Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel implicated as a key regulator of neuronal cell death in stroke. Our research group has previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation was also shown to decrease downstream of TRPM7 activation during anoxia. In the present study we investigated the effects of TRPM7 activation during anoxia, on three regulators of LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced TRPM7activity and cofilin hyperactivation.

TRPM7 (Transient Receptor Potential Melastatin-like 7) is an ion channel necessary for the proper development of many cell types. Insight into the precise role of the channel in different cells has been hampered by the lethality of knocking out the gene in model organisms such as the mouse. Here I examine a zebrafish that has a loss-of-function mutation in the gene encoding Trpm7. First, I show that trpm7 is important for the function of developing dopaminergic neurons in the zebrafish. Second, I examine the interaction between trpm7 and the related gene vmat2 in order to develop a cellular mechanism of trpm7 function in presynaptic dopaminergic neurons. Finally, I investigate the necessity of the kinase and ion channel domains of trpm7 in their ability to promote pigmentation in melanophores as a model cell type. Based on the results from these experiments and observations from other researchers, I form a new hypothesis for Trpm7 function in protein sorting. These studies provide a detailed and novel analysis of the function of an ion channel that is necessary for life.

No description available.

La mucoviscidose est la maladie héréditaire autosomique récessive, rare, létale, la plus fréquente dans la population caucasienne. Cette maladie est causée par des mutations du gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) codant la protéine CFTR. Cette protéine est principalement un canal chlorure (Cl-) AMPc-dépendant localisé dans la membrane apicale des cellules épithéliales. La mutation F508del entraîne un défaut de maturation de la protéine qui est retenue dans le réticulum endoplasmique avant d’être dégradée. Cependant, une faible quantité de protéines malformées échappe à ce système de contrôle et parvient à la membrane plasmique.Des travaux de notre équipe ont montré une augmentation des efflux ioniques dépendants du CFTR dans des lignées cellulaires épithéliales bronchiques (CFBE41o-), exprimant le CFTR sauvage ou le CFTR muté F508del, après un traitement par une hormone: la gonadolibérine (GnRH, Gonadotropin releasing hormone, 1h, 10-9M). Cette augmentation est vraisemblablement due à un nombre plus important de canaux CFTR à la membrane plasmique.L’objectif de cette thèse a été de tester un analogue de la GnRH comme modulateur de l’exportation membranaire et/ou de l’activité canal du CFTR muté, sur des cultures primaires de cellules épithéliales nasales humaines homozygotes pour la mutation F508del. Dans un premier temps, nous avons vérifié la présence du récepteur à la GnRH (R-GnRH) dans notre modèle cellulaire. Puis, nous avons étudié l’effet de l’analogue sur la fonction du CFTR par des techniques d’électrophysiologie. Nous avons observé une augmentation des efflux d’ions Cl- médiés par le canal CFTR après un traitement à l’analogue (2h, 10-12M). Enfin, une étude protéomique nous a permis d’identifier des protéines différentiellement exprimées après traitement. Certaines protéines mises en évidence pourraient appartenir à des voies de signalisation intracellulaires ayant un rôle dans la régulation de la protéine CFTR et être des cibles thérapeutiques.Par ailleurs, le canal CFTR est régulé par le Mg2+ intracellulaire ([Mg2+]i). Le canal TRPM7 est le principal régulateur du [Mg2+]i. La [Mg2+]i a été mesurée et l’expression de TRPM7 vérifiée dans des cellules Hela transfectées avec le CFTR sauvage (wt) ou mutés (G551D et F508del). Nous avons étudié la localisation, la fonction et la régulation de TRPM7 dans nos modèles cellulaires avant de rechercher un possible lien fonctionnel entre le CFTR et TRPM7. Dans les modèles CF, l’expression, la fonction et la localisation du canal TRPM7 sont altérées. Il existerait un lien fonctionnel entre TRPM7 et le CFTR par l’intermédiaire de la diminution du [Mg2+]i impliquant TRPM7 dans la physiopathologie de la mucoviscidose. / Cystic fibrosis is the most common lethal autosomal recessive disease in the Caucasian population. This disease is caused by mutations in the gene encoding the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) protein. This protein is a cAMP-regulated chloride channel expressed at the apical membrane of epithelial cells. The F508del mutation causes a defect in CFTR protein folding preventing its maturation. Some misfolded proteins escape the control system and reach the plasma membrane.We previously showed a rise of CFTR-dependent ion efflux in wt- and F508del-CFTR human bronchial epithelial expressing cells (CFBE41o-) after incubation with GnRH (Gonadotropin releasing hormone; 1h, 10-9M). This increase was probably due to an increased cell surface expression of CFTR.The aim of the present study was to test a GnRH analog as a modulator of CFTR delivery to the plasma membrane and/or activity of CFTR on primary culture of human nasal epithelial cells (F508del/F508del).We checked the GnRH receptor expression in our model. Then, we studied the GnRH effect on CFTR’s function by electrophysiology. We found a significant increase of CFTR dependent chloride efflux in cells pretreated with analogue (2h, 10-12M). Proteomic study enabled us to identify differentially expressed proteins after treatment. Some highlighted proteins could be part of signalling pathways regulating CFTR and could be therapeutic targets.Moreover, CFTR is regulated by intracellular Mg2+ ([Mg2+]i). TRPM7 (Transient Receptor Potential Melastatin 7) is the main channel regulating [Mg2+]i. [Mg2+]i and TRPM7 expression were measured in Hela cells stably expressing wildtype and two CFTR mutants (F508del-CFTR and G551D-CFTR). We studied TRPM7 expression, function and regulation in our cell models before examining a functional link between TRPM7 and CFTR. In CF models, TRPM7 expression, localization and function are altered. There should be a functional link between TRPM7 and CFTR through the decreased [Mg2+]i involving TRPM7 in cystic fibrosis physiopathology.

