Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)]; transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20 mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH. (c) 2011 Elsevier Ltd. All rights reserved.

Simulation of rectifier voltage malfunction on OWECS, four-level converter, HVDC light link: Smart grid context tool

This paper presents a model for the simulation of an offshore wind system having a rectifier input voltage malfunction at one phase. The offshore wind system model comprises a variable-speed wind turbine supported on a floating platform, equipped with a permanent magnet synchronous generator using full-power four-level neutral point clamped converter. The link from the offshore floating platform to the onshore electrical grid is done through a light high voltage direct current submarine cable. The drive train is modeled by a three-mass model. Considerations about the smart grid context are offered for the use of the model in such a context. The rectifier voltage malfunction domino effect is presented as a case study to show capabilities of the model. (C) 2015 Elsevier Ltd. All rights reserved.

Project of a bandgap voltage reference and a temperature sensor for "energy harvest" systems

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e Computadores

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

Regulation of the voltage-gated sodium channel Nav1.7 by ubiquitin ligase Nedd4-2

Background and aim: Neuropathic pain (NP) is a frequent and disabling disorder occurring as a consequence of a direct lesion of the nervous system and recurrently associated with a positive shift toward nervous system excitability. Peripheral nerve activity is mainly carried by voltage-gated sodium channels (VGSC), with Nav1.7 isoform being an important candidate since loss of function mutations of its gene is associated with congenital inability to experience pain. Interestingly, ubiquitin ligases from the Nedd4 family are well known proteins that regulate the turnover of many membrane proteins such as VGSC and we showed Nedd2-2 is downregualted in experimental models of chronic pain. The aim of this study was to investigate the importance of Nedd4-2 in the modulation of Nav1.7 at the membrane. Methods: In vitro: whole cell patch clamp on HEK293 cell line stably expressing Nav1.7 was used to record sodium currents (INa), where the peak current of INa reflects the quantity of functional Nav1.7 expressed at the membrane. The possibility that Nedd4-2 modulates the currents was assessed by investigating the effect of its cotransfection on INa. Biotinylation of cell surface was used to isolate membrane-targeted Nav1.7. Furthermore, as the interaction between Nedd4-2 and Nav isoforms was previously reported to rely on an xPPxYx sequence (PY-motif), we mutated this latter to study its impact in the specific interaction between Nav1.7 and Nedd4-2. GST-fusion proteins composed of the Nav1.7 c terminal 66 amino acids (wild-type or PY mutated) and GST were used to pull-down Nedd4-2 from lysates. Results: Co-transfection of Nav1.7 with Nedd4-2 reduced the Nav1.7 current amplitude by ~80% (n = 36, p <0.001), without modifying the biophysical properties of INa. In addition, we show that the quantity of Nav1.7 at the membrane was decreased when Nedd4-2 was present. This effect was dependent on the PY-motif since mutations in this sequence abolished the down-regulatory effect of Nedd4-2. The importance of this motif was further confirmed by pull down experiments since the PY mutant completely eliminate the interaction with Nedd4-2. Perspectives: Altogether, these results point to the importance of Nedd4-2 as a Nav1.7 regulator through cell surface modulation of this sodium channel. Further experiments in freshly dissociated neurons from wild type and Scn1bflox/Nedd4-2Cre mice are needed to confirm in vivo these preliminary data.

Modification of the Perrine-Baum diagram to improve the calculation of high voltage electric lines

Through the history of Electrical Engineering education, vectorial and phasorial diagrams have been used as a fundamental learning tool. At present, computational power has replaced them by long data lists, the result of solving equation systems by means of numerical methods. In this sense, diagrams have been shifted to an academic background and although theoretically explained, they are not used in a practical way within specific examples. This fact may be against the understanding of the complex behavior of the electrical power systems by students. This article proposes a modification of the classical Perrine-Baum diagram construction to allowing both a more practical representation and a better understanding of the behavior of a high-voltage electric line under different levels of load. This modification allows, at the same time, the forecast of the obsolescence of this behavior and line’s loading capacity. Complementary, we evaluate the impact of this tool in the learning process showing comparative undergraduate results during three academic years

Mutism and Amnesia following High-Voltage Electrical Injury: Psychogenic Symptomatology Triggered by Organic Dysfunction?

