It is well established that atherosclerosis, an inflammatory response to chronic injury in the blood vessel wall, plays a leading role in the development and progression of cardiovascular disease. Mineralocorticoid receptor (MR) over-activation has been implicated in atherosclerosis. In mineralocorticoid-target tissues, 11β- Hydroxysteroid dehydrogenase type 2 (11β-HSD2) inactivates glucocorticoids, conferring aldosterone specificity upon the normally unselective MR. Recent evidence suggests that 11β-HSD2 may also afford protection of MR in the cells of the vasculature, providing possible mechanisms by which MR activation may directly promote atherosclerosis. Consistent with this, Apoe-/-/11β-HSD2-/- double knockout (DKO) mice show accelerated atheroma development. The present thesis tested the hypothesis that inactivation of 11β-HSD2, allowing inappropriate activation of MR in cells of the vasculature, accelerates atherogenesis through promotion of a pro-inflammatory environment with increased endothelial cell expression of adhesion molecules and subsequent macrophage infiltration into plaques. DKO mice received either the MR antagonist eplerenone (200mg/kg/day) or vehicle in normal chow diet from 2 months of age for 12 weeks. Eplerenone significantly decreased atherosclerotic burden in brachiocephalic arteries of DKO mice, an effect that was accompanied by alterations in the cellular composition of plaques such that a more stable collagen- and smooth muscle cell- rich plaque was formed. Eplerenone treatment was also associated with a reduction in vascular inflammation as demonstrated by a significant reduction in macrophage infiltration into DKO plaques. The accelerated atherogenesis in DKO mice was clearly evident by 3 months of age, a time point at which Apoe-/- mice were completely lesion free. By 6 months, some Apoe-/- mice had developed lesions whilst all DKO mice at this age showed much larger plaques. Compared to Apoe-/- mice, the cellular composition of DKO plaques was altered favouring vulnerability and inflammation, with increased macrophage and lipid content and decreased collagen content. To investigate the possible underlying mechanisms responsible for increased inflammatory cell content, the expression of vascular cell adhesion molecule 1 (VCAM-1) was compared in DKO and Apoe-/- brachiocephalic arteries. VCAM-1 immunostaining was significantly greater on the endothelial cells of DKO arteries at 3 months compared to age-matched Apoe-/- mice. At 6 months, DKO and Apoe-/- mice had similar expression of VCAM-1. Finally, mouse aortic endothelial cells (MAECs) were used to investigate the mechanism of adhesion molecule up-regulation in the absence of 11β-HSD2. Both aldosterone and TNF-α, included as a positive control, dramatically increased VCAM-1 expression in MAECs. Spironolactone pre-treatment blocked the effect of aldosterone, suggesting an MR-mediated mechanism. Corticosterone alone had no effect on VCAM-1 expression. However, inhibition of 11β-HSD2 by pre-treatment with glycyrrhetinic acid allowed corticosterone to induce a significant increase in the number of VCAM-1-stained MAECs, demonstrating functional expression of 11β- HSD2 in MAECs. Consistent with 11β-HSD2 involvement, VCAM-1 up-regulation by corticosterone in the presence of glycyrrhetinic acid was reversed by blockade of MR with spironolactone. In conclusion, loss of 11β-HSD2 activity leading to inappropriate activation of MR in atherosclerotic mice promotes plaque vulnerability and increases vascular infiltration of macrophages which accelerates plaque growth, possibly through enhanced MR- mediated endothelial cell expression of VCAM-1.

