Glucocorticoids - cortisol and corticosterone - are steroid hormones synthesised in the adrenal gland that are important mediators of the stress response. Glucocorticoids are also vital in development to aid in organ maturation. Endogenous glucocorticoid levels rapidly rise before birth in all mammals to promote fetal organ maturation. Because preterm birth occurs before this natural rise in glucocorticoid levels, pregnant women at risk of preterm delivery are administered synthetic glucocorticoids to mature the fetal lung and aid neonatal survival. Mice that globally lack the glucocorticoid receptor (GR) die at birth, attributed to lung immaturity. Effects on tissues other than the lung remain less well characterised. Previous work has shown endogenous glucocorticoid action is also essential to mature the mouse fetal heart. Mice globally lacking GR have small, functionally and structurally immature hearts. Mice with tissue-specific deletion of GR in cardiomyocytes and vascular smooth muscle cells (SMGRKO mice; generated using Sm22α-Cre) also have an increased risk of death around the time of birth, suggesting that glucocorticoid maturation of the cardiovascular system is important for neonatal survival. GR expression within the fetal mouse heart initiates at E10.5 but GR in the myocardium is not activated and localised to the nucleus until E15.5. This suggests that mice can respond to glucocorticoid from E10.5. Here, it was hypothesised that antenatal glucocorticoid exposure, prior to the increase in endogenous glucocorticoid levels, would advance fetal heart maturation and this will depend on cardiovascular GR. To investigate the effects of antenatal glucocorticoid treatment on fetal heart maturation in mid-gestation and identify effects mediated by GR, mice with a conditional deletion of GR in cardiomyocytes and vascular smooth muscle cells were studied (SMGRKO mice). Pregnant mice received dexamethasone (dex) in the drinking water from E12.5-E15.5. Levels of Fkbp5 mRNA (a marker of glucocorticoid action) were unchanged between control and SMGRKO mice at E15.5 or following dex treatment. This suggested a lack of response to dex treatment. However, liquid chromatography mass spectrometry measurement confirmed the presence of dex and its active metabolite 6- hydroxydexamethasone (6OHDex) in livers of E15.5 fetuses from dex treated dams (fetal: Dex 0.46 ± 0.1 ng/g, 6OHDex 13.6 ± 0.35 ng/g; dam: Dex 7.96 ± 3.65 ng/g, 6OHDex 4.75 ± 1.2 ng/g). Livers of fetuses exposed to dex had lower levels of the naturally occurring active glucocorticoid, corticosterone, compared to vehicle treated fetuses. This suggests HPA axis suppression in dex exposed fetuses. Maternal liver showed no significant difference in corticosterone levels between dex and vehicle treated mice, suggesting that whilst dex suppressed the HPA axis in fetuses, it did not in the dams. To determine any persistent effects of early antenatal dex treatment on fetal heart, a later time point in gestation, E17.5, was also assessed. At E17.5, 2-days following cessation of dex treatment, dex and its metabolites were undetectable in the fetal and maternal liver. However, corticosterone levels remained reduced in fetal liver at E17.5 in dex exposed animals (vehicle treated: 4.31 ± 0.47 ng/g, Dex treated: 1.72 ± 0.42 ng/g, p < 0.01), whilst levels in the dam liver did not differ from vehicle treated controls. This suggests prolonged HPA axis suppression following dex treatment, which reduced the natural late-gestation rise in glucocorticoids required for fetal organ maturation. To determine whether early antenatal dex treatment could advance fetal heart function, Doppler imaging with a Vevo 770 high frequency ultrasound imager was used. Isovolumetric contraction time, isovolumetric relaxation time and ejection time of the left ventricle were unaltered by dex treatment. However, at E15.5 the mitral deceleration index (MDI), a measure of diastolic function that takes into account loading conditions, was 1.5 fold lower in vehicle treated SMGRKO mice than control (Cre-) littermates (p < 0.05). This reduction in SMGRKO mice suggests glucocorticoids are required within the fetal cardiomyocytes and/or vascular smooth muscle cells to mature the diastolic function of the fetal heart. Dex exposure had no effect on MDI in SMGRKO fetuses, but reduced the MDI by 1.5 fold in control mice to similar levels as in SMGRKO mice (p < 0.05). RNA analysis revealed a trend (p=0.09) for reduced levels of Nr3c1 mRNA (encoding GR) in hearts of E15.5 control (Cre-) fetuses following dex treatment. Although this requires confirmation at the level of GR protein, this finding together with the lack of induction of the GR target, Fkbp5, suggests dex may cause glucocorticoid resistance through down-regulation of GR. At E17.5, 2-days following cessation of dex there were no changes in systolic parameters and the reduction in MDI found at E15.5, following dex, had normalised. Litter size was reduced (close to a 50% reduction) at E17.5 in dex treated mice. This was similar between SMGRKO and control fetuses. The cause of death was not established, but potentially could be due to the reduction in the natural rise in glucocorticoids at E17.5, previously shown to be important for fetal heart maturation. It is therefore possible that mice with more immature hearts may die before reaching E17.5. RNA analysis was undertaken to determine any mechanistic alterations following dex treatment, which could support fetal heart functional alterations