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Sperotto, Rita Leal. "Hidrólise de atp e adp em líquor humano." Universidade Federal de Santa Maria, 2008. http://repositorio.ufsm.br/handle/1/11088.
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Adenine nucleotides hydrolysis is an important step of the neuromodulation of CNS and some of its breakdown products are able to protect the nervous tissue against brain injuries. The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in cerebrospinal fluid (CSF) from patients without neural inflammatory process under different conditions and in the presence of several inhibitors. The samples were chose considering the low protein levels, normal glucose levels, low
leukocyte count and CSF differential count. We chose use the supernatant 1 (S1) for enzyme assay due to the best enzymatic activities in this fraction. The best hydrolyze
temperature was 37°C to ATP and ADP. This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in pH 8.0 in the presence of 5mM Ca+2.
Sodum azide inhibited both nucleotide hydrolysis at concentrations higher than 10 mM. Sodium fluoride inhibited the ATP and ADP hydrolysis at concentrations of 15 mM and 20 mM. The Na+ K+ ATPase inhibitor ouabain, did not affect ATP or ADP hydrolysis. The inhibitor P-type ATPase lanthanum 5mM was ineffective on ATPDase hydrolysis. Suramin (30-300 μM) inhibited ATP and ADP hydrolysis and presented a maximal inhibitory effect of 50% at 300 μM. The results of the present study demonstrated that ATP and ADP hydrolysis from human CSF presented a similar response those obtained from rat synaptosomes.A análise do líquor é de grande importância para a detecção de desordens neurológicas de diversas etiologias. O ATP junto a seus produtos de hidrólise (ADP, AMP e adenosina) desempenha importantes funções junto ao SNC, as quais envolvem ações neuroprotetoras em doenças de etiologias variadas. O presente estudo tem como objetivos verificar a ocorrência de hidrólise de nucleotídeos da adenina em líquor de pacientes sem doença inflamatória do sistema nervoso. A determinação da atividade enzimática da NTPDase foi feita em líquor humano em diferentes condições experimentais e na presença de inibidores. As amostras foram escolhidas de acordo com os baixos níveis protéicos, valores normais de glicose e contagem celular diminuída. Foi escolhido o sobrenadante 1 (S1) por apresentar melhores atividades enzimáticas. A melhor temperatura de hidrólise do ATP e ADP foi 37°C. Esta enzima é cátion dependente, sendo a atividade de hidrólise ótima do ATP em presença de 5 mM Ca+2 e o ADP 5-7 mM de Ca+2, ambos em pH 8.0. A azida sódica alterou a atividade enzimática do ATP e do ADP somente nas concentrações mais altas deste inibidor da ATPase mitocondrial. A ouabaína, um inibidor da Na+/K+ ATPase não afetou a hidrólise do ATP/ADP. O inibidor de ATPase tipo-P lantânio (5 mM) foi ineficaz na hidrólise dos nucleotídeos. O suramin (30-300 XM), inibidor específico de NTPDase, inibiu a hidrólise do ATP/ADP e apresentou máximo efeito inibitório na concentração de 300 XM. Os resultados deste estudo mostraram que a hidrólise de ATP/ADP em líquor humano apresentou uma resposta semelhante àquelas obtidas em sinaptossomas de ratos.
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HATIN, ISABELLE. "L'adenylate translocase : un adp/atp transporteur chez plasmodium falciparum." Paris 7, 1994. http://www.theses.fr/1994PA077245.
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L'adp/atp transporteur catalyse l'echange adp/atp a travers la membrane interne des mitochondries. La proteine fonctionnelle est un dimere de deux sous unites identiques de 30 a 34 kda chaque, codee par le genome nucleaire. Cette proteine appartient a une superfamille de transporteurs mitochondriaux. L'objectif de ce travail etait de montrer la presence d'un gene codant pour un adp atp transporteur chez p. Falciparum. L'utilisation repetee de la pcr, nous a permis d'etablir qu'une sequence nucleotidique chez p. Falciparum code pour un polypeptide de 301 acides amines, homologue aux adt mitochondriales. L'alignement de cette sequence avec les sequences deja publiees pour d'autres organismes eucaryotes montre que l'adp/atp transporteur de p. Falciparum comporte toutes les sequences considerees comme importantes pour sa localisation intramembranaire ainsi que pour sa fonctionalite. Le profil d'hydrophilie revele six domaines hydrophobes, potentiellement transmembranaires. Le northern blot des arn a partir de cultures synchrones, montre un faible taux de transcription du messager specifique de l'adp/atp transporteur chez p. Falciparum. La quantification de ces arnm par hybridation in situ a revele une expression plus faible au stade trophozoite qu'au stade anneau et schizonte. Le gene codant pour ce transporteur chez p. Falciparum est localise sur le chromosome 10. L'immuno-electromicroscopie avec un anticorps polyclonal heterologue montre un marquage uniquement dans le membrane interne de la mitochondrie de p. Falciparum
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Lange, Ulf. "Elektrophysiologische Untersuchungen an rekombinanten kardiovaskulären K ATP -Kanälen Effekte von Nukleotiden, neuartigen K ATP -Kanalöffnern und Blockern /." [S.l. : s.n.], 2005. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11947832.
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Kramarova, Tatiana. "Limiting factors in ATP synthesis." Doctoral thesis, Stockholm : Wenner-Gren Institute for Experimental Biology, Stockholm university, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-987.
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Harrington, R. A. "Evolution of ATP synthase." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603734.
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The hypothesis of modular evolution of the atp operon is re-evaluated by conducting a phylogenetic analysis of the atp operons and constituent genes of all fully sequenced genomes. Although the original hypothesis of modular evolution cannot be conclusively supported, a number of novel observations are made. Foremost is that the atp operon is a mosaic operon, a result of extensive horizontal transfer. In addition, two independent duplication events are proposed for subunits b/b’ of the enzyme, the transmembrane subunits I and z are shown to be more prevalent than previously thought, and an α/β gene pair is proposed as a putative ancestral gene duplication for these catalytic subunits. Further studies investigate the evolution and current importance of the P-loop domain, the nucleotide binding domain found with the α and β subunits of ATP synthase. A database has been developed to annotate the structure-function relationships of protein structural domains on a large-scale basis, including a novel facility to describe these relationships in terms of their taxonomic distribution. It is used in conjunction with the previously created PSIMAP structural interaction database and taxonomy databases to describe the P-loop as consistently one of the most diverse domains in the proteome. It is also represented by nodes at critical positions within both the structural interaction network, and a novel protein structure-function bipartite network, which supports the proposal that it is among the oldest and most successful of all domains. The final chapter presents case studies of three P-loop families, and explains the diversity of function, interaction partners and taxonomic distribution exhibited in terms of their structures and sequences. In particular a novel study comparing and contrasting the pore structures of all P-loop rings is presented, and partially conserved motifs in the G-proteins are described to explain their interactivity.
