essencial para o fenótipo da SMF. Por outro lado, o estresse do RE é uma característica evolutiva do aneurisma da aorta ascendente na SMF concomitante ao agravamento do fenótipo neste tecido / Marfan syndrome (MFS) is the most common connective tissue hereditary disease, caused by mutations in the glycoprotein fibrillin-1, the main structural component of extracellular matrix elastic microfibrils. MFS phenotypic manifestations are systemic and typically involve the ocular, skeletal and cardiovascular systems, the latter a major cause of morbidity/mortality. However, how gene mutation induxes disease is yet unclear. Previous studies suggest endoplasmic reticulum (ER) morphological abnormalities or fibrillin-1 intracellular retention in advanced MFS stages. However, the contribution of mutated fibrillin-1 folding and ER stress to MFS cellular pathophysiology is unknown. Un/misfolded proteins may associate with their intracellular retention and/or increased degradation through ER-associated degradation (ERAD), in addition to inducing the unfolded protein response (UPR), both sharing potential contributions to disease pathophysiology, including MFS. Thus, we studied in embryonic fibroblasts (MEFs) isolated from WT and MFS mice, if mutated fibrillin-1 can be recognized by ER quality control as a misfolded protein, able to induce ER stress due to its intracellular retention. We showed that fibrillin-1 mutation by itself did not promote UPR chaperone markers or oxidant generation. Moreover, it did not sensitize cells to exogenous ER stress nor affected cell survival curves after proteasome inhibition. Furthermore, no intracellular retention of fibrillin-1 was observed in MFS cells, and even after secretory pathway inhibition or ER stress induction, fibrillin-1 secretion inhibition was similar in MFS and wild-type (WT) MEFs. Protein disulfide isomerase (PDI), an important ER redox chaperone, interacts with fibrillin-1 and its silencing induced an increased fibrillin-1 secretion in WT, but not MFS MEFs. Besides, PDI silencing promoted fibrillin-1 extracellular matrix disorganization in WT MEFs, whereas in MFS MEFs, the basal matrix disorganization was not further modified. Parallel in vivo evaluations demonstrated that ER stress is also not induced in 1 and 3 month-old mice MFS, despite evident phenotypical manifestations. However, concomitant to accelerated disease progression at 6 months, ER stress was detectable in ascendant aorta, but not in other disease-affected or unaffected organs. Thus, classic MFS phenotype manifestations do not require loss of ER homeostasis directly induced by mutated fibrillin-1. Contrarily, the latter can evade a PDI-mediated quality control mechanism to be normally secreted. Therefore, evading such PDI-mediated quality control is an essential permissive condition for enabling the MFS phenotype. On the other hand, ER stress is an evolutive feature of MFS ascendant aorta aneurysm concomitant to phenotype progression in this tissue

O melanoma é o tipo de câncer de pele mais letal, apesar de ser o menos incidente. Em virtude de sua alta letalidade e de sua crescente incidência, estudos sobre melanoma são de fundamental importância nos dias de hoje. Assim como células tumorais em geral, células de melanoma apresentam características metabólicas diferenciadas, como, por exemplo, altos níveis de espécies reativas de oxigênio e alta taxa de síntese proteica. Essas modificações no metabolismo dispararam vias de resposta a estresse, como a \"Unfolded Protein Response\" (UPR), contudo, essas células se adaptam a esse estresse constante, que não culmina com a morte das mesmas. Além disso, o padrão de glicosilação em células tumorais também é sabidamente alterado, entre outros motivos, pela expressão diferencial de enzimas da via de glicosilação, como a N-acetilglicosaminiltransferase 5 (MGAT5). Relacionando essas duas características de células de melanoma, propusemo-nos a avaliar se a alta expressão de MGAT5A ( e/ou MGAT5B) funcionaria como uma resposta adaptativa de células de melanoma ao estresse de retículo endoplasmático, e seria, portanto, responsável por manter o equilíbrio diferenciado nessas células. Durante o desenvolvimento desse estudo, foi possível comprovar que a indução de estresse de retículo por meio de tratamento com tunicamicina, um inibidor da N-glicosilação e indutor clássico de UPR, sensibilizou as células de melanoma ao posterior tratamento com cisplatina. Contudo, o tratamento com swainsonina, um inibidor do processamento dos N-glicanos que ocorre no complexo de Golgi, não foi capaz nem de disparar \"Unfolded Protein Response\" nem de induzir morte nessas células e, talvez por esse motivo, não apresentou efeito sensibilizador frente à