Dissertations / Theses on the topic 'TOR Serine-Threonine Kinases'

Orientadores: Sandra Martha Gomes Dias, Marília Meira Dias
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A proliferação celular comanda os processos de embriogênese e de crescimento do organismo, sendo essencial para a correta função de vários tecidos adultos. Apesar de ser importante para a homeostase do organismo, a sua desregulação compõe a força motriz do desenvolvimento tumoral. Somente nos últimos vinte anos começou a ser evidenciada a relação entre as vias de tradução de sinais estimuladas por fatores de crescimento e a reorganização da atividade metabólica, a qual precisa priorizar a biossíntese e o aumento da biomassa, processos essenciais para a divisão celular. Em células tumorais, o consumo de glutamina é aumentando concomitante ao aumento da atividade de glutaminase. Três isoenzimas de glutaminase são expressas na maioria dos tecidos (liver-type glutaminase, kidney-type glutaminase e glutaminase C), todavia pouco se sabe sobre a necessidade específica de cada uma delas para o metabolismo tumoral. Vários artigos recentes têm definido o papel da glutaminólise, ou metabolismo da glutamina e seus subprodutos, na ativação da mTOR. Neste sentido é uma hipótese válida imaginar que mTOR possa contra-regular glutaminase. Desta maneira, resolvemos investigar se mTOR atua na regulação da atividade de glutaminase. Para tanto, realizamos knockdown estável de PTEN em células MDA-MB 231 e verificamos que não o mesmo afetou os níveis protéicos de GAC e KGA, assim como não houve mudança na localização subcelular das isoformas. Cinética enzimática da fração mitocondrial desta linhagem revelou que o knockdown de PTEN levou à uma diminuição do KM da enzima sem alteração de Vmax. De acordo, o tratamento com rapamicina, inibidor da mTOR, elevou o KM para os níveis detectados nas células controles. A atividade de glutaminase de lisado total de MDA-MB 231, NIH 3T3, IMR90 e BJ5TA foi afetada pelo tratamento com rapamicina conforme julgado por ensaios de dose e tempo resposta. Mais, ensaios de privação de glicose, glutamina e de fatores de crescimento levaram à inibição de mTOR e concomitante redução da atividade de glutaminase. Somado a isso, o knockdown estável de TSC2 em MDA-MB 231 e BJ5TA, assim como o knockout de TSC2 em MEF, promoveu superestimulação de mTOR e foi capaz de aumentar a atividade de glutaminase. Dosagem de atividade de glutaminase de células MDA-MB 231 com knockdown de GAC, KGA ou GAC/KGA tratadas com rapamicina indicaram que mTOR possa agir em ambas as isoformas. Curioso foi que apenas células shGAC e shGAC/KGA apresentaram redução da fosforilação de S6K em Thr389 indicando que GAC ou o metabolismo de glutamina via esta isoforma, possa contra-regular mTOR. Em adição, na comparação entre PC3 e DU145, verificamos que DU145 apresentou maior expressão de GAC, maior consumo de glutamina, maior dependência de glutamina em seu crescimento, maior sensibilidade ao inibidor de glutaminase, BPTES, e por fim, se mostrou mais responsiva à metformina, ativador indireto de AMPK. A ativação de AMPK por metformina, um conhecido sensor de estresse energético, mostrou diminuir a atividade de glutaminase em célula de tumor de próstata, DU145, indicando uma potencial ação de AMPK na atividade de glutaminase
Abstract: Cell proliferation is crucial for embryogenesis and organism growth, being also essential for the proper function of several adult tissues. Although important for the homeostasis of the organism, its deregulation composes the driving force of tumor development. In the past twenty years the relationship between the processes of signal translation stimulated by growth factors and the reorganization of metabolic activity has become more evident. Growing cells need to prioritize the biosynthesis and biomass increase, processes essential for cell division. In tumor cells, the glutamine consumption is increased concurrently with the increasing in the glutaminase activity. Three glutaminase isoenzymes are expressed in most tissues (liver- type glutaminase, kidney -type glutaminase and glutaminase C), but not much is known about the necessity of each isoform for the tumor metabolism. Several recent papers have defined the role of glutaminolysis or glutamine metabolism in mTOR activation. So it is a valid hypothesis to speculate that mTOR can counter-regulate glutaminase. Thus, we decided to investigate whether mTOR can control glutaminase activity. To this end, we have made MDA - MB 231 cells stably knocked down for PTEN and verified no alteration in KGA and GAC protein levels, as well as there was no change on their subcellular location. Enzyme kinetics of the MDA-MB 231 mitochondrial fraction revealed that PTEN knockdown led to a decrease in the KM of the enzyme without changing Vmax. Accordingly, the treatment with rapamycin (mTOR inhibitor), led to an increase in KM back to the level detected in control cells. The glutaminase activity of MDA - MB 231, NIH 3T3, IMR90 and BJ5TA total cellular lysates was also affected by rapamycin treatment in a dose- and time-response fashion. Moreover, glucose, glutamine and growth factors deprivation promoted mTOR inhibition and concomitant reduction on glutaminase activity. Glutaminase activity of MDA-MB 231 cells knocked down for GAC, KGA or GAC/KGA and treated with rapamycin indicated that mTOR can regulate both isoforms. Curiously, it was only on GAC or GAC/KGA knocked down cells that we observed a decrease in S6K Thr 389 phosphorylation, which could indicate that GAC or the GAC dependent-glutamine metabolism is a specific mTOR counter-regulator. Accordling, stable TSC2 knockdown in MDA-MB 231 and BJ5TA, as well as TCS2 knockout in MEF cells, promoted overstimulation of mTOR and increasing on glutaminase activity. Moreover, a comparison between PC3 and DU145 revealed that DU145 has higher GAC expression, greater consumption of glutamine, is more dependent on glutamine for its growth, more sensitive to the inhibitor of glutaminase, BPTES, and more responsive to metformin, an indirect AMPK activator. The activation of AMPK by metformin, a known energy stress sensor, led to a decreased glutaminase activity in the prostate tumor cell line DU145 indicating a potential role of AMPK on glutaminase activity
