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Orientadores: Sebastião Roberto Taboga, Ricardo Alexandre Fochi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T10:21:05Z (GMT). No. of bitstreams: 1 Antoniassi_JuliaQuilles_M.pdf: 2795390 bytes, checksum: 43457dd2f5a6928d83f9fa9c05fd3820 (MD5) Previous issue date: 2015 / Resumo: A próstata, glândula do sistema reprodutor que tem origem embrionária a partir do seio urogenital, sendo formada por quatro pares de lobos fortemente associado à uretra: lobo anterior ou glândula coaguladora (GC), lobo dorsal (LD), lobo dorsolateral (LDL) e lobo ventral (LV). A fisiologia prostática é regulada por hormônios esteroides, principalmente andrógenos e estrógenos. Enquanto os andrógenos induzem a diferenciação, desenvolvimento, e atividade secretora, os estrógenos parecem modular os efeitos androgênicos, mantendo o crescimento e fisiologia normal da próstata. Embora o cortisol seja um hormônio largamente utilizado em medicamentos por suas ações anti-inflamatórias e imunossupressoras, pouco se sabe sobre os efeitos colaterais desses medicamentos, principalmente na morfofisiologia prostática. Além disso, sabe-se que esse hormônio está altamente relacionado com o estresse e com alguns distúrbios de comportamento. Desta forma, este projeto visou avaliar, por métodos histológicos, histoquímicos, imunocitoquímicos e estruturais com parâmetros qualitativos e quantitativos, diversos aspectos morfofuncionais da glândula prostática ventral de machos de gerbilo (Meriones unguiculatus), após a aplicação por cinco dias de corticosterona em duas doses diferentes: 0,5mg/kg/dia e 1,5mg/kg/dia, além disso os grupos foram subdivididos em um grupo com um período de 5 dias de descanso após o tratamento e outro grupo que foi morto um dia após o fim do tratamento. Os dados apresentados mostram que a corticosterona possui propriedades antiproliferativas e antiapoptóticas sobre a próstata de gerbilos. Esse hormônio também alterou a frequência dos receptores dos principais hormônios reguladores desse órgão, como andrógenos e estrógenos. Em relação aos receptores de glicocorticóides (GR), foi observada uma redução da sua expressão quando a concentração sérica da corticosterona encontrava-se elevada. As alterações na expressão dos receptores supracitados levaram ao surgimento de displasias prostáticas importantes. Houve uma variação nos efeitos desencadeados por esse hormônio de acordo com a dose aplicada, sendo que uma maior dose apresentou efeitos mais discretos e uma menor dose mostrou ser mais impactante. Os efeitos da corticosterona nos receptores nucleares foram revertidos ou atenuados após o período de descanso, o que não foi observado para os padrões de proliferação e apoptose celular / Abstract: The prostate, gland of reproductive system, has embryonic origin from the urogenital sinus and consists four lobes that are strongly associated with the urethra: anterior lobe or coagulating gland (CG), dorsal lobe (LD), dorsolateral lobe (LDL) and ventral lobe (LV). Steroid hormones, mainly androgens and estrogens, regulate the prostate physiology. While androgens induce differentiation, development and secretory activity, estrogens appear to modulate androgen effects, maintaining normal growth and prostate physiology. Although cortisol is widely used in drugs because their anti-inflammatory and immunosuppressive actions, little is known about the side effects of these drugs, especially in the prostate morphophysiology. Furthermore, it is known that this hormone is highly correlated with stress and some behavioral disorders. Thus, this research aimed evaluate, by histological methods, histochemical, immunocytochemical with structural and qualitative and quantitative parameters, various morphological and functional aspects of the ventral prostate gland of male gerbil (Meriones unguiculatus) after application of corticosterona. This hormone was applicated for five days with two doses of corticosterone: 0.5mg/kg/day and 1.5mg/kg/day, in addition, the groups were divided into a group with a 5-day period of rest after the treatment and another group was killed one day after the end treatment. The data showed that corticosterone has antiproliferative and anti-apoptotic properties on the prostate gerbils. This hormone also changed the frequency of AR, ER?, ER? e GR. We observed a reduction of glucocorticoids receptors expression when serum concentration of corticosterone was elevated. These changes led to the emergence of important prostatic dysplasia. There was a variation in effects triggered by this hormone in accordance with the applied dose, being that a higher dose reported effects more discrete and a lower dose was shown to be more impactful. The effects of