Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "December 1998" Typescript. Vita. Includes bibliographical references (l. 140-148). Also available on the Internet.

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Les canaux chlore volume-sensibles (VRACs) régulent les activités différenciatives, migratoires et prolifératives des fibroblastes et pourraient donc être impliqués dans la pathobiologie de l’hypertension artérielle pulmonaire (HTAP) et la fibrose pulmonaire interstitielle (FPI). Diverses études antérieures ont montré que l’endothéline-1 (ET1) a des propriétés pro-fibrosantes, en plus cette molécule participe au remodelage des artérioles pulmonaires dans l’HTAP. D’autre part, ces pathologies d’HTAP et de fibrose pulmonaire peuvent être antagonisées par la mélatonine et par l’interaction entre protéines de morphogenèse (BMPs: bone morphogenetic proteins) et leur récepteur 2 (BMPR2). <p>La première partie de mon travail s’est intéressée aux effets de la BMP2 et de l’endothéline1 sur les canaux chlore volume-sensibles de fibroblastes pulmonaires. <p>La stimulation hypotonique du courant a été inhibée par la BMP2 en dépendance de la dose appliquée et de la durée d’exposition à la molécule. Un maximum d’effet de la BMP2 a été observé avec une concentration de 10ng/ml pendant 45min de prétraitement. En plus, les courants chlore volume-sensibles, inhibés par la BMP2, se sont restaurés en présence de l’inhibiteur spécifique de la voie de la protéine kinase C (PKC), le GFX. D’autre part, le prétraitement des fibroblastes avec l’ET1 à 100μM pendant 2heures a induit l’apparition d’un courant activable par l’acide lysophosphatidique (ICl-LPA) (marqueur de la différenciation des fibroblastes) et l’expression de l’α-sma (alpha smooth muscle actin, marqueur classique des myofibroblastes). La migration des fibroblastes a été aussi induite en présence de l’ET1, alors que l’inhibition des canaux chlore par le DIDS (Diisothiocyanatostilbene-disulfonic acid) a bloqué cet effet. La BMP2 s’est opposée à l'effet de l’ET1 sur la différenciation des fibroblastes par l’inhibition de l’induction du courant ICl-LPA et de l’expression génique de l’α-sma. En plus, la migration des fibroblastes, induite par l’ET1, a été inhibée par la BMP2. Nous avons aussi montré que l’expression de gène du canal anoctamine6 a été stimulée par l’ET1, alors la BMP2 s’est opposée à cet effet, ce qui suggère que l’anoctamine6 est le canal responsable de la différenciation des fibroblastes marquée par l’apparition du courant ICl-LPA. Il apparaît donc que l’ET1 et la BMP2 ont des effets opposés sur la différenciation et la migration des fibroblastes pulmonaires via leurs effets sur l’activité et l’expression des canaux chlore volume-sensibles. <p>La deuxième partie du travail s’est intéressée à l’effet de la mélatonine sur les canaux chlore volume-sensibles de fibroblastes L929 et aux conséquences de cet effet sur la migration et la prolifération de ces cellules. Le prétraitement des fibroblastes avec 100μM de mélatonine pendant 30min a inhibé significativement l’activation des canaux chlore volume-sensibles. En plus, une concentration de 100 nM pendant une nuit a donné le même effet observé avec la mélatonine à 100μM pendant 30 min. Nous avons aussi constaté que l’inhibition des VRACs par la mélatonine a été dose-dépendante. L’effet de la mélatonine sur les VRACs a été inhibé en présence de l’antagoniste non sélectif des récepteurs de la mélatonine (Luzindole) et l’antagoniste sélectif pour le récepteur 2 (MT2) de la mélatonine (K185). En plus, l’inhibiteur de la voie de la PKC (GFX) a empêché la mélatonine d’agir sur les canaux chlore volume-sensibles. Ces résultats suggèrent que la mélatonine agit sur les VRACs en se fixant sur MT2 et en activant la voie de la PKC. L’inhibition des VRACs par la mélatonine a eu pour conséquence l’inhibition du phénomène de RVD (regulatory volume decrease), qui suit le gonflement hypotonique. Nous avons aussi montré que la migration des fibroblastes L929 a été inhibée par la mélatonine à 100μM et cela via l’inhibition des VRACs, puisque la mélatonine s’est montrée incapable d’induire une inhibition supplémentaire de la migration en présence de l’inhibiteur des canaux chlore volume-sensibles (DIDS). En plus, l’antagoniste non sélectif des récepteurs de la mélatonine (luzindole), l’antagoniste sélectif pour MT2 (K185) et l’inhibiteur de la voie de PKC (GFX) ont provoqué la disparition de l’effet de la mélatonine sur la migration. Cela suggère que la mélatonine agit sur la migration via les voies empruntées pour l’inhibition des VRACs. L’inhibition des VRACs, par la mélatonine et le DIDS, n'a pas induit