La formation de métastases est la cause majeure des décès par cancer. Le développement de métastases est consécutif à une série d‟événements complexes tels que la migration, l‟invasion et la prolifération cellulaire. Le canal potassique SK3 (membre de la famille des SKCa) régule la migration des cellules cancéreuses du sein et favorise le développement de métastases osseuses. Le but du projet était d‟identifier et de caractériser les voies d‟entrées calciques associées à la migration cellulaire dépendante du canal SK3 dans différents cancers (sein, colon et prostate). Nous avons pu mettre en évidence que les canaux Ca2+qui étaient associés au canal SK3 variaient en fonction du cancer et régulaient la migration cellulaire dépendante du canal SK3. De plus, nous avons montré que la localisation de ces complexes KCa/Ca2+ dans les radeaux lipidiques était importante pour leur régulation et leur fonction. Ainsi, la délocalisation de ces complexes hors des radeaux lipidiques par des alkyl-phospholipides est un moyen permettant de moduler la migration des cellules exprimant le canal SK3 et le développement de métastases. / In most cases of cancer, metastasis and not the primary tumor per se is the main cause of mortality. To establish secondary growth in distant organs cancer cells must develop an enhanced propensity to migrate. The key objective of this thesis proposes that some actors of Ca2+ signaling (Orai, and TRPC, STIM) coupled to SK3 channel would form complexes that play a critical role in cell migration of various cancers (breast, colon and prostate). Furthermore we showed that the localization of these channels complexes in lipid-rafts is essential to their regulation and function. Thus, the delocalization of these complexes of lipid-raft outside by alkyl-phospholipids could be a new way to modulate the SK3/Ca2+ dependent cell migration and metastasis development.

Calcium ions (Ca2+) are involved in almost all neuronal functions, providing the link between electrical signals and cellular activity. This work examines the mechanisms by which a neuron can regulate the movement and sequestration of Ca2+ through specific channels such that this ubiquitous ion can encode specific functions. My initial focus was using intracellular calcium ([Ca2+]i) imaging techniques to study the influence of the inhibition of specific voltage gated calcium channels (VGCC) by ethanol on a depolarization induced rise in [Ca2+]i in neurohypophysial nerve terminals. This research took an unexpected turn when I observed an elevation of [Ca2+]i during perfusion with ethanol containing solutions. Control experiments showed this to be an artifactual result not directly attributable to ethanol. It was necessary to track down the source of this artifact in order to proceed with future ethanol experiments. The source of the artifact turned out to be a contaminant leaching from I.V. drip chambers. Due to potential health implications stemming from the use of these drip chambers in a clinical setting as well as potential artifactual results in the ethanol field where these chambers are commonly used, I choose to investigate this phenomenon more rigorously. The agent responsible for this effect was shown to be di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer that has been shown to be carcinogenic in rats and mice. The extraction of this contaminant from the I.V. drip chamber, as measured by spectrophotometry, was time-dependent, and was markedly accelerated by the presence of ethanol in the solution. DEHP added to saline solution caused a rise in [Ca2+]i similar to that elicited by the contaminant containing solution. The rise in calcium required transmembrane flux through membrane channels. Blood levels of DEHP in clinical settings have been shown to exceed the levels which we found to alter [Ca2+]i. This suggests that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics. As part of a collaboration between Steven Treistman and Robert Messing's laboratory at UCSF, I participated in a study of how ethanol regulates N-type calcium channels which are known to be inhibited acutely, and upregulated in the chronic presence of ethanol. Specific mRNA splice variants encoding N-type channels were investigated using ribonuclease protection assays and real-time PCR. Three pairs of N-type specific α-subunit Cav2.2 splice variants were examined, with exposure to ethanol observed to increase expression of one alternative splice form in a linker that lacks six bases encoding the amino acids glutamate and threonine (ΔET). Whole cell electrophysiological recordings that I carried out demonstrated a faster rate of channel activation and a shift in the voltage dependence of