Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 6 uppsatser.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1941. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 24-26).

Existem evidências na literatura que demonstram a existência de células indiferenciadas na capsula adrenal, e que o ACTH poderia estimular estas células. Porém não se sabe quais os genes e vias que desencadeiam esta resposta. Através de animais Cre-Lox induzível por Tamoxifeno, silenciamos o gene Pomc em camundongos adultos e avaliamos o efeito do ACTH nessas células. Foram utilizadas placas de PCR array para análise de genes relacionados com células progenitoras em amostras obtidas pela técnica de rolamento, e validação por PCRq com amostras microdissecadas da zona capsular/subcapsular da adrenal. Após caracterização dos animais com o gene Pomc silenciado e tratamentos com ACTH observamos o aumento da expressão de genes relacionados com as vias Wnt, Igf1 e Notch. Esses dados corroboram evidencias descritas na literatura que mostram a importância dessas vias no desenvolvimento e manutenção do córtex adrenal, e sugerem o envolvimento do ACTH nesses processos que envolvem as células progenitoras do córtex adrenal. / There is evidence in the literature demonstrating the existence of stem cells in the adrenal capsule, and that ACTH could stimulate these cells. However, it remains unknown which genes and pathways that trigger this response. By using a tamoxifen-inducible Cre-Lox mice strain, we knocked-out Pomc gene in adult mice and evaluated the effect of ACTH in these cells. PCR array technique was used to determine the expression level of key genes related to progenitor cells in samples obtained by the technique of \"rolling bearing\". Also, we validated the data by qPCR using samples from microdissected capsular areas of the adrenal gland. After characterization of animal model, the results show that treatment with ACTH increase the expression of genes related to Wnt, Igf1 and Notch pathways. These data corroborate with the literature, reinforcing the importance of these pathways in the development and maintenance of the adrenal cortex, and also suggesting the involvement of ACTH in these processes involving the progenitor cells of the adrenal cortex.

The work in this thesis consisted of initial experiments designed to elucidate the role of hormones in a hormonal dependent tumor. Various aspects of nucleic acid synthesis in a hormone dependent tumor in the presence (growing) and absence (regressing) of the hormone were studied. The rates of nucleic acid synthesis were studied in whole animals by injecting radioactive formate and allowing the animal to incorporate radioactivity for various periods of time. Nucleic acids were extracted by PAS, phenol procedure and separated on a MAK column. Labelling of all species of nucleic acid was decreased in regressing tumors. In order to determine whether estrogen is acting directly on cells or at some indirect physiological level; the ability of cells from growing and regressing tumor to synthesize nucleic acids in vitro was determined. Results of experiments with these cell suspensions demonstrate that cells from the regressing tumor had a decreased ability to synthesize nucleic acids relative to growing tumor. The rate of DNA synthesis was decreased somewhat more than RNA. In preliminary experiments the activity of DNA dependent DNA polymerase and RNA polymerase from regressing tumor was compared with the same enzyme in growing tumor. The specific activity of both RNA and DNA polymerase was decreased in the regressing tumor. In target tissue like uterus stimulation with estradiol results in an increased rate of synthesis of several species of RNA. In the tumor system used in these preliminary experiments, stimulation with estrogens has a greater effect on the synthesis of DNA than RNA. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

