Parathyroid hormone (PTH) secretion maintains free-ionised extracellular calcium (Ca2+o) homeostasis under the control of the calcium-sensing receptor (CaR). In humans and dogs, blood acidosis and alkalosis is associated with increased or suppressed PTH secretion respectively. Furthermore, large (1.0 pH unit) changes in extracellular pH (pHo) alter Ca2+o sensitivity of the CaR in CaR-transfected HEK-293 cells (CaR-HEK). Indeed, it has been found in this laboratory that even pathophysiological acidosis (pH 7.2) renders CaR less sensitive to Ca2+o while pathophysiological alkalosis (pH 7.6) increases its Ca2+o sensitivity, both in CaR-HEK and parathyroid cells. If true in vivo, then CaR’s pHo sensitivity might represent a mechanistic link between metabolic acidosis and hyperparathyroidism in ageing and renal disease. However, in acidosis one might speculate that the additional H+ could displace Ca2+ bound to plasma albumin, thus increasing free-Ca2+ concentration and so compensating for the decreased CaR responsiveness. Therefore, I first demonstrated that a physiologically-relevant concentration of albumin (5% w/v) failed to overcome the inhibitory effect of pH 7.2 or stimulatory effect of pH 7.6 on CaR-induced intracellular Ca2+ (Ca2+i) mobilisation. Determining the molecular basis of CaR pHo sensitivity would help explain cationic activation of CaR and permit the generation of experimental CaR models that specifically lack pHo sensitivity. With extracellular histidine and free cysteine residues the most likely candidates for pHo sensing (given their sidechains’ pK values), all 17 such CaR residues were mutated to non-ionisable residues. However, none of the resulting CaR mutants exhibited significantly decreased CaR pHo sensitivity. Even co-mutation of the two residues whose individual mutation appeared to elicit modest reductions (CaRH429V and CaRH495V) failed to exhibit any change in CaR pHo sensitivity. I conclude therefore, that neither extracellular histidine nor free cysteine residues account for CaR pHo sensitivity. Next, it is known that cytosolic cAMP drives PTH secretion in vivo and that cAMP potentiates Ca2+o-induced Ca2+i mobilisation in CaR-HEK cells. Given the physiological importance of tightly controlled PTH secretion and Ca2+o homeostasis, here I investigated the influence of cAMP on CaR signalling in CaR-HEK cells. Agents that increase cytosolic cAMP levels such as forskolin and isoproterenol potentiated Ca2+o-induced Ca2+i mobilisation and lowered the Ca2+o threshold for Ca2+i mobilisation. Indeed, forskolin lowered the EC50 for Ca2+o on CaR (2.3 ± 0.1 vs. 3.0 ± 0.1 mM control, P<0.001). Forskolin also potentiated CaR-induced ERK phosphorylation; however protein kinase A activation appeared uninvolved in any of these effects. Pertussis toxin, used to block CaR-induced suppression of cAMP accumulation, also lowered the Ca2+o threshold for Ca2+i mobilisation though appeared to do so by increasing efficacy (Emax). Furthermore, mutation of the CaR’s two putative PKA consensus sequences (CaRS899 and CaRS900) to a non-phosphorylatable residue (alanine) failed to alter the potency of Ca2+o for CaR or attenuate the forskolin response. In contrast, phosphomimetic mutation of CaRS899 (to aspartate) did increase CaR sensitivity to Ca2+o. Together this suggests that PKA-mediated CaRS899 phosphorylation could potentiate CaR activity but that this does not occur following Ca2+o treatment in CaR-HEK cells. Together, these data show that cAMP regulates the Ca2+o threshold for Ca2+i mobilisation, thus helping to explain differential efficacy between CaR downstream signals. If true in vivo, this could help explain how multiple physiological signal inputs may be integrated in parathyroid cells.

No description available.

Our previous work in cultured cells has shown that the maintenance of mitochondrial Ca2+ homeostasis is essential for cell survival, and that the anti-apoptotic protein Bcl-2 is able to maintain a threshold level of mitochondrial Ca2+ by the inhibition of permeability transition. To test whether Bcl-2 also affects the mitochondrial Na+-Ca2+ exchange (NCE), a major efflux pathway for mitochondrial Ca2+, studies using transgenic mice that overexpress Bcl-2 in the heart have been performed. NCE activity was determined as the Na+-dependent Ca2+ efflux in the isolated mitochondria. Overexpression of Bcl-2 led to a significant reduction of NCE activity as well as increased resistance to permeability transition in the mitochondria of transgenic heart. This was accompanied by increased matrix Ca2+ level, enhanced formation of NADH and enhanced oxidation of pyruvate, an NAD+-linked substrate. Furthermore, there was induction of cellular Ca2+ transport proteins including the Na+-Ca2+ exchanger of the sarcolemma (NCX). Bcl-2 not only stimulates NCX expression in the sarcolemma but also attenuates the Na+-Ca2+ exchange in the mitochondria. These results are consistent with the protection by Bcl-2 against apoptosis in heart following ischemia/reperfusion.