activation to more negative potentials after chronic alcohol exposure, consistent with increased expression of ΔET variants. These results demonstrate that chronic ethanol exposure not only increases the abundance of N-type calcium channels, but also increases the expression of a Cav2.2 splice variant with kinetics predicted to support a larger and faster rising intracellular calcium signal. This is the first demonstration that ethanol can up-regulate ion channel function through expression of a specific mRNA splice variant, defining a new mechanism underlying the development of drug addiction. Depolarizing a neuron opens voltage gated Ca2+ channels (VGCC), leading to an influx of Ca2+ ions into the cytoplasm, where Ca2+ sensitive signaling cascades are stimulated. How does the ubiquitous calcium ion selectively modulate a large array of neuronal functions? Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca2+ current and the resulting rise and decay of [Ca2+]i, showing that equal amounts of Ca2+ entering through N-type and L-type voltage gated Ca2+ channels result in significantly different [Ca2+]i temporal profiles. When the contribution of N-type channels was reduced, a faster [Ca2+]i decay was observed. Conversely, when the contribution of L-type channels was reduced, [Ca2+]i decay was slower. Potentiating L-type current or inactivating N-type channels both resulted in a more rapid decay of [Ca2+]i. Channel-specific differences in [Ca2+]i decay rates were abolished by depleting intracellular Ca2+ stores suggesting the involvement of Ca2+-induced Ca2+ release (CICR). I was able to conclude that Ca2+ entering through N-type, but not L-type channels, is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR generates a unique intracellular Ca2+ signal depending on the route of entry, potentially encoding the selective activation of a subset of Ca2+ -sensitive processes within the neuron.

Orientador: Everardo Magalhães Carneiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T13:23:02Z (GMT). No. of bitstreams: 1 Trevisan_Amon_M.pdf: 3520740 bytes, checksum: 90ddd0dc662586a0279a2c1e1f000b3b (MD5) Previous issue date: 2010 / Resumo: Os canais de cálcio voltagem-dependentes (CaV) são proteínas de membrana plasmática que conduzem cálcio, e são ativados pela despolarização da mesma promovendo influxo do íon, que serve como um segundo mensageiro intracelular, transformando sinais elétricos em químicos. Esse processo controla diversos eventos intracelulares, como exocitose, endocitose, contração muscular, transmissão sináptica e metabolismo. Em células beta (B), a secreção de insulina estimulada por glicose e/ou leucina ocorre por diversos sinais que levam à despolarização da membrana e influxo de cálcio, que age no processo de acoplamento estímulo/secreção dos grânulos contendo insulina. É possível a existência de efeitos sinérgicos entre o metabolismo da leucina e glicose, permitindo um controle fino da expressão gênica, produção e secreção de insulina em células B. Recentemente demonstramos que animais que sofreram um processo de restrição protéica modificam o mecanismo de secreção de insulina alterando a resposta secretória para diferentes secretagogos (glicose, aminoácidos, etc.). Nesse trabalho, buscamos elucidar o envolvimento dos íons cálcio nos processos de secreção de insulina, bem como avaliar a expressão gênica e protéica das subunidades alfa1 e beta2 do canal nos diferentes grupos estudados. Observamos uma redução na expressão gênica das duas subunidades do CaV em ilhotas de animais desnutridos e uma tendência de recuperação na expressão protéica da subunidade ?1 em animais desnutridos suplementados com leucina. As áreas abaixo das curvas (AUC) de cálcio das ilhotas não apresentaram diferença entre os grupos quando estimulados com alta (16,7 mM) glicose e 40 mM de K+, porém, há um aumento na área abaixo da curva quando estimuladas com tolbutamida no grupo desnutrido suplementado com leucina (LPL). As secreções de insulina frente a inibidores de canal de cálcio apresentaram resultados similares frente a alta glicose e tolbutamida, porém apresentando valores absolutos menores. Ilhotas provenientes de animais LPL apresentaram recuperação da taxa de liberação de insulina quando estimuladas com ácido ketoisocapróico, atingindo valores similares aos controles. Essa recuperação não foi observada quando estimulamos as ilhotas com cloreto de potássio. Observamos também uma redução na área das ilhotas de animais desnutridos, com recuperação a valores equivalentes aos controles quando há suplementação com leucina. Nossos dados sugerem que o manejo dos íons cálcio possa não estar diretamente envolvido na melhora da secreção de insulina por ilhotas