found at E15.5. In contrast to expectation, dex also decreased expression of mRNA encoding the calcium handling proteins SERCA2a, NCX1, and CaV1.2 in E15.5 fetal mouse hearts in both control and SMGRKO mice (p < 0.05), compared with the respective vehicle treated mice. These proteins had previously shown to be induced by glucocorticoid action in cardiomyocytes. However, the similar down-regulation in both genotypes indicates this effect is not dependent on GR in cardiomyocytes. Lowered SERCA2a activity following dex treatment could contribute to the changes in MDI observed in control mice. Similarly, Scnn1a and Kcnj12 mRNA levels, previously found to be induced by glucocorticoids in cardiomyocytes, were down-regulated in the E15.5 fetal heart in vivo following dex. Collectively, these data are consistent with glucocorticoid resistance or down-regulation of glucocorticoid action in E15.5 fetal hearts following dex administration. Mutations in KCNJ12 are associated with long QT syndrome, which is characterised by a delayed repolarisation of the heart following each contraction. An altered relaxation of the fetal heart found in control mice following dex could therefore be due to a prolongation of the cardiac action potential, particularly with a delayed repolarisation, because of lower Kcnj12 expression. At E17.5, there were no significant differences in expression of calcium handling genes or ion channel mRNAs between genotypes or following earlier dex exposure. Thus, effects of dex on mRNA expression level may not persist, which could account for the lack of functional changes observed 2-days following cessation of treatment. Because effects seen in vivo with dex treatment were contrary to those predicted, and to further investigate the effect of dex upon calcium content, an in vitro model of primary fetal E15.5 cardiomyocytes was used. Cardiomyocytes were treated with dex for 24 hours and effects on membrane potential voltage changes and calcium transients measured. Following dex, isolated fetal cardiomyocytes showed an elongated repolarisation phase of the action potential (untreated: 120.45 ± 13.81 ms, Dex: 142.34 ± 12.97 ms, p < 0.01), and duration of calcium transients (untreated: 103.31 ± 13.78 ms, Dex: 120.43 ± 23.36 ms, p < 0.05). This assessment of fetal cardiomyocytes was preliminary work to aid in the understanding of mechanisms of fetal heart functional alterations associated with glucocorticoid regulation. The results suggest glucocorticoids may be important in regulating calcium levels. In summary, dex treatment in mice from E12.5-E15.5 did not advance fetal heart maturation. It reduced litter size at E17.5, irrespective of whether GR was expressed in cardiomyocytes or not. The normal late-gestation increase in endogenous glucocorticoid levels in the fetus was reduced by dex, even after treatment finished. / The suppression of corticosterone levels following antenatal dex may reduce maturation of the heart at E15.5 and could be responsible for the reduction in litter size. Downregulation of GR in the fetal heart, may be a mechanism that results in glucocorticoid resistance following antenatal dex treatment, which could explain the lack of beneficial effects of antenatal dex upon fetal heart maturation in these experiments in mice.

As urbanization spreads, understanding its impact on wildlife is increasingly urgent. By comparing the traits and fitness of individuals within the same species found in both urban and rural habitats (urban adapters), we can better understand the behavioral and physiological coping mechanisms wild birds employ in the face of rapid environmental change. For my dissertation, I investigated the physiological, behavioral, and fitness differences between urban and rural living song sparrows (Melospiza melodia) to explore how song sparrows are adjusting to urban environments. In my first chapter, I investigated urban birds' termination of the glucocorticoid stress response by looking at their ability to reduce circulating levels of glucocorticoid 'stress' hormones and the relative abundance of receptors that provide negative feedback in the hippocampus and hypothalamus. I found that urban males have a lower relative abundance of glucocorticoid receptors and the enzyme 11β-HSD2 in the hippocampus compered too rural, though we found no difference in negative feedback at the periphery, as both urban and rural song sparrows responded similarly to a challenge with synthetic glucocorticoid (dexamethasone). In chapter 2, I asked if increased aggression, which has been rigorously documented in urban males, is also expressed by females, and whether this aggressive signaling is constraining other reproductive behaviors such as maternal care. Indeed, female song sparrows, like males, expressed increased aggressive signaling compared to rural, suggesting urban habitats may favor a more aggressive phenotype. Finally, in Chapter 3, I investigated the consequences of increased male aggression on their social partners and offspring by measuring parental care and nestling outcomes across urban and rural habits. I was unable to establish a trade-off between parental care and aggression in either sex, suggesting this increased aggression is not constraining other reproductive behaviors. In fact, the more aggressive urban males visited the nest significantly more frequently, a trend also seen in urban females during the daylight hours, though the relationship was not significant over a 24-hour period. Additionally, urban birds had