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Sheldon, Jonathan Gary. "Control of ATP turnover." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627507.
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Woźnicka-Misăilă, Aleksandra. "An investigation and characterization of different ADP/ATP Carrier homologs." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV011/document.
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L'objectif principal de ce projet de thèse était d'obtenir de nouvelles données structurales sur les transporteurs ADP/ATP mitochondriaux et de développer des outils pour les approches de micro- et nano-cristallographie appliquées à la biologie structurale des protéines membranaires.Le rôle principal du transporteur ADP/ATP (AAC) est d'importer et d'exporter respectivement de l’ADP3- et l’ATP4- à travers la membrane mitochondriale interne, entre l'espace intermembranaire et la matrice. AAC est le transporteur mitochondrial le mieux caractérisé de toute cette famille de protéines. De nombreuses études ont été menées pour caractériser sa fonction et sa structure. Toutefois, les données structurales n’étant disponibles que pour une conformation de la protéine, de nombreuses questions fondamentales notamment sur les différents états conformationnels adoptés par la protéine au cours du processus de transport restent encore posées. Dans cette thèse, nous avons étudié les 4 isoformes humaines d’AAC. Elles sont impliquées dans diverses maladies génétiques, mais jouent également un rôle dans la cancérogenèse. Cette thèse décrit ainsi en détail la caractérisation structurale et fonctionnelle de ces protéines et leur comparaison. C’est est une étape essentielle pour définir leurs propriétés, et constitue un point de départ précieux dans le développement de nouvelles thérapies.Le domaine de la biologie structurale ne cesse de connaître de nouveaux développements, comme c’est le cas par exemple avec l’avènement de la cristallographie sérielle. Il y a donc un besoin constant de nouvelles approches notamment pour la préparation des échantillons, leur montage sur les lignes de lumière et les collectes de données afin de continuer à améliorer la qualité des données collectées au synchrotron. Ainsi, notre objectif était d'utiliser différents échantillons de protéines membranaires pour développer de nouvelles techniques de cristallisation et de montage d’échantillons sur les lignes de lumière afin de préserver au mieux la qualité des échantillons tout en permettant des collectes de données plus rapides, plus efficaces et plus simplesThe main objective of this PhD project was to gain new structural data on the mitochondrial ADP/ATP carriers and develop tools for micro- and nano-crystallography approaches applied to membrane protein structural biology.The main role of the ADP/ATP carrier (AAC) is to import and export ADP3- and ATP4- respectively between the intermembrane space and the matrix through the inner mitochondrial membrane. AAC is the best characterized among all mitochondrial carriers. Much has been done to investigate its function and structure. However, since structural data are only available for one conformation of the protein some fundamental questions about the different conformational states adopted during the transport process still need to be answered.In this thesis we considered 4 human AAC homologs as a main target. They are involved in different genetic diseases but play also a role in cancerogenesis. This thesis describes and compares in detail the functional and structural characterization of the human AAC isoforms. It was an essential step to give insight into their native properties and is a precious starting point for the drug development field.Since the structural biology field is rapidly developing especially in serial crystallography techniques, there are more and more new applications for samples preparation, mounting and measurements in order to improve the quality of the data collected at the synchrotrons. Hence, our second objective was to use different membrane protein samples to develop new crystal-friendly crystallization set up combined with different sample environment on the beamline toward faster, more efficient and simpler data collection
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Rey, Martial. "Transporteur mitochondrial d'ATP/ADP : étude par échange hydrogène / deutérium couplé à la spectrométrie de masse et caractérisation de mutations pathogènes." Grenoble 1, 2009. http://www.theses.fr/2009GRE10320.
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Le tran'sporteur d'ADP/ATP est une protéine de la membrane interne mitochondriale qui joue un rôle physiologique majeur en catalysant l'échange d'un ADP cytoplasmique contre un A TP néo-synthétisé dans la matrice mitochondriale. Cette protéine peut être inhibée de manière très spécifique par deux poisons naturels, le carboxyatractyloside (CATR) et l'acide bongkrekique (BA) qui stabilisent la protéine dans deux états conformationnels distincts adoptés au cours du mécanisme de transport. Afin de mieux appréhender cette dynamique fonctionnelle, une étude du transporteur d'ADP/ATP bovin en complexe avec le CATR ou le BA dans le détergent Triton X-IOO a été réalisée par échange hydrogène/deutérium couplé à la spectrométrie de masse. Ces travaux se sont déroulés en 4 parties. La première a consisté à adapter cette technique à l'étude des protéines membranaires intégrales. En effet, la présence de détergents, nécessaires au maintien en solution de l'état natif de ces protéines, n'a pas permis jusqu'ici de les étudier par cette approche. Pour pallier cette difficulté, un protocole automatisé de chromatographie liquide permettant l'élimination du Triton X-IOO a été mis au point. Les cinétiques de deutération des différents complexes ont alors pu être analysées dans le deuxième volet de cette étude. Les données obtenues ont permis de proposer des modèles conformationnels du transport de nucléotides au travers de la membrane interne mitochondriale, dans lesquels le transporteur présenterait une cavité ouverte tour à tour vers l'espace intermembranaire et vers la matrice. Afin d'apporter d'autres éléments de réponse sur ce mécanisme de transport et de s'affranchir de différents problèmes liés à l'utilisation des détergents, des essais de deutération du transporteur d'ADP/ATP bovin dans les mitochondries ont été entrepris et représentent le troisième volet de ces travaux. Cette approche, qui nécessite encore plusieurs améliorations, a permis d'obtenir les premières données de deutération d'une protéine membranaire dans son environnement natif. Le transporteur d'ADP/ATP est aussi impliqué dans des pathologies humaines plus ou moins grave. Dans une dernière partie, l'étude