The coronavirus disease 2019 (COVID-19) pandemic is provoking a global public health crisis. Even though the academic world is intensively pursuing new therapies, there is still no “game changer” in the management of COVID 19. The Mammalian Target of Rapamycin (mTOR) is an ancient signaling system that has been proposed as a molecular tool used by coronaviruses and other RNA and DNA viruses in order to replicate and persist in the host cell. In recent years, Intermittent Fasting (IF), a practice consisting on a strict calorie restriction during a prolonged period of time during the day, has gained popularity due to its potential benefits in multiple health systems and in regulating inflammation. IF inhibits the mTOR pathway which is similar to the effects of Rapamycin in some animal models. mTOR inhibition and promotion of autophagy could potentially be the link between the possible direct benefits of IF in COVID-19 due to the interruption of the viral cycle (protein synthesis). Besides, IF has shown to be a strong anti-inflammatory in multiple prior studies, and may play a role in attenuating COVID -19 severity. This review hypothesizes the possible intersection between viral, immunological, and metabolic pathways related to mTOR and the potential mechanisms through which IF may improve clinical outcomes. Future prospective randomized controlled clinical trials to evaluate intermittent fasting (IF) regimens in order to prevent and treat moderate to severe forms of COVID-19 in humans are needed. / Revisión por pares

The mamallian target of rapamycin (mTOR) is a protein implicated in a variety of cellular processes involving growth and division. In the context of the brain, it regulates synaptic plasticity and axon elongation; its dysfunction is implicated in the pathogenesis of multiple complex, heterogeneous neurodegenerative diseases. These include, but are not limited to Alzheimer’s Disease (AD), autism spectrum disorder (ASD), and epilepsy. mTOR boasts a deeply complex and far-reaching signalling cascade, and its activity affects the expression levels of a large number of proteins. As such, investigation of the proteins with whom mTOR interacts is a pertinent endeavor to the advancement of understanding the complex pathogenesis of neurodegenerative disease. The complexity of this endeavor makes it a target well-poised for protein-protein interaction network (PPIN) analysis. Thus, using a previously recorded MS/MS dataset listing proteins whose expression levels change upon rapamycin administration, we set out to identify key proteins and characterize the properties of the mTOR interactome overall using a variety of toplogical measures and analytical techniques. Using such techniques, we found that the in the PPIN created from our data, a certain subset of proteins subjected the network to particular fragility. Namely, the kinless hubs, which have high within-module degree as well as a large participation coefficient, show vulnerability exceeding that of even conventionally defined hub. Some of these kinless hubs exhibit critical structural roles in the PPIN such that their removal damages the overall efficiency of communication within the network at an individually observable level. Work is ongoing to further investigate these proteins and the potential biological implications of their importance in the network described in the present study.

No description available.

No description available.

Specific AA have been demonstrated to activate signaling pathways that regulate<br />translation initiation and to stimulate protein synthesis in mammary tissue. The<br />objectives of this research were to determine the response to Ile, Leu, Met, and Thr in<br />cellular signaling and "-S1 casein fractional synthesis rates (CFSR). An experiment was<br />developed as a composite design. The experiment was replicated in tissue corresponding<br />to 5 cows. Mammary tissue slices (0.12 ± 0.02 g) from lactating dairy cows were<br />incubated 4 h in treatment media enriched with 2H5 Phe. Following incubation, slices<br />were homogenized in lysis buffer and caseins were precipitated by acidification to pH<br />4.6. An aliquot of the pellet was trypsinized and 2H5 Phe enrichment in the 34-<br />NLLRFFVAPFPE-45 peptide of "-S1 casein was measured by MALDI TOF-MS and<br />used to determine CFSR (%/h). Western immunoblotting was performed to identify total<br />and site-specific phosphorylated mammalian target of rapamycin (mTOR, Ser2448),<br />eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein (rp) S6 (Ser235/236),<br />and eukaryotic initiation factor (eIF) 2" (Ser51). Addition of Ile, Leu, Met, or Thr had<br />no effect on eIF2" phosphorylation. Isoleucine positively affected mTOR, and rpS6, and<br />negatively affected eEF2 phosphorylation. Leu had a similar effect on eEF2, but not on<br />mTOR or rpS6, and these two AA inhibited each other. Thr negatively interacted with<br />Ile on mTOR and rpS6, and with Leu on eEF2. Increasing concentrations of Ile, Leu,<br />Met, and Thr caused curvilinear increases in CFSR. The maximum response to Ile, Leu,<br />iii<br />Met, and Thr was at 71, 49, 60, and 65% of DMEM concentrations, respectively. All<br />maximums were above plasma AA concentrations observed in lactating cows fed to meet<br />NRC requirements. The CFSR estimated at those maximums were similar between AA<br />(3.6 ± 0.6 %/h). Individual AA effects on CFSR did not correlate with mTOR signaling.<br />Independent CFSR responses to individual essential AA observed in this study contradict<br />the single-limiting AA theory assumed in current requirement systems. The saturable<br />responses of CFSR to these 4 AA also demonstrate the deficiencies of a fixed postabsorptive<br />AA efficiency approach for determining AA requirements for milk protein<br />synthesis. / Ph. D.

