Macrophage activation plays a crucial role in regulating adipose tissue inflammation and is a major contributor to the pathogenesis of obesity-associated cardiovascular diseases. On various types of stimuli, macrophages respond with either classic (M1) or alternative (M2) activation. M1- and M2-mediated signaling pathways and corresponding cytokine production profiles are not completely understood. The discovery of microRNAs provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function. We have examined the activity of microRNA-223 (miR-223) and its role in controlling macrophage functions in adipose tissue inflammation and systemic insulinresistance. miR-223-/- mice on a high-fat diet exhibited an increased severity of systemic insulin resistance compared with wild-type mice that was accompanied by a marked increase in adipose tissue inflammation. The specific regulatory effects of miR-223 in myeloid cell-mediated regulation of adipose tissue inflammation and insulin resistance were then confirmed by transplantation analysis. Moreover, using bone marrow-derived macrophages, we demonstrated that miR-223 is a novel regulator of macrophage polarization, which suppresses classic pro-inflammatory pathways and enhances the alternative anti-inflammatory responses. In addition, we identified Pknox1 as a genuine miR-223 target gene and an essential regulator for macrophage polarization. For the first time, this study demonstrates that miR-223 acts to inhibit Pknox 1,suppressing pro-inflammatory activation of macrophages; thus, it is a crucial regulator of macrophage polarization and protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance.

The overall aim of the work presented in this thesis was to further characterise aspects of metabolic and immune system cross-talk in human subcutaneous adipose tissue, with a particular emphasis on the potential role of T-lymphocytes in adipose tissue dysfunction and insulin resistance. Chapter 3 characterised macrophage and T-lymphocyte populations residing in adipose tissue from lean through to class I obese men. This work demonstrated that T-lymphocytes display increased activation with increased adiposity and that potential compensatory mechanisms may be present to help counteract adipose tissue inflammation. In Chapter 4, the same participants were exposed to a meal-based stimulus in order to examine the postprandial metabolic and inflammatory responses in blood and adipose tissue. Despite increased glucose and insulin responses in blood with obesity, there were no differences in inflammatory cytokine gene expression responses in adipose tissue. This suggests that mechanisms may be present to limit or dampen inflammatory output from adipose tissue after feeding in individuals with modestly increased adiposity. Chapter 5 examined metabolic and immune system changes to 50 % calorie restriction for 3 days, resulting in reduced serum leptin which was temporally associated with a reduction in blood T-lymphocyte activation. In adipose tissue, however, leptin gene expression/secretion was not reduced and neither was resident T-lymphocyte activation, indicating that there may be local tissue-specific responses of immune cells to caloric restriction. Chapter 6 characterised differences between obese individuals with either normal or impaired glucose tolerance, and their respective responses to 10 days of diet and activity modification. Overall, this thesis highlights key differences in properties of T-lymphocyte populations with increasing levels of adiposity and insulin resistance together with responses in adipose tissue and the immune system in times of feeding, severe calorie restriction and glucose lowering diet and activity.

A inibição de mTOR com rapamicina exacerba a intolerância glicose associada a obesidade, um efeito que pode estar associado ao desenvolvimento de processo pró-inflamatório no tecido adiposo. O tratamento de camundongos com rapamicina exacerbou a intolerância a glicose e inflamação do tecido adiposo induzida por dieta hiperlipídica. In vitro, a inibição dos complexos 1 e 2 da mTOR com rapamicina e torina induziu polarização espontânea de macrófagos para o fenótipo M1 e aumentou a atividade fagocítica de macrófagos M0. Camundongos com ativação constitutiva de mTORC1 exclusivamente em células mielóides foram protegidos do ganho de peso, adiposidade, intolerância a glicose e a insulina induzidos pela ingestão de dieta hiperlipídica e apresentaram um aumento da polarização de macrófagos para o fenótipo M2. Em conjunto, nossos dados indicam que a atividade do complexo 1 da mTOR atenua o desenvolvimento da inflamação do tecido adiposo associada a obesidade por um mecanismo que envolve a polarização de macrófagos para o fenótipo M2. / Pharmacological mTOR inhibition with rapamycin exacerbates the glucose intolerance associated with obesity, such an effect that may be associated to the development of inflammatory process in adipose tissue. Rapamycin treatment exacerbates the glucose intolerance and adipose tissue inflammation induced by high fat diet feeding. In vitro, inhibition of mTOR complexes 1 and 2 with rapamycin and torin induced spontaneous macrophage polarization into a pro-inflammatory M1 phenotype and increased M0 macrophage phagocytosis. Mice with constitutive activation of mTOR complex 1 in myeloid cells were protected from body weight gain, fat accretion, glucose and insulin tolerance induced by the intake of high fat diet and displayed a significant increase in macrophage polarization to a M2 phenotype. Altogether, our findings indicate that the activity of mTOR complex 1 attenuates the development of adipose tissue inflammation induced by high fat feeding, through a mechanism that involves a higher polarization of macrophages to anti-inflammatory M2 phenotype.