Atherosclerosis and post-angioplasty neointimal proliferation, which are leading causes of cardiovascular morbidity and mortality, develop as a result of chronic or acute vascular injury producing inflammatory and proliferative responses in the vessel wall. Glucocorticoids, the stress hormones produced by the adrenal cortex, have anti-inflammatory and anti-proliferative characteristics and can also influence systemic cardiovascular risk factors. The systemic levels of these hormones are controlled by the hypothalamic pituitary adrenal axis. However, there is also a tissue-specific pre-receptor regulation of these hormones by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD); type 1 regenerates active glucocorticoids within the cells and type 2 inactivates glucocorticoids. Whilst it has been shown that the inhibition of 11β-HSD1 has favourable effect on cardiovascular risk factors and the inhibition of 11β-HSD2 results in hypertension; the effect of these enzymes on vascular lesion development is not known. The work described in this thesis tested the hypothesis that 11β-HSD1 inhibition reduces vascular lesion development due to improvement in cardiovascular risk factors, whereas 11β-HSD2 inhibition leads to adverse vascular remodelling. Apolipoprotein-E deficient (ApoE-/-) mice fed on western diet were used to study atherosclerosis, whereas neointimal proliferation was investigated using a well-established mouse model of wire-angioplasty. Vascular lesions were assessed using novel imaging and standard histological techniques. 11β-HSD1 inhibition reduced the size of atherosclerotic lesions and improved markers of plaque stability with a reduction in lipid content and increase in collagen content of the plaques. This was associated with a reduction in weight gain and blood pressure but without any effect on lipid profile. 11β-HSD1 inhibition did not produce any significant effect on neointimal proliferation in C57Bl/6J mice. However in ApoE-/- mice, 11β-HSD1 inhibition reduced neointimal proliferation with corresponding increase in size of patent lumen and with an associated reduction in macrophage content of neointimal lesions. 11β-HSD2 deletion produced an outward remodelling in un-injured vessels but there was no effect on neointimal proliferation after wire-angioplasty. Administration of a selective mineralocorticoid antagonist, eplerenone, reduced neointimal lesions significantly but to a similar degree in both C57Bl/6J and 11β-HSD2-/- mice, associated with a significant reduction in macrophage content of lesions but without any effect on blood pressure. Data in this thesis highlight the potential therapeutic application of 11β-HSD1 inhibition in reducing the size and vulnerability of atherosclerotic plaques and also reduction in neointimal proliferation (and hence post-angioplasty restenosis) in high risk patients with „metabolic syndrome‟ phenotype. The results also indicate that 11β-HSD2 has a limited, if any, role to play in the development of neointimal lesions.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs on immune function. The effect of the immune system on GC action, especially in the periphery, is not as well studied. The liver plays an important role in inflammation and stress in producing the acute phase proteins (APPs) required for the resolution of inflammation as well as in producing systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding effects of stress and inflammation and their interplay in the liver is thus not only useful to expand our understanding of these systems but could also have clinical applications in understanding the side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be developed as an anti-inflammatory drug that displays fewer side effects. The current study investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK), and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects at the protein level, using Western blotting and ELISA assays, the protein activity level, using enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs on immune function. The effect of the immune system on GC action, especially in the periphery, is not as well studied. The liver plays an important role in inflammation and stress in producing the acute phase proteins (APPs) required for the resolution of inflammation as well as in producing systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding effects of stress and inflammation and their interplay in the liver is thus not only useful to expand our understanding of these systems but could also have clinical