Dairy cows inefficiently convert dietary protein to milk protein causing economic and environmental costs. Amino acids (AA), insulin, and glucose significantly enhance muscle protein synthesis efficiencies. The objectives of this research project were 1) to investigate the regulatory effects of essential AA (EAA) and their interactions with insulin, glucose and acetate on mammary protein synthesis rates, 2) to investigate whether branched chain amino acids (BCAA): leucine , isoleucine , and valine , become limiting for milk protein synthesis when Met and Lys supply were not limiting, and 3) to develop a mathematical representation for the EAA and insulin effects on cellular signals for protein synthesis. MAC-T cells were treated with EAA, insulin, glucose, and acetate to observe their individual and interactive effects on phosphorylation of mTOR, rpS6, S6K1, 4EBP1, eEF2, eIF2α, Akt, and AMPK. These signaling effects on protein synthesis rates were examined with mammary tissue slices. A mathematical representation of the insulin and EAA effects was developed. The effects of supplementing BCAA on milk protein synthesis were investigated using nine Holstein cows, assigned to 7 d continuous jugular infusions of saline, Met and Lys, and Met and Lys plus BCAA. Multiple essential amino acids, Leu, Ile, Met, and Thr were able to substantially regulate protein synthesis rates in bovine mammary cells by increasing (P < 0.05) phosphorylation of mTOR, S6k1, 4EBP1, and decreasing (P < 0.10) eEF2 phosphorylation. Insulin considerably (P < 0.10) exerted similar signaling effects in MAC-T cells, independent of EAA. Supplementation of only acetate increased (P = 0.09) mammary cell energy status as indicated by reduced AMPK phosphorylation in MAC-T cells. Neither acetate nor glucose had substantial regulatory effects on mammary protein synthesis rates. Although Met and Lys supplementation increased (P < 0.01) milk protein yields and protein efficiencies, there were no apparent benefits of BCAA supplementation under the feeding circumstances of our study. The developed mathematical model adequately represented the regulatory effects of EAA and insulin. Such mathematical representations of regulatory effects of EAA and their interaction with other nutrients may improve our current AA requirement models to predict AA requirements of dairy cows with increased accuracy. / Ph. D.