Spinal cord injury is a devastating neurological disorder partially characterized by a loss of motor function below the lesion. The dramatic loss of activity results in muscle atrophy and slow-to-fast transformation of contractile elements, producing smaller, weaker and more fatiguable muscles. Functional electrical stimulation (FES), has been proposed in order to induce muscular activity and reverse these changes. FES has primarily been applied in the periphery, either at the surface or implanted in or around a nerve or muscle. Although this can excite nervous tissue and produce muscular contractions, these systems often produce reversed recruitment of motor units leading to inappropriate force generation and increased fatigue. We applied intraspinal microstimulation (ISMS) through fine microwires implanted into the spinal cord of rats. Electrical stimulation through these microwires caused contractions of the quadriceps muscles in both acute and chronically spinalized animals. We showed that muscle recruitment is significantly more gradual with ISMS in intact rats compared to that produced by a standard nerve cuff. Our results further showed that this was due to preferential activation of fatigue resistant muscle fibers. Given this more orderly recruitment of motor units by ISMS, we tested the muscle phenotypes produced by ISMS or nerve cuffs after chronic stimulation. Surprisingly, over a 30 day stimulation period the quadriceps muscles chronically activated by either daily ISMS or nerve cuff stimulation underwent similar fast-to-slow transformations in fiber type and functional properties. This indicates that the recruitment order of motor units does not play the only role in determining the muscle phenotype. Other factors such as the total daily time of activity may be critically important to the phenotypic outcome of skeletal muscle. Finally, we demonstrated that quadriceps force recruitment by ISMS was unchanged following the 30 day stimulation period. In addition, 30 days of chronic ISMS did not cause observable damage in the spinal cord beyond that incurred by the implantation of sham microwires. These studies advance our understanding of the force recruitment properties, neuromuscular plasticity and damage incurred by ISMS and move us closer to developing a clinically viable ISMS procedure.

No description available.

A inibição de mTOR com rapamicina exacerba a intolerância glicose associada a obesidade, um efeito que pode estar associado ao desenvolvimento de processo pró-inflamatório no tecido adiposo. O tratamento de camundongos com rapamicina exacerbou a intolerância a glicose e inflamação do tecido adiposo induzida por dieta hiperlipídica. In vitro, a inibição dos complexos 1 e 2 da mTOR com rapamicina e torina induziu polarização espontânea de macrófagos para o fenótipo M1 e aumentou a atividade fagocítica de macrófagos M0. Camundongos com ativação constitutiva de mTORC1 exclusivamente em células mielóides foram protegidos do ganho de peso, adiposidade, intolerância a glicose e a insulina induzidos pela ingestão de dieta hiperlipídica e apresentaram um aumento da polarização de macrófagos para o fenótipo M2. Em conjunto, nossos dados indicam que a atividade do complexo 1 da mTOR atenua o desenvolvimento da inflamação do tecido adiposo associada a obesidade por um mecanismo que envolve a polarização de macrófagos para o fenótipo M2. / Pharmacological mTOR inhibition with rapamycin exacerbates the glucose intolerance associated with obesity, such an effect that may be associated to the development of inflammatory process in adipose tissue. Rapamycin treatment exacerbates the glucose intolerance and adipose tissue inflammation induced by high fat diet feeding. In vitro, inhibition of mTOR complexes 1 and 2 with rapamycin and torin induced spontaneous macrophage polarization into a pro-inflammatory M1 phenotype and increased M0 macrophage phagocytosis. Mice with constitutive activation of mTOR complex 1 in myeloid cells were protected from body weight gain, fat accretion, glucose and insulin tolerance induced by the intake of high fat diet and displayed a significant increase in macrophage polarization to a M2 phenotype. Altogether, our findings indicate that the activity of mTOR complex 1 attenuates the development of adipose tissue inflammation induced by high fat feeding, through a mechanism that involves a higher polarization of macrophages to anti-inflammatory M2 phenotype.