applications in understanding the side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be developed as an anti-inflammatory drug that displays fewer side effects. The current study investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK), and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects at the protein level, using Western blotting and ELISA assays, the protein activity level, using enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes / AFRIKAANSE OPSOMMING: Kruiskommunikasie bestaan tussen die stres– en die immuunsisteem en hierdie kruiskommunikasie is van farmakologiese belang vir die gebruik van glukokortikoïede (GKe) as anti-inflammatoriese medikasie vir siektes soos asma en artritis. Tot dusver was die fokus van studies oor hierdie kruiskommunikasie hoofsaaklik op die effek van GKe op immuunfunksie. Die effek van die immuunsisteem op GK werking, veral in die periferie, is nie so goed bestudeer nie. Die lewer speel ʼn belangrike rol in inflammasie en stres deurdat dit die akute fase proteïene (AFPs) produseer wat benodig word vir die resolusie van inflammasie en omdat dit ook sistemiese glukose produseer, d.m.v. glukoneogenese, wat benodig word om die stres reaksie te dryf. ’n Beter insig in die effek van stres en inflammasie sowel as hul interaksie in die lewer is dus handig, nie net om ons begrip van hierdie sisteme te verbeter nie, maar ook omdat dit kliniese toepassing kan hê deurdat dit ons begrip van die newe-effekte wat gepaard gaan met die farmakologiese gebruik van GKe verbeter. Verbinding A (CpdA) is geïdentifiseer as ʼn selektiewe glukokortikoïed reseptor (GR) moderator (SERGM) omdat dit die vermoë het om gene te onderdruk maar nie te aktiveer d.m.v. die GR. Hierdie eienskap dui op die potensiaal van CpdA om ontwikkel te word as ʼn anti-inflammatoriese middel met minder newe-effekte. Die huidige studie het die effekte van dexamethasone, ʼn sterk GR agonis, en CpdA, beide in die teenwoordigheid en afwesigheid van interleukin 6 (IL6), op die GR, drie metaboliese ensiem gene wat betrokke is by glukoneogenese, sowel as drie APP gene, ondersoek en vergelyk. Die metaboliese ensiem gene wat ondersoek is, is tirosien aminotransferase (TAT), fosfoenolpirovaat karboksikinase (PEPCK), en gamma glutamieltransferase (GGT), terwyl die APP gene serum amiloïede A (SAA), C-reaktiewe proteïen (CRP), en kortikosteroïed bindings globien (CBG) was. Die studie het die effekte in ʼn muis hepatoma sellyn (BWTG3) op die proteïen vlak, deur van Western blotting en ELISA essays gebruik te maak, die proteïen aktiwiteits vlak, deur van ensiem aktiwiteits essays en vol-sel binding gebruik te maak, sowel as op die mRNA vlak, deur van kwantitatiewe polimerase ketting reaksie (qPCR) gebruik te maak, ondersoek. Die studie toon dat dexamethasone (Dex) en IL6 in die algemeen divergente effekte het op die GR en metaboliese ensieme deurdat Dex GR af-reguleer en die metaboliese ensieme op-reguleer, terwyl IL6 die GR op-reguleer en die metaboliese ensieme af-reguleer, en dat hulle funksies konvergerend is vir die APPs deurdat beide positiewe APPs opreguleer en negatiewe APPs afreguleer. In teenstelling met Dex het CpdA die GR op-gereguleer en die metaboliese ensieme af-gereguleer terwyl dit, soos Dex, die positiewe APPs op-gereguleer en die negatiewe APPs af-gereguleer het. Ons resultate vir Dex en IL6 word ondersteun deur vorige werk in die literatuur. Ons studie is wel uniek omdat dit die ondersoek van drie metaboliese ensieme kombineer met die ondersoek van drie APPs, sowel as GR vlakke in ʼn enkele sisteem onder dieselfde eksperimentele kondisies. Verder het ons resultate met CpdA verskeie nuwe aspekte, soos die af-regulering van metaboliese gene, opgelewer wat bydra tot die groeiende poel van kennis oor hierdie ongewone GR ligand en die moontlike farmakologiese gebruik daarvan.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: This study investigated the effect of several hormones on the rat corticosteroid binding globulin proximal promotor and for the first time showed that modulation occurs at the promotor level and can be correlated with changes in corticosteroid binding globulin mRNA and protein levels. The effect of various physical and psychological stressors on rat liver corticosteroid binding globulin mRNA levels was also tested and it was shown that voluntary running had no effect on rat corticosteroid binding globulin levels but that involuntary swimming and immobilization decreased rat corticosteroid binding globulin mRNA levels. Glucocorticoid responsiveness of the corticosteroid