The key transcription factor sterol regulatory element binding protein-1 (SREBP1) plays a central role in milk fat synthesis. SREBP1 stimulates the transcription of genes encoding lipogenic enzymes. The overall objective of these studies was to investigate the mechanisms of SREBP1 regulation by nutrients. In the first study, chromatin immunoprecipitation (ChIP) accompanied with deep-sequencing was employed to investigate the potential sterol regulatory elements (SRE) in the promoter of SREBP1-target genes. The SRE in three known SREBP1-target genes SREBP1, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) were first validated in a bovine mammary epithelial cell line (MacT) and in bovine mammary tissues. At least one or two SRE binding sites in 24 selected lipogenic genes were identified within 50,000 base pair to the 5'-transcription start site through ChIP-seq. The genes closest to the highest enriched peaks were involved in cell integrity, defense or signal transduction whereas lipogenic genes were not among the top enrichment leading to the questions about the success of the ChIP. The second study was conducted to determine the effect of t10, c12-conjugated linoleic acid (CLA) on insulin induced gene-1 (Insig1), an endoplasmic reticulum (ER) protein that anchors SREBP1 and prevents proteolytic activation of SREBP1. MacT cells were treated with increasing levels of t10, c12-CLA. High concentration of t10, c12-CLA inhibited Insig1 degradation therefore decreased SREBP1 maturation. Furthermore, immunoprecipitation (IP) confirmed that t10, c12-CLA reduced Insig1 proteasomal degradation by disrupting the interaction between Insig1 and UBX domain-containing protein 8 (Ubxd8), which is part of a degradation complex that removes Insig1 from the ER. In the third study, three potential regulators of SREBP1 activation and their pathways were investigated in insulin, t10, c12-CLA or glucose treated MacT cells. Insulin-induced mammalian target of rapamycin (mTOR) signaling stimulated lipogenesis via activation of SREBP1 and the stimulatory effect was based on the regulation on cAMP response element binding protein coactivator 2 (CRTC2) phosphorylation, Lipin1 translocation and glycogen synthase kinase-3 (GSK3)-dependent proteasomal degradation. t10, c12-CLA inhibited SREBP1 through AMP-activated protein kinase (AMPK) phosphorylation, a key protein kinase in energy homeostasis. Glucose stabilized the SREBP1 chaperone protein SCAP and facilitated SREBP1 activation. Overall, SREBP1 activation is under specific regulation of t10, c12-CLA and interacts with multiple major cellular signaling pathways in response to hormonal stimulation and nutrient availability. / Ph. D.

Les TNE-GEPs constituent un groupe de tumeurs hétérogènes pour lesquelles il convient d’élargir les approches thérapeutiques. Les thérapies ciblées, particulièrement l’inhibition de la voie mTOR par l’évérolimus, sont la référence pour les TNE-GEPs pancréatiques. Néanmoins, le bénéfice thérapeutique de l’évérolimus n’a pas été évalué dans le sous-groupe des carcinomes neuroendocrines GEPs peu différenciés (pdCNE-GEPs). A travers un modèle préclinique in vivo, nous montrons que l’inhibition de mTOR pourrait constituer une option thérapeutique pour les pdCNEGEPs. Ensuite, une étude protéomique a mis en évidence de nouveaux facteurs impliqués dans la progression de TNE-GEPs. Certaines protéines identifiées présentent un rôle dans la régulation du cytosquelette. Parmi celles-ci, CRMP2 est un acteur clé de la voie de signalisation des sémaphorines de classe 3 (sema3). Un profil d’expression de ces sema3 montre que l’expression de la sema3F est diminuée dans les TNE-GEPs. La ré-expression de ce facteur dans des modèles cellulaires de TNEGEPs montre que la sema3F induit une baisse de la survie et de la prolifération. In vivo, la sema3F permet de ralentir le développement tumoral. Des études complémentaires sont nécessaires pour mieux comprendre le rôle de la voie de signalisation de la sema3F dans la progression des TNE-GEPs / GEP-NETs are heterogeneous tumors for which therapeutic options are limited and must therefore be enlarged. Targeted therapies, and mainly everolimus-directed mTOR inhibition, constitute standard treatments for GEP-NETs of pancreatic origin. Nevertheless, the therapeutic benefits of everolimus have not been evaluated in the poorly-differentiated GEP neuroendocrine carcinomas (pdGEP-NECs) subgroup. By using a preclinical in vivo model, we demonstrated that mTOR inhibition could be considered as a therapeutic option for pdGEP-NECs. Then, a proteomic study highlighted novel proteins involved in GEP-NETs progression with all identified factors displaying function in cytoskeleton regulation. Among them, CRMP2 is a key member of class 3 semaphorin (sema3) signaling. An expression profile of sema3 revealed that sema3F expression was decreased in GEP-NETs. The re-expression of this protein in TNE-GEPs cellular models showed that sema3F is responsible of the reduction of cell viability and proliferation. In vivo, sema3F hampered tumor development. Further studies are thus needed to better understand the role of the sema3F signaling pathway in the progression of GEP-NETs