d'inhibition significative sur la prolifération des fibroblastes L929, ce qui veut dire que l’inhibition des VRACs est insuffisante pour induire une inhibition significative de la prolifération. Donc, la mélatonine inhibe les canaux chlore volume-sensibles via sa fixation sur MT2 et l’activation de la voie de la PKC. Cela a pour conséquence l’inhibition du RVD et de la migration des fibroblastes L929, mais cette inhibition des VRACs est insuffisante pour inhiber la prolifération de ces cellules. <p>En conclusion, j’ai pu montrer l’importance des canaux chlore volume-sensibles dans la régulation de la physiologie des fibroblastes et leurs interactions avec des médiateurs d’affections pulmonaires à composante fibrosante, telles que l’HTAP et la FPI./<p>Volume-regulated anion channels (VRACs) regulate fibroblast differentiation, migration and proliferation. Fibroblasts have been shown to be involved in several pathologic states including pulmonary arterial hypertension (PAH) and interstitial pulmonary fibrosis (IPF). A number of previous studies have shown that endothelin-1 (ET1) has pro-fibrotic properties and participates in the remodeling of pulmonary arterioles in PAH. On the other hand, PAH and IPF may be controlled by melatonin and bone morphogenetic protein receptor 2 (BMPR2) signaling. <p>The first part of my work described the effects of BMP2 and ET1 on the VRAC in the pulmonary fibroblasts and the consequences of these effects on differentiation and migration of these cells. Pretreatment of fibroblasts with BMP2 inhibited hypotonic current stimulation and this effect was dependent on the BMP2 concentration and on the time of exposition to the molecule. The maximum effect of BMP2 was observed at a concentration of 10ng/ml for 45 min of pretreatment. In addition, volume-sensitive chloride current, inhibited by BMP2, was restored in presence of PKC (protein kinase C) pathway inhibitor (GFX). On the other hand, the pretreatment of fibroblasts with100μM of ET1 for 2 hours, induced the appearance of a lysophosphatidic acid-activable chloride current (ICl-LPA) (a marker of fibroblast differentiation) and stimulated the expression of the smooth muscle actin alpha (α-sma) (the classical marker of myofibroblasts). ET1 also stimulated fibroblast migration, while the inhibition of chloride channels by (DIDS) (Diisothiocyanatostilbene disulfonic acid) bloked this effect. The BMP2 opposed the effect of ET1 on fibroblast differentiation by preventing the induction of ICl-LPA current and α-sma gene expression. In addition, BMP2 inhibited the fibroblast migration induced by ET1. We have also shown that ET1 stimulated anoctamin6 channel gene expression and that BMP2 opposed this effect, which suggests the implication of anoctamin6 on fibroblast differentiation marked by the appearance of ICl-LPA current. Thus, ET1 and BMP2 have opposite effects on pulmonary fibroblast differentiation and migration via their effects on the activity and expression of volume-regulated anion channels. <p>The second part of the work focused on the effect of melatonin, which is a vasorelaxant and antifibrotic agent, on the volume-sensitive chloride channels in L929 fibroblasts and primary rat fibroblasts and on the consequences of this effect on migration and proliferation of these cells. Fibroblast pretreatment with 100μM of melatonin for 30 min significantly inhibited the activation of volume-sensitive chloride channels. In addition, a concentration of 100 nM of melatonin overnight produced the same effect observed with melatonin at 100μM for 30 min. The effect of melatonin on VRAC current was dose-dependent. Inhibition of VRACs by melatonin resulted the inhibition of the RVD phenomenon (Regulatory Volume Decrease) following the hypotonic swelling. The effect of melatonin on VRACs was inhibited in the presence of the non-selective antagonist of melatonin receptors (Luzindole) and the selective antagonist of the melatonin receptor 2 (MT2), the K185. In addition, the PKC pathway inhibitor (GFX) inhibited the effect of melatonin on the volume-sensitive chloride channels. These results suggest that, melatonin acts on the VRACs by binding to MT2 and by activating the PKC pathway. We have also shown that the L929 fibroblast migration was inhibited by melatonin (100μM) via inhibition of VRAC channels, since melatonin was unable to induce further inhibition of migration in the presence of the volume-sensitive chloride channels inhibitor (DIDS). In addition, the non-selective melatonin receptors antagonist (luzindole), the selective antagonist for MT2 (K185) and the PKC pathway inhibitor (GFX), blocked the effect of melatonin on migration, which