Glucocorticoids are the most widely prescribed medications due primarily to their anti-inflammatory and immunosuppressive actions, however, their use is not without side effects. Among these, glucocorticoids cause profound muscle atrophy, yet paradoxically are used as the first line of treatment for muscle wasting disorders such as Duchenne Muscular Dystrophy (DMD) and inflammatory myopathies. In DMD patients, glucocorticoid treatment can improve muscle strength during the first 6 months of treatment and can delay loss of muscle function by up to three years. While recent advancements have been made to understand the effect of glucocorticoids (GCs) on the myofiber, the impact of GCs on skeletal muscle stem cells (MuSCs), the adult stem cells responsible for muscle regeneration, and their role in myogenic differentiation, are relatively unknown. To study the role of glucocorticoid signalling during muscle repair, I developed a conditional null mouse (GRMuSC-/-) model in which glucocorticoid receptor (GR) expression is knocked out specifically in MuSCs (GRMuSC-/-). One-week following acute muscle injury, WT and GRMuSC-/- mice both underwent robust repair assessed by myofiber cross-sectional area (CSA) analysis. However, the GR-/- MuSCs failed to return to quiescence following repair resulting in a significant increase in average myofiber CSA at 28- and 42- days post-injury, as compared to controls. Loss of the GR led to a significant increase in the percentage of PAX7+Ki67+ cycling cells in GRMuSC-/- mice (as compared to controls) at 42 days post injury. In the uninjured contralateral limb, I observed significantly fewer MuSCs in GRMuSC-/- mice with a concomitant increase in fibers with centrally located nuclei, indicating that these PAX7+ MuSCs progressed to differentiation in the absence of direct injury. In an uninjured model, two weeks following loss of GR expression there was an increase in the percentage of BrdU+ and Ki67+ cycling cells in resting GRMuSC-/- tibialis anterior muscles as compared to WT, suggesting that the GR acts to maintain MuSC quiescence. Consistent with this, immunostaining of single EDL myofiber fibers at T2h post-dissociation revealed that loss of GR in MuSCs lead to precocious activation and subsequent proliferation of MuSCs as compared to controls. Bulk RNA-sequencing from in situ fixed MuSCs in resting muscle revealed that the gene signature of GR-/- MuSCs was consistent with cells that have exited from the quiescent state and are activated for differentiation. Despite precocious activation, GR-/- myoblasts differentiate and fuse normally, however the myotubes produced had abnormal morphology and aberrant myonuclear placement in regenerated muscle fibers in vivo.

No description available.

No description available.

No description available.

There were three objectives of this study. The first was to determine if commercially available fluorochromes could be used to determine the glucocorticoid receptor (GR) density and binding affinity (BA) in equine peripheral blood mononuclear cells. The second was to determine if there was a correlation between elevated plasma cortisol and GR density or binding affinity in healthy adult horses. The third objective was to evaluate the HPA axis in adult horses presenting with systemic inflammatory response syndrome (SIRS), and to determine where any alterations in HPA axis function occur in these patients compared to healthy adults. For the first part of the study, peripheral venous blood was collected from 3 healthy research horses on 3 days. Peripheral blood mononuclear cells were isolated using Ficoll gradient centrifugation. Phycoerythrin (PE)-CD44 was then used to extracellularly label leukocytes, and then an intracellular GR antibody was used to determine a baseline measurement of GR density and fluorescein isothiocyanate (FITC)-dexamethasone was used to determine binding affinity via flow cytometric analysis. Comparison of control samples to those for CD44, GR density, and GR binding affinity showed a statistically significant difference for all samples (P<0.0001, P<0.0001, and P<0.0001 respectively). This showed that the CD44, GR antibody, and FITC-dexamethasone could successfully be used to analyze equine peripheral blood mononuclear cells for GR activity. For the second part of the study, an ACTH stimulation test was performed on 8 healthy horses in order to induce an increase in endogenous cortisol production. Plasma cortisol levels, GR density, and GR binding affinity were measured at baseline, 4, 8, and 24 hours after treatment. Median basal cortisol concentration was 4.9, range 3.2-6.1 μg/dl. This initially increased following ACTH