Submitted by Livia Mello (liviacmello@yahoo.com.br) on 2016-09-28T20:16:49Z No. of bitstreams: 1 DissIJ.pdf: 2276989 bytes, checksum: 70bd31f2d0212ca687248df11b43678b (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T16:15:53Z (GMT) No. of bitstreams: 1 DissIJ.pdf: 2276989 bytes, checksum: 70bd31f2d0212ca687248df11b43678b (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T16:16:00Z (GMT) No. of bitstreams: 1 DissIJ.pdf: 2276989 bytes, checksum: 70bd31f2d0212ca687248df11b43678b (MD5) / Made available in DSpace on 2016-10-20T16:16:09Z (GMT). No. of bitstreams: 1 DissIJ.pdf: 2276989 bytes, checksum: 70bd31f2d0212ca687248df11b43678b (MD5) Previous issue date: 2016-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dexamethasone (DEX) is widely used in clinic due to its effectiveness as anti- inflammatory. Nevertheless, its chronic use may cause unwanted metabolic and hemodynamic alterations, which contribute for the development of hypertension. Arterial hypertension may be determined by increases in cardiac output or peripheral resistance and rarefaction may be associated with this response. Microcirculation maintenance is dependent on the balance between anti-apoptotic and apoptotic proteins and vascular endothelial growth factor (VEGF), known to be a key-regulator protein of the physiological angiogenesis, helps to maintain this balance. We recently demonstrated that high doses with DEX-treatment reduce VEGF protein levels, but the mechanisms involved in this response were not evaluated. In the other hand, it has been shown that aerobic training (T) is a good strategy in preventing rarefaction and/or stimulating angiogenesis, however almost nothing is known about the effects of T on microcirculation and hypertension induced by DEX. Therefore, the aim of this study was to investigate the mechanisms induced by T that can contribute to attenuate DEX-induced rarefaction. Wistar rats were subjected to an aerobic exercise protocol on the treadmill or kept sedentary for 8 weeks. Additionally, animals were treated with DEX or saline (50μg/kg, s.c. for 14 days). Groups were: sedentary control (SC), DEX sedentary (SD), trained control (TC) and trained DEX (TD). Body weight (BW) and arterial pressure (AP) were analyzed. After euthanasia, adrenal gland, myocardium, SOL and TA muscles were weighted and normalized by tibia. The cross-sectional area (CSA), capillary:fiber ratio (C:F ratio), capillary density (CD) and protein levels were evaluated in SOL and TA. Treatment with DEX caused reduction in BW and in muscle weight (MW) in TA. DEX treatment also determinated decrease in CSA (TA). Further, C/F and CD were also reduced (-41 and -43%, SOL) and (-30 and 68.6%, TA). Training was able to prevent C:F ratio and CD reduction (72.7 and 81.0%) and (32.9 and 54.2%) induced by DEX- treatment. Furthermore, DEX significantly reduced protein levels in SOL and TA muscles VEGFR-2 (-14.6% and -20.1%), VEGF (-15.6 and -19%), Bcl-2 (-18.4 and20.5%), Bcl-2/Bax ratio (-29.0 and -13.7%) and p-Bax/Bax (-25.4 and -20%), beyond COX-2 in TA (-22.8%). DEX also promoted increase in caspase-3 cleaved (25 and 24.1%, SOL and TA). Moreover, training was able to prevent reduction in proteins levels in DEX-treated groups in SOL and TA: VEGFR-2 (14.7 and 25.2%), VEGF (15.3 and 25.3%), Bcl-2 (21.6 and 35.5%), Bcl-2/Bax ratio (26.1 and 19.9%), p-Bax/Bax (23.7 and 32.1%) and COX-2 (31.5%) and the increase in caspase-3 cleaved (16.0 and 17.8%). In conclusion, these results showed that DEX-induced rarefaction promoted imbalance between apoptotic and angiogenic factors, become one