Background: Mutism and dense retrograde amnesia are found both in organic and dissociative contexts. Moreover, dissociative symptoms may be modulated by right prefrontal activity. A single case, M.R., developed left hemiparesis, mutism and retrograde amnesia after a high-voltage electric shock without evidence of lasting brain lesions. M.R. suddenly recovered from his mutism following a mild brain trauma 2 years later. Methods: M.R.'s neuropsychological pattern and anatomoclinical correlations were studied through (i) language and memory assessment to characterize his deficits, (ii) functional neuroimaging during a standard language paradigm, and (iii) assessment of frontal and left insular connectivity through diffusion tractography imaging and transcranial magnetic stimulation. A control evaluation was repeated after recovery. Findings: M.R. recovered from the left hemiparesis within 90 days of the accident, which indicated a transient right brain impairment. One year later, neurobehavioral, language and memory evaluations strongly suggested a dissociative component in the mutism and retrograde amnesia. Investigations (including MRI, fMRI, diffusion tensor imaging, EEG and r-TMS) were normal. Twenty-seven months after the electrical injury, M.R. had a very mild head injury which was followed by a rapid recovery of speech. However, the retrograde amnesia persisted. Discussion: This case indicates an interaction of both organic and dissociative mechanisms in order to explain the patient's symptoms. The study also illustrates dissociation in the time course of the two different dissociative symptoms in the same patient.

Nitric oxide reduces Cl- absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism.

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na(+) and Cl(-) in the kidney, we asked whether NO regulates net Cl(-) flux (JCl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl(-) absorption. Cl(-) absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced JCl and transepithelial voltage (VT) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N(G)-nitro-l-arginine methyl ester) increased JCl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in JCl, we examined the effect of NO on JCl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive JCl, NO modulates JCl independent of the Na(+)-dependent Cl(-)/HCO3(-) exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H(+)-ATPase and pendrin. In contrast, both total and NO-sensitive JCl and VT were abolished with application of an epithelial Na(+) channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl(-) absorption in the CCD through a mechanism that is ENaC-dependent.

High sensitivity of immature GABAergic neurons to blockers of voltage-gated calcium channels.

The involvement of voltage-gated calcium channels in the survival of immature CNS neurons was studied in aggregating brain cell cultures by examining cell type-specific effects of various channel blockers. Nifedipine (10 microM), a specific blocker of L-type calcium channels, caused a pronounced and irreversible decrease of glutamic acid decarboxylase activity, whereas the activity of choline acetyltransferase was significantly less affected. Flunarizine (1-10 microM, a relatively unspecific ion channel blocker) elicited similar effects, that were attenuated by NMDA. The glia-specific marker enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase, were affected only after treatment with high concentrations of nifedipine (50 microM) or NiCl2 (100 microM, shown to block T-type calcium channels). Nifedipine (50 microM), NiCl2 (100 microM), and flunarizine (5 microM) also caused a significant increase in the soluble nucleosome concentration, indicating increased apoptotic cell death. This effect was prevented by cycloheximide (1 microM). Furthermore, the combined treatment with calcicludine (10 nM, blocking L-type calcium channels) and funnel-web spider toxin-3.3 (100 nM, blocking T-type channels) also caused a significant increase in free nucleosomes as well as a decrease in glutamic acid decarboxylase activity. In contrast, cell viability was not affected by peptide blockers specific for N-, P-, and/or Q-type calcium channels. Highly differentiated cultures showed diminished susceptibility to nifedipine and flunarizine. The present data suggest that the survival of immature neurons, and particularly that of immature GABAergic neurons, requires the sustained entry of Ca2+ through voltage-gated calcium channels.

ENaC inhibition stimulates Cl- secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism.

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ∼66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

Voltage-gated sodium channel expression in mouse DRG after SNI leads to re-evaluation of projections of injured fibers.

