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Ridley, John Malcolm. "Pharmacology of the HERG K⺠channel." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411068.
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Thomson, Steven James. "Deactivation gating and pharmacology of hERG potassium channel." Thesis, University of Leicester, 2012. http://hdl.handle.net/2381/11071.
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hERG (Kv11.1) encodes the α-subunit of the potassium (K+) channel that carries IKr, an important current for repolarisation of the cardiac action potential. Alterations of hERG current, either through inherited mutations that alter gating or through drugs that block the pore, are associated with Long QT syndrome, cardiac arrhythmias and sudden death. The N-terminus has an important role in regulating deactivation, a gating process that is important for timing of the hERG current during cardiac action potentials. Removing the entire N-terminus accelerates deactivation. A crystal structure of part of the N-terminus (residues 26-135) was solved in 1998 and showed it contained a PAS domain, but it did not resolve the structure of the functionally important first 26 residues (NT 1-26). Here we present an NMR structure of residues 1-135. The structure reveals that residues 1-10 are unordered and residues 11-24 form an amphipathic helix one face of which is positively charged. Neutralising the positive charge accelerates deactivation to similar rates as if the whole of the N-terminus has been removed. Neutralising negative charge in the C-terminus also accelerates deactivation. We propose a model where the N and C-termini interact to stabilise the open state of the channel and slow deactivation. Exactly how changes in membrane voltage are transduced into movement of the activation gate is not fully understood. In hERG, the mutation V659A dramatically slows deactivation. Val659 is located in a region where hERG’s activation gate is believed to lie. From the structure of Kv2.1 it can be seen the S4-S5 linker forms a cuff around S6 where the activation gate is thought to be. Using cysteine cross-linking experiments we show that V659C interacts with E544C and Y545C in the S4-S5 linker to lock the channel in the open state. Trapping of drugs in the inner cavity of hERG has been an important model used to help explain why hERG is blocked by so many drugs and with high potency. A series of derivatives of E-4031, a well characterised high-affinity hERG blocker, were made that progressively increased the length of the molecule. Results in this thesis showed these compounds had binding kinetics completely different from E-4031 and none were trapped in the inner cavity. An alternative model of strongly state-dependent drug binding rather than drug-trapping is proposed. Together, the results in this thesis present new insights on the structural basis for deactivation gating and drug binding in hERG channels.
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Pettini, Francesco. "Molecular Dynamics simulation of the hERG channel assisting Precision Medicine in Channelopathies." Doctoral thesis, Università di Siena, 2023. https://hdl.handle.net/11365/1227475.
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In the last two decades, a revolution in biology has shifted the traditional reductive approach to a bottom-up study of virtual models. This discipline, known as System Biology, integrates information coming from individual components, in order to predict the functioning of biological systems, with the idea that complex systems are made up of many independent components that can interact within well-structured networks changing over time, and that the functional properties of biological systems emerge as a consequence of interactions among their components. This paradigm shift is enabled by rapid advancements in technologies providing high-throughput instruments able to analyse in detail biological processes at the single molecule and single cell scale. The vast amount of data produced by these experimental techniques asks for adequate methods of analyses. The present dissertation focues on structural based methods for simulating the functioning of biological molecules, and in particular on the role of Molecular Dynamics simulations. The advantage of Molecular Dynamics simulations is that it is based on physical description of the systems, and consequently it might offer an atomistic description of the process under investigation.
The first chapter of this thesis will provide an introduction on the role of Molecular Genetics and Biology in Medicine, also considering new challenges for the prediction of protein interactions and for development of Precision Medicine. In the Second Chapters, Molecular Dynamics simulations will be discussed, with an emphasis on the methods for data analysis adopted in the research projects presented in the second part of the thesis. The third Chapter will be present the main research project produced during my PhD: the study of inactivation and drug binding in the hERG potassium channel. Side project and parallel collaborations are briefly discussed in the fourth Chapter, followed by concluding remarks.
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Blütner, Carmen. "Der Einfluss des Antiemetikums Droperidol auf den HERG-Ionenkanal /." Hamburg, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000254071.
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Shimizu, Atsuya, Ryoko Niwa, Zhibo Lu, Haruo Honjo, and Kaichiro Kamiya. "Effects of Dronedarone on HERG and KCNQ1/KCNE1 Channels." Research Institute of Environmental Medicine, Nagoya University, 2003. http://hdl.handle.net/2237/7585.
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NIWA, Ryoko, Zhibo LU, Haruo HONJO, and Kaichiro KAMIYA. "Voltage-Dependent Effects of Bepridil on D540K HERG Channels." Research Institute of Environmental Medicine, Nagoya University, 2002. http://hdl.handle.net/2237/2798.
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Niwa, Ryoko, Atsuya Shimizu, Zhibo Lu, Haruo Honjo, and Kaichiro Kamiya. "Voltage-Dependent Effects of Amiodarone on D540K HERG Channels." Research Institute of Environmental Medicine, Nagoya University, 2003. http://hdl.handle.net/2237/7587.
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Du, Chunyun. "The effects of acidosis on the hERG potassium channel." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555619.
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The human ether-a-go-go-related gene (hERG) encodes channels mediating the rapid delayed rectifier K+ current (IKr). IKr participates in cardiac action potential (AP) repolarisation and may also protect the ventricles against premature stimulation. The heart is exposed to acidosis (low pH) in a number of pathological conditions including myocardial ischemia. Extracellular acidosis is known to modulate hERG current (IhERG) function, although a number of aspects of the modulation remain incompletely understood. The aims of this investigation were to establish the effects of acidosis on: (i) IhERG amplitude, kinetics and the response to premature stimulation at mammalian physiological temperature; (ii) the hERG blocking potency of selected anti arrhythmic drugs. Whole-cell patch-clamp recordings of IhERG were made from mammalian cells (CHO or HEK 293) at 37 QC. Lowering external pH from 7.4 to 6.3 reduced the magnitude of IhERG by reducing macroscopic hERG conductance and modulating IhERG kinetics, with positively shifted activation and accelerated deactivation. Results from experiments using an acidic pipette solution showed that the actions of protons occurred from the external surface and not from secondary intracellular acidosis. Experimental and computer simulation work demonstrated that acidosis impairs the protective role of IhERG against premature stimulation. The effects of extracellular acidosis on IhERG kinetics were preserved when the shortened hERG I b isoform was studied, indicating that a full-length N-terminus is not necessary for acidic modulation of hERG channel function. Interestingly, the inhibitory effect of acidosis on IhERG was greater for hERG 1 band hERG lall b than for hERG la. Extracellular acidosis decreased the hERG blocking potency of flecainide, dofetilide and ranolazine, whilst the potency of amiodarone was unaffected. IhERG inactivation was found to be important for ranolazine's inhibitory action and a series of S6 and inner helix residues (Y652, F656, T623, S624 and V625) were identified as contributing to ranolazine binding. I.
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DJUIDJE, ERNESTINE NICAISE. "Design, Synthesis and Biological activities of Benzothiazole, Benzimidazole and Imidazopyrimidine polyphenols as multifunctional molecules against oxidative stress sustained processes." Doctoral thesis, Università degli studi di Ferrara, 2018. http://hdl.handle.net/11392/2488156.
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Reactive oxygen species regulate several essential physiological processes such as cell proliferation, differentiation, vascular tone and inflammation. However, their higher concentrations can have deleterious effects on many molecules including protein, lipid, RNA and DNA which leads to cell destruction causing several diseases including Parkinson’s diseases, inflammatory disease, cardiovascular, cancer, diabetes, Alzheimer’s disease, cataracts, autism and aging. Most of these diseases involve more physiopathological indications. Nevertheless, different pharmacological strategies, such as the use of multifunctional drugs, have been designed to prevent or restore redox imbalances and to treat complex diseases.
The present research project comes from the desire to synthesize multifunctional compounds with the capacity to present or treat multifactorial diseases such as cancer. For this purple, isosteric modification was performed on 2-Phenyl-1H-benzimidazol-5-sulfonic acid (PBSA) and three set of compounds were obtained: benzimidazole, benzothiazole and imidazopyrimidine derivatives. Synthesized compounds were evaluated for their UV-filter, antioxidant, antifungal and antiproliferative activites.
Photoprotective capacity was determined using spectrophotometric transmittance technique. DPPH and FRAP were performed to determine antioxidant activity. Diffusion method in Sabouraud Dextrose Agar (SDA) was used to evaluate anti-dermatophyte activity, while for broth microdilution method in RPMI was used to investigate anti-candida activity. Finally, MTS essay were performed to determine antioroliferative activity.
For benzimidazole derivatives, compound DE 35 was found to be a potential candidate in the development of multifunctional drugs, while for benzothiazole and imidazopyrimidine we respectively have 4g/4k and 14g. In addition, these set of compounds might have possible application as a drug for the treatment of neoplastic diseases such as childhood leukemia, pancreatic cancer and melanoma.Le specie reattive dell'ossigeno (ROS) regolano diversi processi fisiologici essenziali come la proliferazione cellulare, la differenziazione e il tono vascolare. Tuttavia, un'alta concentrazione di ROS può avere effetti indesiderati su molte molecole tra cui proteine, lipidi, RNA e DNA che portano alla distruzione cellulare causando varie pathologie ad esempio: malattie infiammatorie, malattie cardiovascolari, cancro, diabete, cataratta, autismo invecchiamento malattia di Parkinson e malattia Alzheimer. La maggior parte di queste malattie coinvolge più indicazioni fisiopatologiche. Diverse strategie farmacologiche, come l'uso di farmaci multifunzionali, sono state progettate per prevenire o ripristinare gli squilibri di riduzione dell'ossidazione e per trattare malattie complesse. Il presente progetto di ricerca nasce dal desiderio di sintetizzare composti multifunzionali con la capacità di prevenire o curare malattie multifattoriali come il cancro. Per questo motivo, le modifiche isosteriche sono state effettuate sull’ acido 2-fenil-1H-benzimidazol-5-solfonico (PBSA) e sono state ottenute tre serie di composti: derivati benzimidazolici, benzotiazolici e imidazopirimidici. I composti sintetizzati sono poi stati valutati per le loro attività UV filtrante, antiossidante, antifungina e antiproliferativa. La capacità fotoprotettiva è stata determinata utilizzando la tecnica di trasmittanza spettrofotometrica. DPPH e FRAP sono stati eseguiti per determinare l'attività antiossidante. Il metodo di diffusione in Sabouraud Dextrose Agar (SDA) è stato utilizzato per valutare l'attività anti-dermatofiti, mentre il metodo di microdiluizione del brodo RPMI è stato utilizzato per studiare l'attività anti-candida. Infine, è stato eseguito il saggio MTS per determinare l'attività anti-proliferativa. Per i derivati benzimidazolici, il composto DE 35 è risultato essere il potenziale candidato nello sviluppo di farmaci multifunzionali, mentre per i benzotiazolici e l'imidazopirimidici abbiamo rispettivamente 4g / 4k e 14g. Inoltre, questi set di composti potrebbero avere una possibile applicazione come farmaco per il trattamento di malattie neoplastiche come: la leucemia infantile, il cancro del pancreas e il melanoma.
