Tesi: "NCX3" – Grafiati

1

Sokolow, Sophie. "Les souris déficientes pour les échangeurs sodium-calcium (NCX1 et NCX3): deux modèles murins pour l'étude de leurs rôles pysiologiques in vivo ;Implication de NCX3 dans la fonction neuromusculaire". Doctoral thesis, Universite Libre de Bruxelles, 2004. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211196.

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Nous avons généré des souris déficientes pour les gènes codant pour les échangeurs Na/Ca de type I (NCX1) et de type III (NCX3) afin d'étudier, in vivo, le rôle de ces deux protéines.

L‘analyse phénotypique des souris adultes totalement déficientes pour le gène Ncx1 (Ncx1-/-) n'a pu être menée étant donné que ces souris décèdent au cours du développement embryonnaire.

Les souris déficientes pour le gène Ncx3 (Ncx3-/-) sont viables et fertiles. Nous avons analysé l'effet de l'inactivation du gène Ncx3 dans le muscle squelettique et plus particulièrement au niveau de la jonction neuromusculaire.

L'analyse histologique des muscles squelettiques de souris Ncx3-/- a révélé des altérations des fibres musculaires caractérisées par la présence de foyers de fibres nécrotiques et d'infiltrats de cellules mononuclées.

L'analyse électromyographique classique a montré un électromyogramme anormal du muscle gastrocnémien de souris Ncx3-/-, révélant une affection neuromusculaire pré- et post-synaptique caractérisée par (i) la petitesse de l'amplitude de la réponse M au repos, (ii) le décrément après stimulation répétitive à basse fréquence, (iii) l'incrément après stimulation répétitive à haute fréquence et (iv) la facilitation post-exercice. L'électromyographie à fibre unique a révélé une MCD élevée et des blocages anormaux de la transmission neuromusculaire, reflétant une atteinte post-synaptique de la jonction neuromusculaire chez les souris Ncx3-/-. L'ensemble de ces anomalies électromyographiques sont les caractéristiques du syndrome myasthénique de Lambert-Eaton.

Finalement, pour déterminer les conséquences de l'inactivation du gène Ncx3 sur l'activité physique des souris Ncx3-/-, nous avons réalisé des tests comportementaux sur ces souris. Ces tests ont permis de détecter un épuisement et une faiblesse musculaire accrus à l'effort chez ces souris.

En conclusion, nos observations montrent que les souris Ncx3-/- présentent des anomalies électromyographiques similaires à celles du syndrome myasthénique de Lambert-Eaton. Ces résultats suggèrent que l'échangeur NCX3 est peut-être impliqué dans la pathogenèse de certaines formes de cette maladie.

Des études supplémentaires afin de confirmer notre hypothèse devront donc être réalisées.

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We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein which mediates cellular Ca2+ efflux (forward mode) or Ca2+ influx (reverse mode) and thus controls intracellular Ca2+ concentration. NCX3-deficient mice (Ncx3-/-) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities including low compound muscle action potential amplitude, a decremental response at low frequency nerve stimulation, an incremental response and a prominent post-exercise facilitation at high frequency nerve stimulation as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3-/- neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high frequency nerve stimulation. It is suggested that Ca2+ entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca2+ and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca2+ concentrations in the skeletal muscle fibers and at the neuromuscular junction.

Doctorat en sciences biomédicales
info:eu-repo/semantics/nonPublished

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2

Jeffs, Graham J. "The effect of sodium/calcium exchanger 3 (NCX3) knockout on neuronal survival following global cerebral ischaemia in mice". University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2007. http://theses.library.uwa.edu.au/adt-WU2008.0063.

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Cerebral ischaemia is a leading cause of disability and death world-wide. The only effective treatments are thrombolytic therapy (plasminogen activator; tPA) and hypothermia (33?C). However, tPA has limited clinical application due to its short therapeutic time window and its specific application in thrombo-embolic stroke. Moderate hypothermia (33?C) is only being used following cardiac arrest in comatose survivors. Hence more treatments are urgently required. The first step in developing new treatments is the identification and characterisation of a potential therapeutic target. Since brain damage following cerebral ischaemia is associated with disturbances in intracellular calcium homeostasis, the sodium-calcium exchanger (NCX) is a potential therapeutic target due to its ability to regulate intracellular calcium. Currently, however there is uncertainty as to whether the plasma membrane NCX has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue I compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3-/-) and wild-type mice (Ncx3+/+) following global cerebral ischaemia. In order to perform this study I first established a bilateral common carotid occlusion (BCCAO) model of global ischaemia in wild-type C57/BlHsnD mice using controlled ventilation. After trials of several ischaemic time points, 17 minutes was established as the optimum duration of ischaemia to produce selective hippocampal CA1 neuronal loss in the wild-type mice. I then subjected NCX3 knockout and wild-type mice to 17 minutes of ischaemia. Following the 17 minute period of ischaemia, wild-type mice exhibited 80% CA1 neuronal loss and 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed > 95% CA1 neuronal loss and 95% CA2 neuronal loss. Following experiments using a 17 minute duration of global ischaemia, a 15 minute duration of ischaemia was also evaluated. Wild-type mice exposed to a 15 minute period of ischaemia, did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed 45% CA1 neuronal loss and 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient for the NCX3 protein are more susceptible to global cerebral ischaemia than wild-type mice. My findings showing a neuroprotective role for NCX3 following ischaemia, suggest that the exchanger has a positive role in maintaining neuronal intracellular calcium homeostasis. When this function is disrupted, neurons are more susceptible to calcium deregulation, with resultant cell death via calcium mediated pathways. Therefore, improving NCX activity following cerebral ischaemia may provide a therapeutic strategy to reduce neuronal death.

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3

Bauer, Klemens Verfasser], e Christian [Akademischer Betreuer] [Pott. "Einfluss des Natrium-Kalzium-Austauscher (NCX1)-Antagonisten SEA0400 auf Kalzium-Handling und Arrhythmogenese in Abhängigkeit vom NCX1-Expressionsniveau / Klemens Bauer ; Betreuer: Christian Pott". Münster : Universitäts- und Landesbibliothek Münster, 2016. http://d-nb.info/1141577461/34.

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4

Sieber, Matthias. "Modulatoren des Calcineurin-NFATc-Signalweges in humanen TH-Zellen". Phd thesis, Universität Potsdam, 2010. http://opus.kobv.de/ubp/volltexte/2010/4467/.

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Die Ca2+/Calmodulin-aktivierte Serin/Threonin-Phosphatase Calcineurin ist ein Schlüsselmolekül des T-Zell-Rezeptorabhängigen Signalnetzwerkes. Calcineurin aktiviert die Transkriptionsfaktoren der NFATc-Familie durch Dephosphorylierung und reguliert darüber die Expression wichtiger Zytokine und Oberflächenproteine. Die Aktivität von Calcineurin wird durch zahlreiche endogene Proteine moduliert und ist Angriffspunkt der immunsuppressiven Substanzen Cyclosporin A und FK506. In dieser Arbeit wurde der alternative niedermolekulare Calcineurin-NFATc-Inhibitor NCI3 hinsichtlich seiner Effekte auf T-Zell-Rezeptor-abhängige Signalwege charakterisiert. Die Ergebnisse zeigen, daß das Pyrazolopyrimidinderivat NCI3 nichttoxisch und zellmembranpermeabel ist. In T-Zell-Rezeptor-stimulierten primären humanen TH-Zellen unterdrückt NCI3 die Proliferation und IL-2-Produktion (IC50-Wert ~4 µM), da die Dephosphorylierung von NFATc und die anschließende nukleäre Translokation gehemmt wird. NCI3 inhibiert die calcineurinabhängige NFAT- und NF-κB-, aber nicht die AP-1-kontrollierte Reprtergenexpression, in mikromolaren Konzentrationen (IC50-Werte 2 bzw. 7 µM). Im Gegensatz zu Cyclosporin A stört NCI3 nicht die Phosphataseaktivität von Calcineurin, sondern interferiert mit der Calcineurin-NFATc-Bindung. Ein wichtiges endogenes Modulatorprotein für die Calcineurinaktivität ist RCAN1, das vermutlich den Calcineurin-NFATc-Signalweg über einen negativen Rückkopplungsmechanismus reguliert. Hier wurde gezeigt, daß RCAN1 in humanen TH-Zellen exprimiert wird. Die Spleißvariante RCAN1-1 ist in ruhenden T-Zellen basal exprimiert und wird nicht durch T-Zell-Rezeptor-Stimulierung in seiner Expression verändert. RCAN1-4 dagegen ist in ruhenden Zellen kaum zu detektieren und wird stimulierungsabhängig induziert. Durch die Verwendung Calcineurin-NFATc-spezifischer Inhibitoren wie NCI3 wurde gezeigt, daß die RCAN1-4-Induktion durch diesen Signalweg limitiert ist. Die in dieser Arbeit gewonnenen Daten und Erkenntnisse tragen dazu bei, das Verständnis der Funktion und Regulation von Calcineurin in T-Zellen zu vertiefen.
The Ca2+/calmodulin dependent serine/threonine phosphatase calcineurin is a key molecule in the T cell receptor dependent signalling network. Calcineurin dephosphorylates and thereby activates the transcription factors of the NFATc family that, among others, control the expression of important cytokines and cell surface molecules. The activity of Calcineurin is modulated by several endogenous proteins and is inhibited by the immunosuppressants cyclosporine A and FK506. Here, the novel low molecular weight inhibitor NCI3 was characterized in respect to its effects on T cell receptor dependent signalling. The results of this work show, that the pyrazolopyrimidine derivate NCI3 is nontoxic and permeates the cell membrane. Upon TCR stimulation NCI3 suppresses T cell proliferation and IL-2 production of primary human TH cells with IC50 values of ~4 µM by blocking the dephosphorylation and subsequent nuclear translocation of NFATc. NCI3 conse-quently inhibits calcineurin dependent NFAT- and NF-κB-, but not AP-1-controlled reporter gene expression, in micromolar concentrations (IC50 values 2 and 7 µM, respectively). In opposite to cyclosporine A and FK506, NCI3 does not interfere with the phosphatase activity of calcineurin but rather disturbs the calcineurin-NFATc interaction. A major endogenous modulator of calcineurin is the protein RCAN1, which is supposed to regulate calcineurin-NFATc signalling in a negative feedback loop. The presented data show that RCAN1 is expressed in human TH cells. The splice variant RCAN1-1 is basally expressed in resting T cells, and its expression levels are not changed by T cell receptor stimulation. Expression of RCAN1-4, on the other hand, is nearly undetectable in resting TH cells and is induced upon cell stimulation. By using calcineurin-NFATc specific inhibitors such as NCI3 it could be shown that RCAN1-4 induction is limited by this pathway. This work provides a comprehensive characterization of the novel inhibitor NCI3 and insights into the regulation of calcineurin by RCAN1 in human TH cells.