No description available.

A complex bio-mechanism, referred to as calcium homeostasis, regulates plasma ionized calcium (Ca++) concentration in the human body to within a narrow physiologic range which is crucial for maintaining normal physiology and metabolism. Various metabolic disorders and pathologic conditions originate from acute and/or chronic disturbances/disorders in calcium homeostatic system. This system relies on numerous sub-systems which operate in different time-scales ranging from minutes to weeks. In this thesis we focus on a particular sub-system that operates on the time-scale of minutes; the dynamics involves the response of the parathyroid glands to acute changes in plasma Ca++ concentration. We develop a two-pool, linear time-varying model describing the dynamics of the sub-system. We show that this model can predict dynamics observed in clinical tests of induced hypo- and hyper-calcemia in normal humans. In addition, we develop a new protocol for the construction of a Ca-PTH reverse sigmoid curve based on the mathematical model. This protocol removes deficiencies in current protocols in that the resulting curve is invariant with respect to the subject's axis dynamics and calcium clamp test dynamics.

No description available.

O Ca+2 tem sido relatado como um íon de extrema importância celular. Sua concentração intracelular alterada está relacionada com: (i) a ativação de proteínas importantes, dentre elas as de reparação e de apoptose, (ii) as doenças humanas de grande relevância sócio-econômica e (iii) o aumento dos danos celulares. Sabendo-se que a sua concentração intracelular é responsável pela manutenção da viabilidade celular e frente à importância já conhecida deste íon, esta dissertação teve por objetivo, primeiramente, fazer uma busca sobre os dados existentes na literatura para delinear quais são os possíveis mecanismos moleculares pelo qual o Ca+2 afeta os mecanismos de reparação e apoptose. Esta busca norteou posteriores testes e pesquisas que culminaram nos resultados obtidos e apresentados neste trabalho. Para a realização de alguns testes foi desenvolvida uma nova metodologia, o ensaio Poissoner Quantitative Drop Test (PQDT), a qual permite a análise de sobrevivência de diferentes linhagens de Saccharomyces cerevisiae deficientes ou proficientes em determinadas proteínas, frente à exposição crônica a um agente genotóxico. Esta técnica também permite observar efeitos citostáticos e citotóxicos nas células testadas. Além disso, realizou-se uma análise de Biologia de Sistemas das proteínas de S. cerevisiae responsáveis pela homeostase de Ca+2 e encontrou-se uma correlação direta destas com proteínas responsáveis pela reparação de DNA por meio da via de excisão de nucleotídeos ou NER. Uma vez obtidos estes dados, as linhagens de leveduras deficientes nas proteínas tanto de homeostase de Ca+2, Pmr1p, Cod1p, Por1p, como para o gene RAD4 foram construídas. Além dos simples mutantes, foram construídas linhagens duplos mutantes para ambas as vias. Assim, foram realizados ensaios de citotoxicidade, citostaticidade e de parada do ciclo celular frente à exposição ao agente genotóxico 4-NQO. Conforme os resultados obtidos, a homeostase de Ca+2 exerce influência sobre os mecanismos de reparação de DNA, principalmente nas proteínas da via NER, como a Rad4p. As proteínas Pmr1p e Cod1p são as proteínas de homeostase de Ca+2 mais importantes na resposta ao dano celular. Estes resultados propõem que o estresse de retículo endoplasmático e a fosforilação de proteínas de parada de ciclo celular foram os principais processos metabólicos evidenciados nas linhagens de leveduras testadas após a exposição ao agente genotóxico 4-NQO. Os resultados da análise de Biologia de Sistemas e análise ontológica dos genes mostram que existem muitas vias bioquímicas relacionadas com a homeostase de Ca+2. Isso demonstra a importância destes achados e predispõem muitos outros testes para a elucidação completa destas influências. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-05-22T16:41:02Z No. of bitstreams: 1 Dissertacao Nadine Paese Poletto.pdf: 2514929 bytes, checksum: e52f9e75c1db5e89a4fbaf0d3b14215f (MD5) / Made available in DSpace on 2014-05-22T16:41:02Z (GMT). No. of bitstreams: 1 Dissertacao Nadine Paese Poletto.pdf: 2514929 bytes, checksum: e52f9e75c1db5e89a4fbaf0d3b14215f (MD5) / Calcium (Ca2+) has been reported as an important cellular ion. The changes in the intracellular Ca2+ concentrations are related to: (i) the activation of major proteins related to DNA repair and apoptosis; (ii) induction of human diseases; and (iii) increase in cellular damages. Considering that the Ca2+ intracellular concentration is also responsible for the maintenance of cellular viability, the aim of this study was to proceed with a literature data mining to outline the major Ca2+ molecular mechanisms that can affect the cellular activities. This data mining was necessary to define further biological assays described in this work. In this sense, it was developed a new methodology called PDQT, which allows to evaluate the survival of different Saccharomyces cerevisiae strains deficient or proficient for determined proteins in a chronic exposure conditions in the presence of a genotoxic agent. This new method also allows to observe the citotoxic, citostatic, and cell cycle arrests effects induced by a genotoxic agent. Moreover, it was performed a Systems Biology analyses of the major S. cerevisiae Ca2+ homeostasis-associated proteins and it was found a direct association with proteins of the nucleotide excision repair (NER) pathway. The data gathered by Systems Biology analyses were used to construct yeast strains single and double mutants deficient for Ca2+ homeostasisassociated proteins, like Pmr1p, Cod1p, Por1p, and for the NER-associated protein Rad4p. The yeast strains obtained were challenged against the genotoxic agent 4-NQO and citotoxic, citostatic, and cell cycle arrest assays were performed. Taking into account the results, it was observed that Ca2+ homeostasis mechanisms affect the DNA repair pathways, specially the NER proteins like Rad4p. In this sense, both Cod1p and Pmr1p are the most important Ca2+ homeostasis proteins for the damage cellular response. In addition, these results propose that ER stress and the phosphorylation of cell cycle arrest-associated proteins can be the major metabolic processes after 4-NQO exposure. The results obtained by System Biology and gene ontology analyses proved that many biochemical pathways are related to Ca2+ homeostasis, which demonstrate the importance of the results obtained in this work and lead to other studies.