suggests that melatonin acts on migration via the same pathways that inhibit VRAC channels. Inhibition of VRACs by melatonin and DIDS have not shown any significant inhibition of L929 fibroblast proliferation, which means that the VRAC inhibition is not sufficient to induce a significant inhibition of proliferation. Thus, melatonin inhibits volume-sensitive chloride channels via its binding to MT2 and activation of the PKC pathway. This has as consequences the inhibition of RVD and migration of L929 fibroblasts but insufficient to inhibit the proliferation of these cells. <p>In conclusion, I have shown the importance of volume-sensitive chloride channels in the regulation of fibroblast physiology and its interactions with ET-1, BMP and melatonin signaling. These results are compatible with the notion that the participation of fibroblasts in the pathobiology of PAH or IPF is mediated by VRAC channels, which can be activated by ET-1 and inhibited by BMP’s or melatonin. The translational relevance of these findings will have to be investigated on fibroblasts from patients with PAH or IPF, or from animal models of pulmonary hypertension or lung fibrosis.<p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

卵巢是女性生殖系統中一個重要的器官，負責為受精提供卵子，以及合成生殖過程中所必需，同時也在其他生理過程中起重要作用的各種激素。大約有30%的不育源於卵巢的問題，包括無排卵，無月經，月經週期不規律和激素水平異常等。雄激素:雌激素比例過高，卵泡發育異常，無排卵等卵巢功能障礙常見於各種疾病中，例如多囊性卵巢綜合征(PCOS)--一種影響5~10%育齡婦女的內分泌疾病，以及囊性纖維化( CF)--一種由囊性纖維化跨膜電導調節器(CFTR) 基因突變引起的遺傳疾病。然而引起這些卵巢功能障礙的確切機制並不清楚。 / 雌激素是在卵泡雌激素(FSH) 的調節下，在卵巢顆粒細胞中通過芳香化臨的住激素轉化而生成的。在論文第一部分的研究中，我們旨在證明CFTR 在卵巢顆粒細胞中的表達，以及它參與雌激素生成的過程。實驗結果證實了CFTR 在小鼠和人顆粒細胞中的表達，同時表明CFTR 通過一種碳酸氫根離子(HC0₃⁻) 敏感的可溶性腺苦酸環化梅(sAC) ，放大FSH 所刺激的雌激素生成過程。實驗結果顯示，在原代小鼠顆粒細胞中， HC0₃⁻能夠增強FSH 所引起的CREB 磷酸化，芳香化晦表達，以及雌激素的生成，而在抑制CFTR 的情況下，或在CFTR 敲除/DeltaF508 突變小鼠的顆粒細胞中， HCO3-的放大作用顯著降低。CFTR 和芳香化醋的表達水準在人顆粒細胞中具有正相關性，進一步支持CFTR 對雌激素生成的調節作用。在PCOS 患者的顆粒細胞和大鼠PCOS 模型的卵巢中， CFTR 和芳香化醋的表達水準顯著下調。這些結果提示， CFTR 對雌激素生成調節這一機制的缺陷可能參與了CF 和PCOS 中卵巢功能障礙的發病機理。 / 卵泡發育很大程度上依賴於顆粒細胞的增殖'生存和凋亡，這些過程在PCOS 中都會出現異常。論文的第二部分冒在研究顆粒細胞的CFTR 在PCOS 的卵泡發育異常中的作用。實驗結果表明， CFTR 在PCOS 大鼠的囊，性卵泡的顆粒細胞中表達降低，同時伴隨著PCNA 和Bcl-2 的下調，而Bax 和cleaved caspase-3則沒有變化，提示顆粒細胞的增殖和生存/抗凋亡能力降低。敲減或抑制顆粒細胞中的CFTR 導致細胞存活降低， PCNA 和Bcl-2 表達下調，以及細胞凋亡增加，提示CFTR 對顆粒細胞增殖和生存的調節作用。CFTR 通過HC0₃⁻/ sAC/PKA 信號通路，調節基礎及FSH 刺激引起的ERK I!2 磷酸化，及其下游的CyclinD2 和PCNA表達，從而促進顆樹圍胞的增殖。顆粒細胞CFTR 的下調可能通過抑制細胞增殖和降低細胞生存能力，參與了PCOS 中的囊性卵泡的形成過程。 / 綜上所述，本論文證明了CFTR 在卵巢顆粒細胞上的表達，並且參與調節顆粒細胞雌激素生成和細胞的增殖和生存。CFTR 的缺陷或表達下調可能是導致CF和PCOS 的卵巢功能障礙的發病機理。 / The ovary is the female reproductive organ, which produces female gametes, oocytes for fertilization and sex hormones essential to reproduction and important to a wide range of physiological and pathological events as well. About 30% of infertility cases arise from ovarian problems, including anovulation, amenorrhea, irregular menstrual cycle and abnormal hormone levels. Ovarian disorders, such as high androgen to estrogen ratio, abnormal folliculogenesis and anovulation, are often seen in diseases, including polycystic ovarian syndrome (PCOS) and cystic fibrosis (CF). The former is an endocrine disorder affecting 5~10% women of reproductive age, and the latter is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). However, the exact mechanisms underlying the ovarian disorders seen in these diseases are not well understood. / Estrogen biosynthesis is profoundly influenced by follicle-stimulating hormone (FSH) that regulates the conversion of androgen to estrogen in ovarian granulosa cells by the rate-limiting enzyme aromatase. The first part of the study aims to investigate the expression of CFTR in granulosa cells and its involvement in regulating estrogen production. The results demonstrate the expression of CFTR in both mouse and human granulosa cells, and provide evidence demonstrating a previously unsuspected role of CFTR in amplification of FSH-stimulated ovarian estrogen biosynthesis and the involvement of a HC0₃⁻ sensor, the soluble adenylyl cyclase (sAC) in this synthesis. FSH-stimulated CREB phosphorylation, aromatae expression, as well as estradiol production are enhanced by HC0₃⁻ and sAC, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/deltaF508 mutant mice. The fact that CFTR expression is found positively correlated with aromatase expression in human granulosa cells supports its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in polycystic ovarian syndrome (PCOS) rodent models and human patients. These findings suggest that defective CFTR-dependent regulation of estrogen production may underline the ovarian disorders seen in CF and PCOS. / Folliculogenesis largely depends on the proliferation, survival and apoptosis of granulosa cells in the follicles and alteration in which