Understanding the molecular mechanisms that govern productivity and growth of recombinant host cells is essential to devise informed approaches to increase commercial viability and availability of biopharmaceuticals. This work has focused on the roles of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway in CHO cells, the most widely used expression system in the biopharmaceutical industry. mTORC1 is a master regulator of cell growth, protein synthesis and metabolism in response to availability of nutrients, oxygen and growth factors. Therefore, it was hypothesised that increased activity of mTORC1 enhances growth and productivity of recombinant CHO cells. The study of a recombinant GS-CHO cell line in the serum-free suspension batch culture indicated a gradual decrease in the activity of mTORC1, as defined by the decreased extent of site-specific phosphorylation of two widely ascribed downstream target proteins (ribosomal protein S6 kinase 1 (S6K1) and 4E-BP1, an inhibitor of translation initiation). The decline in the activity of mTORC1 paralleled decreased growth rate, recombinant protein specific productivity and global protein translation. To further clarify the role of the mTOR pathway in cell growth and protein production, cells in batch culture were treated with rapamycin, a specific inhibitor of mTORC1. Treatment with rapamycin stalled the growth of the CHO cell line transiently, but recombinant protein specific productivity, longevity of batch culture, and final antibody titre were greater than control. Rapamycin addition produced discriminating effects on downstream signalling targets, implicating distinct roles for these targets in control of growth and protein synthesis. Engineering the mTORC1 pathway by overexpression of specific components of this pathway (S6K1 and Rheb) generated increased growth and extended viability. Greater proliferation was not associated with improved productivity suggesting highly proliferative phenotypes that prioritise cell growth over synthesis and secretion of recombinant antibody in the recombinant GS-CHO cells examined. Therefore, the engineering of mTORC1 pathway may be beneficial to increase robustness or adaptation to stressed conditions (such as serum- free suspension growth, low nutrition availability and hypoxia).

The mammalian target of rapamycin (mTOR) signalling pathway takes part in both extracellular and intracellular signals. It is a major regulator of cell metabolism, growth, proliferation and survival. mTOR also regulates critical processes such as cytoskeletal organization, ribosomal biogenesis, transcription and protein synthesis. The mTOR pathway has been implicated in many diseases such as cancer, neurodegeneration and diabetes, which impact homeostasis and cellular functions. Moreover, mTOR has also been shown to play a critical role in immune cell regulation of T and B cells together with neutrophils and antigen presenting cells, as it integrates signals between them extending to the entire immune microenvironment. The aim of my study was to investigate the role of a component of the mTOR complex 1, Raptor, in myeloid cells. My findings show that the absence of Raptor knock out (KO) does not affect bone marrow derived macrophage (BMDM) differentiation and maturation. However, the absence of Raptor influences BMDM polarisation towards an inflammatory phenotype, at least at the level of transcription as observed by increases in mRNA expression of inflammatory cytokines such as TNFα, IL-12β, and IL-6. This finding was consolidated by an increase in NFκΒ pathway signalling in Raptor KO BMDMs. Downstream intracellular signalling in myeloid cells was affected by deletion of Raptor as I found reduced S6K phosphorylation in Raptor KO BMDMs compared to wild type (WT) BMDMs. As a consequence of Raptor absence in BMDMs, STAT3 phosphorylation was also reduced. Raptor deletion did not impact the PI3K/Akt signalling pathway, but decreased phosphorylation of ERK. BMDMs lacking Raptor had reduced phagocytic activity as they were also observed to migrate less towards a pancreatic cancer cell line. However preliminary experiments in pancreatic cancer models did not indicate a major role for Raptor in the activity of tumour associated myeloid cells. My results demonstrate that Raptor and by implication mTORC1, is involved in macrophage polarisation and function.