possible causes of hypertension. However, also showed that aerobic training is a good strategy to attenuate DEX-induced rarefaction and this response may involve a better balance between apoptotic and angiogenic factors, which contribute for the attenuation of hypertension. / A dexametasona (DEX) é amplamente utilizada em vários casos clínicos devido a sua eficácia como fármaco anti-inflamatório. Por outro lado, a utilização crônica deste medicamento pode causar alterações metabólicas e hemodinâmicas que contribuem para o desenvolvimento da hipertensão. A hipertensão arterial pode ser determinada pelo aumento no débito cardíaco ou da resistência periférica e a rarefação pode estar associada a esta resposta. A manutenção da microcirculação é dependente do equilíbrio entre proteínas anti e apoptóticas e o fator de crescimento endotelial vascular (VEGF) contribui para a manutenção deste equilíbrio. Demonstramos recentemente que altas doses de DEX reduz a produção do VEGF, no entanto, os mecanismos envolvidos nesta resposta não foram avaliados. Por outro lado, tem sido mostrado que o treinamento físico (TF) aeróbio é uma ferramenta importante na prevenção da rarefação e/ou promoção angiogênese, no tratamento da hipertensão, no entanto, quase nada se sabe sobre os efeitos do TF na microcirculação e hipertensão induzida pela DEX. Portanto, o objetivo deste estudo foi investigar se o tratamento crônico com DEX compromete a densidade de vasos por alterar o balanço angiogênico/apoptótico na musculatura esquelética e se o pré-condicionamento físico aeróbio atenua esta resposta. Para isso, 60 ratos wistar foram submetidos a um protocolo de TF aeróbio na esteira ou mantidos sedentários por 8 semanas. Além disso, os animais foram tratados com DEX ou salina (50μg/kg, s.c. por 14 dias). Os grupos foram separados em: sedentário controle (SC), sedentário e tratado com DEX (SD), treinado controle (TC) e treinado tratado com DEX (TD). O peso corporal (PC) e a pressão arterial (PA) foram analisados. Após a eutanásia, a glândula adrenal, miocárdio e os músculos SOL e TA foram pesados e normalizados pela tíbia. Foram avaliados também, área de secção transversa (AST), razão capilar/fibra (C/F), densidade capilar (DC) e análise da produção proteica dos músculos SOL e TA. O tratamento com DEX causou redução tanto no PC quanto no peso muscular do TA, assim como da AST. Além disso, a DEX reduziu significativamente C/F e DC nos músculos (-41 e -43%, SOL) e (- 30 e 68,6%, TA). Por outro lado, o TF aeróbio preveniu a redução da C/F e DC causado pela DEX (72,7 e 81,0%, SOL) e (32,9 e 54,2%, TA). Já, os níveis proteicos no SOL e TA foram reduzidos significativamente pela DEX: VEGFR-2 (-14,6% e -20,1%), VEGF (-15,6 e -19%), Bcl-2 (-18,4 e -20,5%), razão Bcl-2/Bax (-29,0 e -13,7%) e p-Bax/Bax (- 25,4 e -20%), além da COX-2 no TA (-22,8%). A caspase-3 clivada estava aumentada (16,0 e 17,8%, SOL e TA respectivamente). Em contrapartida, o TF aeróbio foi capaz de prevenir a redução dos níveis proteicos causado pela DEX nos músculos SOL e TA, além de prevenir o aumento da caspase-3 clivada. Em conclusão, os resultados deste presente estudo mostrou que a rarefação induzida DEX ocorreu por promover o desbalanço entre fatores angiogênicos e apoptóticos,sendo esse um dos possíveis mecanismos da hipertensão, no entanto, demonstrou que o TF aeróbio é uma boa estratégia para manter o balanço entre fatores angiogênicos e apoptóticos, o qual contribui para a manutenção da microcirculação na musculatura esquelética e, portanto, pode contribuir para a atenuação da hipertensão induzida pela DEX.

Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-11-30Bitstream added on 2014-06-13T18:58:50Z : No. of bitstreams: 1 fruhvald_e_me_botfmvz.pdf: 332018 bytes, checksum: e9ea4cd232ff2adc94ec7372c75727e1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As aves da família Psittacidae são muito visadas pelo tráfico de animais selvagens. O papagaio-verdadeiro (Amazona aestiva) é uma das espécies mais traficadas. Os animais provenientes do tráfico são transportados em espaço restrito, sem alimento ou água e as aves adoecem ou morrem devido ao estresse ou brigas, antes mesmo de chegarem ao consumidor final. O estresse leva a secreção de glicocorticoides que possui diversas funções nos vertebrados além de sofrerem flutuações dos seus níveis basais para preparar o organismo para o início das atividades diárias. Fatores ambientais e necessidades fisiológicas interagem para a manutenção do relógio biológico, que é responsável por determinar essas alterações endócrinas. As análises hormonais através de técnicas não-invasivas têm sido usadas em estudos da biologia da conservação, comportamentais e fisiológicos em aves cativas ou de vida livre devido a sua facilidade de coleta e por não causarem prejuízos ao indivíduo. Os níveis de metabólitos de glicocorticoides presentes nas excretas refletem as concentrações plasmáticas desses hormônios. Com objetivo de determinar o perfil de excreção urofecal de metabólitos de glicocorticoides nos excrementos de A. aestiva, ao longo do dia e relacionar esses achados com as atividades diária desses animais, foram utilizados 22 animais (12 machos e 10 fêmeas) alojados em gaiolas individuais, e suas excretas foram colhidas a cada três horas, por 24 horas consecutivas. Foi observado um pico nas concentrações de metabólitos de glicocorticóides nas primeiras horas do dia, quando também se inicia as atividades diárias das aves; e as concentrações diminuíram gradualmente até atingirem seus níveis basais no final da tarde e se mantiveram baixos até a manhã seguinte / The birds of the Psittacidae family are highly targeted for wildlife trafficking. The Blue-fronted parrot (Amazona aestiva) is one of the most trafficked species. The animals are transported in restricted spaces, without food or water and the birds get sick or die due to stress or fights, before arriving to the final consumer. Stress leads to secretion of glucocorticoids that has several functions in vertebrates and suffer fluctuations of baseline levels to prepare the organism for the start of daily activities. Physiological and environmental factors interact to maintain the biological clock, which is responsible for determining these endocrine changes. The hormonal assays employing non-invasive techniques have been used in studies of conservation biology, behavioral and physiological studies of captive or free-living birds because of the ease of collection and lack of harm to the individual. Levels of glucocorticoid metabolites in feces reflect plasma concentrations of these hormones. In order to define the profile of excretion of glucocorticoids metabolites in A. aestiva droppings throughout the day and correlate these findings with the daily activities of these animals, we used 22 animals (12 males and 10 females) housed individually in cages, and their droppings were collected every three hours for 24 consecutive hours. A peak in the concentration of glucocorticoids metabolites was observed in the first hours of the day at the start of the birds’ daily activities, and the concentrations decreased gradually to basal levels in the late afternoon and remained low until the next morning

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-07T13:15:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Mestrado Verônica Melo CCS.pdf: 1393412 bytes, checksum: ba56180c890511d0089ae952978dcc15 (MD5) / Made available in DSpace on 2017-04-07T13:15:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Mestrado Verônica Melo CCS.pdf: 1393412 bytes, checksum: ba56180c890511d0089ae952978dcc15 (MD5) Previous issue date: 2016-07-01 / Introdução: o uso crônico de glicocorticoides é considerado a principal causa de osteoporose secundária e iatrogênica. Existem poucos estudos associando fraturas ao uso de glicocorticoides na faixa etária pediátrica. Eles poderiam ajudar na criação de abordagens preventivas e terapêuticas. Objetivos: avaliar se o uso de glicocorticoides, nos 12 meses precedentes, associou-se à ocorrência de fraturas em crianças e adolescentes; identificar a frequência de asma e outras doenças; comparar o perfil demográfico, o tipo de trauma, o índice de massa corpórea, a prática de exercício físico, a ingesta de leite e o tabagismo passivo domiciliar nos grupos com e sem fratura; verificar a frequência de deficiência de vitamina D. Métodos: no período de abril a outubro de 2015, um estudo tipo caso controle foi conduzido em crianças e adolescentes vitimadas