La sarcopénie (faible masse musculaire) est associée à l’inflammation silencieuse et à une augmentation du risque d’incapacité physique chez la population vieillissante. Une des interventions non pharmacologiques les mieux appuyées scientifiquement pour ralentir la sarcopénie est l’entraînement en résistance. Pour être en mesure de dépister la sarcopénie et évaluer l’efficacité des interventions qui s’y adressent, nous avons besoin d’outils qui permettent de quantifier la masse musculaire. Bien qu’elles soient issues de technologies avancées, les techniques d’imagerie actuelles quantifient la masse musculaire de façon « statique » ; c’est-à-dire qu’elles illustrent ses caractéristiques à un moment précis dans le temps, de manière figée. Or, la masse musculaire est en réalité le résultat d’une balance entre la dégradation et la synthèse des protéines musculaires, deux processus hautement dynamiques sur le plan métabolique. Récemment, les extracellular heat shock proteins 72 (eHSP72) – protéines de choc thermiques – ont été proposées comme biomarqueur de sarcopénie. En effet, des niveaux élevés d’eHSP72 circulants seraient un indicateur de dégradation musculaire et ont été associés à de faibles valeurs de masse musculaire. Ce biomarqueur offre l’avantage de refléter un processus intracellulaire dynamique et indique vraisemblablement qu’un individu se trouve en situation de perte de masse musculaire. Cette association entre eHSP72 et masse musculaire a été proposée comme étant modulée par l’inflammation systémique dans une population cachectique. Toutefois, l’évolution d’eHSP72 n’a jamais été étudiée chez une population sarcopénique en santé à la suite d’une intervention visant spécifiquement l’augmentation de la masse musculaire. L’adéquation entre l’évolution d’un biomarqueur et celle de son vis-à-vis clinique est importante pour justifier la pertinence de son utilisation en contexte longitudinal. Ce projet de maîtrise vise à : 1) évaluer l’effet d’un entraînement en résistance sur l’évolution du biomarqueur eHSP72 et de la masse maigre d’individus sarcopéniques en santé; 2) déterminer s’il existe des relations entre les changements d’eHSP72, de masse maigre et des niveaux de marqueurs inflammatoires circulants. Au total, 26 hommes sarcopéniques en santé ont complété 16 semaines d’entraînement et les variables suivantes ont été mesurées avant et après l’intervention : a) niveaux plasmatiques d’eHSP72 (ELISA), b) masse maigre (DXA), c) indice de masse musculaire appendiculaire [IMMapp : masse maigre appendiculaire (kg) / taille (m)[indice supérieur 2] ], d) niveaux sériques d’interleukine-6 (IL-6), protéine c-réactive (CRP) et facteur de nécrose tumorale alpha (TNF-α) (ELISA haute sensibilité). Des concentrations d’eHSP72 ont été détectées chez 9/19 (47%) des participants en préintervention. Une diminution des niveaux d’eHSP72 a été observée à la suite de l’intervention (p=0,04) parallèlement à l’augmentation de l’IMMapp et de plusieurs variables de masse maigre (Ps ≤ 0,035). Une tendance intéressante a été soulevée entre l’évolution d’eHSP72 (diminution), hsIL-6 (diminution) et IMMapp (augmentation), sans corrélation significative entre les changements de ces variables, possiblement en raison d’un manque de puissance statistique. Ces résultats sont les premiers à démontrer l’intérêt d’utiliser eHSP72 comme biomarqueur de sarcopénie dans un devis longitudinal puisque son évolution est en adéquation avec celle de la masse maigre et de l’IMMapp, deux mesures cliniques pertinentes chez une population sarcopénique. Toutefois, les technologies ou protocoles utilisés pour son dosage doivent être raffinés, car nos données indiquent un faible taux de détection. Finalement, la tendance observée entre eHSP72, masse maigre et hsIL-6 indique que, même à des niveaux très bas d’inflammation systémique, des interactions sont possibles dans l’évolution de ces variables. Cette hypothèse mérite à notre avis d’être approfondie et possiblement confirmée chez une plus large population.