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Ouille, Aude. "Evaluation des risques torsadogènes en pharmacologie de sécurité : du test hERG à la télémétrie sur animal éveillé, vers une évolution des recommandations ?" Thesis, Tours, 2009. http://www.theses.fr/2009TOUR4016.
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Toutes les molécules en développement préclinique (ICH S7B) doivent être testées pour évaluer leur potentiel torsadogène. Le but de ce travail est d’établir le profil électrophysiologique de molécules torsadogènes connues afin de mieux comprendre le mécanisme de déclenchement des Torsades de Pointes et de déterminer des points clés nous permettant de prédire les molécules à risque. Il existe une base de données, TdPScreen®, combinant données cliniques et tests réalisés sur fibres de Purkinje de chien, qui permet d’attribuer un score pro-arythmique aux molécules testées. Treize molécules connues ont été choisies dans cette base de données, et testées en patch-clamp sur des cellules HEK293 exprimant le canal hERG (IKR), le canal KvLQT1+MinK (IKS), le canal Kir2.1 (IK1), le canal NaV1.5 (INa), ou le canal CaV1.2+? (ICaL). Des investigations in vivo ont également été réalisées, afin de mettre en évidence l’impact du système nerveux autonome sur l’allongement de l’intervalle QT lors d’études de pharmacologie de sécuritéAccording to the ICH S7B guidelines, the torsadogenic risk of new drug candidates must be evaluated before clinical trials. The aim of this work was to establish the electrophysiological profile of known torsadogenic drugs to better understand the mechanism triggering the Torsades de Pointe and defined key points for prediction of proarrhythmic risk. TdPScreen®, a predictive tool, based on clinical data and the model of isolated canine Purkinje fibres allows determination of a proarrhythmic score. Thirteen drugs were chosen in this data base, and tested in patch-clamp on HEK293 cells expressing different channels: hERG (IKR), KvLQT1+MinK (IKS), Kir2.1 (IK1), NaV1.5 (INa), or CaV1.2+? (ICaL). In vivo investigations were also performed, to bring to light the impact of the autonomic nervous system on QT interval prolongation in safety pharmacology
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Wu, Yue. "Mutagenesis studies of charged residues in the hERG K+ channel." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52904.
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The loss-of-function mutations in the human ether-à-go-go–related gene (hERG) K+ channel serve as one of the primary genetic substrates for congenital long QT syndrome (LQTS), and unlike most Kv channels with fast-activating kinetics, hERG channels have unusually slow activation kinetics. Thus, it is a necessary task to understand mechanisms of hERG gating, especially since the charge-rich S4 segments play such an important role in channel opening in response to membrane potential changes. Despite the structural homology to other Kv channels, how individual S4 charges in hERG are positioned and moved under the influence of membrane voltage changes remains controversial. Therefore, this thesis focuses on the molecular basis of individual charge residues underlying S4 movement. To monitor S4 movement associated with the voltage- and time-dependence accessibility by the sulfhydryl-specific agent MTSET, we mutated a series of S4 charges (K525-K538) to uncharged glutamine (Q) in a mutant in which cysteine replaced the isoleucine at position 521 near the top of S4. We found that K525, R528, and R534 mainly secure the S4 position in the resting state. Our results on the rate of S4 movement suggest that R528, R531, and R537 potentially facilitate the transition of S4 from the closed to the active state. Conversely, neutralization of the bottom lysine K538 significantly accelerates the rate of S4 movement, implying an important constraint on S4 by K538. To further investigate this unique role of K538, we measure the gating charge contributions of K538 and D411 (a potential ion-pairing in S1) with the cut-open vaseline gap technique. Our data show that mutations of both charges to neutral residues (K538Q and D411N) accelerate the time dependence of charge movement while both mutant channels are gated with faster kinetics than control over a physiologically relevant range of depolarizations. This highlights the substantial contribution of both residues to slow hERG gating.Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
Graduate
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McPate, Mark John William. "hERG potassium channel electrophysiology and pharmacology in the short QT syndrome." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486078.
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K+ channels mediating the rapid delayed rectifier current (lKr) are encoded by human ether-a-go-go-related gene (hERG) and play an important role in determining cardiac action potential (AP) repolarisation and the QT interval of the electrocardiogram. A gain-of-function hERG mutation (N588K) is associated with variant 1 of the Short QT syndrome (SQT1), which is characterised by short QT intervals «320 ms) and increased risk of cardiac arrhythmias and sudden death. Using whole-cell patch-clamp recordings ofhERG current (lhERG) from Chinese Hamster Ovary cells expressing WildType (WT) or N588K-hERG at 37°C, I investigated the effects of the N588K mutation on hERG channel electrophysiology and pharmacology. The N588K mutation produced a -+60 mV shift in IhERG availability, without concomitant alterations ofthe voltage-dependence of activation or deactivation kinetics OfIhERG. N588K IhERG peaked much earlier than did WT IhERG during guinea-pig and human ventricular AP command waveforms. The N588K mutation also resulted in IhERG peaking earlier during atrial and Purkinje fibre AP commands. MiRP1 co-expression with hERG had little effect on the timing ofpeak repolarising current during AP commands, but did affect maximal IhERG density. Results of experiments using paired AP waveforms raise the possibility that the potentially protective role ofhERG against premature depolarisations may to an extent be compromised for N588K-hERG. The N588K mutation differentially attenuated IhERG block of selected antiarrhythmic drugs. My data indicate that in addition to quinidine, disopyramide and amiodarone may be useful pharmacological treatments for SQT1. Co-expression ofhERG-1a with hERG-1b accelerated WT and N588K IhERG deactivation kinetics and for N588K increased attenuation of inactivation compared to hERG-1a alone. The differences in IhERG-Iallb kinetics as a result of the N588K mutation did not greatly influence currents during AP command waveforms, except that peak repolarising current occurred earlier during the AP than for hERG 1a alone.
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Cilia, Emanuele. "Ruolo dei canali del K+ herG nello sviluppo neuronale nella fisiopatologia dell'epilessia." Doctoral thesis, Università degli studi di Trieste, 2008. http://hdl.handle.net/10077/2572.
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2006/2007Università degli studi di Trieste Riassunto Ruolo dei canali di K+ hERG nello sviluppo neuronale e nella fisiopatologia dell’epilessia Ciclo: XX Coordinatore: Chia.ma Prof.ssa Paola Lorenzon Dottorando: Emanuele Cilia Lo scopo di questa tesi è stato quello di individuare e definire una correlazione fra canali della famiglia ERG e la sindrome epilettica. Le motivazioni che hanno spinto ad affrontare sperimentalmente questo argomento risiedono, da una parte nel crescente coinvolgimento dei canali voltaggio-dipendenti nell’epilessia, dall’altra dal fatto che i canali ERG sono altamente espressi nel Sistema Nervoso Centrale (SNC) di topo e di ratto e sono in grado di controllare l’eccitabilità neuronale. Studi di espressione relativi ai geni e alle proteine di questa famiglia sono stati condotti, nel nostro laboratorio, sul SNC di topo (Guasti et al., 2005). Una prima parte del lavoro oggetto della presente tesi ha avuto pertanto lo scopo di approfondire tali studi di espressione, applicandoli anche a colture organotipiche di midollo spinale, ottenute da topi sia in età embrionale che neonatale. Tali studi, nei quali è stata verificata l’espressione dei canali ERG sia a livello di m-RNA che di proteina hanno evidenziato che tutti i geni (e le proteine) m-erg sono espressi in tali colture, seguendo un preciso pattern spazio-temporale (Furlan et al., 2005). Tali studi hanno inoltre permesso di ipotizzare un ruolo importante della corrente ERG IK(ERG) durante le prime fasi dello sviluppo, essendo espresso in maniera specifica ed età-dipendente solo da alcune specifiche popolazioni neuronali. In seguito, una volta completato lo studio del pattern di espressione dei geni e delle proteine ERG nel SNC di topo e di ratto, è stata avanzata l’ipotesi che i canali ERG potessero essere coinvolti nel fenomeno epilettico. Pertanto, la seconda parte del lavoro oggetto della presente tesi si è basato sulla analisi della modulazione dei geni erg nell’ippocampo di topo durante l’induzione di epilessia sperimentale ottenuta tramite l’inoculo di acido Kainico e Pilocarpina. Il lavoro sperimentale si è articolato in due fasi: nella prima fase è stato asportato l’ippocampo di topi inoculati con farmaci epilettogeni (acido Kainico e Pilocarpina) e di topi inoculati con soluzione fisiologica (considerati topi di controllo); nella seconda fase gli ippocampi sono stati tagliati (ad una determinata distanza dal punto Bregma) e sono state asportate tre fette, su cui sono stati eseguiti esperimenti di Real Time PCR al fine di quantificare l’espressione dei geni m-erg1, m-erg2 e m-erg3. Nessuno dei tre geni m-erg è stato modulato in modo significativo a seguito delle crisi epilettiche indotte né da acido Kainico, né da Pilocarpina. Questi risultati apparentemente negativi ci hanno viceversa stimolato a valutare l’ipotesi opposta, e cioè se alterazioni primitive della funzionalità dei canali ERG potessero essere in grado di rappresentare il “primum movens” della malattia epilettica. Nella terza parte della presente tesi, è stata pertanto condotta un’analisi genetica in famiglie affette da epilessia idiopatica valutando eventuali mutazioni del gene herg3, (KCNH7), al fine di valutare un ruolo patogenetico di tale canale in questo tipo di sindrome. La nostra attenzione si è rivolta verso lo studio di questo gene, perché risulta l’unico, della famiglia ERG, ad essere espresso specificamente nel SNC senza alcuna espressione a livello cardiaco, come accade per herg1. A tale scopo è stato messo a punto un protocollo sperimentale per l’analisi del DNA genomico mediante la tecnica della DHPLC (Denaturyng High Performance Liquid Chromatography) e successivo sequenziamento. Sono stati identificati 3 profili mutati, nelle sequenze relative agli esoni 4 (dominio N-terminale della proteina), 6 (regione comprendente la prima porzione transmembrana) e 13 (porzione C-terminale). Sono state inoltre identificate le specifiche mutazioni nucleotidiche che provocano un cambiamento nella sequenza amminoacidica. In particolare a livello dell’esone 13 è risultata un cambiamento nucleotidico a→g, che determina, a livello amminoacidico, un cambio Serina (AA basico)→Glicina (AA neutro). Nell’esone 4 la mutazione c→a determina, a livello proteico, una sostituzione dell’amminoacido basico Istidina (H) con Asparagina (N), molecola neutra. Il profilo dell’esone 6 evidenzia la sostituzione di base a→g in una porzione intronica tra l’esone 6 e 7, questo sito risulta di minor interesse rispetto agli altri perché non viene espresso. E’ stato infine analizzato l’effetto delle mutazioni trovate a livello dell’esone 4 e dell’esone 13 sulle proprietà elettrofisiologiche e sulla localizzazione cellulare. Questi esperimenti sono stati condotti presso il laboratorio del Prof. E. Wanke (Università di Milano Bicocca). Dall’analisi delle proprietà biofisiche della corrente mediata dai canali codificati dai plasmidi mutagenizzati, è emerso che tutte le correnti mediate dai mutanti presentano uno slittamento della curva di attivazione verso valori più iperpolarizzati. In cellule neuronali, dove queste proteine sono in grado di regolare la frequenza di scarica, ciò potrebbe influenzare le proprietà biofisiche alla base dell’eccitabilità cellulare.