por trauma, com e sem fratura, a partir da análise dos dados coletados. Resultados: foram estudados 104 pacientes, 50 com fratura e 54 com trauma, mas sem fratura. Ao todo, 80,4% eram meninos e 40,4% estavam na faixa etária de 10 a 14 anos. O uso prévio de glicocorticoides ocorreu em 15,4% do total, sem diferença estatisticamente significante entre os dois grupos. Entre 39 pacientes com fratura e que dosaram a vitamina D, 47,2% tinham níveis séricos < 30ng/ml. A prática de exercício físico associou-se a um aumento em 2,2 vezes no risco para fratura. Conclusões: este estudo não mostrou associação entre o uso prévio de glicocorticoides e a ocorrência de fraturas em crianças e adolescentes. A faixa etária de 10 a 14 anos, o trauma grave e o exercício físico associaram-se com um maior risco para fraturas. Cerca de metade de uma amostra dos pacientes com fratura apresentou níveis insuficientes/deficientes de vitamina D, mesmo em região tropical. / Introduction: Osteoporosis is not exclusive to older adults and manifests by fractures. Chronic glucocorticoid use is considered the main cause of secondary and iatrogenic osteoporosis. Few studies have related fractures to the use of glucocorticoids in children and adolescents. Such studies could be useful for the development of preventive and therapeutic strategies. Objectives: To assess whether glucocorticoid use in the past 12 months is associated with the occurrence of fractures in children and adolescents; to identify the frequency of asthma and other diseases; and to compare the demographic profile, type of trauma, body mass index, physical activity, milk intake, and household exposure to cigarette smoke of groups with and without fractures; to verify the frequency of vitamin D insufficiency/deficiency. Methods: A case-control study, conducted from April to October 2015, analyzed the data of trauma children and adolescents with and without fractures. Results: A total of 104 trauma patients were studied, 50 with and 54 without fractures. In all, 80.4% were males, and 40.4% were aged 10 to 14 years. Previous glucocorticoid use occurred in 15.4% of the sample, without significant difference between the groups. Of the 39 fracture patients with measured serum vitamin D levels, 47.2% had levels < 30ng/ml. Physical activity was associated with a 2.2-fold risk of fractures, but without significance in multivariate analysis. Conclusions: This study did not find an association between previous glucocorticoid use and the occurrence of fractures in children and adolescents. In 10- to 14-year-olds, severe trauma and physical activity were associated with higher risk of fractures. About half the fracture sample had insufficient/deficient vitamin D levels, despite residing in a tropical region.

O processo adaptativo a hipóxia implica em modificações nas funções endócrinas e metabólicas. Embora seja bem estabelecido que o metabolismo de carboidratos é profundamente alterado pela hipóxia, muito pouco se sabe acerca dos efeitos in vivo do estresse hipóxico no metabolismo de proteínas na musculatura esquelética. Portanto, o presente trabalho teve como objetivo avaliar os efeitos agudos da hipóxia intermitente nos processos de síntese e degradação protéica em músculos esqueléticos de ratos e a possível participação dos glicocorticóides na resposta induzida pela hipóxia. Para isso, ratos jovens (~80g) foram expostos à hipóxia intermitente aguda (HIA) por 8 horas (6% O2 durante 40 segundos em intervalos de 9 minutos). Os resultados mostram que a HIA é uma situação catabólica que resulta no aumento da glicose e insulina plasmática, na redução do conteúdo do glicogênio hepático (97%), oxidação de glicose muscular (40%) e massa do tecido adiposo branco retroperitoneal e epididimal (30%), sem alterações na massa muscular. No metabolismo protéico, os animais hipóxicos apresentaram aumento da proteólise total em músculos soleus e EDL acompanhada por uma hiperativação dos sistemas proteolíticos dependente de ubiquitina (Ub)-proteassoma e dependente de cálcio, sem que houvesse alterações na síntese protéica. Essa resposta foi associada ao maior conteúdo de proteínas miofibrilares conjugadas à Ub e à ativação da expressão de genes relacionados à atrofia (atrogina-1 e MuRF1) e autofagia (LC3 e GABARAP ). Adicionalmente, em músculos soleus, observou-se redução da fosforilação da Akt (Ser473), uma proteína chave no controle do metabolismo protéico. Verificou-se ainda que a adrenalectomia em animais hipóxicos preveniu a ativação dos sistemas proteolíticos e a transcrição do RNAm dos \"atrogenes\" sem alterar a autofagia. Esses dados mostram que a HIA, durante 8 horas, age como um gatilho catabólico no processo de degradação de proteínas dependente de Ub-proteassoma e cálcio assim como na hiperexpressão de genes relacionados à atrofia e autofagia. A ativação do programa atrófico induzido pela HIA parece ser mediada pelos