de ces mutations a été abordée en réalisant chez la levure Saccharomyces cerevisiae une étude phénotypique et biochimique de plusieurs mutants du transporteur de l'amibe Dictyostelium discoideum correspondant aux mutations humaines. Cette étude a mis en évidence un problème dans le mécanisme intrinsèque de transport, induit par la mutation V291M, qui pourrait être à l'origine de la pathologie associéeThe ADP/ATP carrier is a protein located in the inner membrane of the mitochondria. It plays a major physiological role by catalyzing the exchange of a cytoplasmic ADP against a newly synthesized A TP of the mitochondrial matrix. This protein could be inhibited very specifically by two natural poisons, the carboxyatractyloside (CATR) and the bongkrekic acid (BA), who stabilizes the protein in two distinct conformations involved in the transport mechanism. Ln order to understand this functional dynamic, a study of the ADP/ATP carrier in complex with the CATR or the BA carried out in micelles of detergent (Triton X-lOO) was realized by hydrogenldeuterium exchange coupled to a mass spectrometry analysis. This work was organized in 4 parts. The first part has consisted in an adaptation of this technique to integral membrane proteins. Indeed, the necessity of using detergent to maintain the native state of this kind of protein didn't allow using this approach to study them. To overcome this difficulty, an automatic protocol of liquid chromatography was set up and allows washing out the Triton X-lOO. Ln a second part ofthis work, exchange kinetics were analyzed. The collected data allow us to propose sorne conformational models of the nucleotide transport across the inner membrane. Ln these different models, the mitochondrial carrier would be alternatively open to the intermembrane space or to the matrix with different movements of the peptidic chain. Ln order to bring other data on this mechanism and to avoid several problems due to the detergent, sorne tries of deuteration of the bovine ADP/ATP carrier into the mitochondrial membrane were attempted. This represents the third part of this work. Even if this approach needed sorne amelioration, it provides us the first data of deuteration of a membrane protein in this native environment. The ADP/ATP carrier is also involved in severe human pathologies. Ln a last part, the study of these mutations was investigated. Several mutants of the unique ADP/ATP carrier of the amoeba Dictyostelium discoideum corresponding to human mutants was expressed in the yeast Saccharomyces cerevisiae. Analysis of the phenotype of the strains expressing mutated carriers and the measurement of the biochemical constants of these proteins have pointed out a problem in the intrinsic mechanism of transport due to the mutation V291M and could explain the associated pathology
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Schaffer, Veronika. "Bestimmung der ATP-Freisetzung und des ATP-Abbaus an peripheren Nerven mittels Lumineszenzmessung." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-94932.
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Hendon, Tyler. "IMPACT OF PHOSPHOINOSITIDES ON REGULATION OF K-ATP BY ATP AND HYDROGEN SULFIDE." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5556.
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Hydrogen sulfide (H2S) reduces ischemia reperfusion (IR) injury by stimulating adenosine triphosphate (ATP) sensitive potassium channels (KATP) [1-5]. Demonstrating H2S stimulation is unique to KATP, as other inwardly rectifying potassium (Kir) channels demonstrate inhibition or are unaffected [6]. We recently showed that H2S inhibits Kir2 and Kir3 by decreasing channel sensitivity to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) [6]. Here, we test the hypothesis that H2S regulation of Kir6.2, a pore-forming subunit of the KATP channel, is also dependent on PIP2. Using whole-cell patch-clamp we show that H2S increases the activity of Kir6.2 channels expressed in HEK-293 cells. To study the mechanism, we modulated PIP2 levels by expressing a light- activated phosphatase, or by including high levels of a water-soluble PIP2 analog in the patch pipette. The results suggest that H2S augmentation of Kir6.2 channel activity is increased when PIP2 levels are elevated.
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Ashton, Valerie Lauren. "Probing the conformational changes of the yeast mitochondrial ADP/ATP carrier." Thesis, University of Cambridge, 2013. https://www.repository.cam.ac.uk/handle/1810/245071.
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The mitochondrial ADP/ATP carrier in the inner mitochondrial membrane imports ADP and exports ATP by switching between two conformational states. In the cytoplasmic state, which can be locked by carboxy-atractyloside, the substrate binding site is accessible to the cytoplasm, whereas in the matrix state, which can be locked by bongkrekic acid, the substrate binding site is open to the mitochondrial matrix. Access to the substrate binding site is regulated by salt bridge networks on either side of the central cavity, called the matrix and cytoplasmic salt bridge network. It has been proposed that during transport the salt bridge networks disrupt and form in an alternating way, opening and closing the binding site to opposite sides of the membrane, but experimental evidence has not been obtained for this mechanism. Single cysteine mutations were introduced at the cytoplasmic side of the yeast mitochondrial ADP/ATP carrier, and the mutant carriers were expressed in the cytoplasmic membrane of Lactococcus lactis. They were capable of ADP transport and they could be inhibited by carboxy-atractyloside and bongkrekic acid. The complete inhibition by carboxy-atractyloside demonstrated that the carriers were oriented with the cytoplasmic side to the outside of the cells. To probe the accessibility of the single cysteines, the mutant carriers were locked in either the cytoplasmic or matrix state with the two inhibitors and labelled with the membrane-impermeable sulphydryl reagent eosin-5-maleimide. Specific cysteines that were accessible in the cytoplasmic state had become inaccessible in the matrix state. Subsequent experiments showed that ADP and ATP, but not AMP, led to the occlusion of single cysteines, demonstrating that the cytoplasmic side of the ADP/ATP carrier closes as part of the transport cycle. In addition, cross-linking studies combined with mass spectrometry and electron paramagnetic resonance spectroscopy were tried to probe the closure of the cytoplasmic salt bridge network.
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Shatarat, Amjad. "ATP as a sympathetic neurotransmitter." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12069/.