XX Ciclo
1976
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Vincent, Yohann. "Étude électrophysiologique de canalopathies d’origine génétique causant des troubles du rythme cardiaque." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10152/document.
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L'unité de recherche EA4612 de l'Université Claude Bernard Lyon 1 s'intéresse à la physiopathologie des troubles du rythme cardiaque, en particulier d'origine héréditaire. Nous avons étudié des mutations de gène de canaux ioniques découvertes chez des patients hétérozygotes atteints d'un syndrome du QT long ou de bradycardie sinusale et de fibrillation atriale. La mutation R148W du gène hERG diminue le courant maximal de 29%. Dans un modèle mathématique, ceci allonge la durée du potentiel d'action ventriculaire, ce qui pourrait rendre compte du phénotype QT long des porteurs. La mutation F627L du gène hERG se situe au centre du motif de sélectivité ionique (GFG) de la protéine hERG. Elle cause une perte de la sélectivité ionique du courant, de la propriété d'inactivation et de la sensibilité aux bloqueurs spécifiques. Ainsi, la présence du groupement aromatique de la chaîne latérale semble essentielle au maintien des propriétés du canal. La mutation Q1476R du gène SCN5A provoque un gain de fonction du courant sodique persistant. Dans un modèle de cellule cardiaque ventriculaire humaine, nous montrons une surcharge sodique intracellulaire pouvant protéger de l'allongement de la durée du potentiel d'action ventriculaire. La mutation D600E du gène HCN4 accélère la désactivation, ce qui pourrait causer une bradycardie. Par ailleurs, la mutation abolit la réponse à la suppression de l'adénosine monophosphate cyclique (AMPc) intracellulaire. La mutation V501M du gène HCN4 cause une perte totale de courant à l'état homozygote. A l'état hétérozygote, l'amplitude moyenne du courant est inchangée par rapport au WT. Cependant, un décalage négatif de la courbe d'activation rendrait compte de la bradycardie des patients porteursThe EA4612 unit of the University Lyon 1 focuses on the pathophysiology of heart rhythm disorders, especially hereditary. We studied ion channel gene mutations discovered in heterozygote patients with long QT syndrome or sinus bradycardia and atrial fibrillation.The R148W mutation of the hERG gene decreases the maximum current by 29%. In a mathematical model, this lengthens the duration of the ventricular action potential, which could account for long QT phenotype of the patients. The F627L mutation of the hERG gene is in the center of the ion selectivity filter (GFG) of the hERG protein. It causes a loss of the ionic selectivity of the current, the inactivating property and sensitivity to specific blockers. Thus, the presence of this aromatic group of the side chain seems to be essential to the maintenance of the channel properties. The mutation Q1476R in the SCN5A gene causes a gain-of-function of the persistent sodium current. In a model of human ventricular heart cells, we show an intracellular sodium overload that can protect against the lengthening of the duration of the ventricular action potential. The D600E mutation of the HCN4 gene accelerates deactivation, which could cause bradycardia. Moreover, the mutation abolishes the response to the suppression of intracellular cyclic adenosine monophosphate (cAMP). The V501M mutation of the HCN4 gene causes a total loss of current in the homozygous state. In the heterozygous state, the average amplitude of the current is unchanged from the WT. However, a negative shift of the activation curve would account for bradycardia in patients
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Shehatou, George S. G. "An investigation of the mechanisms of cellular transformation by hERG potassium channels." Thesis, University of Leicester, 2011. http://hdl.handle.net/2381/10299.
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Human ether-à-go-go-related gene 1 (hERG1) potassium channels are expressed in a variety of tumour cells and expression of hERG1 K+ channels in normal cells can induce a transformed phenotype. The transformative potential of hERG1 appears to be extracellular matrix-dependent. hERG1-expressing NIH-3T3 cells maintained a normal cell morphology when plated on collagen-1 and cell migration speeds were not different to those measured for empty vector-transfected NIH-3T3 cells (NIH-VC). However, hERG1-expressing NIH-3T3 cells displayed a transformed morphology and enhanced cell migration speeds when plated on laminin-1 or fibronectin, and this was associated with a reduction in vinculin protein cell content and cytoskeletal rearrangements. I have provided evidence to indicate that the ion flux through the hERG1 pore and its cell-surface localization is important for its oncogenic potential. Unlike for wild-type hERG1, stable expression of a non-conducting G628S hERG1, or a trafficking-deficient A561V hERG1 mutant did not induce a transformed phenotype in NIH-3T3 cells. Pentamidine, a compound which inhibits hERG1 trafficking to the cell-surface, inhibited fibronectin-dependent migration of wild-type hERG1-expressing cells. Although dofetilide, which blocks the ion conductance of hERG1, did not alter the transformative effect of wild-type hERG1 expression in cell grown on fibronectin, chronic application of this hERG1 inhibitor at a therapeutically-relevant concentration (100 nM) did cause a near-complete reversion of hERG1-expressing cells to a normal cell phenotype within 14 days. NIH-3T3 cells transiently transfected with a plasmid encoding both hERG1 and hERG1b exhibited increases in cell proliferation relative to cells expressing either isoform alone, suggesting a potential role for the hERG1b isoform in regulating hERG1 pro-oncogenic effects. In summary, the transforming potential of hERG1 expression appears to be dependent on hERG1 trafficking to the cell-surface and its ion channel functionality. Chronic administration of hERG1-blockers may be able to impair oncogenic progression in hERG1-expressing tumours.
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Süßbrich, Hartmut. "Untersuchungen zur Pharmakologie und physiologischen Rolle von HERG- und I Ks - Kaliumkanälen /." Sindelfingen : Selbstverl, 1997. http://www.gbv.de/dms/bs/toc/233887849.pdf.
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Raghib, Hala, and halaraghib@yahoo com. "Death By QT: A New Safety Challenge." RMIT University. Medical Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080812.162252.
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The HERG gene encodes for the delayed rectifier K+ channel in human cardiac tissue and contributes to the repolarization phase of the ventricular action potential. Defects in its activity underlies a cardiac disorders linked to a prolongation in the QT interval known as acquired long QT syndrome. The channel has structural properties that lead to its unintentional inhibition by various classes of drugs and is a source of drug induced cardiac toxicity. To date, no assay has been set as a standard due to variability across laboratories and the use of animals providing variable results due to differences in the ion channels involved in repolarisation. This thesis focuses on the development of testing assays for HERG using animal-free methodology. In Chapter 2, a human embryonic kidney (HEK293) cell line was cultured and transfected with the human HERG gene using animal-free methodologies. The success of the transfection was confirmed using PCR, patch clamp electrophysiology and a non-radioactive rubidium assay. Using a non-radioactive rubidium assay, drug inhibition on the transfected cell line was measured. The IC50 values obtained for a range of drugs were compared to those obtained using electrophysiological studies in the literature and there was a high correlation (r2 = 0.76). In Chapter 3, a human neuroblastoma cell line (SH-SY5Y) was tested for its validity for testing the effect of drugs on the endogenously expressed HERG K+ channel. The drug IC50 values obtained using the Rb+ assay were well correlated (r2= 0.82) with patch clamp studies in HERG transfected HEK293 cells in the literature. Clomipramine a clinically used antidepressant causes prolongation in the QT interval, however its mechanism of action on cardiac cells leading to this cardiotoxic effect is unclear. In this study, clomipramine was tested using HERG transfected HEK293 cells and the neuroblastoma cell line (SH-SY5Y) using a rubidium assay and whole cell patch clamp. Clomipramine inhibited HERG with an IC50 value of 8.35 µM and 2.18 µM in HERG transfected HEK293 cells and the neuroblastoma cell line (SH-SY5Y) using the rubidium assay respectively. Clomipramine inhibited HERG currents with an IC50 value of 0.50 µM using the patch clamp technique in HEK293 cells. The results indicate that the prolongation in the QT interval caused by clomipramine may involve HERG inhibition. The HERG K+ channel is regulated by several protein kinases including protein kinase A and protein kinase B. In Chapter 5, the specific PKC activator and phorbol ester PDA was used to study HERG regulation by PKC in HERG transfected HEK293 cells. PDA caused a reduction in HERG currents in HEK293 cells. The PKC pseudo substrate inhibitor PKC [19-36] did not inhibit the effect of PDA on HERG currents. The results of the study suggest that (1) PDA could be acting directly on the channel and inhibiting its function or (2) PDA is activating other proteins which are affecting HERG currents in the HERG transfected HEK293 cells.
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KUKAVICA, DENI. "HERG Channel Agonists: A Novel, Substrate-Based Therapeutic Approach for Long QT Syndrome." Doctoral thesis, Università degli studi di Pavia, 2022. https://hdl.handle.net/11571/1468336.
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Long QT Syndrome (LQTS) is a potentially fatal genetic arrhythmia syndrome, characterized by prolongation of ventricular repolarization, which predisposes young and otherwise healthy individuals to sudden cardiac death.
Despite quantum leaps in our understanding of LQTS, the mainstay of treatment remains the use of beta-adrenergic blockers, which are aimed at control of the arrhythmic trigger, and which do not modify the QT interval duration. Since the duration of the QT interval is a potent predictor of outcomes, our group championed a substrate-based approach aimed at shortening the duration of the QT interval using mexiletine, a class I antiarrhythmic for treatment of Long QT Syndrome type 3 (LQT3). Currently, mexiletine is indicated as a standard-of-care for all patients with LQT3, suggesting that in principle shortening the QTc interval may be an effective strategy to treat all forms of LQTS.