stimulation to 5.6, range 4.8-7.4 μg/dl, then showed a significant decrease by 8 hours post ACTH administration to 1.4, range 1.1-2.7 μg/dl (P=0.0221). No correlation was observed between plasma cortisol concentration in healthy horses and GR density or binding affinity (r=-0.145, P=0.428 and r=0.046, P=0.802, respectively). For the third phase of the study, horses (N=10) with systemic inflammatory response syndrome (SIRS) were compared to healthy, age and sex matched controls (N=10) presenting for lameness evaluation or ophthalmologic examination. Blood was collected from SIRS cases and controls on presentation to the Equine Medical Center. A CBC, serum biochemistry, and serum ACTH and cortisol measurements were performed. GR density and binding affinity were also determined. Nonsurvivors had a significantly decreased GR binding affinity (P=0.008) and demonstrated a trend towards an increase in the ACTH:cortisol ratio. ROC analysis was performed for serum ACTH and cortisol concentrations, the ACTH:cortisol ratio, GR density and GR binding affinity, and triglycerides to determine cut-off values associated with nonsurvival. These were then used to analyze this population using Fischer's exact test to determine the odds ratio (OR) associated with nonsurvival for each variable. This revealed that a serum triglyceride concentration greater than 28.5 mg/dl was associated with nonsurvival (OR=117, 95% CI, 1.94-7060). The other variables were not found to be significantly associated with nonsurvival, although a Delta BA% of less than 35.79% was found to be closely associated with nonsurvival (OR=30.33, 95% CI, 0.96-960.5). Additionally, a significant negative correlation was detected between the plasma ACTH concentration and Delta BA% (r=-0.685, P=0.029) and the ACTH:cortisol ratio and the Delta BA% (r=-0.697, P=0.025). This study showed that nonsurviving horses with SIRS had a significantly decreased GR binding affinity compared to survivors, and a tendency toward an increase in their ACTH:cortisol ratios. This confirms that HPA axis dysfunction occurs in adult horses with SIRS as tissue resistance to glucocorticoids, and potentially relative adrenal insufficiency as well. These results suggest that there are horses with SIRS that might benefit from "physiologic" doses of synthetic glucocorticoids to complement their relative adrenal insufficiency in addition to their poor tissue sensitivity. Further research should focus on methods to more rapidly determine which horses might benefit from treatment with glucocorticoids on presentation, as well as to more accurately determine prognosis for survival. / Master of Science

O estresse provoca alterações significativas no metabolismo dos animais, provocando a liberação de hormônios glicocorticoides. Estas alterações do metabolismo têm efeito anabólico sobre o metabolismo proteico muscular, podendo afetar os processos bioquímicos de transformação do músculo em carne. O presente trabalho teve como objetivo geral (i) verificar as relações entre variáveis endócrinas e metabólicas associadas ao estresse e características indicadoras de qualidade da carne, em animais castrados e não-castrados; (ii) avaliar a expressão gênica dos receptores mineralocorticoide (MR) e glicocorticoide (GR) em variáveis endócrinas, metabólicas e relacionadas à qualidade da carne de bovinos Nelore castrados e não-castrados. Para tal, 130 animais foram abatidos entre os anos de 2009 e 2011. Amostras de sangue foram coletadas antes e depois do abate para mensuração das concentrações de ACTH e cortisol. Amostras do músculo Longissimus dorsi foram coletadas durante os abates para mensuração do glicogênio e lactato, bem como, para análises de expressão gênica (RT-qPCR). Para as análises de maciez, foram coletadas amostras maturadas por um, sete e 14 dias. Para expressão gênica foram determinados os genótipos dos animais para três marcadores relacionados ao MR (MR1_1, MR1_2 e MR1_3) e dois ao GR (GR2_1 e GR2_2), por meio de PCR em tempo real. Foi verificado que animais castrados apresentam pH 24 horas menores e carnes mais macias ao sétimo e 14º dias de maturação, bem como, concentrações de cortisol (in vivo e post mortem) e lactato significativamente superiores aos animais não-castrados. O marcador MR1_3 apresenta expressão gênica significativamente diferenciada. Os animais com genótipo GA apresentaram 57,27% mais transcritos quando comparados aos animais GG. A expressão gênica do MR e GR foi significativamente relacionada às concentrações de cortisol in vivo e post mortem, porém não influenciou as concentrações de ACTH (in vivo e post mortem), glicogênio e lactato. A expressão gênica do MR e GR não foi