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Olsen, Jessica M. "β-Adrenergic Signalling Through mTOR." Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-142169.
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Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.
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Schalm, Stefanie. "Molecular mechanism of mTOR downstream signaling." [S.l. : s.n.], 2003. http://www.diss.fu-berlin.de/2003/249/index.html.
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Joyce, Claire Lois. "Tumour cell responses to mTOR inhibition." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610245.
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März, Andreas. "A new player in mTOR regulation." Diss., Ludwig-Maximilians-Universität München, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-139523.
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Lee, John Hung. "Altered mTOR signaling in Huntington's Disease." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/5547.
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Huntington's Disease (HD) is caused by a polyglutamine tract expansion in huntingtin (HTT). Despite HTTs ubiquitous expression, there is selective vulnerability in a specific brain region known as the striatum, the cause of which is poorly understood. Here, we provide evidence that impaired striatal mTORC1 activity underlies varied metabolic and degenerative phenotypes in striatal tissues from HD mouse models and patients, and show that further mTORC1 impairment in mouse models, achieved through the knockdown of Rhes, a striatum-enriched mTORC1 activator, exacerbates disease phenotypes. In contrast, exogenous addition of Rhes or the constitutively active form of the mTORC1 regulator, Rheb, into HD mouse brain, alleviates mitochondrial dysfunction, aberrant cholesterol homeostasis, striatal atrophy, and elicits increased autophagy, and reverses impaired dopamine signaling. Furthermore, while HD has been considered primarily a neurological disease, organs with high metabolic demand, such as heart, are also severely affected. The mechanism by which mHTT disrupts cardiac function remains unknown. I provide evidence that mTORC1 is impaired in HD mouse model hearts, causing hyperactive FoxO1 signaling which may render HD hearts vulnerable to stress induced cardiomyopathy. In sum, my combined work indicates impaired mTORC1 signaling as a primary mechanism underlying the neurodegenerative and heart-related disease phenotypes in HD, and thus presents a rational therapeutic target.
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Ramsbottom, Ben Alan. "Regulation of pol III transcription by mTOR." Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438962.
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Pimentel, Gustavo Duarte 1983. "Caracterização da AMPK/mTOR hipotalâmica na anorexia induzida pelo câncer : Characterization of hypothalamic AMPK/mTOR in cancer-induced anorexia." [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312747.
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Orientador: Jose Barreto Campello CarvalheiraTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-26T11:52:05Z (GMT). No. of bitstreams: 1 Pimentel_GustavoDuarte_D.pdf: 5245849 bytes, checksum: ec5c43602ec0d60564455aae4b1fee3a (MD5) Previous issue date: 2015
Resumo: A teoria das doenças geradas por citocinas inflamatórias trouxe ao longo dos anos indícios que o organismo pode produzir citocinas que desempenham respostas biológicas benéficas ou prejudiciais. Com o passar dos anos ficou claro que a inflamação é um mecanismo chave na fisiopatologia do câncer. Interessantemente, diversos estudos sugerem que a AMPK e mTOR hipotalâmica, importantes moléculas no controle do balanço energética também seja responsável por modular a inflamação e anorexia. Nesse sentido, foi observado: 1) A inibição da AMPK hipotalâmica proporciona redução do peso corporal e da inflamação central e periférica potencializando o crescimento tumoral. Por outro lado, a ativação da AMPK com AICAR, salicilato e vetor viral reverte à anorexia induzida pelo câncer. Entretanto, os efeitos benéficos do AICAR foram bloqueados quando associados com os antagonistas colinérgicos, sugerindo que a AMPK no núcleo ventromedial é responsável pelo controle da anorexia e inflamação. 2) A AMPK no núcleo ventromedial do hipotálamo, principalmente a isoforma alfa 1 ativa a termogênese aumentando a produção de calor na qual converte tecido adiposo branco em bege. Além disso, o uso do antagonista ?3 adrenérgico ou a ativação da AMPK foram capazes de atenuar a produção de calor melhorando a caquexia induzida pelo câncer. 3) Roedores com câncer possuem a via do IKK/mTOR ativada no núcleo arqueado do hipotálamo proporcionando anorexia e caquexia. Por outro lado, o bloqueio da S6K com adenovírus foi capaz de melhorar a anorexia. Portanto, esses achados permitem concluir que o hipotálamo funciona como um centro regulador da anorexia e caquexia induzida pelo câncer, abrindo novos horizontes para o tratamento do câncer
Abstract: The theory of diseases generated by inflammatory cytokine brought over the years evidence that the organism may produce cytokine with beneficial and deleterious responses. Nowadays, it is clear that the inflammation is a key mechanism in cancer pathophysiology. Interestingly, several studies suggest that hypothalamic AMPK and mTOR, important molecules in the energy balance control also is responsible for modulation of both inflammation and anorexia. The studies presented herein observed that: 1) Inhibition of hypothalamic AMPK leads to weight loss and central and systemic inflammation which potentiates the tumor growth. However, AMPK activation with AICAR, salicylate and vector viral might reverse the cancer-mediated anorexia. Nevertheless, benefic effects of AICAR are blunted with a combination of cholinergic antagonists, suggesting that ventromedial of hypothalamus (VMH)-specific AMPK action is responsible for the anorexia and inflammation control. 2) VMH-specific AMPK, particularly the isoform alpha 1 activates thermogenesis increasing heat production which switches the white adipose tissue in beige. Furthermore, ?3 adrenergic antagonist and AMPK activation were able to attenuate the heal generation, block the "browning of WAT" and improve the cancer cachexia. 3) Cancer rats have activated IKK/mTOR pathway in arcuate nucleus (ARC) of the hypothalamus. In contrast, neutralization of S6K through adenovirus was able to improve anorexia. Therefore, our data show evidences that the hypothalamus a key center that integrates a number of mechanisms triggered by cancer-induced anorexia and -cachexia, opening new horizons for the treatment of cancer
Doutorado
Fisiopatologia Médica
Doutor em Ciências
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Wong, Jacky Sui Ki. "The Evaluation of Dual PI3K/mTOR Inhibitors as a Superior Alternative to mTOR Inhibitors in Pre-B Acute Lymphoblastic Leukaemia." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/13644.
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Acute lymphoblastic leukaemia (ALL) is the most common form of cancer in children. Poor long term survival in adults as well as the bleak outlook for relapsed patients highlights the need for new therapeutic strategies for the treatment of ALL. The major regulators of ALL cell proliferation and survival mediate their effects through the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin. It has been previously shown that the mTOR inhibitor RAD001 extended survival in a non-obese diabetic/severe combined immune deficient (NOD/SCID) mouse xenograft model of ALL. The work presented in this thesis examines the effect of the dual PI3K/mTOR inhibitors BEZ235 and BGT226 in ALL. In summary, dual PI3K/mTOR inhibitors demonstrate primarily superior cytostatic effects in vitro but a mixed level of cytotoxicity when compared to RAD001. In addition, the dual PI3K/mTOR inhibitors extended survival in NOD/SCID mice engrafted with ALL xenografts but failed to demonstrate overall superiority over mTOR inhibition alone. This work contributed to the publication of a paper, presented in two separate chapters. Subsequent unpublished work presented in this thesis examined the effects of the dual PI3K/mTOR inhibitors in combination with conventional chemotherapeutic agents. However, the results presented in this thesis indicate that the dual PI3K/mTOR inhibitors do not cooperate well with the tested agents. Furthermore, the cooperation observed with the dual PI3K/mTOR inhibitors in combination with a MEK inhibitor highlights the need to explore strategies to target multiple signalling pathways.
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Gulati, Ruhi. "Developing Viral Strategies to Study mTOR and its Regulators as Mediators of Epileptogenesis." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1563273811946353.
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Payne, Sara Lauren. "Small-molecule inhibitors of mTOR and DNA-PK." Thesis, University of Newcastle Upon Tyne, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627731.
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The phosphatidylinositol-3-kinase related kinase (PIKK) family of proteins consists of five serine-threonine protein kinase members (ATM, ATR, hSMG, DNA-PK and mTOR), each of which have been implicated in the cellular response to DNA damage or cellular stress. Upregulation of the PBKJAKT cell signalling pathway has been demonstrated to be a common driver of malignancy in human cancer. The mammalian target of rapamycin (mTOR) exists in two isoforms, both of which lie within the PBKJAKT pathway and as such are capable of mediating the activity of the signalling pathway. The first reported inhibitor of mTOR was rapamycin, a macrocylic lactone which acts an allosteric inhibitor of the mTORCI complex only. Subsequent drug discovery efforts have been focussed upon the development of ATP-competitive inhibitors of mTOR, which would facilitate the inhibition of both mTOR complexes thereby interrupting the P13K/AKT pathway at two distinct points.
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Peter, Christian. "mTOR signalling and the regulation of FOXP3 expression." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.600239.
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Regulatory T-cells (Treg) are essential for the establishment of self-tolerance and they can be identified by the expression of FOXP3, the "master" transcription factor for the development of Treg. A number of essential Signals, namely TCR engagement, TGF~, as well as the inhibition of the kinase mTOR, have been shown to be crucial for the induction of FOXP3 in the peripheral immune system. Despite recent progress, the molecular mechanisms for induction of FOXP3 remains elusive due, at least in part, to the lack of a cell line that mimics the behavior of primary CD4+ T-cells. Here we report the isolation of a clone B02 from the mouse lymphoma line EL4 that can be induced to express high levels of FOXP3 in up to 50% of the cells. Pharmacological inhibition of mTOR and EAA depletion synergized with TGF~ in the induction, and led to reduced activity of both mTORCl and mTORC2, which seemed to independently contribute to the enhanced FOXP3 expression. Luciferase reporter assays using Foxp3 promoter and enhancer sequences indicated that the mTOR inhibition-mediated increase in FOXP3 expression resulted primarily from enhanced transcriptional activity. In addition to the enhancement of FOXP3 induction, mTOR inhibition reduced the levels of lL-17A, presumably by SOCS-mediated inactivation of STAT3, which could also have accounted for increased Foxp3 transcription. Although the precise targets of mTOR inhibition involved in the regulation of FOXP3 expression have yet to be identified, both luciferase assays and mass spectrometry-based quantitative phospho-proteomics using SILAC labelled B02 cells, have identified promising candidates. This unique EL4 clone could represent a special resource for investigating the signaling events leading to the induction of FOXP3 as well as events downstream of FOXP3 expression.
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Graham, Emma Louise. "The regulation of Pol III transcription by mTOR." Thesis, University of Glasgow, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414344.
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Morris, Katherine Louise. "Investigation of RNA binding proteins regulated by mTOR." Thesis, University of Leicester, 2017. http://hdl.handle.net/2381/39742.
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The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which plays a key role in the transduction of cellular energy signals, in order to coordinate and regulate a wide number of processes including cell growth and proliferation via control of protein synthesis and protein degradation. For a number of human diseases where mTOR signalling is dysregulated, including cancer, the clinical relevance of mTOR inhibitors is clear. However, understanding of the mechanisms by which mTOR controls gene expression is incomplete, with implications for adverse toxicological effects of mTOR inhibitors on clinical outcomes. mTOR has been shown to regulate 5’ TOP mRNA expression, though the exact mechanism remains unclear. It has been postulated that this may involve an intermediary factor such as an RNA binding protein, which acts downstream of mTOR signalling to bind and regulate translation or stability of specific messages. This thesis aimed to address this question through the use of whole cell RNA binding protein capture using oligo‐d(T) affinity isolation and subsequent proteomic analysis, and identify RNA binding proteins with differential binding activity following mTOR inhibition. Following validation of 4 identified mTOR‐dependent RNA binding proteins, characterisation of their specific functions with respect to growth and survival was conducted through depletion studies, identifying a promising candidate for further work; LARP1. Having selected LARP1 from depletion screens, overexpression co‐IP experiments conducted alongside known binding partner PABP and subsequent arrays allowed for preliminary identification of mRNAs to which LARP1 binds. Finally, we showed evidence for differential binding of mRNA subsets between LARP1 and PABP, opening a new caveat for the role of the effector protein LARP1 in mTOR dependent gene expression regulation.
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Alharbi, Z. M. S. "Identification and characterization of novel mTOR splicing isoforms." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1459435/.