We tested the efficacy and safety of 3-nitro-N-(4-phenoxyphenyl) benzamide (ICA-105574), the most potent HERG1 channel agonist, in different models of LQTS. In Phase One, we successfully developed drug-induced models of type 2, type 3, and type 8 LQTS and used them to investigate the ability of ICA-105574 to safely abbreviate the QT interval in different forms of LQTS. Following the successful preliminary demonstration of efficacy and safety, we exploited the opportunity to study in vivo the effects and safety of ICA-105574 using the first successful knock-in large mammal model of a cardiac channelopathy. Using state-of-the art, clinical grade, ultra-high density mapping of ventricular repolarization, we showed that ICA-105574 significantly reduces the arrhythmogenic gradients of ventricular repolarization identified in our knock-in model of LQTS.
These results offer evidence to support the view that development of HERG1 channel agonists, as a novel class of antiarrhythmics, could have clinical application.Long QT Syndrome (LQTS) is a potentially fatal genetic arrhythmia syndrome, characterized by prolongation of ventricular repolarization, which predisposes young and otherwise healthy individuals to sudden cardiac death. Despite quantum leaps in our understanding of LQTS, the mainstay of treatment remains the use of beta-adrenergic blockers, which are aimed at control of the arrhythmic trigger, and which do not modify the QT interval duration. Since the duration of the QT interval is a potent predictor of outcomes, our group championed a substrate-based approach aimed at shortening the duration of the QT interval using mexiletine, a class I antiarrhythmic for treatment of Long QT Syndrome type 3 (LQT3). Currently, mexiletine is indicated as a standard-of-care for all patients with LQT3, suggesting that in principle shortening the QTc interval may be an effective strategy to treat all forms of LQTS. We tested the efficacy and safety of 3-nitro-N-(4-phenoxyphenyl) benzamide (ICA-105574), the most potent HERG1 channel agonist, in different models of LQTS. In Phase One, we successfully developed drug-induced models of type 2, type 3, and type 8 LQTS and used them to investigate the ability of ICA-105574 to safely abbreviate the QT interval in different forms of LQTS. Following the successful preliminary demonstration of efficacy and safety, we exploited the opportunity to study in vivo the effects and safety of ICA-105574 using the first successful knock-in large mammal model of a cardiac channelopathy. Using state-of-the art, clinical grade, ultra-high density mapping of ventricular repolarization, we showed that ICA-105574 significantly reduces the arrhythmogenic gradients of ventricular repolarization identified in our knock-in model of LQTS. These results offer evidence to support the view that development of HERG1 channel agonists, as a novel class of antiarrhythmics, could have clinical application.
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Crottès, David. "Rôle du récepteur Sigma-1 sur la régulation des canaux ioniques impliqués dans la carcinogenèse." Thesis, Nice, 2014. http://www.theses.fr/2014NICE4032/document.
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Le récepteur sigma-1 est une protéine chaperonne active dans des tissus lésés. Le récepteur sigma-1 est principalement exprimé dans le cerveau et joue un rôle neuroprotecteur dans l’ischémie ou les maladies neurodégénératives. Le récepteur sigma-1 est également exprimé dans des lignées cellulaires cancéreuses et des travaux récents suggèrent sa participation dans la prolifération et l’apoptose. Cependant, son rôle dans la carcinogenèse reste à découvrir. Les canaux ioniques sont impliqués dans de nombreux processus physiologiques (rythme cardiaque, influx nerveux, …). Ces protéines membranaires émergent actuellement comme une nouvelle famille de cibles thérapeutiques dans les cancers. Au cours de ma thèse, j’ai montré que le récepteur sigma-1 régule l’activité du canal potassique voltage-dépendent hERG et du canal sodique voltage-dépendent Nav1.5 respectivement dans des cellules leucémiques et des cellules issues de cancer du sein. J’ai également montré que le récepteur sigma-1, à travers son action sur l’adressage du canal hERG, augmente l’invasivité des cellules leucémiques en favorisant leur interaction avec le microenvironnement tumoral. Ces résultats mettent en évidence le rôle du récepteur sigma-1 sur la plasticité électrique des cellules cancéreuses et suggèrent l’intérêt de cette protéine chaperonne comme cible thérapeutique potentielle pour limiter la progression tumoraleThe sigma-1 receptor is a chaperone protein active in damaged tissues. The sigma-1 receptor is mainly expressed into brain and have a neuroprotective role in ischemia and neurodegenerative diseases. The sigma-1 receptor is also expressed into cancer cell lines and recent investigations suggest its involvement into proliferation and apoptosis. However, its role in carcinogenesis remains to delineating. Ion channels are involved in numerous physiological processes (heart beating, nervous influx, …). These membrane proteins currently emerge as a new class of therapeutic targets in cancer. During my thesis, I observed that the sigma-1 receptor regulates voltage-dependent potassium channel hERG and voltage-dependent sodium channel Nav1.5 activities respectively into leukemic and breast cancer cell lines. I also demonstrated that the sigma-1 receptor, through its action on hERG channel, increases leukemia invasiveness by promoting interaction with tumor microenvironment. These results highlight the role of the sigma-1 receptor on cancer cell electrical plasticity and suggest this chaperone protein as a potential therapeutic target to limit tumor progression
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Chang, Michael Woun Yein. "Investigating the characteristics of drug binding to the inner cavity of hERG potassium channels." Thesis, University of Leicester, 2010. http://hdl.handle.net/2381/10252.
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The human Ether à-go-go Related Gene (hERG) channel makes up the pore forming subunit of the IKr channel. This channel is involved in the repolarisation of the cardiac action potential. Reduction in IKr may cause the prolongation of the action potential, leading to fatal arrhythmias. A large variety of potentially therapeutic compounds inhibit the IKr channels, leading to acquired long QT syndrome. Therefore characterisation of the channel and improvements to in silico models are needed to accurately predict potential hERG side effects. The aim of this project was to use a series of analogues to block the hERG K+ channel, using them as molecular rulers to measure the size of the inner cavity. The phenotype of block was examined to see whether increasing length, results in a change in the phenotype of block from a drug trapping type to a foot in the door one. This would have given an estimate of the size of the hERG inner cavity. Excised inside-out patches of hERG channels display a significant rundown with time after excision. The project also investigated the region of hERG responsible for rundown. A chimeric channel between hERG and bEAG, a closely related channel that does not display rundown, was produced. The aim was to produce a channel with hERG properties, but without the rundown characteristic. This chimeric channel could then be used in excised inside-out patch recordings. The series of derivatives were found to have high affinity to the hERG channel. However, they displayed unusual blocking characteristics, inhibiting the channel in the open state, yet unbound in the closed state. It was also found that the C-terminus of hERG appears to be responsible for the rundown characteristic. The exchange for that of bEAG resulted in a channel that had attenuated rundown.
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Paul, Ashok Abraham. "Investigation of cardiac and non-cardiac drugs that modulate cardiac Herg K⺠channels." Thesis, University of Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274632.
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Pier, David. "The oncogenic potential of human ether-a-go-go-related gene (hERG) potassium channels." Thesis, University of Leicester, 2007. http://hdl.handle.net/2381/29962.
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Human ether-a-go-go-related gene product (hERG) is a potassium channel found ubiquitously in embryonic tissue, but only in key excitable tissues in the adult. hERG is expressed in many tumours of different histological origin, and is not found in the healthy tissues from which the tumour originates. The related EAG channel has been shown to possess oncogenic potential. Having investigated the effect of stable, recombinant hERG expression in NIH-3T3 and HEK293 cell-lines, it was found that there was not significant difference in the proliferative rate or serum-dependency of growth at physiological hERG expression levels, compared to mock-transfected cells. However, hERG expression increased the ability of confluent cells to overgrow each other resulting in a significant increase in post-confluent cell density compared with wild-type cells. In contrast to the parental cell-line, hERG-expressing NIH-3T3 cells grew in soft agar, and exhibited increased rates of cell migration. hERG-mediated transformation appeared to be cell density-dependent, with effects on cell shape and cytoskeletal organization only being seen in confluent cultures. None of the effects of hERG expression were affected by the presence of hERG channel-blocking compounds, or co-expression of non-conducting, dominant-negative hERG subunits. However, hERG-mediated overgrowth seemed to be dependent on p38 MAPK, protein kinase C, phosphoinositide 3-kinase and Src signalling, and independent of MEK/ERK signalling. In addition, hERG expression permitted Src-independent cell proliferation not seen in control cell-lines.;In summary, stable expression of hERG results in NIH-3T3 and HEK293 cell-lines exhibiting some of the characteristics of a transformed phenotype. Pharmacological and molecular biological evidence suggests that this effect on cell phenotype is not dependent on a functional channel current. Instead, the hERG channel protein may interact with protein components of key cell signalling pathways, in a similar fashion to the protein-protein interactions observed for the EAG channel.
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Dalibalta, Sarah. "Human ether-a-go-go related gene (hERG) potassium channel gating and drug block." Thesis, University of Leicester, 2008. http://hdl.handle.net/2381/29964.
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hERG encodes the a-subunit of the rapid delayed rectifier potassium current, a crucial current for normal repolarisation of the cardiac action potential. Pharmacological block of hERG is associated with arrhythmias and sudden death. Given its physiological importance, aspects of both the gating and pharmacology of this channel were investigated.;hERG has unusual gating properties characterised by slow activation and deactivation gating. The roles of conserved S6 glycines (Gly648 and Gly657) in hERG as hinges for activation gating were studied. Glycine residues impart flexibility that is thought to be conducive for channel opening. However, mutations at positions 648 and 657 altered gating in a manner consistent with a role in protein packing rather than flexibility. Deactivation gating in hERG is slow due to interactions between the amino-terminus, the voltage sensor, and the pore that stabilise the open state. The pore mutation V659A dramatically slowed channel deactivation and reduced drug block. Replacing Val659 with larger hydrophobic residues resulted in faster deactivation kinetics, but in contrast, V659G hERG was constitutively open. It was concluded that Val659 mutations influence deactivation through hydrophobic interactions with the S4-S5 linker that couples S6 to the voltage sensor. Effects on drug binding correlated with deactivation rates, indicating that Val659 mutations have allosteric rather than direct effects on drug binding.;Tyr652 is thought to be a critical residue for high affinity drug binding. However, this study showed that the contribution of Tyr652 to drug binding varied considerably among 24 compounds tested, with the majority of low affinity blockers being relatively insensitive to the Y652A mutation. Pharmacophore models generated from the results suggest that higher affinity compounds are longer than lower affinity compounds and simultaneously interact with multiple inner cavity residues. The compact structure of low affinity, Y652A-insensitive drugs permits multiple binding modes, making the compounds less reliant on interactions with Tyr652.
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Choveau, Frank. "Couplages moléculaires à l'origine de la dépendance au potentiel des canaux KCNQ1 et hERG." Nantes, 2009. https://archive.bu.univ-nantes.fr/pollux/show/show?id=52ed54bc-eab2-4b97-8586-b8fcd4f57141.