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ATP has been shown to be a sympathetic neurotransmitter in blood vessels. However, its relative importance has been shown to be influenced by the experimental conditions employed such as alteration of the vascular tone. Thus the main aim was to raise the tone of vascular preparations and to further examine sympathetic neurotransmission in these preparations. Porcine whole mesenteries were perfused with physiological buffer and changes in pressure recorded or different sized mesenteric arteries were isolated and set up for isometric recording. Responses to electrical field stimulation (EFS) were obtained under basal and raised tone conditions induced by U46619, a thromboxane A2 mimetic. The nature of the neurotransmitters involved in the mediation of the electrically-evoked responses was assessed using an α1-adrenoceptor antagonist, prazosin and/or the P2X receptor desensitizing agent, α,β-methyleneATP, an α2-adrenoceptor RX811059 antagonist, and a neuropeptide Y Y1 receptor antagonist BIBP3226. In separate experiments, responses to nerve stimulation were investigated in rat mesenteric small arteries pressurized to 90 mmHg. The effects of a selective α1-adrenoceptor antagonist, YM-12617, and selective P2X1 receptor antagonist, NF-449, on the electrically-evoked response were determined. Under basal tone conditions the electrically-evoked contractile responses in porcine whole mesenteric bed and isolated arteries were exclusively mediated by noradrenaline (NA) since they were inhibited by prazosin. However, under conditions of raised tone, the electrically-evoked responses were enhanced and a role for ATP was evident since these responses were sensitive to α,β-methyleneATP. Responses to exogenous NA and α,β-methyleneATP were also enhanced at raised tone indicating a postjunctional mechanism of enhancement. Nifedipine attenuated the enhanced responses to EFS and α,β-methyleneATP suggesting a possible role for L-type calcium channels in the mediation of the enhanced responses. In rat pressurised mesenteric arteries the electrically-evoked vasocontractile responses were sensitive to YM-12617 and NF-449, indicating that NA and ATP were involved in the mediation of these responses. Raising tone with U46619 in these arteries enhanced the electrically-evoked contractile response; under these conditions responses were sensitive to both YM-12617 and NF-449. The present study supports the observation that ATP becomes a more important sympathetic neurotransmitter under conditions of raised tone in contrast to when tone is absent. In porcine mesenteric vascular preparations NA predominates as the main sympathetic neurotransmitter under conditions of basal tone. However, when tone was raised the responses were enhanced and a role for ATP became evident.
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Wild, Julia Stephanie. "An Investigation into ATP Misses." Thesis, University of Canterbury. Engineering Management, 2014. http://hdl.handle.net/10092/8929.
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This project was carried out in order to complete the requirements of the Master of Engineering Management degree at the University of Canterbury. The project objective was to examine the reasons for Attainment to Plan (ATP) misses at the Meadow Fresh Christchurch plant, specifically the Fresh Beverages division. ATP is a measure of how closely the production team follows the daily packing plan, and is a site Key Performance Indicator (KPI). This report describes the action plans that were developed to decrease the number of misses to the target value, an analysis of the success of these plans, and recommendations which were made around the purchase of plant equipment in order to further improve the ATP results.
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Hamilton, Sara M. "ATP and peripheral sensory systems." Thesis, King's College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325538.
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Green-Petersen, Minna. "Mitochondrial alignment in ATP gradients." Thesis, KTH, Tillämpad fysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-189551.
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Johansson, Monika. "The role of nucleoside diphosphate kinase in plant mitochondria /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200674.pdf.
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Weber, Cornelia [Verfasser]. "The challenge of ATP biosensing - application, investigation and further development of ATP microbiosensors / Cornelia Weber." Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2014. http://d-nb.info/1049238877/34.
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Zheng, Jing-Sheng. "ATP receptors and regulation of the ATP-induced calcium ion mobilization response in cardiac myocytes." Case Western Reserve University School of Graduate Studies / OhioLINK, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=case1056573774.
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Bamber, Lisa. "Yeast mitochondrial ADP/ATP carriers are monomers in detergent and in function." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612861.
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Claes, Richard John. "Discovery of inhibitors of the mitosomal ADP/ATP carrier from Entamoeba histolytica." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648445.
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Cerson, Elizabeth. "Structural and functional studies on mitochondrial ADP/ATP carriers of thermophilic organisms." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648816.
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Plante, Hendrick. "Production d'anticorps polyclonaux contre les récepteurs AT1 humain et ATp de poulet." Sherbrooke : Université de Sherbrooke, 1997.
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Hinchy, Elizabeth. "How cellular ATP/ADP ratios and reactive oxygen species affect AMPK signalling." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/270029.
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Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.
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Kowalke, Julia Maria. "Chemosensitivitätstestung beim primären Mammakarzinom : individuelle Kreuzaktivität üblicher Chemotherapeutika ermittelt mit Hilfe des ATP-Tumorchemosensitivitätsassay (ATP-TCA) /." Köln, 2009. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000256392.
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Knust, Tobias [Verfasser], and Peter [Akademischer Betreuer] Graumann. "Regulation of SMC by associate proteins and ATP = Regulation von SMC durch assoziierte Proteine und ATP." Freiburg : Universität, 2011. http://d-nb.info/112345888X/34.
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Douglas, Corsten Perrie Louise Claire. "Studies of the assembly pathway of human ATP synthase." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267744.
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Human mitochondrial ATP synthase is an enzyme containing 18 unlike subunits located in the inner mitochondrial membrane (IMM), where the catalytic F1 domain extends into the mitochondrial matrix and the FO domain, which contains the c8-ring rotor, the a-subunit and the supernumerary subunits, is anchored in the IMM. All the subunits, apart from the a- and A6L-subunits, are encoded in the nucleus and require transport into the mitochondria before being assembled. The a- and A6L-subunits are encoded on the mitochondrial genome. The respiratory complexes generate the proton motive force (PMF), which ATP synthase uses to generate ATP from ADP and Pi. Rotation of the α- and β-subunits with the central stalk γ-, δ- and ε-subunits is prevented by coupling the F1 domain to the FO domain via the peripheral stalk (the OSCP-, F6-, d- and b-subunits). ATP hydrolysis is prevented by the natural inhibitor of the enzyme, IF1, binding to the F1 domain. In addition to the aand, b-subunits, the FO domain contains the c8-ring and six supernumerary subunits not involved in the catalytic activity of ATP synthase. The roles of five of these subunits in the assembly of ATP synthase, the e-, f-, g-, DAPIT- and 6.8 kDa proteolipid-subunits, were investigated by suppressing or disrupting their expression individually. The e-subunit is the first of the supernumerary subunits to assemble, then the g-subunit followed by the f-, 6.8 kDa proteolipid- and DAPIT-subunits. All five supernumerary subunits investigated were required to facilitate the dimerisation and oligomerisation of ATP synthase. The e-, f- and g-subunits were found to be important for maintaining mitochondrial respiratory capacity.