This research was conducted to test the hypothesis that select nutrients including glucose, leucine, arginine and glutamine stimulate conceptus development by activating mTOR (mammalian target of rapamycin; HGNC approved gene name: FRAP1, FK506 binding protein 12-rapamycin associated protein 1) signaling pathway. First, temporal changes in quantities of select nutrients (glucose, amino acids, glutathione, calcium, sodium and potassium) in uterine lumenal fluid from cyclic (Days 3 to 16) and pregnant (Days 10 to 16) ewes were determined. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium and sodium was greater in uterine fluid of pregnant compared to cyclic ewes between Days 10 and 16 after onset of estrus. Of note were remarkable increases in glucose, Arg, Leu and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Second, effects of the estrous cycle, pregnancy, progesterone (P4) and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3 and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters, cationic amino acid transporters (SLC7A1, SLC7A2 and SLC7A3), neutral amino acid transporters (SLC1A4, SLC1A5, SLC3A1, SLC6A14, SLC6A19, SLC7A5, SLC7A6, SLC7A8, SLC38A3, SLC38A6 and SLC43A2) and acidic amino acid transporters (SLC1A1, SLC1A2 and SLC1A3) in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. Among these genes, SLC2A3 and SLC7A6 were detectable only in trophectoderm and endoderm of conceptuses. The abundance of mRNAs for SLC2A1, SLC2A4, SLC5A1, SLC5A11, SLC7A1, SLC7A2, SLC1A4, SLC1A5, SLC43A2 and SLC1A3 changed dynamically in ovine uterine endometria according to day of the estrous cycle and early pregnancy. Expression of mRNAs for SLC2A1, SLC5A11 and SLC7A1 in endometria was induced by P4 and further stimulated by IFNT with shortterm treatment (12 days), while expression of SLC7A1 and SLC1A5 in endometria required long-term treatment (20 days) with P4 and IFNT. Third, effects of the estrous cycle, pregnancy, P4 and IFNT on expression of nitric oxide synthase (NOS1, NOS2 and NOS3), GTP cyclohydrolase (GCH1), ornithine decarboxylase 1(ODC1), insulin-like growth factor II (IGF2), FRAP1 complexes (FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR), regulators (TSC1, TSC2, RHEB) and an effector (EIF4EBP1) of FRAP1 signaling in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. All of these genes were expressed in ovine uterine endometrium and conceptuses. Among these genes, expression of NOS1, IGF2, RHEB and EIF4EBP1 changed dynamically due to day of the estrous cycle and early pregnancy. Progesterone stimulated NOS1 and GCH1 expression while IFNT inhibited NOS1 expression in uterine endometria, and P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Collectively, these results indicate that: 1) the availability of select nutrients in the ovine uterine lumen increases to support the rapid growth and elongation of the conceptus during the peri-implantation stage of pregnancy; 2) P4 and/or IFNT stimulate(s) glucose and amino acid transporters to facilitate their transport from maternal tissues and/or blood into the uterine lumen during early pregnancy; 3) the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptus and both P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1. Expression of NOS, ODC1 and IGF2 appear to be linked to FRAP1 signaling in both uteri and peri-implantation conceptuses.

No description available.

A targeted approach to the development of antifolate therapies has been sought for many years. Central to the success of such development is an understanding of the molecular mechanisms dictating the sensitivity of cells to antifolates and the fundamental differences of these processes between normal and neoplastic phenotypes. This dissertation addressed transcriptional mechanisms and cell-signaling events responsible for the efficacy of antifolate therapies. Transcriptional processes and cell signaling pathways are often aberrant in neoplastic tissues, providing a potential point of distinction between a normal and neoplastic cellular state. Folylpolyglutamate synthetase (FPGS) catalyzes the formation of poly-γ-glutamate derivatives of folates and antifolates, which permits intracellular retention and accumulation of these compounds. The mouse fpgs gene uses two distant promoters to produce functionally distinct isozymes in a tissue-specific pattern. We questioned how the two promoters were differentially controlled. An analysis of DNA methylation and histone post-translational modifications across the length of the mouse fpgs gene showed that epigenetic mechanisms contributed to the tissue-specific control of the upstream (P1), but not the downstream (P2) fpgs promoter. RNAPII complexes and general transcription factors were present over P1 only when P1 was transcribed, but these components were present over P2 in most tissues, and promoter-proximal pausing was evident in brain. Clear promoter occlusion was found over P2 in liver. These studies concluded that tissue-specific coordination of dual promoters required multiple interacting controls. The mammalian target of rapamycin (mTOR) controls protein translation initiation, and is central to a cell-signaling pathway rich in tumor suppressor and oncogenic proteins. mTOR dysregulation is a common feature of several human cancers and inhibition of this protein has been sought as an ideal cancer drug target. We have determined that antifolates inhibiting the two folate-dependent steps of purine synthesis (GART or AICART) activate AMP-dependent protein kinase (AMPK) and inhibit mTOR. The mechanism of AMPK stimulation appears to be mediated by either nucleotide depletion (GART inhibitors), or ZMP accumulation (AICART inhibitors). These studies discovered a new mechanism for antifolates that surprisingly defines them as molecularly targeted therapeutics.