significantly higher reproductive metrics compared to rural, though they also had the added cost of increase brood parasitism by brown-headed cowbirds (Molothrus ater) compared to rural. Overall increased urban aggression was associated with higher reproductive success without any reduction in paternal care. Additionally, we found physiological differences in the glucocorticoid stress response system associated with the differences in habitat but whether theses differences represent mechanisms of acclimation or potential costs of living in urban habitats is not yet clear. / Doctor of Philosophy / As urbanization spreads, understanding its impacts on wild bird conservation is increasingly urgent. By comparing the behaviors and reproductive success of animals living in urban and rural habitats (urban adapters), we can better understand the coping mechanisms wild birds' employ in the face of this form of rapid environmental change. In my dissertation, I compared the physiology, behavior, and reproductive success of urban and rural song sparrows (Melospiza melodia) to explore the changes song sparrows make to survive and reproduce in urban environments. In my first chapter, I investigated how urban birds terminate their stress response by looking at their ability to reduce circulating levels of stress hormones and the relative abundance of "shut down" targets in the brain. In chapter 2, I asked if increased aggression, regularly document in urban males, is also expressed by females, and whether this aggression is constraining other reproductive behaviors. Finally, in Chapter 3, I investigated the consequences of increased male aggression on their social partners and offspring by measuring parental care and nestling outcomes across urban and rural habits. I found that urban males have a lower relative abundance of one type of "shut down" target, and a lower abundance of a potentially protective enzyme in the hippocampus, though we found no difference in how quickly urban and rural birds cleared stress hormone from their blood. Female song sparrows, like males, expressed increased aggressive signaling compared to rural, suggesting urban habitats may favor a more aggressive pattern of behavior. However, I was unable to establish a trade-off between parental care and aggression in either sex, suggesting increased aggression is not constraining other reproductive behaviors. In fact, the more aggressive urban males visited the nest significantly more often, a trend also seen in urban females during the daylight hours, though the relationship was not significant over a 24-hour period. Additionally, urban birds had significantly higher reproductive metrics compared to rural, though they also had the added energetic cost of increased brood parasitism by brown-headed cowbirds (Molothrus ater), compared to rural. Collectively, my results suggest that individuals of this species, the song sparrow, may benefit from livening in low intensity urban habitats and that living in such altered environments favors or permits higher aggression.

Thesis (PhD)--Stellenbosch University, 2011. / See also the post-print version of the article that was published from the PhD - http://hdl.handle.net/10019.1/19557 / ENGLISH ABSTRACT: Glucocorticoid receptor (GR) levels, which modulate the response to glucocorticoids (GCs), vary between tissues and individuals and are altered by physiological and pharmacological effectors. In this study we set out to investigate the effects and implications of differences in GR concentration. Firstly, we established conditions that resulted in three statistically different GR populations in transiently transfected COS-1 cells. We demonstrated, using whole cell saturation ligand binding experiments, that high levels of wild type GR, but not of dimerization deficient GR, exhibited positive cooperative ligand binding with a concomitant increased ligand binding affinity. Furthermore, we established, through co-immunoprecipitation and fluorescent resonance energy transfer, that ligand independent dimerization correlates with positive cooperative ligand binding. This is the first time that positive cooperative ligand binding and increased ligand binding affinity have been explicitly correlated and linked to increased ligand independent dimerization of the GR. The downstream consequences of variation in GR concentration and dimerization included modulation of GR import and export rates, as investigated through live cell as well as immunofluorescent analysis. Furthermore, the nuclear distribution of GR was also influenced by GR dimerization. The major function of the GR is as a transcription factor, which mediates the response to GCs via activation or repression of genes. We have revealed direct influences of GR concentration and dimerization in a number of promoter reporter assays as well as in the transactivation of an endogenous gene. Specifically, cooperative ligand binding was found to be responsible for the GR level dependent potency shift in transrepression of an NF B containing promoter reporter construct via dexamethasone and the shift in the bio-character of Compound A, a dissociative GR agonist. Transactivation potency of dexamethasone as well as the partial agonist bio-character of medroxyprogesterone and mifepristone via a multiple GRE containing promoter reporter construct were influenced directly by cooperative ligand binding. Dimerization of the GR was shown to be crucial for ligand dependent transactivation of a single GRE containing promoter