relacionada às características indicadoras da qualidade da carne. / The stress causes significant changes in the metabolism of the animals causing the release of glucocorticoid hormones. These metabolic changes have anabolic effect on muscle protein metabolism, affecting the biochemical processes of transformation of muscle on meat. This study aimed to (i) examine relationships between endocrine and metabolic variables associated with stress and meat quality characteristics in castrated and non-castrated animals, (ii) evaluate mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in endocrine and metabolic characteristics and related this to meat quality of Nellore castrated and non-castrated animals. To this end, 130 animals were slaughtered between the years 2009 and 2011. Blood samples were collected before and after slaughter to measure concentrations of ACTH and cortisol. Longissimus dorsi muscle samples were collected during slaughter for measurement of glycogen and lactate, as well for gene expression analyzes (RT-qPCR). For the shear force analyzes, samples were aged for one, seven and 14 days. For gene expression analysis, genotypes of three markers related to MR (MR1_1, MR1_2 and MR1_3), and the two related to GR (GR2_1 and GR2_2) were determined via real-time PCR. It was observed that castrated have lower pH value at 24 hours than non-castrated animals, and tender meat on the seventh and 14th day of aging, such as cortisol (in vivo and post mortem) and lactate concentrations significantly superior to non-castrated animals. Gene expression of MR1_3 was significantly different. Animals with GA genotype had 57.27% more transcripts than GG genotype. The gene expression of MR and GR was significantly related to cortisol concentrations in vivo and post mortem, but did not influence the concentrations of ACTH (in vivo and post mortem), glycogen and lactate. The MR and GR gene expression was not related to the meat quality characteristics.

No description available.

Alcohol abuse disorders (AUD) represent a serious worldwide health problem with far reaching social, financial, and interpersonal implications. One of the most devastating facets of these disorders is the propensity to relapse following periods of abstinence. Ethanol withdrawal (EWD) is believed to promote relapse by increasing anxiety and craving, and may contribute to the development of cognitive decline associated with long-term dependence. Clinical data suggest that stress also plays a main role in both the development of AUD as well as relapse to drinking. As a physiological stressor, EtOH elevates levels of stress hormones (cortisol in humans, corticosterone (CORT) in the rat). Both CORT and EtOH have been shown to alter the composition, function, and activity of the N-methyl-D-aspartate (NMDA) receptor, and in particular, the NR2B subunit of this receptor. These alterations have been suggested to mediate EWD, which may negatively impact abstinence rates. This synergistic interaction between EtOH and CORT may present a therapeutic target for the treatment of EWD. In fact, data suggest that blocking the glucocorticoid receptor, which is a main target for CORT, with RU-486 could promote abstinence, as treatment with the drug has been shown to reduce consumption and the development dependence, as well as the severity of EWD and the cognitive deficits following EWD. However, these latter effects have not been validated in models of voluntary EtOH consumption. As there is considerable evidence that active versus passive intake can significantly impact neuroadaptations to ethanol this is an important consideration. These studies sought to characterize consumption and evaluate the development of dependence in a chronic voluntary model of intermittent access (IA) to EtOH. CORT plasma levels and protein expression of the glucocorticoid and NR2B receptors were measured during and/or following exposure. Finally, to assess the role of CORT in EtOH consumption and the development of dependence, the glucocorticoid receptor antagonist ORG-34517 was administered during access to EtOH. IA access to 20% EtOH produced varying levels of consumption (2.0-6.7g/kg/24hr exposure) and blood EtOH levels (6.3-116.9 mg/dl), but did not significantly affect food consumption or weight gain. Baseline CORT levels were found to be predictive of subsequent EtOH consumption and levels of consumption were sufficient to elevate CORT levels following one hour of EtOH exposure. Further, IA to EtOH was sufficient to produce dependence, as measured by elevations in the acoustic startle reflex following 26 hours and five days of withdrawal. No alteration in protein expression was observed regarding either the NR2B or glucocorticoid receptors and exposure to ORG-34517 had no effect on consumption or withdrawal.