de animais desnutridos suplementados com leucina, mesmo havendo uma melhora na expressão protéica da subunidade alpha1 do Cav em ilhotas de animais LPL, através de uma possível modulação pós-transcricional. / Abstract: Voltage-dependent calcium channels (CaV) are plasma membrane proteins that lead calcium, and are activated by depolarization of the same by promoting the influx of this ion, which serves as an intracellular second messenger, turning electrical signals into chemical. This process controls several intracellular events such as exocytosis, endocytosis, muscle contraction, synaptic transmission and metabolism. In beta cells (B), insulin secretion stimulated by glucose and/or leucine occurs by several signs that lead to membrane depolarization and calcium influx, which acts on the coupling process stimulus/secretion of granules containing insulin. It is possible that there are synergistic effects between the metabolism of glucose and leucine, allowing fine control of gene expression, production and insulin secretion in B cells. We have shown that animals which have undergone a process of protein restriction alter the mechanism of insulin secretion by altering the secretory response to different secretagogues (glucose, amino acids, etc.). In this paper we elucidate the involvement of calcium ions in the process of insulin secretion, as well assess the gene and protein expression of alpha1 and beta2 subunits of the channel in different groups. Observed a decrease in gene expression of two subunits of CaV in islets of malnourished animals and a recovery trend in protein expression of ?1 subunit in malnourished animals supplemented with leucine. The areas under the curve (AUC) for calcium of islets did not differ between groups when stimulated with high (16.7 mM) glucose and 40 mM K +, however, there is an increase in area under the curve when stimulated with tolbutamide in undernourished group supplemented with leucine (LPL). The secretions of insulin compared to calcium channel inhibitors showed similar results compared to high glucose and tolbutadima, although a smaller absolute values. Islets from LPL animals showed recovery of the rate of insulin release when stimulated with ketoisocaproic acid (KIC), reaching values similar to controls. This recovery was not observed when we stimulate the islets with potassium chloride. We also observed a reduction in the area of the islets of malnourished animals, with recovery to similar values for controls when supplementation with leucine. Our data suggest that the management of calcium ions can not be directly involved the improvement of insulin secretion by islets of malnourished animals supplemented with leucine, even with an improvement in protein expression of the alpha1 subunit of the Cav in islets of animals LPL, through a possible post-transcriptional modulation. / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientadores: Antonio Carlos Boschiero, Luiz Fernando de Rezende / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:18:46Z (GMT). No. of bitstreams: 1 Santos_GustavoJorge_D.pdf: 3129731 bytes, checksum: b00bd77f6b06be14f135a76b6977ca47 (MD5) Previous issue date: 2014 / Resumo: Introdução: A Cálcio-Calmodulina quinase II (CaMKII) atua tanto na regulação da secreção de insulina com de neurotransmissores pela mesma via de sinalização. Além disso, a CaMKII é conhecida por ser a "molécula da memória", pois sua atividade é fundamental em sua formação. Portanto, hipotetizamos que células ß pancreática tem a capacidade de adquirir e estocar informações contidas em pulsos de cálcio, formando uma memória metabólica. Métodos: Para comprovar nossa hipótese, desenvolvemos um novo paradigma de exposição de células ? a pulsos de 30 mM de glicose, seguido de uma período de consolidação (24 hrs) para excluir qualquer efeito agudo do metabolismo da glicose. Após esse período analizamos a secreção de insulina (RIA), expressão proteica (Western blot), a resposta secretória frente a uma "rampa de glicose" e o Ca2+ citoplasmático induzido por glicose. Resultados: Células ß expostas a pulsos de glicose (30 mM) mostraram maior secreção de insulina estimulada por glucose, evidenciando a memória metabólica a qual foi totalmente dependente a CaMKII. Esse fenômeno foi refletido na expressão proteica de proteínas importantes na sinalização do cálcio e na secreção de insulina. Além disso, células expostas ao regime de pulsos de glucose apresentaram maior expressão do MAFA, um fator de transcrição chave para a função da célula ß. Conclusão: Em suma, assim como neurônios, células ß tumorais (MIN6), ilhotas de camundongos e de humanos são capazes de adquirir, estocar e evocar informações / Abstract: Backgroun: Ca2+/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Memory is the ability to acquire, to store and to evocate any kind of information. In CNS, the process behind this phenomenon in the Long-Term Potentiation (LTP) and is known that it requires Ca2+ to occur. In additional, CaMKII is necessary to store information during LTP. In pancreatic ß-cells, CaMKII plays pivotal role during GSIS process. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in Ca2+ pulses as a form of "metabolic memory", just as neurons store cognitive information. Methods: To test this hypothesis, we developed a novel paradigm of pulsed exposure of mice and human ß-cells to intervals of high glucose, followed by a 24-hour consolidation period to eliminate any acute metabolic effects. After this period, we analyzed insulin secretion (by RIA), protein expression (by Western blot), response to a glucose-ramp and the glucose-induced Ca2+ influx. Results: Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. We also observed, in pulse group, an increase in Ca2+ influx induced by glucose. In additional, metabolic memory was reflected on the protein level by increased expression of proteins involved in GSIS and Ca2+-dependent vesicle secretion, such as GCK, Cav1.2, SNAP25, pCaMKII and pSynapsin. Finally, we observed in human islet elevated levels of the key ß cell transcription factor MAFA. Discussion: Based on or findings we conclude that pancreatic ß cells, either from mice or humans, have the ability to acquire, store and retrieve information. This process is CaMKII-dependent and is due to modifications in the glucose-sensing machinery of the cell, since we observed an increase in GSIS and Ca2+ influx together with an increase in several proteins involved in this process. Our findings suggests that MAFA is the key effector in this memory, since (a) it is a potent activator of insulin gene, (b)is activated by CaMKII and (c) its expression is increased even 24 hours after the last pulse. Conclusion: In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Abstract This study utilized normal and defective epithelial cell cultures and epidermal skin samples to examine intra- and intercellular calcium signaling. The main interests of this thesis were Hailey-Hailey disease (HHD), Darier disease (DD), neurofibromatosis 1 (NF1) and transitional cell carcinoma (TCC). HHD and DD diseases are rare autosomal dominant skin disorders characterized by dissociation of epidermal keratinocytes (acantholysis) at the suprabasal layer of the epidermis. HHD and DD diseases are caused by mutations in the genes encoding the calcium pumps in the Golgi apparatus (hSPCA1) and endoplasmic reticulum (SERCA2b), respectively. Due to these mutations calcium uptake into the Golgi apparatus or ER is diminished, which is believed to cause abnormal cell junction protein processing and dissociation of keratinocytes. This study utilized electron probe microanalysis (EPMA) and demonstrated for the first time that lesional areas of HHD and DD and non-lesional areas of DD epidermis display abnormally low calcium content in the basal cell layer. Furthermore, ATP mediated calcium signaling was impaired in cultured HHD and DD keratinocytes and epidermal ATP receptor localization was disrupted. In conclusion, these results suggest that the low calcium content in the basal cell layer is the reason for suprabasal ruptures in HHD but not necessarily in DD lesions, and that abnormal ATP receptor localization contributes to the calcium signaling defects. NF1 deficient keratinocytes display abnormally low resting cytosolic calcium levels and it has been suggested that the calcium concentration in the lumen of the endoplasmic reticulum is decreased. This study demonstrated that NF1 keratinocytes rely mostly on ATP mediated calcium signaling while normal keratinocytes rely mostly on gap junctional intercellular communication (GJIC). Studies with TCC cells have demonstrated that gap junctions participate in intercellular calcium wave propagation. This thesis demonstrated that the ATP mediated pathway was also operational in calcium wave propagation in normal uroepithelial and TCC cell cultures. Furthermore, impaired calcium wave propagation in the TCC cell culture could be improved through PKC α/βI –isoenzyme inhibition with Gö6976. Gö6976 treatment increased connexin 26 clustering at plasma membrane but did not alter expression level of the protein. This thesis contains a wide repertoire of calcium detection techniques including a new cutting-edge technology for elemental calcium detection of epidermal samples. These techniques can be used for molecular specific analysis of calcium signaling in epithelial cells.