As células dendriticas (DC) da lamina própria constituem um grupo heterogêneo de células essenciais na homeostase do intestino. Nos últimos anos, estudos vem mostrando participação do complexo mTORC2 na regulação de células da imunidade inata. Desta forma, usando animais deficientes em mTORC2 nas DCs nos investigamos o papel deste complexo sob a funcoes das DCs num modelo de colite ulcerativa. Observamos que a ausência de mTORC2 nas DCs diminui a inflamação intestinal e prejudica a migração dessas células para os linfonodos. Consequentemente, estes animais também apresentam redução no numero de linfócitos Th17, Treg e T CD8&#43IFNg&#43. Estes resultados parecem ser independentes da composição da microbiota intestinal. In vitro, células dendriticas derivadas da medula ossea (BMDCs) deficientes em mTORC2 não mostraram deficiência na regulação positiva de CD80 e MHC-II quando estimuladas, mas mostraram menor produção de TNF-a e IL-6. Estas células também apresentaram menor capacidade na diferenciação de Th17. Nossos resultados indicam que mTORC2 participa na regulação das funções das DCs, especialmente na migração e produção de citocinas pos estímulos lnflamatorios. / Dendritic cells from lamina propria encompass a heterogeneous group of cells which play a key role in intestinal homeostasis. In the past few years, it has been shown the participation of mTORC2 in regulating innate immune cells. In our study, we used mTORC- deficient DCs to investigate the role of this complex in DCs functions in ulcerative colitis model. Absence of mTORC2 in DCs reduces the intestinal inflammation, impairs DCs migration which in turn decreases the number of Th17, Tregs and CD8&#43IFNg&#43 T cells. These results seem to be independent of gut microbiota composition. Our in vitro results showed that bone marrow-derived DCs (BMDCs) deficient in mTORC2 can upregulate CD80 and MHC-II after stimulus but present decreased production of TNF-a and IL-6. Those cells also showed deficiency in Th17 differentiation. Our results suggest that mTORC2 play pivotal role in DCs functions highlighting migration and cytokine production.