Lipedema may be considered a model for healthy expandability of subcutaneous adipose tissue (SAT). This condition is characterized by the disproportional and symmetrical SAT accumulation in the lower-body parts and extremities, avoiding the abdominal area. There are no circulating biomarkers facilitating the diagnosis of lipedema. We tested the hypothesis that women living with lipedema present a distinct pattern of circulating parameters compared to age- and BMI-matched women. In 26 women (Age 48.3 ± 13.9 years, BMI 32.6 ± 5.8 kg/m2; lipedema group: n=13; control group: n=13), we assessed circulating parameters of glucose and lipid metabolism, inflammation, oxidative stress, sex hormones and a proteomics panel. We find that women with lipedema have better glucose metabolism regulation represented by lower HbA1c (5.55 ± 0.62%) compared to controls (6.73 ± 0.85%; p<0.001); and higher adiponectin levels (lipedema: 4.69 ± 1.99 mmol/l; control: 3.28 ± 1.00 mmol/l; p=0.038). Despite normal glycemic parameters, women with lipedema have significantly higher levels of total cholesterol (5.84 ± 0.70 mmol/L vs 4.55 ± 0.77 mmol/L in control; p<0.001), LDL-C (3.38 ± 0.68 mmol/L vs 2.38 ± 0.66 mmol/L in control; p=0.002), as well as higher circulating inflammation (top 6 based on p-values: TNFSF14, CASP8, ENRAGE, EIF4EBP1 , ADA, MCP-1) and oxidative stress markers (malondialdehyde, superoxide dismutase and catalase). Our findings suggest that the expected association between activation of inflammatory and oxidative stress pathways and impaired glucose metabolism are counterbalanced by protective factors in lipedema.

Este estudo, in vivo, visou a avaliar a influência do auxiliar químico no selamento dos canais radiculares obturados com o sistema RealSeal® e preparados para a colocação de pino protético, pela análise da resposta dos tecidos periapicais de dentes de cães. Foram testados os produtos químicos hipoclorito de sódio a 1% e a clorexidina gel a 2%. A pesquisa foi desenvolvida em 6 cães, 10 dentes em cada um deles, num total de 60 dentes e 120 canais divididos em sete grupos, sendo três grupos teste, três grupos controle negativo e um grupo controle positivo. Todos os dentes permaneceram selados por 24 horas após terem sido tratados. Decorrido este prazo, os selamentos provisórios dos dentes pertencentes aos grupos teste e controle positivo foram removidos, permanecendo sem selamento por 90 dias. Os dentes dos grupos controle negativo foram mantidos com selamento durante todo o período experimental, quando os animais foram mortos e suas mandíbulas removidas. O processamento histológico foi realizado com hematoxilina e eosina. A análise histológica, com auxílio de um microscópio óptico, baseou-se na avaliação da situação histológica dos tecidos periapicais. A análise estatística foi realizada a partir do teste não paramétrico de Kruskal-Wallis com localização de diferenças pelos procedimentos de post-hoc, de Mann-Whitney e Willcoxon. O nível de significância considerado adequado foi de 5%. Os grupos experimentais e controle negativo apresentaram menor grau de inflamação dos tecidos periapicais em comparação com o grupo controle positivo no qual se observou, em todos os espécimes, presença de infiltrado polimorfonuclear neutrófilo e/ou eosinófilo, com presença de áreas de formação de abscesso (p=0,001). Como conseqüência das análises descritas foi concluído que os auxiliares químicos da instrumentação do canal, hipoclorito de sódio a 1%, clorexidina gel a 2% e o soro fisiológico, associados ao EDTA a 17%, não interferiram na resposta do tecido periapical, parecendo, também, não terem interferido na imbricação do sistema RealSeal® nas paredes do canal radicular, no período experimental de 90 dias. Pode-se inferir, ainda, que o material obturador remanescente após o preparo para o pino protético foi eficaz na vedação da zona apical do canal radicular, no espaço do tempo experimental. / The aim of this study was to assess the influence of the cleaning protocol on sealing of root canal obturation with RealSeal following post space preparation, by means of periapical tissue response analysis. 1% sodium hypochlorite and 2% chlorhexidine were the tested products. For this purpose, six dogs were used, 10 teeth in each dog, comprising a total of 60 teeth and 120 root canals, were randomly divided into seven groups, being three test groups, three negative controls and a positive control. Following obturation, all teeth were kept totally sealed during 24 hours to allow sealer setting. Then the positive control and the three test groups had their seal removed, and obturation material exposed to the oral environment for 90 days. Negative controls were kept sealed during the whole experimental period. The animals were killed and their mandibles removed. Histological procedures were adopted as to HE staining. Optical histological analysis at magnifications varying from 25x to 400x considered the periapical tissue status at various sites by a blinded senior observer. Non-parametric Kruskal-Wallis and a Mann-Whitney and Willcoxon post-hoc were adopted at α= 0.05. Experimental groups and negative control had lower degree of inflammation when compared with the positive control, which showed consistently neutrophil and eosinophil infiltrates and areas of abscess (p=0.001). Both sodium hypochlorite and chlorhexidine behaved similarly, not interfering with the periapical tissue response. The remaining filling material was efficient to seal the apical portion of the root canal, regardless of the cleaning protocol.