has been found in PCOS. The second part of the study aims to investigate the possible involvement of granulosa cell CFTR in the impaired folliculogenesis in PCOS. The results show that downregulation of CFTR is found in the cystic follicles, which is accompanied by reduced expression of PCNA and Bcl-2, but not Bax and cleaved caspase-3, in the ovaries of PCOS rat models, indicating reduced cell proliferation and survival/anti-apoptotic ability. Knockdown or inhibition of CFTR in granulosa cell culture results in reduced cell viability, downregulation of PCNA and Bcl-2 and increase of apoptosis, supporting a role of CFTR in regulating granulosa cell proliferation and survival. CFTR exerts its effect on granuloa cell proliferation by modulating basal and FSH-stimulated ERKl/2 phosphorylation and the expression of its downstream target CyclinD2 and PCNA through the HC0₃⁻/sAC/PKA pathway. These findings suggest that downregulation of CFTR may play a role in the formation of cystic follicles by inhibiting granulosa cell proliferation and reducing cell survival ability, therefore providing a possible mechanism for the abnormal folliculogenesis in PCOS. / In conclusion, the present study has demonstrated the expression of CFTR in the ovarian granulosa cell and its role in regulation of granulosa cell proliferation, survival and estrogen production. Defect of CFTR in CF and downregulation of CFTR in PCOS may contribute to the abnonnal honnone profile and impaired folliculogenesis in both disease conditions. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Hui. / "October 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-137). / 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 --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / LIST OF PUBLICATIONS --- p.viii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvi / Chapter 1 --- CHAPTER I: Introduction --- p.1 / Chapter 1.1 --- The ovary --- p.1 / Chapter 1.1.1 --- Structure and function of the ovary --- p.1 / Chapter 1.1.2 --- Follicle development --- p.5 / Chapter 1.1.3 --- Ovulation and luteinization --- p.7 / Chapter 1.1.4 --- Ovarian hormone biosynthesis --- p.10 / Chapter 1.2 --- Diseases with ovarian dysfunction --- p.14 / Chapter 1.2.1 --- Polycystic ovarian syndrome (PCOS) --- p.14 / Chapter 1.2.1.1 --- Introduction to PCOS --- p.14 / Chapter 1.2.1.2 --- Diagnostic criteria --- p.14 / Chapter 1.2.1.3 --- Abnormal hormone profile in PCOS --- p.16 / Chapter 1.2.1.4 --- Abnormal folliculogenesis in PCOS --- p.18 / Chapter 1.2.1.5 --- Etiology --- p.22 / Chapter 1.2.2 --- Cystic Fibrosis (CF) --- p.24 / Chapter 1.2.2.1 --- Introduction to CF --- p.24 / Chapter 1.2.2.2 --- Cause and pathogenesis of CF --- p.25 / Chapter 1.2.2.3 --- Ovarian disorder in CF --- p.27 / Chapter 1.3 --- CFTR in reproduction --- p.29 / Chapter 1.3.1 --- Introduction to CFTR --- p.29 / Chapter 1.3.2 --- Channel function --- p.30 / Chapter 1.3.3 --- Protein regulator function --- p.32 / Chapter 1.3.4 --- Regulation of CFTR expression --- p.34 / Chapter 1.3.5 --- Role of CFTR in reproduction --- p.35 / Chapter 1.3.6 --- CFTR in the ovary --- p.39 / Chapter 1.4 --- General hypothesis and aims --- p.39 / Chapter 1.4.1 --- General hypothesis --- p.39 / Chapter 1.4.2 --- Aims of the study --- p.40 / Chapter 2 --- CHAPTER II: General Methods --- p.42 / Chapter 2.1 --- Meterials --- p.42 / Chapter 2.1.1 --- Animals --- p.42 / Chapter 2.1.2 --- Chemicals and reagents --- p.42 / Chapter 2.1.3 --- Antibodies --- p.44 / Chapter 2.1.4 --- Primers --- p.45 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Determination of estrous cycle --- p.45 / Chapter 2.2.2 --- Granulosa cell culture --- p.46 / Chapter 2.2.3 --- PCGS rat model --- p.47 / Chapter 2.2.4 --- Collection of human granulosa cells --- p.47 / Chapter 2.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.2.6 --- Western blot --- p.50 / Chapter 2.2.7 --- Histological studies --- p.53 / Chapter 2.2.8 --- siRNA transfection --- p.55 / Chapter 2.2.9 --- Intracellular pH measurement --- p.56 / Chapter 2.2.10 --- Whole-cell patch clamp recording --- p.57 / Chapter 2.2.11 --- Statistics --- p.57 / Chapter 3 --- CHAPTER III: Result I - The Role of CFTR in FSH-stimulated Estrogen Production: Implication in Cystic Fibrosis and PCGS --- p.59 / Chapter 3.1 --- Summary --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Methods --- p.63 / Chapter 3.3.1 --- Intracellular cAMP assay --- p.63 / Chapter 3.3.2 --- Nuclei isolation and nuclear cAMP measurement --- p.63 / Chapter 3.3.3 --- CREB phosphorylation assay --- p.64 / Chapter 3.3.4 --- Estradiol enzyme immunoassay --- p.64 / Chapter 3.4 --- Results --- p.64 / Chapter 