Les glioblastomes sont des tumeurs au pronostic défavorable. L’échec des thérapies est lié à la présence de cellules souches cancéreuses (CSCs), résistantes aux traitements ; la caractérisation de ces cellules et l’identification de biomarqueurs sont donc primordiales. Le calcium contrôle de nombreux processus cellulaires ; parmi les éléments majeurs de la signalisation calcique, la Calmoduline (CaM) est impliquée dans différentes pathologies, dont des cancers, et est un puissant régulateur de l’état physiologique d’une cellule. CaM interagit avec de nombreuses protéines impliquées dans la régulation de l’homéostasie calcique de la cellule. Nous avons cherché à identifier et caractériser des antagonistes de CaM, inhibant différentiellement ces interactions. L’utilisation de ces antagonistes en tant que perturbateurs de l’homéostasie calcique a permis de mettre en évidence un marqueur caractérisé des CSCs de glioblastomes. D’autre part, l’étude comparée du protéome membranaire de CSCs issues de glioblastomes a permis de mettre en évidence la surexpression de clusters de différenciation et protéines impliquées dans la signalisation calcique. Ces protéines sont de potentiels marqueurs moléculaires des CSCs de glioblastome. / Glioblastomas are malignant tumor of poor prognosis. Therapeutic failure might be supported by cancer stem cells (CSCs); characterization of these cells and biomarkers identification are of most importance. Calcium controls numerous cellular process; beyond major elements of calcium signaling, Calmodulin (CaM) is involved in different pathologies and tumors, and is a powerful regulator of cell physiological state. CaM interacts with a plethora of proteins involved in cell calcium homeostasis regulation. We aimed at identifying and characterizing CaM antagonists, capable of differentially inhibiting these interactions. The use of these antagonists in calcium homeostasis disturbance led to the identification of a characterized marker of glioblastomas CSCs. In another approach, the comparative study of glioblastomas CSCs membrane proteome uncovered the overexpression of differentiation clusters and proteins involved in calcium signaling. These proteins are potential molecular biomarkers for glioblastomes CSCs.

Muitos subtipos de receptores são ativados pelo mesmo ligante, mas estão acoplados a diferentes mensageiros secundários podendo produzir sinalização divergente em uma célula, enquanto receptores ativados por diferentes ligantes, mas que compartilham o mesmo mensageiro secundário, podem produzir sinalização convergente. Para examinar as bases mecanísticas que influenciam a proliferação e a diferenciação celular determinamos as funções de liberação intracelular de Ca2+ e a excitabilidade celular mediada pelos receptores purinérgicos e colinérgicos utilizando imageamento de cálcio por microscopia confocal. Para tanto, caracterizamos a participação dos subtipos P2X1-7 e P2Y1,2,4,6 de receptores purinérgicos aos níveis dos transcritos de mRNA e de expressão protéica, assim como pela atividade de induzir os transientes de [Ca2+]i, aumento na concentração livre de cálcio intracelular, durante a diferenciação neuronal de células P19 de carcinoma embrionário, que foram utilizadas como modelo in vitro para o desenvolvimento neuronal precoce. Em células embriônicas os receptores P2Y1,2, P2X4 ou os heteromultímeros de P2X com farmacologia semelhante ao do receptor P2X4 foram os responsáveis pelos transientes de [Ca2+]i induzidos pelo ATP e seus análogos. Ao término da diferenciação neuronal, os receptores P2Y2,6 e P2X2 foram os principais mediadores das respostas de [Ca2+]i. Obtivemos evidências do envolvimento destes receptores na indução da proliferação tanto de células embriônicas como de progenitores neuronais, por ensaios de incorporação de BrdU, e da indução da diferenciação neuronal das células progenitoras, na presença de vários agonistas e antagonistas de receptores purinérgicos. Como resultado desses estudos, a regulação da proliferação e diferenciação celular foi principalmente devida