Este estudo, in vivo, visou a avaliar a influência do auxiliar químico no selamento dos canais radiculares obturados com o sistema RealSeal® e preparados para a colocação de pino protético, pela análise da resposta dos tecidos periapicais de dentes de cães. Foram testados os produtos químicos hipoclorito de sódio a 1% e a clorexidina gel a 2%. A pesquisa foi desenvolvida em 6 cães, 10 dentes em cada um deles, num total de 60 dentes e 120 canais divididos em sete grupos, sendo três grupos teste, três grupos controle negativo e um grupo controle positivo. Todos os dentes permaneceram selados por 24 horas após terem sido tratados. Decorrido este prazo, os selamentos provisórios dos dentes pertencentes aos grupos teste e controle positivo foram removidos, permanecendo sem selamento por 90 dias. Os dentes dos grupos controle negativo foram mantidos com selamento durante todo o período experimental, quando os animais foram mortos e suas mandíbulas removidas. O processamento histológico foi realizado com hematoxilina e eosina. A análise histológica, com auxílio de um microscópio óptico, baseou-se na avaliação da situação histológica dos tecidos periapicais. A análise estatística foi realizada a partir do teste não paramétrico de Kruskal-Wallis com localização de diferenças pelos procedimentos de post-hoc, de Mann-Whitney e Willcoxon. O nível de significância considerado adequado foi de 5%. Os grupos experimentais e controle negativo apresentaram menor grau de inflamação dos tecidos periapicais em comparação com o grupo controle positivo no qual se observou, em todos os espécimes, presença de infiltrado polimorfonuclear neutrófilo e/ou eosinófilo, com presença de áreas de formação de abscesso (p=0,001). Como conseqüência das análises descritas foi concluído que os auxiliares químicos da instrumentação do canal, hipoclorito de sódio a 1%, clorexidina gel a 2% e o soro fisiológico, associados ao EDTA a 17%, não interferiram na resposta do tecido periapical, parecendo, também, não terem interferido na imbricação do sistema RealSeal® nas paredes do canal radicular, no período experimental de 90 dias. Pode-se inferir, ainda, que o material obturador remanescente após o preparo para o pino protético foi eficaz na vedação da zona apical do canal radicular, no espaço do tempo experimental. / The aim of this study was to assess the influence of the cleaning protocol on sealing of root canal obturation with RealSeal following post space preparation, by means of periapical tissue response analysis. 1% sodium hypochlorite and 2% chlorhexidine were the tested products. For this purpose, six dogs were used, 10 teeth in each dog, comprising a total of 60 teeth and 120 root canals, were randomly divided into seven groups, being three test groups, three negative controls and a positive control. Following obturation, all teeth were kept totally sealed during 24 hours to allow sealer setting. Then the positive control and the three test groups had their seal removed, and obturation material exposed to the oral environment for 90 days. Negative controls were kept sealed during the whole experimental period. The animals were killed and their mandibles removed. Histological procedures were adopted as to HE staining. Optical histological analysis at magnifications varying from 25x to 400x considered the periapical tissue status at various sites by a blinded senior observer. Non-parametric Kruskal-Wallis and a Mann-Whitney and Willcoxon post-hoc were adopted at α= 0.05. Experimental groups and negative control had lower degree of inflammation when compared with the positive control, which showed consistently neutrophil and eosinophil infiltrates and areas of abscess (p=0.001). Both sodium hypochlorite and chlorhexidine behaved similarly, not interfering with the periapical tissue response. The remaining filling material was efficient to seal the apical portion of the root canal, regardless of the cleaning protocol.

