Capturing and holding of white rhinoceros (Ceratotherium simum) for the reintroduction to new reserves or breeding in zoos often involves a risk of mortality. Non-invasive techniques to monitor the stress experienced by these animals may guide the selection of management techniques that reduce risks to animal well-being. The aim of the study was to evaluate the biological relevance of a developed technique to monitor stress hormone metabolites in faecal samples of wild-caught and captive-bred white rhinoceros. Faecal corticosterone concentrations were measured via radioimmunoassay (125I RIA), in seven white rhinoceros (3 males and 4 females), at three sites, before and after an adrenocorticotropic hormone (ACTH) challenge test and control saline injection. Administration of ACTH resulted in a significant increase in faecal corticosterone concentrations (up to 350% above pre-ACTH baseline) within 1-2 days of injection, returning to baseline 4 days post-injection. It was found that individual baseline corticosterone concentrations fluctuate naturally and vary between individual animals, suggesting that an adequate baseline period of faecal sampling is needed in order to accurately assess responses to ACTH stimulation. Furthermore, the technique proved sensitive enough to detect elevations in faecal corticosterone concentrations due to environmental stressors. Data of faecal corticoid concentrations were correlated with gastrointestinal transit (GIT) times before and after ACTH and saline treatment by using art glitter as a digestive marker. This showed that gut passage times correlated to the ACTH-induced time to peak. Overall the results confirm that measurements of faecal corticosterone metabolites with the validated 125I RIA is a useful diagnostic tool to monitor adrenocortical activity in white rhinoceros. This study can therefore provide a methodology for examining chronically heightened adrenal activity in these animals and consequently be used to inform management strategies that aim to improve the welfare of white rhinoceros in captivity. / Dissertation (MSc)--University of Pretoria, 2011. / Anatomy and Physiology / unrestricted

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.

Neste estudo, testamos dois novos moduladores seletivos do receptor de glicocorticóide, nomeados L5 e L7, em comparação com o dexametasona, sobre aspectos estruturais, funcionais e moleculares no músculo sóleo. Ratos Wistar foram tratados com doses progressivas de dexametasona, L5 e L7 em 1 ou 7 dias. A massa corporal e a ingestão alimentar apresentaram queda após o tratamento com dexametasona em todas as doses; os tratamentos com L5/L7 mostraram resposta semelhante aos controles. O peso do músculo foi diminuído pelo dexametasona, efeito não observado nos tratamentos com L5/L7. Apenas o tratamento com dexametasona causou uma diminuição na área de secção transversa dos tipos de fibra muscular analisada. A força tetânica do sóleo foi diminuída pela dexametasona, nos tratamentos com L5/L7 este parâmetro também não foi afetado. A expressão gênica de MAFbx/Atrogin-1 e MuRF-1 foi elevada pela dexametasona; por outro lado, L5/L7 não elevaram a expressão destes genes. Concluímos que o L5/L7, em contraste com o dexametasona, preveniu o músculo esquelético da atrofia. / In this study, we have tested two new selective modulators named L5 and L7 along with dexamethasone in skeletal muscle structural, functional and molecular aspects. Male Wistar rats were treated with progressive doses of dexamethasone, L5 and L7 for 1 and 7 days. While body weight and food intake were decreased by the dexamethasone treatment in all doses, L5/L7 treatments induced gain in body weight similarly to controls. Muscle weight was decreased by dexamethasone, while L5/L7 were ineffective. Only the dexamethasone treatment caused a decrease in the analyzed cross sectional area of the skeletal muscle fiber types. Soleus tetanic force was decreased by the dexamethasone treatment, while L5/L7 treatments did not alter this parameter. MAFbx/Atrogin-1 and MuRF-1 gene expressions were elevated by dexamethasone; on the other hand, L5/L7 did not modulate any expression of those genes. We conclude that L5/L7, in contrast to dexamethasone, spare skeletal muscle from structural and functional loss, and molecular changes, reinforcing their role as a therapeutic device.