cisplatina. Além disso, foi observado que as linhagens tumorigênicas apresentam maior expressão de MGAT5A em comparação à linhagem não-tumorigênica melan-a. As tentativas de realização de silenciamento de MGAT5A não foram exitosas. Informações relacionando estresse de retículo e N-glicosilação aberrante em células tumorais ainda serão foco de estudo em nosso grupo. Com os resultados apresentados, é possível concluir que o equilíbrio diferencial dos níveis de estresse de retículo em que se encontram as linhagens tumorigênicas do nosso modelo é importante para a sobrevivência das mesmas. Além disso, é de nosso interesse avaliar a dependência de células tumorais das vias ativadas pela superexpressão de MGAT5A, caso ela realmente exista / Melanoma is the most lethal skin cancer, despite being the least prevalent. Due to its lethality and resistance to a variety of known chemotherapeutic drugs, studies on melanoma are paramount. Tumor cells in general, and melanoma cells particularly, commonly present a disturbed metabolic rate, e.g., altered metabolism of reactive oxygen species and increased rates of protein synthesis. Altogether these perturbations trigger the Unfolded Protein Response (UPR); however, tumor cells are adapted to these conditions and are able to survive. Besides, glycosylation of tumor cells is commonly altered, due to differentiated expression rates of N-glycosylation enzymes, like N-acetylglucosaminyltransferase 5 (MGAT5). Considering these information together, we proposed that the sustained overexpression of MGAT5A (and/or MGAT5B) observed in Tm1 and Tm5 melanoma cells is part of an adaptive response to reticulum stress, maintaining an unstable equilibrium in tumor cells. In this work, we observed that the induction of endoplasmic reticulum stress caused by tunicamycin treatment, a N-glycosylation inhibitor and UPR inducer, sensitized melanoma cells to further cisplatin treatment. In contrast, swainsonine treatment, an inhibitor of Golgi N-glycan processing pathway, did not cause cell death nor UPR signaling, and this may be the reason why this treatment did not sensitize cells to cisplatin treatment. MGAT5A silencing was not successful yet. Altogether, the results above show that the unstable equilibrium under which Tm1 and Tm5 tumor cells are seems necessary for their survival. Therefore, it seems that upon malignant transformation, melanoma cells present dependence of MGAT5A expression. Its our interest exploit this melanoma model to understand the concept of oncogenic dependence for MGAT5A expression in the case of melanomas, if it exists

Les ligands des récepteurs nucléaires PPAR[gamma] (15-deoxy-[delta]12,14-Prostaglandine J2 (15d-PGJ2) et les thiazolidinediones (TZDs) : troglitazone (TGZ), ciglitazone (CGZ)) exercent un effet antiprolifératif sur les lignées cancéreuses mammaires. Plusieurs études suggèrent que les effets anticancéreux sont liés à des effets PPAR[gamma]-indépendants. Notre travail s?inscrit dans la compréhension de tels mécanismes d?action. Notre étude a montré une induction du facteur de transcription EGR1 (Early Growth Response gene 1) par certains ligands de PPAR[gamma] (TGZ, CGZ, 15d-PGJ2 et [delta]2-TGZ (agoniste inactif de PPAR[gamma])) dans les cellules cancéreuses mammaires hormono-dépendantes MCF7. Cet effet est précoce et PPAR[gamma]-indépendant. Il est lié à une libération quasi immédiate de calcium intracellulaire suivie de l?activation des ERK1/2. L?induction d?EGR1 a aussi lieu dans les cellules hormono-indépendantes MDA-MB-231 exposées à la [delta]2-TGZ. Cependant, l?induction d?EGR1 ne joue qu?un rôle partiel dans l?effet antiprolifératif. Les données d?une analyse par puce à ADN ont suggéré l?induction d?un stress du réticulum endoplasmique (RE) dans les cellules MCF7 exposées à la [delta]2-TGZ. Des analyses complémentaires ont confirmé que la [delta]2-TGZ induit un tel stress dans les cellules MCF7 et MDA-MB-231. Cependant, le rôle du stress du RE dans l?effet antiprolifératif de la [delta]2-TGZ reste à déterminer. Nous avons enfin testé l?hypothèse d?un lien entre EGR1 et stress du RE. En effet, EGR1 est aussi induit précocement par d?autres inducteurs de stress du RE. Les diverses situations où l?induction d?EGR1 est inhibée suggèrent une régulation possible de l?expression du facteur de transcription ATF3 par EGR1 / The ligands of PPAR[gamma] nuclear receptors (15-deoxy-[delta]12,14-Prostaglandin J2 (15d-PGJ2) and thiazolidinediones (TZDs) : troglitazone (TGZ), ciglitazone (CGZ)) show antiproliferative effects on breast cancer cell lines. Several studies suggest that the anti-cancer effects are PPAR[gamma]-independent. Our work is focused on the comprehension of such mechanisms of action. Our study has shown the induction of