La glande surrénale est un organe endocrinien d’une importance vitale de par son rôle dans le maintien de l’homéostasie corporelle. Pour assurer cette fonction, le cortex surrénalien est divisé en différentes zones qui produisent des hormones spécifiques. Les mécanismes de la mise en place de cette zonation au niveau embryonnaire ainsi que de son maintien tout au long de vie sont encore inconnus de nos jours. Les gènes Rspo1 et 3 sont exprimés de manière très spécifique au niveau de la capsule mais ils codent pour des protéines secrétées qui agissent sur les cellules adjacentes de la zone glomérulée afin d’induire l’activation de la voie canonique Wnt/β-caténine. La délétion du gène Rspo3 pendant le développement embryonnaire entraine un défaut d’activation des voies Shh et Wnt/β-caténine et donc en conséquence un défaut de la mise en place de la zonation. Sa fonction reste également essentielle au cours de la vie adulte puisqu’elle assure à la fois le maintien de l’homéostasie tissulaire et de la zone glomérulée. L’absence du gène Rspo1, n’affecte pas le développement ni la zonation ou l’homéostasie de la glande surrénale. Par contre, si on l’exprime de façon ectopique dans tous le cortex surrénalien, entrainant une activation anormale de la voie Wnt/β-caténine dans cette zone, on peut observer une hyperplasie des glandes surrénales. A partir de 1 an d’âge, cette hyperplasie surrénalienne entraine une formation de tumeurs. Ce travail démontre donc que la capsule par le biais du gène Rspondin 3 agit comme un centre de signalisation capable de contrôler à la fois l’homéostasie par le remplacement des cellules endommagés et le maintien de la zonation de la glande surrénale / The adrenal gland is an endocrine organ of vital importance because of its role in the maintenance of body homeostasis. To ensure this function, the adrenal cortex is divided into different areas that produce specific hormones. The mechanisms of the establishment of this zonation at the embryonic level as well as its maintenance throughout life are still unknown today. The Rspo1 and 3 genes are expressed very specifically at the capsule level but they encode secreted proteins that act on the adjacent cells of the zona glomerulosa in order to induce the activation of the Wnt / β-catenin canonical pathway. The deletion of the Rspo3 gene during embryonic development leads to a lack of activation of the Shh and Wnt / β-catenin pathways and hence a lack of zonation. Its function is also essential during adult life since it ensures both the maintenance of tissue homeostasis and the glomerular zone. The absence of the Rspo1 gene does not affect development, zonation or homeostasis of the adrenal gland. On the other hand, its ectopical expression in all the adrenal cortex leads to an abnormal activation of the Wnt / β-catenin pathway in this area and thus to an hyperplasia of the adrenal glands. From 1 year of age, this adrenal hyperplasia leads to the formation of tumors. This work demonstrates that the capsule through the Rspondin 3 gene acts as a signaling center capable of controlling both homeostasis by replacing damaged cells and maintaining the zonation of the adrenal gland

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21006号 / 医博第4352号 / 新制||医||1028(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 高橋 淳, 教授 濵﨑 洋子, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cortisol, a hormone produced by a hormone produced by the adrenal gland, is responsible for many regulatory functions in the body. Cortisol release is mediated by adrenocorticotrophic hormone, or ACTH, through the hypothalamus-pituitary-adrenal or HPA axis. This HPA axis is the major release pathway used during acute stress, during which the levels of ACTH parallel those of cortisol. However, in states of chronic stress, the level of ACTH drops dramatically, while cortisol remains high. This study focuses on the pathway of cortisol release during these chronic stress states, specifically examining the role of IL-6 with respect to STATs and the Jak/STAT pathway. It has been shown that IL-6 increases cortisol levels, and that IL-6 utilizes the Jak/STAT pathway. Also, the steroidogenic acute regulatory (StAR) promoter contains multiple STAT binding sites. Thus, STATs could be mediating the effects of IL-6 in the chronic release of cortisol by inducing expression of StAR. Experiments were performed to identify whether IL-6 has a direct effect on StAR promoter activity, StAR mRNA and StAR protein levels. Electromobility Shift Assays (EMSA) were performed to show that STATs bind to the full STAT site within the StAR promoter region. Various experiments were also carried out in the presence of IL-6 alone or, congruently with either a Jak (AG490) or STAT3 (Piceatannol) inhibitor, to show the effects of STATs and the Jak/STAT pathway on StAR. Luciferase assays were performed in order to observe the effects on induction of the StAR promoter. RT-PCR and western blots were also performed to observe the effect of Jak/STAT inhibition on both StAR mRNA levels and StAR protein levels. These experiments showed a marked decrease in the IL-6-stimulated StAR promoter activity, mRNA and protein expression when treated with wither Jak or STAT inhibitor. Therefore, IL-6 regulates expression of StAR through utilization of the Jak/STAT pathway; which phosphorylates and subsequently dimerizes STAT, allowing STAT to translocate to the nucleus and bind to the StAR promoter, thus increasing StAR expression and thereby inducing synthesis of cortisol.