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mTOR (mammalian target of rapamycin) is a serine/threonine protein kinase which belongs to the family of phosphoinositide 3-kinase related kinases (PIKK), which also includes ATR, ATM, DNA-PK, SMG1 and TRRAP. mTOR contains several conserved protein-protein interaction modules at the N-terminus and a protein kinase domain at the C-terminus. The regulatory interactions of mTOR are mainly mediated by HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, and TOR1) repeats and FAT (FRAP, ATM and TRRAP) domains. In contrast to other PIKK family members, mTOR possesses a unique FRB (FKBP12/Rapamycin Binding) domain which mediates the interaction with the FKBP12/rapamycin inhibitory complex. mTOR is a key component of two distinct multi-protein complexes in mammalian cells, termed mTOR complex 1 (TORC1) and mTOR complex 2 (mTORC2). A diverse range of extracellular and intracellular signals stimulate both mTOR complexes to regulate cell growth, survival and proliferation. Dysregulation of mTOR signalling has been implicated in various human pathologies, including cancer, inflammation, neurodegenerative and metabolic disorders. Therefore, mTOR is an attractive target for drug discovery. In cancer research, mTOR inhibitors have shown a potent anti-proliferative activity in pre-clinical studies and several of these are currently being evaluated in clinical trials. There is only one mTOR gene in higher vertebrates, which is known to encode two splicing isoforms: mTORα and mTORβ. In contrast to the full length mTORα protein, mTORβ lacks most of its protein-protein interaction modules, HEAT and FAT, but retains domains responsible for FKBP12/rapamycin binding, protein kinase activity and regulation. Importantly, mTORβ was shown to shorten considerably the G1 phase of the cell cycle, to stimulate cell proliferation and to possess oncogenic potential in cell-based and xenograft studies. Like other PIKK family members, there is increasing evidence of the presence of mTOR splicing isoforms. The main aim of this study was to identify new mTOR splicing isoforms and to determine their molecular characterization. In addition we aim to explore the ability of these isoforms to phosphorylate known mTOR targets such as 4E-BP1. Furthermore, we aim to study the role of the new isoforms in the mTOR–mediated cellular processes, such as cell proliferation, and the contribution of the identified isoforms in the oncogenic characteristics of cells. In this study, we have employed bioinformatics, biochemical, cell and molecular techniques to identify and characterize two novel mTOR splicing isoforms, denoted mTORδ and mTORγ. When compared to mTORα, the mTORδ splice variant contains only the N-terminal HEAT repeats and a unique C-terminal region, while the mTORγ isoform possesses a 12 amino acid deletion in the kinase domain. The existence of the mTORδ isoform was confirmed at mRNA and protein levels by identifying corresponding EST clones and detecting the splice variant with specific anti–mTORδ antibodies. Furthermore, we have found several EST clones corresponding to the mTORγ splicing variant. In contrast to mTORα and the mTOR activated mutant S2215Y, the stably expressed mTORδ and mTORγ lack the kinase activity in vitro. It was also found that stable overexpression of mTORδ and mTORγ splice variants in HEK293T cells inhibits cell proliferation and colony formation in soft agar. These findings suggest that identified novel isoforms have the potential to regulate the mTOR signalling pathway in a dominant–negative manner.
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Earwaker, Philip L. "Resistance mechanisms to mTOR inhibition in renal cancer." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:dc500011-d486-43cc-a0a7-87b9d6d9e682.
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Introduction: Advanced clear cell renal cell cancer (CCRCC) is incurable, but molecularly targeted treatments have improved the prognosis. One such molecular target is the mammalian target of rapamycin (mTOR) and two rapalogues (everolimus and temsirolimus) have been licensed for the treatment of advanced CCRCC, but resistance to treatment is a significant problem. Novel mTOR kinase inhibitors show more complete blockade of mTOR downstream targets, and greater in vitro anti proliferative activity, although resistance remains a problem. Hypotheses: 1) CCRCC cell lines, resistant to mTOR kinase inhibition could be generated in vitro through continuous culture in drug, and 2) mediators of resistance could be identified through screening cells at the mRNA and protein/phosphoprotein level, or by examining the signalling in resistant cells to identify the re-activation of key mTOR targets. Methods: Resistant cells were created by continuous culture in the PI3K-mTOR kinase inhibitor BEZ235. Resistance was assessed by CellTiter-Glo viability assays and clonogenic assays. mRNA microarray and antibody arrays were conducted. Intra-cellular signalling was assessed by western blotting. The functional relevance of identified markers of resistance were assessed with small molecule inhibitors and siRNA protein depletion. Results: BEZ235 resistant cells were created and had a 14 fold higher growth inhibitory 50 (GI50) concentration compared to sensitive cells (99 nM vs. 7 nM). Array screening of the cells identified markers, but not mediators of resistance (MET, Abl, MEK/ERK and the Notch pathway). In BEZ235 resistant cells, the downstream mTOR target 4E-BP1 was rephosphorylated, despite evidence of on-going blockade of another mTOR target, S6. Recovery of 4E-BP1 phosphorylation was associated with increased protein expression of the mTOR binding partner RAPTOR, and could be reduced by depletion of RAPTOR, or by the addition of rapamycin. Both of these interventions partially reversed BEZ235 resistance. Conclusions: RAPTOR protein up-regulation, represents a novel mechanism of resistance to mTOR kinase inhibition and can be partially overcome by rapamycin. The combination of BEZ235 and rapamycin warrants further investigation to evaluate its potential to overcome resistance to mTOR kinase inhibition in RCC.
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Hare, Stephen. "The development and characterisation of everolimus resistant breast cancer cells." Thesis, Brunel University, 2018. http://bura.brunel.ac.uk/handle/2438/17466.
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The mTOR inhibitor and rapalogue everolimus was approved use in 2012, in HR+, HER-2-, post-menopausal patients, who had previously failed aromatase inhibitor treatment. mTOR pathway activation has been associated with resistance to breast cancer therapies, but how cells may become resistant to mTOR therapies themselves in breast cancer is currently not well explored, due to the relative recentness of everolimus approval. Drug resistance across all areas of cancer research is a major clinical issue, often leading to the spread of a patient's cancer. This project set out to create in vitro breast cancer models that were resistant to everolimus, and thus explore any changes that had developed in these models, help determine the mechanisms behind resistance and discover drugs/drug combinations that could overcome resistance. Cell lines T47D and MDA-MB-361 were subsequently developed into everolimus resistant lines (EveR) over the course of 4-6 months using an on/off exposure routine. The exact mechanism behind the everolimus resistance was not fully determined but EveR cells did show multiple intriguing characteristics. An increase in dormancy and stem-cell like phenotype was noted, as revealed by a decrease in cell cycle progression and an increase in increase ALDH activity. mTORC2 components and signalling was up-regulated although siRNA down-regulation of PKCα did not decrease everolimus resistance, suggesting other mTORC2 targets may be involved. The rapalogue 'receptor', FKBP12, was up-regulated which was accompanied by an increased growth inhibition by the rapalogue, temsirolimus, possible due to temsirolimus lower binding affinity for FKBP12 compared to everolimus. No resistance to the dual mTOR/PI3K inhibitor BEZ-235 was observed, in line with similar published work. The combination of vitamin D/calcitriol and everolimus had no added effect compared to everolimus alone, in parental cells, but the addition of 1μM calcitriol did drastically lower EveR cell resistance to everolimus. Future work focusing on the exact nature of calcitriol's interaction with the mTOR pathway is required to advance calcitriols role as a breast cancer therapeutic. Research with everolimus resistant breast cancer patients has not yet been published on, but the work presented here aims to help guide such studies, when they are carried out in the future.
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Suárez, Rodríguez Cristina. "Valor pronóstico de las alteraciones genéticas de la vía mTOR en pacientes con cáncer renal metastásico tratados con inhibidores de mTOR." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/664121.
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El cáncer renal supone el 2% de todos los nuevos casos de cáncer en el adulto y su incidencia va en aumento en los últimos años.
El mejor conocimiento de la biología molecular del cáncer renal ha llevado al descubrimiento y utilización de dos grandes grupos de fármacos: los inhibidores de la vía de la angiogénesis y los inhibidores de la vía mTOR, a los que recientemente se ha unido la inmunoterapia.
Existen dos inhibidores de mTOR (imTOR) aprobados en el tratamiento de cáncer renal: temsirolimus y everolimus.
Diversos estudios han demostrado que las alteraciones genéticas en la vía mTOR confieren un peor pronóstico a estos pacientes, con mayor riesgo de recaída tras la nefrectomía y mayor riesgo de progresión y de muerte en pacientes metastásicos.
En esta tesis se han analizado las mutaciones en genes de la vía mTOR en muestras tumorales de 90 pacientes tratados con imTOR. Dentro de estas alteraciones se incluyen PI3CA (gen de la proteína PI3K), AKT, PTEN, mTOR, TSC1 y TSC2.
El objetivo principal de este estudio ha sido correlacionar la presencia de dichas mutaciones con el pronóstico de los pacientes en términos de supervivencia global desde el inicio de enfermedad metastásica (SGM), supervivencia global desde el inicio de del tratamiento con imTOR (SGMT) y supervivencia libre de progresión desde el inicio del tratamiento con imTOR (SLPT).
Entre los objetivos secundarios estaban estimar el porcentaje de pacientes con mutaciones de la vía mTOR en dicha serie y estimar la SLP y la SG ajustada de acuerdo con los subgrupos definidos por la IMDC (International Metastatic Renal Cell Carcinoma Database Consortium).
En la muestra analizada se identificaron mutaciones en esta vía en el 33% de los pacientes. El 17.8% de las muestras analizadas presentaban una mutación en mTOR, el 5.6% en PIK3CA, el 3.3% en PTEN, 5.6% en TSC1 y 4.4% en TSC2. No se objetivó ninguna mutación en AKT (AKT1, AKT2, AKT3).
No se observaron diferencias estadísticamente significativas en SGM, SGMT ni SLPT entre los pacientes con cáncer renal metastásico tratados con imTOR que presentaban mutaciones genéticas en los genes de la vía mTOR y aquellos que no.
La SG para los grupos de riesgo favorable, intermedio y mal pronóstico del IMDC fueron de 43.7, 22.2 y 8.7 meses respectivamente. La mediana de SLP durante el tratamiento con imTOR fue 5.4 meses.
Este es el primer trabajo que analiza el valor pronóstico de la presencia o no de mutaciones en la vía mTOR en pacientes tratados con inhibidores de mTOR. Los resultados de este estudio indican que en pacientes tratados con inhibidores de mTOR la presencia de mutaciones en esta vía no parece tener un efecto significativo sobre la supervivencia libre de progresión y la supervivencia global, como se demuestra con la estimación de HR ajustada de riesgo de recaída y muerte. Estos datos contradicen el estado actual del conocimiento que indica un peor pronóstico asociado a la presencia de estas mutaciones.
Dado que todos los pacientes de este estudio habían sido tratados con imTOR, esto podría ser un signo indirecto de la eficacia de estos fármacos en este tipo de pacientes.Renal cell carcinoma (RCC) accounts for 2% of all new cases of cancer in adults and its incidence is increasing in recent years. The best knowledge of the molecular biology of RCC has led to the discovery and use of two large groups of drugs: the inhibitors of the angiogenesis pathway and the inhibitors of the mTOR pathway, to which immunotherapy has recently been added. There are two approved mTOR inhibitors (imTOR) in the treatment of renal cancer: temsirolimus and everolimus. Several studies have shown that genetic alterations in the mTOR pathway confer a worse prognosis in these patients, with a higher risk of relapse after nephrectomy and higher risk of progression and death in metastatic patients. In this thesis mutations in genes of the mTOR pathway in tumor samples of 90 patients treated with imTOR have been analyzed. These alterations include PI3CA (PI3K protein gene), AKT, PTEN, mTOR, TSC1 and TSC2. The primary endpoint of this study was to correlate the presence of these mutations with the prognosis of patients in terms of overall survival from the diagnosis of metastatic disease (OS), overall survival from the start of imTOR treatment (OST) and progression free survival from the beginning of the treatment with imTOR (PFS). Secondary objectives were to estimate the percentage of patients with mTOR pathway mutations and estimate the PFS and the adjusted OS based on the IMDC (International Metastatic Renal Cell Carcinoma Database Consortium) subgroups. Mutations were identified in the mTOR pathway were found in 33% of patients. 17.8% of the analyzed samples had a mutation in mTOR, 5.6% in PIK3CA, 3.3% in PTEN, 5.6% in TSC1 and 4.4% in TSC2 respectively. No mutation was found in AKT (AKT1, AKT2, AKT3). No statistically significant differences were observed in OS, OST or PFS between patients with metastatic renal cancer treated with imTOR who had genetic mutations in the genes of the mTOR pathway and those who did not. The OS for the groups of favorable, intermediate and poor prognosis of the IMDC were 43.7, 22.2 and 8.7 months respectively. The median PFS during the treatment with imTOR was 5.4 months. This is the first work that analyzes the prognostic value of the presence or not of mutations in the mTOR pathway in patients treated with mTOR inhibitors. The results of this study indicate that in patients treated with mTOR inhibitors the presence of mutations in this pathway does not seem to have a significant effect on progression-free survival and overall survival, as demonstrated by the estimated risk adjusted HR relapse and death. These data contradict the current state of knowledge indicating a worse prognosis associated with the presence of these mutations. Since all the patients in this study had been treated with imTOR, this could be an indirect sign of the efficacy of these drugs in this type of patients.