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as global endocrine and physiological effectors. In recent years the zebrafish has been utilized as a favorable animal model as a step towards dissecting and better understanding these postembryonic developmental processes. One of the more powerful methods utilized in zebrafish has been the identification of new gene functions through the use of mutant screens. The nutria mutant was recovered from one such screen to identify postembryonic defects in pigment pattern, growth and metamorphosis. These mutants exhibited a pigment cell defect, touch unresponsiveness and severe growth retardation. Here I will discuss my work towards dissecting the underlying developmental processes governing the phenotypic changes in nutria mutants. I characterize gross alterations in skeletal development in nutria mutants that lead to accelerated endochondral ossification but delayed intramembranous ossification. I show that the nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and a kinase. I find trpm7 expression in the fish-specific, ion homeostasis-regulating gland known as the corpuscles of Stannius (CS), and in the mesonephric kidney. I show that mutants also develop kidney stones. Together these results suggest a role for trpm7 activity in regulation of physiological ion homeostasis. Next I confirm that role by identifying late-embryonic and early larval defects in the CS and the kidney, two organs that regulate physiological ion homeostasis. I demonstrate the early larval detection of kidney stones in trpm7 mutants and show that their appearance is presaged by decreased levels of total calcium and magnesium. Furthermore I establish a link between trpm7 function in the CS and stanniocalcin1 (stc1), a potent molecular regulator of calcium homeostasis. Finally, using transgenic overexpression and morpholino-oligonucleotide knockdown, I demonstrate that stc1 modulates calcium and magnesium levels in trpm7 mutant and wild-type backgrounds. Together these analyses establish postembryonic roles for trpm7 function in growth, skeletogenesis, kidney function, and physiological ion homeostasis. / text