Mestrado
Genetica Animal e Evolução
Mestra em Genética e Biologia Molecular

Orientador: Gonçalo Amarante Guimarães Pereira
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A produção de energia por meio de fontes renováveis é uma exigência atual para se atingir uma economia sustentável. Os organismos fotossintetizantes surgem nesse contexto como ferramentas importantes na produção de compostos carbônicos ricos em energia, com destaque para microalgas em que tais compostos podem atingir até 80% do peso seco. Entretanto, um fator ainda desfavorável para sua utilização é o seu baixo rendimento na produção de biomassa. A espécie Chlamydomonas reinhardtii, por exemplo, é capaz de duplicar apenas algumas vezes durante 24 horas. As vias que controlam o crescimento celular, portanto, são alvos promissores para modificação genética. Dentre essas vias, à via de sinalização sensível à rapamicina aparece como um controlador central. Com o intuito de entender melhor como esse controle é exercido ao nível da expressão gênica global, foi utilizado a ferramenta de sequenciamento de RNA em larga escala para obtenção dos transcriptomas de culturas (sincronizadas) sob inibição dessa via e na condição controle, em oito momentos ao longo de um ciclo celular de 24h. O controle exercido por essa via sobre o metabolismo e sobre o ciclo celular foi o foco das análises. Foi encontrado que a inibição da via da TOR é capaz de gerar uma resposta de direcionamento parcial do metabolismo para a produção de TAG em detrimento de moléculas complexas como proteínas. Esse direcionamento foi considerado parcial devido à ocorrência concomitante de reações catabólicas. Outros dados obtidos sugerem que a via da TOR, além de regular o metabolismo de uma maneira geral e diversas funções celulares, também exerce influência sobre o progresso do ciclo celular e sua inibição resulta no atraso do desenvolvimento das fases do ciclo. Diversos fatores reguladores da transcrição envolvidos no desenvolvimento, no crescimento e na regulação do ciclo celular, foram encontrados diferencialmente expressos e constituem possíveis genes chave no controle do crescimento. Eles representam alvos em potencial para modificação genética com intuito de otimizar as taxas de crescimento na primeira etapa do sistema de produção. Na busca de alternativas aos processos atuais de indução do acúmulo de cadeias carbônicas, os efeitos da combinação rapamicina e via da TOR representam uma abordagem interessante para pesquisas futuras para viabilização da utilização de microalgas como fonte de energia. Este estudo possibilitou um melhor entendimento da atuação da via da TOR no crescimento e progresso do ciclo celular em C. reinhardtii ao nível de expressão gênica
Abstract: The energy production through renewable sources is an actual demand for achieving a sustainable economy. In this context, photosynthesizing organisms come to light as important tools for the production of energy-rich carbonic compounds, especially the microalgae, in which these compounds can reach up to 80% of the dry weight. However, an unfavorable factor for its utilization is the low yield of biomass production. The species Chlamydomonas reinhardtii, for instance, is capable of achieving only some duplication after 24 hours. The pathways that control cell growth are therefore promising targets for genetic modification. Among them, the rapamycin-sensitive signaling pathway emerges as a central controller. With the aim of better understanding how this control is fulfilled by the means of global gene expression, the high throughput RNA sequencing technology was used. With it, the synchronized cultures transcriptome under the inhibition of this pathway and in the control condition, of eight points during a cellular cycle of 24 hours, were obtained. The metabolism and the cell cycle control by the TOR pathway was the main focus of the analysis. It was found that the inhibition of this pathway is capable to partially draw the metabolism towards TAG production to the detriment of producing more complex chains as proteins. This directing was considered partial due to the concomitant occurrence of catabolic reactions. Other data suggested that the TOR pathway, apart from the metabolism regulation in a general way and regulation of many other cellular functions, also influence the cell cycle progression and its inhibition retards the development of cell phases. Several transcription regulators involved in development, growth and cell cycle regulation were found out to be differentially expressed and are likely to constitute key genes in growth control. They represent potential targets for genetic modification aiming the optimization of growth rate in the first step of the production system. In the search for alternatives to the current process of inducing carbon chain accumulation, the effects of the combination between rapamycin and TOR pathway represent an interesting approach for future research intending to turn the utilization of microalgae as an energy source into a feasible option. This study enabled a better understanding of the role of the TOR pathway in growth and cell cycle progression of C. reinhardtii at the level of gene expression
Mestrado
Genetica de Microorganismos
Mestre em Genética e Biologia Molecular

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

Orientador: Gabriel Forato Anhê