aos subtipos de receptores P2Y1 e P2Y2, já que estes efeitos foram eliminados após a depleção dos depósitos intracelulares de cálcio e pela demonstração de que estes eram os possíveis receptores funcionais. Entre os receptores colinérgicos, fornecemos evidências para a expressão de receptores nicotínicos (nAChRs) e muscarínicos (mAChRs) funcionais durante a diferenciação de células P19. Detectamos a expressão e a atividade dos subtipos de nAChRs formados pelos subtipos α2-α7, β2, β4 e M1-M3 e M5 de mAChRs durante a diferenciação neuronal. As respostas de [Ca2+]i induzidas pelos agonistas dos nAChRs foram observadas em células P19 embriônicas e neuronais. As respostas de [Ca2+]i mediadas pelos receptores muscarínicos, em níveis próximos aos basais em células embriônicas, aumentaram durante a diferenciação. As elevações na [Ca2+]i induzidas pelos nAChRs em células indiferenciadas foram devidas ao influxo de Ca2+ do meio extracelular. Em células diferenciadas em neurônios, os aumentos de transientes de [Ca2+]i induzidos pelos nAChRs foram parcialmente inibidas após o pré-tratamento das células com a rianodina, enquanto as respostas de [Ca2+]i mediadas pelos mAChR não foram afetadas na presença deste composto, sugerindo uma contribuição da liberação de Ca2+ a partir dos depósitos de Ca2+ sensíveis à rianodina para as elevações mediadas pelos nAChRs. Demonstramos também, que a nicotina, agindo através dos nAChRs, inibiu a proliferação em células embriônicas, porém, a induziu em células progenitoras neuronais pela mobilização de Ca2+ dos depósitos intracelulares. A muscarina induziu em células embriônicas o aumento na proliferação via mAChRs acoplados às proteínas Gαq/11, e promoveu a diferenciação neuronal via M2 mAChRs em células precursoras neuronais. Estes dados sugeriram que a acetilcolina agindo via mAChR funciona como um mitógeno que ativa as proteínas quinases de trifosfato de inositol (IP3) e que poderia estar envolvida na síntese de DNA durante os estágios iniciais da neurogênese. Nós ainda provemos evidências que as oscilações de [Ca2+]i são características para cada estágio da diferenciação e são iniciadas pela liberação de Ca2+ mediada pelo IP3. As análises da determinação do fenótipo neuronal na presença de vários inibidores da transdução do sinal induzido pelo cálcio residem na liberação de Ca2+ induzida pelo IP3 é necessária para o progresso da diferenciação neuronal. Assim, os sinais espontâneos de [Ca2+]i são propriedades intrínsecas das células em diferenciação. A modulação de sua freqüência e amplitudes especifica a aquisição de um fenótipo de célula neuronal. / Various receptors subtypes are activated by the same ligand although coupled to different second messengers. These receptors act either by inducing divergent signaling in one cell, whereas in another cell different receptors may stimulate the very same pathways producing convergent signaling. We have characterized intracellular Ca2+- release and -influx mediated by purinergic and cholinergic receptors using calcium imaging by confocal microscopy to evaluate the mechanistic bases which influence cell proliferation and differentiation We have characterized the participation of purinergic subtypes P2X1-7 and P2Y1,2,4,6 receptor subtypes at mRNA transcription and protein expression levels as well as receptor-induced changes in free intracellular calcium concentration ([Ca2+]i) during differentiation of P19 embryonal carcinoma cells as an in vitro model for early neuronal development. The participation of individual P2X and P2Y receptor subtypes in the differentiation process was studied by employing different available purinergic receptor agonists and antagonists. In embryonic cells, P2Y1,2, P2X4 receptors, or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP-analog-induced [Ca2+]i transients. Following completion of neuronal differentiation, P2Y2,6 receptors and P2X2 subtypes were the major mediators of the [Ca2+]i-response. Regulation of cell proliferation and differentiation of P19 embryonic and progenitor cells was mostly due