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Schollenberger, Daniel [Verfasser]. "Immunhistochemische Analyse der Expressionsniveaus von mTOR und p-mTOR in Metastasen von Nierenzellkarzinomen unter der Berücksichtigung ihrer prognostischen Relevanz / Daniel Schollenberger." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1230796495/34.
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Silva, Hamilton Augusto Roschel da. "Efeito agudo de diferentes velocidades de exercício excêntrico na sinalização da hipertrofia muscular." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/39/39132/tde-22062012-103944/.
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Atualmente, alguns pesquisadores tem se dedicado ao estudo do efeito da manipulação do treinamento de força sobre a ativação das vias de sinalização intracelular para hipertrofia. Tem-se sugerido que o grau de tensão muscular desempenhe um papel importante nesta sinalização. Dentre os diferentes tipos de ações musculares, as ações excêntricas (AE) reconhecidamente proporcionam maior grau de tensão à estrutura do músculo esquelético. Em particular, AE de alta velocidade parecem exercer um efeito interessante sobre os ganhos de hipertrofia muscular. Porém, pouco se sabe sobre o efeito da manipulação da velocidade sobre as vias de sinalização da hipertrofia. Assim, o presente estudo teve como objetivo verificar o efeito agudo da AE de alta e baixa velocidade sobre a sinalização para hipertrofia muscular. Vinte sujeitos foram aleatoriamente divididos em dois grupos. Um realizou cinco séries de oito AE máximas à 20º/s (EXC20) e o outro à 210o/s (EXC210) do exercício extensão de joelhos. Amostras do músculo vasto lateral foram obtidas antes, imediatamente após e duas horas após o exercício. As análises de quantificação protéica de Akt e p70S6K totais não apresentaram diferenças significantes intra ou inter grupos. A avaliação da fosforilação das mesmas proteínas revelou um efeito principal de tempo, indicando um aumento da fosforilação nos tempos imediatamente após e duas horas após o término do exercício em relação à amostra controle (p<0,05), porém não foram observadas diferenças entre os grupos. Para os dados de expressão gênica de MGF e mTOR, não foram observadas diferenças intra ou inter grupos. Em conclusão, a manipulação aguda da velocidade das AE parece não influenciar a fosforilação ou expressão gênica das proteínas em questãoRecently, many studies have focused on the effects of strength training variables manipulation on the activation of intracellular signaling pathways for skeletal muscle hypertrophy. It has been suggested that the muscle tension plays a major role in such process. Eccentric muscle actions (EE) are notorious for imposing a greater amount of tension on the active muscle. In particular, EE performed at high velocities seems to exert an interesting effect on hypertrophy gains. However, little is known about the effect of EE velocity manipulation on hypertrophy pathways signaling. Thus, the present study aimed to investigate the acute effect of low and high velocity EE on muscle hypertrophy signaling. Twenty subjects were randomly assigned to either a slow velocity group 20o/s (ECC20) or fast group 210o/s (ECC210). Muscle biopsy samples were taken before, immediately after and two hours after the completion of five sets of eight maximal repetitions at the designated velocity, knee extension exercise. Akt and p70S6K analysis did not reveal any differences inter or intra groups. Akt and p70S6K phosphorylation results indicated a main effect for time (p<0,05), with increased phosphorylation values for immediately after and two hours after time points in comparison to control samples. MGF and mTOR mRNA analysis did not return any inter or intra groups differences. In conclusion, the acute manipulation of EE velocity does not seem to differently influence the phosphorylation or expression of the proteins studied
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Alqurashi, Naif. "The Therapeutic Potential of mTOR signalling Pathway as Drug Target in Human Colorectal Cancer." Thesis, Griffith University, 2015. http://hdl.handle.net/10072/367346.
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The mammalian target of rapamycin (mTOR), a downstream effector of PI3K/Akt signalling, is a critical regulator of cellular processes in response to oncogenic signals. In mammalian cells, mTOR exists in 2 large physically and functionally distinct signalling complexes, i.e.: mTORC1 and mTORC2. Each complex has its own set of downstream targets, which modulate cellular growth, proliferation, and survival in normal cells and cancer. Deregulation of mTOR signalling often occurs in a variety of human malignant diseases, making it a validated target in the development of treatments for cancer. In colorectal cancer, both in vitro and in vivo studies have demonstrated various roles for different genes, including those related to the mTOR pathway in the pathogenesis of cancer. Clinical trials have already started to examine the feasibility of mTOR inhibitors as therapeutic drugs for cancer. In this study, our objectives were firstly to investigate the expression levels of mTOR in human pathological samples of colorectal cancer and subsequently investigate the effect of mTOR inhibitors in human colorectal cancer cell lines. Finally, the study sought to identify potential miRNAs with putative targets in the mTOR pathway.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
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Houssaini, Amal. "La voie de signalisation Akt/mTOR : rôle physiopathologique etcible thérapeutique dans l’hypertension artérielle pulmonaire expérimentale." Thesis, Paris Est, 2012. http://www.theses.fr/2012PEST0069.
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Les travaux de la thèse portent sur l'implication de la voie de signalisation Akt (sérine/thréonine kinase Akt) et mTOR (mammalian target of rapamycin) dans la physiopathologie de l'hypertension artérielle pulmonaire (HTAP) expérimentale. L'HTAP résulte d'une prolifération exagérée des cellules constitutives des vaisseaux pulmonaires, principalement les cellules musculaires lisses artérielles pulmonaires (CML-AP). De nombreux effecteurs biologiques et physiques préalablement identifiés agissent sur les CML-AP et participent à l'hyperplasie de celles-ci. Nous montrons que ces nombreux effecteurs convergent vers une voie de signalisation intracellulaire commune, la voie Akt/mTOR, qui de fait représente une cible thérapeutique pour le traitement de l'HTAP, et pourrait conditionner l'hyperplasie des CML-AP. mTOR est présent dans la cellule sous forme de deux complexes, mTORC1 et mTORC2, qui phosphorylent des substrats variés contrôlant la prolifération cellulaire. Les effecteurs de mTORC1 incluent les S6 kinases (S6K1 and S6K2) et les "eIF4E-binding proteins" (4EBP) alors que mTORC2 active la sérine/thréonine kinase Akt et parmi les kinases sous-jacentes, la kinase GSK3. La première étude est consacrée à l'évaluation des effets des inhibiteurs de protéases du VIH (ritonavir, amprenavir, nelfinavir) sur la progression de HTAP expérimentale, induite par la monocrotaline ou l'hypoxie. Nous montrons que ces deux formes d'HTAP sont associées à une activation de la voie Akt/mTOR dans les artères pulmonaires. Les traitements respectifs par les trois inhibiteurs des protéases du VIH durant 3 semaines induisent une réversibilité de l'HTAP, de l'hypertrophie ventriculaire droite et du remodelage des vaisseaux pulmonaires, de même qu'une inhibition de la phosphorylation d'Akt, de S6K et de GSK3. La prolifération des CML-AP induite par le PDGF ou le SVF 5%, associée à une augmentation de p-Akt et p-GSK3, est également bloquée par les inhibiteurs des protéases, de façon similaire et non additive à celle d'inhibiteurs spécifiques de la PI3 kinase et de GSK3. La conclusion est que ces traitements antirétroviraux inihibent la progression de l'HTAP en inhibant la voir Akt/mTOR dans les CML-AP. Cette proposition permettrait d'expliquer l'effet suspecté en clinique des traitements antirétroviraux sur l'HTAP compliquant l'infection par le VIH.Dans la seconde étude, nous montrons que les CML-AP en culture prélevées à partir de rats ayant développé une HTAP induite par la monocrotaline proliférent de façon exagérée en comparaison avec les cellules de rats témoins. Ce phénotype prolifératif est observé en présence de nombreux facteurs mitogènes parmi lesquels le SVF 5%, le PDGF, la sérotonine ou 5-HT, l'IGF1 ou l'IL1-beta, et est associé à une activation des substrats de mTORC1 et mTORC2. Le traitement in vitro par la rapamycine des CML-AP de rats avec HTAP établie permet d'inhiber la prolifération de ces cellules et de bloquer à la fois mTORC1 et mTORC2. De même, le traitement par la rapamycine de rats porteurs de l'HTAP préétablie pendant une semaine permet de normaliser la prolifération des CML-AP in vitro et in vivo et d'inhiber mTORC1 et mTORC2, effets non observés par l'Imatinib ou la Fluoxetine. De plus, le traitement des rats par la rapamycine prévient ou corrige l'HTAP induite par la monocrotaline de façon plus importante que l'Imatinib ou la Fluoxétine.Ces résultats indiquent donc que l'activation de la voie Akt/mTOR, très étroitement associée au développement de l'HTAP expérimentale, pourrait expliquer le phénotype prolifératif anormal des CML-AP inhérent à la pathologie, et ainsi représenter une cible thérapeutique de choix pour le traitement de l'HTAP humaineThe major objectives of research described in this thesis is focused on the cell signaling pathway of Akt (serine/threonine kinase Akt) and mTOR (mammalian target of rapamycin) in the patho-physiology of experimental pulmonary arterial hypertension (PAH). PAH occurs as a result ofhyperplasia of the components of pulmonary vessels, principally the pulmonary arterial smooth muscle cells (PA-SMCs). Numerous previously identified biological and physical effectors act on the PA-SMCs and participate in PA-SMC hyperplasia. Here we show studied that these different effectors converge into a common intracellular signaling pathway, Akt/mTOR signaling pathway, which represents actually a therapeutic target for PAH treatment, and could be involved in the hyperplasia of PA-SMCs. In cells mTOR, is presented in the form of two complexes, mTORC1 and mTORC2, which phosphorylate various substrates controlling the cellular proliferation. The effectors of mTORC1 include the S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4EBP), meanwhile mTORC2 activates the serine/threonine kinase Akt and the underlying kinases, e.g. GSK3 kinase.The first study is devoted to evaluate the effects of the protease inhibitors of HIV (ritonavir, amprenavir, nelfinavir) on experimental PAH development induced by monocrotaline or hypoxia. We studied that the two forms of PAH are associated with an activation of Akt/mTOR signaling pathway in pulmonary arteries. The treatment by the three protease inhibitors of HIV during 3 weeks causes reversibility in experimental PAH with decreased right ventricular hypertrophy and pulmonary vascular remodeling as well as inhibition of phosphorylation of Akt, S6K and GSK3. The proliferation of PA-SMCs stimulated by PDGF or FCS 5%, which is associated with an increased p-Akt and p-GSK3, is also blocked by the proteases inhibitors, in a similar and non additive way like the specific inhibitors of PI3 kinase and GSK3. We conclude that the antiretroviral treatments significantly inhibits PAH development by inhibiting Akt/mTOR signaling pathway in PA-SMCs. This proposition allows explaining the effect of antiretroviral treatments of PAH accompanied with HIV in patients.In the second study, we studied that the cultured PA-SMCs extracted from the rats with monocrotaline induced-PAH(MCT-PAH) proliferates faster as compared to control. This proliferative phenotype is observed in the presence of different mitogenic factors including FCS 5%, PDGF, 5-HT, IGF1 or IL-1β, and is associated with an activation of the substrates of mTORC1 and mTORC2. Treatment with rapamycin in the PA-SMCs extracted from the rats with PAH in vitro inhibits the proliferation and also blocks the activation of mTORC1 and mTORC2. The treatment by rapamycin in the rats with PAH during one week allows normalizing the proliferation of PA-SMCs in vitro and inhibiting the activation of mTORC1 and mTORC2 in vivo. These effects were not observed when treated with imatinib or fluoxetine. Moreover, treatment with rapamycin prevents or reverse MCT induced PAH more significantly than that by imatinib or fluoxetine.These results indicate that the activation of Akt/mTOR signaling pathway isclosely related to experimental PAH development, which can explain the abnormal proliferative phenotype of PA-SMCs involved in the patho-physiology of PAH, and represent a therapeutic target for the treatment of PAH in human
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Boudra, Rafik. "Rôle de la Nucléophosmine (NPM1) dans la physiopathologie prostatique." Thesis, Clermont-Ferrand 2, 2015. http://www.theses.fr/2015CLF22598/document.
