Dans les leucémies aiguës myéloïdes (LAM), l'activation anormale de nombreuses voies de signalisation intracellulaires favorise la croissance et la survie des cellules tumorales. L'amélioration des connaissances biologiques de ces pathologies hétérogènes, dont le pronostic est réservé, devrait permettre le développement de thérapies ciblées. La kinase oncogénique mTOR est présente au sein de deux complexes, parmi lesquels mTORC1, activé constitutivement dans la majorité des blastes primaires de patients porteurs de LAM, qui contrôle la synthèse protéique, et mTORC2 activé constitutivement dans 50% des LAM. Les inhibiteurs allostériques de mTORC1 (la rapamycine et ses dérivés) n'inhibent pas la phosphorylation du répresseur traductionnel 4E-BP1, ne diminuent pas la traduction et induisent peu d'apoptose in vitro dans les LAM. Utilisés en monothérapie, leur effet est décevant. De plus ces inhibiteurs n'agissent pas sur le complexe mTORC2. J'ai étudié l'effet d'un inhibiteur catalytique de mTOR, l'AZD8055, actif sur les deux complexes. In vitro, l'AZD8055 inhibe efficacement la signalisation en aval de mTORC1 et de mTORC2, dont les sites de phosphorylation de 4E-BP1 résistants à la rapamycine, ainsi que la synthèse protéique. Il diminue la prolifération, bloque le cycle cellulaire en phase G0G1 et induit une apoptose caspase-dépendante dans les blastes primaires de LAM. Il diminue également la clonogénicité des progéniteurs leucémiques, sans affecter celle des cellules CD34+ normales. Dans un modèle murin de xéno-transplantation, l'AZD8055 inhibe la croissance tumorale et améliore la survie des souris traitées. Je me suis également intéressée à la régulation de l'activité de mTORC1 par les acides aminés. Dans les cellules de mammifères, l'activation de mTORC1 nécessite la présence de glutamine et de leucine qui agissent en coopération via deux transporteurs membranaires, SLC1A5 et SLC7A5/SLC3A2. J'ai montré que la privation en glutamine, obtenue par l'activité glutaminase de la drogue L-asparaginase ou par l'utilisation de milieux de culture spécifiques dépourvus sélectivement en acides aminés, inhibe l'activation de mTORC1 et induit de l'apoptose dans diverses lignées leucémiques et dans les blastes primaires de LAM. La L-asparaginase inhibe la synthèse protéique et ses effets fonctionnels sont liés à son activité glutaminase. J'ai pu également constater une augmentation de l'expression protéique de la glutamine synthase induite par la Lasparaginase, dont l'inhibition majore l'apoptose induite par la L-asparaginase dans certaines lignées leucémiques. J'ai également étudié l'effet de l'inhibition spécifique par un shARN inductible du transporteur SLC1A5, qui permet l'import de glutamine. L'inhibition de SLC1A5 bloque la réactivation de mTORC1 par l'association leucine/glutamine après privation et induit de l'apoptose dans la lignée leucémique MOLM14. Cette inhibition diminue la croissance tumorale dans un modèle de xénogreffe