binding globulin promoter was investigated further by using truncated contructs of the corticosteroid binding globulin proximal promoter. Glucocorticoid responsiveness was delineated to between -296 and -145bp from the transcription start site an area that contains putative binding sites for D-site binding protein, hepatic nuclear factor-3 and CAAT/enhancer binding protein suggesting that these transcription factors may be involved in glucocorticoid responsiveness of the corticosteroid binding globulin proximal promoter. The dissociative glucocorticoid activity of medroxyprogesterone acetate and Compound A, both putative dissociated glucocorticoids, was compared to standard glucocorticoids by examining transactivation of glucocorticoid response element-containing reporter constructs and transrepression of corticosteroid binding globulin gene expression in hepatic cell lines. Results showed that medroxyprogesterone acetate, but not Compound A, trans activates only in the presence, but not in the absence, of co-transfected glucocorticoid receptor. Medroxyprogesterone acetate down modulated dexamethasone transactivation while the modulatory effect of Compound A depends on the order of addition of Compound A. If added together Compound A has no effect on dexamethasone transactivation, however, if Compound A was added before dexamethasone, Compound A significantly decreased dexamethasone transactivation. Both medroxyprogesterone acetate and Compound A, like glucocorticoids, transrepressed the rat corticosteroid binding globulin proximal promoter. The potency of repression was similar but Compound A repressed with a higher efficacy than medroxyprogesterone acetate. We conclude that Compound A is a completely dissociated glucocorticoid in contrast to medroxyprogesterone acetate that displays only partial dissociation, which is dependent on glucocorticoid receptor levels. / AFRIKAANSE OPSOMMING: Tydens hierdie ondersoek is die effek van verskeie hormone op die rot kortikosteroied bindings globulien proksimale promoter ondersoek en vir die eerste keer is getoon dat modulering plaasvind op promoter-vlak en dat repressie korrileer met die verandering in kortikosteroied bindings globulien mRNA-en proteinvlakke. Die effek van verskeie fisiese en fisiologiese stressors op rotlewer kortikosteroied bindings globulien-mRNAvlakke is ook getoets en daar is getoon dat willekeurige hardloop geen effek op rot kortikosteroied bindings globulien-mRNA-vlakke het nie maar dat gedwonge swem en immobilisering rot kortikosteroied bindings globulien-mRNA-vlakke verlaag. Glukokortikoied responsiewiteit van die kortikosteroied bindings globulien proksimale promoter is verder ondersoek deur verkorte konstrukte van die kortikosteroied bindings globulien te toets. Glukokotikoied responsiewiteit is afgebaken tot tussen -296 en - 145bp vanaf die transkripsie beginplek 'n area wat beweerde bindings setels vir D-setel bindings protein, hepatosiet faktoor-3 en CCAAT-bindings protein-2 bevat en dus suggereer dat hierdie transkripsie faktore betrokke mag wees met glukokortikoied effekte op die kortikosteroied bindings globulien-proksimale promoter. Die dissosiatiewe glukokortikoied aktiwiteit van medroksiprogesteroon asetaat en Verbinding A, beide beweerde dissosiatiewe glukokortikoiede, relatief tot standaard glukokortikoiede is vergelyk deur transaktivering van glukokortikoied reseptor e1elment-bevattende konstrukte en onderdrukking van kortikosteroied bindings globulien geen ekspressie in lewersellyne te bestudeer. Medroksiprogesteroon asetaat, maar nie Verbinding A nie, transaktiveer slegs in die teenwoordigheid, maar nie in die afwesigheid, van ko-getransfekteerde glukokortikoied reseptore. Medroksiprogesteroon asetaat moduleer deksametasoon transaktivering afwaarts terwyl die modulerende effek van Verbinding A afhanklik van die orde van Verbinding A byvoeging is. Indien saam bygevoeg het Verbinding A geen effek op deksametasoon transaktivering nie, maar indien Verbinding A voor deksametasoon bygevoeg word verlaag Verbinding A deksametasoon transaktivering. Beide medroksiprogesteroon asetaat and Verbinding A, soos glukokortikoiede, onderdruk die rot kortikosteroied bindings globulien-proksimale promoter. Die sterkte van onderdrukking is dieselfde maar Verbinding A onderdruk met 'n hoër effektiwiteit as medroksiprogesteroon asetaat. Ons toon dat Verbinding A 'n totale dissosiatiewe glukokortikoied is in teenstelling met medroksiprogesteroon asetaat, wat slegs gedeeltelik dissosiatief is afhangende van glukokortikoied reseptor-vlakke.