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Le syndrome du QT long est une anomalie de la repolarisation cardiaque provoquant des troubles du rythme et parfois la mort subite. Il existe sous 2 formes : congénitale et acquise. La forme congénitale est le plus souvent due à une dysfonction d'un canal potassique à 6 domaines transmembranaires dépendant du potentiel : KCNQ1. Actuellement, les mécanismes moléculaires régissant la sensibilité au potentiel de ce canal ne sont pas clairement identifiés. Nous montrons que la boucle S4-S5 est un ligand qui se fixe sur le domaine S6 stabilisant la fermeture du canal : des peptides mimant la boucle S4-S5 inhibent l’activité de KCNQ1. A l'opposé, ceux mimant le domaine S6 l'activent. L'activité accrue de KCNQ1 induite par les peptides S6 pourrait compenser la dysfonction du canal observée dans le syndrome du QT long et donc représenter une nouvelle thérapie. L'inhibition du canal potassique hERG par de nombreux médicaments à visée non cardiaque peut induire la forme acquise du QT long. Cette inhibition serait favorisée par le piégeage de ces molécules dans le pore du canal suite à la fermeture de la porte d'activation. Nous avons bloqué la porte d'activation à l'état ouvert pour évaluer cette hypothèse. De plus, si l'activation et l'inactivation sont couplées, une inhibition réduite de hERG pourrait être due à l'inactivation et non pas l'activation. Nous montrons que (i) l'activation et l'inactivation sont deux processus distincts, et que (ii) la sensibilité de hERG n'est pas réduite par le blocage de la porte d'activation, infirmant l'hypothèse du piégeageThe long QT syndrome is a cardiac abnormality of cardiac leading to altered ventricular repolarization that can lead to arrhythmias, and sudden death. This syndrome exists in 2 forms: congenital and acquired. The congenital form is mostly due to a dysfunction of the six-transmembrane-domain voltage-gated potassium channel: KCNQ1. Currently, the molecular mechanisms controlling the voltage-dependency of KCNQ1 are not clearly identified. We show that the S4-S5 linker acts as a ligand binding to the S6 and stabilizing the channel closed state: peptides mimicking the S4-S5 linker inhibit KCNQ1 whereas those mimicking the S6 domain increase upregulate KCNQ1 activity. This increase in KCNQ1 activity by S6 peptides could compensate for the channel dysfunction observed in the long QT syndrome and thus represent a new therapytherapeutic approach. Off target inhibition of hERG potassium channel by many drugs can induce the acquired form of the long QT syndrome. This inhibition may be favored by the trapping of these molecules in the channel pore of the channel following the closure of the gate activation. To check this hypothesis, we blocked this gate in the open state. Moreover, if the activation and inactivation are coupled, a reduced inhibition of hERG may be due to inactivation rather than activation. We show that (i) activation and inactivation are not coupled, and that (ii) stabilizing of the activation gate is responsible for this inhibition
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Cockerill, Sarah Louise. "Second messenger modulation of the human ether a go-go related gene (HERG) potassium channel." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/29947.
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HERG (human ether-a-go-go related gene) encodes the major pore-funding subunit of IKr, a current which is vital for normal repolarisation of the cardiac action potential. Attenuation of IKr can lead to long QT syndrome, which can predispose individuals to arrhythmias and sudden cardiac death. Given the physiological importance of HERG potassium currents, it is important to understand how they are regulated by intracellular signalling pathways. Whole cell voltage clamp and calcium imaging techniques were used to investigate modulation of HERG channels expressed in HEK 293 cells by second messenger pathways. Stimulating protein kinase C (PKC) by Gaq/11-coupled muscarinic receptor stimulation to elevate diacylglycerol (DAG) and calcium, using OAG (an analogue of DAG), or elevating calcium using ionomycin resulted in a sustained decrease of HERG current. This HERG current response is likely to be mediated by a or b (calcium-sensitive) isoforms of PKC. 32P labelled phosphate incorporation into HERG in OAG treated and non-treated cells was used to determine if PKC directly phosphorylates the channel. Two protein bands at 155 and 135 kDa, corresponding to mature and core glycosylated forms of HERG respectively, were observed in untreated cells, indicating phosphorylation under basal conditions. Stimulation of PKC significantly increased phosphorylation of both bands. HERG currents were also attenuated by activation of cAMP dependent protein kinase (PKA). However, in contrast to PKC, PKA stimulation resulted in a net dephosphorylation of HERG. Overall, the phosphorylation assays suggest HERG channel phosphorylation is dynamically regulated by PKC, PKA and protein phosphatases. In the course of this study it was also found that HERG is directly blocked by caffeine. Caffeine block is open/inactivation state dependent. Caffeine binds within the inner cavity, to sites that include Phe656 and Tyr652.
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Lu, Yu. "Electrophysiological and anti-arrhythmic properties of HERG K+ channels expressed in a mammalian cell line." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620679.
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Bellocq-Lacoustète, Chloé. "Implications physiopathologiques des canaux potassiques HERG et KvLQT1 dans les anomalies de la repolarisation cardiaque." Nantes, 2004. http://www.theses.fr/2004NANT2032.
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Les courants potassiques I Kr et I Ks sont essentiels à la phase de repolarisation du potentiel d'action cardiaque. La canal KvLQT1 s'associe avec sa protéine régulatrice Mink pour former le complexe macromoléculaire à l'origine du courant I Ks. Le courant I Kr est généré par le canal potassique HERG. Des mutations induisant une perte de la fonction des protéines KvLQT1 et HERG sont associées respectivement au syndrome du QT long congénital de type 1 et 2. Le mécanisme sous-jacent est une diminution des courants repolarisants I Ks (LQT1) ou I Kr (LQT2) à l'origine d'un allongement de la durée du potentiel d'action cardiaque. Inversement, une mutation du canal HERG induisant une augmentation du courant I Kr , a récemment été associée au syndrome du QT court. Dans ce travail, nous explorons, à travers l'identification et la caractérisation de plusieurs nouvelles mutations des canaux HERG et KvLQT1, certains des mécanismes physiopathologiques à l'origine de ces canalopathiesHERG and KvLQT1 are two voltage-gated K+ channels respectively responsible for the I Kr and I Ks potassium currents needed for the repolarizing phase of cardiac action potential. KvLQT1 channels are integrated into a macromolecular complex in association with Mink regulatory subunits. The existence and identity of HERG partners are still discussed. Mutations associated with the loss of KvLQT1 or HERG function are respectively responsible for type 1 and type 2 long QT syndrome. The underlying mechanism is a I Ks (LQT1) or I Kr (LQT2) repolarizing currents decrease associated with action potential duration lengthening. Conversely, a new HERG mutation, inducing a gain of channel function, has recently been described in families with short QT syndrome. In this work, by identifying and characterizing new HERG and KvLQT1 mutations, we have explored some of the physiopathological mechanisms responsible for these channelopathies
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Bérubé, Jocelyn. "Effets de changements des conditions environnementales survenant lors de l'ischémie cardiaque sur le canal potassique HERG." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0025/MQ51111.pdf.
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Wilson, Stacey. "Modulation of the hERG potassium channel function by extracellular acidosis : single channel effects and underlying basis." Thesis, University of Bristol, 2018. http://hdl.handle.net/1983/7ef42e09-9a08-4da4-8762-e6797cbad57e.
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Human ether-à-go-go-related gene (hERG) potassium channels underlie the rapid delayed rectifier K+ current (IKr) and play an important role in repolarisation of cardiac action potentials (APs). Pathological events such as cardiac ischaemia can lead to a decrease in extracellular pH (acidosis). Extracellular acidosis is known to modulate the hERG current (IhERG) but the underlying mechanism(s) are not completely known. The aims of this study were: (1) to establish the effects of acidosis on macroscopic and single-channel IhERG, investigating both hERG1a and hERG1b isoforms; (2) to use an amino acid modifying reagent and site-directed mutagenesis to probe the molecular basis of proton modulation of IhERG, focusing on the hERG1a isoform. Whole-cell and cell-attached patch-clamp recordings were made at ambient temperature of wild-type IhERG from mammalian cell lines (HEK-293 or CHO). When external pH (pHe) was reduced from 7.4 to 6.3, macroscopic IhERG amplitude and conductance decreased, activation was positively shifted, and deactivation kinetics were accelerated. Results obtained in the cell-attached configuration showed a reduction at pHe 6.3 in single-channel IhERG amplitude and conductance, decreased open- and burst-durations and increased closed-time durations. These effects at the single-channel level account for the modulation of macroscopic IhERG by acidic pHe. The first known single-channel recordings from hERG1b showed that this isoform retained sensitivity to acidic pHe, indicating that N-terminal differences between the two isoforms are not critical for proton sensitivity. Experiments completed with a range of extracellular pH values (4.5 – 8.0) revealed that different features of IhERG have distinct pKa values, suggesting multiple sites of proton modulation. Titratable residues located in the pore region of the hERG1a channel were mutated to determine if they were responsible for pH sensitivity. The double mutation E575Q/H578N appeared to remove the proton reduction of channel conductance, thus identifying a novel proton sensor on the hERG channel.
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Melgari, Dario. "Inhibition of the hERG potassium channel by flecainide and ivabradine : binding sites and mechanism of block." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.688218.
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Human ether-a-go-go-related gene (hERO) potassium (K+) channels carry the rapid delayed rectifier K+ current (IKr) which contributes to the repolarisation of cardiac action potentials. lKr plays a protective role against premature beats and is highly prone to pharmacological inhibition that can lead to acquired Long QT syndrome. Flecainide (a class le antiarrhythmic agent) is used in the treatment of atrial fibrillation; it inhibits IhERg/IKr at clinically relevant concentration and it has been occasionally associated with life-threatening ventricular tachycardia (Torsade de Pointes). Ivabradine is a specific bradycardic agent with a high selectivity for hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels. However, some evidence has raised the possibility that it may affect IKr. The principal aims of this study were to investigate the mechanism of interaction and the molecular determinants of pharmacological block of hERG by flecainide and ivabradine. In an additional set of experiments, the modulation by extracellular potassium ([K+]o) of the response of hERG to premature stimulations was examined.
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Asher, Viren. "The expression of EAG and HERG potassium channels in ovarian cancer and their role in cell proliferation." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594215.