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La Nucléophosmine (NPM1/B23) est une petite chaperonne moléculaire impliquée dans de nombreux processus cellulaires, tels que la régulation de l’expression génique ou le contrôle du cycle cellulaire. De nombreuses études rapportent une surexpression de NPM1 dans divers types de tumeurs solides incluant les cancers de la prostate, et son rôle proH prolifératif dans des lignées cellulaires tumorales d’origines variées est bien établi. La première partie de notre travail s’est attaché à évaluer le potentiel oncogénique de NPM1 dans l’épithélium prostatique in vivo. Pour cela, nous avons généré un modèle de souris transgéniques qui surexpriment NPM1 spécifiquement dans l’épithélium de la prostate. Ces animaux présentent une hyperplasie prostatique associée à une augmentation de l’index prolifératif de l’épithélium. Nos expériences révèlent que NPM1 pourrait lever la quiescence des cellules épithéliales différenciées en dérégulant l’expression de gènes clés de la régulation du cycle cellulaire, comme la Cycline E ou p27kip1. Bien que ces souris ne développent pas de lésions néoplasiques, ces données suggèrent que NPM1 participe à la carcinogenèse prostatique en association avec d’autres lésions oncogéniques. La seconde partie du travail visait à comprendre la nature des mécanismes qui supportent la surexpression de NPM1 dans les tumeurs prostatiques. Des données récentes de la littérature indiquent un enrichissement de la protéine kinase mTOR au niveau du promoteur proximal de NPM1 dans des foies de souris. Pour déterminer s’il existe un lien fonctionnel entre mTOR et NPM1, nous avons tiré parti d’un modèle de fibroblastes embryonnaires de souris invalidés pour le suppresseur de tumeur PTEN dont l’inactivation mène à une hyperactivité de mTOR. Dans ce contexte, les taux d’ARNm et de protéines NPM1 sont augmentés par rapport aux cellules sauvages. Nos résultats montrent également que mTOR contrôle l’expression de NPM1 i) en se fixant sur son promoteur et en stimulant l’expression du gène et ii) en stabilisant l’ARNm de NPM1. Nous avons confirmé le lien entre NPM1 et mTOR in vivo grâce à notre modèle de souris invalidées pour PTEN dans l’épithélium prostatique. Enfin, nous avons montré que l’expression de NPM1 est nécessaire pour transduire les effets prolifératifs de la voie PI3K/AKT/mTOR. Ces données placent donc NPM1 comme nouvel effecteur en aval de cette voie de signalisation, faisant de cette protéine une potentielle cible thérapeutique dans les tumeurs présentant une perte de PTENNucleophosmin (NPM1/B23) is a small molecular chaperone involved in a large array of cellular processes, including the regulation of gene expression and the control of the cell cycle. Several studies have reported the overexpression of NPM1 in solid tumors from various histological origin, including prostate cancer, and its proliferative impact on several human cancer cell line is being well described. The first part of our work aimed at assessing the NPM1 oncogenic properties in the prostate gland in vivo. To do so, we generated a new transgenic mouse model that overexpresses NPM1 specifically in the prostatic epithelium. These mice harbor prostatic hyperplasia associated with an increase of the ki67 proliferative index. Our molecular investigations revealed that NPM1 could be an inhibitor of the quiescent state of epithelial cells through a dysregulation of key cell-cycle controlers such as Cyclin E or p27kip1. Although these mice do not develop neoplastic lesions, our data suggest that NPM1 overexpression accelerate prostate cancer progression when associated with other oncogenic alterations. The second part of the work aimed at understanding the mechanisms underlying NPM1 overexpression in prostate tumors. The serine/threonine Kinase mTOR was recently shown to bind to the proximal promoter of NPM1 in the mouse liver. In order to characterize a fonctionnal link between NPM1 and mTOR, we took advantage of murine embryonic fibroblast (MEF) deleted for PTEN, since these cells display a constitutive mTOR activity. In such cells, NPM1 protein and mRNA levels are increased compared to wild type MEF. We also demonstrated that mTOR controls NPM1 expression i) through its binding to NPM1 promoter, thus stimulating NPM1 gene expression and ii) by stabilizing NPM1 mRNA. We have confirmed the functional link between NPM1 and mTOR in vivo in a mouse model deleted for PTEN specifically in the prostatic epithelium. Finally, we have shown that NPM1 expression is necessary for the proliferation of PTEN knock-out MEF. These data set NPM1 as a new downstream effector of the PI3K/AKT/mTOR pathway, and suggest that it could be a new potential therapeutic target in PTEN negative human prostate cancer
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Kalender, Adem. "Regulation of the mTOR/S6K pathway by cellular energy /." [S.l.] : [s.n.], 2009. http://edoc.unibas.ch/diss/DissB_8784.
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Seymour, Lyndsey A. "Characterising the signalling mechanism of the mTOR-dependent phosphatase." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/55115/.
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The mechanistic Target of Rapamycin Complex 1 (mTORC1) complex is central in the regulation of many crucial cellular processes including translation, transcription, proliferation and autophagy. Deregulation of the complex is evident in a number of diseases including Tuberous Sclerosis, Alzheimer's Disease and cancer. Whilst the signalling events leading to activation of mTORC1 are well understood, the inhibitory phosphatase activity that prevents aberrant signalling has received comparatively little attention. In yeast, phosphatases are an integral part of TORC1 signalling. Poor nitrogen supply leads to activation of the phosphatases Pph21/22 and Sit4 and subsequent dephosphorylation of TORC1 substrates. Under these conditions, the phosphatase negative regulatory protein Tap42 is sequestered by Tip41. In good nitrogen supply, TORC1 phosphorylatesTip41 leading to release of Tap42 and subsequent inhibition of Pph21/22 and Sit4. This allows the accumulation of phosphorylated TORC1 substrates. This thesis investigated the role of Tip41 in mTORC1 signalling. Purification of Tip41 identified direct interaction with PP2Ac (human Pph21/22). As overexpression of Tip41 resulted in inhibition of mTORC1 signalling, Tip41 is proposed as a bona fide positive regulatory subunit of PP2Ac. Further investigation indicated that hypophosphorylated PP2A-rjp4i may directly oppose Rheb-mediated activation of mTORC1 thus promoting Raptor degradation. In addition, a specific nuclear isoform of Tip41 was identified, which may specifically regulate the transcription factor HIF1. Studies using the adenoviral protein E40RF4 also identified the PP2ABa complex in regulation of mTORC1 signalling. The data in this thesis show that PP2ABa acts downstream of the TSC 1/2 complex to inhibit mTORC1. Results also indicate that PP2ABa may be negatively regulated by ubiquitin-mediated proteasomal degradation of Ba in an mTORC1-specific manner. Therefore PP2ABa may be subject to an mTORC1 feedback mechanism that is required for activation of downstream substrates. These data indicate that phosphatase activity is critical in regulation of mTORC1, reflecting the mechanism in yeast.
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Sully, Katherine L. "Inhibition of mammalian target of rapamycin (mTOR) in epidermis." Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/3360.
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Cutaneous squamous cell carcinoma (cSCC) is one of the most common Caucasian skin cancers and is particularly prevalent following chronic ultraviolet (UV) radiation exposure and in immunosuppressed patients. Recent use of rapamycin as an immunosuppressant significantly reduces SCC after organ transplantation. However the mechanism remains unclear. Rapamycin inhibits mammalian target of rapamycin (mTOR) kinase, downstream from phosphatidylinositol-3 kinase (PI3K) and Akt kinases previously implicated in SCC. The aim was to find the effects of rapamycin on PI3K-Akt-mTOR signalling in epidermis in order to understand rapamycin’s tumour suppressing activity in skin. Rapamycin increases epidermal Akt phosphorylation via inhibition of an mTOR complex 1 (mTORC1)-dependent negative feedback loop to insulin receptor substrate-1. In a skin experimental model, rapamycin selectively increases phosphorylation of Akt1, the epidermal Akt isoform down-regulated in SCC and also down-regulated by UV. Epidermal Akt2, up-regulated in tumours and by UV, is unaffected. Rapamycin enhances restoration of Akt1 phosphorylation in skin recovering from UV radiation, suggesting a mechanism for rapamycin’s anti-tumour activity in epidermis in spite of its efficient immunosuppressive properties. As rapamycin targets mTORC1, newer classes of mTOR inhibitors active against mTORC1 and mTORC2 are under development. While comparing the two drug classes it was found that rapamycin unexpectedly increases epidermal mTORC2 activity. Since mTORC2 signalling influences lipid synthesis and epidermis requires extensive lipogenesis for formation of its protective barrier, the relationship between epidermal mTORC2 signalling, lipogenesis and barrier to UV was explored. Rapamycin increased epidermal lipid levels, but this increase was not sufficient to protect against UV-induced DNA damage. 3 In conclusion, rapamycin treatment can increase PI3K/Akt1 and mTORC2 signalling and lipid levels in epidermis. Rapamycin can increase epidermal Akt1 phosphorylation during UV recovery, which may contribute to the anti-cancer action of rapamycin in skin. Rapamycin’s potential to increase epidermal lipid levels makes it an interesting possible therapeutic for treating skin disorders with dyslipidemia.
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Kawashima, Naomasa. "The interaction of mTOR and autophagy in salivary glands." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/the-interaction-of-mtor-and-autophagy-in-salivary-glands(a5382fc9-c83b-4046-bff4-98c1b4ed91c5).html.
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Radiation therapy to treat head and neck cancer damages salivary glands, leading to decreased salivary flow which can cause xerostomia (perception of dry mouth), mucositis and dysphagia. These symptoms of salivary gland hypofunction are a major cause of patients stopping their treatment and greatly decrease their quality of life after treatment. Protecting from damage is essential to avoid these problems so this thesis examined some of the damage mechanisms present in salivary glands. Two intracellular processes seem to be particularly relevant. Mammalian Target of Rapamycin (mTOR) is a growth pathway often activated in cancers, repair and regeneration whereas autophagy is a self-digestion of cellular contents often associated with damage that helps to protect cells from apoptosis. Normally the two processes are linked by an enzyme UNC-like kinase (ULK) in a mutually exclusive way so that growth and degradation do not occur at the same time. However, both mTOR and autophagy have been shown to have beneficial effects after irradiation. Therefore, we decided to study the interactions between mTOR and autophagy in order to find an efficient way to uncouple mTOR and autophagy to protect irradiated salivary glands. Since ULK was an important link between mTOR and autophagy, an interesting new ULK inhibitor MRT67307 was used on salivary glands. In the first part of this study, we evaluated the effects of MRT67307 on cell cultures in order to collect enough data before trying the drug in vivo. Initially NIH 3T3 cells, a well-studied cell model, were cultured to verify the effects of MRT67307. As previously reported, the drug blocked starvation- and Torin 1 (an mTOR inhibitor)- induced autophagy. The next step was to test the effects of the drug on salivary acinar cells which were known to be very sensitive to irradiation. SMG-C6 cells were chosen since they were previously derived from rat submandibular acinar cells. In these cells, in contrast to NIH 3T3 cells, Torin 1 failed to upregulate autophagy, suggesting that mTOR and autophagy were not linked by ULK. This finding was interesting and novel and was further tested in primary-cultured cells (ie in vitro) from mouse submandibular glands. Again, administration of Torin 1 inhibited mTOR but did not activate autophagy and MRT67307 had no effect on marker of autophagy (LC3-I/LC3-II ratios). It could be inferred from these experiments that MRT67307 is a useful tool in examining mTOR/autophagy interactions through ULK1 and that in salivary glands autophagy and mTOR could be activated simultaneously. In the second part of this study, we carried out whole body irradiation of mice to study damage, mTOR-autophagy interactions and saliva flow variation in irradiated salivary glands. A dose escalation study appeared to cause minimal damage to salivary glands when the maximum dose of 11 Gy was given. To determine if salivary hypofunction had occurred whole mouth saliva was collected under temporary gaseous anaesthesia by the administration of pilocarpine (I.P.). Surprisingly, despite minimal histological indications of damage an increase in salivary function occurred. Biochemical analyses of the salivary glands indicated autophagy was transiently and weakly activated a few hours after irradiation whereas mTOR activity occurred a few days later. The use of a whole body irradiator limited the dose of irradiation to the salivary glands. Thus as a model system, transient mTOR activation probably had a beneficial effect, since pilocarpine stimulated saliva flow experiment showed a transient increase of saliva flow. However, this model system did not yield the expected salivary gland damage seen in other studies so instead another model of salivary gland damage, ductal ligation was studied. The third part of this study attempted to use autophagy-inhibitors in vivo, using the ligated salivary gland since autophagy activation was weak and did not last in irradiated salivary gland. Whole body injection of autophagy inhibitors chloroquine and MRT67307 (at two different doses and injection intervals) did not appear to have any beneficial effect on submandibular glands, except a slight delay of atrophy in both chloroquine and MRT67307 treated glands. Autophagy appeared to be mainly mTOR independent since MRT67307 failed to inhibit autophagy. This thesis contains novel data to indicate that autophagy and mTOR are independent of each in mouse submandibular glands. To the best of our knowledge, this is the first time that MRT67307 was used in vivo and no paper demonstrates an mTOR independent activation of autophagy in salivary glands. It provides the basis for further studies to protect salivary glands from irradiation damage by upregulating both mTOR and autophagy simultaneously, something that has not, so far, been tested.