O Ca+2 tem sido relatado como um íon de extrema importância celular. Sua concentração intracelular alterada está relacionada com: (i) a ativação de proteínas importantes, dentre elas as de reparação e de apoptose, (ii) as doenças humanas de grande relevância sócio-econômica e (iii) o aumento dos danos celulares. Sabendo-se que a sua concentração intracelular é responsável pela manutenção da viabilidade celular e frente à importância já conhecida deste íon, esta dissertação teve por objetivo, primeiramente, fazer uma busca sobre os dados existentes na literatura para delinear quais são os possíveis mecanismos moleculares pelo qual o Ca+2 afeta os mecanismos de reparação e apoptose. Esta busca norteou posteriores testes e pesquisas que culminaram nos resultados obtidos e apresentados neste trabalho. Para a realização de alguns testes foi desenvolvida uma nova metodologia, o ensaio Poissoner Quantitative Drop Test (PQDT), a qual permite a análise de sobrevivência de diferentes linhagens de Saccharomyces cerevisiae deficientes ou proficientes em determinadas proteínas, frente à exposição crônica a um agente genotóxico. Esta técnica também permite observar efeitos citostáticos e citotóxicos nas células testadas. Além disso, realizou-se uma análise de Biologia de Sistemas das proteínas de S. cerevisiae responsáveis pela homeostase de Ca+2 e encontrou-se uma correlação direta destas com proteínas responsáveis pela reparação de DNA por meio da via de excisão de nucleotídeos ou NER. Uma vez obtidos estes dados, as linhagens de leveduras deficientes nas proteínas tanto de homeostase de Ca+2, Pmr1p, Cod1p, Por1p, como para o gene RAD4 foram construídas. Além dos simples mutantes, foram construídas linhagens duplos mutantes para ambas as vias. Assim, foram realizados ensaios de citotoxicidade, citostaticidade e de parada do ciclo celular frente à exposição ao agente genotóxico 4-NQO. Conforme os resultados obtidos, a homeostase de Ca+2 exerce influência sobre os mecanismos de reparação de DNA, principalmente nas proteínas da via NER, como a Rad4p. As proteínas Pmr1p e Cod1p são as proteínas de homeostase de Ca+2 mais importantes na resposta ao dano celular. Estes resultados propõem que o estresse de retículo endoplasmático e a fosforilação de proteínas de parada de ciclo celular foram os principais processos metabólicos evidenciados nas linhagens de leveduras testadas após a exposição ao agente genotóxico 4-NQO. Os resultados da análise de Biologia de Sistemas e análise ontológica dos genes mostram que existem muitas vias bioquímicas relacionadas com a homeostase de Ca+2. Isso demonstra a importância destes achados e predispõem muitos outros testes para a elucidação completa destas influências. / Calcium (Ca2+) has been reported as an important cellular ion. The changes in the intracellular Ca2+ concentrations are related to: (i) the activation of major proteins related to DNA repair and apoptosis; (ii) induction of human diseases; and (iii) increase in cellular damages. Considering that the Ca2+ intracellular concentration is also responsible for the maintenance of cellular viability, the aim of this study was to proceed with a literature data mining to outline the major Ca2+ molecular mechanisms that can affect the cellular activities. This data mining was necessary to define further biological assays described in this work. In this sense, it was developed a new methodology called PDQT, which allows to evaluate the survival of different Saccharomyces cerevisiae strains deficient or proficient for determined proteins in a chronic exposure conditions in the presence of a genotoxic agent. This new method also allows to observe the citotoxic, citostatic, and cell cycle arrests effects induced by a genotoxic agent. Moreover, it was performed a Systems Biology analyses of the major S. cerevisiae Ca2+ homeostasis-associated proteins and it was found a direct association with proteins of the nucleotide excision repair (NER) pathway. The data gathered by Systems Biology analyses were used to construct yeast strains single and double mutants deficient for Ca2+ homeostasisassociated proteins, like Pmr1p, Cod1p, Por1p, and for the NER-associated protein Rad4p. The yeast strains obtained were challenged against the genotoxic agent 4-NQO and citotoxic, citostatic, and cell cycle arrest assays were performed. Taking into account the results, it was observed that Ca2+ homeostasis mechanisms affect the DNA repair pathways, specially the NER proteins like Rad4p. In this sense, both Cod1p and Pmr1p are the most important Ca2+ homeostasis proteins for the damage cellular response. In addition, these results propose that ER stress and the phosphorylation of cell cycle arrest-associated proteins can be the major metabolic processes after 4-NQO exposure. The results obtained by System Biology and gene ontology analyses proved that many biochemical pathways are related to Ca2+ homeostasis, which demonstrate the importance of the results obtained in this work and lead to other studies.