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Vários trabalhos têm demonstrado uma relação entre sono e memória. Desta forma, tem sido descrito um papel importante do sono na consolidação da memória e um efeito negativo pela privação do mesmo. O hipocampo é uma região importante para a formação e consolidação da memória espacial, e contém uma alta expressão de receptores para corticosteróides. As ações dos corticosteróides no hipocampo são fundamentais para a aquisição de memória e dependem de um balanço adequado entre receptores de Glicocorticóides (RGc) e Mineralocorticóides (RMn). Assim é descrito na literatura que um aumento na expressão de RMn é promotor de aquisição de memória, enquanto que um aumento na expressão de RGc produz um efeito negativo. Apesar dos níveis circulantes de glicocorticóides na privação de sono paradoxal (PSP), não serem responsáveis pelo enfraquecimento de memória, não existem dados sobre a expressão dos receptores para corticosteróides no hipocampo, após PSP. Neste trabalho tivemos como objetivo investigar a expressão de receptores de Glicocorticóides no hipocampo, bem como avaliar aprendizado e memória em ratos privados de sono paradoxal. Ratos Wistar machos (250- 350g) foram submetidos à PSP, utilizando-se o método de múltiplas plataformas por um período de 96 horas. Após 96h de privação os animais foram anestesiados e perfundidos. Secções de 25 &#956;m na área do hipocampo foram obtidas e reagidas com anticorpos para receptores de Glicocortidóides. Avaliamos as áreas CA1, CA3 e Giro Denteado. O aprendizado e memória espacial foram avaliados através do teste do labirinto aquático de oito braços, antes e após o período de privação de sono. Avaliou-se a latência de escape e o número de erros obtidos. O grupo PSP apresentou um aumento na expressão de RGc nas regiões: CA1 e Giro Denteado, não se observando diferença significativa na região CA3. A PSP prévia aos testes de aprendizado e memória não provocou alterações significativas. A privação de sono pós-aprendizado também não produziu diferenças estatisticamente significativas, mas um aumento no tempo de latência de escape e número de erros sugere um enfraquecimento na consolidação da memória. O aumento na expressão de RGc nas áreas estudadas, pode ser consequente a uma alteração no balanço entre os receptores para corticosteróides no hipocampo e ser responsável por alterações no aprendizado e memória em ratos PSP. / Several studies have shown a relation between sleep and memory. In this way, an important role in memory consolidation by sleep and a negative effect induced by sleep deprivation have been described. Hippocampus is a region responsible for consolidation of spatial memory and contains a high expression of corticosteroids receptors. In the hippocampus, the corticosteroids actions are crucial for memory acquisition and depend on an adequate balance between Glucocorticoid (GR) and Mineralocorticoid receptors (MR). Studies have demonstrated that an increased expression of MR promotes memory acquisition while an increased expression of GR has negatives effects. In spite of the circulating levels of glucocorticoids in paradoxical sleep deprivation (PSD) are not responsible for the PSD induced memory impairments, do not exist studies about the expression of the GR and MR in hippocampus after PSD. In this study we investigate the expression of GR in the hippocampus and evaluate learning and memory in PSD rats. Wistar male rats (250-350g) were paradoxical sleep deprived by the multiple platform method for 96 hours. After 96h of sleep deprivation, the animals were anesthetized and perfused. Slices of 25 micron of the area of the hippocampus were obtained and reacted with antibodies against GR. We evaluated the areas CA1, CA3 and dentate gyrus (GD). Learning and spatial memory were evaluated in Radial water maze before and after PSD. We evaluated the escape latency and the number of errors obtained. PSD group showed an increased expression of GR in CA1 and GD. However, in the CA3 area there was no significant difference in expression. The PSD prior to the tests of learning and memory did not provoke significant alterations. The sleep deprivation after learning also did not produce statistically significant differences, but an increase in the time of escape latency and number of errors suggests impairment in the memory consolidation. The increase in the RGc expression in the studied areas can be consequent to an alteration in the balance between corticosteroid receptors in the hippocampus and be responsible for alterations in the learning and memory in PSD rats.