During fetal development, the lung is filled with fluid that is secreted by an active Cltransport promoting lung growth. The basolateral Na+,K+,2Cl- cotransporter (NKCC1) participates in Cl- secretion. The apical Cl- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that stimulate alveolar Na+ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this study was to analyze the influence of glucocorticoids on pulmonary Cl- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Clchannels and are likely causing the decline of CFTR during late gestation in preparation for perinatal lung transition.

O uso crônico de altas doses de glicocorticoides (GC) aumenta tecidos adiposos (TA) centrais. Essa pesquisa visou verificar os efeitos do uso contínuo de GC em doses mais baixas sobre diferentes TAs. Ratos Wistar machos com 10 semanas de vida foram divididos em 2 grupos: controle (CON) e cortisol (CORT) e foram implantados com minibomba osmótica, pela qual o grupo CORT recebeu 0,6 mg/kg/d de cortisol e o grupo CON recebeu salina, por 6 semanas. Os resultados mostram um aumento da gordura subcutânea (SC) abdominal dos animais CORT, acompanhada de: aumento da atividade máxima das enzimas ATP-citrato-liase e glicose-6-fosfato desidrogenase; redução da expressão da enzima AMPK; aumento da expressão da enzima 11βHSD1 e das triglicérides circulantes. Esses dados sugerem que a intervenção aumentou a SC abdominal devido maior atividade de enzimas lipogênicas e de um possível aumento da captação de lipídeos séricos por esse tecido, o que pode ter resultado do aumento da concentração local de GC provocado pela expressão aumentada da 11βHSD1. / Chronic use of high doses of glucocorticoids (GC) increases central adipose tissue (AT). This research aimed to verify the effects of continuous use of lower doses of GC on different TAs. Male Wistar rats, 10 weeks old, were divided into 2 groups: control (CON) and Cortisol (CORT), and were implanted with osmotic minipump, whereby CORT group received 0.6 mg/kg/d of cortisol and CON group received saline, for 6 weeks. The results show an increase in abdominal subcutaneous fat (SC) of CORT animals accompanied by: increased maximum activity of ATP citrate lyase and glucose 6-phosphate dehydrogenase enzymes; reduced expression of AMPK enzyme; increased expression of 11βHSD1 enzyme and circulating triglycerides. These data suggest that the intervention increased abdominal SC due to increased activity of lipogenic enzymes and a possible increase in the uptake of serum lipids through this tissue, which may have resulted from increased local GC concentration caused by increased expression of 11βHSD1.

Afecções do sistema respiratório estão entre as mais frequentes em equinos, sendo uma das principais causas de baixo desempenho em animais atletas. Dentre essas doenças pode-se citar as afecções inflamatórias das vias aéreas, que apresentam uma prevalência considerável e afetam a saúde e vida atlética do animal. Um dos principais meios de atuação terapêutica no tratamento dessas doenças é a administração de medicamentos corticoides, sendo a dexametasona um dos principais fármacos dessa classe utilizados na medicina equina. No tratamento de doenças semelhantes em humanos preconiza-se a administração dos fármacos por via inalatória, otimizando assim o seu efeito terapêutico e diminuindo os efeitos colaterais. Em equinos existem trabalhos que mostram a deposição pulmonar de medicamentos quando administrados por via inalatória, no entanto, não foram encontrados estudos nesse sentido utilizando a dexametasona. O objetivo desse estudo foi avaliar a deposição pulmonar de dexametasona quando administrada por via inalatória em equinos, bem como fatores que possam interferir no seu nível de deposição e a concentração plasmática do fármaco quando administrada por essa via. Para isso foram utilizados seis equinos que foram submetidos à inalação com dexametasona duas vezes, cada uma utilizando um veículo diferente (aquoso ou oleoso) na formulação do fármaco, e quatro animais foram utilizados para o grupo controle, sendo submetidos à inalação somente com o veículo. Após cada inalação foi realizado um lavado broncoalveolar (LAB) e coletas sanguíneas seriadas para quantificação da dexametasona. Após a inalação o fármaco foi identificado nas amostras de LAB dos animais nos grupos tratados. Não houve diferença significativa entre os veículos utilizados e houve diferença entre os animais agitados e os animais calmos, sendo que os primeiros apresentaram uma concentração significativamente maior de dexametasona no LAB. O fármaco não foi identificado no plasma dos animais. A dexametasona quando administrada via nebulização pneumática atinge a região de bronquíolos e alvéolos respiratórios em equinos, principalmente os de comportamento agitado, e não atinge níveis plasmáticos significativos / Disorders of the respiratory system are among the most frequent deseases in horses and one of the main causes of low performance in athletic animals. Inflammatory diseases of the airways should be mentioned due