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Background Ovarian cancer is the second most common cancer of the female genital tract in the United Kingdom (UK), accounting for 6% of female deaths due to cancer. This cancer is associated with poor survival and there is a need for new treatments in addition to existing chemotherapy to improve survival. Potassium (K) channels have shown to be promising therapeutic targets in the treatment of various cancers. We sought to determine the expression of EAG and HERG potassium channels in ovarian cancer and establish their role in cell proliferation. Material and Methods The Trent Research and Ethics Committee granted initial approval for the study. Informed consent was obtained from all patients undergoing surgery for ovarian cancer and oopherectomy for benign causes for their participation in the study. The tissues were prospectively collected and analysed anonymously. Immunoflurescence. Immunohistochemistry, Western blotting and Reverse transcriptase Polymerase chain reaction experiments were used to determine the expression of EAG and HERG potassium channels in ovarian cancer and normal ovaries. The effect of the EAG blockers (imipramine and clofilium) and HERG blockers (E-4031 and ergtoxin) on SK~OV-3 ovarian cancer cell line proliferation was assessed using the MTS assay with further investigation of their role in the cell cycle and apoptosis determined by flow cytometry. Results EAG and HERG potassium channels have significant (P<0.001) higher expression in patients with ovarian cancer compared to normal ovarian cells and high expression of EAG channels is significantly associated with poor survival (P=0.016) unlike HERG channel expression where there was no correlation with survival. There was also a significant association of EAG staining with high tumour grade (p=0.014) and presence of residual disease (p=0.011). Proliferation of SK-OV-3 cells was significantly (p<0.001) inhibited after treatment with voltage gated K+ channel blockers. There was significant inhibition of proliferation of SK-OV- 3 ovarian cancer cells by imipramine (p<0.001) and ergtoxin (p<0.05) at 72 hours of culture. Incubation of cells with ergtoxin led to the accumulation of cells in the S and G2IM phase while cells accumulated in S phase after incubation with E-403 1, with no effect on apoptosis.imipramine did not affect the cell cycle but increased the proportion ofSK-OV-3 cells undergoing early apoptosis. Conclusion Both EAG and HERG channels are expressed in ovarian cancer and have a role in cell proliferation. Higher expression of EAG channel is associated with poor prognosis suggesting its role as a poor prognostic marker in patients with ovarian cancer. HERG channels affect the cell cycle while EAG channels are implicated in the inhibition of apoptosis of ovarian cancer cells. EAG channels have the potential to be used as new therapeutic targets in patients with ovarian cancer with use of anti-EAG monoclonal antibody.
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Yoshida, Hidetada. "Characterization of a novel missense mutation in the pore of HERG in a patient with long QTsyndrome." Kyoto University, 2001. http://hdl.handle.net/2433/150536.
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Danielsson, Christian. "Role of the hERG-channel in arrhythmia and teratogenicity studies in animal models and the human embryonic heart /." Stockholm, 2010. http://diss.kib.ki.se/2010/978-91-7409-831-0/.
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Vasseur, Lucie. "Optimisation de la production et de la purification du canal hERG en vue d’une caractérisation biophysique et structurale." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT127/document.
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La protéine humaine hERG (human ether-à-go-go related gene) s’associe en homo-tétramère pour former le canal potassique voltage-dépendant Kv11.1. C’est un acteur majeur de la repolarisation du potentiel d’action cardiaque par sa capacité à externaliser le potassium du cardiomyocyte. L’altération de sa fonction induit le syndrome du QT long à l’origine d’arythmies cardiaques et pouvant conduire à un arrêt du cœur. Ce syndrome parfois génétique provient le plus souvent d’une inhibition pharmacologique. De nombreux médicaments ont montré leur capacité à inhiber hERG en se fixant dans la lumière du canal. L’étude des interactions moléculaires entre hERG et médicaments intéresse les scientifiques depuis de nombreuses années. Très récemment, la première structure atomique de hERG à l’état ouvert par cryo-microscopie électronique a permis une avancée majeure dans la compréhension de l’agencement du pore du canal. De nombreuses questions restent malgré tout non résolues concernant les mécanismes de liaison des ligands. Plus encore, le développement d’approches biophysiques à partir de canal purifié permettraient de caractériser et d’anticiper des interactions avec les médicaments. Dans cette perspective, nous avons testé plusieurs stratégies pour obtenir le canal hERG purifié dans une forme stable, homogène et fonctionnelle. Notre étude est basée sur une construction simplifiée et chimérique du canal hERG, la version hERG(S1-coil). Chaque étape permettant la production et la purification d’une protéine membranaire a été optimisée en testant différentes techniques proposées par la littérature. Nous avons comparé les rendements d’expression du canal dans différents systèmes recombinants procaryotes ou eucaryotes. La quantité de protéine totale et le pourcentage de protéine fonctionnelle dans les membranes ont été étudiés. Dans un deuxième temps, le canal a été solubilisé puis purifié. Nous avons comparé les rendements de solubilisation et la stabilité protéique en fonction du type de détergent. En parallèle, nous avons mis au point des moyens techniques pour évaluer la fonction du canal au fur et à mesure du processus de production et purification. Le canal hERG(S1-coil) tétramérique et fonctionnel a finalement été identifié dans la fraction purifiée. Cependant, des optimisations sont encore à apporter pour conserver l’agencement tétramérique et empêcher l’agrégation au cours du temps avant de pouvoir envisager des études biophysiques et structurales. A terme, ces travaux pourraient profiter à la production et à la purification d’autres protéines membranaires oligomériquesThe human protein hERG (human ether-à-go-go related gene) assembles as homo-tetramer to form the voltage-gated potassium channel Kv11.1. This channel is involved in repolarization of the cardiac action potential by regulating the potassium release from cardiomyocytes. hERG malfunction was found to cause long QT syndrome, a disorder that predisposes affected patients to arrhythmias and sudden death. This can be due to congenital mutation in the hERG gene and, most frequently, it is caused by pharmacological agents. Several drugs are known to block the channel ion pathway, resulting in off-target inhibition of hERG. Consequently, understanding the molecular basis of drug binding to hERG has become a high priority. The recent determination of a near-atomic resolution structure of the opened channel, using cryo-electron microscopy, provides insights into how this channel work. But several questions are still unanswered to understand the mechanisms of hERG function and drug binding. Moreover, new biophysical protocols with the purified hERG channel would help scientists and industries to anticipate drug side effects. In this context, we investigated strategies to purify a stable, homogenous and functional hERG channel. Our study was based on a shorter and chimeric hERG channel, the hERG(S1-coil) version. We optimized each step from production to purification of membrane proteins by testing experimental protocols found in the literature. In this thesis project, we first compared production rates of the channel in several prokaryote and eukaryotes recombinant systems. Total protein produced and the percent of functional channel were investigated in membranes from each recombinant system. Then, the channel was extracted from membranes before purification. Solubilizing rates and channel stability were compared depending on detergents. In another hand, we also developed protocols to investigate the channel stability and function along production and purification. A tetrameric and functional channel was finally purified and identified by this strategy. More work however is still needed to improve channel homogeneity and stability before to be suitable for biophysical and structural studies. In the future, this work could also help investigations in production and purification of other oligomeric membrane proteins
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Macdonald, Logan Campbell Alexander. "Gating and pore block of the human ether-à-go-go related gene (hERG) voltage-gated potassium channel (Kv11.1)." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62066.
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Ion channels are integral membrane proteins that form an aqueous pore through the cell lipid bilayer, and allow ions to traverse the membrane at rates approaching limits set by diffusion. Selectivity and gating differences amongst members of this protein family enable complex physiological processes such as action potentials. The diversity in ion channel selectivity and gating is endowed through structural permutations of protein structure that slightly alter factors such as the rate at which a channel activates or the width of the pore region and thus the type of ions it interacts with. This thesis investigates structural bases for the anomalous gating and drug interaction behaviour exhibited by the human ether-à-go-go related gene (hERG) voltage-gated potassium channel (VGKC). The unique gating kinetics of hERG allow it to fulfill its role as the rapid delayed rectifier potassium current of the cardiac action potential and the unique susceptibility to drug block can compromise this function. Chapter 2 describes how slow deactivation of hERG can be largely attributed to cytosolic domain interaction with channel gating, an interaction that serves to establish a mode shift of the channel gating charge, shifting the deactivation gating pathway to more hyperpolarized potentials. Chapter 3 demonstrates that an interaction between an acidic residue at the bottom of the S1 and a basic residue at the bottom of the S4 stabilizes the closed state of the channel and slows activation. Through gating currents and fluorescence experiments, we propose a model of hERG gating in which this unique interaction stabilizes an early closed state of the channel. Chapter 4 investigates the role of cation-π interactions in hERG drug block, testing the importance of the two most significant residues for drug interaction, Y652 and F656. Using unnatural amino acid mutagenesis, this final study shows that cation-π interactions do not appear to play a major role in drug interaction with the hERG pore.Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
Graduate
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Riochet, David. "Regulation des canaux potassiques par l'expression de proteines membranaires." Nantes, 1999. http://www.theses.fr/1999NANT16VS.
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Bahrke, Anna Sophia [Verfasser], and Michael [Akademischer Betreuer] Brunner. "Effekt des hERG-Aktivators NS1643 auf die kardiale Repolarisation transgener Kaninchen mit Long QT Syndrom in-vivo und in-vitro." Freiburg : Universität, 2010. http://d-nb.info/1123465339/34.
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Thomas, Gaëlle. "Ciblage de l'entité HER2/HER3 par des anticorps monoclonaux pour le traitement des cancers du pancréas exprimant faiblement HER2." Thesis, Montpellier 1, 2013. http://www.theses.fr/2013MON1T011.
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Avec un taux de survie à 5 ans inférieur à 5%, l'adénocarcinome pancréatique (PDAC) est l'un des cancers les plus agressifs et pour lequel les thérapies existantes sont largement insuffisantes. Ce cancer est caractérisé par un tissu fibreux dense et un stroma très développé, en constante interaction avec la tumeur, favorisant le développement d'un environnement propice à la progression tumorale. Actuellement, la gemcitabine est la seule chimiothérapie approuvée capable de prolonger légèrement la survie des patients. Une thérapie ciblée dirigée contre l'EGFR, l'erlotinib, a prouvé que le ciblage de cette famille pourrait être une stratégie intéressante dans cette pathologie mais qu'une sélection des patients était indispensable pour augmenter les réponses thérapeutiques. Deux récepteurs de cette famille, HER2 et HER3, dimérisent pour former une entité particulièrement agressive et impliquée dans la croissance tumorale des PDACs. Diverses techniques récemment développées sont utilisées pour quantifier ces dimères, afin d'étudier leur rôle, mais également pour tenter de les cibler, et empêcher leur signalisation dans différents cancers. A l'heure actuelle, le pertuzumab est le seul anticorps monoclonal dirigé contre le récepteur HER2, capable de bloquer sa dimérisation, et utilisé en clinique. Dans un premier temps, nous nous sommes intéressés au rôle de HER3 en tant que cible et marqueur pronostique de l'effet du pertuzumab sur les PDAC exprimant faiblement HER2. Puis dans une deuxième partie, nous avons étudié et comparé les effets de différents anticorps monoclonaux dirigés contre HER2 et/ou HER3, sur la prolifération tumorale de tumeurs du pancréas. L'ensemble de ces résultats a permis d'établir que le ciblage du dimère HER2/HER3 s'avère être une stratégie prometteuse pour inhiber la croissance des tumeurs du pancréas exprimant faiblement HER2, et que le récepteur HER3 pourrait être un marqueur pronostique de l'effet du pertuzumabWith a 5-year survival lower than 5%, PDAC is one of the worst cancers in terms of mortality, and for which existing therapies are unsatisfying. This cancer is characterized by a dense fibrotic tissue and an over-developed stroma, in continual interaction with the tumor, promoting the development of an ideal environment for tumor progression.To date, gemcitabine is the only approved chemotherapy able to slightly increase patients' survival. The use of erlotinib, an EGFR targeting therapy, underlined that EGFR family targeting could be an interesting treatment strategy in this pathology, but that a better patients' selection is essential to increase therapeutic response. Two receptors of this family, HER2 and HER3, are able to dimerize to consititute an aggressive entity, involved in PDAC tumoral growth. Various recently developed techniques are used to quantify those dimers, in order to study their role, but also to target them and block their signaling in cancer cells. Pertuzumab is currently the only HER2-targeting monoclonal antibody able to block its dimerization and used in clinic. We first evaluated the role of HER3 as therapeutic target and prognostic marker of pertuzumab efficacy on HER2-low expressing PDACs. We then studied and compared therapeutic effects on pancreatic tumor proliferation of different antibodies targeting HER2 and/or HER3. Taken together, those results demonstrated that HER2/HER3 dimers targeting is a promising strategy to inhibit low-HER2 expressing pancreatic tumor growth, and that HER3 could be a pronostic marker for pertuzumab efficacy in those cancers
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Xu, Duo, and Duo Xu. "Hera and Her Sanctuaries." Thesis, The University of Arizona, 2017. http://hdl.handle.net/10150/624103.