3.4.1 --- Functional expression of CFTR in granulosa cells --- p.64 / Chapter 3.4.2 --- Expression and localization of sAC in granulosa cells and its involvement in BC03f CFTR-dependent cAMP production --- p.66 / Chapter 3.4.3 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated CREB phosphorylation --- p.67 / Chapter 3.4.4 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated aromatase expression and estradiol production --- p.68 / Chapter 3.4.5 --- Impaired CREB phosphorylation aromatase expression and estradiol production by granulosa cells from CFTR-deficient mice --- p.70 / Chapter 3.4.6 --- Reduced CFTR and aromatase expression in human PCOS granulosa cells and rat PCOS ovaries --- p.71 / Chapter 3.5 --- Discussion --- p.87 / Chapter 4 --- CHAPTER IV: Result II - The Role of CFTR in Granulosa Cell Proliferation and survival in PCOS --- p.91 / Chapter 4.1 --- Summary --- p.91 / Chapter 4.2 --- Introduction --- p.92 / Chapter 4.3 --- Methods --- p.95 / Chapter 4.3.1 --- Cell viability assay (MTT and MTS assay) --- p.95 / Chapter 4.3.2 --- ERKI/2 phosphorylation assay --- p.95 / Chapter 4.4 --- Results --- p.96 / Chapter 4.4.1 --- Reduced CFTR expression in PCOS rat models --- p.96 / Chapter 4.4.2 --- Downregulation of genes related to proliferation and survival in PCOS --- p.96 / Chapter 4.4.3 --- CFTR affect viability of granulosa cells --- p.97 / Chapter 4.4.4 --- CFTR regulate cell cycle protein and promote proliferation via HC0₃⁻/sAC/PKA and ERK pathway --- p.98 / Chapter 4.4.5 --- CFTR regulates apoptosis-related protein expression --- p.100 / Chapter 4.5 --- Discussion --- p.114 / Chapter 5 --- CHAPTER V: General Discussion --- p.119 / Chapter 5.1 --- Role of CFTR in ovarian function --- p.119 / Chapter 5.2 --- Role of CFTR/HC0₃⁻/sAC in modulating FSH signaling in the ovary --- p.120 / Chapter 5.3 --- CFTR/HC0₃⁻/sAC as a general modulator in receptor-mediated signaling cascades --- p.122 / Chapter 5.4 --- Concluding remarks --- p.123 / REFERENCES --- p.124 / APPENDICES --- p.138

Orientador: Ana Maria Lima de Azeredo Espin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:46:46Z (GMT). No. of bitstreams: 1 Lopes_AlbertoMouraMendes_M.pdf: 2739543 bytes, checksum: 13578f8ab684d7f2c1af2020305008a8 (MD5) Previous issue date: 2013 / Resumo: O Brasil possui o maior rebanho bovino comercial do mundo, sendo este um dos setores de geração de renda mais importantes para o país. As infestações por ecto e endoparasitas estão entre os principais problemas que levam à diminuição da produtividade. Dentre eles, a Cochliomyia hominivorax (Coquerel), conhecida como mosca-da-bicheira, destaca-se como uma das principais causadoras de miíases, gerando prejuízos de aproximadamente US$ 1,7 bilhões/ano ao país. Produtos sistêmicos como as lactonas macrocíclicas/macrolactonas, ativadoras, dentre outros, do canal de cloreto (GLUCL?), vêm sendo utilizados intensivamente como medida preventiva e de tratamento nas infestações por C. hominivorax e diversos parasitas, e a intensa/prolongada utilização das lactonas macrocíclicas tem levado ao surgimento da resistência a tais produtos. A identificação dos mecanismos moleculares da resistência às lactonas macrocíclicas em outras espécies indica a ocorrência de possíveis alterações no canal de cloreto associados à resistência em C. hominivorax. Utilizando-se de sequências do canal de cloreto de outras espécies, previamente depositadas no banco de dados, além de sequências obtidas na caracterização do transcriptoma de C. hominivorax, o objetivo deste projeto é amplificar e sequenciar a região codificante do gene da subunidade desse canal de cloreto dependente de L-glutamato (GluCl?) em C. hominivorax. Além disso, serão comparadas as sequências de indivíduos resistentes e suscetíveis às macrolactonas visando à identificação da(s) mutação (ões) que confere(m) resistência a tais produtos e, consequentemente, o diagnóstico molecular da resistência / Abstract: Brazil has the largest commercial cattle herd in the world, being one of the most important sectors of income generation for the country. Infestations by ecto- and endo parasites are among the major problems that lead to low productivity. Among them, the Cochliomyia hominivorax (Coquerel), known as the New World Screwworm fly, stands out as a major cause of myiasis, resulting in losses of approximately US$ 1.7 billion each year to the country. Systemic products such as macrocyclic lactones/macrolactones (MLs), mainly the MLs member ivermectin, have been extensively used as a preventive measure and treatment in infestations by C. hominivorax and various parasites, and the intense/prolonged use of macrocyclic lactones