to its considerable prevalence and to affect the health and athletic life of the animal. One of the main ways of therapeutic action of these diseases is the administration of corticosteroid drugs, being dexamethasone one of the main drugs used in equine medicine. In the treatment of similar diseases in humans it is recommended the administration of drugs by inhalation, which optimizes its therapeutic effect and reduces side effects. In horses, there are studies that show the pulmonary deposition of drugs when administered by inhalation, however, there are no studies using dexamethasone. The aim of this study was to evaluate the pulmonary deposition of dexamethasone when administered by inhalation in horses, as well as factors that may interfere with their level of deposition and the plasma concentration of the drug when administered by inhalation. To that, it was used six horses that were submitted to inhalation of dexamethasone twice, each one using a different vehicle (aqueous or oily) in the drug formulation, and four animals were used as control group, being submmited to inhalation just with the vehicle. After each inhalation a bronchoalveolar lavage (BAL) and serial blood collections for quantification of dexamethasone were performed. After the inhalation, the drug was identified in BAL samples from the animals of the treated groups. There was no significant difference between the vehicles used and there was difference between the agitated animals and calm animals, and the first ones had a significantly higher concentration of dexamethasone in the BAL. The drug was not identified in the plasma of animals. Dexamethasone, when administered via nebulization air, reaches the bronchioles region and respiratory alveoli in horses, especially the agitated behavior ones, and it does not affects plasma levels

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which controls the neuroendocrine response to stress. Following stressful stimuli, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus act synergistically to stimulate adrenocortiotrophin hormone (ACTH) secretion from corticotroph cells. ACTH is released into the circulation where it stimulates the secretion of glucocorticoids from the adrenal cortex. The HPA axis is kept in fine balance through an elegant negative feedback system where elevation of plasma glucocorticoids results in inhibition at the level of both the pituitary and the hypothalamus. During acute stress, glucocorticoids can be beneficial however chronic elevation of glucocorticoids can have many adverse effects on health. Corticotroph cells are electrically excitable and have been shown to fire single-spike action potentials as well as complex bursting patterns. Stimulation of corticotrophs with physiological concentrations of CRH/AVP results in a robust increase in firing frequency and a transition from spiking to bursting. Intracellular Ca2+ increases to a greater extent during bursting which has been proposed to drive hormone secretion. There is evidence to suggest that large conductance calcium- and voltage-gated potassium (BK) channels promote bursting behaviour in anterior pituitary cells. Glucocorticoids have been shown to regulate ACTH secretion and also modulate BK channel activity. However, the effects of glucocorticoids on native corticotroph excitability are currently unknown. The aim of this study was to first characterise the electrical properties of corticotrophs under basal conditions and following exposure to CRH/AVP. Secondly, to investigate the regulation of corticotroph excitability by glucocorticoids. Finally, establish the role of the BK channel in regulating bursting behaviour and CORT regulation in corticotroph cells. Corticotroph cells were acutely isolated by trypsin digestion from mice aged 2-5 months constitutively expressing GFP under control of the POMC promoter (POMC-GFP). Mice used for pituitary cell culture were male unless otherwise stated. Cells were maintained in a serum free media and electrophysiological recordings obtained 24-96 hours post-isolation. Current clamp recordings were obtained from corticotrophs using the perforated patch technique. Although spontaneous activity of corticotroph cells was variable, they displayed predominantly single-spike action potentials under basal conditions. Stimulation with physiological concentrations of CRH and AVP (0.2 nM and 2 nM respectively) resulted in a membrane depolarisation accompanied by an increase in firing frequency and a transition to bursting. Individually, CRH and AVP were able to increase corticotroph excitability. However, only CRH was able to drive an increase in bursting suggesting that bursting is primarily regulated through the cAMP/PKA pathway. Experiments were performed to investigate the modulation of corticotroph activity by glucocorticoid negative feedback. Acute exposure (< 10 min) to corticosterone resulted in a decrease in spontaneous activity as well as shortening the response to CRH/AVP. Pretreatment of corticotrophs with 100 nM corticosterone (90 min) resulted