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Ho, Wei Meng. "Single molecule characterisation of ATP Synthase." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533849.
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Klinker, Henrike. "ATP-dependent nucleosome sliding by ISWI." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-180992.
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Bowers, Keith Cyril. "Pathophysiology of ATP in single cardiomyocytes." Thesis, University of Liverpool, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316576.
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Manning, Benjamin J. "ATP-Dependent Heterochromatin Remodeling: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/795.
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Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure.
First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin.
Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.
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Manning, Benjamin J. "ATP-Dependent Heterochromatin Remodeling: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/795.
Full textAbstract:
Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure.
First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin.
Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.
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Kimura, Yasuhisa. "Analysis of ATP hydrolysis activities of ABC transporters involved in multidrug resistance and K[ATP] channel regulation." Kyoto University, 2005. http://hdl.handle.net/2433/59289.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第11833号
農博第1523号
新制||農||918(附属図書館)
学位論文||H17||N4082(農学部図書室)
UT51-2005-K499
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 和光, 教授 植田 充美, 教授 矢﨑 一史
学位規則第4条第1項該当
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Macedo, Denise Vaz de 1959. "Envolvimento do carreador ADP/ATP nos processos de permeabilização da membrana mitocondrial interna." [s.n.], 1993. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314434.
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Orientador : Lucia Pereira da SilvaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-07-18T12:24:41Z (GMT). No. of bitstreams: 1 Macedo_DeniseVazde_D.pdf: 4866200 bytes, checksum: 168aa5c20312bef638bfa207ebc599b4 (MD5) Previous issue date: 1993
Resumo: Neste trabalho, apresentamos evidências experimentais suficientes para propor o envolvimento direto do carreador ADP/ATP no processo de abertura do poro dependente de Ca2+, responsável pelo fenômeno de transição de permeabilidade da membrana mitocondrial interna. Comparando a proteção conferida pelo ADP ¿ substrato do carreador ADP/ATP, ditiotreitol ¿ redutor de grupamentos sulfidrila e butil hidroxitolueno - sequestrador de radicais livres, mostramos que o ADP sempre foi o mais efetivo contra o dano mitocondrial, quando presente no meio de reação desde o inicio. Esta proteção conferida pelo ADP parece ser contra os efeitos específicos do Ca2+ sobre a membrana, independente do agente liberador utilizado ser um oxidante ou fosfato inorgânico. Esses resultados descartaram a possibilidade de um ataque de radicais de oxigênio aos lipídeos ou proteínas da membrana como o evento primário que dispara a permeabilização mitocondrial. Quando pré-incubamos mitocôndrias desenergizadas com Ca2+, mostramos uma diminuição no conteúdo de translocases ativas na membrana, sensível à presença de ciclosporina A. Estes dados indicaram um envolvimento direto do carreador ADP/ATP na abertura do poro dependente de Ca2+. Nossos resultados descartaram também a oxidação de grupos tiólicos desta proteína como a responsável por sua inativação. Nos experimentos com partículas sub-mitocondriais, demonstramos pela primeira vez a abertura do poro dependente de Ca2+, sensível à ciclosporina A também na membrana invertida das partículas, o que descarta definitivamente a interação da ciclofilina com o carreador ADP/ATP como o mecanismo responsável pela abertura do poro dependente de Ca2+. Esses experimentos também forneceram evidências da existência de dois sítios de ligação para Ca2+ na membrana, com efeitos opostos sobre sua abertura. Os resultados apresentados neste trabalho, no seu conjunto, nos permitiram apresentar a nossa hipótese para o mecanismo molecular de abertura do poro dependente de Ca2+, modulado pelo carreador ADP/ATP. Sugerimos que a ligação do Ca2+ ao carreador, quando este está no estado conformacional "c" induz a dissociação da estrutura dimérica funcional desta proteína, transformando gradativamente o próprio carreador ADP/ATP no poro dependente de Ca2+. Palavras Chave: transição de permeabilidade, poro dependente de Ca2+, carreador ADP/ATP, ciclosporina A, permeabilização da membrana mitocondrial interna
Abstract: In this work we presented sufficient experimental evidences to propose the direct involvement of the ADP/ATP carrier in the permeabilization processes of the inner mitochondrial membrane. Comparing the protection conferred by ADP - a subtrate of the ADP/ATP carrier, dithiothreitol - a disulfide reductant and butylhydroperoxide - a radical scavenger, it was found that ADP was always the most effective against the mitochondrial damage, when present in the incubation medium from the beginning. Our results also indicate that the protection of ADP is against the specific Ca2+ effect in the membrane, independently an pyridine nucleotide oxidant t-butylhydroperoxide or inorganic phosphate were used and discard the possibility of an attack of oxygen radicals on lipids or proteins of the mitochondrial membrane as the primary event that triggers the permeability transition of the inner mitochondrial membrane. Experiments where deenergized mitochondria were preincubated with Ca2+ showed a decrease on the content of active ADP/ATP carrier, indicating a direct involvement of this protein in the formation of an unspecific Ca2+ dependent pore. They also discard the -SH oxidation as a cause of the carrier inactivation. Our experiments with submitochondrial particles provide good evidence for then existence of two binding sites for Ca2+ in the mitochondrial membrane. These resulte also discard cyclophilin as mediator of the pore opening. Key Words: permeability transition, pore Ca2+-dependent, ADP/ATP carrier, cyclosporin A, mitochondrial inner membrane
Doutorado
Bioquimica
Doutor em Ciências
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Moiseeva, Vera. "Caractérisation de l'état oligomérique du transporteur mitochondrial ADP/ATP dans des membranes natives." Thesis, Grenoble, 2012. http://www.theses.fr/2012GRENY018.