to P2Y1 and P2Y2 receptor activation, as these effects were abolished following depletion of intracellular calcium stores, and they are probably the unique functional P2Y receptors at these stages of differentiation. We also provide evidence for expression of functional nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) during neuronal differentiation of P19 cells. We have detected expression and activity of nAChRs formed by the subunits α2-α7, β2, β4, and M1-M3 and M5 mAChR subtypes along the differentiation process. Receptor response in terms of nicotinic agonist-evoked Ca2+ flux was observed in embryonic and neuronal-differentiated cells. However, mAChRs-induced calcium responses, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced [Ca2+]i response in undifferentiated cells was due to Ca2+ influx. However, in differentiated P19 neurons the nAChR-induced [Ca2+]i response was partially inhibited following pretreatment of the cells with ryanodine, while the mAChR-induced response remained unaffected, suggesting the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. In the present study we have also shown that nicotine, acting via nAChRs, inhibited proliferation in embryonic cells, but induced cell division of progenitor cells by Ca2+ mobilization from internal stores. Stimulation of progenitor cells by muscarine led to an increase in DNA synthesis mainly resulting from activation of Gαq/11-coupled mAChRs. Muscarine as well promoted differentiation of neural precursor cells by activation of M2 mAChRs subtypes. These data suggest that acetylcholine, acting via mAChRs, functions as a mitogen during early neurogenesis. We also provide evidence that oscillations of [Ca2+]i as characteristics for the respective stage of differentiation are initiated by triphosphate inositol (IP3)-mediated Ca2+-release. Neuronal cell fate determination analysis in the presence of various inhibitors of calcium-induced signal transduction underlined that IP3-mediated Ca2+-release is necessary for neuronal differentiation progress. Thus, spontaneous Ca2+-signals are an intrinsic property of differentiating neural precursor cells. Modulation of their frequency and amplitude is believed to direct the acquisition of a defined neuronal phenotype.

L'élévation de la concentration du calcium (Ca2+) cytosolique est responsable de l’activation plaquettaire. Cette élévation est due à l'entrée du Ca2+ à partir du milieu extérieur (influx) ou sa translocation (mobilisation) dans le cytosol depuis ses réserves internes. Les SERCAs (Sarco/Endoplasmic Reticulum Ca2+ ATPases) pompent le Ca2+ depuis le cytosol vers les réserves internes, maintenant le Ca2+ cytosolique bas (100 nM) et les plaquettes au repos. D'autre part elles assurent une concentration calcique élevée (≥1 mM) dans les réserves calciques permettant sa mobilisation, et enfin modulent l'intensité et la forme du signal calcique lors de l'activation. Mais les rôles respectifs des SERCAs plaquettaires, SERCA2b et SERCA3, sont encore mal définis. D’où l’intérêt de mon projet qui a été de déterminer si SERCA3 avait un rôle fonctionnel précis et spécifique. Nous avons observé sur des souris SERCA3-/- un défaut de l'hémostase et s’accompagne d’une résistance à la thrombose dû à un déficit de sécrétion d'ADP, entrainant un défaut d'agrégation et d'adhérence. SERCA3 semble contrôler une voie de sécrétion initiale d'ADP capable d’agir en synergie avec une faible activation plaquettaire, aboutissant à un renforcement de la sécrétion et de l'agrégation. De plus, l’utilisation des inhibiteurs pharmacologiques spécifiques de SERCA2b (thapsigargine) ou SERCA3 (tBHQ), a montré que la sécrétion initiale d'ADP n'était pas dépendante de la mobilisation des réserves SERCA2b mais dépendait spécifiquement des réserves SERCA3. Nous avons retrouvé la même voie de sécrétion d'ADP dépendante de SERCA3 dans les plaquettes humaines. Nous avons en particulier montré par le suivi d'une cohorte de patientes atteintes d'obésité morbide, un déficit d'agrégation, une faible mobilisation calcique et un taux