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A obesidade é caracterizada pela deposição ectópica de gordura no fígado. Este acúmulo de gordura hepática (NAFLD) pode gerar consequências graves, como a hepatite não alcoólica (NASH), fator de ricos para carcino hepatocelular (HCC). A morte de hepatócitos, evento chave na evolução da NAFLD para NASH, é causada pelo excesso de nutrientes e é dependente do estresse de retículo endoplasmático (RE). O estresse no RE resulta no acúmulo de proteínas não processadas desencadeia a "unfolded protein response" (UPR), podendo gerar apoptose. A mTOR é formada basicamente por dois complexos: mTOR1 e mTOR2; ambos são sensíveis a nutrientes, a insulina e a rapamicina. O complexo mTOR2/Rictor catalisa a fosforilação da AKT, aumentando a sinalização da insulina. Deste modo, o objetivo deste trabalho foi avaliar a relação entre ativação da mTOR, do estresse de RE e da apoptose em hepatócito expostos a ácidos graxos livres. Observamos que a apoptose causada pelo palmitato ativa o estresse de RE de maneira tempo dependente. Não observamos alterações na fosforilação de proteínas alvo específicas para o complexo mTOR1. No entanto, a fosforilação geral da mTOR foi estimulada pelo palmitato. Altas doses de rapamicina inibiram a apoptose e do estresse de RE causado pelo palmitato, sugerindo a participação do complexo mTOR2. Estes resultados ainda foram confirmados pelo silenciamento gênico da Rictor. A fosforilação em serina 473 da AKT apresenta um caráter transitório, elevando-se em tempos que precedem morte e o estresse de RE, e diminuindo em tempos prolongados concomitantemente à apoptose. A inibição da AKT pelo "AKT inhibitor" gerou diminuição da apoptose, do estresse de RE e da incorporação lipídica na linhagem de hepatoma. Estes dados sugerem que a AKT, como alvo preferencial da mTOR2 é necessária para geração de morte e da UPR. A glicose (33.3mM) gera morte as células HepG2 e esta é inibida com baixas doses de rapamicina, mostrando possível atividade via mTOR1 nesta resposta. De outro modo, a frutose (4.5mM) que também desencadeia apoptose das células de hepatoma, tem seu efeito inibido por doses maiores de rapamicina, indicando atividade mTOR2 neste processo. No entanto, a possibilidade de diferentes monossacarídeos recrutarem complexos diferentes de mTOR para desencadear apoptose ainda precisa ser melhor explorada
Abstract: Obesity is characterized by fat ectopic deposition in liver. This hepatic fat accumulation our non-alcoholic fat liver disease (NAFLD) can have serious consequences such as non-alcoholic hepatitis (NASH), that is a factor to liver cancer. The cell death of hepatocytes is an important event in the development to NAFLD to NASH, all that are caused by excess nutrients and dependent of endoplasmic reticulum (ER) stress. The ER stress is caused by accumulation of unfolded proteins triggers the unfolded protein response (UPR), which mau cause apoptosis. mTOR is basically formed by two complexes: mTOR1 and mTOR2, both are sensitive to nutrients, insulin and rapamycin. The mTOR2/Rictor complex catalyse AKT phosphorylation increasing the insulin pathway. All together, the aim of this study was evaluate the relationship between mTOR, ER stress and apoptosis in liver cells exposed to free fatty acids. We observed that apoptosis caused by palmitate activates ER stress in a manner dependent on time. We din¿t observed changes in phosphorylation of specific target proteins to mTOR1 complex. However, a general phosphorylation of mTOR was stimulated by palmitate. High doses of rapamycin inhibited apoptosis and ER stress caused by palmitate, suggesting the participation of the mTOR2 complex. These results were further confirmed by gene silencing of Rictor. The AKT phospholylation in serine 473 has a transitional character, rising in times that preceding cell death and ER stress, and decreasing concomitantly apoptosis in prolonged times. Inhibition of AKT by AKT inhibitor caused a decrease in apoptosis, ER stress and lipid incorporation in hepatoma cell line. These data suggest that AKT, preferential targets of mTOR2 is required for generation death and UPR. Glucose (33.3mM) generates HepG2 cell death and this is inhibited by low doses on rapamycin, showing possible mTOR1 activity. Otherwise, fructose (4.5mM) also triggers apoptosis of hepatoma cells; its effect is inhibited by higher doses of rapamycin, indicating mTOR2 activity in this process. However, the possibility of different monosaccharide recruit different complexes of mTOR to trigger apoptosis should be further explored
Doutorado
Farmacologia
Doutor em Farmacologia

Retinitis Pigmentosa (RP) is an inherited photoreceptor degenerative disease that leads to blindness and affects about 1 in 4000 people worldwide. The disease is predominantly caused by mutations in genes expressed exclusively in the night active rod photoreceptors; however, blindness results from the secondary loss of the day active cone photoreceptors, the mechanism of which remains elusive. Here, we show that the mammalian target of rapamycin complex 1 (mTORC1) is required to delay the progression of cone death during disease and that constitutive activation of mTORC1 is sufficient to maintain cone function and promote cone survival in RP. Activation of mTORC1 increased expression of genes that promote glucose uptake, retention and utilization, leading to increased NADPH levels; a key metabolite for cones. This protective effect was conserved in two mouse models of RP, indicating that the secondary loss of cones can be delayed by an approach that is independent of the primary mutation in rods. However, since mTORC1 is a negative regulator of autophagy, its constitutive activation led to an unwarranted secondary effect of shortage of amino acids due to incomplete digestion of autophagic cargo, which reduces the efficiency of cone survival over time. Moderate activation of mTORC1, which promotes expression of glycolytic genes, as well as maintains autophagy, provided more sustained cone survival. Together, our work addresses a long-standing question of non-autonomous cone death in RP and presents a novel, mutation-independent approach to extend vision in a disease that remains incurable.