BACKGROUND: Dysregulation of voltage-gated sodium channels (Na(v)s) is believed to play a major role in nerve fiber hyperexcitability associated with neuropathic pain. A complete transcriptional characterization of the different isoforms of Na(v)s under normal and pathological conditions had never been performed on mice, despite their widespread use in pain research. Na(v)s mRNA levels in mouse dorsal root ganglia (DRG) were studied in the spared nerve injury (SNI) and spinal nerve ligation (SNL) models of neuropathic pain. In the SNI model, injured and non-injured neurons were intermingled in lumbar DRG, which were pooled to increase the tissue available for experiments. RESULTS: A strong downregulation was observed for every Na(v)s isoform expressed except for Na(v)1.2; even Na(v)1.3, known to be upregulated in rat neuropathic pain models, was lower in the SNI mouse model. This suggests differences between these two species. In the SNL model, where the cell bodies of injured and non-injured fibers are anatomically separated between different DRG, most Na(v)s were observed to be downregulated in the L5 DRG receiving axotomized fibers. Transcription was then investigated independently in the L3, L4 and L5 DRG in the SNI model, and an important downregulation of many Na(v)s isoforms was observed in the L3 DRG, suggesting the presence of numerous injured neurons there after SNI. Consequently, the proportion of axotomized neurons in the L3, L4 and L5 DRG after SNI was characterized by studying the expression of activating transcription factor 3 (ATF3). Using this marker of nerve injury confirmed that most injured fibers find their cell bodies in the L3 and L4 DRG after SNI in C57BL/6 J mice; this contrasts with their L4 and L5 DRG localization in rats. The spared sural nerve, through which pain hypersensitivity is measured in behavioral studies, mostly projects into the L4 and L5 DRG. CONCLUSIONS: The complex regulation of Na(v)s, together with the anatomical rostral shift of the DRG harboring injured fibers in C57BL/6 J mice, emphasize that caution is necessary and preliminary anatomical experiments should be carried out for gene and protein expression studies after SNI in mouse strains.

Regulation of Nav1.7 and Nav1.8 voltage-gated sodium channels by Nedd4-2 and β-subunits and its implication in neuropathic pain