Exercise has been considered a cornerstone of diabetes prevention and treatment for decades, but the benefits of resistance training are less clear. Nineteen non-diabetic subjects (10 metabolic syndrome, 9 sedentary controls) underwent 8 weeks of supervised resistance training. After training, strength and V̇ O2max increased by 10% in both groups. Percent body fat decreased in subjects with the metabolic syndrome. Additionally, lean body mass increased in both groups (p<0.05). Expression of glucose transporter protein-4 (GLUT4), the principle insulin-responsive glucose transporter, increased significantly in both groups. 5-adenosine monophosphateactivated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) expression increased in both groups, indicating increased protein synthesis and mitochondrial biogenesis. Markers of insulin resistance measured by a euglycemic hyperinsulinemic clamp did not improve in subjects with the metabolic syndrome but increased significantly in control subjects (13%). Resistance training upregulates intracellular signaling pathways that may be beneficial for ameliorating the metabolic syndrome.

This thesis will be organized into two chapters discussing the role of hypoxia in the human placenta. The goal of this thesis is to characterize pyruvate kinase M2, mammalian target of rapamycin, mitochondrial function, and cell invasion in hypoxic conditions in the trophoblast. Understanding the mechanisms of placental metabolism can lead to further treatments for placental diseases. Chapter one covers the background of intrauterine growth restriction, hypoxia, placental metabolism, and pyruvate kinase M2 (PKM2). Little is currently understood about the role of the mitochondria in placental diseases. Expression of PKM2, trophoblast cell invasion, and mitochondrial function is shown to be inhibited by hypoxia. PKM2 inhibition decreases trophoblast cell invasion and nuclear expression of PKM2, but increases mitochondrial function. Studying how hypoxia affects the placenta during placental diseases can help clarify the mechanisms by which these diseases occur. Chapter two further characterizes the background of intrauterine growth restriction and hypoxia. It also covers the background of mammalian target of rapamycin. The objective of this chapter was to assess activated mTOR in the trophoblast in hypoxia. Decreased placental and fetal weights, as well as trophoblast cell invasion were observed in hypoxia. A decrease in the activation of mTOR was also found in the hypoxic placenta. This study could provide insight into the physiological relevance of the pathways and could be targeted to help alleviate placental diseases.

Orientador: Luis Gustavo Modelli de Andrade / Resumo: Introdução: Os inibidores do alvo de rapamicina em mamíferos (mTORI) podem conferir vantagens cardioprotetoras. Em modelos animais, o mTORI pode prevenir a aterogênese pela regulação da homeostase do colesterol e pela redução da resposta inflamatória. Além disso, a administração de mTORI pode levar à redução da massa ventricular esquerda. O objetivo deste estudo é comparar a espessura médio intimal da carotída (cIMT) e a massa ventricular esquerda indexada (LVMi) entre o grupo de tacrolimus associado a micofenolato (grupo micofenolato) e tacrolimo associado a sirolimo (grupo sirolimo) em baixas doses. A cIMT é considerada um marcador substituto da aterosclerose. Métodos: Nós avaliamos a cIMT e a LVMi no início e aos 6 e 12 meses após o transplante renal. Foram randomizados prospectivamente todos os receptores de transplante renal com mais de 60 anos para um dos dois grupos: tacrolimus / sirolimus (n = 21) ou tacrolimus / micofenolato (n = 23). A cIMT foi avaliada por ultrassonografia na parede da artéria carótida comum e o LVMi pelo ecocardiograma. Resultados: Os níveis de colesterol total e de lipoproteína de alta densidade (HDL) foram maiores no grupo do sirolimus aos 6 e 12 meses. O cIMT diminuiu com o tempo aos 6 e 12 meses no grupo do sirolimus (p = 0,012); esta diminuição continuou a ser significativa em um modelo ajustado para idade, sexo, presença de diabetes, uso de estatina e tabagismo. Houve redução ao longo do tempo na massa ventricular esquerda indexada, mas não ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Background: Mammalian target of rapamycin inhibitors (mTORI) may confer cardioprotective advantages. In animal models, the mTORI may prevent atherogenesis by the regulation of homeostasis of cholesterol and by a reduced inflammatory response. In addition, the administration of mTORI may lead to reduction of left ventricular mass. The aim of this study is to compare the carotid intima-media thickness (cIMT) and left ventricular mass index (LVMi) between de novo tacrolimus/mycophenolate and tacrolimus/sirolimus at low doses. The cIMT is considered a surrogate marker of atherosclerosis. Methods: We evaluated cIMT and LVMi at baseline and at 6 and 12 months after kidney transplantation. We prospectively randomly assigned kidney transplant recipients older than 60 years of age to one of two groups: tacrolimus/sirolimus (n=21) or tacrolimus/mycophenolate (n=23). The cIMT was evaluated by using ultrasound in the common carotid artery wall in both sides ant the LVMi by echocardiogram. Results: The total and high-density lipoprotein cholesterol levels were higher in the sirolimus group at 6 and 12 months. The cIMT decreased over time at 6 and 12 months in the sirolimus group (p = 0.012); this decrease continued to be significant in a model adjusted for age, sex, presence of diabetes, statin use and smoking. There was a reduction over time in LVMi, but there were no differences between groups suggesting absence of sirolimus class effect. Conclusions: The use of sirolimus plus tacrolimu... (Complete abstract click electronic access below) / Mestre