glicocorticóides sendo esta resposta provavelmente importante para o fornecimento de aminoácidos do músculo para o fígado e manutenção da hiperglicemia. Portanto, essa resposta catabólica se sustentada pode levar à perda de massa muscular esquelética em situações de exposição prolongada à hipóxia intermitente. / The adaptive process to hypoxia involves changes in endocrine and metabolic functions. Although it is well established that the carbohydrate metabolism is profoundly altered by hypoxia, the in vivo effects of hypoxic stress on protein metabolism in skeletal muscle is still poorly understood. Thus, the main goal of the present work was to investigate the acute effects of intermittent hypoxia on the processes of synthesis and protein degradation in rat skeletal muscles and the possible role of glucocorticoids in the responses induced by hypoxia. For this, young rats (~80g) were exposed to acute intermittent hypoxia (AIH) for 8 hours (6% O2 for 40 seconds at 9 minutes intervals). The data show that AIH is a catabolic situation that increases plasma levels of glucose and insulin, reduces the content of liver glycogen (97%), the muscle glucose oxidation (40%) and the retroperitoneal and epididymal white adipose tissue mass (30%), without changing muscle mass. With respect to the protein metabolism, AIH rats showed an increase in the rates of overall proteolysis in soleus and EDL muscle, which were accompanied by a hyperactivity of the ubiquitin (Ub)-proteasome and calcium-dependent proteolytic systems, without changes in rates of protein synthesis. This response was associated with a high content of myofibrillar proteins Ub-conjugates and mRNA levels of atrophy-related genes (atrogin-1 and MuRF1) and autophagic genes (LC3 and GABARAP). Furthermore, in soleus muscle, we observed reduction of Akt phosphorylation (Ser473), a key protein in the control of protein metabolism. It was also found that adrenalectomy prevented activation of proteolytic systems and gene transcription of \"atrogenes\" in hypoxic rats, while the autophagic genes were not affected. The data suggest that the AIH, during 8 hours, acts as a catabolic trigger activating the Ub-proteassome and calcium-dependent proteolytic processes, as well as the expression of atrophy-related genes and autophagy. The activation of atrophy program induced by hypoxia seems to be mediated by glucocorticoids, and is probably important for the provision of amino acids from muscle to the liver which maintains the hyperglycemia. Therefore, this catabolic response, if sustained, can lead to the loss of skeletal muscle mass in situations of prolonged exposure to intermittent hypoxia.

Não é claro como o receptor de glicocorticóide (GR) contrarregula a atividade do STAT5 na transição da gestação para a lactação. Dexras1 é uma pequena proteína G ativada por dexametasona (DEX), que regula morfologia celular, crescimento, etc. Neste estudo detectamos a expressão de Dexras1 em células beta de ilhotas pancreáticas. DEX induz a expressão de Dexras1 em células RINm5F, que está aumentada na gestação e diminuída na lactação. A expressão protéica de 11<font face=\"Symbol\">&#946HSD1, enzima ativadora de glicocorticóides (GCs), segue esse perfil. A ligação tanto do GR como do STAT5, analisada por ChIP assay, ao promotor do gene da Dexras1 aumentada por DEX é revertida por prolactina (PRL), e está diminuída e aumentada na gestação e lactação, respectivamente. DEX induz a associação ao GR, fosforilação e translocação nuclear do STAT5b. O silenciamento gênico de Dexras1 promoveu aumento da secreção de insulina, e aumentou os níveis de pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 e PKA. Sendo assim, a regulação de Dexras1 por PRL e GCs contribui para a secreção de insulina característica do periparto. / It is not clear how glucocorticoid receptor (GR) counteracts STAT5 activity during the transition of pregnancy to lactation. Dexras1 is a small G protein activated by dexamethasone (DEX) that controls cell morphology, growth, etc. In the present study we detected Dexras1 expression in pancreatic beta cell. DEX induces Dexras1 expression in RINm5F cells, which is increased in pregnancy and decreased in lactation. The expression of 11<font face=\"Symbol\">&#946HSD1, the glucocorticoids (GCs) activating enzyme, followed Dexras1 profile in pancreatic islet. Both GR and STAT5b bindings to Dexras1 gene promoter, analyzed by ChIP assay, are increased by DEX and PRL counteracts this effect. Both bindings are decreased in pregnancy and increased in lactation. DEX induces STAT5b association to GR, phosphorylation and nuclear translocation. Dexras1 knockdown using small interference RNA (si-RNA) promoted an increase in insulin secretion, as well as increased levels of pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 and PKA. Thus, Dexras1 regulation by PRL and GCs contributes to insulin secretion during peripartum.