Tanto hormônios glicocorticóides (GCs) como o gene supressor de tumor TP53, medeiam a resposta celular a uma diversidade de condições fisiológicas de estresse, sendo reguladores fundamentais do processo de vida/morte de diversos tipos celulares. A interação funcional entre estes fatores vem sendo explorada, recentemente, revelando que GCs exercem um efeito dual sobre p53. O modelo celular ST1/P7 de glioma de rato é particularmente interessante para investigar o papel de p53 na ação de GCs, já que estas linhagens apresentam respostas distintas a GCs. O tratamento com Hidrocortisona (Hy) leva as células ST1 a uma complexa reversão fenotípica tumoral&#8594;normal, enquanto as células P7 são altamente resistentes ao tratamento. Foi possível observar que a ativação de p53 por Hy ocorre apenas em células ST1, mas não em P7. Esta ativação é mediada pela indução de fosforilação da Ser15 de p53 e seu acúmulo nuclear, o que resulta no aumento de sua ligação a elementos responsivos a p53 no DNA e na sua capacidade de transativação de p53, levando a um aumento da expressão de alguns de seus genes-alvo. Contudo, o bloqueio de p53 através de siRNA não foi suficiente para alterar a resposta de células ST1 a GCs, indicando que a regulação positiva de p53 por GCs pode ser um evento secundário, mas não essencial, para a resposta anti-tumoral exercida por estes hormônios em células ST1. / Both glucocorticoid hormones (GCs) and the TP53 tumor suppressor gene mediate cellular responses to a diversity of physiological stress conditions, acting as crucial regulators of the life/death process in a wide variety of cell types. The ST1/P7 rat glioma model cell system is particularly interesting to investigate the role of p53 in the action of GCs, since these cell lines display opposite responses to GCs. Treatment with Hydrocortisone (Hy) leads ST1 cells to a complete tumoral&#8594;normal phenotypic reversion, while P7 cells are highly resistant to this treatment. It was possible to observe that activation of p53 by Hy occurs only in ST1 cells, but not in GC-resistant P7 cells. This activation is mediated by induction of phosphorylation of the Ser15 residue of p53 and its accumulation in the nucleus, resulting in increased binding of p53 to its responsive elements on the DNA and in activation of its transactivating potential, leading to increased expression of some of its target genes. However, blocking of p53 through siRNA was not sufficient to alter ST1 cells response to GCs, indicating that the positive regulation of p53 by GCs may be a secondary, non-essential, event for the anti-tumor response exerted by these hormones in ST1 cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22322号 / 医博第4563号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 椛島 健治, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les formes oligomériques du peptideβ-amyloïde (oAβ) sont toxiques pour les synapses et engendrent la perte de mémoire lors de la phase précoce de la maladie d’Alzheimer (MA). La MA est aussi associée à une dérégulation de l’axe du stress engendrant une augmentation des glucocorticoïdes (GCs) qui activent les récepteurs associés (GRs). Nous avons montré que, dans un modèle murin de la MA, les Tg2576 (Tg+), l’inhibition des GRs prévient les déficits de mémoire et de plasticité synaptique (Lanté et al. 2015). Nous avons continué à étudier le rôle des GRs dans la physiopathologie de la MA. La dérégulation de l’axe du stress dans les souris Tg+ est caractérisée par des niveaux élevés de GCs et la perte de la boucle de rétroaction négative. Aussi, nous avons croisé les souris Tg+ avec des souris GR floxées pour générer des double mutants GRlox/lox Tg+. Ces souris exhibaient plusieurs phénotypes non-anticipés et nous avons décidé de mettre fin à cette lignée de souris. Nous avons aussi analysé la relation fonctionnelle spécifique entre les GRs et oAβ à la synapse en utilisant un traitement aigu d’oAβ. Dans des cultures de neurones, ce traitement a favorisé une augmentation des niveaux de GRs à la synapse. Aussi, nous avons montré que bloquer l’activité des GRs par pharmacologie ou par ablation génétique neutralise l’effet inhibiteur d’oAβ sur la potentialisation synaptique étudiée sur tranches d’hippoccampe. En conclusion, nos résultats sur souris Tg+ suggèrent la présence d’une dérégulation en début de MA. Aussi, nous mettons en évidence une relation fonctionnelle entre oAß et GRs à la synapse, les GRs jouant en rôle clé dans la synapto-toxicité induite par oAß. / Strong evidence shows that oligomeric forms of the amyloid-ß peptide (oAß) cause synapse dysfunction promoting loss of hippocampus-dependent memories in the early phase of Alzheimer’s disease (AD). AD is also associated with Hypothalamus-Pituitary-Adrenal (HPA) axis dysfunction which results in an increase of glucocorticoids (CORT) activating glucocorticoid receptors (GRs). We showed that subchronic GR antagonist in 4 month Tg2576 (Tg+) mice could rescue the synaptic deficit and memory impairment (Lanté et al., 2015).In this context, we studied the contribution of GRs to AD physiopathology. Dysregulated HPA axis was characterized by increased CORT levels at 4 and 6 months of age and by loss of CORT feedback inhibition in the Tg+ mice. We further crossed the Tg+ with GRlox/lox to produce GRlox/loxTg+ mice. These mice innately exhibited high CORT levels from weaning period and due to other several unforeseen reasons, we discontinued using this new mouse model. Instead, to identify the functional relationship between the GRs and oAß at synapses, we shifted to acute oAß treatment in neurons in vitro and ex-vivo hippocampus slices. In neuron cultures, GR levels increased in the post synaptic density upon acute oAß treatment. Further, treatment of oAß on ex-vivo hippocampus slices after either pharmacological blocking of GR or genetic ablation, prevented the oAβ-dependent LTP impairment. To conclude, our results with the Tg+ mice suggest that a neuroendocrine dysregulation occurs during the onset of AD pathology. Additionally, we have evidence for a functional relationship between oAß and GRs with GRs at the synapse playing an important role in acute Aß-induced synapto-toxicity.