La voie de signalisation mTORC1 hypothalamique (mammalian target of rapamycincomplexe 1) intègre les signaux hormonaux et nutritionnels. La disponibilité des nutrimentsmodule les espèces réactives derivées de l’oxygène (ROS) qui régulent l’activité des neuronesà propiomélanocortine (POMC). La modulation de la prise alimentaire induite par les ROSpourrait impliquer mTORC1.Des souris C57Bl6J et wild-type (WT) ou invalidées pour S6K1 (S6K1-KO), principaleprotéine cible de mTORC1, ou invalidées pour raptor, protéine clé de mTORC1,sélectivement au niveau des neurones anorexigènes POMC (POMC-raptor-KO) ont ététraitées par injections intracérébroventriculaires (ICV) d’H2O2 ou d’honokiol (piégeur deROS), uniques ou combinées avec un inhibiteur de mTOR (rapamycine) ou un activateur demTOR (leptine).L’H2O2 ICV induit une augmentation de l’activité hypothalamique mTORC1, de l’activationneuronale du noyau arqué, de l’expression des ROS dans les neurones POMC, associée à unediminution de la prise alimentaire et du poids. Cet effet anorexigène est diminué chez lessouris S6K1-KO, chez les C57Bl6J après administration de rapamycine, et chez les POMCraptor-KO.L’honokiol ICV bloque l’effet anorexigène de la leptine, suggérant que cet effet soitdépendant des ROS. La leptine ICV entraine une augmentation des ROS dans les neuronesPOMC des souris C57Bl6J et POMC-raptor-WT, mais pas chez les POMC-raptor-KO.Nos résultats montrent que la régulation de la prise alimentaire induite par les ROS nécessiteune voie mTORC1 fonctionnelle et que l’effet anorexigène de la leptine nécessite uneaugmentation de ROS, mTORC1 dépendante, au niveau des neurones POMC. / The mechanistic target of rapamycin complex 1 (mTORC1) pathway is an importanthypothalamic integrator of nutrients and hormones. Nutrient availability also affects thereactive oxygen species (ROS) in propiomelanocortin (POMC) neurons and regulatesneuronal activity. We hypothesize that modulation of mTORC1 activity mediates ROS effectson food intake.To this purpose, C57Bl6J mice or WT mice and their KO littermates either deficient for themTORC1 downstream target S6K1 or for the mTORC1 component raptor specifically inPOMC neurons (POMC-raptor-KO) were treated with an intracerebroventricular (ICV)injection of the ROS producer H2O2 or the ROS scavenger honokiol, alone or in combinationwith the mTOR inhibitor rapamycin or the mTOR activator leptin.ICV H2O2 induced phosphorylation of S6K1 within the hypothalamus, increased expressionof c-fos, a marker of neuronal activity, in the arcuate nucleus and increased ROS in POMCneurons. These effects were associated with a significant decrease in food intake. Theanorexigenic effect of ICV H2O2 was not seen in S6K1-KO mice, in C57Bl6J mice cotreatedwith rapamycin (an mTOR inhibitor) and in POMC-raptor-KO mice.Similarly, ICV honokiol administration combined with a leptin injection blunted theanorexigenic effect of leptin, suggesting that leptin requires ROS formation to reduce FI. ICVadministration of leptin increased ROS in POMC neurons in C57Bl6J and POMC-raptor-WTmice, but not in POMC-raptor-KO mice.Our results demonstrate that ROS modulators require a functional mTORC1 pathway toregulate food intake and that leptin needs an mTORC1-dependent increase in ROS levels inPOMC neurons to decrease food intake.

Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer. / Revisión por pares