Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-04-24T12:44:41Z No. of bitstreams: 1 KARINA_VIDARTE_GASPARY_DIS.pdf: 1111674 bytes, checksum: e2b2dc4b1b1eea8b10007b7c07f592ec (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-05-11T17:57:10Z (GMT) No. of bitstreams: 1 KARINA_VIDARTE_GASPARY_DIS.pdf: 1111674 bytes, checksum: e2b2dc4b1b1eea8b10007b7c07f592ec (MD5) / Made available in DSpace on 2018-05-11T18:03:07Z (GMT). No. of bitstreams: 1 KARINA_VIDARTE_GASPARY_DIS.pdf: 1111674 bytes, checksum: e2b2dc4b1b1eea8b10007b7c07f592ec (MD5) Previous issue date: 2018-02-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Memory and learning allow animals to adapt and modify their behavior towards new experiences. Some factors may result in memory deficits such as: neurodegenerative diseases, changes due to intoxication and the use of drugs. Zebrafish can be used to model complex human behavioral traits such as learning and memory. This study aims to establish a protocol for evaluating the object recognition memory or object location tasks in zebrafish. We evaluated novel object recognition memory and analyzed the exploration time of novel and familiar objects in the training and test sessions. There was a preference of the animal to explore the new object in comparison to the familiar object (61% of exploration time). We also evaluated the object location task and measured the exploration time of each object in the familiar and novel object locations. There was a preference of the animal to explore the object in the novel location in comparison to the object in the familiar location (63% of exploration time). We also evaluated the effect of the non-competitive NMDA receptor antagonist MK-801 on the object recognition and object location memory in zebrafish. In this regimen treatment, control (water only) and treated animals (5 ?M MK-801) presented a significant preference in exploring the familiar object in comparison to the new object (66 and 68% of exploration time, respectively); however, 10 ?M MK-801-treated animals did not show differences in the exploration time of the objects. In the object location task, the animals treated with the 5 or 10 ?M MK-801 did not show a significant preference for the familiar or novel location whereas the control group had a higher preference in exploring the object in the familiar location (64% of exploration time). Therefore, it is possible to suggest that 5 ?M MK-801 impaired the memory formation in an object location task, which is in agreement with previous studies demonstrating the cognitive deficit induced by MK-801 treatment in aversive and spatial memory. Considering the different responses of the control group between original task and in the regimen treatment, we evaluated the impact of habituation on cortisol levels of animals in three different protocols: 1) habituated at the experiment apparatus for 3 days (Condition 1 ? C1), 2) habituated at the experiment apparatus for 3 days plus treatment tank exposure at fourth day (Condition 2 - C2), 3) habituated at the treatment tank exposure and experiment apparatus for 3 days and exposed to treatment tank again at fourth day (Condition 3 ? C3). The results showed higher levels of cortisol in animals submitted to C2 and C3 conditions compared to animals submitted to C1. These results demonstrated that the animals submitted to treatment tank exposure have a different performance in object recognition and object location memory due to stress responses. Therefore, these tasks are prone to evaluate memory in physiological and pathological conditions, but its use is limited to perform pharmacological studies due to sensitivity to stress caused by manipulation. / A mem?ria permite que os animais possam adaptar e modificar seu comportamento diante de novas experi?ncias. Uma s?rie de fatores pode resultar num d?ficit de mem?ria como: doen?as neurodegenerativas, altera??es decorrentes de intoxica??es e uso de f?rmacos. O peixe-zebra vem sendo usado para modelar caracter?sticas comportamentais humanas complexas, como o aprendizado e mem?ria. Este estudo tem como objetivo estabelecer um protocolo para avaliar tarefas de mem?ria de reconhecimento de objetos ou de localiza??o de objetos no peixe-zebra. N?s avaliamos a mem?ria de reconhecimento de objeto novo e analisamos o tempo de explora??o de objetos novos e familiares nas sess?es de treino e teste. Houve uma prefer?ncia do animal em explorar o novo objeto em compara??o com o objeto familiar (61% do tempo de explora??o). Tamb?m avaliamos a tarefa de localiza??o de objeto e medimos o tempo de explora??o de cada objeto nos locais familiares e novos. Houve uma prefer?ncia do animal para explorar o objeto na localiza??o nova em compara??o com o objeto na localiza??o familiar (63% do tempo de explora??o). Tamb?m avaliamos o efeito do antagonista n?o competitivo do receptor NMDA MK-801 na mem?ria de reconhecimento de objeto novo e na localiza??o de objeto em peixe-zebra. Neste tratamento, o controle (somente ?gua) e os animais tratados (MK-801 5 ?M) apresentaram uma prefer?ncia significativa na explora??o do objeto familiar em compara??o com o novo objeto (66 e 68% do tempo de explora??o, respectivamente); no entanto, animais tratados com MK-801 10 ?M n?o mostraram diferen?as no tempo de explora??o dos objetos. Na tarefa de localiza??o de objeto, os animais tratados com MK-801 5 ou 10 ?M n?o mostraram prefer?ncia significativa pelo objeto na localiza??o familiar ou nova, enquanto que o grupo controle teve uma prefer?ncia maior em explorar o objeto na localiza??o familiar (64% de tempo de explora??o). Portanto, sugere-se que o MK-801 5 ?M prejudicou a forma??o da mem?ria em uma tarefa de localiza??o de objeto, o que est? de acordo com estudos pr?vios, demonstrando o d?ficit cognitivo induzido pelo MK-801 em mem?rias aversivas ou espaciais. Considerando as diferentes respostas do grupo controle entre a tarefa original e no tratamento, avaliamos o impacto da habitua??o nos n?veis de cortisol dos animais em tr?s protocolos diferentes: 1) habituado ao aparato experimental por 3 dias (Condi??o 1 - C1) 2) habituado ao aparato experimental por 3 dias mais a exposi??o ao aqu?rio de tratamento no quarto dia (Condi??o 2 - C2), 3) habituado na exposi??o ao aqu?rio de tratamento e no aparato experimental por 3 dias e exposto ao aqu?rio de tratamento novamente no quarto dia (Condi??o 3 - C3). Os resultados mostraram n?veis mais elevados de cortisol em animais submetidos a condi??es C2 e C3 em compara??o com animais submetidos a C1. Esses resultados demonstraram que os animais submetidos ? exposi??o ao aqu?rio de tratamento apresentam desempenho diferente nas tarefas de mem?ria de reconhecimento de objetos e de localiza??o do objeto devido ao estresse. Portanto, essas tarefas s?o adequadas para avaliar a mem?ria em condi??es fisiol?gicas e patol?gicas, mas seu uso ? limitado em estudos farmacol?gicos devido ? sensibilidade ao estresse causado pela manipula??o.