the transcription factor EGR1 (Early Growth Response gene 1) by some PPAR[gamma] ligands (TGZ, CGZ, 15d-PGJ2, and [delta]2-TGZ (PPAR[gamma] inactive agonist)) in the hormone-dependent breast cancer cells MCF7. This early effect is PPAR[gamma]-independent. It is associated with the almost immediate release of intracellular calcium followed by the activation of ERK1/2. EGR1 induction also occurs in the hormone-independent breast cancer cells MDA-MB-231 treated with [delta]2-TGZ. However, EGR1 induction plays only a partial role in the antiproliferative effect. Data analysis of DNA array has suggested the induction of an endoplasmic reticulum stress (ER) in MCF7 cells treated with [delta]2-TGZ. Complementary data have confirmed this result in MCF7 cells and in MDA-MB-231 cells. However, the role of ER stress in the antiproliferative effect is still to be determined. Finally, we have tested the hypothesis of a link between EGR1 and ER stress. Indeed, EGR1 is also early induced by other ER stress inductors. Diverse conditions where EGR1 is inhibited suggest a possible regulation of ATF3 expression by EGR1

Une carence en donneurs de méthyle (CDM) est fréquente dans les maladies inflammatoires chroniques intestinales (MICI) et induit un stress du réticulum endoplasmique (RE). La désacétylase SIRT1 (Sirtuine 1) régule la réponse cellulaire suite à un stress nutritionnel par le biais d’un stress du RE. Nous avons étudié pour la première fois l’impact d’une CDM sur le stress du RE et les effets de l'activation de SIRT1 dans un modèle expérimental de colite chez le rat. Pour restaurer l’homéostasie du réticulum endoplasmique, la cellule met en place la réponse UPR « Unfolded Protein Response ».Les quantités de formes actives des acteurs de l’UPR (effecteurs et régulateurs), ainsi que du facteur de transcription (HSF1) et SIRT1 ont été étudiés par western blot, immunoprécipitation et RT-qPCR sur des tissus coliques (rats et humains) et sur des lysats cellulaires. Les effets de l'activation de SIRT1 ont été étudiés in vitro et in vivo. Une CDM aggrave la colite induite par le DSS cliniquement, endoscopiquement et histologiquement. Une CDM induit un stress du RE en augmentant le niveau d’expression des formes active des acteurs de l’UPR : p-PERK, p-eIF-2a, p-IRE-1a, ATF6, XBP1-S et l’ARNm d’ATF4. Ceci est accompagné d’une diminution dramatique du niveau d’expression des protéines chaperonnes, d’une réduction du niveau d'expression de SIRT1 et d’une hyperacétylation de HSF1. L’ajout de la vitamine B12, de la S-Adénosine Methionine (SAM), ou un activateur de SIRT1 (SRT1720) réduit l'UPR in vitro. L’immunohistochimie a montré une diminution de l’expression de SIRT1 dans l'épithélium du côlon de patients atteints de MICI. Chez le rat, l’activation de SIRT1 prévient la colite en réduisant le niveau acétylé de HSF1 et en augmentant le niveau d’expression des molécules chaperonnes. L'activation de SIRT1 prévient la colite en réduisant le stress du RE causé par la CDM. Ces résultats suggèrent que SIRT1 pourrait être une cible thérapeutique dans les MICI où les CDM sont fréquentes / Methyl donor deficiency (MDD) aggravates experimental colitis in rats and decreases SIRT1 activity in cell models. Endoplasmic reticulum (ER) stress plays a key role in inflammatory bowel disease (IBD) pathogenesis. We investigated whether the influence of MDD on colitis resulted from an ER stress response triggered by decreased SIRT1 expression. The Unfolded protein response (UPR), chaperone proteins, heat-shock factor protein 1 (HSF1) and SIRT1 were examined in rats with MDD and dextran sulfate sodium (DSS)- induced colitis, in a Caco-2 cell model with stable expression of transcobalamin-oleosin chimera (TO), which impairs cellular availability of vitamin B12, and in human IBD. The effects of SIRT1 activation were studied both in vitro and in vivo. MDD aggravated DSS-induced colitis clinically, endoscopically and histologically. MDD activated ER stress pathways, with increased p-PERK, p-eiF-2a, p-IRE-1a, ATF6, XBP1-S protein and ATF4 mRNA expression levels in rats. This was accompanied by reduced SIRT1 expression level and greater acetylation of HSF1, in relation with a dramatic decrease of chaperons (BIP, HSP27 and HSP90). Adding either vitamin B12, Sadenosylmethionine, or a SIRT1 activator (SRT1720) reduced the UPR in vitro. In rats, SIRT1 activation by SRT1720 prevented colitis by reducing acetylation of HSF1 and increasing the expression of BIP, HSP27 and HSP90. Immunohistochemistry showed impaired expression of SIRT1 in the colonic epithelium of IBD patients. SIRT1 is a master regulator of ER stress and severity of experimental colitis in case of MDD. It could deserve further interest as a therapeutic target of IBD