Background: Rats exposed to chronic predator scent stress mimic the phenotype of complex post-traumatic stress disorder (PTSD) in humans, including altered adrenal morphology and function. High- and low-anxiety phenotypes have been described in rats exposed to predator scent stress (PSS). This study aimed to determine whether these high- and low-anxiety phenotypes correlate with changes in adrenal histomorphology and corticosteroid production. Methods: Rats were exposed to PSS for ten days. Thirty days later, the rats’ anxiety index (AI) was assessed with an elevated plus-maze test. Based on differences in AI, the rats were segregated into low- (AI ≤ 0.8, n = 9) and high- (AI > 0.8, n = 10) anxiety phenotypes. Plasma corticosterone (CORT) concentrations were measured by ELISA. Adrenal CORT, desoxyCORT, and 11-dehydroCORT were measured by high-performance liquid chromatography. After staining with hematoxylin and eosin, adrenal histomorphometric changes were evaluated by measuring the thickness of the functional zones of the adrenal cortex. Results: Decreased plasma CORT concentrations, as well as decreased adrenal CORT, desoxyCORT and 11-dehydroCORT concentrations, were observed in high- but not in low-anxietyphenotypes. These decreases were associated with increases in AI. PSS led to a significant decrease in the thickness of the zona fasciculata and an increase in the thickness of the zona intermedia. The increase in the thickness of the zona intermedia was more pronounced in low-anxiety than in high-anxiety rats. A decrease in the adrenal capsule thickness was observed only in low-anxiety rats. The nucleus diameter of cells in the zona fasciculata of high-anxiety rats was significantly smaller than that of control or low-anxiety rats. Conclusion: Phenotype-associated changes in adrenal function and histomorphology were observed in a rat model of complex post-traumatic stress disorder.

O carcinoma do córtex da supra-renal (ACC) é uma neoplasia rara e de prognóstico sombrio. Embora estudos moleculares tenham explorado diversos aspectos relacionados à tumorigênese destas neoplasias, o conhecimento das vias relacionadas à disseminação metastática é restrito. O objetivo do presente estudo é avaliar a expressão de genes relacionados a metástases em uma coorte de pacientes portadores de tumores do córtex da supra-renal metastáticos e não-metastáticos, a fim de identificar vias envolvidas na disseminação metastática destas neoplasias, novos marcadores prognósticos e eventuais alvos terapêuticos. Os perfis de expressão de 27 tumores do córtex da supra-renal de 15 pacientes adultos (8 ACC e 7 adenomas) e 12 pediátricos (5 metastáticos e 7 não-metastáticos) foram avaliados por um array de expressão contendo um painel de 113 genes que sabidamente estão envolvidos no processo de disseminação metastática de diversas neoplasias humanas. A análise de grupamentos mostrou que adenoma dos pacientes adultos forma um grupo distinto dos demais tumores (ACC de adultos e tumores pediátricos). Os genes MMP11e DENR foram identificados como diferencialmente expressos quando se compararam os adenomas e ACC de adultos. Na comparação dos tumores pediátricos nenhum gene foi diferencialmente expresso. Assim como a análise de grupamento, a PCA utilizando grupo selecionado de genes também não foi capaz partir os tumores pediátricos em subgrupos pela evolução. A expressão dos genes MMP2, TIMP3 e FN1 também foram avaliados por RT-PCR e foram concordantes com os dados gerados pelo array de expressão. O papel da LOH como causa da redução da expressão de TIMP3 foi estudado com tipagem de microssatélites. Em alguns casos, foi identificada LOH da região 22q13. Porém, em outros casos em que a expressão do TIMP3 foi bastante reduzida, não houve LOH. Em resumo, foram identificados aspectos moleculares importantes envolvidos na disseminação e metástases de neoplasias do córtex da supra-renal de adultos e crianças, bem como características biológicas deste processo. Diferentes padrões de expressão identificados em tumores metastáticos e não-metastáticos podem ajudar na predição do prognóstico / Adrenocortical carcinoma (ACC) is a rare neoplasm with a poor prognosis. Although molecular studies have uncovered many aspects of ACC tumorigenesis, little is known about molecular pathways involved in metastatic spread. The objective of our study is to analyze the expression profile of metastasis-related genes in a cohort of metastatic and nonmetastatic adrenocortical tumors in order to identify genes involved in the metastatic spread, as well as to find new prognostic markers. The expression profiles of 27 adrenocortical tumors from 15 adults (8 ACC and 7 adenomas) and 12 children (5 metastatic and 7 non-metastatic) were evaluated by an array of 113 known to be involved in human metastasis. Cluster analysis showed adult adrenocortical adenomas form a group distinct from other adrenocortical tumors (adult carcinomas and pediatric tumors). The comparison of adult adenoma and ACC revealed that MMP11 and DENR were differentially expressed between these two groups while no gene was differentially expressed among pediatric adrenocortical tumors. Similarly to cluster analysis, Principal component analysis failed to identify partition amongst pediatric tumors categorized by their evolution. The expression data of MMP2, TIMP3 and FN1 genes by RT-PCR agreed with those generated by the arrays. LOH of 22q12.3 region was detected in some cases in which TIMP3 down regulation was verified (but not in all cases). In conclusion, we have identified important aspects of molecular pathways and biological characteristics involved in metastatic spread of adrenocortical tumors. Distinctive patterns of gene expression between metastatic and nonmetastatic tumors may help in prognosis prediction