A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum, afetando em média 2-3% dos indivíduos com idade superior a 65 anos. Além dos sintomas motores altamente debilitantes, os sintomas não-motores podem preceder em muitos anos o aparecimento dos sintomas motores, podendo caracterizar uma fase prodrômica da DP. Uma potencial estratégia farmacológica é a introdução de agentes neuroprotetores no estágio inicial da doença, a fim de prevenir a morte neuronal decorrente do progresso da neurodegeneração. A N-acetilcisteína (NAC) é um antioxidante, anti-inflamatório e modulador glutamatérgico que mostrou vários benefícios terapêuticos em transtornos psiquiátricos. Neste estudo, foi avaliado o efeito da NAC na prevenção dos danos induzidos por 6-Hidroxidopamina (6-OHDA) sobre parâmetros motores, cognitivos e morfológicos em um modelo de DP em larvas de peixes-zebra. Nossos resultados mostraram que NAC foi capaz de prevenir os déficits motores e cognitivos e as alterações morfológicas causadas pela exposição a 6-OHDA, o que reforça a relevância de seus efeitos neuroprotetores. Por atuar em diferentes alvos relevantes na fisiopatologia da DP, NAC é um potencial candidato para prevenção e tratamento de DP. / Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting on average 2-3% of the individuals older than 65 years. In addition to its highly debilitating motor symptoms, non-motor symptoms may precede by many years their motor counterparts, which may characterize a prodromal phase of PD. A potential pharmacological strategy is to introduce neuroprotective agents at an earlier stage in order to prevent further neuronal death. N-acetylcysteine (NAC) is an antioxidant, anti-inflammatory and glutamatergic modulator that has shown various therapeutic benefits in brain disorders. In this study, it was evaluated the effect of NAC to prevent the damage induced by 6-OHDA on motor, cognitive and morphological parameters in a PD model in larval zebrafish. NAC was able to prevent the motor and cognitive deficits and morphological alterations caused by exposure to 6-OHDA, which reinforce the relevance of its neuroprotective effects. By acting on different relevant targets in the pathophysiology of PD, NAC is a potential candidate for prevention and treatment of PD.

Pang Yefei. / "August 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 161-197). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

卵泡刺激素（FSH）和促黃體激素（LH）是脊椎動物體內的促性腺激素（GTH）。它們通過其相應的GTH受體（GTHR）－ FSH受體（FSHR）及LH/絨毛膜性腺激素受體（LHCGR），來調控雌性脊椎動物的主要性腺活動，如卵泡生成和類固醇生成。因此，GTHR的表達水平可控制卵泡細胞對於GTH的反應程度，從而影響脊椎動物的繁殖能力。 / 然而，跟哺乳動物中的資料相比，這些受體的表達調控機制在硬骨魚類中仍然很模糊。此前，我們已經證明了斑馬魚卵泡之fshr和lhcgr的表達譜差異，顯示出lhcgr的表達滯後於fshr的表達。此表達時間之差異引申出兩條有趣的問題：一）甚麼激素能分別調節fshr和lhcgr的表達？ 二）這些調控的機制是甚麼？因此，我們發起本研究來解答這些問題。 / 利用培養出來的斑馬魚卵泡細胞，我們展示了雌二醇（E2）是一個有力的GTHR調控激素。雖然E2同時刺激了fshr和lhcgr的表達，但E2對於lhcgr的表達調控效力遠遠比對fshr的高。由於雌激素核受體（nER）的特異拮抗劑（ICI 182,780）能完全抵消E2的效果，表明了E2是通過傳統的nER來直接促進了lhcgr的表達。有趣的是，不能穿越細胞膜的雌二醇-牛血清白蛋白偶聯複合物（E2-BSA）能完全模仿E2的效果，因此我們的證據提出這些nER可能位於細胞膜上。此外，我們運用各種藥劑發現了多種信號分子跟E2調控GTHR的能力有關，包括cAMP、PKA、PI3K、PKC、MEK、MAPK及p38 MAPK。當中以cAMP-PKA的信號傳導最有可能在E2的雙相調控效果起了直接作用，而E2的行動也極依賴其他信號分子的允許作用。 / 除了E2，人絨毛膜促性腺激素（hCG; LH的類似物）、垂體腺苷酸環化酶激活多肽（PACAP）、表皮生長因子（EGF）和胰島素樣生長因子-I（IGF-I）也能有效地調節斑馬魚卵泡細胞的GTHR表達。hCG能大幅下調其受體lhcgr的表達，顯示hCG能令卵泡細胞對GTH脫敏。與此同時，PACAP能瞬時模仿hCG的行動，表明了PACAP很可能是hCG的瞬態下游信號。EGF是一個強烈抑制lhcgr表達的因子，而IGF-I是一個潛在的fshr表達增強因子，均說明了旁分泌因子對GTHR表達調控有關鍵作用。除了這些激素或因子的獨立調控作用，我們進一步發現了E2的效果可能會被它們覆蓋或調節。它們對nER的調控作用可能會造成這種現象。PACAP瞬時減少了esr2a及esr2b的表達量，而EGF則顯著地下調了esr2a。 / 作為第一個在硬骨魚卵巢中對GTHR調控的全面研究，它無疑豐富了我們對卵泡生成過程中GTH的功能及GTHR表達調控的認識。此外，我們成功將目前的研究平台應用於雙酚A（BPA）的研究，進一步展示了本研究平台的潛力，有助於我們未來對各種內分泌干擾物（EDC）的作用機制進行研究。 / Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the gonadotropins (GTHs), which bind to their cognate GTH receptors (GTHRs), FSH receptor (FSHR) and LH/choriogonadotropin receptor (LHCGR), to mediate major gonadal events in female vertebrates, including folliculogenesis and steroidogenesis. The expression level of GTHRs, therefore, controls the responsiveness of follicle cells to GTHs and hence governs the vertebrate reproduction. / However, compared with the information in mammals, the expression control of these receptors in teleosts remains largely unknown. Previously, we have demonstrated the differential expression profiles of fshr and lhcgr in the zebrafish folliculogenesis, showing that lhcgr expression lags behind fshr expression. This temporal difference between fshr and lhcgr expression has raised two interesting questions: 1) What hormones regulate the differential expression of fshr and lhcgr? and 2) What are the control mechanisms of these regulations? The present study was initiated to answer these questions. / With the primary zebrafish follicle cell cultures, we demonstrated that estradiol (E2) was a potent differential regulator of GTHRs. Although E2 increased both fshr and lhcgr expression, the up-regulatory potency of E2 on lhcgr was much greater than that on fshr. E2 directly promoted lhcgr expression via classical nuclear estrogen receptors (nERs) since nER-specific antagonist (ICI 182,780) completely abolished the E2 effect. Interestingly, our evidence suggested that these nERs could be localized on the plasma membrane because the