Retinitis Pigmentosa (RP) is an inherited photoreceptor degenerative disease that leads to blindness and affects about 1 in 4000 people worldwide. The disease is predominantly caused by mutations in genes expressed exclusively in the night active rod photoreceptors; however, blindness results from the secondary loss of the day active cone photoreceptors, the mechanism of which remains elusive. Here, we show that the mammalian target of rapamycin complex 1 (mTORC1) is required to delay the progression of cone death during disease and that constitutive activation of mTORC1 is sufficient to maintain cone function and promote cone survival in RP. Activation of mTORC1 increased expression of genes that promote glucose uptake, retention and utilization, leading to increased NADPH levels; a key metabolite for cones. This protective effect was conserved in two mouse models of RP, indicating that the secondary loss of cones can be delayed by an approach that is independent of the primary mutation in rods. However, since mTORC1 is a negative regulator of autophagy, its constitutive activation led to an unwarranted secondary effect of shortage of amino acids due to incomplete digestion of autophagic cargo, which reduces the efficiency of cone survival over time. Moderate activation of mTORC1, which promotes expression of glycolytic genes, as well as maintains autophagy, provided more sustained cone survival. Together, our work addresses a long-standing question of non-autonomous cone death in RP and presents a novel, mutation-independent approach to extend vision in a disease that remains incurable.

Orientador: Jose Barreto Campello Carvalheira
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A proteína mTOR é um proteína reguladora chave de vários processos celulares, dentre eles proliferação, crescimento e sobrevivência celular. Fatores de crescimento, oxigênio, status energético e a presença de aminoácidos são fundamentais para que todos esses processos ocorram normalmente. Descobertas realizadas nas últimas décadas mostraram que a via da mTOR encontra-se ativada em vários processos celulares, incluindo formação tumoral e angiogênese. A leucina é um aminoácido de cadeia ramificada que tem o maior potencial em ativar a via da mTOR. Devido sua capacidade de promover a síntese proteica e ganho de massa muscular, seu uso é constantemente estimulado em pacientes com câncer. No entanto, seus efeitos no crescimento tumoral não está claro. Dessa forma, realizamos um estudo cujo objetivo principal foi investigar os efeitos da dieta suplementada com leucina na modulação do crescimento tumoral em diferentes linhagens de células tumorais que se diferenciem em relação à ativação constitutiva da via IRS1/Akt/mTOR. Estudos in vivo e in vitro realizados demonstraram que as células que se diferenciam em relação à ativação da via IRS1/AKT/mTOR respondem de maneira distinta à suplementação com leucina. Linhagens de células tumorais que possuem a via da mTOR constitutivamente ativada, PC-3 e MCF-7, quando suplementadas com doses elevadas de leucina in vitro reduziram a proliferação celular e causaram retenção das células na fase G1 do ciclo celular. Já o xenoenxerto tumoral da PC-3 reduziu sua proliferação e aumentou a morte celular quando os animais foram suplementados com leucina na dieta. Nós também observamos aumento da atividade da mTOR e da p70S6K em todas as linhagens celulares quando suplementadas com leucina. O aumento da atividade da proteína mTOR foi acompanhado de redução na fosforilação de AKTser473 nas células que possuíam a via da PI3K hiperativada (PC-3 e MCF-7). Esse fato pode estar ocorrendo devido a ativação das alças de contraregulação ocasionadas pela estimulação excessiva provocada pela suplementação com leucina, naquelas linhagens celulares que já possuem a via hiperativada. Fato este comprovado pelo aumento da fosforilação em serina 307 da proteína IRS1. Dessa forma, nossos resultados sugerem que a ativação da via da mTOR é central para determinar a sensibilidade de tumores à dieta suplementada com leucina, podendo modular o desenvolvimento tumoral naquelas células que já possuem a via IRS1/AKT/mTOR constitutivamente ativada. O mecanismo pelo qual a leucina pode retardar o desenvolvimento tumoral em células que possuem a via da mTOR hiperativada parece estar relacionado com o eixo de regulação negativa p70S6K-PI3K, com consequente redução da fosforilação de AKT e liberação das vias apoptóticas nos tecidos tumorais
Abstract: mTOR is a key regulatory protein in various cellular processes including proliferation, cell growth and survival. Growth factors, oxygen, energy status and amino acids are all essential to these processes. New findings in the last few decades have shown that the mTOR pathway is activated in many cellular processes, including tumorigenesis and angiogenesis. The branched chain amino acid leucine has the greatest potential to activate the mTOR pathway. Due to its ability to promote protein synthesis and muscle mass gain, use of leucine is frequently utilized in patients with cancer. However, the effect of leucine on tumor growth is not clear. The aim of this study is therefore to investigate the effect of diet-supplemented leucine on the modulation of tumor growth in several tumor cell lines that differ in the constitutive activation status of the insulin receptor substrate 1 (IRS1)/AKT/mTOR pathway. Both