Energy balance refers to the relationship between energy intake and energy expenditure. Epidemiological studies have established a clear association between energy balance and cancer, however the underlying mechanisms are unclear. The objective of the current study was to evaluate the impact of caloric consumption on epithelial carcinogenesis and identify potential mechanisms of inhibition or enhancement. Using ICR female mice, we demonstrated that positive energy balance enhanced, while negative energy balance inhibited susceptibility to multistage carcinogenesis in mouse skin. We next evaluated diet-induced changes in the epidermal proliferative response. Calorie restriction (CR) significantly reduced epidermal hyperproliferation, in the presence and absence of tumor promotion, as compared to diet-induced obesity (DIO). Additional studies were conducted to determine the impact of dietary manipulation on TPA-induced growth factor signaling. CR reduced, while DIO increased insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation, which subsequently modulated signaling downstream to Akt and mTOR. These diet-induced changes in growth factor signaling were confirmed under steady-state conditions in multiple epithelial tissues (i.e., skin, liver and dorsolateral prostate) in multiple mouse strains (FVB/N, C57BL/6 and ICR). Further analyses demonstrated that caloric consumption directly correlated with levels of cell cycle progression related proteins and inversely correlated with levels of cell cycle inhibitory proteins. Genetic reduction of circulating IGF-1, liver IGF-1 deficient (LID) mouse model, inhibited two-stage skin carcinogenesis, reduced epidermal hyperproliferation and attenuated IGF-1R and EGFR growth factor signaling during tumor promotion, similar to CR, suggesting a potential for IGF-1R and EGFR crosstalk. Further studies, demonstrated that IGF-1 induced EGFR activation in cultured mouse keratinocytes, possibly due to IGF-1R and EGFR heterodimerization or IGF-1 induced changes in EGFR mRNA expression. In vivo, CR reduced, while DIO increased IGF-1R and EGFR association during tumor promotion. Furthermore, CR attenuated EGFR ligand mRNA expression both in the presence and absence of TPA treatment. Collectively, these findings suggest that dietary energy balance modulates epithelial carcinogenesis, at least in part due to diet-induced changes in levels of circulating IGF-1, which then modulate IGF-1R and EGFR crosstalk and downstream signaling to cell cycle related proteins, subsequently altering epidermal hyperproliferation. / text

A vital factor for foetal development is the nutrient transport at placental level. This is because any disturbances in the maternal compartments, for example due to maternal stress or nutritional status, which will affect foetal development, will involve the foetal-placental barrier. Moreover, numerous studies have linked other factors such as preterm labour as the leading cause of perinatal morbidity and mortality in the developed world. To this date, despite a numerous epidemiological and clinical studies that identify potential risk factors for the mother as well as the foetus, there is no comprehensive analysis at all these levels taken from the same cohort of patients. Our working hypothesis is that for a successful pregnancy certain events at nutritional, biochemical, genetic and molecular level could be tightly linked. Therefore, in this study we followed a “holistic” approach investigating how maternal stress, nutrition, placental mTOR and glucocorticoid receptor (GR) signalling can influence pregnancy outcome. We have decided to map in detail the components of these two signalling pathways as they appear to cross-talk as well as been implicated in stress responses. The largest part of the questionnaire was focused on the nutritional status with questions targeting the maternal dietary habits before, as well as during, pregnancy. The collection of data took place at the Department of Obstetrics and Gynecology, University of Crete Medical School. With regards to this profile, key findings included the significant reduction in the intake of alcohol, caffeine-containing and sugar-containing refreshments, whereas passive smoking during pregnancy stayed the same. Another major finding of this part of the study was the effects of maternal stress on foetal weight and how pregnancy planning was implicated in this complex relation. In our cohort, women with negative attitudes during pregnancy gave birth to infants with significantly lower birth weights (2.5Kg) than those women showing positive or neutral attitudes towards their pregnancy (2.9Kg). We then assessed how maternal stress might affect this signalling cascade using two placental models (BeWo and JEG-3 cell lines) mimicking a stress milieu in vitro. Treatment of these cell lines with cortisol (100nM and 1000nM) significantly downregulated Deptor and upregulated GAS5 at mRNA level. In an attempt to dissect further a potential gene-environment interaction, we have assessed how 4 well-characterised polymorphisms (ThtIII 1, Bcl I, ER22/23EK, N363S) of the GR gene might affect foetal and placental weight. We have demonstrated that only the maternal ThtIII 1 polymorphism was suggestive of a nature-nurture interaction since only in ThtIII 1 II (CC), maternal stress attitude predicts foetal weight-reduction, but not in ThtIII 1 (GC) independent of confounders such as BMI, pregnancy planning or fast food eating during pregnancy. This is the first time that a gene-environment interaction between a common GR polymorphism and foetal weight was noted. Finally, one of the most important findings of our study came from the preclinical studies using placental tissues. Quantitative PCR revealed that the major transcripts in the human placenta are GRα, GAS5 (decoy for GR DNA binding) and Deptor. We have shown for first time that there are marked differences in the relative mRNA abundance of these components between term and preterm labour as well as colocalisation of GRα with GAS5. With regards to placental regulation these data conclusively demonstrate that: a) there is evidence of gene-environment interaction between maternal stress, pregnancy planning, glucocorticoid receptor polymorphisms and foetal weight and b) potential cross-talk of mTOR and glucocorticoid signalling. We propose that measuring maternal stress levels in addition to circulating cortisol and mapping for known GR polymorphisms could become a useful non-invasive tool of diagnostic and prognostic value, with implications for preterm labour.