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Pereira, Carla Sofia Domingues. "Via de sinalização mTOR no carcinoma urotelial da bexiga." Master's thesis, [s.n.], 2014. http://hdl.handle.net/10284/4673.
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Trabalho de Projeto apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Análises Laboratoriais Especializadas, área de especialização em Análise BiomédicaMundialmente, o cancro da bexiga (CB) é o 7º tipo de cancro mais frequente em homens e o 17 º mais frequente em mulheres. É uma doença com etiologia multifatorial associada a vários agentes ambientais e genéticos, dos quais o tabagismo é o fator de risco mais importante. Os carcinomas uroteliais da bexiga (CUBs) são geralmente superficiais em 70% a 80% dos pacientes e invasores em 20% a 30%. Os CUBs não-invasores têm uma alta taxa de recorrência e progressão e os invasores e metastáticos representam a principal causa de morbidade e mortalidade entre os pacientes com CB. A via fosfatidilinositol-3cinase (PI3K) /AKT (Proteína cinase b) -alvo da rapamicina em mamíferos (m-TOR) é uma importante via envolvida no crescimento celular, tumorogénese, invasão celular e resposta a drogas. Esta via é frequentemente ativada em muitas neoplasias e o descontrolo da sinalização PI3K-AKT-mTOR no CB pode contribuir para o crescimento do tumor, angiogénese e metastização. Neste trabalho, foram estudados 96 casos de tumores diagnosticados como CUBs de vários graus e estádios e foi avaliada a imunoexpressão do phospho-mTOR (Ser2448) e do phospho-S6 (Ser235/236). Em relação à expressão do p-mTOR verificou-se que mais de 50% dos casos de CUB não apresentavam ou apresentavam uma baixa expressão desta proteína sendo que não foi encontrada associação deste com estádio ou grau de diferenciação tumoral. Em relação à proteína p-S6 a sua expressão nos CUB foi igualmente nula ou baixa, no entanto encontrou-se uma associação estatisticamente significativa com o estádio e grau de diferenciação, em duas formas de avaliação qualitativa. Estes dois marcadores imunohistoquímicos, quando analisados em conjunto apresentaram uma correlação positiva moderada nos CUB, no entanto, estudos futuros são necessários para avaliar a sua validade como marcadores biológicos e, eventualmente, alvos terapêuticos. Worldwide, bladder cancer (BC) is the 7th most frequent type of cancer in men and the 17th most common in women. It is a disease with multifactorial etiology associated with multiple genetic and environmental agents, of which smoking is the most important risk factor. The urothelial carcinomas of the bladder (CUBs) are generally superficial in 70% to 80% of patients and invasive in 20% to 30%. The non-invasive CUBs have a high rate of recurrence and progression, and the metastatic and invaders ones are the leading cause of morbidity and mortality among patients with CB. The 3 cinase via phosphatidylinositol PI3K/AKT (cinase protein b) target of rapamycin in mammals (m-TOR) is an important pathway involved in cell growth, tumorigenesis, cell invasion and drug response. This pathway is frequently activated in many tumors and uncontrollable signaling PI3K/AKT/mTOR pathway in BC may contribute to tumor growth, angiogenesis and metastasis. In this study, 96 cases of tumors diagnosed as CUBs of various grades and stages were studied, and it was evaluated the immunoreactivity of phospho-mTOR (Ser2448) and phospho-S6 (Ser235/236). Concerning the p-mTOR expression, it was observed that more than 50% of CUB didn`t express or had a low expression of this protein, and it wasn`t found any association between this and the state or grade of the tumor. In what concerns the p-S6 protein, it was equally low or absent its expression in the CUB, although it was found a statistical association with the state and grade of the tumor, in two forms of qualitative evaluation. When analyzed, together, these two markers presented a positive moderated correlation in the CUB, however future studies are necessary to access its validity as biological markers and, eventually, therapeutic targets.
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Mota, Filipa Dias Alves da. "Carcinoma urotelial da bexiga e via de sinalização mTOR." Master's thesis, [s.n.], 2012. http://hdl.handle.net/10284/3742.
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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasO carcinoma da bexiga é o quinto tumor maligno mais comum, representando cerca de 3,2% de todos os cancros no mundo. A sua patogénese envolve alterações genéticas somáticas induzidas por agentes carcinogéneos ambientais, tais como o tabaco, as aminas aromáticas ou o arsénio. Apesar da caracterização elaborada dos fatores de risco, o carcinoma da bexiga ainda é um problema epidemiológico importante, cuja incidência continua a aumentar a cada ano. A carcinogénese pode ocorrer através da ativação de protooncogenes ou através da perda de genes supressores de tumor, ambos os quais têm sido documentados no carcinoma urotelial (CUB). O CUB, a forma mais comum de carcinoma da bexiga pode ser não-invasor ou invasor, Apesar do carcinoma da bexiga recidivar frequentemente, apresentam um baixo risco de progressão para a doença invasiva e um prognóstico geral favorável. Em contraste, os CUB invasivos caracterizam-se por serem uma doença agressiva, muitas vezes com uma taxa de sobrevida de 5 anos reduzida, apesar do tratamento com cistectomia radical e quimioterapia adjuvante. Ainda que se tenha evoluído nas estratégias cirúrgicas e quimioterapêuticas, a sobrevida na doença invasiva e metastática não apresentou melhorias significativas nas últimas décadas, desde a introdução do BCG na década de 1970 e do MVAC na década de 1980. Neste contexto urge a necessidade de novos alvos terapêuticos. A proteína alvo da rapamicina nos mamíferos (mTOR) desempenha um papel importante na regulação da tradução de proteínas, crescimento celular e metabolismo. Alterações nesta via de sinalização são comuns no cancro (amplificação/mutação da PI3K, perda de função do PTEN, sobrexpressão do AKT e amplificação/sobrexpressão do S6K1 e eIF4E), e, portanto, o mTOR constitui um alvo terapêutico cada vez mais relevante noâmbito destadoença. A rapamicina e os seus análogos provaram ser benéficos como agentes anticancerígenos, numa ampla gama e ensaios pré-clínicos, isoladamente ou combinados com outros inibidores de outras vias de sinalização, como terapêutica para vários tipos de cancro. No entanto, são necessários mais estudos para validar o mTOR como agente terapêutico para o carcinoma da bexiga. Bladder carcinoma is the fifth most common malignancy, accounting for about 3.2% of all cancers worldwide. Its pathogenesis involves somatic genetic changes induced by environmental carcinogens such as tobacco, aromatic amines or arsenic. Despite the characterization established risk factors, carcinoma of the bladder is still an important epidemiologic problem whose incidence continues to increase every year. Carcinogenesis may occur through activation of protooncogenes or through loss of tu-mor suppressor genes, both of which have been documented in urothelial carcinoma (CUB). CUB, the most common form of bladder carcinoma, is divided into noninvasive and invasive with non-invasive lesions divided into low and high grade. Despite frequente reccurences, these injuries often have a low risk of progression to invasive disease and a generally favorable prognosis. In contrast, the invasive subtipe of CUB is characterized as being an aggressive disease, often with a reduced 5-year survival, de-spite treatment with adjuvant chemotherapy and radical cystectomy. Although its sur-gical and chemotherapeutic strategies have evolved, survival in invasive and metastatic disease showed no significant improvements in the last decades, since the introduction of BCG in the 1970s and in the 1980s MVAC. In this context there is an urgent need for new therapeutic targets. Protein mammalian target of rapamycin (mTOR) plays an important role in the regula-tion of protein translation, cell growth and metabolism. Changes in this signaling path-way are common in cancer (amplification / mutation of PI3K, loss of PTEN function, overexpression of AKT and amplification / overexpression of S6K1 and eIF4E) and thus mTOR is an increasingly important therapeutic target in this disease. Rapamycin and its analogs proved to be beneficial as anticancer agents in a broad range in preclinical trials, alone or combined with other inhibitors of other signaling path-ways, such as therapy for various cancers. However, more studies are needed to validate mTOR as a therapeutic agent for bladder carcinoma.
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Bozorgi, Sophie Shaghayegh. "Role of mTOR in salivary gland atrophy and regeneration." Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/role-of-mtor-in-salivary-gland-atrophy-and-regeneration(261e1f87-673f-498e-891b-3f57395c04ff).html.
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The mammalian target of rapamycin (mTOR) is a protein kinase whose dysfunction has been identified in many diseases ranging from cancer to Down’s syndrome. Previous studies have examined salivary gland atrophy and observed the submandibular gland’s ability to regenerate from atrophy, however the mechanism underlying this process is still unknown. The current study aims to investigate the effects of blocking mTOR signaling in atrophic salivary glands and blocking mTOR signalling in submandibular glands regenerating from atrophy. The first part of the study revealed that inhibition of mTOR delays ligation-induced atrophy of salivary glands and that furthermore, mTOR could only be inhibited for shorter periods of 3 days, whereas 5 or 7 days of ligation and rapamycin treatment cause glands to re-express active mTOR and show considerable signs of atrophy. The second part of the study aimed to find out the reasoning behind the reactivation of mTOR following 5 or 7 days, despite the presence of mTOR inhibitors. It concluded that 2nd generation mTOR inhibitors also failed to block mTOR activation following 7 days of atrophy. Proteomic and microarray analysis were performed and gave rise to possible future enquiries. This study then exposed the role of mTOR in salivary gland regeneration following atrophy, revealing that mTORC1, specifically its substrates, are needed for a full regeneration. Inhibiting mTOR during periods of atrophy and allowing phosphorylation of mTORC1 substrates during periods of regeneration, is a treatment method which could be of importance. The final part of the study observed samples of atrophic human salivary glands in order to find evidence of aberrant mTOR activity. It caused three realisations, firstly that mTOR is one of the driving forces of atrophic processes as once atrophy is severe, most acinar cells are lost and mTOR is no longer as active. Secondly, that autophagy coincides with salivary gland atrophy in humans. And thirdly, that some salivary gland functions might possibly be intrinsically linked to ageing. This leads to the suggestion that the future of treating salivary gland atrophy in humans could lie in using mTOR inhibitors, whether they be localised treatment in the form of intraductal injection of rapamycin loaded nanoparticles to get localised targeting whilst reducing whole body toxicity or in the form of combination therapies that combine mTOR inhbitiors with the addition of another drug that inhibits autophagy and counteracts any toxic effects of mTOR inhibition.
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Obayashi, Yoko. "Novel physiological function of proline and mTOR regulator tuberin." Kyoto University, 2018. http://hdl.handle.net/2433/232154.
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Jobim, Paulo Fernandes Costa. "Papel de mTOR na formação e reconsolidação da memória." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2011. http://hdl.handle.net/10183/35905.
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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.
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Mushaben, Elizabeth M. "BMPR2 and mTOR Signaling Pathways in Inflammatory Lung Diseases." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1352485302.
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KHAPRE, ROHINI VISHAL. "CIRCADIAN REGULATION OF mTOR SIGNALING VIA BMAL1 DEPENDENT MECHANISM." Cleveland State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1399025275.
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Pothiraju, Deepika [Verfasser]. "Targeting PI3K/mTOR pathway in hepatocellular carcinoma / Deepika Pothiraju." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2011. http://d-nb.info/1013286243/34.
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Caccamo, Antonella Maria Giovanna. "The role of mTOR signaling in Alzheimer s disease." Doctoral thesis, Università di Catania, 2016. http://hdl.handle.net/10761/3767.
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The majority (>95%) of Alzheimer s disease (AD) cases are sporadic and of unknown causes. The single major risk factor for AD is aging and molecular changes that occur in the brain as a function of age may facilitate the development of AD. However, little is known as to how the aging process facilitates the development of AD.