corticosterone on nuclear receptors have been reversed or attenuated after rest period, which was not observed for apoptosis and cell proliferation patterns / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Le stress chronique apparait comme une cause importante de troubles de la fertilité chez la femelle. L’hypercorticisme (sécrétion excessive de glucocorticoïdes (GC) surrénaliens) induit par le stress agit sur l’axe de reproduction pour perturber le cycle œstrien et l’ovulation. Chez la femelle, l’axe de reproduction comprend 3 grandes structures : l’hypothalamus, l’hypophyse et l’ovaire. Pour bloquer l’activité de cet axe, les GC agissent via un récepteur spécifique (GR) pour induire une signalisation rapide non génomique et/ou une signalisation génomique tardive. Malgré de nombreuses recherches, les connaissances sur le mode d’action des GC sur cet axe restent fragmentaires. Au cours de ce travail de thèse, l’utilisation de différents modèles cellulaires et animaux nous a permis de mettre en évidence des mécanismes d’action des GC originaux sur l’axe hypothalamo-hypophysaire. Au niveau hypothalamique, nous avons décrit un nouveau dialogue entre les œstrogènes et les GC, qui favorise l’expression d’un neuropeptide hypothalamique inhibiteur, la dynorphine A. Ce neuropeptide pourrait ainsi participer au blocage des sécrétions pulsatiles de la GnRH (Gonadotropin-releasing Hormone) hypothalamique et des gonadotropines hypophysaires indispensables à l’ovulation. Au niveau hypophysaire, nous avons mis en évidence une action paradoxale des GC sur les cellules gonadotropes. En absence de GnRH, les GC stimulent une nouvelle voie de signalisation non génomique, initiée à la membrane plasmique par un GR palmitoylé. Cette signalisation rapide implique la calcium/calmoduline kinase II (CaMKII) ainsi qu’une de ses cibles, la synapsine-Ia. Néanmoins, en présence de GnRH, les GC bloquent la signalisation induite par la GnRH, en empêchant l’activation de la CaMKII, pouvant être à l’origine de l’inhibition de la sécrétion de la gonadotropine LH (Luteinizing Hormone).De nouvelles recherches sont à poursuivre pour approfondir nos connaissances sur les différents modes d’action des GC pouvant être spécifiquement mis en jeu au sein de chacune des cellules, et à l’origine des effets variés des GC au sein de l’organisme. Une meilleure compréhension des mécanismes moléculaires responsables de l’altération de la fertilité, en particulier durant un stress chronique, est essentielle pour envisager l’émergence de nouvelles cibles thérapeutiques innovantes. / The chronic stress is an important cause of fertility disorders in female. Stress-induced hypercorticism (excessive secretion of glucocorticoids (GC)) acts on the reproductive axis to disrupt the estrous cycle and ovulation. In female, the reproductive axis comprises 3 structures: the hypothalamus, the pituitary and the ovary. To block the activity of this axis, GC act through a specific receptor (GR) to promote rapid non genomic and/or late genomic signaling. Despite extensive researches, knowledge on the mechanism of action of GC on this axis remains elusive. During my PhD, the use of different cellular and animal models allowed to highlight new mechanisms of GC actions on the hypothalamic-pituitary axis. At the hypothalamic level, we described a new genomic cross-talk between estrogen and GC to promote the expression of an inhibitory hypothalamic neuropeptide, dynorphin A. This neuropeptide could then participate in disrupting the pulsatile secretion of GnRH (Gonadotropin-releasing hormone) and pituitary gonadotropins which are essential for ovulation. At the pituitary level, we demonstrated a paradoxical action of GC on gonadotrope cells. In the absence of GnRH, GC stimulate a new non genomic pathway initiated at the plasma membrane through a palmitoylated GR. This rapid signaling involves calcium/calmodulin kinase II (CaMKII) as well as one of its targets, synapsin-Ia. Nevertheless, in the presence of GnRH, GC interfere with GnRH-induced signaling by preventing CaMKII activation, which may be responsible for the inhibition of LH (Luteinizing Hormone) release.Further researches are required to improve our knowledge on cell-specific mechanisms of action of GC that could explain the diversity of their activities. A better understanding of the molecular mechanisms responsible for fertility dysfunction, especially during chronic stress, is essential for the development of new and innovative therapeutic targets.