The body initiates an immune response to danger signals. The Danger model of the immune system postulates that danger signals are produced by exogenous molecules from foreign invaders, such as bacteria, and endogenous molecules released from damaged or injured cells. The response involves antigen recognition leading to up-regulation of cytokines and cell surface markers, followed by the recruitment of antigen presenting cells and T-helper cells which determine how the immune system responds. Endogenous danger signals include Hsp72 and HMGB-1. This thesis describes the development of specific antibodies and ELISAs for use in the quantification and detection of intra-cellular Hsp72 from cell extracts, and released Hsp72 from cell cultures which enabled the confirmation of physiological levels of Hsp72 from model systems. The ability of endogenous Hsp72 to stimulate an immune response was demonstrated and this response was not solely due to LPS contamination of recombinant protein preparations. Hsp72 was able to augment the response to LPS. In the presence of another endogenous danger signal, HMGB-1, relative amounts of Hsp72 were shown to augment a pro-inflammatory response whilst being able to maintain an anti-inflammatory response demonstrating Hsp72 has the ability to modulate the immune response. Hsp72 was also shown to be able to stimulate an immune response by binding to cell surface receptors, which could be blocked by specific peptides corresponding to known receptors. These include some receptors not utilised by LPS. The proportion of these different danger signals has consequences for the progression and outcome of an immune response and this may well be modulated by imposition of a supplemental or future stress at different points. In the most severe case, this can lead to death through sepsis following trauma.

In recent years, compelling evidence has accumulated suggesting heat shock proteins (HSPs) which are generally believed to be localised and functioning mainly within eukaryotic cells as cyto-protective molecular chaperones, are also localised in the extracellular milieu. Depending on their localisation, on the cell surface (membrance-bound or embedded), or in the peripheral circulation, extracellular HSPs may induce apoptotic cell death, or in contrast protect cells from cell damage and/or cell death when exposed to cellular stress, or may even elicit a stimulatory effect on the innate immune response including cell activiation and cytokine secretion. Hence, the localisation of intracellular and extracellular HSPs appears to be critical in determining their roles in terms of stimulating cell death, cyto-protection, or immune activiation under normal physiological conditions and following exposure to stress stimuli. This thesis describes the intracellular expression, up-regulation, and cell surface localisation of endogenous HSPs: HSP27, Hsp60, Hsp72 and Hsp90 by flow cytometry, florescence microscopy and Western blotting, under control conditions and in response to environmental stress using in vitro and ex vivo models with the intention of determining their physiological roles. The ability of extracellularly administered HSPs (Hsp70 and Hsp72) to protect cultured U937 cells in vitro or peripheral primary human leukogytes or erythrocytes ex vivo from various stress stimuli was demonstrated and was found to be dependent on surface binding and/or internalisation via scavenger receptors (SRs) or phosphatidylserine (PS), which could be blocked by receptor specific ligands. Extracellular HSPs were also shown to be able to stimulate an immune response through the induction of U937 monocyte differentiation into macrophages as evidenced through the up-regulation of the surface receptors: CD36, SR-A1 and CD91 analysed by flow cytometry. These proteins were able to stimulate TNF-x and IL-10 production and secretion by U937 macrophages, shown by ELISA, and chemotatic properties were demonstrated using Boyden chambers. The cyto-protective and immune regulatory effects of extracellular HSPs have potential therapeutic value as treatments in a wide variety of clinical situations.