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Le passage sélectif de molécules à travers la membrane interne des mitochondries est essentiel aux processus métaboliques des cellules eucaryotes. Cette communication cellulaire est assurée par des protéines transmembranaires de la famille des transporteurs mitochondriaux (MCF). Le transporteur ADP/ATP (AAC) est le membre le plus connu et le mieux caractérisé de cette famille. Il est responsable de l'import d'ADP dans la matrice mitochondriale et de l'export d'ATP après synthèse vers le cytosol. La structure d'AAC est connue mais plusieurs questions restent ouvertes concernant le mécanisme du transport, la sélectivité et l'état oligomérique, controversé, de la protéine. Pendant plusieurs années des études biochimiques réalisées sur la protéine solubilisée en détergent étaient en faveur d'une organisation dimérique du transporteur, mais la structure d'AAC, monomérique a remis en cause ce dogme. Afin de caractériser l'organisation oligomérique d'AAC in vivo, nous avons combiné plusieurs approches. Nous avons réalisé des expériences de FRET (Fluorescence Resonance Energy Transfer) directement sur des cellules mammifères ou bactériennes (E. coli) surexprimant la protéine AAC fusionnée avec des sondes FRET. En parallèle, nous avons mis au point des tests fonctionnels afin de contrôler l'état des mitochondries et l'activité du transporteur dans ces cellules. Enfin nous avons étudié la stoechiométrie de liaison de l'inhibiteur carboxyatractyloside grâce à des mesures de respiration sur des mitochondries extraites de foie de rat et placées dans différents états métaboliques. L'ensemble des résultats présentés dans ce manuscrit ont permis de montrer que 1) l'unité fonctionnelle d'AAC est monomérique 2) l'organisation structurale d'AAC dans les membranes natives dépend de l'état métabolique des mitochondries et peut être associée à des phénomènes de régulationThe transport of small molecules through the inner mitochondrial membrane is essential in eukaryotic metabolism and is selectively controlled by a family of integral membrane proteins, the Mitochondrial Carrier Family (MCF). The ADP/ATP carrier (AAC), which is responsible for the import of ADP to the matrix of mitochondria and the export of newly synthesized ATP toward the cytosol, is the best-known and characterized MCF member. Although its structure sheds light on several aspects of the carrier activity, additional investigations are still required to decipher the whole transport mechanism, to understand the specificity and to characterize the controversial oligomeric state of the protein. For many years, based on studies mainly carried on detergent solubilized AAC the general consensus has been in favor of a dimeric organization of the carrier. The AAC three-dimensional structure, monomeric, broke this dogma. In order to get a precise insight into the in vivo oligomeric organization of AAC we combined several approaches. Fluorescence resonance energy transfer (FRET) measurements were performed directly on mammalian and E.coli cells expressing AAC labeled with several types of FRET probes. In parallel, different functional assays were established to control the state of the mitochondria in these cells and the transport activity of these AAC fusions. Lastly, measurements of the respiration rate coupled to the titration of the inhibitory effect of carboxyatractyloside on isolated rat liver mitochondria were used to investigate the organization of AAC in native mitochondria within two regimes of oxidative phosphorylation. Taken together the results described herein revealed that 1) AAC can function mechanistically as a monomer, 2) the organization of AAC in native membranes might be related to the state of the mitochondria and be involved in regulation
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DI, MARINO DANIELE. "Molecular dynamics and docking simulations of the ADP/ATP mitochondrial carrier: structural-dynamical insights for the inactivation of pathological mutants and detection of potential ATP binding sites." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2010. http://hdl.handle.net/2108/1174.
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Il carrier mitocondriale ADP/ATP (AAC) è stato cristallizato in complesso con il suo inibitore carboxyatractyloside (CATR). La proteina è composta da un fascio di sei eliche trans membrana che forma un canale all'interno della membrane mitocondriale interna che si presenta chiuso verso il lato matriciale ed aperto verso lo spazio intermembrana, conformazione dovuta alla presenza di una prolina sulle eliche dispari che forma una piegatura dell' elica. Il ruolo di questa proteina è di importare ADP nella matrice mitocondriale ed esportare ATP nel citosol. L' insorgenza di alcune patologie è stata associata ad un malfunzionamento di questa proteina. Al fine di capire meglio le proprietà dinamico/strutturali del trasportatore sono stati eseguiti due diversi esperimenti computazionali, per analizzare sia il meccanismo di trasporto che il ruolo di determinate mutazioni patologiche.
In un primo esperimento sono state condotte tre simulazioni di Dinamica Molecolare di 20 ns della protein wild type, del mutante patologico Ala113Pro e del doppio mutante Ala113Pro/Val180Met immerse in un doppio strato lipidico, al fine di capire il ruolo della seconda mutazione Val180Met capace di restaurare il corretto funzionamento del mutante Ala113Pro . L' analisi delle componenti principali ha evidenziato per i tre sistemi che il moto della proteina è caratterizzato dal movimento dei loops matriciali e delle eliche dispari che presentano una proline conservata nella regione centrale dell 'elica. L' analisi del moto mostra un comportamento diverso del singolo mutante rispetto a wild type e doppio mutante. La singola mutazione induce una regolarizzazione dellâ elica H3, che viene persa in seguito allâ introduzione della seconda mutazione. Questo è direttamente correlato alla distribuzione della rete di ponti salini che coinvolge i residui Arg79, Asp134, Arg234 coinvolti nellâ 'interazione con il substrato. Infatti, nella simulazione del wild type sono visibili due ponti salini stabili lungo tutta la dinamica e cruciali per il legame con il substrato, Arg79:Asp134 e Arg234:Asp134. Uno di questi ponti salini è perso nella dinamica del singolo mutante e viene ristabilito nella dinamica del doppio mutante, che si comporta come il wild type. Questo causa nel singolo mutante un errato assetto del sito di legame dellâ 'ADP, spiegando il malfunzionamento del carrier.