reporter construct, while ligand independent transactivation of both single and multiple GRE containing constructs was significantly increased due to an increase in GR concentration. The endogenous GC responsive glucocorticoid induced leucine zipper (GILZ) gene demonstrated significant ligand independent transactivation at GR levels, which displayed ligand independent dimerization. An increase in GR concentration resulted in an increase in efficacy through all promoter reporter constructs as well as the endogenous GILZ gene. Positive cooperative binding and the concomitant increase in ligand binding affinity to the GR at high levels may be a crucial factor in determining both the efficacy and potency of the GC response. Considering the significant differences in GR concentrations expressed by different tissues and by individuals within the same tissue, our findings may explain the interindividual as well as tissue specific responses to GC treatment and suggest an important mechanism of action through which the GR is primed to responsed to subsaturating GC concentrations and displays a significant level of ligand independent activity. / AFRIKAANSE OPSOMMING: Glukokortikoïed reseptor (GR) vlakke, wat die gedrag van glukokortikoïede (GCs) moduleer, wissel tussen weefsels en onder individue en word verander deur fisiologiese en farmakologiese effektore. In hierdie studie ondersoek ons die gevolge en implikasies van verskille in GR konsentrasie. Eerstens het ons die kondisies vasgestel wat benodig word om drie statisties verskillende GR populasies te vestig in kortstondige getransfekteerde COS-1 selle. Ons het getoon, met behulp van die heel sel versadigings ligand bindings eksperimente, dat hoë vlakke van wilde-tipe GR, maar nie van dimeriserings gebrekkige GR, positiewe koöperatiewe ligand binding, met 'n gepaardgaande toename in ligand bindings affiniteit, toon. Verder het ons bevestig, deur ko-immunopresipitasie en fluoressente resonansie energie-oordrag, dat ligand onafhanklike dimerisering korreleer met positiewe koöperatiewe ligand binding. Dit is die eerste keer dat positiewe koöperatiewe ligand binding en verhoogde ligand bindings affiniteit uitdruklik gekorreleer en gekoppel word aan verhoogde ligand onafhanklike dimerisering van die GR. Die daarop nagevolge van variasie in GR konsentrasie en dimerisering sluit in modulasie van die invoer en uitvoer tempo van die GR, soos ondersoek deur lewendige sel sowel as immunofluorescente analise. Verder is die verspreiding van die GR in die kern ook beïnvloed deur GR dimerisering. Die belangrikste funksie van die GR is as 'n transkripsie faktor, wat die respons van GCS bemiddel via aktivering of onderdrukking van gene. Ons het die direkte invloed van GR konsentrasie en dimerisering in 'n aantal promotor verslaggewer essais sowel as in die transaktivering van endogene gene onthul. Spesifiek, is gevind dat koöperatiewe ligand binding verantwoordelik is vir die GR vlak afhanklike verskuiwing in transrepressie potensie van 'n NF B bevattende promotor verslaggewer konstruk via deksametasoon en die verskuiwing van die biokarakter van verbinding A,' dissosiatiewe GR agonis. Transaktiverings potensie van deksametasoon, asook die gedeeltelike agonis bio-karakter van medroksie-progesteroon en mifepristoon, via 'n veelvoudige GRE bevattende promotor verslaggewer konstruk is direk beïnvloed deur koöperatiewe ligand binding. Dimerisering van die GR is getoon om deurslaggewend vir ligand afhanklike transaktivering van 'n enkele GRE bevattende promotor verslaggewer konstruk te wees, terwyl ligand onafhanklike transaktivering van beide enkel-en veelvoudige GRE bevattende konstrukte aansienlik toegeneem het as gevolg van toename in GR konsentrasie. Die endogene GC responsiewe glukokortikoïed geïnduseerde leusien rits (GILZ) gene het beduidende ligand onafhanklike transaktivering gedemonstreer op GR vlakke wat ligand onafhanklike dimerisering toon. 'n toename in GR konsentrasie het gelei tot toename in die effektiwiteit van al die promotor verslaggewer konstrukte, sowel as die endogene GILZ gene. Positiewe koöperatiewe ligand binding en die gepaardgaande toename in ligand bindings affiniteit van die GR by hoë vlakke kan 'n belangrike faktor wees in die bepaling van sowel die effektiwiteit as die potensie van die GC respons. As die aansienlike verskille in GR konsentrasies van verskillende weefsels en tussen verskillende individue in dieselfde weefsel in ag geneem word, kan ons bevindings die inter-individuele sowel as weefsel spesifieke response op GC behandeling verduidelik en stel dit 'n belangrike meganisme van aksie voor waardeur die GR voorberei word om op sub-versadigings konsentrasies van GC te reageer deur 'n beduidende vlak van ligand onafhanklike aktiwiteit te toon.