The inducible heat shock protein Hsp72 inhibits apoptosis and promotes long term survival after a number of stresses but the mechanism by which this is achieved remains unclear. A role for Hsp72 in modulating apoptosis mediated through members of the TNF-receptor super family other than TNF-R1 has not been clearly established. Given the observations of high levels of Hsp72 in tumours of poor prognosis, we set out to determine whether Hsp72 could specifically modulate apoptosis induced through the death receptor pathways mediated by Fas and the TRAIL receptors. Both these pathways are of relevance in tumour surveillance. (For complete abstract open document)

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Justificativa: O processo de envelhecimento está associado com o aumento demasiado na produção das espécies reativas de oxigênio (EROS). Dentre as estratégias que as células desenvolveram ao longo da evolução para combater o estresse celular, destacam-se as proteínas de choque térmico (HSP). Em especial, a HSPA1A (HSP72) é importante para recuperação de proteínas danificadas, impedindo a agregação das mesmas no citoplasma. O processo de envelhecimento parece estar associado a um declínio nos níveis das HSPs, resultando na perda do controle da proteostase, atrofia muscular, resistência à insulina e incapacidade de reparo após dano. Já é demonstrado na literatura que o exercício físico aumenta a expressão de HSPA1A, o que, possivelmente, exerce função protetora durante o envelhecimento e reduz a inflamação sistêmica clássica encontrada em idosos. Mais recentemente, tem se verificado que HSPA1A pode ser encontrada tanto dentro quanto fora das células, apresentando funções diferentes. Intracelularmente (iHSP70), apresenta função protetora, anti-inflamatória e anti-apoptótica, enquanto que no meio extracelular (eHSP70) apresenta um importante papel imunológico, com função pró-inflamatória e pró-apoptótica. Objetivos: A presente dissertação esta dividida em três partes distintas, denominados como estudos 1, 2 e 3. No primeiro estudo, o objetivo foi comparar a resposta ao choque térmico (leucócitos expostos a temperatura elevada – 42º C por 2 horas) em diferentes populações: indivíduos de meia-idade, idosos e idosos com diabetes do tipo 2. No segundo estudo, o objetivo foi verificar a influência de um protocolo de treinamento de força na relação da HSPA1A extra e intracelular em leucócitos, do dano oxidativo, das adaptações neuromusculares e morfológicas, em indivíduos de meia-idade Finalmente, no último estudo, serão mostrados dados preliminares da resposta de indivíduos idosos ao mesmo protocolo e parâmetros do estudo dois. Metodologia: No primeiro estudo os voluntários foram divididos em três grupos: Meia Idade (49,36±3,61 anos), Idosos (63,57±3,25 anos) e Idosos Diabéticos (68,9± 7,8 anos; HbA1c 7±0,67 %). Foram realizadas coletas de sangue para posterior indução ao choque térmico e avaliação da eHSP70. Nos estudos dois e três, indivíduos de meia-idade (40-59 anos) e idosos (60-75 anos), respectivamente, foram randomicamente alocados nos grupos Controle ou Treinado. O treinamento durou doze semanas com frequência de três vezes por semana. Cada sessão de treinamento incluía nove exercícios de força tanto para membros inferiores quanto superiores e três exercícios funcionais (subir um lance de escadas, sentar e levantar e subir e descer de uma caixa). Antes e após esse período foram realizadas coletas de sangue para posterior análise da HSPA1A extracelular e intracelular em leucócitos, avaliação de peroxidação lipídica (TBARS), atividade antioxidante (SOD e CAT), nitritos e do perfil lipídico. Foram realizadas avaliações de composição corporal, força e teste de consumo máximo de oxigênio Resultados: A resposta ao choque térmico (avaliada pela capacidade das células em exportar eHSP70 quando submetidas a uma temperatura elevada) parece ser igual entre indivíduos de meia-idade e idosos saudáveis. Curiosamente, em indivíduos idosos e com diabetes, esta resposta parece estar bloqueada, indicando que a resistência à insulina e o diabetes tem um papel dominante na capacidade da resposta ao estresse. Quando indivíduos de meia-idade (estudo 2) foram submetidos ao treinamento de força, os mesmos responderam com ganhos de força, melhora da capacidade funcional e aumentos na massa muscular. No entanto, a resposta ao choque térmico (eHSP70 e iHSP70) não foi modificada pelo treinamento, possivelmente pelo fato de que esta se encontrava normal. Os dados preliminares do estudo 3 mostram que, em idosos saudáveis, ocorreram ganhos de força, melhora da capacidade funcional, redução do tecido adiposo visceral (VAT) e melhora da resposta ao choque térmico após o treinamento, no entanto, considerando o baixo n amostral, essa resposta ainda deve ser confirmada em estudos posteriores Conclusão: A ausência de resposta ao choque térmico em Diabéticos está relacionada com resistência à insulina e inflamação, de modo que o treinamento de força parece ser uma alternativa eficaz para contornar esse quadro. Em relação ao