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5

Germaud, Nathalie. "Polymorphisme du gène NCR3/NKp30 et variabilité de la fonction des cellules Natural Killer humaines". Phd thesis, Université René Descartes - Paris V, 2012. http://tel.archives-ouvertes.fr/tel-00789417.

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Les cellules Natural Killer (NK) sont non, seulement de précieux effecteurs cytotoxiques de la réponse immunitaire innée dirigée contre les tumeurs et les infections, mais aussi d'importants immunorégulateurs. Leur activation dépend d'une balance complexe entre des signaux émanant de multiples récepteurs, tantôt inhibiteurs, tantôt activateurs. Parmi les récepteurs activateurs, NCR3 représente un acteur important de la lyse tumorale et de l'interaction avec les cellules dendritiques. Dans le but de caractériser la variabilité interindividuelle de la réponse NK et d'établir une référence pour des études ultérieures dans un contexte pathologique, nous avons examiné, dans un échantillon de 43 donneurs sains, les corrélations entre la variabilité fonctionnelle des cellules NK en réponse à la stimulation de leur récepteur NKp30 et le niveau d'expression des transcrits de NCR3 ainsi que celui de la protéine correspondante, au regard du polymorphisme génomique. Nous avons mis en évidence une étroite corrélation entre l'expression membranaire de NKp30 et NKp46 et la fonction cytotoxique NK, mais pas avec la sécrétion de cytokines. Nous avons retrouvé l'effet déjà connu du variant rs986475 sur l'expression de l'un des transcrits alternatifs de NCR3, T3. Nous avons également identifié un autre variant, rs11575836, influençant le niveau de transcrits T1, en relation avec la cytotoxicité induite par la signalisation NKp30. Cette étude pointe le doigt sur la spécificité des voies de signalisation des fonctions NK et le réseau complexe de gènes impliqués dans leur régulation. Nous avons par ailleurs évalué la variabilité génétique de NCR3 dans la myasthénie auto-immune où les cellules NK pourraient jouer un rôle. Le re-séquençage du gène NCR3 n'a pas révélé d'association avec un polymorphisme commun mais a permis d'identifier deux mutations rares, " faux-sens ", retrouvées uniquement chez des patients myasthéniques. L'une d'elle, L19R, est non-conservative et représente un candidat particulièrement intéressant à examiner en détail du fait de sa localisation dans une région très conservée dans la phylogénie. Même si de nombreux points restent à élucider, ces résultats indiquent qu'il devrait être possible de relier de façon globale et intégrative le polymorphisme de l'ADN, ainsi que l'expression des transcrits et des protéines, à la réponse fonctionnelle des cellules NK

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6

Baaklini, Sabrina. "Compréhension de la résistance humaine au paludisme : des études génétiques aux approches fonctionnelles". Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0384/document.

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La sévérité du paludisme est influencée par des interactions complexes entre de nombreux facteurs dont la génétique de l’hôte. Plusieurs études de liaison génétique menées dans différentes ethnies africaines ont montré une liaison entre le locus 6p21 et le paludisme simple. De plus, différents variants au sein des gènes TNF et NCR3, retrouvés dans ce locus, ont été indépendamment associés à ce phénotype au Burkina Faso.Ainsi, nous nous sommes tout d’abord intéressés aux polymorphismes du TNF. Nos résultats montrent que les variants TNF-308, TNF-244, et TNF-238 sont associés à la parasitémie maximale ou aux accès simples au Congo. Les approches moléculaires indiquent que le TNF-244 a un effet cis-régulateur avec une activité promotrice réduite en présence du variant A ainsi qu’une fixation altérée de protéines nucléaires en présence de ce même variant. Enfin, nos analyses bio-informatiques suggèrent que le TNF-244 et le TNF-238 agissent en synergie pour modifier le site de fixation d’au moins un facteur de transcription.Nous avons ensuite confirmé l’association du NCR3-412 avec le paludisme simple et le nombre d’accès fébrile au Congo. Les analyses fonctionnelles montrent que ce SNP a aussi un effet cis-régulateur avec une activité promotrice accrue en présence de l’allèle G et une liaison altérée de deux complexes protéiques en présence de l’allèle C. Les approches in silico et in vitro indiquent que les facteurs STAT4 et RUNX3 sont ceux dont la fixation est altérée.NCR3-412 altérant la résistance à la forme simple du paludisme, nous avons souhaité déterminer s’il est aussi impliqué dans la résistance au paludisme sévère mais nous n’avons détecté aucune association
The severity of malaria is influenced by complex interactions between many factors including host genetics. Numerous genetic studies conducted in different African ethnic groups have shown a significant linkage between the 6p21 locus and mild malaria attack. In addition to their linkage, several polymorphisms found under the linkage peak, and more precisely within TNF and NCR3, were also independently associated with different sub-phenotypes of mild malaria in Burkina Faso.Thus, we first focused on TNF polymorphisms. Among the 4 polymorphisms analyzed, we found associations between TNF-238, TNF-244, TNF-308 and either mild malaria attack or maximum parasitemia. Molecular approaches showed that TNF-244 has a cis-regulatory effect. Indeed, we observe a decreased promoter activity and an altered binding of nuclear proteins in the presence of the A variant. In addition, our bioinformatics analyses suggested a cooperative effect of TNF-244 and TNF-238 in modifying the binding of at least one transcription factor.We then confirmed the association of NCR3-412 with both mild malaria and the number of febrile episodes in Congo. Functional analyses have shown that this SNP has also a cis-regulatory effect with a decreased promoter activity and an altered binding of two nuclear protein complexes in the presence of the C allele. Finally, in silico and in vitro approaches indicated that STAT4 and RUNX3 are the two transcription factors affected.As NCR3-412 is associated with resistance to mild malaria, we therefore investigated whether this SNP is also involved in severe malaria resistance, but we did not detect any association neither with severe anemia nor with cerebral malaria

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7

Semeraro, Michaela. "Neuroblastoma and gastrointestinal stromal tumor as a target for natural killer lymphocytes : the role of ncr3/nkp30". Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T045/document.

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Depuis la formulation de la théorie de l’immuno-surveillance en 1957 par Burnet et Thomas, le monde scientifique s’est efforcé d’identifier les cellules immunitaires impliquées dans ce processus. Les lymphocytes Natural Killer (NK) constituent une composant majeure de l’immuno-surveillance innée dans plusieurs cancers hématologiques et solides. L’activité des lymphocytes NK passe principalement par une grande variété de récepteurs avec un rôle activateur ou inhibiteur. Parmi les récepteurs activateurs présents à la surface des lymphocytes NK, le récepteur NCR3/NKp30 a un rôle majeur dans la toxicité directe contre la cellule cible et dans l’activation des cellules dendritiques.Les tumeurs stromales gastrointestinales (GIST) et le Neuroblastome (NB) sont deux tumeurs sensibles à l’immuno-surveillance par les lymphocytes NK. Dans une étude récente notre équipe a démontré que l’épissage alternatif du gène NCR3/NKp30 peut être déterminant dans la fonction NK et dans la survie des patients atteints de GIST.Afin de caractériser les lymphocytes infiltrant le GIST, nous avons effectué une recherche visant à analyser l’infiltrat des lymphocytes CD3+, des lymphocytes T régulateurs (Treg) et des lymphocytes NK dans des tumeurs GIST localisés, et corréler ces résultats à la survie des patients. Nous avons mis en évidence que, avant traitement, les lymphocytes NK sont surtout localisés au niveau des fibres trabéculaires qui entourent la tumeur, alors que les lymphocytes T sont localisé à l’intérieur de la tumeur en contact avec les cellules tumorales qui expriment HLA-I.Nous avons aussi observé que les cellules NK ont un phénotype plutôt CD56bright et migrent à l’intérieur de la tumeur après traitement par Imatinib. L’analyse de survie a mis en évidence que les lymphocytes NK et T peuvent prédire la survie sans progression (PFS). Ces résultats mettent en évidence l’importance de l’infiltrat immunitaire dans la prédiction du risque de rechute dans le GIST et surlignent l’importance de viser une réponse immunitaire dans les protocoles thérapeutiques.Nous avons ensuite déterminé la proportion de lymphocytes NK dans le sang périphérique et dans la moelle dans une cohorte de Neuroblastome (NB) localisé et métastatique : une infiltration plus important par les NK CD56bright a été observé chez les patients présentant une maladie métastatique et chez les patients avec une réponse mineure au traitement d’induction. De plus, les NK présents dans les échantillons de moelle osseuse infiltrés par les neuroblastes, présentaient une expression plus basse du récepteur NKp30. L’expression du ligand de NKp30, B7-H6, a été mise en évidence sur les neuroblastes infiltrant la moelle osseuse, et sa forme soluble, sB7-H6, a été retrouvée être positivement corrélée à l’extension de maladie et inversement à la réponse au traitement d’induction. L’analyse de l’épissage alternatif du gène NCR3/NKp30 a permis de mettre en évidence l’impact des isoformes NKp30 sur la survie sans progression chez les patients atteints de NB de haut risque en maladie minimale résiduelle après chimiothérapie d’induction. En particulier, les patients présentant un taux élevé de l’isoforme pro-inflammatoire (NKp30b) par rapport à l’isoforme immunosuppressive (NKp30c), présentent une meilleure survie sans évènement. Nous avons aussi démontré le rôle des monocytes dans l’amplification de la réponse NKp30 dépendant. Les résultats de notre recherche dans le GIST et dans le NB, deux maladies différentes mais toutes les deux sensibles aux lymphocytes NK, surlignent l’importance d’intégrer de nouvelles options thérapeutiques aptes à cibler le système immunitaire
Since Burnet and Thomas formulated in 1957 the cancer immunosurveillance theory, the scientific world has made tremendous progress to identify the immune cells involved in this process. Natural Killer (NK) cells have emerged as a major component of the innate immunosurveillance of several hematological and solid malignancies. The activity of NK-cells is mainly mediated through their wide variety of receptors with activating and inhibitory functions. Among the versatile receptors present on NK cells, the activating receptor NCR3/NKp30 is a major receptor involved in both direct killing of target cells and mutual NK and dendritic cell activation.Gastrointestinal stromal tumors (GIST) and Neuroblastoma (NB) are known to be tumors sensitive to NK immunosurveillance. In a recent study we showed that alternative splicing of NCR3/NKp30 gene can affect NK cell function and GIST patient’s outcome.In order to better characterize the GIST tumor-infiltrating lymphocytes, we analyzed the CD3+, T regulatory (Treg) and NK lymphocytes infiltration within primary localized GIST tumors and we determined their prognostic value. We described that, before treatment, NK cells are mainly localized in fibrous trabeculae while T lymphocytes are in the tumor nests in HLA-I positive tumor cells contact. Moreover infiltrating NK cells displayed a secreting CD56bright phenotype, and accumulate in tumor nests after Imatinib (IM) treatment. Importantly CD3+ and NK lymphocytes independently predicted progression free survival (PFS). These results highlight the importance of the immune infiltrate in re-define the GIST risk stratification and allow enhancing the immune response in the therapeutic decisions.We next investigated the proportions of NK cells in blood and bone marrow (BM) in a cohort of localized and metastatic NB; a high proportion of CD56bright NK cells was associated with metastatic NB and with poor response to induction treatment within the metastatic NB. Moreover, infiltrated BM presented NKp30 down regulation. The expression of the NKp30 ligand, B7-H6, was found on BM neuroblasts, while the soluble protein, sB7-H6 correlated with resistance to treatment. Furthermore the transcriptional status of NKp30/NCR3 dictated the event-free survival rates of HR-NBs with minimal residual disease post-induction chemotherapy: in particular patients presenting a high proportion of the immunosuppressive isoform (NKp30c) compared to the pro-inflammatory isoform (NKp30b), presented a worse outcome. We further demonstrated the significant role of monocytes to amplify the NKp30 activation response.These researches in GIST and NB, two different but at the meantime NK-sensitive diseases support the effort to define new immunological therapeutic approaches and to determine their optimal use