in vitro and in vivo experiments demonstrated different cell proliferation responses when cells were exposed to high doses of leucine. Tumor cell lines PC-3 and MCF-7, which have a constitutively activated mTOR signaling, displayed reduced cell proliferation and G1 phase cell cycle arrest when supplemented with high doses of leucine in vitro. Likewise, leucine-supplemented PC-3 cell tumor xenografts displayed reduced proliferation and increased cell death. We also observed increased activity of mTOR and its downstream substrate p70S6K in all cell lines supplemented with leucine. Increased mTOR activity was accompanied by a reduction in AKT serine 473 (ser473) phosphorylation in cell lines with a hyperactivated PI3K pathway (PC-3 and MCF-7). This most likely occurred because leucine supplementation further increased mTOR and p70S6K activity, triggering the inhibitory p70S6K/IRS1 axis. In fact, we found increased IRS1 ser307 phosphorylation in hyperactivated cell lines (PC-3 and MCF-7) supplemented with high doses of leucine. Therefore, our results suggest that mTOR pathway activation is central to determining the sensitivity of tumors to leucine supplementation. Furthermore, this could affect the response to leucine-supplemented therapies of those tumors in which the PI3K pathway is constitutively activated. The mechanism for this appears to be related to the negative p70S6K/IRS1 regulation axis, with consequent reduction of AKT phosphorylation and the release of apoptotic pathways in tumor tissues
Doutorado
Fisiopatologia Médica
Doutora em Ciências

Recent studies suggest adipose tissue plays a critical role in regulating whole body energy homeostasis in both animals and humans. In particular, activating brown adipose tissue (BAT) activity is now appreciated as a potential therapeutic strategy against obesity and metabolic disease. However, the signaling circuits that coordinate nutrient uptake and BAT function are poorly understood. Here, I investigated the role of the nutrient-sensing mTOR signaling pathway in BAT by conditionally deleting Rictor, which encodes an essential component of mTOR Complex 2 (mTORC2) either in brown adipocyte precursors or mature brown adipocytes. In general, inhibiting BAT mTORC2 reduces glucose uptake and de novo lipogenesis pathways while increases lipid uptake and oxidation pathways indicating a switch in fuel utilization. Moreover, several key thermogenic factors (Ucp1, Pgc1α, and Irf4) are elevated in Rictor-deficient BAT, resulting in enhanced thermogenesis. Accordingly, mice with mTORC2 loss in BAT are protected from HFD-induced obesity and metabolic disease at thermoneutrality. In vitro culture experiments further suggest that mTORC2 cell-autonomously regulates the BAT thermogenic program, especially Ucp1 expression, which depends on FoxO1 activity. Mechanistically, mTORC2 appears to inhibit FoxO1 by facilitating its lysine-acetylation but not through the canonical AKT-mediated phosphorylation pathway. Finally, I also provide evidence that β-adrenergic signaling which normally triggers thermogenesis also induces FoxO1 deacetylation in BAT. Based on these data, I propose a model in which mTORC2 functions in BAT as a critical signaling hub for coordinating nutrient uptake, fuel utilization, and thermogenic gene expression. These data provide a foundation for future studies into the mTORC2-FoxO1 signaling axis in different metabolic tissues and physiological conditions.

Recent studies suggest adipose tissue plays a critical role in regulating whole body energy homeostasis in both animals and humans. In particular, activating brown adipose tissue (BAT) activity is now appreciated as a potential therapeutic strategy against obesity and metabolic disease. However, the signaling circuits that coordinate nutrient uptake and BAT function are poorly understood. Here, I investigated the role of the nutrient-sensing mTOR signaling pathway in BAT by conditionally deleting Rictor, which encodes an essential component of mTOR Complex 2 (mTORC2) either in brown adipocyte precursors or mature brown adipocytes. In general, inhibiting BAT mTORC2 reduces glucose uptake and de novo lipogenesis pathways while increases lipid uptake and oxidation pathways indicating a switch in fuel utilization. Moreover, several key thermogenic factors (Ucp1, Pgc1α, and Irf4) are elevated in Rictor-deficient BAT, resulting in enhanced thermogenesis. Accordingly, mice with mTORC2 loss in BAT are protected from HFD-induced obesity and metabolic disease at thermoneutrality. In vitro culture experiments further suggest that mTORC2 cell-autonomously regulates the BAT thermogenic program, especially Ucp1 expression, which depends on FoxO1 activity. Mechanistically, mTORC2 appears to inhibit FoxO1 by facilitating its lysine-acetylation but not through the canonical AKT-mediated phosphorylation pathway. Finally, I also provide evidence that β-adrenergic signaling which normally triggers thermogenesis also induces FoxO1 deacetylation in BAT. Based on these data, I propose a model in which mTORC2 functions in BAT as a critical signaling hub for coordinating nutrient uptake, fuel utilization, and thermogenic gene expression. These data provide a foundation for future studies into the mTORC2-FoxO1 signaling axis in different metabolic tissues and physiological conditions.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.

Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related deaths. Systemic therapies are the main treatment options for HCC patients with advanced disease (∼ 80% of all cases). However, only very moderate clinical responses are achieved with most of the conventional therapies. Thus, more effective therapeutic strategies are much needed. The PI3K/Akt/mTOR signaling pathway, which plays a critical role in controlling cell proliferation and survival, is aberrantly activated in ∼ 45% HCC, suggesting it to be a potential target for HCC treatment. Moreover, emerging evidences indicate that activation of the PI3K/Akt/mTOR pathway may be associated with resistance to many cytotoxic chemotherapies, including microtubule targeting agents. In this study, by gene expression profiling and gene ontology analysis, "microtubule-related cellular assembly" was identified to be the major biological/functional process involved in HCC development, suggesting that microtubule is also an important therapeutic target for HCC. With these understandings, it is hypothesize in this thesis that combined targeting of a key component ofthe PI3K/Akt/mTOR pathway, namely the mammalian target of rapamycin (mTOR) and the microtubule would be an effective therapeutic strategy for HCC. The objectives of the thesis are to examine the therapeutic potential of microtubule targeting, mTOR targeting, and combined targeting of the microtubule and mTOR in both in vitro and in vivo models of HCC.
In summary, the PI3K/Akt/mTOR pathway and the microtubule represent promising therapeutic targets for HCC treatment. The findings from this thesis offer a rationale for combining mTOR inhibitors with microtubule targeting agents for effective HCC treatment.
In the second part, the effect of mTOR inhibition, either alone or in combination with an additional microtubule targeting agent (vinblastine) was investigated in HCC. Temsirolimus, an mTOR inhibitor, suppressed HCC cell proliferation in as early as 24 hrs with an IC50 of 1.27+/-0.06muM (Huh7), 8.77+/-0.76muM (HepG2), and 52.95+/-17.14muM (Hep3B). Vinblastine (1nM) alone caused 30--50% growth inhibition in 3 HCC cell lines. In these HCC cell lines, it was found that temsirolimus/vinblastine combination resulted in an additive to synergistic effect (when compared to single agents alone) with maximum growth inhibition of 80--90% as early as 24 hrs upon treatment. This marked growth inhibition was accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage (a hallmark for apoptosis). Moreover, the combination specifically caused concerted down-regulation of several important anti-apoptotic and survival proteins (survivin, Bcl-2 and Mcl-1), which was not observed in single agent treatments. It was hypothesized that inhibition of these key anti-apoptotic/survival proteins may represent a novel mechanistic action of this highly effective combination approach of dual targeting of mTOR and microtubule by temsirolimus/vinblastine in HCC cells. Indeed, transient over-expression of each of these genes (survivin, Bcl-2 or Mcl-1) in HCC cells did partially rescue the growth inhibitory effect of the temsirolimus/vinblastine combination. More importantly, this novel combination significantly suppressed the growth of HCC xenografts in nude mice (when compared with single agents alone).
In the third part, the anti-tumor effect of another mTOR inhibitor everolimus in combination with microtubule targeting agents, vinblastine and patupilone (a microtubule-stabilizing agent), was investigated in HCC cells. Everolimus/vinblastine combination resulted in an additive to synergistic effect accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage. The combination also caused concerted down-regulation of anti-apoptotic and survival proteins (survivin, Bel-2 and Mel-1) as observed with the temsirolimus/vinblastine combination. However, everolimus only moderately enhanced the sensitivity of patupilone for reasons unknown.
Taxanes are the major chemotherapeutic agents that target the microtubule. In the first part of the thesis, the anti-tumor activity of two taxanes, paclitaxel and docetaxel (which are known to stabilize microtubules) was examined and compared with doxorubicin (a DNA intercalating agent). Across all three HCC cell lines tested, it was found that the microtubule targeting agents, taxanes, were more efficacious than doxorubicin. This supports the initial finding that microtubule assembly process is functionally important in HCC. Recent studies demonstrated that using nanoparticles for drug delivery can greatly enhance therapeutic efficacy and reduce side-effects. Therefore, the nanoparticle albumin-bound (nab)-paclitaxel was employed to further evaluate the therapeutic efficacy of such a delivery strategy in HCC models. In all three HCC cell lines tested, nab-paclitaxel was found to be the most effective agent, with an average IC50 value of 0.16--10.42nM, when compared to non-conjugated taxanes (paclitaxel, docetaxel) and doxorubicin. In vitro analysis showed that nab-paclitaxel was able to induce cell cycle arrest at G2/M phase and apoptosis in HCC cells. In vivo study demonstrated that nab-paclitaxel readily inhibited the growth of HCC xenografts with lower toxicity when compared to paclitaxel, docetaxel and doxorubicin. Moreover, specific silencing of a key regulatory protein for microtubule dynamics, Stathmin 1, by siRNA significantly enhanced the effect of nab-paclitaxel in HCC cells, resulting in synergistic growth inhibition in vitro.