faible de SERCA3 plaquettaire, revenus à la normale après retour à un poids normal après chirurgie bariatrique. Surtout nous avons retrouvé, dans les plaquettes de ces patientes obèses, un défaut de sécrétion d'ADP associé au défaut de SERCA3. Il s'agit du premier défaut de SERCA3 plaquettaire lié à une pathologie humaine. Nous avons ensuite montré que la sécrétion initiale d'ADP était rapide (5 sec) et entièrement dépendante de SERCA3. A l'aide d'une sonde calcique fluorescente membranaire (FURA2-NearMem-AM), nous avons démontré l’existence d’une mobilisation calcique juxta-membranaire spécifique de SERCA3, indépendante de l'ADP, correspondant donc à une sécrétion primaire. Cette mobilisation SERCA3 s'est avérée indépendante d'IP3, mais dépendante du NAADP, qui mobilise spécifiquement les réserves SERCA3 et non SERCA2b. En conclusion, nous avons mis en évidence une nouvelle voie d'activation plaquettaire, indépendante de l'IP3 mais dépendante du NAADP qui libère le Ca2+ des stocks internes dépendants de SERCA3 et spécifiquement engagés dans la libération précoce d'ADP lors de l'activation plaquettaire. Ces données identifient de nouvelles cibles avec un intérêt thérapeutiques anti-thrombotiques potentiel. / The elevation of cytosolic calcium (Ca2 +) is responsible for platelet activation. This elevation is due to the entry of Ca2 + from the extracellular medium (influx) where its translocation (mobilization) into the cytosol from its Ca2+ stores. SERCAs (Sarco / EndoplasmicReticulumCa2 + ATPases) pump Ca2 + from the cytosol to the Ca2+ stores, maintaining low cytosolic Ca2 + (100 nM) and platelets at resting state. On the other hand, they ensure a high calcium concentration (≥ 1 mM) in the Ca2+ stores allowing its mobilization, and finally modulate the intensity and the shape of the Ca2 signal during the activation. However, the respective roles of SERCA platelets, SERCA2b and SERCA3, are still poorly defined. Hence the interest of my project which was to determine if SERCA3 had a precise and specific functional role. We observed in SERCA3 - / - mice a defect in hemostasis that is accompanied by resistance to thrombosis due to ADP secretion deficiency, resulting in a lack of aggregation and adhesion. SERCA3 seems to control an initial pathway of ADP secretion able to acting in synergy with low platelet activation, resulting in increased secretion and aggregation. In addition, the use of specific pharmacological inhibitors of SERCA2b (thapsigargine) or SERCA3 (tBHQ) showed that the initial secretion of ADP was not dependent on the mobilization of SERCA2b stores, but was specifically dependent on SERCA3 stores. We found the same SERCA3-dependent ADP secretion pathway in human platelets. In particular, we observed a defect of platelet aggregation, low Ca2+ mobilization and low platelet SERCA3 levels in a cohort of patients with morbid obesity compared to control subjects. Platelet functions and SERCA3 levels are restored after weight loss by a surgery bariatric. Above all, we found in the platelets of these obese patients, a defect of secretion of ADP associated with the defect of SERCA3. This is the first defect of platelet SERCA3 related to human pathology. We then showed that the initial secretion of ADP was rapid (5 sec) and entirely dependent on SERCA3. Using a membrane fluorescent Ca2+ probe (FURA2-NearMem-AM), we have demonstrated the existence of a juxta-membrane-specific calcium mobilization specific to SERCA3, independently of ADP, corresponding to primary secretion. This SERCA3 mobilization proved to be independent of IP3, but dependent on the NAADP, which specifically mobilizes the SERCA3 and not SERCA2b reserves. In conclusion, we have demonstrated a new platelet activation pathway, independent of IP3 but dependent on NAADP, which releases Ca2 + from SERCA3 dependent stores and specifically involved in the early release of ADP during platelet activation. These data identify new targets with a potential interest of anti-thrombotic therapy.

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