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)), designed to mimic the actions of the endogenous hormone progesterone (Prog), are extensively used by women as contraceptives and in hormone replacement therapy (HRT). A number of reports have indicated that these synthetic progestins affect immune function in the female genital tract thereby increasing the risk of acquiring sexual transmitted infections. Despite these findings, very little is known about their mechanism of action at the cellular level, in particular their steroid receptor-mediated effects on cytokine gene expression. In the first part of this thesis, the effect of Prog, MPA and NET-A on the expression of the endogenous pro-inflammatory cytokine gene, interleukin (IL)-12p40, and anti-inflammatory cytokine gene, IL-10, was investigated in a human ectocervical epithelial cell line, Ect1/E6E7. Quantitative realtime PCR (qPCR) showed that all three ligands significantly upregulated the tumor necrosis factor alpha (TNF )-induced IL-12p40 gene expression, while IL-10 gene expression was downregulated. Moreover, by reducing the glucocorticoid receptor (GR) levels with siRNA, these effects were shown to be mediated by the GR. A more detailed investigation into the molecular mechanism of the progestogen-induced upregulation of IL-12p40 gene expression, using chromatin immunoprecipitation (ChIP), siRNA, co-immunoprecipitation and re-ChIP analyses, showed that the progestogen-bound GR is recruited to the CCAAT enhancer binding protein (C/EBP)- regulatory element of the IL-12p40 promoter, most likely via an interaction with the transcription factor C/EBP . Similar experiments for the progestogen-induced downregulation of IL-10 gene expression showed that the progestogen-bound GR is recruited to the signal transducer and activator of transcription (STAT)-3 regulatory element of the IL-10 promoter, most likely via an interaction with the transcription factor STAT-3. The second part of this study elucidated the influence of the HIV-1 accessory viral protein R (Vpr) on progestogen-induced regulation of IL-12p40, IL-12p35 and IL-10 in the Ect1/E6E7 cell line. Results showed that in these cells, the overexpression of Vpr significantly modulated the effects of Prog, MPA and NET-A on the mRNA expression of IL- 12p40 and IL-10, while only the NET-A effect was modulated on IL-12p35. Moreover, reducing the GR protein levels by siRNA suggested that the GR is required by Vpr to mediate its effects. Taken together, these results show that Prog, MPA and NET-A promote the pro-inflammatory milieu in the ectocervical environment, and that during HIV-1 infections, this milieu is modulated. Furthermore, the results suggest that the use of MPA or NET in vivo may cause chronic inflammation of the ectocervical environment, which may have important implications for ectocervical immune function, and hence susceptibility to infections such as HIV-1. / AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), ontwerp om die funksies van die natuurlike hormone progesteroon (Prog) na te boots, word wêreldwyd deur vroue as voorbehoedmiddels sowel as vir hormoon vervangingsterapie (HVT) gebruik. Daar is verskeie aanduidings dat hierdie sintetiese progestiene die immuunfunksie in die vroulike geslagskanaal kan beïnvloed en ook die moontlike vatbaarheid van seksueel oordraagbare infeksies kan verhoog. Ten spyte hiervan, is baie min bekend oor hulle meganisme van werking op ‘n molekulêre vlak, veral in die besonder hul effek op sitokinien geenuitdrukking. Die effek van Prog, MPA en NET-A op die geenuitdrukking van ’n endogene pro-inflammatoriese sitokinien, interleukin (IL)-12, en ’n anti-inflammatoriese sitokinien, IL-10, asook die onderliggend meganisme van werking, in ’n menslike ektoservikale sellyn, Ect1/E6E7, is in die eerste deel van hierdie studie ondersoek. Kwantitatiewe “realtime” polimerisasie ketting reaksie (PKR) het getoon dat al drie die ligande die tumor nekrosis faktor alfa (TNF- )-geïnduseerde IL-12p40 geenuitdrukking opreguleer en IL-10 geenuitdrukking onderdruk. Verder is gevind dat induksie van IL-12p40 en inhibisie van IL-10 deur Prog, MPA en NET-A deur die glukokortikoïed reseptor (GR) gedryf word, aangesien volledige opheffing van die effekte op hierdie sitokinien gene waargeneem is wanneer die GR proteïen vlakke deur middel van kort inmengende ribonukleïensuur (siRNS) verminder is. 'n Meer beskrywende ondersoek in die molekulêre meganisme is uitgevoer deur gebruik te maak van chromatien immunopresipitasie (ChIP), siRNS, mede-immunopresipitasie en her-ChIP analises. Hierdie resultate het voorgestel dat die progestogeen (Prog en die sintetiese progestiene)-gebonde GR tot die CCAAT verbeterende bindings protein (C/EBP)- regulatoriese element van die IL-12p40 promotor betrek word en dat die transkripsie faktor C/EBP benodig word om transkripsie van die IL-12p40 geen te aktiveer. Met betrekking tot IL-10, het die resultate voorgestel dat die progestogeen-gebonde GR tot die sein transduksie en aktiveerder van transkripsie (STAT)-3 regulatoriese element van die IL-10 promotor betrek word en dat die transkripsie faktor STAT-3 benodig word om transkripsie van die IL-10 geen te onderdruk. Die tweede deel van die studie het die invloed van die MIV-1 aksesorale virale proteïen R (Vpr) op sitokinien geenuitdrukking, spesifiek die progestogeen-geïnduseerde regulering van IL-12p40, IL-10 en IL-12p35, in die Ect1/E6E7 sellyn ondersoek. Resultate het getoon dat ooruitdrukking van Vpr in hierdie sellyn die effekte van Prog, MPA en NET-A op die mRNS uitdrukking van IL-12p40 en IL-10, en slegs die NET-A effek op IL-12p35, aansienlik moduleer. Vermindering van die GR proteïen vlakke deur middel van siRNS het getoon dat Vpr die GR benodig om hierdie veranderinge mee te bring. In samevatting, die resultate van hierdie proefskrif stel voor dat Prog, MPA en NET-A die pro-inflammatoriese milieu in die ektoservikale omgewing bevorder, en dat hierdie milieu gedurende MIV-1 infeksies verander. Verder, die resultate van hierdie studie impliseer dat die gebruik van MPA en NET in vivo nadelige lokale immuunonderdrukkende effekte mag hê wat kan lei tot kroniese inflammasie van die ektoservikale omgewing en ‘n moontlike verhoging in die vatbaarheid van infeksies soos MIV-1.