Este estudo, in vivo, visou a avaliar a influência do auxiliar químico no selamento dos canais radiculares obturados com o sistema RealSeal® e preparados para a colocação de pino protético, pela análise da resposta dos tecidos periapicais de dentes de cães. Foram testados os produtos químicos hipoclorito de sódio a 1% e a clorexidina gel a 2%. A pesquisa foi desenvolvida em 6 cães, 10 dentes em cada um deles, num total de 60 dentes e 120 canais divididos em sete grupos, sendo três grupos teste, três grupos controle negativo e um grupo controle positivo. Todos os dentes permaneceram selados por 24 horas após terem sido tratados. Decorrido este prazo, os selamentos provisórios dos dentes pertencentes aos grupos teste e controle positivo foram removidos, permanecendo sem selamento por 90 dias. Os dentes dos grupos controle negativo foram mantidos com selamento durante todo o período experimental, quando os animais foram mortos e suas mandíbulas removidas. O processamento histológico foi realizado com hematoxilina e eosina. A análise histológica, com auxílio de um microscópio óptico, baseou-se na avaliação da situação histológica dos tecidos periapicais. A análise estatística foi realizada a partir do teste não paramétrico de Kruskal-Wallis com localização de diferenças pelos procedimentos de post-hoc, de Mann-Whitney e Willcoxon. O nível de significância considerado adequado foi de 5%. Os grupos experimentais e controle negativo apresentaram menor grau de inflamação dos tecidos periapicais em comparação com o grupo controle positivo no qual se observou, em todos os espécimes, presença de infiltrado polimorfonuclear neutrófilo e/ou eosinófilo, com presença de áreas de formação de abscesso (p=0,001). Como conseqüência das análises descritas foi concluído que os auxiliares químicos da instrumentação do canal, hipoclorito de sódio a 1%, clorexidina gel a 2% e o soro fisiológico, associados ao EDTA a 17%, não interferiram na resposta do tecido periapical, parecendo, também, não terem interferido na imbricação do sistema RealSeal® nas paredes do canal radicular, no período experimental de 90 dias. Pode-se inferir, ainda, que o material obturador remanescente após o preparo para o pino protético foi eficaz na vedação da zona apical do canal radicular, no espaço do tempo experimental. / The aim of this study was to assess the influence of the cleaning protocol on sealing of root canal obturation with RealSeal following post space preparation, by means of periapical tissue response analysis. 1% sodium hypochlorite and 2% chlorhexidine were the tested products. For this purpose, six dogs were used, 10 teeth in each dog, comprising a total of 60 teeth and 120 root canals, were randomly divided into seven groups, being three test groups, three negative controls and a positive control. Following obturation, all teeth were kept totally sealed during 24 hours to allow sealer setting. Then the positive control and the three test groups had their seal removed, and obturation material exposed to the oral environment for 90 days. Negative controls were kept sealed during the whole experimental period. The animals were killed and their mandibles removed. Histological procedures were adopted as to HE staining. Optical histological analysis at magnifications varying from 25x to 400x considered the periapical tissue status at various sites by a blinded senior observer. Non-parametric Kruskal-Wallis and a Mann-Whitney and Willcoxon post-hoc were adopted at α= 0.05. Experimental groups and negative control had lower degree of inflammation when compared with the positive control, which showed consistently neutrophil and eosinophil infiltrates and areas of abscess (p=0.001). Both sodium hypochlorite and chlorhexidine behaved similarly, not interfering with the periapical tissue response. The remaining filling material was efficient to seal the apical portion of the root canal, regardless of the cleaning protocol.