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8

Yoo, Edwin. "Inflammatory cytokines induce human bronchial smooth muscle cell proliferation via an NCX-1 dependent mechanism". Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/fullcit?p1477952.

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Thesis (M.S.)--University of California, San Diego, 2010.
Title from first page of PDF file (viewed July 16, 2010). Available via ProQuest Digital Dissertations. Includes bibliographical references (leaves 36-40).

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9

Burr, Adam R. "Sodium dysregulation coupled with calcium entry leads to muscular dystrophy in mice". University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1414750156.

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10

Choudhury, Moinuddin Hasan. "Biopacemaking : new targets and new mechanisms". Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/biopacemaking-new-targets-and-new-mechanisms(b35ec222-29eb-432f-9b6d-238f3cbcd72d).html.

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Background: Biopacemaking is the attempt to replicate sinoatrial node (SAN)-like pacemaker activity in other areas of the heart by manipulating genes involved in pacemaking. Application of this could emulate the electronic pacemaker without the need for implantation of permanent hardware, or directly repair dysfunctional SAN tissue in human disease. We upregulated the transcription factors Tbx18, Tbx3 and the membrane ion exchanger NCX1 in bradycardic subsidiary atrial pacemaker (SAP) tissue which we used as a model of SAN dysfunction. We aimed to show that one or more of these gene targets could improve pacemaker function and alter the molecular character of SAP tissue and thus could potentially be used for the repair of dysfunctional SAN tissue. Methods: SAP tissue was isolated from the right atria of rats and kept beating in culture at 37°C for 48 hours. Recombinant adenoviruses were injected into SAP preparations to upregulate Tbx18, Tbx3 and NCX1 individually. Beating rate, overdrive suppression and pharmacological response to If blockade and β-adrenergic stimulation were measured along with molecular changes in pacemaker and atrial genes and proteins using RT-qPCR and immunohistochemistry. Results: Tbx18 upregulation significantly increased SAP beating rate after 48 hours of culture (a final rate of 141 ± 9 bpm in uninfected SAP tissue versus 215 ± 16 bpm in Ad-Tbx18 infected SAP tissue, p<0.01). It induced upregulation of HCN2 (p<0.01) and RYR2 (p<0.05), downregulation of HCN4 (p<0.05) and no change HCN1, Tbx3, Kv1.5, Kir2.1, Nav1.5, NCX1, Cx43, Cx45, Cav1.2 or Cav3.1. There was also no change in overdrive suppression and no change in response to pharmacology. No increase in beating rate was seen with either Tbx3 or NCX1 upregulation. Tbx3 preparations induced downregulation of the atrial genes Kir2.1 (p<0.01) and Nav1.5 (p<0.05), along with HCN1 (p<0.05), HCN4 (p<0.01), Tbx18 (p<0.05) and NCX1 (p<0.01), upregulated Cx43 (p<0.05) and showed no change in Cx45, RYR2, Kv1.5. NCX1 preparations demonstrated reduced overdrive suppression (p<0.05). Conclusion: Tbx18 showed the most potential for biopacemaking in SAP tissue, however both Tbx3 and NCX1 could be applied as secondary targets to fine tune biopacemaker function. Future work would focus on applying these targets to dysfunctional SAN tissue in larger animals.

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Nguyen, Thy Ngoc. "Prédisposition génétique au paludisme à Plasmodium falciparum : études d'association et analyses fonctionnelles de variants génétiques candidats situés dans des régions liées génétiquement au paludisme". Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4116.

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Dans cette thèse, nous avons étudié l'influence de plusieurs variants génétiques situés dans les régions chromosomiques 5q31-q33, 6p21, et 17p12, pour lesquelles une liaison génétique avec des phénotypes de paludisme a été montrée.Les gènes NCR3 et TNF, qui sont situés dans la région chromosomique 6p21, ont été associés au paludisme dans une population vivant au Burkina Faso. Nous avons répliqué ces études dans une population congolaise afin deconfirmer les associations des polymorphismes avec les accès palustres simples et la parasitémie symptomatique. Nos résultats montrent que le polymorphismeNCR3-412 est associé avec les accès palustres simples au Congo, et que les polymorphismes TNF-308, TNF-244, et TNF-238 sont associés avec les accès palustres simples ou la parasitémie symptomatique. En outre, nos analyses bioinformatiques suggèrent que les polymorphismes TNF-244 et TNF-238 agissent en synergie pour modifier le site de fixation pour au moins un facteur de transcription.Les deux gènes HS3ST3A1 et HS3ST3B1, qui sont situés dans la région chromosomique 17p12, sont impliqués dans la biosynthèse des heparanes sulfates. Dans cette étude, nous avons étudié l'association d’un polymorphisme situé dans le promoteur de HS3ST3A1 avec les accès palustres simples et la parasitémie symptomatique, et n’avons détecté aucune association. Nous avons étudié en outre le gène NDST1, situé dans la région chromosomique 5q31-q33, et qui code également pour une enzyme impliquée dans la voie héparane sulfate. Des résultats préliminaires encourageants soutiennent l'hypothèse que la variation génétique de NDST1 influence la parasitémie asymptomatique
In this thesis, we investigated the influence of some genetic variants located within chromosomes 5q31-q33, 6p21, and 17p12, which have been shown to be linked to malaria phenotypes. The genes NCR3 and TNF, which are located in the chromosomal region 6p21, have been reported to be associated with malaria in Burkina Faso population. We have replicated those studies in Congolese population to evaluate the associations of the SNPs in those genes with mild malaria attack and Plasmodium parasitemia. The results showed that the variant NCR3-412 is associated with mild malaria in Congo, and TNF-308, TNF-244, and TNF-238 are associated with mild malaria attack, maximum parasitemia, or both. In addition, bioinformatic studies suggest that TNF-244 and TNF-238 synergise to alter the binding of transcription factors.The two genes HS3ST3A1 and HS3ST3B1, which are located in chromosomal regions 17p12, are involved in the heparan sulfate proteoglycan biosynthesis. In this study, we further investigated the association of the polymorphisms in these genes with mild malaria attack and maximum parasitemia. However no association was found. We further studied the NDST1 gene, which is located within chromosome 5q31-q33, and which encodes the bifunctional enzyme N-deacetylase/ N-sulfotransferase 1, and also participates in the heparan sulfate synthesis . Encouraging results support the hypothesis that NDST1 variation influence controlling parasitemia. Further association and functional studies are needed to validate the role of NDST1 in malaria infection. More generally, the enzymes involved in the heparan sulfate pathway might play a key role in controlling malaria infection

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Sanka, Michel. "Compréhension des mécanismes moléculaires et des facteurs génétiques impliqués dans le paludisme sévère : analyse des profils transcriptomiques et processus biologiques caractéristiques du neuropaludisme et méta-analyse sur des gènes associés à la résistance au paludisme". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0615/document.