have led to the emergence of resistance to such products. The macrolactones target-sites are the cys-loop ligand-gated ion channels receptors, specially the glutamate-gated chloride channel subunit (GLUCL?). The identification of the molecular mechanisms of macrocyclic lactones resistance in other species indicates that possible alterations in chloride channels and GLYR? and nAchR?7 are associated with macrolactones resistance in C. hominivorax. Using the glutamate-gated chloride channel subunit sequences from other species, previously deposited in the database, and sequences obtained in the characterization of the transcriptome of C. hominivorax, the aim of this project was to amplify and sequence the coding region of the glutamate-gated chloride channel subunit gene (GluCl?) in C. hominivorax. In addition, sequences will be compared among putative ivermectin resistant and susceptible individuals in order to identify mutation(s) conferring resistance to such product and thereby the molecular diagnosis of resistance / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Abstract Functional defects in the muscle specific chloride channel ClC-1 result in reduced chloride conductance and electrical hyperexcitability, which in turn impairs muscle relaxation and leads to myotonia. The gene CLCN 1 codes for ClC-1 in humans, and mutations in CLCN 1 cause the disease known as myotonia congenita. Worldwide over 80 mutations in CLCN1 have been described, but only three were found in patients in Northern Finland. These included two missense mutations and a nonsense mutation. The behavior and localization of the normal and mutated ClC-1 mRNA and protein were analyzed in muscle cell cultures. In intact muscle the ClC-1 protein was seen in the sarcolemma, but after myofiber isolation the protein was located intracellularly. Sarcolemmal localization was restored when myofibers were electrically stimulated or treated with a protein kinase C inhibitor. When mutated ClC-1 proteins were examined in a myofiber cell culture system, retardation in the ER was observed with the two missense mutations. The nonsense mutation did not have an effect on the transport from the ER to the Golgi elements, but the mutated ClC-1 was degraded more rapidly than the wild type ClC-1, at least in myotubes. Both retardation and degradation of the mutated ClC-1 are likely to result in too few channels present at the plasma membrane of the muscle cell to maintain normal physiological function. A very strict quality control in muscle cells was observed. The behavior and survival of multinuclear skeletal muscle cells is dependent on innervation and muscle activity, and the balance between the phosphorylation and dephosphorylation pathways modulates the function of muscle chloride channels. / Tiivistelmä Lihasspesifisen kloridikanavan ClC-1:n toiminnalliset virheet johtavat alentuneeseen kloridin johtumiseen solukalvon läpi ja lihassolun ylieksitoitumiseen. Tämän seurauksena lihaksen rentoutuminen vaikeutuu ja havaitaan myotoniaa, lihasjäykkyyttä. Pohjoissuomalaisesta potilasmateriaalista tautiin johtavia geenimutaatioita löytyi kolme erilaista. Poikkeuksellista havainnoissa on erilaisten mutaatioiden vähyys, mikä on tyypillistä suomalaiselle tautiperinnölle. Yhteensä tämän kloridikanavan mutaatioita on julkaistu yli 80 erilaista. Tutkiessamme normaalin ja mutatoidun ClC-1 lRNA:n ja proteiinin käyttäytymistä ja sijaintia lihassoluviljelmissä. Havaitsimme eron lihasleikkeiden ja eristettyjen myofiibereiden välillä. Lihasleikkeissä ClC-1 paikantui solun pinnalle sarkolemmalle, mutta eristetyissä myofiibereissä lähinnä solun sisälle. Stimuloimalla eristettyjä myofiibereitä sähkövirralla tai käsittelemällä proteiini kinaasi C inhibiittorilla, saimme kloridikanava-proteiinin siirtymään takaisin solun pinnalle. Proteiinitasolla kuljetuksessa on havaittavissa eroja. Aminohappomuutokseen johtavat pistemutaatiot aiheuttivat proteiinin jäämisen endoplasmiseen kalvostoon, kun taas ennenaikaisen stop-kodonin johdosta lyhentynyt proteiini kuljetetaan eteenpäin Golgin laitteeseen. Myotuubeissa tämä lyhentynyt proteiini kuitenkin hajotettiin nopeammin kuin normaali kloridikanavaproteiini. Sekä kuljetuksen hidastuminen että nopeampi hajotus johtavat tilanteeseen, jossa lihassolun solukalvolla on liian vähän kloridikanavia ylläpitämään lihaksen normaalia fysiologista toimintaa. Monitumaisten lihassolujen laaduntarkkailu havaittiin vielä monitahoisemmaksi kuin yksitumaisilla. Monitumainen lihassolu on riippuvainen hermoärsytyksestä ja lihasaktiivisuudesta. Lisäksi fosforylaatioon liittyvä signalointi on tärkeää ClC-1 proteiinin oikealle paikantumiselle lihassolussa.