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Vários trabalhos têm demonstrado uma relação entre sono e memória. Desta forma, tem sido descrito um papel importante do sono na consolidação da memória e um efeito negativo pela privação do mesmo. O hipocampo é uma região importante para a formação e consolidação da memória espacial, e contém uma alta expressão de receptores para corticosteróides. As ações dos corticosteróides no hipocampo são fundamentais para a aquisição de memória e dependem de um balanço adequado entre receptores de Glicocorticóides (RGc) e Mineralocorticóides (RMn). Assim é descrito na literatura que um aumento na expressão de RMn é promotor de aquisição de memória, enquanto que um aumento na expressão de RGc produz um efeito negativo. Apesar dos níveis circulantes de glicocorticóides na privação de sono paradoxal (PSP), não serem responsáveis pelo enfraquecimento de memória, não existem dados sobre a expressão dos receptores para corticosteróides no hipocampo, após PSP. Neste trabalho tivemos como objetivo investigar a expressão de receptores de Glicocorticóides no hipocampo, bem como avaliar aprendizado e memória em ratos privados de sono paradoxal. Ratos Wistar machos (250- 350g) foram submetidos à PSP, utilizando-se o método de múltiplas plataformas por um período de 96 horas. Após 96h de privação os animais foram anestesiados e perfundidos. Secções de 25 &#956;m na área do hipocampo foram obtidas e reagidas com anticorpos para receptores de Glicocortidóides. Avaliamos as áreas CA1, CA3 e Giro Denteado. O aprendizado e memória espacial foram avaliados através do teste do labirinto aquático de oito braços, antes e após o período de privação de sono. Avaliou-se a latência de escape e o número de erros obtidos. O grupo PSP apresentou um aumento na expressão de RGc nas regiões: CA1 e Giro Denteado, não se observando diferença significativa na região CA3. A PSP prévia aos testes de aprendizado e memória não provocou alterações significativas. A privação de sono pós-aprendizado também não produziu diferenças estatisticamente significativas, mas um aumento no tempo de latência de escape e número de erros sugere um enfraquecimento na consolidação da memória. O aumento na expressão de RGc nas áreas estudadas, pode ser consequente a uma alteração no balanço entre os receptores para corticosteróides no hipocampo e ser responsável por alterações no aprendizado e memória em ratos PSP. / Several studies have shown a relation between sleep and memory. In this way, an important role in memory consolidation by sleep and a negative effect induced by sleep deprivation have been described. Hippocampus is a region responsible for consolidation of spatial memory and contains a high expression of corticosteroids receptors. In the hippocampus, the corticosteroids actions are crucial for memory acquisition and depend on an adequate balance between Glucocorticoid (GR) and Mineralocorticoid receptors (MR). Studies have demonstrated that an increased expression of MR promotes memory acquisition while an increased expression of GR has negatives effects. In spite of the circulating levels of glucocorticoids in paradoxical sleep deprivation (PSD) are not responsible for the PSD induced memory impairments, do not exist studies about the expression of the GR and MR in hippocampus after PSD. In this study we investigate the expression of GR in the hippocampus and evaluate learning and memory in PSD rats. Wistar male rats (250-350g) were paradoxical sleep deprived by the multiple platform method for 96 hours. After 96h of sleep deprivation, the animals were anesthetized and perfused. Slices of 25 micron of the area of the hippocampus were obtained and reacted with antibodies against GR. We evaluated the areas CA1, CA3 and dentate gyrus (GD). Learning and spatial memory were evaluated in Radial water maze before and after PSD. We evaluated the escape latency and the number of errors obtained. PSD group showed an increased expression of GR in CA1 and GD. However, in the CA3 area there was no significant difference in expression. The PSD prior to the tests of learning and memory did not provoke significant alterations. The sleep deprivation after learning also did not produce statistically significant differences, but an increase in the time of escape latency and number of errors suggests impairment in the memory consolidation. The increase in the RGc expression in the studied areas can be consequent to an alteration in the balance between corticosteroid receptors in the hippocampus and be responsible for alterations in the learning and memory in PSD rats.