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Le paludisme est l'une des maladies infectieuses les plus dévastatrices qui a affecté environ 214 millions de personnes dans le monde. Elle est causée par l'infection par le parasite plasmodium, dont P. falciparum et P. vivax sont les plus représentés. Le développement asexué du parasite dans le sang provoque la physiopathologie de la maladie dont l'évolution passe du paludisme simple au paludisme grave, notamment le neuropaludisme. Nos travaux ont d'abord porté sur l'analyse du transcriptome, par la technologie des microarrays, des cellules sanguines d'une cohorte constituée au Sénégal. L'analyse des résultats a permis d'identifier un ensemble de gènes dont l'expression permettait de distinguer le profil transcriptomique du neuropaludisme de ceux du paludisme simple et des autres formes de paludisme grave. Ces gènes sont enrichis en voies biologiques impliquées dans l'activation des récepteurs des lymphocytes B et T mais aussi des TLR et des récepteurs Fcgamma. On y trouve aussi plusieurs gènes candidats qui ont déjà été testés pour leur résistance au paludisme, dont RNASE3 et IL18R. Nous avons aussi réalisé, avec une partie de cette même cohorte sénégalaise, une étude d’association cas-contrôles qui n’a pas permis de détecter d’association entre le polymorphisme NCR3-412 et le paludisme sévère. Enfin l’approche basée sur une métaanalyse a permis de confirmer son implication dans le paludisme simple, ainsi que celle du polymorphisme LTA+252 dans le paludisme sévère, contrairement au LTA+80, au TNF-238 et au TNF-308. L’ensemble des travaux contribuent à une meilleure compréhension des facteurs génétiques et génomiques impliqués dans la résistance de l’hôte au paludisme
Malaria is one of the most devastating infectious diseases that has affected an estimated 214 million people worldwide and caused nearly 600,000 deaths in 2015. It is caused by infection with the plasmodium parasite, P. falciparum and P. vivax are the most represented. The asexual development of the parasite in the blood causes the pathophysiology of the disease which can evolve from mild malaria to severe malaria, including cerebral malaria. Our work first focused on the analysis of the microarray transcriptome of blood cells of a cohort composed in Senegal. The analysis of the results allow to identify a set of genes whose expression permit to distinguish the transcriptomic profile of cerebral malaria from those of mild malaria and other forms of severe malaria. These genes are enriched in biological pathways involved in the activation of B and T lymphocyte receptors also TLRs and Fcgamma receptors. These genes also include several candidate proteins that have already been tested for resistance to malaria, including RNASE3 and IL1RN

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Lee, Junyoung. "FURTHERING PHARMACOLOGICAL AND PHYSIOLOGICAL ASSESSMENT OF THE GLUTAMATERGIC RECEPTORS AT THE DROSOPHILA NEUROMUSCULAR JUNCTION". UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_theses/619.

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Drosophila larval neuromuscular junctions (NMJs) serve as a model for synaptic physiology. The molecular sequence of the postsynaptic glutamate receptors has been described; however, the pharmacological profile has not been fully elucidated. Despite the postsynaptic molecular sequence used to classify the receptors as a kainate subtype, they do not respond pharmacologically as such. Kainate does not depolarize the muscle, but dampens evoked EPSP amplitudes. Quantal responses show a decreased amplitude and area under the voltage curve indicative of reduced postsynaptic receptor sensitivity to glutamate transmission. ATPA, a kainate receptor agonist, did not mimic kainate’s action. The metabotropic glutamate receptor agonist t-ACPD had no effect. Domoic acid, a quisqualate receptor antagonist, blocks the postsynaptic receptors without depolarizing the muscle, which supports the presence of quisqualate subtype receptors. The results suggest a direct postsynaptic action of kainate due to partial antagonist action on the quisqualate receptors. There does not appear to be presynaptic auto-regulation via a kainate receptor subtype or a metabotropic auto-receptor. A complete pharmacological profiling of the known receptor subtypes at this NMJ has not yet occurred; however, this study aids in furthering the ongoing investigations to provide a clearer picture of pharmokinetic profile and specificity of the receptor subtypes.

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Afridi, Sarwat. "Influence de variants génétiques candidats sur des phenotypes liés au paludisme à Plasmodium falciparum et effet fonctionnel du polymorphisme NCR3-412 associés au paludisme". Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4037.

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Le paludisme est une cause majeure de morbidité et de mortalité, plus particulièrement en Afrique sub-saharienne. De très nombreuses observations sont en faveur de l'existence de facteurs génétiques contrôlant le devenir de l'infection palustre. Il est très probable que certains variants génétiques de gènes candidats du paludisme affectent la résistance du paludisme à travers leur effet sur la réponse immunitaire acquise. Afin de vérifier cette hypothèse, nous avons étudié, dans une population vivant au Burkina Faso, des variants génétiques de HBB, IL4, IL12B, TNF, LTA, FCGR2A et NCR3 dont l'association avec des phénotypes liés à la résistance au paludisme a été publiée; nous avons évalué leur influence sur les niveaux d'IgG dirigés contre les antigènes de Plasmodium falciparum en utilisant un test d'association basé sur les familles. Ainsi, nous avons détecté, l'effet de l'hémoglobine C, FCGR2A-H131, le TNF-857T, et TNF1304A sur les niveaux des sous-classes d'IgG anti-P. falciparum. Ces résultats constituent une base utile pour des études ultérieures du contrôle génétique de la réponse immunitaire chez des individus vivant dans une zone d'endémie. Un autre projet a porté sur l'étude fonctionnelle du polymorphisme NCR3-412, dont nous avions montré l'association avec les accès palustres simples. Nos résultats basés sur des techniques moléculaires montrent l'effet de ce polymorphisme situé dans le promoteur sur la liaison de protéines nucléaires
Malaria is the major cause of morbidity and mortality especially in the Sub-Saharan Africa. There is a growing body of evidence for genetic factors controlling the outcome of malaria infection. It is thought that some genetic variants of malaria candidate genes affect malaria resistance through their effect on the acquired immune response. In order to verify this hypothesis, we worked on genetic variants of HBB, IL4, IL12B, TNF, LTA, FCGR2A and NCR3, which have been associated with malaria resistance phenotypes, to determine their influence on levels of anti-P. falciparum IgG in urban population of Burkina Faso. Using family-based association analysis, we detected the effect of Hemoglobin C, FCGR2A-H131, TNF-857T, and TNF1304A on the levels of anti-P. falciparum IgG. This study can pave the way towards further comprehension of genetic control of an individual's immune response against malaria. Another project focused on functional study of polymorphism NCR3-412, which has already been associated to mild malaria. We investigated the functional effect of this polymorphism located in the promoter by using molecular techniques and showed the effect of this polymorphism on the binding of nuclear proteins

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Madariaga, Venegas Francisco Alejandro. "Efecto antibiofilm de la aspirina liberadora de óxido nítrico NCX-4040 en presencia de fluconazol, sobre Candida albicans aisladas de pacientes con estomatitis protésica". Tesis, Universidad de Chile, 2016. http://repositorio.uchile.cl/handle/2250/140230.

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Memoria para optar al título de Químico Farmacéutico
La creciente resistencia a los fármacos antifúngicos es un problema de salud pública mundial. Según estadísticas de la Organización Mundial de la Salud (OMS), las infecciones por estos patógenos han aumentado por diversas causas entre las que se encuentran el aumento de pacientes inmunocomprometidos y el uso masivo de antibióticos de amplio espectro. Lo más alarmante es la alta tasa de morbilidad que exhibe este tipo de infecciones, las que se asocian con una alta tasa de resistencia a los fármacos actualmente ocupados. Uno de los mecanismos de resistencia más relevantes es la formación de biofilms, que se definen como comunidades microbianas rodeadas por una matriz de sustancias poliméricas extracelulares, la cual les otorgaría una protección mecánica frente a la acción de antimicrobianos. Una de las patologías donde la formación de biofilms está directamente asociada con el fracaso terapéutico es la estomatitis protésica, una inflamación crónica de la mucosa oral en contacto con una prótesis removible y que tiene una directa asociación con la presencia de biofilms de levaduras del género Candida. Por estas razones es de gran interés investigar novedosas estrategias farmacológicas para reducir o revertir los biofilms. Debido al escaso desarrollo de nuevas moléculas antifúngicas, es que se ha buscado potenciar el efecto de los antifúngicos convencionales con fármacos “no antimicrobianos”. En este sentido, se ha descrito que los antiinflamatorios no esteroidales (AINES), que inhiben la formación de prostaglandinas, poseen efecto antibiofilm. Esto estaría relacionado con el rol de la Prostaglandina E2 en los procesos claves involucrados en la formación de biofilms; entre ellos, la morfogénesis de Candida y la adhesión a superficies abióticas. Entre los AINES destaca la aspirina que es la que ha presentado mejor efecto antibiofilm. Otra molécula de interés como agente antimicrobiano, es el óxido nítrico (NO), cuya acción se ha ensayado con compuestos dadores de NO como el nitroprusiato de sodio o el isosorbide mononitrato. Estos han presentado efectos antibiofilm presumiblemente por los efectos antimicrobianos que presentan el NO y por su capacidad como inmunomodulador. Por estas razones en esta investigación se evaluó el efecto antibiofilm de la aspirina liberadora de óxido nítrico, cuya estructura aparece en la Figura 1; en este caso el efecto sobre los biofilms sería dual. En este trabajo se determinó la susceptibilidad a fluconazol de los aislados clínicos de C. albicans y se cuantificó el efecto antibiofilm de la aspirina liberadora de óxido nítrico. Para este efecto, se evaluaron los siguientes parámetros: adhesión, morfogénesis, morfología de los biofilms y viabilidad de los biofilms en presencia de fluconazol y aspirina liberadora de óxido nítrico. Este último fármaco fue eficaz sobre los biofilms, pero su efecto no fue sinérgico en presencia de fluconazol, confirmando que fluconazol no presenta efecto antifúngico frente a los biofilms. Estos resultados son prometedores y nuevos estudios están en progreso para confirmar su posible uso en este tipo de patologías
The growing resistance to antifungal drugs is a global public health problem. According to statistics from the World Health Organization (WHO), infections with these pathogens have increased for various reasons, among which are the increase of immunocompromised patients and the widespread use of broadspectrum antibiotics. Most alarming is the high rate of morbidity exhibiting these infections, which are associated with a high rate of resistance to drugs currently occupied. One of the most important mechanisms of resistance is the formation of biofilms, which are defined as microbial communities surrounded by a matrix of extracellular polymeric substances, which would provide them with mechanical protection against the action of antimicrobials. One of the conditions where the formation of biofilms is directly associated with treatment failure is denture stomatitis, a chronic inflammation of the oral mucosa in contact with a removable prosthesis and has a direct association with the presence of biofilms of Candida yeasts. For these reasons it is of great interest to investigate novel pharmacological strategies to reduce or reverse the biofilms. Due to the limited development of new antifungal molecules it is that they have sought to enhance the effect of conventional antifungals with "no antimicrobial" drugs. In this regard, it described those nonsteroidal anti-inflammatory drugs (NSAIDs) which inhibit the formation of prostaglandins, have antibiofilm effect. This would be related to the role of Prostaglandin E2 in the key processes involved in the formation of biofilms; including Candida morphogenesis and adherence to abiotic surfaces. Among the highlights NSAIDs aspirin presents the antibiofilm best effect. Another molecule of interest as an antimicrobial agent is nitric oxide (NO), whose action has been tested with NO donor compounds such as sodium nitroprusside or isosorbide mononitrate. These have presented antibiofilm effects presumably by antimicrobial effects of NO and its ability as an immunomodulatory agent. For these reasons in this investigation antibiofilm effect of aspirin releasing nitric oxide, was evaluated; in this case the effect on biofilms would be dual. Therefore, we evaluated fluconazole susceptibility of C. albicans clinical isolates and quantified the antibiofilm effect of aspirin releasing nitric oxide. For this purpose, adhesion, morphogenesis, morphology and viability of biofilms in the presence of fluconazole biofilms and aspirin releasing nitric oxide were evaluated. The latter drug was effective against biofilms, but its effect was not synergistic with those of fluconazole. This confirmed that fluconazole have no effect against biofilms. These results are promising and further studies are in progress to confirm their possible use in this type of pathology