Overwhelming evidence shows that reducing the activity of the mammalian target of rapamycin (mTOR) increases lifespan and health-span in several genetically different species. mTOR is a ubiquitously expressed protein kinase that plays a key role in regulating protein synthesis and cell growth. mTOR also is a negative regulator of autophagy induction. By simultaneously regulating protein synthesis and degradation, mTOR is key in controlling protein homeostasis, a process that is altered in AD and other proteinopathies. Another known function of mTOR signaling is the regulation of synaptic plasticity and function.
Using two widely used animal models of AD, known as Tg2576 and 3xTg-AD mice, we employed multidisciplinary approaches to dissect the role of mTOR signaling in AD. We found that genetic reduction of mTOR signaling reduced amyloid-â deposits and tau pathology and rescued memory deficits in Tg2576 mice. Mechanistically, the reduction in mTOR signaling led to an increase in autophagy induction and restored the hippocampal gene expression signature of the Tg2576 mice to wild type levels. Consistent with these findings, we also found that that genetic reduction of the ribosomal protein S6 kinase 1 (S6K1), a protein kinase directly downstream of mTOR, improved synaptic plasticity and spatial memory deficits, and reduced the accumulation of amyloid-â and tau, in 3xTg-AD mice. Mechanistically, these changes were linked to reduced translation of tau and the beta-site APP cleaving enzyme 1 (BACE-1), a key enzyme in the generation of amyloid-â.
Given the overwhelming evidence showing that reducing mTOR signaling increases lifespan and health span, the data presented here have profound clinical implications for aging and AD and provide the molecular basis for how aging may contribute to AD pathology. Our results implicate hyperactive mTOR/S6K1 signaling as a previous unidentified signaling pathway underlying gene-expression dysregulation, synaptic and cognitive deficits in Alzheimer s disease.
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Machado, Camila de Oliveira Freitas. "Estudo do envolvimento da proteína colibistina no controle do início da tradução." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-15122014-142323/.
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A proteína colibistina (CB), uma Rho GEF neuro-especifica, apresenta papel importante na formação e funcionamento das sinapses inibitórias do sistema nervoso central por interagir com a proteína scaffold gefirina e com receptores GABAA e promover o agrupamento e transporte dessas proteínas para a membrana pós-sináptica. Recentemente, nosso grupo de pesquisa identificou interação de CB com um complexo proteico que controla o início da tradução em eucariotos (complexo eIF3), o que sugeriu pela primeira vez que essa proteína pode estar envolvida também na regulação da tradução em células neurais. Ainda, já havia sido descrito que gefirina, parceira funcional de CB, interage com mTOR, uma quinase que desempenha papel fundamental no controle do início da tradução. Contudo, até o momento não havia estudos adicionais investigando o papel de CB neste cenário. Assim sendo, o presente trabalho teve como objetivo investigar o envolvimento da proteína CB no controle do início da síntese proteica mediada pela via de sinalização mTORC1. Foram utilizados dois modelos experimentais: i) um sistema de expressão heteróloga - superexpressão de CB em células HEK293T, e ii) um modelo endógeno de expressão - células neuroprogenitoras derivadas de células-tronco pluripotentes induzidas (iNPCs) provenientes de indivíduos controles e de um paciente com deleção no gene da CB. Por meio de experimentos de coimunoprecipação nós verificamos que CB interage fisicamente com mTOR nos dois modelos experimentais utilizados. Ainda, nossos resultados mostraram que CB parece modular a atividade da via mTORC1, e nas iNPCs derivadas do paciente a ausência de CB leva a um aumento na ativação desta via de sinalização. Em concordância com esses resultados, nós observamos aumento em neo-síntese proteica nas iNPCs provenientes do paciente, o que pode ser um mecanismo patofisiológico contribuindo para as alterações cognitivas e comportamentais observadas no paciente. Embora estudos adicionais sejam necessários para melhor entender os mecanismos moleculares deste controle de início de tradução mediado por CB, nós sugerimos um modelo no qual CB, por interagir fisicamente com mTOR e eIF3, sequestra estas proteínas e impede que mTOR ative seus alvos e desencadeie a formação do complexo de inicio de tradução. Em conclusão, nossos resultados oferecem novas evidências do envolvimento de CB no controle da síntese proteicaCollybistin (CB), a neural specific RhoGEF, plays key roles in inhibitory synapse formation and function that cluster and localize the scaffold protein gephyrin and GABAA receptors to the neural postsynaptic membrane. We have recently reported that CB interacts with a protein complex that controls translation initiation in eukaryotic cells (eIF3 complex), which suggested for the first time that this protein may also act as regulator of protein synthesis in neural cells. Moreover, it has been previously described that gephyrin, the CB functional partner, interacts with mTOR, a kinase that plays a pivotal role in the control of translation initiation. However, until now there were no further studies investigating the role of CB in this scenario. The purpose of this study was to investigate if CB is involved in the control of translational initiation mediated by the mTORC1 signaling pathway. Two experimental models were used: i) a heterologous expression system - overexpression of CB in HEK293T cells, and ii) an endogenous expression model - neural progenitor cells derived from induced pluripotent stem cells (iNPCs) from control individuals and a patient with a deletion of the entire CB gene. We performed coimmunoprecipitation experiments and verified that CB physically interacts with mTOR both in 293T cells and in control iNPCs. In addition, our results show that CB appears to modulate the activity of mTORC1 pathway, and the absence of CB leads to increased mTORC1 signaling activation in patient\' iNPCs. In agreement with these results, we observed increased de novo cap-dependent translation in patient cells, which could be a pathophysiological mechanism contributing to cognitive and behavioral abnormalities observed in the patient. Although further studies are needed to better understand the molecular details of CB-mediated translational control, we suggest a model whereby CB, by physically interacting with mTOR and eIF3, sequesters these proteins, thereby preventing both the ability of mTOR to activate its targets and the formation of the translational initiation complex. In conclusion, our results offer new insights into the role of CB in protein synthesis control
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Rocha, Alisson Luiz da. "Overreaching não funcional em modelo animal: adaptações inflamatórias e hipertróficas do músculo cardíaco." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/17/17152/tde-23032018-134837/.
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O overreaching não funcional (NFOR) induzido pelo exercício excêntrico (EE) em modelo animal está associado com a diminuição de desempenho físico, dano no DNA (amostras de músculo esquelético e soro), estresse oxidativo (amostras de músculo esquelético e soro), inflamação crônica de baixo grau (amostras de músculo esquelético e soro) e prejuízo da via de sinalização da insulina (amostras de músculo esquelético). No entanto, as adaptações do músculo cardíaco em resposta ao estado de NFOR induzido ou não pela predominância do EE ainda não foram investigadas. Além disso, sabe-se que a mTOR (mammalian target of rapamycin) possui um efeito protetor no músculo cardíaco, suprimindo o aumento de citocinas pró-inflamatórias, que estão relacionadas à disfunções cardíacas. Assim, o presente estudo teve como objetivo comparar os efeitos do NFOR em declive com outros dois protocolos de mesmo volume e intensidade, mas realizados sem inclinação e em aclive, no conteúdo das proteínas relacionadas às vias moleculares inflamatória e hipertrófica, no conteúdo de fibrose intersticial e na expresão gênica em músculo cardíaco de camundongos. Os animais foram divididos em 6 grupos: Naíve (N; camundongos sedentários), Controle (C; camundongos sedentários submetidos aos testes físicos), Treinado (TR; camundongos submetidos ao protocolo de treinamento), Overtraining em declive (OTR/down; camundongos submetidos ao protocolo de OT com corrida na descida), Overtraining sem inclinação (OTR; camundongos submetidos ao protocolo de OT com corrida sem inclinação) e Overtraining em aclive (OTR/up; camundongos submetidos ao protocolo de OT com corrida na subida). Em relação aos parâmetros metabólicos, o grupo OTR/down apresentou menor variação de peso corporal na semana 8. Todos os grupos que passaram pelo protocolo de OT demonstraram queda de desempenho ao final da semana 8, aumento no conteúdo de tecido conjuntivo no ventrículo esquerdo e menor ativação da proteína AMPKalfa. O grupo OTR/down apresentou menor conteúdo da proteína mTOR e S6RP, e aumento na expressão do gene beta-MHC. Pode-se concluir que os protocolos de OT provocaram indícios de hipertrofia patológica no ventrículo esquerdo, sendo esse efeito mais pronunciado no grupo OTR/down.Nonfunctional overreaching (NFOR) induced by eccentric exercise (EE) in animal model is associated with performance decrement, DNA damage (muscle and serum samples), oxidative stress (muscle and serum samples), low grade chronic inflammation (muscle and serum samples) and insulin signaling impairment (muscle and serum samples). However, the adaptations of cardiac muscle in response to NFOR induced or not induced by EE are unknown. In addition, the mammalian target of rapamycin (mTOR) has a protector effect in the cardiac muscle, suppressing the increase of the proinflammatory cytokines that is related to cardiac dysfunctions. Thus, the main aim of present study was to compare the effects of NFOR based on EE (downhill running) with other two protocols with similar intensity and volume, but performed in uphill and without inclination, on the protein contents related to hypertrophic and inflammatory signaling, on the content of interstitial fibrotic tissue and on genes expression in mice cardiac muscle. The animals were divided on six groups: Naïve (N; sedentary mice), Control (C; sedentary mice submitted to physical tests), Trained (TR; mice submitted to training protocol), Overtraining in downhill (OTR/down; mice submitted to the overtraining protocol in downhill), Overtraining without inclination (OTR; mice submitted to the overtraining protocol without inclination), and Overtraining in uphill (OTR/up; mice submitted to the overtraining protocol in uphill). Regarding metabolic parameters, OTR/down group presented reduced body weight variation at week 8. All OT groups presented performance drop at end of week 8, increased connective tissue content in left ventricle and reduced AMPKalpha activation. OTR/down group presented reduced mTOR and S6RP protein content, and increased betaMHC gene expression. The conclusion is that OT protocols provoked signs of pathological hypertrophy in left ventricle, being this effect more pronounced in OTR/down group.
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Reita, Damien. "Evaluation préclinique d'une nouvelle combinaison thérapeutique associant l'irinotécan à un inhibiteur de mTOR pour le traitement des tumeurs coliques." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ071.
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Positionnée en aval des voies PI3K/AKT et RAS/MAPK, la protéine kinase mTOR joue un rôle déterminant dans le développement et la progression tumorale des cancers colorectaux où elle est fortement surexprimée. Par ailleurs, les cancers colorectaux comme toutes les tumeurs solides, ont un microenvironnement hypoxique. L’adaptation des cellules tumorales à l’hypoxie est notamment régulée par la voie PI3K/AKT/mTOR ainsi que par les facteurs de transcription HIFs dont l’expression protéique et l’activité transcriptionnelle est en partie régulée par mTOR. Dans cette étude, nous avons montré que l’inhibition verticale et complète de l’axe PI3K/AKT/mTOR/HIF-1α par l’utilisation combinée d’irinotecan à faible dose et d’inhibiteurs catalytiques de mTOR inhibe significativement la prolifération cellulaire de lignées coliques humaines, la croissance tumorale et le développement de métastases de xénogreffes de tumeurs coliques dérivées de patients.En parallèle, une étude de cohorte de tumeurs coliques humaines de stade III par Tissue Micro Array montre que les facteurs HIFs sont fortement exprimés dans l’épithélium et le stroma de cancers du côlon de stade III, qu’une faible expression nucléaire de HIF-1α dans les cellules épithéliales confère une mauvaise survie aux patients et qu’elle a une valeur prédictive de moins bonne réponse au traitement 5-FUDownstream of the PI3K/AKT and RAS/MAPK pathways, mTOR protein kinase plays a decisive role in the development and tumor progression of colorectal cancers. Furthermore, the microenvironment of colorectal cancers is hypoxic. The adaptation of the tumor cells to hypoxia is regulated by the PI3K/AKT /mTOR pathway as well as by the HIFs transcription factors whose protein expression and transcriptional activity is partially regulated by mTOR. In this study, we showed that the vertical and complete inhibition of the PI3K / AKT / mTOR /HIF-1α axis by the combined use of low-dose irinotecan and mTOR catalytic inhibitors significantly inhibits human colon cancer cell proliferation, as well as the growth and metastatic development of xenografted human colon tumors. In parallel, a Tissue Micro Array study on a cohort of stage III human colic tumors shows that the HIFs are strongly expressed in the epithelium and stroma of the tumors and a low nuclear expression of HIF-1α in epithelial cells provides with poor survival to patients and has a predictive value of worse response to 5-FU treatment
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Nierhoff, Ann-Kathrin [Verfasser], and Hagen Sjard [Akademischer Betreuer] Bachmann. "Bedeutung von genetischen Polymorphismen im MTOR-Gen bei Patienten nach Nierentransplantation unter und ohne mTOR-Inhibitor-Therapie / Ann-Kathrin Nierhoff ; Betreuer: Hagen Sjard Bachmann." Duisburg, 2019. http://d-nb.info/1191690873/34.