efeito do treinamento, em sujeitos de Meia Idade, ele mostra-se eficaz para reduzir as chances de sarcopenia e dinapenia e possíveis doenças relacionadas ao processo do envelhecimento. Os dados preliminares em idosos saudáveis demonstram que, além de diminuir as chances de sarcopenia e dinapenia, o treinamento parece eficaz para reduzir inflamação sistêmica, visto que houve redução do VAT e aumento da resposta ao choque pós-treinamento. Este projeto é financiado pelo CNPq, Edital Universal (Processo nº 482398/2013-2). / Introduction: The aging process is associated with increase in the production of reactive oxygen species (ROS). Among the strategies that the cells developed during the evolution to counteract the cellular stress, we highlight the heat shock proteins (HSP). In particular, HSPA1A (HSP72) is important for the recovery of damaged proteins, preventing them from aggregating into the cytoplasm. The aging process appears to be associated with a decline in HSP levels, resulting in loss of proteostasis control, muscle atrophy, insulin resistance, and inability to repair after damage. It has been demonstrated in the literature that exercise increases the expression of HSPA1A, which possibly exerts protective function during aging and reduces the classical systemic inflammation found in the elderly. More recently, it has been found that HSPA1A can be found both inside and outside the cells, exhibiting different functions. Intracellularly (iHSP70), it has a protective, anti-inflammatory and anti-apoptotic function, whereas in the extracellular environment (eHSP70) it has an important immunological role, with pro-inflammatory and pro-apoptotic function. Objectives: This dissertation is divided into three distinct parts, called studies 1, 2 and 3. In the first study, the objective was to compare the response to heat shock (leukocytes exposed to high temperature - 42º C for 2 hours) in different populations: middle-aged, elderly and elderly individuals with type 2 diabetes. In the second study, the objective was to verify the influence of a resistance training protocol on the relationship of extra and intracellular HSPA1A in leukocytes, oxidative damage, neuromuscular and morphological adaptations in middle-aged individuals. Finally, in the last study, preliminary data from the response of elderly subjects to the same protocol and parameters of study two will be shown Methods: In the first study the volunteers were divided into three groups: Middle Age (49.36 ± 3.61 years), Elderly (63.57 ± 3.25 years) and Diabetic Elderly (68.9 ± 7.8 years; HbA1c 7 ± 0.67%). Blood samples were collected for further induction of heat shock and evaluation of eHSP70. In studies two and three, individuals of middle age (40-59 years) and elderly (60-75 years), respectively, were randomly assigned to the Control or Trained groups. The training lasted twelve weeks three times a week. Each training session included nine strength exercises for both lower and upper limbs and three functional exercises (climbing a flight of stairs, sitting and getting up and going up and down a box). Blood samples were collected for analysis of extracellular and intracellular HSPA1A in leukocytes, lipid peroxidation (TBARS), antioxidant activity (SOD and CAT), nitrite and lipid profile. Body composition, strength and maximal oxygen consumption tests were performed. Results: The response to heat shock (assessed by the ability of cells to export eHSP70 when submit to elevated temperature) appears to be equal between middle-aged and healthy elderly individuals. Interestingly, in elderly individuals with diabetes, this response appears to be blocked, indicating that insulin resistance and diabetes play a dominant role in the ability to respond to stress. When middle-aged individuals (study 2) underwent strength training, they responded with strength gains, improved functional capacity, and increases in muscle mass. However, the response to heat shock (eHSP70 and iHSP70) was not modified by training, possibly because the response was normal. Preliminary data from study 3 show that, in healthy elderly, strength gains, functional capacity improvement, visceral adipose tissue (VAT) reduction and improved heat shock response after training occurred, however, considering the low n sample , this response has yet to be confirmed in later studies. Conclusion: The lack of response to heat shock in diabetics is related to insulin resistance and inflammation, so resistance training seems to be an effective alternative to circumvent this condition. Regarding the training effect, in middleaged subjects, it is effective in reducing the chances of sarcopenia and dynapenia and possible diseases related to the aging process. Preliminary data in healthy older adults demonstrate that, in addition to decreasing the chances of sarcopenia and dynapenia, training seems effective in reducing systemic inflammation, since there was a reduction in VAT and increased post-training shock response. This project is funded by the CNPq, Edital Universal (Process nº 482398/2013-2).