A melatonina (MEL) é um hormônio que participa do controle de uma série de processos fisiológicos em situações de higidez e durante processos de defesa. Além da produção noturna pela glândula pineal, células do sistema imunológico também produzem MEL. Diversos estudos demonstram que a produção de MEL pela pineal e células imunes é controlada por agentes endógenos (TNF, catecolaminas) e exógenos (LPS, zimosan) que ativam/controlam o desenvolvimento de processos de defesa. Em macrófagos, a produção de MEL induzida por zimosan, por exemplo, aumenta a fagocitose destas células. Na glândula pineal, glicocorticoides podem tanto potencializar quanto reduzir a síntese de MEL dependendo do padrão de estimulação adrenérgica imposto sobre a glândula. Contudo, não existem relatos sobre os efeitos dos glicocorticoides sobre a produção de MEL por células imunocompetentes. Tendo em vista a importância da produção de MEL no funcionamento adequado de macrófagos, o presente estudo investigou os efeitos da corticosterona (CORT) sobre a produção de MEL induzida por diferentes estímulos em macrófagos da linhagem RAW 264.7. Constatamos que os efeitos induzidos pela CORT sobre a produção de MEL dependem do contexto aos quais essas células foram submetidas. Tanto CORT quanto zimosan aumentam a produção de MEL. Todavia, quando as células são pré-incubadas com CORT, a produção de MEL induzida pelo zimosan é inibida. Em ambos os casos, os efeitos da CORT parecem ser dependentes da ativação dos receptores de glicocorticoides (GR). Com relação à produção de MEL induzida por zimosan, o efeito dos GRs está associado com a inibição da via do NF?B. Este trabalho pode ser relevante para a compreensão dos efeitos da CORT sobre o funcionamento das células imunes em condições de estresse crônico, uso excessivo de corticoides e diante de desafios imunológicos / Melatonin (MEL) is a hormone that participates in the control of a series of physiological processes in healthiness situations and during defense processes. In addition to the nocturnal production by the pineal gland, cells of the immune system also produce MEL. Several studies have shown that the production of MEL by pineal and immune cells is controlled by endogenous (TNF, catecholamines) and exogenous (LPS, zymosan) agents that activate / control the development of defense processes. In macrophages, the production of MEL induced by zymosan, for example, increases the phagocytosis of these cells. In the pineal gland, glucocorticoids may both potentiate and reduce MEL synthesis depending on the adrenergic stimulation pattern imposed on the gland, however there are no reports on the effect of glucocorticoids on the production of MEL by immunocompetent cells. The present study investigated the effects of corticosterone (CORT) on the production of MEL induced by different stimuli in RAW 264.7 macrophages. We found that the effects induced by CORT on the production of MEL depend on the context to which these cells were submitted. Both CORT and zymosan increase MEL production, however, when cells are preincubated with CORT, the production of MEL induced by zymosan is inhibited. In both cases, the effects of CORT appear to be dependent on the activation of glucocorticoid receptors (GR). With respect to zymosan induced MEL production, the effect of GRs is associated with the inhibition of the NF?B pathway. This work might support the understanding of the effects of CORT on the functioning of immune cells under conditions of chronic stress, excessive use of corticosteroids and immunological challenges