Zhou, Qian.
Advisers: Winnie Yeo; Vivian Lui; Nathalie Wong.
Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 148-164).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Trabalho de Projeto do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Neurodegenerative diseases are devastating conditions for both patients and caregivers and their sporadic forms are increasingly common with population ageing. Autophagy has conquered a relevant role in the study of pathological mechanisms associated with neurodegenerative disorders and their management. In this review, the latest results on the study of autophagic modulation in neurodegenerative processes are compiled and put in a broader context. Autophagy was shown to be impaired in both human neuronal tissues of affected patients and cellular and animal models of disease as it was evident that pharmacological enhancement of autophagy alleviates neurodegeneration in animal models of such diseases. A few studies however evidenced a more complex network where overactive or deregulated autophagic flux might be harmful. The disclosure of the autophagic machinery has proven to be one of the most important breakthroughs in modern medicine and thus modulating autophagy to relief neurodegeneration is a central field for present and future research and future therapeutic options for neurodegenerative diseases. ....................................................................................... ...................................................................................... ....................................................................................... ........................................................................................ .......................................................................................
As doenças neurodegenerativas constituem situações especialmente desgastantes para doentes e cuidadores e as suas formas esporádicas crescem em prevalência a par do envelhecimento da população. A autofagia é um processo celular de reconhecida importância tanto no processo etiológico como no tratamento desta patologia. Nesta revisão são compilados os resultados mais recentes sobre a modulação do fluxo autofágico nos processos neurodegenerativos. Observou-se que a autofagia está desregulada em células nervosas de doentes e em modelos celulares e animais de degeneração neuronal, sendo que foi evidente que a estimulação farmacológica da mesma melhorou o desempenho físico e cognitivo de modelos animais para estas doenças. Contudo, alguns estudos evidenciaram um processo mais complexo em que o fluxo autofágico aumentado ou desregulado pode funcionar como agente patológico. A descoberta da maquinaria autofágica tem-se revelado um dos avanços mais importantes da medicina moderna e portanto a modulação da autofagia no alívio da neurodegeneração é uma área central na investigação presente e futura e no desenvolvimento de futuras estratégias terapêuticas. ........................................................................................ (o resumo em inglês e a sua versão portuguesa encontram-se em acordo com as normas estabelecidas previamente pela FMUC e onde não constava um mínimo de 1500 caracteres; também o número limite de palavras-chave, aqui de cinco, não estava previsto e no trabalho encontram-se oito)

Trabalho de Projeto do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Neste trabalho fazemos uma revisão da literatura mais recente sobre o tema da restrição calórica na sua relação com o equilíbrio de nutrientes. Daqui resulta a ideia de que é no contexto da investigação do equilíbrio de nutrientes que a restrição calórica joga o seu papel central, e não como estratégia isolada. Estes estudos, cada vez mais pormenorizados, avançam no sentido de que o contributo das proporções ideais de nutrientes na longevidade é o mais importante. O entrecruzamento de vários mecanismos e vias de transdução de sinais de nutrientes e de energia está implicado na proporção dietética testada desde as leveduras, a moscas da fruta e a mamíferos. Daqui resulta uma explicação plausível dos resultados da “dieta tradicional de Okinawa”. A via de sinalização da glicose, nuclear nas dietas de restrição calórica, e as vias de sinalização de aminoácidos, mediante o entrecruzamento das vias GCN2 (General Control Nonderepressible 2) e mTOR (Mammalian/Mechanistic Target Of Rapamycin), estão implicadas na regulação da longevidade verificada nos regimes de restrição de proteínas com alto teor de hidratos de carbono LPHC (Low Protein, High Carb), numa proporção dietética semelhante à verificada na dieta tradicional de Okinawa.Neste trabalho fazemos uma revisão da literatura mais recente sobre o tema da restrição calórica na sua relação com o equilíbrio de nutrientes. Daqui resulta a ideia de que é no contexto da investigação do equilíbrio de nutrientes que a restrição calórica joga o seu papel central, e não como estratégia isolada. Estes estudos, cada vez mais pormenorizados, avançam no sentido de que o contributo das proporções ideais de nutrientes na longevidade é o mais importante. O entrecruzamento de vários mecanismos e vias de transdução de sinais de nutrientes e de energia está implicado na proporção dietética testada desde as leveduras, a moscas da fruta e a mamíferos. Daqui resulta uma explicação plausível dos resultados da “dieta tradicional de Okinawa”. A via de sinalização da glicose, nuclear nas dietas de restrição calórica, e as vias de sinalização de aminoácidos, mediante o entrecruzamento das vias GCN2 (General Control Nonderepressible 2) e mTOR (Mammalian/Mechanistic Target Of Rapamycin), estão implicadas na regulação da longevidade verificada nos regimes de restrição de proteínas com alto teor de hidratos de carbono LPHC (Low Protein, High Carb), numa proporção dietética semelhante à verificada na dieta tradicional de Okinawa.