Background Idiopathic pulmonary fibrosis (IPF) is one of a number of interstitial lung diseases (ILDs) that result in extensive and chronic pulmonary fibrosis. In IPF pathology, immunological dysfunction has been identified as a contributing factor to the ongoing fibrotic process, implicating cells and mechanisms of both the innate and humoral immune response. Due to the complex and diverse range of cells and mediators involved in IPF, the pathology is still poorly understood. Evidence of complement activation through the classical pathway in IPF lungs implies a role for IgG in the pathology. The active IgG in IPF may be autoreactive in nature, as IgG that target antigens of alveolar epithelial cells have been. Two autoantibodies in IPF, anti-periplakin IgG and anti-Hsp72 IgG, have been associated with poorer prognoses in IPF patients. The association of anti-Hsp72 IgG with IPF patient outcomes has not been validated and little work has been done to study the underlying mechanisms of autoantibodies in IPF pathogenesis. Hypothesis Anti-Hsp72 IgG is associated with poorer outcomes in IPF, and may induce alveolar macrophages to exhibit a pro-fibrotic phenotype. Aims The aims were to:  Optimise an ELISA for anti-Hsp72 IgG detection and determine any association of anti-Hsp72 IgG with IPF patient outcomes  Determine the location of anti-Hsp72 IgG producing cells and detect if Hsp72-IgG complexes are present in IPF patients’ lungs  Explore a potential underlying pro-fibrotic mechanism through which anti-Hps72 IgG modulates macrophage function. Results The presence of anti-Hsp72 IgG was determined in ILD patient and healthy control bronchoalveolar lavage fluid (BALf) and serum. A novel anti-Hsp72 IgG ELISA was developed and optimised and then compared against a commercial anti-Hsp72 IgGAM ELISA which became available during the PhD. Progression in IPF was defined by a decrease of ≥10% vital capacity (VC) over twelve months. Serum anti-Hsp72 IgG(AM) did not associate with changes in VC over 12 months. In contrast, BALf anti-Hsp72 IgG(AM) concentrations were elevated in IPF non-progressors. Patients with high BALf anti-Hsp72 IgGAM, had improved survival compared patient with low anti-Hsp72 IgGAM (adjusted HR 0.39, 95% CI 0.16-0.92; p=0.032) In contrast there was no association between anti-Hsp72 IgG and survival. Detection of anti-Hsp72 IgG subtypes in the serum and BALf of IPF patients revealed no significant difference in anti-Hsp72 IgG subtype detection levels between progressors and non-progressors. BALf anti-Hsp72 IgG1 levels were associated with a significantly lower rate of decline in VC over twelve months than patients with no detectable anti-Hsp72 IgG1. The presence of Hsp72-IgG complexes was confirmed by detection in purified IgG from IPF patient BALf. Immuno-histological detection of C4d deposition in the lungs of IPF patients coincided in areas of Hsp72 expression in alveolar epithelium. Summary These findings do not validate serum and-Hsp72 IgG as a biomarker for IPF. They support a role for anti-Hsp72 IgG in IPF, but associate with decreased rates of lung function decline and increased patient survival. Data also suggests that the decreased rate of decline may be related to specific anti-Hsp72 IgG subtype expression. The immune-histological data further suggests that anti-Hsp72 IgG may be targeting Hsp72 expressed by lung epithelium. Therefore these findings support a role for immunological dysfunction in IPF, but further work is required to determine the underlying mechanism.