Les canaux anioniques ont toujours été sous-estimés par rapport aux canaux cationiques principalement en raison des difficultés à caractériser leurs identités moléculaires et par l'absence d'inhibiteurs sélectifs puissants. C’est une classe comportant une grande hétérogénéité et une importante complexité comprenant entre autres le CFTR activé par l’AMP cyclique, les anoctamines activées par le calcium intracellulaire et les canaux VRAC activés lors d’un gonflement cellulaire. L’identité moléculaire d’une protéine essentielle aux courants ioniques déclenchés par le gonflement cellulaire a été découverte en 2014, permettant de pouvoir enfin investiguer le fonctionnement et le rôle de ces canaux. Dans une première partie, nous avons démontré l’absence de spécificité des inhibiteurs des principaux canaux chlorures. Cette étude met en exergue le besoin de développer de nouveaux inhibiteurs permettant de discriminer les différents canaux chlorures, mais aussi la nécessité de coupler l’approche pharmacologique à une approche génétique. Dans une deuxième partie, nous avons mis en évidence la perméabilité des canaux anioniques sensibles au gonflement cellulaire au glutathion, principal antioxydant cellulaire. Nos résultats suggèrent donc que les canaux VRAC pourraient être impliqués dans la régulation du stress oxydatif. Dans la troisième partie de ma thèse, nous avons montré que le facteur de croissance TGFβ1 stimule l’ouverture des canaux anioniques sensibles au gonflement cellulaire. Cette activation s’accompagne d’une perte de glutathion et d’une accumulation intracellulaire d’espèces réactives de l’oxygène. Cette dernière est à l’origine d’une transition épithélio-mésenchymateuse des cellules rénales. L’inhibition pharmacologique ainsi que la répression génétique des canaux VRAC réduit significativement les effets du TGFβ1. Enfin dans la dernière partie, nous avons caractérisé le rôle des canaux anioniques sensible au gonflement cellulaire dans la sécrétion de facteurs pro-inflammatoires par les macrophages stimulés par un choc hypotonique. En collaboration, nous avons montré que le blocage de la régulation du volume cellulaire, sous le contrôle de ces canaux, entraine une inhibition de l’activation de l’inflammasome et donc de la sécrétion des facteurs pro-inflammatoires. Bien que les mécanismes de signalisation cellulaire restent à déterminer, ces travaux mettent en lumière l’importance des canaux anioniques sensibles au gonflement cellulaire dans la transition épithéliomésenchymateuse et l’inflammasome. Il apparait que ces canaux pourraient être des cibles thérapeutiques prometteuses dans un certain nombre de pathologies et qu’il est primordial de poursuivre les investigations dans ce domaine. / Anion channels have always been underestimated compared to cationic channels mainly because of the difficulty in characterizing their molecular identities and the absence of potent selective inhibitors. It is a class with great heterogeneity and complexity, including CFTR activated by cAMP, anoctamines activated by intracellulaire calcium and VRAC channels activated by cell swelling. The molecular identity of an essential protein to the ionic currents triggered by cell swelling was discovered in 2014, allowing the scientist community to investigate the role of these channels. In a first part, we highlighted the lack of specificity of the inhibitors of different chloride channels. This study shed lights on the need to develop new inhibitors to discriminate the different chloride channels, but also the need to couple the pharmacological approach to a genetic approach. In a second part, we have demonstrated the glutathione permeability of Volume Regulated Anion Channels. Our results therefore suggest that VRAC channels might be involved in the regulation of oxidative stress as glutathione is the main antioxidant within the cell. In the third part of my thesis, we have shown that TGFβ1 growth factor stimulates the opening of Volume Regulated Anion Channels which is correlated with both a loss of glutathione and an intracellular accumulation of reactive oxygen species. The latter is at the origin of an epitheliomesenchymal transition of renal cells. The pharmacological inhibition as well as the genetic repression of the VRAC channels significantly reduces the effects of TGFβ1. Finally, in the last part, we characterized the role of Volume Regulated Anion Channels in the secretion of pro-inflammatory factors by macrophages stimulated by hypotonic shock. In collaboration, we have shown that blocking the regulation of cell volume, under the control of these channels, leads to an inhibition of the inflammasome and therefore of the secretion of pro-inflammatory factors. Although cell signaling mechanisms remain to be determined, this work highlights the importance of VRAC in the epithelial-mesenchymal transition and inflammasome. It appears that these channels could be promising therapeutic targets in a certain number of pathologies and that it is essential to continue the investigations in this field.

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ∼60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 μM calyculin A, or 1 μM N6-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl- channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

In this study, four anion transporter (AT) blockers, DIDS (4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid), 9-AC (anthracene-9-carboxylic acid), NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid), and IAA-94 (indanyloxy acetic acid) were selected to evaluate ATs as potential target sites for insect and nematode control. All the AT blockers showed slowly developing toxicity against second-stage larvae of <i>Meloidogyne incognita</i> (Kofoid and White 1919) Chitwood 1949 and adults of <i>Caenorhabditis elegans</i> Maupas 1900 but not against third-stage larvae of <i>Heterorhabditis bacteriophora</i> Poinar 1975 even at 200 ppm. Symptoms of AT blocker toxicity observed in <i>C. elegans</i> adults were increased pharyngeal muscle contractions and decreased locomotion. Exposure of <i>C. elegans</i> as fourth-stage larvae to double-stranded RNA (dsRNA) of <i>ceclc-1</i> and <i>ceclc-2</i> (VGCC genes coding for CeClC-1 and CeClC-2, respectively) either alone or together for 24 h decreased their expression in F1 progeny in a time-dependent manner. Reduction in expression of <i>ceclc-2</i> alone or together with <i>ceclc-1</i> significantly increased pharyngeal contractions and decreased locomotion in significantly higher percentage of F1 progeny. The above findings suggested AT blockers nematicidal activity primarily comes from inhibition of CeClC-2 channels, while inhibition of CeClC-1 channels may enhance this activity. All the AT blockers showed slowly developing toxicity against adults of a susceptible strain (Oregon-R) of <i>Drosophila melanogaster</i> Meigen 1830, while DIDS, was equally toxic to dieldrin-resistant rdl flies. All AT blockers, except 9-AC, at 100 µM showed significant excitatory effect on desheathed central nervous system (CNS) of third-instar larvae of <i>Drosophila</i>, while DIDS showed a modest excitatory effect on ascending peripheral nerves. Feeding adult flies on 10% sugar solution mixed with 100 ppm of DIDS for 6 h decreased the midgut pH by 2 units approximately. All the AT blockers inhibited the growth of larvae (in weight), increased the developmental time, and decreased survival when <i>Ostrinia nubilalis</i> (Hübner 1796) second-instar larvae were fed for seven days. All the AT blockers decreased the midgut alkalinity and inhibited chloride ion transport from midgut lumen into epithelia in fifth-instar larvae when fed for 3 h on treated diet. Positive correlations observed among growth, midgut alkalinity, and midgut chloride transport in AT blocker-fed larvae suggested that inhibition of chloride/bicarbonate exchangers by AT blockers may have contributed to midgut alkalinity decrease affecting the digestion and resulting in observed lethal and sublethal effects. / Ph. D.