Inoltre, abbiamo descritto le interazioni tra il lato matriciale del trasportatore e il substrato ATP attraverso l' utilizzo di simulazioni di dinamica molecolare classica e docking proteina-ligando. Dalla dinamica molecolare di 20 ns del wild type sono state estratte 15 strutture rappresentative della proteina attraverso il clustering, e per ognuna di queste strutture sono state effettuate 50 runs di docking, per un totale di 750 (MD-docking) i risultati sono stati analizzati in comparazione con quelli ottenuti dalle 750 runs di docking effettuate sulla struttura X-Ray (X-docking). L' analisi mostra la presenza di un unico sito di interazione sulla struttura X-Ray, mantenuto anche nelle strutture estratte dalla dinamica. L' MD-docking mostra la presenza di un secondo sito di legame, non presente nell' X-docking. Il meccanismo di interazione tra la proteina e il substrato ATP è stato studiato analizzando la composizione e l' arrangiamento 3D delle 2 tasche di interazione individuate, e l' orientazione del substrato allâ interno di esse. E' stata quindi proposta una relazione diretta tra la struttura tripartite del carrier e la presenza di più siti di legame per l' ATP.The mitochondrial adenosine diphosphate/adenosine triphosphate, ADP/ATP carrier (AAC) has been crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). The protein is composed by a six trans-membrane helix bundle, defining the nucleotide translocation pathway, that is closed towards the matrix side due to sharp kinks in the odd-numbered helices. The role of the protein is to import ADP in the mitochondrial matrix and export ATP in the cytosol. Several disease have been associated to a malfunctioning of the protein. To better understand the structural/dynamical properties of the carrier, two different computational experiments have been performed, in order to understand both the translocation mechanism and the role of known pathological mutations. In a first experiment Molecular Dynamics simulations of the wild type bovine ADP/ATP mitochondrial carrier, and of the single Ala113Pro and double Ala113Pro/Val180Met mutants, embedded in a lipid bilayer, have been carried out for 20 ns to shed a light on the structural-dynamical changes induced by the Val180Met mutation restoring the carrier function in the Ala113Pro pathologic mutant. Principal component analysis indicates that, for the three systems, the protein dynamics is mainly characterized by the motion of the matrix loops and of the odd-numbered helices having a conserved proline in their central region. Analysis of the motions shows a different behaviour of single pathological mutant with respect of the other two systems. The single mutation induces a regularization and rigidity of the H3 helix, lost upon the introduction of the second mutation. This is directly correlated to the salt bridge distribution involving residues: Arg79, Asp134, Arg234; hypothesized to interact with the substrate. In fact, in the wild type simulation two stable inter-helices salt bridges, crucial for substrate binding, are present almost over all the simulation time. In line with the impaired ADP transport, one salt interaction is completely lost in the single mutant trajectory but reappears in the double mutant simulation, where a salt bridge network, as observed in the wild type, is restored. This causes a wrong assembly of the geometry of the binding site, explaining the impaired transport of the single mutant. Further, we describe the interaction between the matrix side of the AAC transporter and the ATP molecule using classical molecular dynamics simulation (MD) and protein-ligand docking procedure. From the 20 ns MD trajectory of the wild type protein, 15 structures have been extracted through clustering analysis and for each carrier conformation 50 docking runs have been carried out for a total of 750 (MD-docking). The results have been compared with 750 docking runs performed on the X-ray structure (X-docking). The docking procedure shows the presence of a single interaction site in the X-ray structure that is conserved in the structures extracted from the MD trajectory. MD-docking shows the presence of a second binding site, not found in the X-docking. The interaction strategy between the AAC transporter and the ATP molecule has been analyzed investigating the composition and 3D arrangement of the interaction pockets, together with the orientations of the substrate into them. A relationship between sequence repeats and the ATP binding sites in the AAC carrier structure is proposed.
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Taher, Mohammed Ahmed A. "Hormonal regulation of ATP binding cassette transporters." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971837139.
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Qiu, Feng. "Regulation of Pannexin 1 Channels by ATP." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/394.
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Pannexins represent a recently discovered second family of gap junction proteins in vertebrates. However, instead of forming intercellular gap junction channels like connexins, pannexins operate as unpaired pannexons, allowing the flux of molecules from the cytoplasm to the extracellular space and vice versa. Pannexins appear to play a vital role in the local control loop of blood perfusion and oxygen delivery. The properties of Panx1 channels indicate that this protein is the most probable candidate for an ATP release channel and is involved in the propagation of intercellular calcium waves. It is also proposed to mediate the large pore formation of the P2X7 receptor death complex. Prolonged activation of this receptor can lead to cell death. There must be some mechanisms to stop this ATP-induced ATP release and opening of the lethal pore. Here we describe a negative feedback loop controlling pannexin 1 channel activity. ATP, permeant to pannexin 1 channels, was found to inhibit its permeation pathway when applied extracellularly. ATP analogues, including BzATP, suramine, and BBG were even more effective inhibitors of pannexin 1 currents than ATP. These compounds also attenuated the uptake of dyes by erythrocytes, which express pannexin 1. The rank order of the compounds in attenuation of pannexin 1 currents was similar to their binding affinities to the P2X7 receptor, except that receptor agonists and antagonists both were inhibitory to the channel. The ATP inhibitory effect is largely decreased when R75 on the first extracellular loop of Pannexin1 is mutated to alanine, strongly indicating that the ATP regulates this channel through binding. To further investigate the structural property of the ATP binding, we did alanine scanning mutagenesis of the extracellular loops and found that mutations on W74, S237, S240, I247 and L266 on the extracellular loops severely impair the BzATP inhibitory effect indicating that they might be direct binding partners for the ligands. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 have largely decreased BzATP sensitivity. Mutations on other residues didn't change the BzATP sensitivity compared to the wild type except for some nonfunctional mutants. All these data demonstrate that some amino acid residues on the extracellular loop of Pannexin 1 mediate ATP sensitivity. However, how these residues form the ATP-binding pocket remains to be elucidated.
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Yang, Yuanjing. "ATP modulatory actions on hippocampal synaptic transmission." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ59415.pdf.
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Hauser, Dominik Roland Johannes. "Purinderivate als mögliche ATP-kompetitive Kinase Inhibitoren /." [S.l. : s.n.], 2004. http://www.gbv.de/dms/bs/toc/396990371.pdf.
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Bélanger, Danny. "Heterologous functional interactions of P2X ATP receptors." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81596.
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Part I. In this work we show that P2X3 currents are acutely modulated by the GPCRs mGluR5 and P2Y2, and by the neurotrophin TrkA receptor, expressed in nociceptors, in the recombinant Xenopus oocyte system. The intracellular C-terminal domain of P2X 3 plays an important role in its functional coupling to TrkA. Preliminary studies suggest a role for PKC in the P2X3-TrkA cross-talk, but other routes may also contribute. Part II. Neurogenic and pharmacological stimulation of vascular smooth muscle P2X1 elicits a contractile response that we found was potentiated by serotonin acting through 5HT2A. We also found in Xenopus oocytes that P2X 1 currents in the desensitized state are potentiated by M1 ACh receptors and by phorbol ester stimulation of PKC. Part III. We have shown in Boue-Grabot et al. (2003) that there was an intracellular negative cross-talk and physical interaction between P2X2 and 5HT3A receptors. We also found a functional interaction between P2X2 and GABAA alpha2beta 3 receptor subtypes in HEK293 mammalian cells and in Xenopus oocytes; and we confirmed the findings of Sokolova et al. , (2001) in primary cultures of DRG neurons. (Abstract shortened by UMI.)