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Paula, Gabrielle Silveira de. "Efeitos da alimentação/digestão e do jejum prolongado sobre a função cardíaca de cascavéis, Crotalus durissus terrificus". Universidade Federal de São Carlos, 2012. https://repositorio.ufscar.br/handle/ufscar/1351.

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Universidade Federal de Minas Gerais
Some snakes have the ability to survive long periods without food and are capable to ingest large meal size. The ingestion of proportionally large preys triggers an expressive increase on the oxidative metabolic demand (SDA Specific Dinamic Action) which can become several times higher than the resting metabolic rate. The two described extreme situations might lead to cardiac changes to adapt structure and function in order to afford these two opposite physiological demands. During long food deprivation (Phase III), the depletion of the body structure may affect the heart muscle. That should be reverted to the SDA and prevent an overload on cardiovascular system. The standard cardiac function of South-american Rattlesnake, Crotalus durissus terrificus, was described as well as the changes caused by long term food deprivation and SDA. The importance of sodium-calcium exchanger (NCX), functionality of sarcoplasmic reticulum, extracellular calcium dependence and the effect of adrenergic stimulation were tested in adult animals at 30°C, under three different metabolic states: postabsorptive, SDA peak and food deprivation. The ventricular mass does not change after food deprivation and SDA. The force of contraction was higher in the base of the heart if compared to the apex, but there is no difference among the experimental groups and it is probably reflex of the fiber orientation in each region of the ventricle. The sarcoplasmatic reticulum is functional in all groups, but the dependence of reticular calcium is lower during starvation compared to the other groups. Muscle contraction is mostly supported by the extracellular Ca2+. The NCX have minor contribution to force generation (20%) but has a major role pumping calcium out of the cell (faster than SERCA). The increase in extracellular Ca2+ concentration during digestion can augment twitch force and would represent a contractile advantage to support the increased cardiac work without the development of hypertrophy. The adrenergic stimulation produced sustained increase in Fc for a wide range of stimulation frequencies in all the tested groups.
O coração possui a importante capacidade de se remodelar diante de alterações nas demandas funcionais. Serpentes apresentam a capacidade de ingestão de grandes presas e a capacidade de sobreviver a grandes períodos de privação alimentar. A ingestão de grandes massas de alimento demanda uma elevação metabólica e leva a uma compensação na massa ventricular e um aumento na força de contração do miocárdio, evitando uma sobrecarga no sistema cardiovascular. Durante o jejum ocorre depleção da estrutura corpórea podendo também atingir o músculo cardíaco. Para descrever a função cardíaca da Cascavél Sul-americana, Crotalus durissus terrificus, e as possíveis alterações diante dos extremos metabólicos, foi testada a importância do trocador Na+/Ca2+ (NCX), a funcionalidade do retículo sarcoplasmático (RS), a dependência do Ca2+ extracelular e o efeito da estimulação adrenérgica em animais adultos à temperatura constante de 30°C em período pós-absortivo, pico de SDA e após jejum prolongado. Observou-se que a massa ventricular se mantém estável mesmo durante os extremos metabólicos. Há uma diferença na geração de tensão entre as tiras da base e ápice ventriculares, presente nos 3 grupos alimentares e que pode se dar em função da orientação das fibras nas duas regiões do ventrículo. O teste com rianodina mostrou que o RS é funcional nos 3 grupos porém a dependência do cálcio proveniente do RS é menor após o jejum prolongado quando comparado aos demais grupos. O cálcio extracelular é o principal responsável pela contração do miócito cardíaco e o NCX, que fornece uma pequena parte de cálcio para a contração, é o principal bombeador de cálcio para fora da célula e é mais rápido do que a Ca2+- ATPase do RS. O meio extracelular é a principal fonte de cálcio ativador da contração sendo que o aumento de cálcio circulante disponível para a contração nos animais durante a digestão ofereceria uma vantagem contrátil para este grupo sem necessidade de hipertrofia. A adrenalina produziu um aumento substancial na Fc capaz e ser mantido por diferentes frequências de estimulação nos 3 grupos testados.

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Nguidjoe, Evrard. "Etude de la fonction de la cellule bêta pancréatique dans un modèle de souris présentant une mutation nulle partielle de l'échangeur sodium/calcium". Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209833.

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Précédemment, nous avons montré que la surexpression de l'échangeur Na/Ca NCX1), une protéine responsable de la sortie de calcium (Ca2+) des cellules, augmentait la mort cellulaire programmée ou « apoptose » et réduisait la prolifération des cellules β. Afin d’étudier plus en profondeur le rôle de l’échangeur dans les cellules β in vivo, nous avons développé et caractérisé des souris présentant une inactivation de NCX1.

Des méthodes biologiques et morphologiques (imagerie du Ca2+, capture de Ca2+, métabolisme du glucose, sécrétion d'insuline et morphométrie par comptage de points) ont été utilisées pour évaluer la fonction de la cellule β in vitro. Les taux de glucose et d'insuline dans le sang ont été mesurés afin de déterminer le métabolisme du glucose et la sensibilité à l’insuline in vivo. Des îlots ont été transplantés sous la capsule rénale pour évaluer leur capacité à corriger le diabète chez les souris rendues diabétiques par l’alloxane.

L'inactivation hétérozygote de Ncx1 chez les souris provoque une augmentation de la sécrétion d’insuline induite par le glucose avec un renforcement important à la fois de la première et de la deuxième phase. Ces résultats s’accompagnent d’une augmentation de la masse et de la prolifération des cellules β. La mutation augmente également le contenu en insuline, l’immunomarquage de la proinsuline, la capture de Ca2+ induite par le glucose et la résistance à l'hypoxie des cellules β. En outre, les îlots de souris Ncx1+/- montrent une capacité à compenser le diabète 2 à 4 fois plus élevé que les îlots de souris Ncx1+/+ lorsque transplantés chez des souris diabétiques.

En conclusion, l’inactivation de l'échangeur Na/Ca conduit à une augmentation de la fonction de la cellule β, de sa prolifération, de sa masse et de sa résistance au stress physiologique, à savoir à divers changements de fonction des cellules β opposés aux principales anomalies rencontrées dans le diabète de type 2 (Type 2 Diabetes Mellitus,T2DM). Ceci nous procure un modèle unique pour la prévention et le traitement du dysfonctionnement des cellules β dans le T2DM et pour la transplantation d'îlots.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

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Vasconcelos, Eliton da Silva. "Análise da expressão de proteínas envolvidas no manejo de cálcio cardíaco nos répteis píton (Python molurus), cascavel sul-americana (Crotalus durissus terrificus) e jacaré-de-papo-amarelo (Caiman latirostris) em jejum e nos períodos digestivo e pós-absortivo". Universidade Federal de São Carlos, 2014. https://repositorio.ufscar.br/handle/ufscar/1264.