Le muscle squelettique fait preuve d'une remarquable plasticité en réponse aux changements physiologiques, comme l'activité physique, et aux situations pathologiques. Il subit notamment une atrophie sévère lors de la cachexie qui accompagne diverses pathologies chroniques comme le cancer, le SIDA, etc. L'atrophie musculaire est aussi une composante de la sarcopénie qui survient lors du vieillissement normal, et se caractérise par un déclin de la force et de la masse musculaire. L'atrophie musculaire, qui entraîne une augmentation de la mortalité et diminue l'efficacité des traitements, constitue donc un problème de santé majeur.La fonte musculaire se caractérise par une altération de l'équilibre entre synthèse et dégradation protéiques dans les fibres adultes. Des taux particulièrement élevés de cytokines circulantes, dont le TNFα, qui affectent l'homéostasie du muscle via différentes voies de signalisation, semblent être à l'origine de l'atrophie. Les mécanismes de la réponse atrophique musculaire à ces taux circulants élevés sont cependant mal définis. Le TNFα a des effets complexes. Il peut activer de multiples voies de signalisation, parmi lesquelles l'induction de la synthèse de sphingolipides, et plus particulièrement de céramides, par la voie de novo et par l'activation des sphingomyélinases. Au niveau musculaire, les céramides sont connus pour leurs effets sur la signalisation de l'insuline, sur l'apoptose et sur la différenciation myogénique. Par contre, leur implication dans le cadre de l'atrophie n'avait jamais été prise en compte. L'objectif de ce travail a été dans un premier temps de démontrer le rôle des céramides dans l'atrophie. Dans un deuxième temps, nous avons caractérisé la voie de signalisation par laquelle l'augmentation intramusculaire de céramide induite par le TNFα aboutit à une chute de la synthèse protéique, couplée à une augmentation de la protéolyse. Dans ce but, nous avons mis au point des modèles in vitro d'atrophie, impliquant des myotubes traités par des concentrations physiologiques de TNF. Nous avons en parallèle étudié un modèle in vivo de cachexie induite chez la souris par l'implantation d'un adénocarcinome C26. L'analyse des sphingolipides nous a permis de montrer l'augmentation des taux de céramides concomitante à l'atrophie générée in vitro et in vivo. Le rôle des céramides dans l'atrophie a été démontré par l'effet protecteur des inhibiteurs de leur synthèse, dans les modèles in vitro et in vivo. Nous montrons de plus dans un modèle in vitro que les effets atrophiques des céramides sont dus à l'inhibition de la voie de signalisation Phospholipase D/mTOR/Akt. Nos résultats nous ont permis de prouver le rôle des sphingolipides dans le contrôle de l'homéostasie protéique du muscle. La modulation du métabolisme des sphingolipides apparaît donc comme une nouvelle cible thérapeutique prometteuse dans le traitement de la perte musculaire associée à diverses pathologies.