in a membrane hyperpolarisation and a decrease in spontaneous firing frequency. Following corticosterone pretreatment, CRH/AVP failed to induce a significant transition from spiking to bursting. Increasing the pretreatment time to 150 minutes resulted in a further suppression of both spontaneous and CRH/AVPevoked activity. Fast activation of BK channels during the upstroke of an action potential has been proposed to promote bursting behaviour in other pituitary cells. Corticotrophs treated with a BK channel blocker (1 μM paxilline) or isolated from BK-/- mice showed no significant difference in basal activity but displayed a reduction in CRH/AVPevoked bursting activity. In both cases, bursting was significantly reduced but not completely abolished. Corticosterone treatment of BK-/- cells resulted in a further decrease in both firing frequency and bursting behaviour. Taken together, these results suggest that although BK channels play an important role in bursting, they are not the only component. Comparisons of male and female corticotrophs revealed subtle differences in their properties. Following CRH/AVP stimulation, male cells displayed a high degree of bursting activity whereas female cells exhibited predominantly an increase in singlespike action potential frequency. Treatment of female corticotrophs with corticosterone (150 min) resulted in a significant reduction in firing frequency but no measurable change in bursting behaviour. BK-/- cells from female mice showed no difference in bursting activity following CRH/AVP compared to wild types. This data suggests that modulation of firing frequency is the more important component in female corticotroph cells. In conclusion, CRH/AVP is proposed to drive ACTH secretion in male corticotroph cells through an increase in bursting activity. Corticosterone pretreatment suppresses both spontaneous and CRH/AVP-evoked activity. It is possible that corticosterone regulates corticotroph excitability through two mechanisms. Corticosterone suppresses bursting activity following CRH/AVP stimulation through multiple targets which might include the BK channel. Additionally, corticosterone reduces firing frequency through a mechanism independent of BK channels. It is important to further characterise the physiology of corticotroph cells and how ACTH secretion is regulated through their electrical excitability. This would lead to a greater understanding of the role of corticotrophs in the HPA axis. Further study of corticotrophs could potentially lead to pharmacological manipulation of the stress response and novel treatments for stress-related disorders.

Stress contributes to the development of major depressive disorder (MDD) and post-traumatic stress disorder (PTSD), and an intermediary factor between stress and psychiatric disorders may be epigenetics. Studies have shown altered DNA methylation (DNAm) in animal models of and humans with stress exposure and in individuals with PTSD and MDD. The availability of genome-wide experimental platforms has given us new tools to investigate DNAm, and in this dissertation these techniques have been used to further our current understanding of the epigenetics of stress. We performed a genome-wide investigation in mice exposed to chronic stress that exhibit depressive- and anxious-like behaviors, examining DNAm changes within the dentate gyrus, a sub-region of the hippocampus that contributes to the stress response. Using the Methyl-Seq method, an intergenic region of chromosome X was shown to be differentially methylated with chronic stress, and this finding replicated in two additional cohorts of mice. In postmortem brain tissue of humans with MDD, an increase in DNAm within this intergenic region was also found. Animal models do not fully capture the complexity of stress and psychiatric disorders in humans, but comparable studies in humans are limited by the difficulty of obtaining brain tissues. Instead, these studies have used peripheral tissues to examine DNAm changes related to stress and psychiatric disorders. To address the usefulness of these peripheral tissues, we employed the Illumina 450K and EPIC arrays to establish a resource that compares DNAm of the brain to that of blood, buccal, and saliva tissues. Glucocorticoids (GCs) play an essential role in the stress response, and their dysregulation is seen in individuals with MDD and PTSD. To determine the role of GCs in stress-mediated epigenetic changes, buccal samples were obtained before and after individuals were given GCs in the context of oral surgery, and DNAm was analyzed using the Illumina EPIC array. Five CpGs were altered following this exposure, to a genome-wide significant degree. Further analysis revealed FDR-significant CpG changes to be in genes involved in steroid hormone biosynthesis and in genes differentially expressed with GC exposure. Collectively, these results exemplify the complexity of DNAm changes associated with the stress response and provide potential avenues for elucidating their impact on psychiatric disorders.