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The Sarcoplasmic reticulum calcium ATPase (SERCA), phospholamban (PLB) and the Na+-Ca2+ exchanger (NCX) proteins are essential for the cardiac calcium management and myocardial contractility in vertebrates. The increase in metabolic rate generated from mechanical and physiological process of digestion is known as specific dynamic action (SDA) and represents the energy cost of processing, digestion and absorption of food. In several groups of reptiles, feeding generates a rapid increase in rates of gas exchange, whose peak usually occurs one or two days after feeding, before suffering a slower decline and returning to preprandial values. Ingestion of large quantity of food demands a metabolic elevation and leads to indemnities related in ventricular mass and an increase in myocardial force contraction, avoiding an overload on the cardiovascular system. During the fasting, depletion of body mass occurs and it can also reach the heart muscle. To describe the importance and the changes in SERCA2, PLB and NCX proteins on the effects of feeding/digestion in reptiles broad-snouted caiman (Caiman latirostris), burmese phyton (Python molurus) and also by prolonged fasting in South-American rattlesnake (Crotalus durissus terrificus) Western blotting technique was used. We observed the homology existence between the proteins of reptiles in relation to mammals. The SDA increased expression of SERCA2 protein in three species of reptiles. The SDA can also have unduced the expression of an isoform and/or reduced phosphorylation at some active sites of SERCA2 in ventricular tissue of P. molurus. A direct relationship between the expression of SERCA2 with the PLB expression in C. durissus was observed. However in P. molurus and C. latirostris this relation does not exist. The three reptiles expressed the PLB with molecular mass of 50 kDa, whereas the mouse expressed 25 kDa. The vast evolutionary distance that separates mammals of reptiles may have caused the rise of isoforms between these groups, explaining this difference. The increased expression of NCX along with lower expression of SERCA2 and PLB in C. durissus in food group Fasting compared to the other two groups suggests a higher calcium mobilization in this feeding regime, which would be advantageous from the energy point of view. The expression of NCX with different molecular mass between the three species, a smaller number of consensus regions with the animals phylogenetically more distant and the great plasticity of NCX gene to form distinct proteins, suggest the expression of proteins isoforms with the reptiles studied in this research.
As proteínas Ca2+-ATPase do Retículo Sarcoplasmático (SERCA2), Fosfolambam (PLB) e o Trocador Na+/Ca2+ (NCX) são fundamentais para o manejo do cálcio e a contratilidade miocárdica nos vertebrados. A elevação da taxa metabólica gerada a partir dos processos mecânicos e fisiológicos da digestão é conhecida como "ação dinâmica específica" (SDA) e representa o custo energético do processamento, digestão e absorção dos alimentos. Em vários grupos de répteis, a alimentação gera um rápido aumento nas taxas de troca gasosa, cujo pico ocorre geralmente um dia ou dois após a alimentação, antes de sofrer um declínio mais lento e retornar aos valores pré-prandiais. A ingestão de grandes massas de alimento demanda uma elevação metabólica e leva a uma compensação na massa ventricular e um aumento na força de contração do miocárdio, evitando uma sobrecarga no sistema cardiovascular. Durante o jejum ocorre depleção da estrutura corpórea podendo também atingir o músculo cardíaco. Para descrever a importância e as mudanças nas proteínas SERCA2, PLB e NCX diante os efeitos da alimentação/digestão nos répteis cascavel sul-americana (Crotalus durissus terrificus), na píton (Python molurus) e também do jejum prolongado no jacaré-de-papo-amarelo (Caiman latirostris), utilizada a técnica molecular Western blotting. Observou-se a existência de homologia entre as proteínas dos répteis com relação aos mamíferos. A SDA levou a uma maior expressão da proteína SERCA2 nas três espécies de répteis. A SDA também pode ter induzido a expressão de uma isoforma e/ou uma menor fosforilação em alguns sítios ativos da SERCA2 no tecido ventricular de P. molurus. Uma relação direta entre a expressão da SERCA2 com a expressão do PLB foi verificada somente em C. durissus, não havendo tal relação em P. molurus e C. latirostris. As três espécies de répteis expressaram o PLB com mesma massa molecular, de 50 kDa, e diferente da do rato, que é de 25 kDa. A grande distância evolutiva que separa os répteis dos mamíferos pode ter ocasionado o surgimento de isoformas entre esses grupos, explicando tal diferença. O aumento da expressão do NCX em C. latirostris do grupo alimentar Digestão sugere uma maior mobilização do cálcio e um maior inotropismo de forma frequência-específica através dessa proteína. A maior expressão do NCX juntamente com a menor expressão da SERCA2 e do PLB em C. durissus no grupo alimentar Jejum em relação aos outros dois grupos alimentares sugere uma maior mobilização do cálcio pelo NCX nesse regime de alimentação, o que seria vantajoso do ponto de vista energético. A expressão do NCX com diferentes massas moleculares entre as três espécies estudadas, um menor número de regiões conservadas em animais filogeneticamente mais distantes, e a grande plasticidade do gene da proteína NCX em formar proteínas distintas, sugere a expressão de proteínas isoformas nos répteis estudados.

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19

Liu, Li. "Roles of PMCA Isoforms in Ca²⁺-Homeostasis and Contractility of Bladder Smooth Muscle: Evidence from PMCA Gene-Ablated Mice". Cincinnati, Ohio : University of Cincinnati, 2007. http://rave.ohiolink.edu/etdc/view.cgi?acc_num=ucin1178307168.

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Thesis (Ph.D.)--University of Cincinnati, 2007.
Advisor: Richard J. Paul. Title from electronic thesis title page (viewed Apr. 4, 2009). Keywords: PMCA (human gene symbols; ATP2B); SERCA2 (human gene symbols; ATP2A2); NCX; bladder smooth muscle; Ca²⁺ homeostasis; gene-altered mice. Ca²⁺ waves; Ca²⁺ sparks; Fura-PE3; Fluo-4; Indo-1; multi-photon microscopy. Includes abstract. Includes bibliographical references.

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20

de, Moissac Danielle. "Structure-function studies of the sodium-calcium exchanger isoforms, NCX1 and NCX2". 2009. http://hdl.handle.net/1993/3158.

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The sodium-calcium exchanger (NCX) is a countertransporter of Na+ and Ca2+ across most cell membranes. It has been identified as an essential component of Ca2+ homeostasis in physiological and disease conditions in both cardiovascular and neurological settings. The exchanger not only transports Na+ and Ca2+, but is also regulated by these ions. Although ionic regulatory profiles differ between NCX isoforms, similar regulatory domains have been identified. Previous structure-function studies have determined key residues within these domains, particularly in the eXchanger Inhibitory Peptide region (XIP) and the Ca2+ binding domains (CBD1/2), which have a direct impact on ionic regulation of the outward exchange currents. Recent structural studies of the Ca2+ binding domains of NCX1 suggest a mechanism by which Ca2+ binding would not only be essential for activation of current but may also influence Na+-dependent inactivation. The alternative splice region is located within the Ca2+ binding domain and may play a role in mediating these regulatory phenotypes. Previous studies have demonstrated that specific combinations of the mutually-exclusive and cassette exons are associated with profound effects on ionic regulation in NCX1. This study focuses on examining the mechanisms by which the alternative splice region, in combination with specific regulatory domains, modulates exchange activity in two isoforms, NCX1 and NCX2. Chimaeric and mutant constructs in the alternative splice region were expressed in Xenopus oocytes and outward Na+-Ca2+ exchange activity was assessed using the giant, excised patch clamp technique. Substitution of the region corresponding to the mutually exclusive exon in either exchanger greatly reduced the extent of Na+-dependent inactivation, independently of intracellular Ca2+ concentrations. However, replacement of both the region corresponding to the mutually exclusive exon A and the XIP region reestablishes a wild-type profile in NCX2. The first mutually exclusive exon is therefore critical in determining Na+ and Ca2+-dependent regulatory properties. Furthermore, non-conserved residues within the XIP region may be essential in maintaining the structural stability of the Na+-dependent inactive state of NCX1, and by interacting with the mutually exclusive exon, may contribute to the structure-function relationship and the distinct regulatory phenotype of each Na+-Ca2+ exchanger variant and isoform.

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21

Tsai, Wei-Joe, e 蔡維哲. "Reaction, magnetic properties , spectroscopies and crystal structures of Hg(2-NCH3NCTPPH)Cl、Co(2-NCH3-21-CNNCTPP)(NCS)、Co(2-NCH3-21-CH2C6H5NCTPP)Cl". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/s93r38.

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碩士
國立中興大學
化學系所
106
We use Methyl iodide as 2-N substituted alkylation reagent to react with N-confused porphyrin to get 2-NCH3NCTPPH (2). After 2-N substituted alkylation, using HgCl2, CoCl2 ∙ 6H2O and Co(SCN)2 to react seperately with 2-NCH3NCTPPH (2) to get three NCP metal complexes, diamagnetic of Hg(2-NCH3 -NCTPPH)Cl (3)、paramagnetic of Co(2-NCH3-21-CNNCTPP)(NCS) (4) and Co(2-NCH3-21-CH2C6H5NCTPP)Cl (5). Then we use X-ray Single-Crystal Diffractometer, NMR spectroscopy and, etc., to determine the crystal structures and analyze their physical properties.

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22

Chen, Szu-Fan, e 陳思帆. "Study of the regulation of NCX1 activity by creatine kinase". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/23637463824604718609.

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碩士
國立陽明大學
生命科學暨基因體科學研究所
98
Na+/Ca2+ exchanger (NCX) is an essential component for maintaining Ca2+ homeostasis. Three mammalian NCX isoforms have been identified, including NCX1, NCX2, and NCX3. NCX1 plays an important role in cardiac muscle contraction; inhibition of NCX1 activity contributes to cardioprotection against ischemia/reperfusion injury. In our previous results two CK isoforms, sMiCK and CKM interact with NCX1 in HEK293T cells and cardiac myocytes. In addition, the decreased reverse-mode NCX1 activity under energy-compromised conditions is recovered by expression of sMiCK and CKM.In this study, I established the methods for measurement of the forward-mode NCX1 activity in HEK293T cells and demonstrated CKM and sMiCK also recovered the forward-mode NCX1 activity that decreased under energy-compromised conditions. Furthermore, sMiCK protected mitochondria from fragmentation but had no effects on the mitochondrial Ca2+ homeostasis under energy-compromised conditions. In conclusion, CKM and sMiCK can regulate both reverse- and forward-mode NCX1 activity under energy- compromised conditions.

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23

Ho, Pei-Yun, e 何佩芸. "Monitoring interactions among integrinαIIbβ3、NHE1、NCX1 that trigger calcium oscillations by FLIM-FRET technology". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/85697334784946103616.

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碩士
國立陽明大學
生醫光電工程研究所
95
Chinese hamster ovary cells expressing exogenous human integrinαIIbβ3 on their plasma membrane (CHOαIIbβ3) exhibited active calcium oscillations when plated on substrates coated with fibrinogens or disintegrins. In these cells, profound targeting of sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 from intracellular vesicles to the plasma membrane was noticed, where they co-localized with integrins. Such molecular interactions could be clearly demonstrated using FRET-FLIM technique based on fluorescence lifetime measurements. Further more, targeting and interaction of the ion exchangers with integrinαIIbβ3, and their functional coupling to initiate calcium influx, were dependent on lipid microdomain, or lipid rafts.

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24

Chen, Shao-Hong, e 陳紹弘. "A Study of the Sodium Calcium Ion Exchange Mechanism in NCX". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/06903241036428583046.