Glucocorticoids are effective immunosuppressive and anti-inflammatory agents, but some aspects of stress appear to be proinflammatory. This study investigates this apparent paradox as it applies to stress exposure and the development of arthritis in a rat strain that has subnormal hypothalamic-pituitary-adrenal (HPA) responsiveness. Female Lewis rats were subjected to 1 week of rotating, psychological stressors for 5 h daily, beginning 7 days following inoculation with type [I collagen. The collagen-induced arthritis (CIA) group exposed lo stress showed reduced ankle width increase (p < 0.001) and decreased hindlimb severity scores (p < 0.001). At sacrifice, 2 days following stress termination, no differences in either measure remained and there was no difference in hind paw volume. However, the area of the tibia invaded by stroma, as quantitated by image analysis, was reduced in the stressed rats (p < 0.05). In animals exposed to stress, adrenal weights were increased (p < 0.005) and plasma corticosterone levels were elevated at sacrifice (p < 0.02). Both injected groups had significantly larger adrenal (p < 0.005) and lower thymus weights (p < 0.05) than did uninjected controls. Likewise, both CIA groups had reduced glucocorticoid receptor immunoreactivity in synovial membranes compared to controls (p < 0.001), suggesting that the Lewis rat's HPA deficiency may be intensified by glucocorticoid receptor downregulation during the induction of CIA. These data indicate that the responsiveness of the HPA axis to psychological stress in this strain is sufficient to alter disease progression.

MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The norepinephrine transporter (NET) and glucocorticoid receptors are closely related to the homeostatic integration and regulation after stress. Our previous studies demonstrated that NET mRNA and protein levels in rats are regulated by chronic stress and by administration of corticosterone, which is mediated through glucocorticoid receptors. Whether miRNAs are intermediaries in the regulation of these proteins remains to be elucidated. This study was undertaken to determine possible regulatory effects of miRNAs on the expression of NET and glucocorticoid receptors in the noradrenergic neuronal cell line. Using computational target prediction, we identified several candidate miRNAs potentially targeting NET and glucocorticoid receptors. Western blot results showed that over-expression of miR-181a and miR-29b significantly repressed protein levels of NET, which is accompanied by a reduced [3H] norepinephrine uptake, and glucocorticoid receptors in PC12 cells. Luciferase reporter assays verified that both miR-181a and miR-29b bind the 3′UTR of mRNA of NET and glucocorticoid receptors. Furthermore, exposure of PC12 cells to corticosterone markedly reduced the endogenous levels of miR-29b, which was not reversed by the application of glucocorticoid receptor antagonist mifepristone. These observations indicate that miR-181a and miR-29b can function as the negative regulators of NET and glucocorticoid receptor translation in vitro. This regulatory effect may be related to stress-induced up-regulation of the noradrenergic phenotype, a phenomenon observed in stress models and depressive patients. (Figure presented.) This study demonstrated that miR-29b and miR-181a, two short non-coding RNAs that provide global regulation of gene expression, markedly repressed protein levels of norepinephrine (NE) transporter and glucocorticoid receptor (GR), as well as NE uptake by binding the 3′UTR of their mRNAs in PC12 cells. Also, exposure of cells to corticosterone significantly reduced miR-29b levels through a GR-independent way.

Previously, we have found glucocorticoids up-regulate norepinephrine (NE) transporter (NET) expression in vitro. However, the underlying transcriptional mechanism is poorly understood. In this study, the role of glucocorticoids on the transcriptional regulation of NET was investigated. Exposure of neuroblastoma SK-N-BE(2)M17 cells to dexamethasone (Dex) significantly increased NET mRNA and protein levels in a time- and dose-dependent manner. This effect was attenuated by glucocorticoid receptor (GR) antagonist mifepristone, suggesting that up-regulation of NET by Dex was mediated by the GR. In reporter gene assays, exposure of cells to Dex resulted in dose-dependent increases of luciferase activity that were also prevented by mifepristone. Serial deletions of the NET promoter delineated Dex-responsiveness to a -301 to -148 bp region containing a CCAAT/enhancer binding protein-β (C/EBP-β) response element. Co-immunoprecipitation experiments demonstrated that Dex treatment caused the interaction of the GR with C/EBP-β. Chromatin immunoprecipitation (ChIP) assay revealed that Dex exposure resulted in binding of both GR and C/EBP-β to the NET promoter. Further experiments showed that mutation of the C/EBP-β response element abrogated C/EBP-β- and GR-mediated transactivation of NET. These findings demonstrate that Dex-induced increase in NET expression is mediated by the GR via a non-conventional transcriptional mechanism involving interaction of C/EBP-β with a C/EBP-β response element.

Our previous studies demonstrated that chronic social defeat (CSD) up-regulated expression of the serotonin transporter (SERT) and norepinephrine transporter (NET) in the brain, which was mediated by corticosteroid receptors. In the present study we first analyzed the alterations of corticosteroid receptors in different brain regions after the CSD paradigm. The results showed that CSD significantly reduced glucocorticoid receptor (GR) protein levels in the CA1 and dentate gyrus of the hippocampus, as well as in central and basolateral nuclei of the amygdala, which was accompanied by the translocation of GR from cytoplasm to nuclei. CSD also markedly reduced GR mRNA levels and MR immunoreactivity in the CA1, CA3 and dentate gyrus areas of the hippocampus. Conversely, CSD pronouncedly enhanced GR mRNA and protein levels in the dorsal raphe nucleus and locus coeruleus relative to the control. As an extension of our previous studies, in situ hybridization and immunohistochemical staining demonstrated that CSD regimen caused a notable increase of SERT mRNA levels in the dorsal raphe nucleus and increased SERT immunoreactivities in CA1 and CA3 of the hippocampus, as well as those in the basolateral nuclei of the amygdala. Likewise, CSD regimen resulted in an evident enhancement of NET immunoreactivity in the CA1 of the hippocampus and in the basolateral nuclei of the amygdala. Our current findings suggest that GR expressional alterations in response to CSD are complex and brain region-specific, which may correspond to their different functions in these regions.