Os ritmos biológicos circadianos garantem a relação temporal entre os seres e o ambiente. A regulação metabólica se relaciona à ritmicidade dos ciclos de claro-escuro, com importância das ações da melatonina e dos glicocorticoides no fígado. Investigamos a participação dos glicocorticoides no ajuste metabólico mediado pela secreção rítmica de melatonina e no contexto inflamatório hepático. Ratos Wistar machos pinealectomizados tratados ou não com RU 486 tiveram o padrão circadiano circulante de corticosterona, insulina e glicose aferido ao longo de 8ZTs e no ZT10. Neste ponto, analisou-se a expressão de RNAm e protéica envolvidas em ações metabólicas e inflamatórias. Testes in vivo de tolerância à glicose e ao piruvato e a fosforilação da Akt hepática no ZT10 em resposta à insulina, em conjunto às alterações de peso e composição corpórea, conteúdo de glicogênio e histologia do fígado, apontam maior influência da corticosterona na gliconeogênese e correlação de ambos os hormônios nas alterações energéticas e na instalação de um quadro pró-inflamatório hepático. / Circadian biological rhythms guarantee the temporal relationship between living beings and the environment. Metabolic regulation relates to the rhythm to the light-dark cycle, with importance of the actions of melatonin and glucocorticoids in the liver. We investigated the participation of glucocorticoids in the metabolic adjustment mediated by rhythmic secretion of melatonin on the liver inflammatory context. Male Wistar rats pinealectomized treated or not with RU 486 had the circadian levels of circulating corticosterone, insulin and glucose measured along 8ZTs and on ZT10. At this point, we analyzed mRNA and protein expression related to metabolic and inflammatory actions. In vivo tests to glucose and pyruvate tolerance and rating the phosphorylation of Akt in response to insulin in liver at ZT10, with the changes in weight and body composition, glycogen content and liver histology, indicate influence of corticosterone in gluconeogenesis and correlation of both hormones in energy alterations and on the installation of a hepatic pro-inflammatory process.

Abstract To investigate the turnover of type IV collagen in skeletal muscle in conditions where muscle function is impaired, type IV collagen and proteins regulating its degradation were studied during 1, 3 and 7 days of immobilization, 3- and 10-day glucocorticoid treatment and in neuromuscular diseases. In addition, fibrillar type I and III collagens were studied during immobilization and in neuromuscular diseases. The mRNA levels of type I, III and IV collagens were decreased during immobilization and during 10-day dexamethasone treatment. Gene expression and quantity of (pro)MMP-2 was increased during immobilization but decreased during dexamethasone treatment. The expression of TIMP-2 was decreased both during immobilization and dexamethasone treatment. Decreased gene expression and increased degradation caused decreased concentration of type IV collagen, suggesting net degradation of type IV collagen during immobilization. While the gene expression and degradation were decreased during dexamethasone treatment, the amount of type IV collagen was not changed. Dexamethasone thus seemed to slow down the turnover of type IV collagen. Decreased mRNA levels of collagens and prolyl 4-hydroxylase suggest decreased biosynthesis of collagens during immobilization. The mRNA levels of collagens I, III and IV were increased in polyneuropathy and polymyositis. The concentration and staining intensity of type IV collagen was increased in polyneuropathy, as was also the quantity and staining intensity of (pro)MMP-9. The results suggest accumulation of type IV collagen in the basement membranes of muscle cells and capillaries in polyneuropathy muscles. Lengthened position during immobilization partly prevented the atrophy and changes in collagen metabolism in plantarflexors. Endurance running was effective in preventing muscle atrophy during dexamethasone treatment, but exercise did however fail to prevent the changes observed in type IV collagen synthesis and degradation.