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碩士
國立新竹教育大學
應用數學系碩士班
103
Biological sodium calcium ion exchange channel (NCX) removes calcium ions very rapidly from cell inside in exchange with sodium ions from outside. The Poisson-Fermi theory is used to analyze the binding potentials of NCX. It allows us to mathematically investigate the sodium-calcium ion exchange mechanism in NCX. Numerical results have been shown to agree with the experimental results of the sodium-calcium ion exchange.

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25

Chan, Lally Lai Yee. "Interaction between NCX and SERPA in Ca²⁺ signaling in human endothelial cells". Thesis, 2004. http://hdl.handle.net/2429/15452.

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The interaction between sodium-calcium-exchanger (NCX) and the endoplasmic reticulum (ER) with respect to Ca²⁺ signaling was studied using fura-2 fluorescence imaging microscopy in human endothelial cells. The inflammatory agonist histamine was used to increase the intracellular Ca²⁺ concentration. Under resting conditions, the endothelial NCX serves to unload some of the Ca²⁺ content accumulated in the ER. This unloading is important in order to maintain the buffer barrier function of the peripheral ER. Application of histamine (1 uM) in the presence of extracellular Ca²⁺ caused a long lasting Ca²⁺ response. This maintained response is dependent on the state of ER Ca²⁺ content, which is at least partly refilled by Ca²⁺ entry via NCX working in the reverse mode during the course of agonist stimulation. After cessation of agonist stimulation, a major part of the increased Ca²⁺ is cleared by sarcoplasmic/endoplasmic reticulum Ca²⁺ - ATPase (SERCA) and NCX working in a serial configuration. In summary, in human endothelial cells, NCX can unload or refill the ER and thereby modulate the Ca²⁺ level.

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26

Pauls, Paul. "Die Rolle des kardialen Na+/Ca2+-Austauschers in der Entstehung von Vorhofrhythmusstörungen: Studien an isolierten Atriomyozyten aus Mausmodellen mit veränderter NCX-Expression". Doctoral thesis, 2018. https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-20181011654.

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Hintergrund: Das Vorhofflimmern ist die häufigste Rhythmusstörung des Menschen. Eine gesteigerte Expression des Na+/Ca2+-Austauschers (NCX) ist dabei eine von zahlreichen elektrischen Remodeling-Mechanismen, die bei diesem Krankheitsbild beobachtet werden. Der unabhängige, also in Abwesenheit anderer Remodeling-Mechanismen bestehende Einfluss einer gesteigerten NCX-Aktivität auf die atriale Proarrhythmie ist allerdings immer noch unzureichend untersucht. Der zugrundeliegende Mechanismus des Vorhofflimmerns basiert zumindest teilweise auf einer spontanen atrialen Aktivität in Form der Ausbildung von spontanen Aktionspotentialen (sAPs). Dabei führt eine spontane Ca2+-Freisetzung aus dem sarkoplasmatischen Retikulum (SR) zu einer NCX-vermittelten Ca2+-Extrusion, die einen von NCX getragenen Einwärtsstrom (INCX) genereriert, welcher wiederum eine Depolarisation des Membranpotentials (DAD, delayed afterdeplorazition) nach sich zieht. Diese kann, sofern sie das kritische Membranpotential zur Reaktivierung des Natrium-Stroms (INa) überschreitet, ein sAP induzieren, welches letztendlich das Vorhofflimmern auslösen kann. Methoden: In der vorliegenden Arbeit wurden isolierte atriale Herzmuskelzellen von homozygoten NCX-Überexpressor (OE) und heterozygoten NCX-Knockout (KO) Mäusen und deren korrespondierenden Wildtypen (WTOE und WTKO) mit der Ca2+-imaging- und der patch-clamp-Methode, sowie mit molekularbiologischen und histologischen Methoden untersucht. Ergebnisse: Eine gesteigerte bzw. verringerte atriale NCX-Expression und –Aktivität wurde mithilfe eines Immunoblots, durch eine veränderte NCX-vermittelte Ca2+-Extrusionskapazität und durch Messungen des INCX bestätigt. Das Vorhofgewicht, die Zellgröße, die elektrische Zellkapazität und Fibrosefärbungen ergaben keinen Anhalt für ein strukturelles Remodeling in den untersuchten Mauslinien. Es gab weder Unterschiede in der Anzahl von spontanen Ca2+-Freisetzungen aus dem SR noch in der SR-Ca2+-Beladung. Die Anzahl von DADs, die während eines proarrhythmischen Stimulations-Protokolls auftraten, war im OE im Vergleich zum WTOE und im KO im Vergleich zum WTKO unverändert. Allerdings wurden DADs im OE im Vergleich zum WTOE signifikant häufiger in sAPs übersetzt. Komplementär dazu wurden im KO im Vergleich zum WTKO DADs tendenziell aber statistisch nicht signifikant seltener in sAPs übersetzt. Unter Verwendung eines aggressiveren proarrhythmischen Protokolls in KO und WTKO zeigte sich eine signifikant geringere Übersetzung von DADs in sAPs. Im OE zeigte sich eine erhöhte DAD-Amplitude im Vergleich zum WTOE und komplementär dazu eine Verringerung der DAD-Amplitude im KO im Vergleich zum WTKO. Messungen der L-Typ Ca2+-Ströme (ICa,L) zeigten einen vergrößerten Strom im OE und einen verringerten im KO, bezogen auf WTOE bzw. WTKO. Versuche mit einem Ca2+-Chelator ließen auf eine direkte funktionelle Interaktion zwischen NCX und L-Typ Ca2+-Kanälen (LTCC) schließen. Der K+-Strom (IKtot) war im OE verstärkt und im KO verringert im Vergleich zu den korrespondierenden Wildtypen. Schlussfolgerung: In atrialen murinen Herzmuskelzellen führte eine gesteigerte NCX-Aktivität zu einer erhöhten Anfälligkeit gegenüber sAPs, während eine verminderte NCX-Aktivität zu einem 101 Schutz vor dem Auftreten von sAPs führte. Die vorliegende Arbeit legt die folgenden Mechanismen nahe: Eine gegebene spontane Ca2+-Freisetzung induziert über NCX eine DAD, wobei die Amplitude der DAD direkt abhängig von der NCX-Aktivität ist. Sobald eine DAD die Reizschwelle für die Aktivierung von Na+-Kanälen überschreitet, wird ein sAP ausgelöst. Bei, wie in dieser Arbeit beobachtet, unveränderter Anzahl von spontanen Ca2+-Freisetzungen und damit auch von DADs, machte daher im OE die höhere DAD-Amplitude das Überschreiten der spannungsabhängigen Reizschwelle von sAPs wahrscheinlicher. KO Zellen waren umgekehrt dazu durch den komplementären Mechanismus geschützt, da eine geringere DAD-Amplitude seltener die Schwelle zur Induktion eines sAPs erreichte. Die in dieser Arbeit beschriebene Interaktion zwischen NCX und ICa,L kann als additiver Mechanismus interpretiert werden, durch den im OE bei höherer NCX-Aktivität eine leichtere Reaktivierung von ICa,L erfolgt, was die Induktion eines sAPs möglicherweise zusätzlich unterstützt. Die Befunde der vorliegenden Arbeit zeigen, dass die NCX-Aktivität als unabhängiger Proarrhythmiefaktor, zumindest in den in dieser Arbeit untersuchten Modellen, die Entstehung von atrialen sAPs beeinflussen kann. Insbesondere die Befunde im KO lassen eine Inhibition von NCX zur Behandlung des Vorhofflimmerns als zukünftige experimentelle Therapiestrategie möglich erscheinen.

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Yi, Yung-Hsiang, e 易永祥. "Membrane targeting and interaction of NHE1 and NCX1 with integrinaIIbb3 by lipid microdomain induce a calcium influx that triggers calcium oscillation". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/p73e33.

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博士
國立陽明大學
微生物及免疫學研究所
97
Abstract The cyclic calcium release-and-uptake during calcium oscillation is thought to result from calcium-induced calcium release (CICR); however, it is unclear, especially in non-excitable cells, how the initial calcium mobilization occurs that triggers CICR. We report here a novel mechanism, other than conventional calcium channels or phospholipase C-inositol trisphosphate system, for initiating calcium oscillation downstream of integrin signaling. Upon integrinαIIbβ3’s binding to fibrinogen ligand or the disintegrin rhodostomin, sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 are actively transported to the plasma membrane and they become physically coupled to integrinαIIbβ3. Lipid raft-dependent mechanisms modulate the membrane targeting and formation of NHE1/integrinαIIbβ3/NCX1 protein complex. NHE1 and NCX1 within such protein complex are functionally coupled, such that a local increase of sodium concentration caused by NHE1 can drive NCX1 to generate sodium efflux in exchange for calcium influx. The resulting calcium increase inside the cell can then trigger CICR, as a prelude to calcium oscillation downstream of integrinαIIbβ3 signaling. Fluorescence resonance energy transfer based on fluorescence lifetime measurements is employed here to monitor the intermolecular interactions among NHE1/integrinαIIbβ3/NCX1, which could not be properly detected using conventional biochemical assays.

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28

Ouazzani, Chahdi Amine. "La combinaison de l'UDCA ou du NCX-1000 avec des antioxydants liposolubles procure une meilleure protection aux hépatocytes de souris contre la toxicité de l'amiodarone". Thèse, 2004. http://hdl.handle.net/1866/15477.

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29

Souza, Juliane Pereira de. "Clonagem e expressão de proteínas recombinantes humanas envolvidas em processos imunes". Master's thesis, 2021. http://hdl.handle.net/10400.26/37611.