Justificativa: O processo de envelhecimento está associado com o aumento demasiado na produção das espécies reativas de oxigênio (EROS). Dentre as estratégias que as células desenvolveram ao longo da evolução para combater o estresse celular, destacam-se as proteínas de choque térmico (HSP). Em especial, a HSPA1A (HSP72) é importante para recuperação de proteínas danificadas, impedindo a agregação das mesmas no citoplasma. O processo de envelhecimento parece estar associado a um declínio nos níveis das HSPs, resultando na perda do controle da proteostase, atrofia muscular, resistência à insulina e incapacidade de reparo após dano. Já é demonstrado na literatura que o exercício físico aumenta a expressão de HSPA1A, o que, possivelmente, exerce função protetora durante o envelhecimento e reduz a inflamação sistêmica clássica encontrada em idosos. Mais recentemente, tem se verificado que HSPA1A pode ser encontrada tanto dentro quanto fora das células, apresentando funções diferentes. Intracelularmente (iHSP70), apresenta função protetora, anti-inflamatória e anti-apoptótica, enquanto que no meio extracelular (eHSP70) apresenta um importante papel imunológico, com função pró-inflamatória e pró-apoptótica. Objetivos: A presente dissertação esta dividida em três partes distintas, denominados como estudos 1, 2 e 3. No primeiro estudo, o objetivo foi comparar a resposta ao choque térmico (leucócitos expostos a temperatura elevada – 42º C por 2 horas) em diferentes populações: indivíduos de meia-idade, idosos e idosos com diabetes do tipo 2. No segundo estudo, o objetivo foi verificar a influência de um protocolo de treinamento de força na relação da HSPA1A extra e intracelular em leucócitos, do dano oxidativo, das adaptações neuromusculares e morfológicas, em indivíduos de meia-idade Finalmente, no último estudo, serão mostrados dados preliminares da resposta de indivíduos idosos ao mesmo protocolo e parâmetros do estudo dois. Metodologia: No primeiro estudo os voluntários foram divididos em três grupos: Meia Idade (49,36±3,61 anos), Idosos (63,57±3,25 anos) e Idosos Diabéticos (68,9± 7,8 anos; HbA1c 7±0,67 %). Foram realizadas coletas de sangue para posterior indução ao choque térmico e avaliação da eHSP70. Nos estudos dois e três, indivíduos de meia-idade (40-59 anos) e idosos (60-75 anos), respectivamente, foram randomicamente alocados nos grupos Controle ou Treinado. O treinamento durou doze semanas com frequência de três vezes por semana. Cada sessão de treinamento incluía nove exercícios de força tanto para membros inferiores quanto superiores e três exercícios funcionais (subir um lance de escadas, sentar e levantar e subir e descer de uma caixa). Antes e após esse período foram realizadas coletas de sangue para posterior análise da HSPA1A extracelular e intracelular em leucócitos, avaliação de peroxidação lipídica (TBARS), atividade antioxidante (SOD e CAT), nitritos e do perfil lipídico. Foram realizadas avaliações de composição corporal, força e teste de consumo máximo de oxigênio Resultados: A resposta ao choque térmico (avaliada pela capacidade das células em exportar eHSP70 quando submetidas a uma temperatura elevada) parece ser igual entre indivíduos de meia-idade e idosos saudáveis. Curiosamente, em indivíduos idosos e com diabetes, esta resposta parece estar bloqueada, indicando que a resistência à insulina e o diabetes tem um papel dominante na capacidade da resposta ao estresse. Quando indivíduos de meia-idade (estudo 2) foram submetidos ao treinamento de força, os mesmos responderam com ganhos de força, melhora da capacidade funcional e aumentos na massa muscular. No entanto, a resposta ao choque térmico (eHSP70 e iHSP70) não foi modificada pelo treinamento, possivelmente pelo fato de que esta se encontrava normal. Os dados preliminares do estudo 3 mostram que, em idosos saudáveis, ocorreram ganhos de força, melhora da capacidade funcional, redução do tecido adiposo visceral (VAT) e melhora da resposta ao choque térmico após o treinamento, no entanto, considerando o baixo n amostral, essa resposta ainda deve ser confirmada em estudos posteriores Conclusão: A ausência de resposta ao choque térmico em Diabéticos está relacionada com resistência à insulina e inflamação, de modo que o treinamento de força parece ser uma alternativa eficaz para contornar esse quadro. Em relação ao efeito do treinamento, em sujeitos de Meia Idade, ele mostra-se eficaz para reduzir as chances de sarcopenia e dinapenia e possíveis doenças relacionadas ao processo do envelhecimento. Os dados preliminares em idosos saudáveis demonstram que, além de diminuir as chances de sarcopenia e dinapenia, o treinamento parece eficaz para reduzir inflamação sistêmica, visto