Nous avons évalué les conséquences sur un plan endocrinien et cognitif de l’administration d’un stress aigu chez la souris BalbC jeune (6 mois) ou d’âge moyen (16 mois). Le stress aigu a été appliqué 5, 60 ou 120 minutes avant la phase de rappel dans une épreuve de mémoire contextuelle. La concentration en corticostérone hippocampique a été mesurée avant et après le stress. Suite à l'administration du stress, elle augmente rapidement (dès 15 minutes post-stress) et est inversement liée au rappel mnésique. En effet, plus la concentration de corticostérone hippocampique est élevée, plus faible est la réponse hippocampe-dépendante. Les effets cognitif et endocrinien du stress ont été reproduits par l'administration de corticostérone dans l'hippocampe et ont été bloqués par la métyrapone. Le vieillissement accroit l’anxiété et induit des conséquences sur le plan mnésique comparable à celles du stress chez la souris jeune. De plus, la concentration de corticostérone hippocampique, basale ou en réponse au stress, est plus élevée et durable chez les animaux âgés, par rapport aux jeunes. Par ailleurs, l’administration de diazépam 30 minutes avant le rappel chez la souris d’âge-moyen atténue les effets délétères du stress sur la mémoire et la concentration hippocampique de corticostérone. Ces données suggèrent que la perturbation de l'axe HPA chez la souris âgée joue un rôle clef dans les troubles de mémoire hippocampe-dépendants induits par le vieillissement. / The cognitive and endocrinal consequences of an acute stress were studied in young (6 months) and middle-aged (16 months) BalbC mice. Acute stress was delivered 5, 60 or 120 minutes before the retrieval phase of a contextual memory task. Hippocampal corticosterone concentration was measured before and after stress delivery by microdialysis. The acute stress induces a rapid hippocampal corticosterone rise in relation with a cognitive impairment. Indeed, when the level of hippocampal corticosterone is high, the cognitive performance of hippocampal-dependent memory is impaired. Moreover, the cognitive and endocrinal effects of stress were mimicked using corticosterone microinjection in the hippocampus, whereas metyrapone (an inhibitor of corticosterone synthesis) blocked it. The consequences of aging on memory retrieval are similar to stress effects in young mice. Furthermore, pre and post-stress hippocampal corticosterone concentration is higher in middle-aged as compared to young mice. In addition, the increase in hippocampal corticosterone is longer in middle-aged in contrast to young mice. Finally, the administration of diazepam in middle-aged mice (30 minutes before the retrieval test phase) attenuates the deleterious effects of an acute stress on memory retrieval and the increase in hippocampal corticosterone concentration. In conclusion, our data suggest that HPA axis dysregulation, observed in middle-aged subjects, plays a key role in episodic-like memory impairments induced by aging.

Glucocorticoids known to be such powerful agents that cell growth, differentiation and cell death are influenced in the brain of mammals throughout life. Despite this, relatively little toxicological information regarding prenatal exposure is available. The aim of this study was to determine the effect of prenatal hydrocortisone exposure on cell viability and cell morphology in chick embryonic neurons. Four different histological staining techniques namely, Hematoxylin and Eosin (H&E), Cresyl Fast Violet, Silver impregnation and a combination of Gold Chloride and Toluidine Blue were used to evaluate chick embryo neural tissue exposed to 0.137ƒÝM or 0.685ƒÝM hydrocortisone on day 3.75 (Carnegie stage 16) and day 5.5 (Carnegie stage 18) of development. Histological processing was optimized and neural tissue evaluated for any changes in neuron morphology and cell number. Specific ultrastructural changes to membraneous structures were evaluated by transmission electron microscopy (TEM). Fixation procedures that resulted in little to no disruption of these structures were optimized and used in studies evaluating the effect of hydrocortisone on neuron morphology. Primary chick embryonic neuronal cultures were prepared and increasing concentrations of hydrocortisone (26.3nM, 0.16ƒÝM, 0.63ƒÝM, 3.8ƒÝM, and 22.8ƒÝM) added. Fluorescence microscopy was applied to the in vitro hydrocortisone exposed primary neuronal cultures. A combination of fluorescein diacetate (FDA) and propidium iodide (PI) was used to evaluate the effect of hydrocortisone on cell viability, whereas dichlorodihydrofluorescein diacetate (DCH2FDA) was used to visualize reactive oxygen species (ROS) generation in neurons. Histological evaluation of the neural tissue of chick embryos exposed to 0.137ƒÝM and 0.685ƒÝM hydrocortisone showed reduced neuron density and morphological changes associated with cell death. Glutaraldehyde with added magnesium