membrane-impermeable form of estrogen (E2-BSA) fully mimicked the actions of E2. Furthermore, by applying various pharmaceutical agents, we revealed the involvement of multiple signaling molecules, including cAMP, PKA, PI3K, PKC, MEK, MAPK and p38 MAPK. The cAMP-PKA pathway likely played a direct role in the biphasic actions of E2 while the E2 actions were also greatly dependent on the permissive actions of other signaling molecules. / Apart from the sex steroid E2, human chorionic gonadotropin (hCG; as a LH analogue), pituitary adenlyate cyclase-activating peptide (PACAP), epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) also significantly regulated GTHR expression in the zebrafish follicle cells. hCG drastically down-regulated its receptor, lhcgr, suggesting that hCG could desensitize the follicle cells to respond to GTH. Meanwhile, PACAP transiently mimicked the actions of hCG, indicating that PACAP was likely a transient downstream mediator of hCG. EGF was another strong suppressor of lhcgr expression while IGF-I was a potential fshr expression enhancer, which highlighted the crucial roles of paracrine factors in the regulation of GTHRs. In addition to the regulatory effect of these individual hormones or factors, we further revealed that the E2 action could be overridden or modulated by them. Their regulatory effects on the expression of nERs might contribute to this phenomenon. PACAP transiently reduced esr2a and esr2b expression while EGF significantly down-regulated esr2a. / As the first comprehensive study of GTHR regulation in the teleost ovary, the present study certainly enriched our knowledge in the functions of GTHs and the expression control of GTHRs during folliculogenesis. By applying the current research platform on the study of bisphenol A (BPA), an endocrine-disrupting chemical (EDC), the present study further highlighted the potential of this research platform to contribute to the future action mechanism studies of various EDCs. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Ka Cheuk. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 159-212). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgement --- p.v / Table of contents --- p.vi / List of figures and tables --- p.xii / Symbols and abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Hypothalamic-pituitary-gonadal axis / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Gonadotropin-releasing hormone --- p.1 / Chapter 1.2 --- Folliculogenesis / Chapter 1.2.1 --- Structure of ovarian follicles --- p.2 / Chapter 1.2.2 --- Stages of folliculogenesis --- p.3 / Chapter 1.3 --- Gonadotropins and gonadotropin receptors / Chapter 1.3.1 --- History of teleost gonadotropin and gonadotropin receptors --- p.5 / Chapter 1.3.2 --- Structure --- p.6 / Chapter 1.3.3 --- Function --- p.7 / Chapter 1.3.4 --- GTH-GTHR specificity --- p.9 / Chapter 1.3.5 --- Signal transduction --- p.10 / Chapter 1.3.6 --- Expression profile of gonadotropin receptors --- p.11 / Chapter 1.3.7 --- Regulation of gonadotropin receptors --- p.12 / Chapter 1.4 --- Objectives and significances of the project --- p.14 / Chapter 1.5 --- Figure legends --- p.16 / Chapter 1.6 --- Figures --- p.18 / Chapter Chapter 2 --- Differential Regulation of Gonadotropin Receptors (fshr and lhcgr) by Estradiol in the Zebrafish Ovary Involves Nuclear Estrogen Receptors That Are Likely Located on the Plasma Membrane / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Animals --- p.25 / Chapter 2.2.2 --- Hormones and chemicals --- p.26 / Chapter 2.2.3 --- Primary follicle cell culture and drug treatment --- p.26 / Chapter 2.2.4 --- Ovarian fragment incubation --- p.27 / Chapter 2.2.5 --- Total RNA extraction and real-time qPCR --- p.27 / Chapter 2.2.6 --- Western blot analysis --- p.27 / Chapter 2.2.7 --- SEAP reporter gene assay --- p.28 / Chapter 2.2.8 --- Data analysis --- p.28 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Differential stimulation of fshr and lhcgr expression in ovarian fragments and follicle cells by estradiol but not testosterone --- p.28 / Chapter 2.3.2 --- Potentiation of follicle cell responsiveness to hCG by E2 pretreatment --- p.30 / Chapter 2.3.4 --- Evidence for transcription but not translation-dependent up-regulation of lhcgr by E2 --- p.30 / Chapter 2.3.5 --- Evidence for the involvement of nuclear estrogen receptors but not G protein-coupled estrogen receptor 1 (Gper) in E2-stimulated lhcgr expression --- p.31 / Chapter 2.3.6 --- Evidence for possible localization of estrogen receptors on the plasma membrane --- p.32 / Chapter 2.3.7 --- MAPK dependence of E2 effect on lhcgr expression --- p.32 / Chapter 2.4 --- Discussion --- p.33 / Chapter 2.5 --- Table --- p.38 / Chapter 2.6 --- Figure legends --- p.39 / Chapter 2.7 --- Figures --- p.43 / Chapter Chapter 3 --- Signal Transduction Mechanisms of the Biphasic Estrogen Actions in the Regulation of Gonadotropin Receptors (fshr and lhcgr) in the Zebrafish Ovary / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Animals --- p.52 / Chapter 3.2.2 --- Hormones and chemicals --- p.52 / Chapter 3.2.3 --- Primary cell culture and drug treatment --- p.52 / Chapter 3.2.4 --- Total RNA extraction and real-time qPCR --- p.52 / Chapter 3.2.5 --- Fractionation of follicle cells --- p.52 / Chapter 3.2.6 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Statistical analysis --- p.53 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Biphasic roles of cAMP-PKA pathway --- p.53 / Chapter 3.3.2 --- Effects of p38 MAPK inhibition --- p.54 / Chapter 3.3.3 --- Effects of PKC and PI3K inhibition --- p.54 / Chapter 3.4 --- Discussion --- p.55 / Chapter 3.5 --- Figure legends --- p.59 / Chapter 3.6 --- Figures --- p.61 / Chapter Chapter 4 --- Gonadotropin (hCG) and pituitary adenylate cyclase-activating peptide (PACAP) down-regulate basal and E2-stimulated gonadotropin receptors (fshr and lhcgr) in the zebrafish ovary via a cAMP-dependent but PKA-independent pathway / Chapter 4.1 --- Introduction --- p.66 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Animals --- p.69 / Chapter 4.2.2 --- Hormones and chemicals --- p.69 / Chapter 4.2.3 --- Primary cell culture and drug treatment --- p.69 / Chapter 4.2.4 --- Total RNA extraction and real-time qPCR --- p.69 / Chapter 4.2.5 --- Statistical analysis --- p.69 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Down-regulation of fshr and lhcgr by hCG --- p.69 / Chapter 4.3.2 --- Differential regulation of fshr and lhcgr by PACAP --- p.70 / Chapter 4.3.3 --- Inhibition of E2-regulated fshr and lhcgr expression by hCG --- p.71 / Chapter 4.3.4 --- Suppressive effects of PACAP on E2-induced fshr and lhcgr expression --- p.71 / Chapter 4.3.5 --- Role of cAMP in hCG and PACAP actions --- p.72 / Chapter 4.4 --- Discussion --- p.73 / Chapter 4.5 --- Figure legends --- p.78 / Chapter 4.6 --- Figures --- p.80 / Chapter Chapter 5 --- Paracrine regulation of gonadotropin receptors (fshr and lhcgr) by ovarian growth factors: epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) / Chapter 5.1 --- Introduction --- p.85 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- Animals --- p.88 / Chapter 5.2.2 --- Hormones and chemicals --- p.88 / Chapter 5.2.3 --- Primary cell culture and drug treatment --- p.88 / Chapter 5.2.4 --- Total RNA extraction and real-time qPCR --- p.88 / Chapter 5.2.5 --- Statistical analysis --- p.88 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Biphasic down-regulation of lhcgr by EGF --- p.89 / Chapter 5.3.2 --- Evidence for EGFR involvement --- p.89 / Chapter 5.3.3 --- Minor role of MEK-MAPK3/1 pathway in the EGF effect on lhcgr expression --- p.90 / Chapter 5.3.4 --- Up-regulation of fshr by IGF-I --- p.90 / Chapter 5.3.5 --- Evidence for IGF-IR involvement --- p.91 / Chapter 5.3.6 --- Role of PI3K-Akt pathway in IGF-I action --- p.91 / Chapter 5.3.7 --- Role of EGF and EGFR in E2-induced GTHR expression --- p.91 / Chapter 5.3.8 --- Role of IGF-I and IGF-IR in E2-induced GTHR expression --- p.91 / Chapter 5.4 --- Discussion --- p.92 / Chapter 5.5 --- Figure legends --- p.98 / Chapter 5.6 --- Figures --- p.100 / Chapter Chapter 6 --- Regulation of estrogen receptor subtypes (esr1, esr2a and esr2b): a possible mechanism to modulate estradiol-stimulated lhcgr expression in the zebrafish ovary / Chapter 6.1 --- Introduction --- p.107 / Chapter 6.2 --- Materials and methods / Chapter 6.2.1 --- Animals --- p.110 / Chapter 6.2.2 --- Hormones and chemicals --- p.110 / Chapter 6.2.3 --- Staging ovarian follicles --- p.110 / Chapter 6.2.4 --- Primary cell culture and drug treatment --- p.110 / Chapter 6.2.5 --- Total RNA extraction and real-time qPCR --- p.110 / Chapter 6.2.6 --- Statistical analysis --- p.111 / Chapter 6.3 --- Results / Chapter 6.3.1 --- Expression profiles of estrogen receptors (ERs) in zebrafish folliculogenesis --- p.111 / Chapter 6.3.2 --- Homologous regulation of nERs by E2 --- p.111 / Chapter 6.3.3 --- Regulation of nERs by endocrine hormones (hCG and PACAP) --- p.112 / Chapter 6.3.4 --- Regulation of nERs by ovarian paracrine growth factors (EGF and IGF-I) --- p.112 / Chapter 6.3.5 --- Role of cAMP in nER regulation --- p.113 / Chapter 6.3.6 --- Role of PKA in nER regulation --- p.113 / Chapter 6.4 --- Discussion --- p.114 / Chapter 6.5 --- Figure legends --- p.119 / Chapter 6.6 --- Figures --- p.121 / Chapter Chapter 7 --- Estrogenic Action Mechanisms of Bisphenol A / Chapter 7.1 --- Introduction --- p.127 / Chapter 7.2 --- Materials and methods / Chapter 7.2.1 --- Animals --- p.129 / Chapter 7.2.2 --- Hormones and chemicals --- p.129 / Chapter 7.2.3 --- Primary cell culture and drug treatment --- p.129 / Chapter 7.2.4 --- Total RNA extraction and real-time qPCR --- p.129 / Chapter 7.2.5 --- Statistical analysis --- p.130 / Chapter 7.3 --- Results / Chapter 7.3.1 --- Expression of fshr and lhcgr interfered by BPA --- p.130 / Chapter 7.3.2 --- Signaling mechanism of BPA-induced lhcgr up-regulation --- p.130 / Chapter 7.3.3 --- Dependence of transcription and translation in BPA-induced lhcgr expression --- p.131 / Chapter 7.3.4 --- Evidence for the involvement of nuclear estrogen receptors in the BPA actions --- p.131 / Chapter 7.3.5 --- Interference on E2-induced lhcgr expression by BPA --- p.131 / Chapter 7.4 --- Discussion --- p.132 / Chapter 7.5 --- Figure legends --- p.136 / Chapter 7.6 --- Figures --- p.138 / Chapter Chapter 8: --- General Discussion / Chapter 8.1 --- Estradiol as a differential regulator of gonadotropin receptors --- p.143 / Chapter 8.2 --- Conserved role of estradiol with differential action mechanisms in lhcgr regulation of mammals and teleosts --- p.144 / Chapter 8.3 --- Involvement of classical estrogen receptors that are likely located on the plasma membrane --- p.145 / Chapter 8.4 --- Biphasic response of lhcgr to estradiol and the underlying signal transduction mechanisms --- p.145 / Chapter 8.5 --- Desensitization of follicle cells to gonadotropins by hCG --- p.146 / Chapter 8.6 --- Paracrine control of gonadotropin receptors by ovarian growth factors --- p.147 / Chapter 8.7 --- Interaction of the estrogen action with other endocrine and paracrine signals --- p.148 / Chapter 8.8 --- Action mechanism studies of an endocrine-disrupting chemical: bisphenol A --- p.150 / Chapter 8.9 --- Conclusion --- p.151 / Chapter 8.10 --- Figure legends --- p.153 / Chapter 8.11 --- Figures --- p.155 / References --- p.159

O consumo desenfreado de etanol traz consequências negativas, que envolvem problemas fisiopatológicos e de socialização do indivíduo, além de questões de saúde pública. Seu consumo por mulheres grávidas acarreta em danos ao desenvolvimento e formação cerebral do feto, causando a Síndrome Alcoólica Fetal (SAF). Uma forma mais abrangente deste distúrbio é a FASD (Fetal Alcohol Spectrum Disorder), que inclui formas brandas de SAF, bem como qualquer desordem neurológica ou congênita em decorrência do etanol. O objetivo do estudo é avaliar as alterações comportamentais e neuroquímicas em peixezebra adulto após a exposição a diferentes concentrações de etanol na fase inicial do seu desenvolvimento. Para a indução desta desordem, utilizamos ovos de peixe-zebra com 24h pós-fertilização, expostos a diferentes concentrações de etanol: 0%, 0,1%, 0,25%, 0,5% e 1%, durante duas horas. Acompanhamos o desenvolvimento destes animais até a fase adulta (4 meses), onde foram realizadas diferentes tarefas comportamentais: exploração do aparato novel tank e interação social, além de captação de glutamato, como medida neuroquímica. Como resultados mais relevantes, destacam-se a exploração de todo o aparato pelo grupo controle e uma diminuição concentração-dependente da exploração pelos grupos expostos previamente ao etanol; maior permanência no fundo do novel tank nas concentrações elevadas de etanol; uma diminuição concentração-dependente do tempo de permanência próximo ao cardume de acordo com o aumento da concentração de etanol. Quanto às análises bioquímicas, a captação de glutamato mostrou uma diminuição significativa na função de transporte deste neurotransmissor nos animais tratados com concentrações mais elevadas de etanol. A posterior intervenção farmacológica com buspirona reverteu o perfil comportamental previamente observado pelo efeito do etanol. Considerando as análises bioquímicas, a captação de glutamato mostrou uma diminuição significativa na função de transporte deste neurotransmissor nos animais tratados com concentrações intermediárias (0.25 e 0,5%)de etanol. Concluímos que a exposição prévia a diferentes concentrações de etanol na fase embrionária leva a alterações comportamentais na fase adulta, tais como diminuição de exploração frente a novidades, ansiedade e diminuição de interação social. / The excessive consumption of ethanol has negative consequences, which involve pathophysiological and individual socialization issues, and public health issues. Its consumption by pregnant women causes damage to brain development and formation of the fetus, causing fetal alcohol syndrome (FAS). A broader form of this disorder is the FASD (Fetal Alcohol Spectrum Disorder), which includes milder forms of FAS, as well as any neurological disorder or congenital due to ethanol. This project proposes to assess the behavioral and neurochemical alterations in adult zebrafish after exposure to different concentrations of ethanol in the initial phase of its development. To induce this disorder, we use zebrafish eggs and 24h post-fertilization exposed to different concentrations of ethanol, 0%, 0.1%, 0.25%, 0.5% and 1%, for two hours. We observed the development of these animals to juvenile and adulthood, which will be performed different behavioral tasks: exploration of novel apparatus tank and social interaction, as well as glutamate uptake, as measured neurochemistry. Among the most relevant results, we highlight the exploration of the whole apparatus for the control group and a concentration-dependent decrease of exploration by groups previously exposed to ethanol. It was observed high time spent in the bottom of the novel tank in high concentrations of ethanol. Furthermore, elevated spend time close to social interaction chamber was observed, with a dose-dependent decrease of this time in accordance with the increase of ethanol concentration. Further pharmacological intervention with buspirone reversed the behavioral profile previously observed the effect of ethanol. Considering biochemical analysis, glutamate uptake showed a significant decrease in the transport function of this neurotransmitter in animals treated with intermediated doses of ethanol. We conclude that prior exposure to different concentrations of ethanol in infancy leads to behavioral changes in adulthood, such as decreased operating front the news, anxiety and decreased social interaction.