que houve redução do VAT e aumento da resposta ao choque pós-treinamento. Este projeto é financiado pelo CNPq, Edital Universal (Processo nº 482398/2013-2). / Introduction: The aging process is associated with increase in the production of reactive oxygen species (ROS). Among the strategies that the cells developed during the evolution to counteract the cellular stress, we highlight the heat shock proteins (HSP). In particular, HSPA1A (HSP72) is important for the recovery of damaged proteins, preventing them from aggregating into the cytoplasm. The aging process appears to be associated with a decline in HSP levels, resulting in loss of proteostasis control, muscle atrophy, insulin resistance, and inability to repair after damage. It has been demonstrated in the literature that exercise increases the expression of HSPA1A, which possibly exerts protective function during aging and reduces the classical systemic inflammation found in the elderly. More recently, it has been found that HSPA1A can be found both inside and outside the cells, exhibiting different functions. Intracellularly (iHSP70), it has a protective, anti-inflammatory and anti-apoptotic function, whereas in the extracellular environment (eHSP70) it has an important immunological role, with pro-inflammatory and pro-apoptotic function. Objectives: This dissertation is divided into three distinct parts, called studies 1, 2 and 3. In the first study, the objective was to compare the response to heat shock (leukocytes exposed to high temperature - 42º C for 2 hours) in different populations: middle-aged, elderly and elderly individuals with type 2 diabetes. In the second study, the objective was to verify the influence of a resistance training protocol on the relationship of extra and intracellular HSPA1A in leukocytes, oxidative damage, neuromuscular and morphological adaptations in middle-aged individuals. Finally, in the last study, preliminary data from the response of elderly subjects to the same protocol and parameters of study two will be shown Methods: In the first study the volunteers were divided into three groups: Middle Age (49.36 ± 3.61 years), Elderly (63.57 ± 3.25 years) and Diabetic Elderly (68.9 ± 7.8 years; HbA1c 7 ± 0.67%). Blood samples were collected for further induction of heat shock and evaluation of eHSP70. In studies two and three, individuals of middle age (40-59 years) and elderly (60-75 years), respectively, were randomly assigned to the Control or Trained groups. The training lasted twelve weeks three times a week. Each training session included nine strength exercises for both lower and upper limbs and three functional exercises (climbing a flight of stairs, sitting and getting up and going up and down a box). Blood samples were collected for analysis of extracellular and intracellular HSPA1A in leukocytes, lipid peroxidation (TBARS), antioxidant activity (SOD and CAT), nitrite and lipid profile. Body composition, strength and maximal oxygen consumption tests were performed. Results: The response to heat shock (assessed by the ability of cells to export eHSP70 when submit to elevated temperature) appears to be equal between middle-aged and healthy elderly individuals. Interestingly, in elderly individuals with diabetes, this response appears to be blocked, indicating that insulin resistance and diabetes play a dominant role in the ability to respond to stress. When middle-aged individuals (study 2) underwent strength training, they responded with strength gains, improved functional capacity, and increases in muscle mass. However, the response to heat shock (eHSP70 and iHSP70) was not modified by training, possibly because the response was normal. Preliminary data from study 3 show that, in healthy elderly, strength gains, functional capacity improvement, visceral adipose tissue (VAT) reduction and improved heat shock response after training occurred, however, considering the low n sample , this response has yet to be confirmed in later studies. Conclusion: The lack of response to heat shock in diabetics is related to insulin resistance and inflammation, so resistance training seems to be an effective alternative to circumvent this condition. Regarding the training effect, in middleaged subjects, it is effective in reducing the chances of sarcopenia and dynapenia and possible diseases related to the aging process. Preliminary data in healthy older adults demonstrate that, in addition to decreasing the chances of sarcopenia and dynapenia, training seems effective in reducing systemic inflammation, since there was a reduction in VAT and increased post-training shock response. This project is funded by the CNPq, Edital Universal (Process nº 482398/2013-2).