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Hera is among the most puzzling figures in Greek religion who has not been discussed fully in terms of the relationship between her personalities in myths, literature and art and her sanctuaries. Scholars have attempted to discern reasons behind the dichotomy that she was represented as a jealous wife and she was at the same time worshipped solemnly. But, these explanations are limited only to speculative evidences. Also, scholars, working on Hera, tend to focus on only one aspect of the goddess, whether it is archaeology, philology or religion.
I propose to conduct a holistic analysis of Hera, from her depictions in literature, art and myths to her major sanctuaries in the Greek world. I aim to look at multiple evidences: textual, iconographical, archaeological evidences. In short, the dichotomy of Hera helps consolidate the formation and the rising of Greek poleis, and it also empowers and encourages ancient Greek women to go through life crises.
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Chavan, Swapnil. "Towards new computational tools for predicting toxicity." Doctoral thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-51336.
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The toxicological screening of the numerous chemicals that we are exposed to requires significant cost and the use of animals. Accordingly, more efficient methods for the evaluation of toxicity are required to reduce cost and the number of animals used. Computational strategies have the potential to reduce both the cost and the use of animal testing in toxicity screening. The ultimate goal of this thesis is to develop computational models for the prediction of toxicological endpoints that can serve as an alternative to animal testing. In Paper I, an attempt was made to construct a global quantitative structure-activity relationship (QSAR)model for the acute toxicity endpoint (LD50 values) using the Munro database that represents a broad chemical landscape. Such a model could be used for acute toxicity screening of chemicals of diverse structures. Paper II focuses on the use of acute toxicity data to support the prediction of chronic toxicity. The results of this study suggest that for related chemicals having acute toxicities within a similar range, their lowest observed effect levels (LOELs) can be used in read-across strategies to fill gaps in chronic toxicity data. In Paper III a k-nearest neighbor (k-NN) classification model was developed to predict human ether-a-go-go related gene (hERG)-derived toxicity. The results suggest that the model has potential for use in identifying compounds with hERG-liabilities, e.g. in drug development.
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Crottès, David. "Rôle du récepteur Sigma-1 sur la régulation des canaux ioniques impliqués dans la carcinogenèse." Electronic Thesis or Diss., Nice, 2014. http://www.theses.fr/2014NICE4032.
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Le récepteur sigma-1 est une protéine chaperonne active dans des tissus lésés. Le récepteur sigma-1 est principalement exprimé dans le cerveau et joue un rôle neuroprotecteur dans l’ischémie ou les maladies neurodégénératives. Le récepteur sigma-1 est également exprimé dans des lignées cellulaires cancéreuses et des travaux récents suggèrent sa participation dans la prolifération et l’apoptose. Cependant, son rôle dans la carcinogenèse reste à découvrir. Les canaux ioniques sont impliqués dans de nombreux processus physiologiques (rythme cardiaque, influx nerveux, …). Ces protéines membranaires émergent actuellement comme une nouvelle famille de cibles thérapeutiques dans les cancers. Au cours de ma thèse, j’ai montré que le récepteur sigma-1 régule l’activité du canal potassique voltage-dépendent hERG et du canal sodique voltage-dépendent Nav1.5 respectivement dans des cellules leucémiques et des cellules issues de cancer du sein. J’ai également montré que le récepteur sigma-1, à travers son action sur l’adressage du canal hERG, augmente l’invasivité des cellules leucémiques en favorisant leur interaction avec le microenvironnement tumoral. Ces résultats mettent en évidence le rôle du récepteur sigma-1 sur la plasticité électrique des cellules cancéreuses et suggèrent l’intérêt de cette protéine chaperonne comme cible thérapeutique potentielle pour limiter la progression tumoraleThe sigma-1 receptor is a chaperone protein active in damaged tissues. The sigma-1 receptor is mainly expressed into brain and have a neuroprotective role in ischemia and neurodegenerative diseases. The sigma-1 receptor is also expressed into cancer cell lines and recent investigations suggest its involvement into proliferation and apoptosis. However, its role in carcinogenesis remains to delineating. Ion channels are involved in numerous physiological processes (heart beating, nervous influx, …). These membrane proteins currently emerge as a new class of therapeutic targets in cancer. During my thesis, I observed that the sigma-1 receptor regulates voltage-dependent potassium channel hERG and voltage-dependent sodium channel Nav1.5 activities respectively into leukemic and breast cancer cell lines. I also demonstrated that the sigma-1 receptor, through its action on hERG channel, increases leukemia invasiveness by promoting interaction with tumor microenvironment. These results highlight the role of the sigma-1 receptor on cancer cell electrical plasticity and suggest this chaperone protein as a potential therapeutic target to limit tumor progression
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Carstens, Johanna J. "Identification of the modulators of cardiac ion channel function." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/2163.
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Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009.The human ether-à-go-go-related gene (HERG) encodes the protein underlying the cardiac potassium current IKr. Mutations in HERG may produce defective channels and cause Long QT Syndrome (LQTS), a cardiac disease affecting 1 in 2500 people. The disease is characterised by a prolonged QT interval on a surface electrocardiogram and has a symptomatic variability of sudden cardiac death in childhood to asymptomatic longevity. We hypothesised that genetic variation in the proteins that interact with HERG might modify the clinical expression of LQTS. Yeast two-hybrid methodology was used to screen a human cardiac cDNA library in order to identify putative HERG N-terminus ligands. Successive selection stages reduced the number of putative HERG ligandcontaining colonies (preys) from 268 to 8. Putative prey ligands were sequenced and identified by BLAST-search. False positive ligands were excluded based on their function and subcellular location. Three strong candidate ligands were identified: Rhoassociated coiled-coil containing kinase 1 (ROCK1), γ-sarcoglycan (SGCG) and microtubule-associated protein 1A (MAP1A). In vitro co-immunoprecipitation (Co-IP) and mammalian two-hybrid (M2H) analyses were used to validate these proposed interactions, but failed to do so. This should be further investigated. Analysis of confirmed interactions will shed light on their functional role and might contribute to understanding the symptomatic variability seen in LQTS.
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Göstring, Lovisa. "Cellular Studies of HER-family Specific Affibody Molecules." Doctoral thesis, Uppsala universitet, Enheten för biomedicinsk strålningsvetenskap, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-156730.
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The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.
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Österberg, Fredrik. "Exploring Ligand Binding in HIV-1 Protease and K+ Channels Using Computational Methods." Doctoral thesis, Uppsala universitet, Strukturell molekylärbiologi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6167.
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Understanding protein-ligand interactions is highly important in drug development. In the present work the objective is to comprehend the link between structure and function using molecular modelling. Specifically, this thesis has been focused on implementation of receptor flexibility in molecular docking and studying structure-activity relationships of potassium ion channels and their blockers. In ligand docking simulations protein motion and heterogeneity of structural waters are approximated using an ensemble of protein structures. Four methods of combining multiple target structures within a single grid-based lookup table of interaction energies are tested. Two weighted average methods permit consistent and accurate ligand docking using a single grid representation of the target protein structures. Quaternary ammonium ions (QAIs) are well known K+ channel blockers. Conformations around C–N bonds at the quaternary centre in tetraalkylammonium ions in water solution are investigated using quantum mechanical methods. Relative solvation free energies of QAIs are further estimated from molecular dynamics simulations. The torsion barrier for a two-step interconversion between the conformations D2d and S4 is calculated to be 9.5 kcal mol–1. Furthermore D2d is found to be more stable than the S4 conformation which is in agreement with experimental studies. External QAI binding to the K+ channel KcsA is also studied. Computer simulations and relative binding free energies of the KcsA complexes with QAIs are calculated. This is done with the molecular dynamics free energy perturbation approach together with automated ligand docking. In agreement with experiment, the Et4N+ blocker in D2d symmetry has better binding than the other QAIs. Binding of blockers to the human cardiac hERG potassium channel is studied using a combination of homology modelling, automated docking and molecular dynamics simulations. The calculations reproduce the relative binding affinities of a set of drug derivatives very well and indicate that both polar interactions near the intracellular opening of the selectivity filter as well as hydrophobic complementarity in the region around F656 are important for blocker binding. Hence, the derived model of hERG should be useful for further interpretations of structure-activity relationships.
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Hashimoto, Kenji. "Investigating a role of HER3 in anti-HER2 target therapy in breast cancer." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:39025871-f32f-4e38-bd14-c13dbc9301f6.