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Kellenberger, Antonia Judit [Verfasser], and Elke [Akademischer Betreuer] Lütjen-Drecoll. "Die Beteiligung von mTOR an der Regulation des Haarzyklus und Einfluss von Bimatoprost auf die mTOR Aktivierung / Antonia Judit Kellenberger. Betreuer: Elke Lütjen-Drecoll." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2011. http://d-nb.info/1056066474/34.
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Moorthy, Ganesh. "Clinical Pharmacokinetics of the Novel Combination of BEZ235, PI3K/mTOR Inhibitor, and Everolimus, mTOR Inhibitor: Phase I Clinical Studies and Non-clinical Mechanistic Assessment." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1439296033.
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Vicier, Cecile. "Implication de la pseudokinase dans la réponse aux inhibiteurs de mTor." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS320.
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MTor est une protéine centrale de la voie de signalisation PI3K/AKT/mTor et est impliquée dans la croissance, la prolifération et la survie cellulaire. Cette protéine joue un rôle majeur particulièrement dans la prolifération tumorale. Ainsi des inhibiteurs de mTor ont été développés à partir des années 1980, notamment la rapamycine, et deux sont utilisés actuellement en clinique: l’évérolimus et le temsirolimus. Ces deux inhibiteurs font partie des différentes possibilités thérapeutiques dans les cancers du sein et du rein métastatiques. Les patients traités présentent des réponses variées avec des cas de patients très bons répondeurs et des cas de patients résistants d'emblée ou après exposition au traitement. Face à cette notion de résistance, nous avons voulu caractériser les mécanismes d’adaptation de la cellule tumorale après exposition aux anti-mtor. L’analyse de l’expression génomique de huit lignées cellulaires tumorales variées après traitement par la rapamycine a montré une modulation de l’expression de certains gènes dont celle de la pseudokinase TRIB3, diminuée dans toutes les lignées. Cette donnée in vitro est aussi retrouvée chez les patients. En effet après traitement par évérolimus, nous avons pu observer une diminution de l’expression du gène TRIB3 au niveau sanguin. Nous avons donc cherché à comprendre le rôle de cette pseudokinase dans la réponse aux anti-mTor. Nos résultats mettent en évidence, d’une part, que la rapamycine régule l’expression de TRIB3 en agissant sur son promoteur via une interaction avec GCF2. D’autre part, l’hyperexpression de TRIB3 limite les effets anti-tumoraux de la rapamycine dans différentes lignées cellulaires tumorales. Pour étudier plus en détails ce mécanisme, nous avons cherché à déterminer les partenaires de TRIB3. Par des approches de protéomique haut-débit, nous avons mis en évidence un lien avec des protéines impliquées dans l’épissage. Ainsi la rapamycine semble inhiber la machinerie d’épissage via la diminution d’expression de TRIB3. Ce travail relève l’intérêt de TRIB3 dans la réponse aux anti-mTor comme un potentiel biomarqueur et illustre également le mode d’action de la rapamycineMTor is a central protein of the PI3K/AKT/mTor signaling pathway and is involved in growth, proliferation and cell survival. This protein plays a major role particularly in tumor proliferation. Thus, mTor inhibitors have been developed since the 1980s, including rapamycin, and two are currently used in daily practice: everolimus and temsirolimus. These two inhibitors are part of the different therapeutic possibilities in metastatic breast and kidney cancers. Patients treated present different responses with cases of very good responders and cases of resistant patients either immediately or after exposure to treatment. According to the concept of resistance, we wanted to characterize the mechanisms of adaptation of the tumor cell after exposure to mTor inhibitors. Analysis of the genomic expression of eight variant tumor cell lines after treatment with rapamycin showed a modulation of the expression of different genes including the pseudokinase TRIB3, decreased in all the lines. This in vitro data is also found in patients. Indeed, after treatment with everolimus, we observed a decreased expression of the TRIB3 gene in blood. We therefore sought to understand the role of this pseudokinase in the response to mTor inhibitors. Our results show, that rapamycin regulates the expression of TRIB3 through a GCF2-dependent inhibition of promoter activity. Moreover, TRIB3 overexpression limits the anti-tumor effects of rapamycin in different tumor cell lines. To investigate this mechanism, we sought to identify TRIB3 partners. By high-throughput proteomic approaches, we have demonstrated a link with proteins involved in splicing. Thus, rapamycin appears to inhibit splicing machinery with the decreased expression of TRIB3. This work highlights the interest of TRIB3 in the response to mTor inhibitors as a potential biomarker and investigates also how rapamycin acts on cells
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Silva, Adriana Caldo. "Efeitos do overreaching não funcional sobre a via da mTOR no tecido hepático de camundongos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17152/tde-04012017-113714/.
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O propósito do presente estudo foi verificar os efeitos do overtraining (OT) nas proteínas relacionadas com a via de sinalização da mammalian target of the rapamycin complex 1 (mTORC1), no conteúdo proteico de sterol regulatory element binding protein-1 (SREBP-1) e nas características morfológicas do fígado de camundongos C57BL/6. Os animais foram divididos em grupo controle (CT), overtraining em declive (OTR/down), overtraining em aclive (OTR/up) e overtraining sem inclinação (OTR). Teste do rotarod, incremental, exaustivo e força de preensão foram utilizados para avaliação de performance. Após 36 horas o teste de força de preensão, os fígados foram removidos e utilizados para immunoblotting ou análises histológicas. A fosforilação da proteína kinase B (pAkt; Ser473), mammalian target of the rapamycin (pmTOR; Ser2448), 70-kDa ribosomal protein S6 kinase 1 (pS6K1; Thr389) e da AMP-activated protein kinase (pAMPK; Thr172) foram significativamente maiores no grupo OTR/down quando comparado com os grupos CT e OTR. A fosforilação da 4E-binding protein-1 (p4E-BP1; Thr37/46) foi significativamente maior no grupo OTR/down quando comparado com o grupo CT. Os níveis proteicos de sterol regulatory element binding protein- 1 (SREBP-1; p125 precursor) foram significativamente maiores nos grupos OTR/down e OTR/up quando comparados com o grupo CT. Enquanto o grupo OTR/down apresentou sinais de esteatose com inchaço celular acompanhado de inflamação aguda, os grupos OTR/up e OTR demonstraram evidências de injúria hepática, com a presença de núcleos picnóticos, hepatócitos em balão e vacúolos citoplasmáticos. Conclui-se que o protocolo de OTR/down aumenta a modulação da via de sinalização da mTOR e induz a sinais de esteatose hepática.The purpose of this study was to verify the effects of overtraining (OT) on the proteins related to the mammalian target of the rapamycin complex 1 (mTORC1) signaling pathway, the protein content of the sterol regulatory element binding protein-1 (SREBP-1) and the morphological characteristics in the livers of C57BL/6 mice. Rodents were divided into control (CT), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR) groups. Rotarod, incremental load, exhaustive and grip force tests were used to evaluate performance. Thirty-six hours after the grip force test, the livers were removed and used for immunoblotting or histological analysis. The phosphorylation of the protein kinase B (pAkt; Ser473), mammalian target of the rapamycin (pmTOR; Ser2448), 70-kDa ribosomal protein S6 kinase 1 (pS6K1; Thr389) and AMP-activated protein kinase (pAMPK; Thr172) were significantly higher in the OTR/down group when compared to the CT and OTR groups. The phosphorylation of the 4Ebinding protein-1 (p4E-BP1; Thr37/46) was significantly higher in the OTR/down group when compared to the CT group. The protein levels of the sterol regulatory element binding protein-1 (SREBP-1; p125 precursor) were significantly higher in the OTR/down and OTR/up groups when compared to the CT group. While the OTR/down group presented signs of steatosis with cell swelling accompanied by acute inflammation, the OTR/up and OTR groups demonstrated evidences of injury in liver, with the presence of pyknotic nuclei, ballooned hepatocytes and cytoplasmic vacuoles. In conclusion, the OTR/down protocol up-modulated the mTOR signaling pathway and induced signs of hepatic steatosis.
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Dowling, Ryan. "The role of mTOR signalling in translational control and cancer." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66869.
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Abstract The serine/threonine kinase mTOR plays a critical role in the regulation of cellular proliferation, growth, differentiation, autophagy, and cytoskeletal organization. Two mTOR containing complexes exist in mammalian cells: mTORC1 and mTORC2. mTORC1 mediates its effects through the control of mRNA translation initiation via phosphorylation of its two major downstream targets: the 4E-BPs and S6Ks. In order to study the individual roles of S6Ks and 4E-BPs downstream of mTORC1, we utilized new active-site mTOR inhibitors and cells deficient for either 4E-BPs or S6Ks. Surprisingly, we found that 4E-BPs and S6Ks play distinct roles in mediating the activity of mTORC1, with the 4E-BPs mediating cell proliferation and the S6Ks regulating cell growth. The effects of active-site mTOR inhibitors on cell proliferation, cell cycle progression, and protein synthesis were highly attenuated in cells lacking expression of 4E-BPs (4E-BP DKO), while these processes were significantly inhibited in wild-type cells. However, the growth of 4E-BP DKO cells was equally sensitive to active site mTOR inhibitors, as compared to their wild-type counterparts. In contrast, the growth of S6K1/2 null cells was refractory to the effects of active site mTOR inhibitors, whereas these cells were fully sensitive to the effects of the inhibitors on proliferation and cell cycle progression. These data support a model whereby the mTORC1 proliferative and growth signals diverge in mammalian cells to provide a system that allows for increased flexibility in the maintenance of full body homeostasis. mTOR is often over-activated in human cancers and as a result, has emerged as a target for anti-cancer therapies. The anti-diabetic drug metformin was recently identified as a potential anti-cancer agent. To elucidate its mechanism of action, we studied its effects on breast cancer cell proliferation, mTOR signalling and mRNA translation. In bRésumémTOR est une sérine/thréonine kinase qui joue un rôle primordial dans lecontrôle de plusieurs fonctions cellulaires, incluant la prolifération, la croissance, ladifférentiation, l'autophagie et la réorganisation du cytosquelette. Les cellulesmammifères possèdent deux complexes protéiques contenant mTOR, soit mTORC1 andmTORC2. mTORC1 exerce son action en contrôlant l'initiation de la traduction desARN messagers via la phosphorylation de ses principaux effecteurs : les 4E-BP et lesS6K. Dans le but de mieux comprendre les fonctions spécifiques des 4E-BP et des S6Ken aval de la signalisation cellulaire initiée par mTORC1, nous avons utilisé de nouveauxinhibiteurs spécifiques au site actif de mTOR, ainsi que des cellules n'exprimant pas les4E-BP ou les S6K. Nous avons découvert que les 4E-BP et les S6K jouent des rôlesdistinct en aval de mTORC1, les 4E-BP étant principalement impliquées dans laprolifération cellulaire, alors que les S6K jouent un rôle dans le contrôle de la croissancecellulaire. Les effets des inhibiteurs spécifiques au site actif de mTOR sur la proliférationcellulaire, la progression à travers le cycle cellulaire et la synthèse protéique sontfortement atténués dans les cellules nulles pour les 4E-BP, alors qu'ils sont inhibés demanière importante dans les cellules de type sauvage. Cependant, la croissance descellules 4E-BP nulles, en présence des inhibiteurs spécifiques au site actif de mTOR, estla même que celle observée pour les cellules de type sauvage. À l'opposé, la croissancedes cellules S6K nulles n'est pas affectée par ces inhibiteurs, tandis que les inhibiteursconservent leurs effets négatifs sur la prolifération et la progression au travers du cyclecellulaire dans ces mêmes cellules. Ces résultats sont en accord avec un modèle danslequel la signalisation en aval de mTORC1, pour promouvoir la prolifération et lacroissance cellulair
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Kaeuferle, Theresa. "Identification of microRNAs regulating adipocyte differentiation via mTOR nutrient-signaling." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-182836.
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Tam, Ka-ho Chris, and 譚家豪. "Targeting mTOR as a novel therapeutic strategy for hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B41508476.