O desmame precoce (DP) consiste na abrupta substituição do leite pela dieta sólida e este padrão de alimentação pode ter impacto sobre o crescimento do estômago. Esta situação é também estressante para os filhotes e eleva os níveis de corticosterona que age ligando-se ao receptor de glicocorticoide (GR). Estudamos a expressão e a distribuição do GR na mucosa gástrica de ratos amamentados (C) e em DP. A expressão de GR foi maior aos 17 dias no grupo C e aumentou do 17º para o 18º dia no grupo em DP (p<0,05). O DP diminuiu o nível de GR, principalmente aos 18 dias (p<0,05), porém não alterou sua distribuição tecidual. Em termos de localização subcelular o GR, ficou mais concentrado no citoplasma no C (p<0,05), enquanto no DP, a distribuição foi similar entre os compartimentos, com uma redução no citoplasma (p<0,05), e um sutil aumento no núcleo. Sugerimos que a resposta de GR ao DP indica a alteração um elemento essencial na atividade da corticosterona, e essa modificação pode ser importante na coordenação do crescimento da mucosa gástrica durante o desmame precoce. / Early weaning (EW) is the abrupt change from suckling (S) to solid food and it can impair stomach development. This is a stressful situation for pups and it augments corticosterone levels, which acts through glucocorticoid receptor (GR). We studied GR expression, tissue and subcellular distribution in the gastric mucosa of S and EW pups. GR expression was higher at 17 d in S pups (p<0,05), whereas in EW group, it increased from the 17th to 18th d (p<0,05). GR protein levels decreased throughout EW, mainly at 18 d (p<0,05). However, EW did not alter tissue distribution of GR along the gastric gland. As for GR subcellular distribution, we found that in S group GR was more concentrated in the cytoplasm, (p<0,05), whereas in EW pups, GR was similarly distributed between compartments, though we detected a decrease in the cytoplasm (p<0.05) and a slight increase in the nucleus. We suggest that GR response to EW indicates the change of an essential element of corticosterone cascade, and such alteration might be important in the coordination of gastric mucosa growth.

O estresse é um fenômeno conservado e observado evolutivamente, que tem como objetivo assegurar a sobrevivência do indivíduo. Porém quando o organismo perde a capacidade de se autorregular, torna-se uma ameaça. Algumas psicopatologias, como ansiedade e depressão, sugerem o envolvimento dos sistemas CRF e noradrenérgico e de níveis elevados de GCs. Avaliamos nesse trabalho algumas alterações morfofisiológicas e comportamentais decorrentes da exposição do estresse crônico imprevisível (EI) em ratos machos. Avaliados 24 h após o último estímulo estressor, os animais submetidos ao EI apresentaram níveis elevados de corticosterona plasmática, de RNAm de CRF2 e expressão de GR em regiões encefálicas (LSi e VmH e LSi, CeA, BST e PVH, respectivamente). Essas alterações morfofisiológicas foram, em parte, decorrentes da ação de GCs e de NE. Não foram observadas alterações comportamentais quanto à anedonia e ansiedade. Dessa maneira, podemos dizer que o EI utilizado nesse estudo, foi capaz de induzir algumas alterações morfofisiológicas, porém não comportamentais. / While acute stress initiates neuronal responses that prepare an organism to adapt to challenges, chronic stress may lead to maladaptative responses that could result in diseases. Evidence suggests the involvement of CRF system and high corticosterone levels in stress-related psychiatric disorders such as anxiety and major depressive disorders. The aim of this work was to investigate whether chronic unpredictable stress (CUS) could modulate de CRF system, GR expression in the CNS and behavior in male rats. Results showed an increase in corticosterone plasmatic levels, CRF2 mRNA and GR expression in specific regions of the CNS (LSi e VmH e LSi, CeA, BST e PVH, respectively), associated with the limbic system at 24 h after the last stress session. The chronic treatment with an inhibitor of GCs synthesis (metyrapone) and adrenergic receptor antagonists (atenolol and phentolamine) prevented the CUS effects in CRF2 mRNA levels and GR expression. No anxiety or depression-like behavior was observed in rats submitted to CUS. We conclude that CUS cause biochemical alterations since the increase CRF2 mRNA levels and GR expression in limbic region, but these changes were not able to cause behavioral changes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Medical College of Ohio, 2003. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Edwin Sanchez. Includes abstract. Document formatted into pages: iv, 126 p. Title from title page of PDF document. Includes bibliographical references (p. 100-124).