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Background HER2-positive breast cancer is a poor prognostic subgroup, even if treated with anti-HER2 directed therapy. Trastuzumab is an important HER2-targeting antibody but only limited patients respond to this drug, and acquired resistance is a common problem. HER3 has been shown to be a key candidate in mediating resistance to trastuzumab and other ErbB inhibitors. The aims of the project are to investigate the resistance mechanisms and the relevant biomarkers in relation to trastuzumab treatment and resistance in HER2-positive breast cancer, in particular, HER3 subcellular localisation and HER3 phosphorylation. Methods Effects of trastuzumab on HER3 subcellular localisation and HER3 phosphorylation in relation to MET receptor were studied using western blots, nuclear fractionation, confocal microscopy, and immunoprecipitation in a panel of HER2-positive cell lines, including SKBr3 and BT474 breast cancer cells in which trastuzumab resistance was induced by long-term drug exposure. Effects of drug and knockdown experiments were tested by cell viability and proliferation assays. HER3 and MET expression was assessed by immunohistochemistry in xenograft tumours and human tissue samples, and clinical impact was assessed in different cohorts of HER2-positive breast cancer patients. Results Acquired trastuzumab resistant SKBr3 cells showed an increase of nuclear HER3100kD, which was derived from C-terminus of HER3. Nuclear HER3100kD could be due to the proteolytic cleavage of HER3 since it was reduced by ADAM17 or gamma-secretase inhibitor. In a panel of HER2-positive cell lines and xenograft samples, nuclear HER3 was observed only in the resistant cells. In addition, nuclear HER3 was associated with poor progression-free and overall survivals in HER2-positive breast cancer patients. It was also found that HER3 phosphorylation was maintained in acquired trastuzumab resistant cells, which was contributed by the ligand independent interaction of MET and HER3. Higher MET expression was associated with better overall survival in HER2-positive, breast cancer patients who were not treated with trastuzumab. Conclusions Nuclear HER3 was found in trastuzumab resistant cells and appeared to result from HER3 proteolytic cleavage mediated by ADAM17 and gamma-secretase. Further studies are required to investigate its mechanism and to identify the HER3 cleavage sites. MET was a key factor in maintaining HER3 phosphorylation during trastuzumab resistance. Lastly, nuclear HER3 and MET could be two potential biomarkers in HER2-positive breast cancer.
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Sollome, James Jerome. "Heregulin Activates a Novel HER2/HER3-MTK1-GIT1/ERK1/2 MAPK Signaling Pathway." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/315554.
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Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In the studies presented herein, MTK1 is shown to be required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced extracellular acidification and cell migration in MCF-7 breast cancer cells. Furthermore, it was shown that HRG stimulation leads to association of MTK1 with tyrosine phosphorylated HER3 in MCF-7 and T-47D breast cancer cells. The MTK1/HER3 association was dependent on HER2 activation and was decreased by pre-treatment with the HER2 inhibitor, lapatinib. Furthermore, HER2 does not directly associate with MTK1, but phosphorylates HER3 transiently. MTK1 also has a role in the ERK1/2 MAPK signaling pathway in response to heregulin (HRG) stimulation in T-47D and MCF-7 breast cancer cells. In addition to MTK1, Shc, Grb2 and GIT1 proteins are all involved in the ERK1/2 MAPK pathway in response to growth factor stimulation. MTK1 was also shown to associate with activated ERK1/2, GIT1, Shc, Grb2 and p85 of PI3K in response to heregulin stimulation. ERK1/2 kinase activity is involved in aberrant signaling that leads breast cancer progression. GIT1 is a scaffolding protein that is linked to growth factor mediated ERK1/2 signaling in cell migration. Moreover, we also identify the actin interacting region (AIR) on MTK1 and disruption of actin cytoskeletal polymerization with cytochalasin D inhibited the interaction between HER3 and MTK1, indicating that f-actin (which is needed for cell migration) is required for the MTK1/HER3 association. Additionally, HRG stimulation leads to extracellar acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.
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Sampera, Borràs Aïda 1990. "Characterization of molecular mechanisms of acquired resistance to Trastuzumab in gastric cancer." Doctoral thesis, Universitat Pompeu Fabra, 2018. http://hdl.handle.net/10803/665367.
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The HER2 receptor is overexpressed in 7-34% of gastric cancer (GC) tumors, being a potential therapeutic target. Trastuzumab, an anti-HER2 monoclonal antibody, in combination with platinum therapy was approved in 2010 as first-line treatment for advanced GC tumors overexpressing HER2. Despite the clinical benefit of trastuzumab, development of acquired resistance to treatment limits the efficacy of trastuzumab.
To study the mechanisms of trastuzumab acquired resistance we generated, upon chronic exposure to trastuzumab, a trastuzumab-resistant cell line derived from the human HER2-positive GC cell NCI-N87, hereafter named NTR2.4. The resistant phenotype of NTR2.4 cell line was associated with an increased basal activation of SRC, MAPK/ERK and PI3K/mTOR HER2 downstream pathways that persisted upon exposure to trastuzumab. Trastuzumab treatment induced a compensatory upregulation of HER3 and an increased expression of the HER family ligands EGF, AREG, HB-EGF, and NRG1 in NTR2.4 compared with the trastuzumab-sensitive NCI-N87 cell line. Ligand stimulation induced resistance to trastuzumab in NCI-N87 cells. These results were replicated in another model of trastuzumab-resistance, OTR6 cell line, suggesting an important role of HER receptor plasticity and HER family ligand in trastuzumab efficacy and resistance. Analysis of clinical samples from HER2-positive GC patients treated with trastuzumab showed higher EGF concentration in serum samples and increased EGF mRNA expression in tumor biopsies at trastuzumab progression compared with samples previous to trastuzumab treatment.
Treatment with Pan-HER, a novel antibody mixture against EGFR, HER2, and HER3, was effective in both trastuzumab-sensitive and trastuzumab-resistant cell lines and also in the presence of ligands. Altogether, our results suggested that Pan-HER would be a potential candidate to overcome trastuzumab resistance in advanced GC patients.El receptor HER2 està sobreexpressat en 7-34% dels tumors de càncer gàstric (CG), essent una diana terapèutica clau. Trastuzumab, un anticòs monoclonal anti-HER2, en combinació amb la teràpia basada en platí, va ser aprovat al 2010 com el tractament de primera línia per a tumors de CG avançats i amb sobreexpressió de HER2. Malgrat el benefici clínic del trastuzumab, el desenvolupament de resistència adquirida en limiten la seva eficàcia. Per tal d’estudiar els mecanismes de resistència adquirida al trastuzumab vam generar, a través d’una exposició crònica al trastuzumab, una línia cel·lular resistent a trastuzumab derivada de la línia cel·lular humana HER2 positiva i sensible al trastuzumab NCI-N87, anomenada NTR2.4. El fenotip resistent de la línia cel·lular NTR2.4 es va associar a una major activació basal de SRC i de les vies senyalitzadores de HER2, MAPK/ERK i PI3K/mTOR, malgrat el tractament amb trastuzumab. Trastuzumab va induir sobre-regulació de HER3 i una major expressió dels lligands de la família HER: EGF, AREG, HB-EGF i NRG1 en NTR2.4 en comparació amb la seva línia sensible al trastuzumab, NCI-N87. L’estimulació amb lligands va induir resistència a trastuzumab a la línia cel·lular NCI-N87. Aquests resultats es van replicar en un altre model de resistència a trastuzumab, la línia cel·lular OTR6, suggerint un paper important de la plasticitat dels receptors HER i dels lligands de la família HER en l’eficàcia i resistència al trastuzumab. L'anàlisi de mostres clíniques de sèrum pacients de CG HER2 positius tractats amb trastuzumab va demostrar que la concentració d'EGF en sèrum i els nivells de mRNA del biòpsies tumorals era superior a la progressió al trastuzumab en comparació amb les mostres prèvies al tractament amb trastuzumab. El tractament amb Pan-HER, basat en varis anticossos contra els receptors EGFR, HER2 i HER3, va ser efectiu tant en les línies sensibles com en les línies resistents al trastuzumab, fins i tot en presència de lligands. En conjunt, els nostres resultats suggereix Pan-HER com un potencial candidat per a superar la resistència a trastuzumab en pacients amb CG avançat.
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Maslak, Kerstin [Verfasser]. "Untersuchung zur synergistischen Wirkung von Lapatinib (GW 2016), einem potenten dualen HER1-HER2-Tyrosinkinaseinhibitor, und Fulvestrant, einem Östrogenrezeptorantagonisten, in HER2 und nicht HER2 überexprimierenden Brustkrebszellen / Kerstin Maslak." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2012. http://d-nb.info/1030290946/34.
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Saeidi, Shirin. "Hero of her own story : gender and state formation in contemporary Iran." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610745.
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Delord, Jean-Pierre. "Rôle de Her2 dans un modèle in vivo de maladie résiduelle de cancer de l'ovaire." Toulouse 3, 2007. http://www.theses.fr/2007TOU30017.
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L'échec à réduire la mortalité des patientes atteintes de carcinomes ovariens est certainement le résultat d'une dissémination précoce des cellules cancéreuses sur des sites intra-abdominaux secondaires. Bien que, dans de nombreux cas, la première ligne de chimiothérapie peut conduire à constater un état de rémission complète apparente, l'immense majorité des patientes rechutent dans les 18 à 28 mois suivant le diagnostic. Cette rechute est liée à la persistance de cellules carcinomateuses créant une maladie résiduelle (maladie infra-clinique ou encore maladie micrométastatique) typiquement péritonéale. C'est la repopulation tumorale à partir de cette maladie résiduelle qui est à l'origine d'une rechute clinique devenant rapidement symptomatique et conduisant le plus souvent au décès des patientes. L'activité élevée du Topotecan dans les xénogreffes intra-péritonéales de cellules ovariennes (OVCAR-3) nous a permis de créer un modèle pré-clinique de la maladie infra-clinique. Ce modèle de maladie résiduelle de carcinome ovarien chez la souris mime parfaitement l'histoire naturelle des carcinomes ovariens observés en clinique. Ce modèle nous a permis d'évaluer l'effet du traitement au Trastuzumab (TZ, anticorps monoclonale anti-HER2) sur l'amélioration de l'intervalle libre et la survie globale des souris en situation de la maladie résiduelle péritonéale. Nous avons montré qu'un traitement avec du Trastuzumab commencé tôt aboutit dans la plupart des cas à une augmentation très significative de la survie des animaux au point d'observer leur guérison. Cet effet se caractérise au niveau cellulaire par une légère inhibition de la voie de transduction du signal de prolifération (MAPK) et par une inhibition claire de l'expression de la voie de survie (AKT) des cellules OVCAR-3 ne montrant de façon interressante aucune amplification ni sur-expression d'HER2. .Biological effect of HER2 in a model human ovarian carcinoma l malignancies in women. Although first-line chemotherapy induces complete clinical remission in many cases of epithelial ovarian cancer, relapse usually occurs 18–28 months from diagnosis due to micrometastases. The first part of our study was designed to aimed to evaluate the effect of trastuzumab on disease-free and overall survival in a specially designed murine model of ovarian cancer (OVCAR-3), which mimicked the natural history of human micrometastatic disease. Trastuzumab can cure the mice if started soon after induction chemotherapy. It can modestly inhibit the proliferation through mitogen-activated protein kinase (MAPK) signal transduction and clearly inhibit AKT phosphorylation, which is involved in survival pathway. As OVCAR-3 cell lines show no HER2 amplification nor overexpression, these results warrant further studies to assess the efficacy of trastuzumab in the early stage of relapse in cancer models other than those overexpressing HER2. .