Orientador: Renata Fracácio Francisco / Abstract: Metals are known for their toxic potential to the aquatic environment, as well as being bioaccumulative and persistent elements, which increases the concern with the concentration allowed by legislation, considering protection of biota Often, these compounds are released in the water bodies in concentrations which may not cause acute effects but may compromise the viability of biological systems in the long term, considering the continuous exposure to said elements and concentrations. In this context, liver is an important organ for the evaluation of metals toxicity due to its function of excretion and its potential to accumulate exogenous substances. In gonads metals can trigger disturbances in the production, release, transport, metabolism and action of natural hormones, which can lead to fecundity loss, males feminization and consequently imbalance to living organisms, compromising the viability of the species. Exogenous substances that act in this way are known as endocrine disrupters and their influences on individual organisms and aquatic life are not well established. One ways to identify them is through histological and histochemical analysis. Metals copper and nickel originate from natural and also artificial sources due to the generation of domestic and industrial waste, depending on the anthropic activities. They can be considered toxic or essential depending on the organism, the environment and its concentrations. In addition, they have the potential to be endocri... (Complete abstract click electronic access below) / Resumo: Os metais são conhecidos pelo seu potencial tóxico ao ambiente aquático, além de serem elementos bioacumulativos e persistentes, o que aumenta a preocupação com a concentração permitida pela legislação, considerando-se proteção da biota Muitas vezes, estes compostos são liberados nos corpos hídricos em concentrações subletais as quais não provocam efeitos agudo, mas , podem comprometer a viabilidade dos sistemas biológicos a longo prazo, considerando-se a contínua exposição aos referidos elementos e concentrações Nesse contexto, fígado é um importante órgão para avaliação da toxicidade de metais devido a sua função de excreção e seu potencial para acumular substâncias exógenas. Nas gônadas os metais podem desencadear perturbações na produção, liberação, transporte, metabolismo e ação dos hormônios naturais, que podem levar a perda de fecundidade, feminização de machos e consequentemente desequilíbrio aos organismos vivos, comprometendo a viabilidade das espécies. Substâncias exógenas que atuam dessa maneira são conhecidos como interferentes endócrinos e suas influências sobre os organismos individualmente e a vida aquática ainda não são bem estabelecidos. Uma das maneiras de identificá-los é através de análises em nível histológico e histoquímico. Os metais cobre e níquel originam-se de fontes naturais e também artificiais devido a geração de resíduos domésticos e industriais, em função das atividades antrópicas. Podem ser considerados tóxicos ou essenciais dependendo do orga... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Preliminary analyses of three kinds of promoter activity showed that a-actin gene promoter was chosen to initiate the hormone transcription for the first consideration. We have fused the cDNAs encoding the intact somatolactins in frame to a zebrafish a-actin gene promoter to generate transgenic zebrafish lines co-injected with a GFP protein driven by the same promoter. The transgenic zebrafish were selected from GFP expression and confirmed by genomic PCR and Southern blot analysis, then maintained as transgenic founders. Measurement of the transgenes' expressions and the expressions of marker genes in different pathways by using real-time PCR provided a general understanding of SLs' actions. The data obtained indicated that the over-expressing of SLalpha and SLbeta in vivo significantly enhance the transcriptions of the insulin-like growth factors, IGF1 (5.46-fold and 6.77-fold), IGF2a (4.38-fold and 4.35-fold) and IGF2b (2.83-fold and 3.94-fold), but down-regulated IGF3 (a novel member found specifically in gonad) in larvae. However, the stimulation by administration of recombinant proteins (SLalpha and SLbeta) only showed a slight induction of the mRNA levels of IGFs (IGF1, IGF2a and IGF2b) on ZFL cells in vitro. / Somatolactin (SL) is a novel member of pituitary polypeptide hormone found only in fish; it shares significant structural homology with prolactin and growth hormone. Since somatolactin receptor (SLR) was first defined as GHR1 and orthologous to the growth hormone receptor GHR2, SL and GH may share similar actions in growth and development. Recently, two SLs have been identified as SLalpha and SLbeta with similar structures, freshwater fish have these two isoforms found in the same species and only one isoform (SLalpha) is found in marine species. The two isoforms of SL may have different functions and physiological