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2016-10-18T14:55:15Z No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / Made available in DSpace on 2016-10-18T14:55:15Z (GMT). No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / O carvacrol, um monoterpeno fenólico encontrado nos óleos essenciais de diversas plantas do gênero Origanum, já demonstrou causar hipotensão e vasodilatação em diferentes leitos vasculares de ratos normotensos, porém, seu efeito em ratos hipertensos ainda não foi elucidado. O objetivo deste estudo foi investigar os efeitos cardiovasculares do carvacrol em ratos espontaneamente hipertensos (SHR) e comparar com normotensos Wistar, utilizando ensaios farmacológicos in vitro (estudos funcionais e celulares) e in vivo. Nos ensaios funcionais in vitro, anéis de artéria mesentérica superior isolada de animais hipertensos e normotensos foram précontraídos com FEN (1μM) e o efeito de carvacrol (10-8-10-3M) foi observado. Em SHR, este monoterpeno induziu vasodilatação dependente de concentração (pD2=5,13 ± 0,05; Emáx=115,14 ± 5,46%; N=8) e, após a remoção do endotélio funcional, a potência da droga foi alterada significantemente (pD2=4,91 ± 0,05 N=9; p<0,01), sugerindo que a resposta vasodilatadora induzida por carvacrol, provavelmente, envolve uma via dependente e outra independente do endotélio vascular, porém, esta última parece ser a majoritária e, por isso, os ensaios seguintes foram realizados na ausência do endotélio vascular. Interessantemente, quando comparada com animais normotensos, a potência farmacológica de carvacrol foi reduzida significantemente (pD2=4,91 ± 0,05; N=9; p<0,05). Em anéis de ratos hipertensos, carvacrol reduziu o influxo de Ca2+ por canais Cav tipo-L, SOC e ROC, estes resultados foram semelhantes aos obtidos em ratos normotensos. Em ratos hipertensos, mas não em normotensos, a potência farmacológica do carvacrol em anéis pré-contraídos com FEN e na presença de diferentes inibidores de canais TRP (íon Gd3+, 10-5M; 2-APB, 10-6M ou 10-5M; BCTC, 2μM; 9-fenantrol, 10-5M; ou HC03003-1, 10-5M), foi reduzida em relação ao controle na ausência destes bloqueadores, sugerindo que os canais sensíveis à estes bloqueadores (TRPC1-7, TRPM2, M4 e TRPM8, TRPV1 e TRPA1), provavelmente, estão participando dos efeitos vasculares mediados por carvacrol e podem estar envolvidos no processo hipertensivo. Em estudos de patch-clamp em células de artéria mesentérica dispersas de ratos hipertensos, carvacrol (300μM) reduziu as correntes de entrada de Ba2+ por Cav tipo-L e este efeito foi semelhante em ratos normotensos. Além disso, em células de ratos hipertensos, o Mg2+ (2,5mM), bloqueador do TRPM6 e TRPM7, reduziu as densidades de ITRPM de entrada e saída, assim como carvacrol (100μM e 300μM), na ausência ou presença do 2-APB (100μM), bloqueador de TRPM7. A presença do 2-APB provocou inibição adicional nas densidades de ITRPM pelo carvacrol (100μM, mas não 300μM). Altas concentrações intracelulares de Mg2+ reduziram the magnitude of ITRPM7. Foi evidenciado que a ITRPM no controle é menor em ratos hipertensos que em normotensos. Estes dados obtidos e os relatados na literatura são sugestivos para provável inibição de ITRPM7 por carvacrol em células mesentéricas nativas. O efeito anti-hipertensivo do carvacrol foi avaliado por administração via orogástrica (50mg/kg/dia) durante 20 dias foi capaz de reduzir a pressão arterial média dos animais SHR tratados, no 20º dia do tratamento. O tratamento subcrônico com carvacrol não alterou os pesos cardíaco e corpóreo, nem a reatividade vascular. Em conclusão, esses dados sugerem que carvacrol possui atividade anti-hipertensiva em animais SHR, que pode ser devido ao seu efeito vasodilatador em anéis de artéria mesentérica superior isolada, provavelmente, por inibição do influxo de Ca2+ por Cav tipo-L, ROC, SOC e/ou canais TRPC1, 3 ou 6, além da inibição de correntes tipo-TRPM7 em miócitos mesentéricos.

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A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum na população, sendo seus mecanismos estudados em modelos animais. Há evidências de que alguns sistemas de comunicação celular podem modular o desenvolvimento da DP. Os canais de potencial transiente (TRPs) e receptores purinérgicos parecem envolvidos com a neurodegeneração e podem contribuir no desenvolvimento da DP, por outro lado, as cininas parecem estar relacionadas com a neuroproteção. O objetivo deste estudo foi avaliar a expressão e o envolvimento destes receptores de membrana no modelo da DP induzida por 6-OHDA em camundongos (C57Bl/6) injetados com agonista (BK) e antagonista (HOE-140) do receptor B2, e antagonista de TRPM7 (Carvacrol); e em nocautes do receptores B2, pelas técnicas de imuno-histoquímica e Western blotting. Os dados revelaram modulação destes receptores no modelo, e neuroproteção após o bloqueio do TRPM7. Assim, podemos sugerir que todos os receptores avaliados podem estar envolvidos na indução do modelo por 6-OHDA, sendo possíveis alvos terapêuticos para a DP. / Parkinson\'s disease (PD) is a second most common neurodegenerative disease in the population, and the mechanisms involved in these are studied in animal models. There is evidence that some systems can modulate the development of PD. Transient potential channels (TRPs) and purinergic receptors seem to be involved in neurodegeneration and may contribute to the development of PD; on the other hand, kininas appear to be related to neuroprotection. The aim of this study was to evaluate the expression and the involvement of these membrane receptors in the 6-OHDA-induced PD model in mice (C57Bl / 6) injected with agonist (BK) and antagonist (HOE-140) of B2 receptor; antagonist of TRPM7 (Carvacrol); and in B2 knockout knockouts, by immunohistochemistry and Western blotting techniques. The data revealed modulation of these receptors in the model, and neuroprotection after TRPM7 blockade. Thus, we can suggest that all the receptors evaluated may be involved in the induction of the 6-OHDA model, and can be possible therapeutic targets for PD.