Orientador: Chung Man Chin / Banca: Iracilda Zeppone Carlos / Banca: Gustavo Henrique Goulart Trossini / Resumo: A inflamação é caracterizada como uma resposta do organismo frente à uma agressão, a qual pode resultar em dor, edema e em alguns casos levar à disfunção do órgão ou tecido. Durante o processo inflamatório ocorre a liberação de citocinas, pequenas proteínas transcritas e traduzidas pelo metabolismo intrínseco celular, que realizam uma série de comunicações intra e intercelulares. Uma importante citocina é o fator de necrose tumoral alfa (TNF-α), que atua na manutenção do quadro inflamatório em situações de inflamação crônica, sendo super expressa em diversas doenças inflamatórias. A terapia anti fator de necrose tumoral α (anti-TNF-α) tornou-se uma abordagem interessante no tratamento de doenças inflamatórias crônicas, principalmente daqueles pacientes que não respondem ao tratamento convencional. Entre os fármacos utilizados no tratamento das doenças inflamatórias crônicas, encontram-se os glicocorticoides (GCs), anti-inflamatórios de natureza esteroide. Estes atuam no processo reacional de defesa do organismo minimizando o dano causado pelo agente infeccioso. Porém o uso prolongado de GCs está associado a diversos efeitos adversos, como osteoporose, síndrome metabólica, doenças cardiovasculares, catarata, entre outros, limitando o uso desses fármacos em terapias prolongadas. Dessa forma, nesse trabalho, buscou-se através da estratégia de hibridação molecular, a síntese de novos derivados anti-inflamatórios esteroides, com propriedades de modulação da citocina TNF-α, a fim de se alcançar um sinergismo de ação útil no tratamento de doenças inflamatórias crônicas. Nesse contexto, foram sintetizados e caracterizados seis compostos, série Lapdesf GL-FT, derivados dos glicocorticoides prednisolona (compostos I-III) e budesonida (compostos IV-VI), ligados ao grupamento ftalimida, responsável pela inibição da síntese de TNF-α. Os compostos ... / Abstract: Inflammation is characterized as a organism response against an aggression, which can result in pain, edema and in some cases lead to organ or tissue dysfunction. During the inflammatory process are released of cytokines, small proteins transcribed and translated by intrinsic cellular metabolism, performing a variety of communication intra and intercellular. An important cytokine is tumor necrosis factor alpha (TNF-α), which acts in maintaining the inflammatory in situations of chronic inflammation, being over expressed in many inflammatory diseases. Therapy anti-tumor necrosis factor α (anti-TNF-α) has become an interesting approach in the treatment of chronic inflammatory diseases, especially those patients who do not respond to conventional treatment. Among the drugs used in the treatment of chronic inflammatory diseases are glucocorticoids (GCs), anti-inflammatory steroids. These act in the reactive process of organism defense minimizing the damage caused by the infectious agent. However, prolonged use of GCs is associated with various side effects such as osteoporosis, metabolic syndrome, cardiovascular disease, cataracts, among others, limiting the use of these drugs in prolonged therapies. Thus, in this work, we realized the viability of design and synthesis by molecular hybridization strategy of novel anti-inflammatory steroids with modulating properties of cytokine TNF-α in order to achieve a synergism of action useful in the treatment of diseases chronic inflammatory diseases. In this context, were synthesized and characterized six compounds Lapdesf series GL-FT derived from glucocorticoids prednisolone (compounds I-III) and budesonide (compounds IV-VI), connected to the phthalimide group responsible for inhibition of TNF-α. The obtained compounds were evaluated for anti-inflammatory activity in a model of paw edema and distal ulcerative colitis. Derivatives I and IV showed higher ... / Mestre