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A expressão de proteínas recombinantes, em escala laboratorial, tem sido uma das aplicações mais visíveis da biotecnologia. A expressão heteróloga de proteínas depende da tecnologia do DNA recombinante, que, por meio da manipulação do material genético, permite a obtenção de quase todas as proteínas nas quantidades desejadas. Esta tese sobre clonagem e expressão de proteínas recombinantes humanas envolvidas em processos imunes apresenta o trabalho desenvolvido para clonar e expressar o Receptor 3 de Citotoxicidade Natural humano (NCR3), uma proteína de superfície expressa por células Natural Killer, um dos braços do sistema imunológico inato. Outro objetivo foi expressar também o ligante natural do NCR3, NCR3 Ligante 1 (NCR3LG1). Essas duas proteínas estão envolvidas na ativação da resposta inata do sistema imunológico contra proteínas tumorais que expressam NCR3LG1. Uma estratégia tradicional de amplificação por PCR baseada em primers foi utilizada para amplificar as regiões a serem clonadas dos genes de interesse de plasmídeos comerciais e cloná-los no vetor pET28a(+) para a expressão controlada das proteínas, com tag His6, por lac mediada pelo promotor T7 em células Escherichia coli BL21(DE3) Hi-ControlTM. O hNCR3 foi expresso com sucesso por indução com IPTG e foi recuperado dos corpúsculos de inclusão por purificação com refolding de proteína em coluna. O hNCR3LG1 foi expresso usando condições semelhantes, e foi recuperado do conteúdo celular por cromatografia de afinidade simples à base de níquel. Verificou-se que as proteínas produzidas estabelecem um complexo de 1: 1 in vitro, conforme determinado por uma abordagem simples de eletroforese em gel nativo, mantendo assim a funcionalidade desejada.
Recombinant protein expression, at a laboratory scale, has been one of the most visible applications of biotechnology. Protein heterologous expression depends on recombinant DNA technology, that, by manipulating the genetic material, allows obtaining almost any protein is the desired amounts. This thesis on cloning and expression of human recombinant proteins involved in immune processes presents the work developed to clone and express the human Natural Cytotoxicity Receptor 3 (NCR3), a surface protein expressed by Natural Killer cells, one of the arms of the innate immune system. Another goal was also to express NCR3's natural ligand, NCR3 Ligand 1 (NCR3LG1). These two proteins are involved in activating the immune system innate response against NCR3LG1-expressing tumoral proteins. A traditional primer-based PCR amplification strategy was employed to amplify the regions to be cloned of the genes of interest from commercial plasmids and clone them into the pET28a(+) vector for T7 promoter-mediated lac-controlled expression of His6 tag-fused proteins in Escherichia coli BL21(DE3) Hi-Control TM cells. hNCR3 was successfully expressed by IPTG-induction and was recovered from purification bodies by on column- protein refolding. hNCR3LG1 was expressed using similar conditions and was recovered from cell contents by nickel-based simple affinity chromatography. The produced proteins were found to establish a 1:1 complex in vitro, as determined by a simple native gel electrophoresis approach, thus retaining the desired functionality.

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Maurer, Ulrike Kerstin. "Die Rolle des späten Natrium-Stroms bei der Kalzium-Calmodulin-abhängigen ProteinkinaseIIδC (CaMKIIδC)-induzierten Herzinsuffizienz und beim chronischen Vorhofflimmern". Doctoral thesis, 2012. http://hdl.handle.net/11858/00-1735-0000-000D-EFD4-3.

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Christians, Claus. "Die Genregulation des myokardialen Na+/Ca2+-Austauschers". Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0001-BBE7-C.

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Die Regulation der Expression des myokardialen Na+/Ca2+-Austauschers spielt eine entscheidende Rolle in der Genese von kontraktiler Dysfunktion und Arrhythmie bei der Herzinsuffizienz. Ein besseres Verständnis dieser Regulationsprozesse könnte neue Angriffspunkte für Präventions- und Therapiestrategien liefern. In der vorliegenden Arbeit wurden die Effekte von α1-adrenerger-Stimulation und mechanischer Last, auf die mRNA-Transkription des NCX 1-Gens und weiterer Gene des Kalziumtransportes untersucht. Es wurden elektrisch stimulierte (1 Hz, 1,75 mmol/l Ca2+, optimale Vorlast), rechtsventrikuläre Trabekelpräparate des Kaninchens nach 3, 6, und 10 h kontinuierlicher α1-adrenerger Stimulation durch 10 umol/L PE untersucht. Diese Trabekel zeigten eine Abnahme der NCX- mRNA im Vergleich zur Kontrolle (gleiche Bedingungen, kein PE) auf 77,5 ± 3,2% nach 10h (p<0,05). Dieser Effekt war sowohl in Anwesenheit des selektiven α1-Rezeptorblocker Prazosin (Praz) (13 umol/L), als auch des PKC-Inhibitors GF 109203X (1 umol/l) nicht zu beobachten. Darüber hinaus konnte keine Regulation des NCX durch PE in nicht gedehnten Trabekeln beobachtet werden. Da vermutet wurde, dass der α1-adrenerge Effekt Ca2+-abhängig sein könnte, wurde die Wirkung erhöhter extrazellulärer Ca2+-Konzentrationen (3 mmol/l) untersucht und eine Abnahme der NCX-mRNA auf 77,4 ± 7,8 % (p<0.02) beobachtet. Des Weiteren wurden in Zellkulturexperimenten über 24 h, isolierte Kardiomyozyten von der Ratte und dem Kaninchen α1-adrenerger Stimulation durch 10 umol/l Phenylephrin ausgesetzt. Die mRNA der Gene von NCX 1, SERCA 2a und BNP wurden mit der real-time PCR bestimmt und gegen das Transkriptionsprodukt des GaPDH-Gens normiert. Bei den Kaninchenmyozyten waren keine signifikante Expressionsänderungen der Gene NCX 1 und SERCA 2a gegenüber der Kontrolle (Kultur ohne PE) zu beobachten, während die mRNA von BNP auf 257,7 ± 47,9% (p<0,03) der Kontrolle anstieg. In Rattenmyozyten wurden signifikante Veränderungen für NCX 1 (117.8 ± 7,9%, p<0,035), SERCA 2a (89,3 ± 3,4%, p<0,012), und BNP (284,2 ± 82,4%, p<0.016) beobachtet. Die genannten Befunde lassen erkennen, dass α1-adrenerge Stimulation die NCX-Transkription in isometrisch kontrahierenden, multizellulären Herzmuskelstreifen bei optimaler Vorlast über die Aktivierung der PKC vermindert. Die verminderte NCX-Transkription scheint durch Kalzium vermittelt und hängt von den Lastbedingungen ab. Die vorliegenden Ergebnisse lassen vermuten, dass endokrine und mechanische Faktoren über eine ineinander greifende intrazelluläre Signalkaskade und Endstrecke, die Regulation der NCX 1-Expression beeinflussen. Unterschiedlich starke Veränderungen dieser Faktoren könnten die unterschiedlichen Phänotypen der Herzinsuffizienz erklären und nach besserem Verständnis neue Möglichkeiten für Prävention und Therapie eröffnen.

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32

Bellmann, Sarah. "Die Bedeutung der Ca2+/Calmodulin-abhängigen Proteinkinase IIδ für die zytosolische Natrium- und Kalziumüberladung sowie Arrhythmogenese in Herzmuskelzellen". Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-000D-F0E3-D.

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33

Raizman, Joshua E. "The effect of NCX1.1 inhibition in primary cardiac myofibroblast cellular motility, contraction, and proliferation". Thesis, 2006. http://hdl.handle.net/1993/239.

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Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.
May 2006

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34

Haddad, Yara. "Interventions thérapeutiques prometteuses dans un modèle in vivo de stéatohépatite non alcoolique". Thèse, 2008. http://hdl.handle.net/1866/3506.

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La stéatohépatite non alcoolique (NASH) est une pathologie du foie dont l’amplitude et les répercussions sont de plus en plus préoccupantes dans le monde médical ou biomédical. Elle est associée à l’obésité, au syndrome métabolique et au diabète sucré de type II. La recherche de la thérapie optimale pour le NASH est un domaine en plein essor puisqu’aucun traitement n’est suffisamment efficace à ce jour. La présente étude fait le point sur de nouvelles possibilités de traitements qui se sont avérés efficaces pour contrer les différentes lésions métaboliques et cellulaires rencontrées dans un modèle in vivo chez le rat où le NASH est induit par l’ingestion d’une diète riche en gras. Cette étude démontre, tout d’abord, que les traitements durant six semaines avec l’acide ursodéoxycholique (UDCA) et son dérivé le NCX 1000, possédant des propriétés donatrices de monoxyde d’azote, à doses équimolaires, protègent de manière équivalente le foie contre le stress oxydatif, l’hyperinsulinémie, l’inflammation et la fibrose causés par la stéatohépatite. De plus, la combinaison d’une plus faible dose de NCX 1000 avec un antioxydant lipophile tel que la vitamine E offre une protection similaire, particulièrement au niveau des paramètres du stress oxydatif. Par ailleurs, l’étude illustre aussi que la silibinine, composé polyphénolique actif du chardon marie (Silybum marianum) et utilisé en traitement pendant 5 semaines, possède un pouvoir hépatoprotecteur, des propriétés antioxydantes et un effet hypoinsulinémique dans ce modèle de stéatohépatite d’origine nutritionnelle. Le potentiel thérapeutique de ces composés en fait des candidats de choix pour le traitement du NASH qui méritent de faire l’objet d’études cliniques poussées.
Nonalcoholic steatohepatitis (NASH) is a serious liver condition related to the metabolic syndrome, obesity, and type II diabetes mellitus whose prevalence is drastically rising in developed countries and worldwide. Several remedies were investigated for the treatment of NASH but an efficient therapy has yet to be developed. In the present study, we explored novel therapeutic possibilities that were thought to be effective for the treatment of experimental high-fat diet-induced NASH the in rat. Our results show that a chronic six week treatment with a high dose of NCX 1000, a derivative of ursodeoxycholic acid (UDCA) with nitric oxide (NO) donating properties, is efficient at reversing steatosis, oxidative stress, inflammation, insulin resistance and fibrosis; major hallmarks of experimental NASH. We also demonstrated that the mother molecule, UDCA, is as efficacious in controlling the same parameters at equimolar doses. Moreover, our study demonstrates that NCX 1000 at lower doses can exert similar potent properties when combined with lipophilic antioxidants like vitamin E. On the other hand, we found that a 5-week treatment with silibinin, the major active component of milk thistle extract, improved liver steatosis and inflammation and decreased NASH-induced oxidative stress, insulin resistance, and fibrosis. These compounds have therefore the potential for being developed for the treatment of NASH. Clinical evidences are needed.

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