<p>Castration resistant prostate cancer (CRPC) causes significant morbidity and mortality around the world and improving treatment options for patients with CRPC is a major concern for biomedical research. Because of the importance of activating mutations in the PI3K/AKT/mTOR pathway in prostate cancer, several mTOR inhibitors have been tested for efficacy in CRPC but despite promising preclinical findings, the results of clinical trials have been disappointing. The findings of several groups, including a clinical trial of RAD001 conducted at Duke, suggest that feedback upregulation of PI3K and autophagy may be potential mechanisms for resistance of CRPC to mTOR inhibitor therapy. </p><p> The main goal of this dissertation was to explore these mechanisms in vitro and to determine if combinations of PI3K inhibitors and different classes of mTOR inhibitors can overcome resistance to mTOR inhibitor monotherapy. In particular, we used immunoblotting, reverse phase protein microarrays, polysome profile analysis, cell cycle analysis, and several techniques for determining cell survival and proliferation to explore the differences in survival, proliferation, autophagy, and activity of the AKT, translation initiation, and autophagy cell signaling networks between prostate cancer cell lines treated with different combinations of mTOR and PI3K inhibitors. Our findings revealed that the combination of PI3K and mTOR inhibition leads to a synergistic inhibition of prostate cancer cell survival and cytostasis that is correlated decreased translation rates, hypophosphorylation of 4E-BP1, autophagy, and an uncoupling of normal signaling between AKT and mTOR. We were able produce an effect on cell survival similar to treatment with high doses of mTOR/PI3K inhibitor combinations by inhibiting cap-dependent translation using a non-phosphorylatable mutant of 4E-BP1. In contrast, knocking down two major autophagy genes had little to no effect on the survival of prostate cancer cells treated with PI3K/mTOR inhibitors but did protect from cell death caused by the UPR activator tunicamycin. </p><p> We conclude that treatment strategies that target PI3K, mTORC1 and mTORC2 simultaneously have the potential to be clinically useful in CRPC, probably due to the increased inhibition of eIF4E activity and cap-dependent translation when compared to monotherapy with allosteric mTORC1 inhibitors. Although autophagic cell death can be induced in prostate cancer cells, the autophagy observed after inhibition of PI3K and mTOR does not appear to contribute to cell death and is not a major resistance mechanism under these conditions. Nevertheless, we did observe different roles for autophagy in the survival of cells exposed to different types of stressors, and further elucidation of autophagy signaling networks may yet provide useful clinical targets.</p> / Dissertation

Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.

No description available.

No description available.

Orientador: Luis Gustavo Modelli de Andrade / Resumo: Introdução: O transplante renal é a melhor opção de tratamento para doença renal crônica estadio V. Melhora substancial da sobrevida do enxerto no primeiro ano ocorreu principalmente pela drástica redução dos índices de rejeição aguda com o uso de imunossupressores potentes, destacando-se os inibidores de calcineurina. Porém essa não se acompanhou de melhora da sobrevida do paciente e do enxerto em longo prazo. A mortalidade cardiovascular continua sendo a principal causa de morte no transplante renal. A hipertrofia do ventrículo esquerdo (HVE) e a aterosclerose são muito prevalentes nessa população e constituem fatores de risco para eventos cardiovasculares. Os inibidores da mTOR (mammalian target of rapamycin inhibitor) parecem ser drogas promissoras na redução da HVE e na redução e prevenção de aterosclerose no campo experimental, porém seu efeito nos pacientes transplantados renais ainda é controverso.Objetivo: Comparar a ação do everolimo com o tacrolimo na redução do índice de massa do ventrículo esquerdo (iMVE) e da espessura médio intimal das carótidas (EMIC) em pacientes transplantados renais.Material e Métodos: Ensaio clínico prospectivo, randomizado, unicêntrico, cego para o cardiologista que realizou os ecocardiogramas. Os pacientes receberam imunossupressão inicial com Tacrolimo (Tac), Micofenolato sódico (MFS) e Presnisona (PDN). Após 12±4 semanas, pacientes que preencheram critérios de inclusão e exclusão foram randomizados 1:1 nos grupos: TACRO (controle) ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Kidney transplantation is the gold standard treatment for end-stage renal disease. Allograft survival after one year of transplantation has had a significant improvement. However, there is a lack of improvement in patient and allograft long term survival. The mTOR inhibitors (mammalian target of rapamycin inhibitor) are the newest drugs available to prevent allograft rejection and they seem to have potential benefits in reducing myocardial hypertrophy and atherosclerosis in experimental studies. This benefits for kidney transplanted patients is still controversial. Objective: To compare the effect of everolimus to the tacrolimus in reducing left ventricular mass index (LVMi) and carotid intima-media thickness (IMT) after one year in kidney transplanted patients. Material and Methods: This randomized, open-label, controlled trial compared the effect of everolimus to tacrolimus in reducing the LVMi and the IMT in kidney transplanted patients after one year of these immunosuppressive therapies. After initial immunosuppression with tacrolimus, mycophenolate sodium (MFS) and prednisone (PDN), patients were randomly assigned at 12±4 weeks in a 1:1 ratio to undergo conversion from tacrolimus to everolimus (EVERO group) and maintenance of MFS and PDN or continue on standard tacrolimus-based treatment (TACRO group). Each patient was randomized only after a kidney biopsy with no evidence of rejection or inflammation. Clinical and laboratorial data were collected on random... (Complete abstract click electronic access below) / Doutor