chloride (MgCl2) as stabilizing chemical and potassium permangenate were two fixatives that caused minimal disruption to neural tissue. These two fixating methods were applied to control neural tissue as well as tissues exposed to 0.137ƒÝM and 0.685ƒÝM hydrocortisone. When evaluated by TEM, the control tissue appeared to be intact with no displacement. Exposure of neurons to 0.137ƒÝM hydrocortisone appeared to have severe effects on the morphology of the mitochondria, endoplasmic reticulum (ER), nuclear and plasma membranes. More extensive damage was noted with 0.685ƒÝM hydrocortisone, leaving almost no cellular structure. Both concentrations of hydrocortisone indicated cell death associated with apoptosis and necrosis. In vitro studies using primary cultures of chick neurons indicated that hydrocortisone is non-toxic at low concentrations (26.3nM ¡V 3.8ƒÝM) with the percentage viability ranging between 73% and 88%. A more toxic effect was seen at high concentrations (22.8ƒÝM). Cell death at the higher concentrations (22.8ƒÝM and 3.8ƒÝM) of hydrocortisone occurred due to ROS generation, as indicated by DCH2FDA fluorescence In conclusion, hydrocortisone indicated neurotoxicity at high concentrations of exposure. Although cell death could be detected, the exact mechanism (apoptosis or necrosis) still needs to be investigated. Since the developing brain is so susceptible to chemical insults care should be taken when administering this drug to pregnant mothers or young children. / Dissertation (MSc (Cell Biology)))--University of Pretoria, 2007. / Anatomy / unrestricted

Les glucocorticoïdes (GCs) ont des effets diabétogènes avérés. Précédemment, notre équipe a également pu montrer que les GCs, en association avec le corégulateur transcriptionnel PGC-1α, sont impliqués dans la programmation fœtale du diabète de type 2 (DT2). Le DT2 est une maladie métabolique, conséquence à la fois de l’insulinorésistance et d’un défaut de sécrétion d’insuline en partie dû à la diminution de la masse des cellules β. Au laboratoire nous nous intéressons donc d’une part aux mécanismes sous-jacents des effets diabétogènes des GCs et d’autre part, aux mécanismes permettant d’améliorer la sécrétion d’insuline en restaurant une masse fonctionnelle de cellules β. Dans la première partie de cette thèse, nous avons montré que PGC-1α, dont l’expression est stimulée par les GCs dans les cellules β, induit un double stress énergétique et oxydatif impliqué dans l’altération de la sécrétion d’insuline. Dans la deuxième partie, nous avons montré grâce à un model murin d’insulinorésistance sévère par surexposition aux GCs, que l’adaptation compensatrice de la masse fonctionnelle des cellules β se fait par un processus de néogenèse, impliquant la réexpression du facteur Ngn3. Ce processus, indépendant de l’effet des GCs sur le pancréas, alimente l’hypothèse d’un facteur circulant libéré par les organes insulinorésistants pour instruire le pancréas endocrine et initier la néogenèse des cellules β. En conclusion, nos travaux associent indirectement les GCs : 1/ à un effet délétère sur la sécrétion et impliquant PGC-1α et 2/ à un effet bénéfique sur la masse β et impliquant Ngn3. Ces deux voies constituent des perspectives thérapeutiques intéressantes du DT2. / Glucocorticoids (GCs) are hormones secreted in response to stress and that display diabetogenic effects. Previously, our team was able to demonstrate that GCs, in combination with the transcriptional co-regulator PGC-1α, are involved in fetal programming of type 2 diabetes (T2D). T2D is a metabolic disease characterized by fasting hyperglycemia, consequence of both insulin resistance and an insulin secretory defect, partly due to the decrease of the mass of β cells. In the laboratory we are therefore interested in understanding the mechanisms underlying diabetogenic effects of GCs, and mechanisms that improve insulin secretion and functional β-cell mass. In the first part of this thesis, we have shown that PGC-1α, whose expression is strongly stimulated by GCs in β cells, induces both energy and oxidative stress involved in impaired insulin secretion. In the second part of this thesis, we demonstrated through a murine model of massive GCs overexposure – which induces severe insulin resistance – that the adaptation of the functional β-cell mass in order to counteract insulin resistance occurs through a neogenesis process, involving the re-expression of Ngn3 factor. This process is independent of the effect of GCs on the pancreas. We hypothesize that a circulating factor released by insulin-resistant organs will instruct the endocrine pancreas to initiate β-cells neogenesis. In conclusion, our work indirectly associate GCs: 1/ to a deleterious effect on the secretion involving PGC-1α and 2/ to a beneficial effect on the β-cell mass and involving Ngn3. These two pathways are interesting therapeutic perspectives for curing T2D.