Swelling-activated Cl- current (ICl,swell) is an outwardly rectifying Cl- current that influences cardiac electric activities and acts as a potential effector of mechanoelectrical feedback that antagonizes the effects of stretch-activated cation channels. Persistent activation of ICl,swell has been observed in multiple models of cardiovascular diseases. Previously we showed that angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are involved in the activation of ICl, swell by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ETA receptor blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell and the relationship between AngII and ET-1 signaling. Under isosmotic conditions, ET-1 elicited an outwardly rectifying Cl- current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB and by osmotic shrinkage. Selective ETA blockade (BQ123), but not ETB blockade (BQ788), fully suppressed the ET-1-induced current. ET-1-induced ICl,swell was abolished by blockade of EGFR kinase (AG1478) and PI-3K inhibitors (LY294002 and wortmannin), which also suppress beta1-integrin stretch- and swelling-induced ICl,swell. ET-1-induced ICl,swell was abrogated by ebselen, a membrane-permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O, suggesting that ROS were required intermediates in ET-1-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin or mitochondrial complex I with rotenone both completely suppressed ET-1-induced ROS generation and activation of ICl,swell, indicating that ROS from both NOX and mitochondria were required to activate ICl,swell, and complete block by inhibitors of either ROS source suggests mitochondrial and NOX must act in series rather than in parallel. ICl,swell elicited by antimycin A, which stimulates superoxide production by mitochondrial complex III, was insensitive to NOX inhibitor apocynin and the NOX fusion peptide inhibitor gp91ds-tat. Activation of ICl,swell induced by diazoxide, which stimulates mitochondrial ROS production by opening mitochondrial KATP channels, was not affected by gp91ds-tat. These data suggests that mitochondrial ROS is downstream from NOX in the regulation of ICl,swell. Mitochondrial ROS production that is enhanced by NOX ROS is likely to be responsible for the activation of ICl,swell by ET-1. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors (PD 98059 and U0216) on the activation of ICl,swell elicited by ET-1, EGF, and H2O2. ERK inhibitors partially blocked ET-1-induced ICl,swell but fully inhibited activation of ICl,swell in response to EGF. However, ERK inhibitors did not affect ICl,swell elicited by exogenous H2O2. We also established the the relationship of ET-1 to AngII and osmotic swelling in the regulation of ET-1 ICl,swell. ETA blockade abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade did not effect ET-1-induced ICl,swell, suggesting that ET-1 signaling is downstream from AngII and osmotic swelling. HL-1 cell is a murine atrial cell line that retain phenotypic characteristics of adult cardiomyocytes. We showed that osmotic swelling and ET-1 turned on DCPIB-sensitive outwardly rectifying Cl- current in HL-1 cells with both physiological and symmetrical Cl- gradients. The swelling-induced current was suppressed by gp91ds-tat and rotenone but insensitive to apocynin. Blockade of ETA receptor (BQ123) and NOX (gp91ds-tat) completely inhibited ET-1-induced ICl,swell in HL-1 cells. These data indicate that ICl,swell is present in HL-1 cell and regulated by similar mechanisms as in native cells. Finally, we confirmed the production of ROS by ET-1 signaling by flow cytometry of HL-1 cells using the nominally H2O2-selective fluorescent probe C H2DCFDA-AM. Exposure to ET-1 increased ROS production, as did H2O2, a positive control. ET-1-induced ROS production was fully suppressed by both gp91ds-tat and rotenone. HL-1 cell ROS production also was stimulated by the mitochondrial complex III inhibitor antimycin A, and antimycin A-induced ROS production was blocked by rotenone but not by gp91ds-tat. These data suggest that ET-1 ETA receptor signaling elicits ICl,swell by sequentially stimulating ROS production by NOX and mitochondria. ETA receptor signaling is down stream from AngII in the osmotic swelling-induced activation of ICl,swell and is upstream from EGFR kinase and PI-3K. Endothelin signaling is likely to be an important means of activating ROS production and ICl,swell in a variety of cardiovascular diseases.