La Leucémie Myéloïde Chronique (LMC) est une maladie clonale caractérisée par la présence du chromosome Philadelphie codant pour Bcr-Abl, une tyrosine kinase constitutivement active responsable de la leucémogenèse. Bien que très efficaces, les inhibiteurs de tyrosine kinase (ITKs) restent cependant inactifs sur les cellules souches leucémiques. Ce travail de thèse montre que la signalisation calcique, connue pour réguler de nombreux processus dans les cellules saines et cancéreuses, est importante dans la signalisation cellulaire au décours de la LMC. Le rôle des entrées calciques dépendantes des stocks (SOCEs) médiées par STIM1 (STromal Interaction Molecule 1) et les canaux Orai1 et TRPC1 ainsi que des entrées calciques induites par la thrombine a été étudié dans la leucémogenèse. Nous avons observé une diminution de ces entrées dans les cellules exprimant Bcr-Abl pouvant être expliquée par le changement de stœchiométrie Orai1/STIM1. Ceci entraîne la diminution de l'activation de NFAT (Nuclear Factor of Activated T-cells) ainsi que des conséquences sur la prolifération et la migration cellulaire mais pas sur l'apoptose. De plus, les SOCEs sont restaurées dans les cellules cancéreuses après traitement à l'Imatinib, le principal ITK. Nous proposons alors que l'expression de Bcr-Abl joue un rôle sur l'homéostasie calcique en entraînant une dérégulation générale des fonctions cellulaires dans les cellules leucémiques notamment via la voie PKC (Protein Kinase C). Ainsi, ces résultats montrent une dérégulation des entrées calciques dans les cellules exprimant Bcr-Abl, suggérant que la signalisation calcique puisse être une cible thérapeutique en parallèle avec les ITKs. / Chronic Myeloid Leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia chromosome encoding for Bcr-Abl, a constitutively active tyrosine kinase responsible for leukemogenesis. Although Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the therapy of Ph+ leukemia, the complete eradication of CML is limited by the emergence of resistance in hematopoietic stem cells. This thesis proposes that calcium (Ca2+) signaling pathways, known to govern a large number of functions in normal and cancer cells, may be important in CML cell signaling. Therefore, we studied the role of Store Operated-Calcium entry (SOCE) (i.e. STromal Interaction Molecule 1 (STIM1), Orai1 and TRPC1 channels) and thrombin induced Ca2+ entry in leukemogenesis. We found a decrease in both calcium entries in Bcr-Abl-expressing cells compared to normal cells. The reduced SOCE seems related to a change in stoichiometry of Orai1/STIM1. This leads to a reduction of the Nuclear Factor of Activated T-cells (NFAT) translocation and functional consequences on cell proliferation and migration but not on apoptosis. Moreover, we showed that SOCE is restored in malignant cells after treatment with Imatinib, the main TKI. We proposed that Bcr-Abl expression could impact on Ca2+ homeostasis enhancing a general disorganization of cell functions in leukemia cells notably via Protein Kinase C (PKC) pathway. Altogether this work shows a deregulation of Ca2+ entry in Bcr-Abl-expressing cells, suggesting that the Ca2+ signaling pathway could be a therapeutic target in parallel with TKIs.

No description available.