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Carvalho, Lucas de Lima. "InibiÃÃo do mTOR agrava a mucosite intestinal induzida por irinotecano." Universidade Federal do CearÃ, 2016. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=18610.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoINTRODUÃÃO. O cÃncer colorretal (CCR) à uma das neoplasias mais prevalentes em todo o mundo, sendo uma das principais causas de Ãbito por cÃncer. No Brasil, apresenta-se como o terceiro cÃncer mais frequente nos homens e segundo nas mulheres. Nas Ãltimas dÃcadas novas drogas incorporadas à protocolos clÃnicos para o tratamento do CCRm tem proporcionado um aumento significativo na sobrevida de pacientes com esse tipo de malignidade. Dentre estas drogas o irinotecano ocupa lugar de destaque e à comumente utilizado como primeira linha no tratamento do CCR metastÃtico. Entretanto, como todo tratamento quimioterÃpico hà um Ãnus, um quarto dos pacientes em tratamento com irinotecano apresentam uma grave mucosite intestinal como efeito colateral mais notÃrio. A desregulaÃÃo de vias de sinalizaÃÃo celular certamente à uma das principais razÃes para o desenvolvimento do cÃncer. A proteÃna alvo da rapamicina em mamÃferos (mTOR) à responsÃvel pelo crescimento celular e o eixo compreendido por PI3K/Akt/mTOR està frequentemente desregulada em vÃrios tipos de cÃncer. Dessa forma, esse trabalho focou em investigar o papel do mTOR durante a mucosite intestinal experimental induzida pelo irinotecano. MÃTODOS. Camundongos machos C57BL/6 (n=6/grupo) foram administrados intraperitonialmente com salina ou irinotecano (75 mg/kg ou 45 mg/kg) durante quatro dias consecutivos e tratados com rapamicina ou everolimo (inibidores de mTOR, 1,5 mg/kg e 3 mg/kg, respectivamente) seguidos da administraÃÃo de irinotecano. Periodicamente foram avaliados a variaÃÃo de peso corpÃreo e escores para avaliaÃÃo do grau de diarreia. ApÃs a eutanÃsia dos animais, amostras de Ãleo foram coletadas para determinaÃÃo da atividade de mieloperoxidase (MPO), histopatologia e anÃlise morfomÃtrica e dosagem dos nÃveis de citocinas [KC (quimiocina anÃloga da IL-8 humana), IL-1β, TGF-β e IFN-γ]. A expressÃo gÃnica do RNAm das proteÃnas da via PI3K/Akt/mTOR foi realizada por qRT-PCR. TambÃm foi colhido sangue dos animais para avaliaÃÃo da contagem total de leucÃcitos. Os testes de ANOVA/Bonferroni ou Kruskal-Wallis/Dunn foram usados para as anÃlises estatÃsticas. P<0,05 foi aceito como significativo. O presente trabalho foi aprovado pelo comità de Ãtica da UFC (nÃmero de protocolo NÂ100/14). RESULTADOS. Os animais injetados com irinotecano 75 mg/kg apresentaram um aumento na expressÃo gÃnica do RNAm de PI3K, Akt e mTOR evidenciando a participaÃÃo da via na iatrogenia. O irinotecano 75 mg/kg reduziu significativamente o peso corpÃreo, contagem total de leucÃcitos, razÃo vilo/cripta e promoveu uma lesÃo intestinal que lhe à caracterÃstico quando comparado ao grupo controle salina. AlÃm disso, de forma geral, a inibiÃÃo do mTOR com rapamicina ou everolimo agravou a maioria desses parÃmetros, tais como perda de peso, diarreia, histopatologia, infiltraÃÃo de neutrÃfilos e mediadores inflamatÃrios como TGF-β e IFN-γ. CONCLUSÃO. A inibiÃÃo do mTOR agrava a mucosite intestinal induzida por irinotecano provavelmente pelo aumento da resposta pro-inflamatÃria via TGF-β e IFN-γ com participaÃÃo importante das CÃlulas de Paneth. Apoio financeiro: CNPq.
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Abelaira, Helena Mendes. "O papel da via mTOR no efeito antidepressivo da cetamina." reponame:Repositório Institucional da UNESC, 2017. http://repositorio.unesc.net/handle/1/5002.
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Tese apresentada ao Programa de Pós-Graduação em Ciências da Saúde da Universidade do Extremo Sul Catarinense - UNESC, como requisito parcial para a obtenção do título de Doutor em Ciências da Saúde.Estudos recentes indicam que a via de sinalização da proteína alvo da rapamicina em mamíferos (mTOR) está relacionada com a fisiopatologia do transtorno depressivo maior (TDM). A cetamina, um antagonista do receptor N-metil-D-asparto (NMDA), foi identificada como uma nova terapia para o TDM, no entanto, o seu mecanismo de ação como antidepressivo não é ainda totalmente compreendido. Evidências indicam que a ativação da via mTOR no cortéx frontal (CF) é requerida para os efeitos antidepressivos da cetamina. Assim, o presente estudo teve como objetivo avaliar os efeitos comportamentais e neuroquímicos em diferentes regiões cerebrais após a administração de cetamina e a inibição da via de sinalização mTOR no CF de ratos. Para este fim, ratos Wistar machos adultos foram divididos em 4 grupos experimentais: 1) veículo + sal; 2) veículo + rapamicina; 3) veículo + cetamina 15 mg/kg; 4) rapamicina + cetamina 15 mg/kg. O inibidor farmacológico da via mTOR, a rapamicina na dose de 0,2 nmol ou veículo (grupo controle), foram administrados diretamente no CF 1 hora antes do teste do nado forçado (TNF) e 30 minutos antes da administração intraperitoneal de cetamina ou salina. Após o TNF as estruturas cerebrais: CF, amígdala, hipocampo e núcleo acumbens (NAc) e soro foram retirados para as análises bioquímicas (parâmetros de estresse oxidativo e nitrosativo, níveis de citocinas e níveis de proteínas relacionadas com a via mTOR e estresse de retículo (ER)). Os resultados mostraram que a rapamicina foi capaz de bloquear os efeitos antidepressivos da cetamina no tempo de imobilidade no TNF. A cetamina aumentou os níveis das espécies reativas ao ácido tiobarbitúrico (TBARS), carbonilação de proteínas, níveis de nitrito/nitrato e atividade da mieloperoxidase (MPO) e diminuiu a atividade das enzimas antioxidantes superóxido dismutase (SOD) e catalase (CAT) em algumas áreas cerebrais analisadas. No entanto, a inibição da via de sinalização mTOR pela rapamicina no CF, foi capaz de proteger contra o estresse oxidativo através da diminuição do dano a proteínas e lipídeos e o aumento das enzimas antioxidantes. Os níveis do fator de necrose tumoral alfa (TNF-α) aumentaram no soro após a administração de cetamina. Os níveis da proteína mTOR foram reduzidos no grupo rapamicina tratado com salina e cetamina no CF. Houve uma redução nos níveis de p4EBP1 no grupo rapamicina tratado com cetamina no CF e NAc e ainda, os níveis da peEF2K foram aumentados no CF no grupo veículo tratado com cetamina e diminuídos no grupo rapamicina tratado com cetamina. Os níveis de PERK e IRE1- alfa foram diminuídos no CF no grupo rapamicina tratado com cetamina. Os achados do presente estudo sugerem que a inibição da via de sinalização mTOR pode estar envolvida, pelo menos em parte, com o mecanismo de ação da cetamina; e que o efeito antidepressivo da cetamina sobre os níveis das proteínas do ER podem ser mediados pela via de sinalização mTOR em áreas cerebrais envolvidas com a regulação do humor. Sugere-se também que a inibição da via mTOR pode proteger o cérebro contra o estresse oxidativo e nitrosativo induzidos pela cetamina.
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Market, Marisa Rae. "TGFβ Causes Postoperative Natural Killer Cell Paralysis Through mTOR Inhibition." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40945.
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Background: Life-prolonging tumour removal surgery is associated with increased metastasis and disease recurrence. Natural Killer (NK) cells are critical for the anti-tumour immune response. Postoperatively, NK cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed and this dysfunction has been linked to increased metastases/poor patient outcomes. NK cell activity depends on the integration of signals through receptors and can be modulated by soluble factors, including transforming growth factor- beta (TGFβ). The postoperative period is characterized by the expansion of myeloid-derived suppressor cells (sxMDSCs), which inhibit NK cell effector functions. I hypothesize that impaired NK cell IFNγ production is due to altered signaling pathways caused by sxMDSC-derived TGFβ.
Methods: Postoperative changes in NK cell receptor expression, receptor-dependent phosphorylation of downstream targets, and rIL-2/12-stimulated IFNγ production were assessed using newly developed whole blood assays utilizing peripheral blood samples from cancer surgery patients. Isolated healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma or isolated sxMDSCs and NK cell phenotype and function were assessed. NK cells were also cultured with plasma in the presence/absence of a TGFβ blocking monoclonal antibody (mAb) or a TGFβ RI small molecule inhibitor (smi). Single-cell RNA-sequencing was performed on six colorectal cancer surgery patients at baseline and on POD1. S6 phosphorylation was used as a proxy for mammalian target of rapamycin complex (mTORC) 1 activity to investigate the mechanism of TGFβ-mediated NK cell dysfunction.
Results: Intracellular NK cell IFNγ, activating receptors CD132 (IL-2R), CD212 (IL-12R), NKG2D, and DNAM-1, and the phosphorylation of downstream targets STAT5, STAT4, p38 MAPK, and S6 were significantly reduced on POD1. TGFβ was increased in patient plasma on POD1. The dysfunctional phenotype could be phenocopied in healthy NK cells through the addition of rTGFβ1 or by incubation with POD1 plasma. This dysfunctional phenotype could be prevented with the addition of an anti-TGFβ mAb or a TGFβ RI smi in culture. RNA-sequencing revealed a reduction in transcripts associated with mTOR effector functions, suggesting an impairment in mTOR. S6 phosphorylation was maintained with the addition of TGFβ-specific therapies. The hyporesponsive NK cell phenotype was reproduced upon culture of healthy NK cells with sxMDSCs and sxMDSCs were shown to produce soluble TGFβ in culture. Conclusion: Surgically stressed NK cells display a dysfunctional phenotype, which could be prevented in vitro through the addition of TGFβ-specific blocking therapies. sxMDSCs produced TGFβ and co- incubation induced dysfunction in healthy NK cells. The recovery of impaired S6 phosphorylation with TGFβ-specific therapies suggests that TGFβ is inducing NK cell dysfunction via inhibition of mTORC1 activity. The perioperative period of immunosuppression presents a window of opportunity for novel
therapeutics to prevent metastases and cancer recurrence among cancer surgery patients.
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Rato, Leila Sofia Coelho. "Vimentin interacts with the Akt/mTOR pathway mediating cell growth." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22372.
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Mestrado em Bioquímica - Bioquímica ClínicaA vimentina é uma proteína da classe III dos filamentos intermédios que promove processos tais como proliferação, migração e invasão celular através da interação com diferentes vias de sinalização. No entanto, o papel da vimentina no crescimento celular é ainda pouco conhecido. Neste estudo, observamos que fibroblastos isolados de embriões de ratinhos sem vimentina (Vim -/- MEFs) eram mais pequenos que o tipo normal (WT). Assim, o objetivo deste estudo era entender de que forma a vimentina regula o crescimento celular. Com recurso a modelos in vitro, técnicas de microscopia e técnicas bioquímicas descobrimos que Vim -/- MEFs tinham menor volume e concentração de proteínas quando comparadas com WT MEFs. Adicionalmente, a síntese proteica e ativação de mTORC1 estavam significativamente reduzidas em Vim -/- MEFs. Através de co-imunoprecipitação, descobrimos que a vimentina interage com os complexos mTORC2 e TSC. Assim, postulamos que a vimentina regula o crescimento celular por interação com proteínas da via de sinalização AKT/mTO
Vimentin is a type III intermediate filament protein that takes part in cell proliferation, migration and invasion, by acting as a signalling scaffold. The role of vimentin in cell growth, however, is poorly understood. We observed that vimentin knockout mouse embryonic fibroblasts (Vim -/- MEFs) were smaller than the wild type (WT). Therefore, this work aimed to understanding how vimentin regulates cell growth. Using in vitro models, imaging techniques and biochemical approaches, we have found that the volume and protein concentration of Vim -/- MEFs is lower when compared to WT MEFs. Further, protein synthesis and mammalian target of rapamycin complex 1 (mTORC1) activation was attenuated in Vim -/- MEFs. By co-immunoprecipitation we found that vimentin interacts with mammalian target of rapamycin complex 2 (mTORC2) and tuberous sclerosis protein complex (TSC) after insulin stimulation. Consequently, we postulate that vimentin regulates cell growth by interacting with proteins of the AKT/mTOR pathway