In this paper we reviewed the most recent literature on the subject of caloric restriction in its relation to the macronutrients balance. From the review comes the suggestion that it is in the context of nutrient balance and ideal ratios that caloric restriction plays its central role, and not as an isolated strategy. This idea corresponds to a scientific shift towards the detailed investigation of the contribution of optimal nutrient ratios on longevity. The interweaving of various mechanisms and signal transduction pathways of nutrient and energy is involved in the dietary proportion tested in yeast, fruit flies and mammals. From the investigation of these mechanisms comes a plausible explanation of the results of "traditional Okinawa diet". The signaling pathways of glucose, nuclear in energy-restricted diets, and of amino acids detection, through the intersection of GCN2 (General Control Nonderepressible 2) and mTOR (Mammalian/Mechanistic Target Of Rapamycin) pathways, are implicated in the regulation of longevity recorded in the LPHC restriction systems (Low Protein, High Carb), a dietary proportion similar to that of the traditional Okinawa diet.In this paper we reviewed the most recent literature on the subject of caloric restriction in its relation to the macronutrients balance. From the review comes the suggestion that it is in the context of nutrient balance and ideal ratios that caloric restriction plays its central role, and not as an isolated strategy. This idea corresponds to a scientific shift towards the detailed investigation of the contribution of optimal nutrient ratios on longevity. The interweaving of various mechanisms and signal transduction pathways of nutrient and energy is involved in the dietary proportion tested in yeast, fruit flies and mammals. From the investigation of these mechanisms comes a plausible explanation of the results of "traditional Okinawa diet". The signaling pathways of glucose, nuclear in energy-restricted diets, and of amino acids detection, through the intersection of GCN2 (General Control Nonderepressible 2) and mTOR (Mammalian/Mechanistic Target Of Rapamycin) pathways, are implicated in the regulation of longevity recorded in the LPHC restriction systems (Low Protein, High Carb), a dietary proportion similar to that of the traditional Okinawa diet.

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-alpha4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Trabalho final de mestrado integrado em Medicina, apresentado à Faculdade de Medicina da Universidade de Coimbra.
O envelhecimento e a longevidade humana são, fenómenos complexos determinados por vias fisiológicas influenciadas por múltiplos fatores - genéticos, comportamentais, dietéticos, ambientais, e estocásticos. O aumento da esperança média de vida nas últimas décadas traduziu-se num envelhecimento da população com um enorme impacto social e económico. O envelhecimento determina modificações no organismo humano que conduzem a uma maior vulnerabilidade e maior incidência de patologias crónicas características do idoso. Os mecanismos fisiopatológicos relacionados com o envelhecimento e abordados neste trabalho, como a informação genética, o stress oxidativo, a atividade do eixo mTOR (do inglês mammalian target of rampamycin) e a atividade das telomerases, parecem ser determinantes neste processo, segundos vários estudos de investigação conduzidos em animais. Apesar da investigação em seres humanos também demonstrar esta relação, são necessários mais estudos em vertebrados superiores, como os mamíferos e primatas não-humanos, sobretudo na área do mTOR. É evidente neste trabalho de revisão, a interligação dos mecanismos determinantes do envelhecimento e da longevidade e as possíveis intervenções preventivas, incluindo a dieta, a restrição calórica e os seus miméticos e o exercício físico. A qualidade da dieta e o exercício físico têm um papel consensual na prevenção do desenvolvimento de patologias características do envelhecimento e na promoção de um envelhecimento com qualidade de vida, com independência funcional e livre de comorbilidades. Têm sido registados alguns avanços sobre a influência positiva da restrição calórica (RC), sobretudo quando associada a uma dieta equilibrada, no atraso do envelhecimento. Neste estudo podemos concluir que a melhor forma de promover um envelhecimento saudável e um aumento da longevidade, é a conjugação de várias intervenções terapêuticas, incluindo uma dieta de qualidade, prática regular de exercício físico adequado ao indivíduo e à idade e possivelmente algum nível de RC. De referir ainda, o importante papel que os biomarcadores de envelhecimento poderão vir a ter no futuro para monitorizar a aplicação destas e outras terapias/intervenções.
Human aging and human longevity are complex phenomena determined by physiological pathways, influenced by multiple factors - genetic, behavioral, dietary, environmental, and stochastic. The increase in life expectancy in recent decades has led to population aging, with an enormous social and economic impact. Aging determines changes in the human body leading to greater vulnerability and greater incidence of chronic conditions characteristic of the elderly. The pathophysiological mechanisms of aging discussed in this work, such as genetic information, oxidative stress, the activity of mTOR pathway and the telomerase activity, seem to be crucial in this process, according to several research studies conducted on animals. Although research in humans also shows this relationship, further studies are needed in higher vertebrates, such as mammals and non-human primates, especially in mTOR area. The interconnection of the determining mechanisms of aging and longevity and possible preventive therapeutic interventions, including diet, caloric restriction and its mimetics and physical activity are evident in this review. Diet composition and physical exercise have an agreed role in preventing the development of age-associated diseases and promoting healthy aging with quality of life, functional independence and without comorbidities. Some advances about the positive influence of caloric restriction (CR), especially when combined with a balanced diet, on aging delay, have been registered. In this study we can conclude that, the best way to promote healthy aging and increasing longevity, is a combination of several therapeutic interventions, including a healthy diet, regular practice of exercise appropriate to the individual and possibly some level of CR. It should be noted, the important role that biomarkers of aging are likely to have in the future to monitor the implementation of these and other therapies / interventions.