actions. To investigate the roles of SLs on vertebrate development and embryogenesis, we generated transgenic fish models with "all zebrafish" elements in origin to study the physiological functions of SLs in zebrafish. / The ectopic expression of somatolactins also results in up-regulating gene expression of insulin, leptin, sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FAS), as well as the expression of vitellogenin and melanocyte-stimulating hormone (MSH) levels while causing reduction of catalase (CAT) and glutathione S-transferase (GST) levels in larvae. The results here represent the similar function between SLalpha and SLbeta and reveal more details in fish of the endocrinology system involvement in growth development, glucose synthesis, lipid metabolism, reproduction, pigmentation and antioxidant defense system through the actions of SLs. / Three different gene promoters of zebrafish have been isolated to initiate the ectopic expression of somatolactins in vivo, which including a constitutional beta-actin gene promoter, a liver specific transferrin gene promoter and a zinc ion inducible metallothionein (MT) gene promoter. The promoter activities were tested in fish cell-line by using luciferase reporter assay. In MT gene promoter, two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5'-flanking region (1,514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn2+ and the Zn 2+ induction was dose-dependent. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn2+ or Cd2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein. / Wan, Guohui. / Adviser: Chan King Ming. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 139-163). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Finally, as the first step to study the regulation of Kit system, we found that IGF-I was a potent regulatory factor that up-regulated the expression of kitlga in zebrafish follicle cells. The stimulation involved transcription but not translation, indicating that the kitlga gene is a direct downstream target of IGF-I. The effect of IGF-I on kitlga was exerted via PI3K-Akt but not MAPK pathway. In contrast, the MAPK pathway may play a negative role in controlling kitlga expression. / Kit ligand (also named stem cell factor, SCF) is a pleiotropic growth factor with diverse biological functions. It exerts effects on target cells by binding to its cognate tyrosine kinase receptor, Kit. In mammals, accumulated evidence has demonstrated important roles for Kit ligand and Kit in gametogenesis, melanogenesis and haematopoiesis. However, very little is known about Kit system in other vertebrates. In the present study, we used zebrafish as the model to investigate the expression, regulation and function of the Kit system in the ovary. / On the other hand, cAMP is involved in regulating the expression of kitlga in zebrafish follicle cells. Two cAMP-activated effectors, PKA and Epac, have reverse effects. PKA promotes but Epac inhibits the expression of kitlga, which was identified by the respective activator. The effect of forskolin and H89 on IGF-I-induced expression of kitlga suggests a cross-talk between the two signaling pathways. Both hCG and PACAP inhibited IGF-I-induced kitlga expression, indicating that they may have negative regulation through cAMP signaling pathways in the full-grown follicles. (Abstract shortened by UMI.) / The zebrafish has two homologues of Kit ligand (kitlga and kitlgb) and Kit (kita and kitb ) instead of one copy for each as in mammals. The present study proposed the origin of these homologues in the zebrafish by phylogenetic and chromosome synteny analyses, and provided further evidence for neo- or subfunctionalization for both Kit ligands and Kit receptors in the zebrafish ovary. All four Kit system members exhibited distinct and significant changes in mRNA expression during folliculogenesis, particularly in the periovulatory period before and after final oocyte maturation and ovulation. / Then we further studied the spatial localization of each member within the follicle. The present study demonstrated that kitlga and kitb are exclusively expressed in the follicle layer, while kitlgb and kita only in the oocyte. Using CHO cell line as a bioreactor, we produced recombinant zebrafish Kitlga and Kitlgb. Analysis in mammalian COS-1 cells and zebrafish primary follicle cells confirmed their biological activity and binding specifity. Two opposite paracrine pathways of Kit system in the zebrafish ovary have been shown. Kitlga from the follicle cells preferably activates Kita in the oocyte in spite of the weak response of Kitb to it. Kitlgb from the oocyte, however, exclusively activates Kitb in the follicle cells without any effects on Kita. / Yao, Kai. / Adviser: Ge Wei. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 136-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O conteúdo de zinco (Zn) reativo cerebral é importante para o equilíbrio da sinaptofisiologia neural. A prova disto é que um aumento nos seus níveis, após evento hipóxico-isquêmico, resulta em neurotoxicidade, o que tem estimulado o tratamento desta disfunção cerebral com quelantes de Zn, tal como o dietilditiocarabamato (DEDTC). No caso do DEDTC, o uso deste composto sobre esta disfunção deve ser analisado com cuidado, pois ele também apresenta muitos efeitos colaterais sobre o sistema nervoso central. Desta forma, para atender este propósito, é necessário antes obter uma concentração de DEDTC com menores efeitos colaterais. Por esta razão, no presente trabalho, nós decidimos usar um modelo vertebrado mais simples, tal como o peixe-zebra, o qual permitiria a triagem, em larga escala, dos efeitos de DEDTC sobre o Zn reativo. Entretanto, jamais foi mostrada a presença de Zn reativo no cérebro de peixe-zebra. Com isto, através de marcações histológicas, nós conseguimos mostrar pela primeira vez a distribuição citoarquitectônica de Zn reativo em neurônios glutamatérgicos, bem como o número desses neurônios contendo Zn no cérebro de peixe-zebra. Isto nos permitiu avaliar o efeito de diferentes concentrações de DEDTC sobre o conteúdo de Zn cerebral do peixe-zebra, o qual foi intensamente quelado por elevadas quantidades do composto, induzindo comportamentos tipo-crise. Neste mesmo estudo nós obtemos também uma concentração de DEDTC com poucos efeitos colaterais que poderia exercer neuroproteção sobre o aumento de Zn reativo induzido pela hipóxia-isquemia. Assim, após a padronização de um modelo de hipóxia em peixe-zebra, que demonstra danos relacionados à isquemia, nós testamos se essa concentração de DEDTC poderia ser neuroprotetora sobre este modelo. Contudo, DEDTC apresentou efeitos pró-oxidantes, embora ele tenha atenuado o elevado conteúdo de Zn reativo induzido pela hipóxia. Portanto, mesmo que o DEDTC tenha falhado, este modelo, agora, está apto para a triagem de outros fármacos com potencial ação sobre o alterado conteúdo de Zn reativo que ocorre em eventos hipóxicos-isquêmicos. / The content of brain reactive zinc (Zn) is important for the synaptophysiology in the central nervous system (CNS). This is evidenced in hypoxic-ischemic events, when an increase in their levels results in neurotoxicity. Consequently, this has stimulated the treatment of cerebral ischemia with Zn chelators, such as diethyldithiocarbamate (DEDTC). In the case of DEDTC, the use of this compound in this dysfunction should be examined carefully, because it also has many side effects on the (CNS). Thus, to meet this, it is necessary first to obtain a concentration of DEDTC with negligible side effects. Here, we decided to use a simpler vertebrate model, such as zebrafish, which would allow large-scale screening of DEDTC effects on reactive Zn. However, the presence of reactive Zn has never been shown in zebrafish brain. Then, using histological markers, we were able to show for the first time the cytoarchitectonic distribution of reactive Zn in glutamatergic neurons as well as the number of these neurons containing Zn in the zebrafish brain. This allowed us to evaluate the effect of different DEDTC concentrations on the brain content of Zn in zebrafish. As a result, high levels of the compound did strongly chelate the metal, inducing seizure-like behaviors. In this study we also obtained a DEDTC concentration with few side effects that could exert neuroprotection on the increased reactive Zn induced by hypoxia-ischemia. Then, after the standardization of an ischemic-sensitive model of hypoxia in zebrafish, we tested if this DEDTC concentration could be neuroprotective on this model. Nevertheless, DEDTC showed pro-oxidant effects, though it had mitigated the elevated content of reactive Zn induced hypoxia. Therefore, despite the DEDTC have failed as neuroprotective drug, this model enables the screening of other chemical agents with potential action on the increased content of reactive Zn that occurs in hypoxic-ischemic events.

Orientador: Renata Fracácio Francisco / Resumo: Chemicals with potential to cause endocrine disruption in vertebrates and invertebrates have been detected at low concentrations in the world's aquatic environments. Therefore, the search for removal methodologies in aquatic environments became a worldwide interest. The aim of this research was to investigate three materials (powered activated carbon - PAC, powered natural zeolites - ZP and aquatic humic substances - AHS) in chemical and ecotoxicological remediation of 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) in water through chemical and biological tests in vitro and in vivo. An environmentally relevant concentration of hormones (30 ng.L-1) was used during laboratory tests. The results obtained through an extensive list of morphological, reproductive and histological parameters showed the significant impact of HSFs on the development and maintenance of exposed fish. The superior efficiency of PAC was verified in relation to the other substrates in the endocrine disrupting chemicals (EDCs) removal in water. Nevertheless, their properties did not guarantee the same performance to the environmental samples, neither allow biological injuries monitored to be recovered after the period of depuration investigated. An additional study also allowed the development of a histochemical protocol capable of identifying the production of vitellogenin (VTG) prompted by exposure to the synthetic steroid EE2. / Doutor