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22352号 / 医博第4593号 / 新制||医||1042(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 稲垣 暢也, 教授 森田 智視, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Rheumatoid arthritis is the most common joint disease of autoimmune origin. It is accompanied by inflammatory conditions that lead to irreversible changes in the joints, their deformities ending with permanent disability. Treatment of the disease involves routine regimens, surgical, as well as pharmacological treatment, which is necessary for advanced forms. Glucocorticoids play an important role in the therapeutic intervention in the course and progression of the disease. In spite of their anti-inflammatory effect, which is a key to improving the condition of the patient, they have a number of side effects in the long term- use. In this study, we have focused on the impact of these drugs on microRNA expression changes in arthritic patients treated with pulsed doses of glucocorticoids. MicroRNAs are nowadays widely studied due to their possible use as biomarkers in monitoring disease progression and the effect of treatment. MiRNA expression analysis was performed by quantitative real-time PCR array of 754 miRNAs with reverse transcription using stem-loop primers that allow amplification of short sequences that microRNAs are. Data analysis revealed 29 miRNAs differentially expressed at the significance level p ≤ 0.05, 14 miRNAs were at significance level p ≤ 0.025 (respectively 7 miRNAs at p ≤ 0.005...

5 ABSTRACT Candidate: Lenka Polláková1 Supervisor: prof. RNDr. Jiří Vlček, CSc.1 Consultant: doc. MUDr. Tomáš Soukup, Ph.D.2 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University 2 2nd Department of Internal Gastroenterology, University Hospital in Hradec Králové Title of the master thesis: Analysis of non-cardiac adverse event of glucocorticoid pulse therapy Intravenous glucocorticoid pulse therapy (PT GC) is effective in life threatening flares of rheumatic diseases. However, due to GC's pleiotropic effect, higher doses and additive nongenomic mechanism in pulse regimen, it is not free of complications (1). The aim of theoretical part was to describe from literature research the relevance of PT GC, its non- cardiac adverse events (AE) in rheumatic patients and their influencing factors. The aim of experimental part of the study was to analyze the occurrence of non-cardiac AE in real-life setting, analyze risk factors of potential adverse drug reactions (ADR) and its complications and analyze the risk minimalization management in real-life setting. Patients were administered 1000 mg methylprednisolone in 3 to 5 doses on alternating days. Analysis includes 277 rheumatic patients with 325 pulse therapy courses. Data were collected retrospectively from their...

Implantation of dental sponges under the skin of lab rodents has been used to evaluate whether acute stress enhances leukocyte infiltration to a surgical site. First, I replicated this technique in house sparrows to test whether transient stressors cause similar immunoredistribution (i.e., movement of immune cells out of circulation and to the periphery) in a wild animal. As placement into captivity alone may serve as a stressor to wild animals, I compared sponge infiltration over different periods of captivity. Second, I compared how domestication affects immunoredistribution by comparing results of wild sparrows to domesticated zebra finches. Zebra finches were chosen because they are widely used for evolutionary ecology research, and they share a similar diet and comparable body size and lifestyle to house sparrows. Birds were randomly assigned to treatment groups of either a restraint stressor or no restraint stressor treatment prior to implantation. In the first chapter birds were also divided into one of three groups: sponge implantation at capture, after short duration captivity (1 or 2 days), or long duration captivity (1 month). Total leukocyte infiltration into the sponge varied among captive groups. Birds implanted at capture had greater leukocyte infiltration to the sponge compared to birds kept in captivity 1 or 2 days before implantation. Birds placed into captivity for 1 month before implantation showed similar sponge infiltration relative to the immediate implant group. However, time in captivity altered the dominant type of leukocytes present in the sponge at explant with lymphocytes decreasing with time in captivity and granulocytes increasing. Domestication affected cell infiltrates with domesticated species exhibiting more infiltration of heterophils and monocytes while wild house sparrows exhibited more infiltration of lymphocytes and basophils. My data indicates that in house sparrows, time in captivity affects the magnitude and character of immune responses to surgery and more importantly data are suggestive of immunoredistribution. My data also indicate that domestication has an impact on infiltrating cell types.

Chronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer’s disease (AD) and promote AD pathomechanisms in animal models. These include overproduction of synaptotoxic amyloid-β (Aβ) peptides and intraneuronal accumulation of microtubule-associated protein Tau. Tau accumulation is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in stress/GC-induced Aβ overproduction remains an open question. Here, I find that hippocampal Rab35 levels are decreased not only by stress/GCs, but also by aging, another AD risk factor. Moreover, I show that Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking events for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Additionally, I show that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by GCs. Finally, I begin to investigate how GCs and/or Rab35 affect the intercellular spread of Aβ and Tau through exosomes. I describe methods for purifying exosomes and measuring their secretion from neurons, astrocytes, and microglial cells in order to determine the effects of stress/GCs and Rab35 on this process. These studies identify Rab35 as a key regulator of Alzheimer’s disease-related protein trafficking, and suggest that its downregulation contributes to stress- and AD-related pathomechanisms.

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Visceral adipose tissue (AT) mass increases risk for cardiovascular disease and diabetes. Glucocorticoids (GCs) cause preferential expansion of visceral compared to subcutaneous AT through poorly understood mechanisms. GCs are necessary for adipogenesis, the differentiation of adipose stem cells (ASCs) to mature adipocytes. However, this process may be impaired in visceral depots. Insufficient adipogenesis can lead to excessive hypertrophy of existing adipocytes. This hypertrophic expansion increases cell death and inflammation, driving AT dysfunction. To better understand the genes and pathways by which high GCs cause preferential expansion of visceral fat we performed transcriptomic profiling (microarray) on paired samples of visceral (Omental, Om) and abdominal subcutaneous (Abdsc) AT explants cultured with the GC receptor agonist, dexamethasone (Dex), for 7 days. Gene set enrichment analysis showed the transforming growth factor beta (TGFβ) signaling pathway, most notably the secreted anti-adipogenic factors, TGFβ and activin A, was highly enriched in Om and suppressed less by Dex. We hypothesized that Om AT and ASCs secrete factors that inhibit adipogenesis in an autocrine/paracrine manner. Conditioned media (CM) from Om tissue and ASCs suppressed differentiation by 70-80% compared to control; Dex attenuated this anti-adipogenic effect. Both TGFβ and activin A levels were 4-5 fold higher in CM from Om compared to Abdsc ASCs. Both factors signal via cell surface receptors that increase SMAD2 phosphorylation (P-SMAD2), basal levels of which were 3-4 fold higher in Om ASCs. Additionally, CM from Om ASCs increased P-SMAD2. siRNA mediated knockdown of activin A improved differentiation of Om ASCs, but did not reach levels observed in Abdsc. Blocking TGFβ and activin A signaling using SB431542 robustly increased adipogenesis of Om ASCs and prevented the anti-adipogenic effect of CM. GCs decreased production of TGFβ and activin A, but both remained higher in OmCM. Overnight Dex treatment decreased P-SMAD2 and increased the expression of the TGFβ co-receptor, TGFBR3, which decreases TGFβ signaling, in Abdsc ASCs. GCs failed to decrease P-SMAD2 and increased TGFBR3 in Om ASCs only at high concentrations. Taken together, these data implicate GC-TGFβ crosstalk as a determinant of depot differences in adipogenic capacity and hypertrophic vs. healthy hyperplastic expansion of AT. / 2019-11-01T00:00:00Z