Novas informações assimiladas pelo sistema nervoso primeiramente ficam em um estado de labilidade para depois se estabilizarem através de um processo conhecido como consolidação, que envolve síntese de proteínas. Depois da reativação, uma memória previamente consolidada retorna ao seu estado de labilidade, e para que volte a ser estável, é necessário que haja novamente síntese de proteínas. Este segundo processo é chamado de reconsolidação. Recentemente os mecanismos moleculares e celulares envolvidos na regulação da síntese protéica relacionados à formação de memória de longa duração vêm sendo esclarecidos. A proteína alvo da rapamicina em mamíferos (mTOR) modula a plasticidade sináptica pela regulação da fosforilação de dois alvos: a proteína ribossomal S6K e a proteína de ligação 4E. A amígdala basolateral e o hipocampo dorsal são parte integrante do sistema neural envolvido na formação e expressão de diversos tipos de memórias. Estudos indicam que a via de sinalização da mTOR no hipocampo tem um papel importante na consolidação da memória de ratos submetidos a tarefa de esquiva inibitória e reconhecimento de objetos e na reconsolidação da memória de medo contextual condicionado. Contudo, estudos anteriores não avaliaram o efeito da inibição de mTOR amigdalar sobre a memória de esquiva inibitória e reconhecimento de objetos. O objetivo do presente trabalho é investigar o efeito da inibição de mTOR na amígdala basolateral por rapamicina na consolidação e reconsolidação da memória de esquiva inibitória e reconhecimento de objetos e comparar estes resultados com a inibição de mTOR no hipocampo. Ratos Wistar machos foram submetidos à cirurgia estereotáxica para implantação de cânulas na amígdala basolateral e hipocampo dorsal. Os animais foram submetidos à tarefa de esquiva inibitória, um modelo animal de memória de caráter aversivo, e a tarefa de reconhecimento de objetos, um modelo animal de memória de caráter pouco aversivo. Para investigar o efeito da inibição de mTOR na consolidação e reconsolidação da memória, os animais receberam microinfusões de rapamicina intra-amigdalar e intra-hipocampal em diferentes tempos em torno do treino e do teste. Nós demonstramos que a via de sinalização de mTOR na amígdala basolateral é necessária para consolidação da memória de esquiva inibitória e de reconhecimento de objetos. Nós também mostramos que a reativação torna a memória novamente suscetível e sensível à inibição de mTOR por rapamicina. / Memory formation requires protein synthesis, but only recently the cellular and molecular mechanisms involved in the regulation of protein synthesis related to the formation of long term memory has been elucidated. During memory formation, new information is acquired by the central nervous system as an initially fragile trace that over time becomes stable through a process known as consolidation. After reactivation, previously consolidated memories might return to a labile state, requiring a new round of protein synthesis to be restabilized. This second process is called reconsolidation. The basolateral amygdala and dorsal hippocampus are part of the neural systems involved in the formation and expression of several types of memory. One key regulator of protein synthesis is mTOR, a protein critical for different forms of synaptic plasticity by regulation of two targets: S6K and 4EBP. Evidence indicates that the mTOR signaling pathway in hippocampus has an important role in consolidation in rats of inhibitory avoidance and object recognition in rats, as well as in reconsolidation of contextual fear conditioning. However, previous studies have not examinated the effect of amygdalar mTOR inhibition on reconsolidation of inhibitory avoidance and object recognition. The aim of the present study was to evaluate the effect of amygdalar mTOR inhibition by rapamycin on consolidation and reconsolidation of inhibitory avoidance and object recognition, and compare the results with those obtained with hippocampal mTOR inhibition. Male rats Wistar underwent stereotaxic surgeries for cannulae implantation above the basolateral amygdala or dorsal hippocampus. After recovery, the animals were trained in inhibitory avoidance, an aversive memory task, or object recognition, a less aversive task. To investigate the effect of mTOR inhibition on memory consolidation and reconsolidation, we administered rapamycin, a specific mTOR inhibitor, into the basolateral amygdala or the dorsal hippocampus before or after training or reactivation. Our results provide evidence that mTOR in the basolateral amygdala and hippocampus might play a role in inhibitory avoidance and object recognition memory formation and reconsolidation.