Le rôle central de la voie mTOR (mammalian Target Of Rapamycin) dans l’homéostasie cellulaire suscite un réel intérêt dans de nombreux domaines thérapeutiques. Les inhibiteurs de mTOR (mTORi) sont utilisés en prévention du rejet de greffe par leur action d’inhibition de l’activation lymphocytaire T et l’induction de lymphocytes T régulateurs (Treg). En cancérologie, les mTORi sont utilisés pour leur action anti-proliférative et anti-angiogénique. Cependant, l’efficacité clinique de ces traitements reste modeste. Bien que des mécanismes de résistance aux mTORi intrinsèques à la cellule tumorale ont été identifiés, leur impact sur le système immunitaire émerge comme un facteur essentiel dans leur efficacité. Notre hypothèse est donc que l’efficacité anti-tumorale des mTORi serait également liée à la modulation de l’immunité anti-tumorale induite par ces traitements.Nous avons réalisé un immunomonitoring, du taux de Treg et de la réponse lymphocytaire Th1 anti-tumorale au sein d’une cohorte prospective de patients atteints de cancer rénal métastatique traités par éverolimus (mTORi).Nous avons observé une augmentation des Treg et de leur fonction suppressive à partir du 2ème mois de traitement dans la quasi-totalité des patients. Paradoxalement, une augmentation des réponses Th1 anti-tumorales a également été observée chez ces patients. Au moment de la progression tumorale, la majorité des patients ont présenté une forte augmentation des Treg associée à une perte de la réponse Th1. Ainsi, nous avons pu définir différents profils immunitaires basés sur la modulation de ces deux paramètres. Les patients présentant une diminution précoce des Treg associée à une augmentation de la réponse Th1 avaient une meilleure survie sans progression par rapport aux autres patients (13.2 vs 4 mois p= 0.02). De façon consistante, cette immunomodulation a été confirmée chez des patients atteints de tumeur neuroendocrine traités par éverolimus. De plus, l’impact du traitement sur les Treg et la réponse Th1 n’était pas influencé par la pharmacocinétique du médicament. Par la suite, nous avons étudié in vivo, chez des souris porteuses de différentes tumeurs, l’impact des mTORi sur la réponse immunitaire anti-tumorale. Comme chez les patients, nous avons observé une augmentation des Treg chez les souris traitées par mTORi. Ainsi, l’utilisation d’anticorps déplétants les Treg ou de souris transgéniques ont permis de démontrer le rôle délétère des Treg sur l’efficacité anti-tumorale des mTORi. Ces résultats suggèrent fortement que les mTORi ont un double effet sur le système immunitaire dans un contexte tumoral, un premier néfaste par l’induction d’un environnement immunosuppresseur et le second positif par l’augmentation d’une réponse Th1 anti-tumorale.Dans un deuxième travail, nous avons exploré l’effet positif des mTORi sur le système immunitaire afin d’optimiser les immunothérapies anti-tumorales. Nous avons montré que l’administration de temsirolimus (mTORi) améliore l’efficacité anti-tumorale d’un vaccin thérapeutique chez la souris. L’effet synergique de cette combinaison était lié à une augmentation de l’infiltration tumorale des LT CD8 anti-tumoraux de phénotype centro-mémoire. L’efficacité de cette combinaison a été nettement améliorée par l’addition d’un antagoniste de CCR4 permettant d’éliminer les Treg induits par le temsirolimus.En conclusion, les mTORi demeurent une stratégie prometteuse en cancérologie malgré la réduction de leur utilisation due à l’arrivée des nouvelles immunothérapies, comme les checkpoints inhibiteurs (notamment dans le cancer du rein) et d’autres thérapies ciblées (inhibiteurs de cycline dans le cancer du sein). En revanche, le futur développement des mTORi devra s’appuyer sur des biomarqueurs prenant en compte l’impact de ces traitements sur le système immunitaire et sur des combinaisons thérapeutiques notamment avec les immunothérapies / The key role of the mTOR (mammalian Target of Rapamycin) pathway in cellular homeostasis raises a real interest in many therapeutics areas. Inhibitors of mTOR (mTORi) are used for graft rejection prevention to inhibit the T lymphocyte (LT) activation and induce regulatory T cells (LTreg). In cancerology, they are used for their antiproliferative and antiangiogenic actions. However, the clinical efficacy of those treatments is low. Although several mTORi resistance mechanisms linked to the tumor cell have been identified, their impact on the immune system appears as a key factor in their efficiency. Thus, our hypothesis is that the clinical efficacy of mTORi could also be dependent of an anti-tumoral immunity modulation resulting from those treatments.We performed an immunomonitoring of the Treg rate and the tumor specific Th1 anti-tumor response among a prospective cohort of patients with a metastatic renal carcinoma treated by everolimus (mTORi). We observed that the Treg rate and the suppressive function increase after the second month of treatment for almost all the patients. Paradoxically, an increase of the Th1 anti-tumoral response has also been observed for these patients. At disease progression, a majority of the patients has shown an important increase of Treg with a decrease of the Th1 response. Thus, we have been able to define different immune profiles based on the modulation of these two parameters. Progression free survival of patients with an earlier decrease of Treg and an increase of Th1 response was significantly longer compared to other patients (13.2 vs 4 months p=0.02). This immunomodulation has been consistently confirmed with patients affected by neuroendocrine tumor and treated with everolimus. The treatment impact on Treg and the Th1 response was not related to the pharmacokinetic of the drug. Thereafter, we have studied in vivo the impact of mTORi on the anti-tumoral immune response with mice affected by different tumors. We have observed the same increase of Treg in mice treated with mTORi as the increase observed in the patients. Thus, using antibodies depleting the Treg or transgenic mice allowed us to confirm the deleterious role of Treg on the anti-tumor mTORi efficacy. Those results strongly suggest that mTORi have a double effect on the immune system, a harmful impact by the induction of an immunosuppressive environment and a positive impact by the increase of the anti-tumor Th1 response.In a second time, we have studied the positive effect of mTORi on the immune system in order to optimize the anti-tumoral immunotherapies. We have shown that the administration of temsirolimus (mTORi) improves the anti-tumoral efficiency of a therapeutic vaccination on mice. Indeed, the synergic effect of this combination is tied with an augmentation of tumor infiltrate anti-tumor T CD8 with a central memory phenotype. The efficiency of this combination has been greatly improved by the addition of an antagonist CCR4 allowing the elimination of Treg generated by temsirolimus.In conclusion, mTORi remain a promising strategy in cancerology even if their use is likely to be reduced as new immunotherapies such as checkpoint inhibitors (renal carcinoma) or other targeted therapies (cycline dependent kinase inhibitors in breast cancer) appeared. In the future, the development of mTORi must integrate biomarkers taking into account the impact of these treatments on the immune system, and therapeutic combination such as the immunotherapy