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Chabot-Doré, Anne-Julie. "Metabotropic regulation of ATP-gated P2X3 receptors." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101708.
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Among the ATP-gated channels, the P2X3 subtype is exclusively expressed in nociceptors of dorsal root ganglia (DRG) and trigeminal ganglia, where it plays a major role in enhanced pain sensation observed in chronic pain states. We tested the hypothesis that P2X3 receptors are modulated by metabotropic receptors, such as 5-HT2A, mG1uR5 and trkA, leading to increased P2X3-mediated currents. Double fluorescence labeling confirmed that P2X3-expressing neurons are labeled by the lectin IB4 and we showed that 5-HT2A and mGluR5 receptors, but not trkA, are expressed in a fraction of IB4-positive neurons. Using confocal microscopy, we examined the subcellular distribution of P2X3 and we observed that 5-HT induced a translocation of P2X3 labeling in a significant number of neurons. In Xenopus oocytes, we recorded a short-lasting and kinase-dependent potentiation of P2X3 currents by activation of co-expressed 5-HT2A and mGluR5 receptors. The data presented here show that both 5-HT2A and mG1uR5 are potential modulators of P2X3 receptors in a subset of nociceptors in DRG.
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Dyer, Mark Richard. "Nuclear genes for mammalian mitochondrial ATP synthase." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.256750.
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Contin, Marco. "ATP concentration in the soil microbial biomass." Thesis, Coventry University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270692.
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Huckstepp, Robert T. R. "ATP and mechanisms of central CO2 chemosensitivity." Thesis, University of Warwick, 2009. http://wrap.warwick.ac.uk/2760/.
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ATP release from the surface of the ventro-lateral medulla (VLM) is integral to the hypercapnic response in vivo and can be seen in vitro. By employing horizontal slices of the ventral medulla containing the ventral chemosensitive nuclei, I have developed a model that consistently evokes hypercapnia-induced ATP release in vitro. Using this preparation I have studied CO2-triggered ATP release by means of microelectrode biosensors. I conclude that it is the change in PCO2 itself, and not associated pH changes that accompany it, that is directly responsible for eliciting ATP release from the surface of the VLM. In addition ATP release from this region may have a role in the response to hypocapnia as well as hypercapnia. Using pharmacological agents I have demonstrated that gating of connexin hemichannels mediates ATP release. The dorso-ventral distribution of Cx26 ascertained via quantitative PCR and immunofluorescence makes this hemichannel the most likely candidate. Dye loading the cells responsible for ATP release with carboxyfluorescein, which co-localised with Cx26, revealed these cells reside in the pia mater and subpial astrocytes. Application of gap-junction antagonists, with selectivity towards connexin 26, greatly reduced ATP release in response to elevated CO2 in vitro and in vivo and reduced the tone of ATP at the VLM surface. Moreover, by loading Cx26 expressing HeLa cells with ATP, I was able to recapitulate the entire in vivo response. Therefore I propose that ATP is released from sub-pial astrocytes and leptomeningeal cells through connexin 26 hemichannels in response to alterations in PCO2. Here Cx26 performs a dual role, as both the chemosensory transducer and the conduit for ATP release.
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Smith, Andrew James. "Membrane trafficking of ATP-sensitive potassium channels." Thesis, University of Leeds, 2005. http://etheses.whiterose.ac.uk/365/.
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ATP-sensitive potassium (KATp) channels are known to play a vital role in the regulation of insulin secretion from pancreatic 0-cells. Changes in the ratio of [ATP]/[ADP] within the cell are known to regulate the activity of channels, but very little is known how the number of channels at the cell surface is regulated. The number of channels in the plasma membrane could be regulated in two ways; firstly by regulating the overall population of channels within the cells by increasing/decreasing the rates of channel synthesis or degradation, and secondly by regulating the insertion and removal of channels from the plasma membrane. The aim of the current study is to investigate the involvement of both of these mechanisms in regulating the cell surface density of KATp channels. It is shown that a sudden decrease in glucose concentration causes a rapid stimulation of KATp channel synthesis as shown by both immunocytochemistry and protein chemistry in both INS-le and isolated mouse pancreatic ß-cells. The intensity of fluorescence associated with Kir6.2 and SUR1 was - 2.5 fold greater in cells incubated with 3 mM compared to 25 mM glucose. This sudden increase in channel numbers is due to an increase in the rate of translation of pre-existing mRNA and may be mediated by the activation of AMP-activated protein kinase. Despite the - 2.5 fold increase in channel numbers only a small, but non significant, difference in cell surface density was observed as determined by patch-clamp. The internalisation of KATp channels with an extracellular HA-epitope was also investigated in stably transfected HEK293 cells. Channels were seen to internalise rapidly from the cell surface into a perinuclear compartment. The trafficking itinerary of these channels has been found to include the sorting endosome, late endosome and elements of the trans-Golgi network. Upon inhibition of protein kinase-C activity the internalised channels are redirected into a pathway which allows rapid recycling of the channels. Trafficking and function of KATP channels has also been shown to be disrupted by mutations of Kir6.2 known to cause congenital hyperinsulinism. In summary, it has been demonstrated that both regulated expression and trafficking are likely to be involved in determining the cell surface density of pancreatic KATP channels.
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Gray, Christopher H. "ATP-binding cassette transporters of Paracoccidiodes brasiliensis." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342042.
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Cozens, A. L. "ATP synthase genes in cyanobacteria and chloroplasts." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383121.
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Rabindran, Ray 1964. "Inhibition of tau kinase activity by ATP." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/47691.
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Smith, Katherine Sue. "Autocrine/paracrine regulation of ATP citrate lyase /." The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487683401444039.
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Thomas, Collin Ernest. "Extracellular ATP : transport, metabolism, and physiological significance /." Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.
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