Androgens are well documented as important regulators of male health, primarily in the maintenance and development of male sexual characteristics. However, a decline in circulating androgens has also been associated with co-morbidities such as obesity, cardiac disease and metabolic syndrome. Previous research has focussed upon the body wide impact of adrenal androgens, however whilst androgen receptor (AR) is abundantly expressed in the adrenal cortex of both rodents and humans, surprisingly little is known about androgen action on the adrenal cortex itself. This gap in our understanding is at least in part due to the perceived lack of suitable animal models. Rodents have largely been overlooked as a model system as their adrenals are unable to produce androgens due to lack of 17α Hydroxylase and 17, 20 lyse activity and they therefore do not have a zona reticularis. However, historical studies using castrated mice showed that removal of androgens leads to the redevelopment of an additional cortex zone known as the transient X-zone. The foetal adrenal is thought to give rise the adult adrenal cortex in human and rodents. These foetal cells are maintained for a period postnatally and regress differently depending on species and sex. In the human this zone is known as the ‘foetal zone’, and the rodent homologue termed the ‘X-zone’. The mechanisms underpinning the regression of the X-zone and its purpose and maintenance postnatally still aren’t clearly understood. To provide a comprehensive overview of androgen signalling in the adrenal cortex, multiple mouse models were utilised. First, Cre/loxP technology was used to ablate AR specifically from the adrenal cortex. Further androgen manipulation was achieved through castration (removal of androgens) and human chorionic gonadotropin (hCG) treatment (increased androgens). The initial study investigates the impacts on the male mouse adrenal. Histology analysis revealed the presence of an X-zone in all experimental cohorts following loss of AR or circulating androgens, confirmed by 20- α-hydroxysteroid dehydrogenase (20 alpha-HSD) expression. These data demonstrate that androgens signalling via AR is required for X-zone regression during puberty. However, interrogation of morphology of hCG treated cohorts revealed no phenotypic changes compared to controls, this demonstrates that hyper stimulation with androgens does not negatively impact the adrenal cortex or influence X-zone morphology. Differences in X-zone morphology and 20 alpha-HSD localization prompted cortex measurements which revealed significant differences in X-zone depth and cell density depending on ablation of AR, circulating androgens or both. This suggests that androgens and androgen receptor are working together and also independently to regulate the adrenal cortex. This result was strengthened through analysis of steroid enzyme genes and cortex markers, which revealed that normal AKR1B7 expression was absent following loss of androgens but not androgen receptor. A final part of this study examined the impacts long term androgen receptor ablation and long term castration in ageing animals. A final part of this study examined the impacts long term androgen receptor ablation and long term castration in ageing animals. These results demonstrate that following prolonged loss of androgens that there is no major disruption to the adrenal cortex. Morphology analysis and X-zone measurements revealed that X-zone regression was occurring in mice with long term castration, characterized by a reduction in size and pockets of vacuolization throughout the X-zone. This phenotype is also observed in ageing females with X-zone regression via vacuolization. These data suggest that following prolonged loss of androgens, the male adrenal is feminized and behaves as such. In contrast, AR ablation only, results in an enlarged adrenal with large spindle cell lesions and X-zone expansion confirmed by X-zone measurements. Initial experiments have demonstrated that androgens can work independently of AR to regulate the adrenal cortex. Together these data suggests that AR is required to control the appropriate action of circulating androgens in the adrenal cortex, with loss of AR resulting in off target signalling from circulating androgens in the adrenal leading to spindle cell hyperplasia, X-zone expansion and X-zone mislocation. A second set of studies were carried out to determine the role of androgen signalling in the female adrenal, specifically, if loss of AR leads to the absence of normal X-zone regression during pregnancy. To answer this question the same selective AR ablation model was used. Analysis of litters comparing observed and expected genetic distribution revealed significantly fewer females being born carrying complete ablation of adrenal AR. Morphology analysis of these mice revealed severe cortex disruption and spindle cell hyperplasia similar to that observed in mutant males. Investigation of adrenals following pregnancy revealed that X-zone regression still occurred despite loss of AR. This result shows that X-zone regression in the female is under different regulation compared to male adrenal and occurs via an androgen-independent signalling mechanism. However, loss of AR still leads to anatomical dysregulation of the adrenal cortex. AR ablation revealed changes in glucocorticoid receptor (GR) expression in the adrenal cortex. To dissect this relationship further a final study was conducted, attempting to ablate GR from the adrenal cortex also using the Cyp11a1 Cre. Initial observations of these mice revealed excessive hair loss through barbering, curved spines and stressed behaviour when monitored in the cage under normal conditions. Immunohistochemistry was used to confirm GR ablation in the adrenal cortex, however, to our surprise, GR expressing cells were not steroidogenic and thus were not targeted by the Cre recombinase. Despite no GR ablation in the adrenal, morphology analysis revealed severe disruption to the adrenal cortex. The Cyp11a1 Cre not only targets the adrenal but is expressed in the hindbrain. To determine if GR ablation in the hindbrain explains the phenotype, we next used PCR analysis interrogating hindbrain genomic DNA to determine if there was recombination of GR. Results confirmed GR recombination in the hindbrain. Due to the observation of stressed behaviour and adrenal cortex disruption, we wanted to determine if this was a result of hyperactivity of the adrenal cortex. Serum corticosterone was analysed and was elevated in these animals. These data revealed that GR ablation in the hindbrain results in adrenal cortex disruption and an elevated stress response, potentially highlighting a new model to investigate stress disorders and their impact on the hypothalamic-pituitary-adrenal axis. Together this data defines new roles for AR signalling in the adrenal cortex and the role of the hindbrain GR signalling in regulating adrenal morphology and function.