In mammals, the presence of excitable cells in muscles, heart and nervous system is crucial and allows fast conduction of numerous biological information over long distances through the generation of action potentials (AP). Voltage-gated sodium channels (Navs) are key players in the generation and propagation of AP as they are responsible for the rising phase of the AP. Navs are heteromeric proteins composed of a large pore-forming a-subunit (Nav) and smaller ß-auxiliary subunits. There are ten genes encoding for Navl.l to Nav1.9 and NaX channels, each possessing its own specific biophysical properties. The excitable cells express differential combinations of Navs isoforms, generating a distinct electrophysiological signature. Noteworthy, only when anchored at the membrane are Navs functional and are participating in sodium conductance. In addition to the intrinsic properties of Navs, numerous regulatory proteins influence the sodium current. Some proteins will enhance stabilization of membrane Navs while others will favour internalization. Maintaining equilibrium between the two is of crucial importance for controlling cellular excitability. The E3 ubiquitin ligase Nedd4-2 is a well-characterized enzyme that negatively regulates the turnover of many membrane proteins including Navs. On the other hand, ß-subunits are known since long to stabilize Navs membrane anchoring. Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of Navs expressed in dorsal root ganglion (DRG) sensory neurons as highlighted in different animal models of neuropathic pain. Among Navs, Nav1.7 and Nav1.8 are abundantly and specifically expressed in DRG sensory neurons and have been recurrently incriminated in nociception and neuropathic pain development. Using the spared nerve injury (SNI) experimental model of neuropathic pain in mice, I observed a specific reduction of Nedd4-2 in DRG sensory neurons. This decrease subsequently led to an upregulation of Nav1.7 and Nav1.8 protein and current, in the axon and the DRG neurons, respectively, and was sufficient to generate neuropathic pain-associated hyperexcitability. Knocking out Nedd4-2 specifically in nociceptive neurons led to the same increase of Nav1.7 and Nav1.8 concomitantly with an increased thermal sensitivity in mice. Conversely, rescuing Nedd4-2 downregulation using viral vector transfer attenuated neuropathic pain mechanical hypersensitivity. This study demonstrates the significant role of Nedd4-2 in regulating cellular excitability in vivo and its involvement in neuropathic pain development. The role of ß-subunits in neuropathic pain was already demonstrated in our research group. Because of their stabilization role, the increase of ßl, ß2 and ß3 subunits in DRGs after SNI led to increased Navs anchored at the membrane. Here, I report a novel mechanism of regulation of a-subunits by ß- subunits in vitro; ßl and ß3-subunits modulate the glycosylation pattern of Nav1.7, which might account for stabilization of its membrane expression. This opens new perspectives for investigation Navs state of glycosylation in ß-subunits dependent diseases, such as in neuropathic pain. - Chez les mammifères, la présence de cellules excitables dans les muscles, le coeur et le système nerveux est cruciale; elle permet la conduction rapide de nombreuses informations sur de longues distances grâce à la génération de potentiels d'action (PA). Les canaux sodiques voltage-dépendants (Navs) sont des participants importants dans la génération et la propagation des PA car ils sont responsables de la phase initiale de dépolarisation du PA. Les Navs sont des protéines hétéromériques composées d'une grande sous-unité a (formant le pore du canal) et de petites sous-unités ß accompagnatrices. Il existe dix gènes qui codent pour les canaux sodiques, du Nav 1.1 au Nav 1.9 ainsi que NaX, chacun possédant des propriétés biophysiques spécifiques. Les cellules excitables expriment différentes combinaisons des différents isoformes de Navs, qui engendrent une signature électrophysiologique distincte. Les Navs ne sont fonctionnels et ne participent à la conductibilité du Na+, que s'ils sont ancrés à la membrane plasmique. En plus des propriétés intrinsèques des Navs, de nombreuses protéines régulatrices influencent également le courant sodique. Certaines protéines vont favoriser l'ancrage et la stabilisation des Navs exprimés à la membrane, alors que d'autres vont plutôt favoriser leur internalisation. Maintenir l'équilibre des deux processus est crucial pour contrôler l'excitabilité cellulaire. Dans ce contexte, Nedd4-2, de la famille des E3 ubiquitin ligase, est une enzyme bien caractérisée qui régule l'internalisation de nombreuses protéines, notamment celle des Navs. Inversement, les sous-unités ß sont connues depuis longtemps pour stabiliser l'ancrage des Navs à la membrane. La douleur neuropathique périphérique est une condition débilitante résultant d'une atteinte à un nerf. Elle est caractérisée par la dérégulation des Navs exprimés dans les neurones sensoriels du ganglion spinal (DRG). Ceci a été démontré à de multiples occasions dans divers modèles animaux de douleur neuropathique. Parmi les Navs, Nav1.7 et Nav1.8 sont abondamment et spécifiquement exprimés dans les neurones sensoriels des DRG et ont été impliqués de façon récurrente dans le développement de la douleur neuropathique. En utilisant le modèle animal de douleur neuropathique d'épargne du nerf sural (spared nerve injury, SNI) chez la souris, j'ai observé une réduction spécifique des Nedd4-2 dans les neurones sensoriels du DRG. Cette diminution avait pour conséquence l'augmentation de l'expression des protéines et des courants de Nav 1.7 et Nav 1.8, respectivement dans l'axone et les neurones du DRG, et était donc suffisante pour créer l'hyperexcitabilité associée à la douleur neuropathique. L'invalidation pour le gène codant pour Nedd4-2 dans une lignée de souris génétiquement modifiées a conduit à de similaires augmentations de Nav1.7 et Nav1.8, parallèlement à une augmentation à la sensibilité thermique. A l'opposé, rétablir une expression normale de Nedd4-2 en utilisant un vecteur viral a eu pour effet de contrecarrer le développement de l'hypersensibilité mécanique lié à ce modèle de douleur neuropathique. Cette étude démontre le rôle important de Nedd4-2 dans la régulation de l'excitabilité cellulaire in vivo et son implication dans le développement des douleurs neuropathiques. Le rôle des sous-unités ß dans les douleurs neuropathiques a déjà été démontré dans notre groupe de recherche. A cause de leur rôle stabilisateur, l'augmentation des sous-unités ßl, ß2 et ß3 dans les DRG après SNI, conduit à une augmentation des Navs ancrés à la membrane. Dans mon travail de thèse, j'ai observé un nouveau mécanisme de régulation des sous-unités a par les sous-unités ß in vitro. Les sous-unités ßl et ß3 régulent l'état de glycosylation du canal Nav1.7, et stabilisent son expression membranaire. Ceci ouvre de nouvelles perspectives dans l'investigation de l'état de glycosylation des Navs dans des maladies impliquant les sous-unités ß, notamment les douleurs neuropathiques.

Iowa Motorcycle Ride Meter, HR-1019, 1980

The Iowa Department of Transportation (Iowa DOT) through the Highway Division is responsible for the design, construction and maintenance of roadways that will provide a high level of serviceability to the motorist. First, the motorist expects to be able to get where he wants to go, but now he also demands a minimum level of comfort. In the construction of new roadways, the public is quick to express dissatisfaction with rough pavements. The Highway Division of the Iowa DOT (formerly Iowa State Highway Commission) has a specification which requires a "smooth-riding surface". For over 40 years, new portland cement concrete (pcc) pavement has been checked with a 10-foot rolling straightedge. The contractor is required to grind, saw or mill off all high spots that deviate more than 1/8" from the 10-foot straight line. Unfortunately, there are instances where a roadway that will meet the above criteria does not provide a "smooth-riding surface". The roadway may have monger undulations (swales) that result in an undesirable ride. The objective of this project was to develop a repeatable, reliable time stable, lightweight test unit to measure the riding quality of pcc pavement at normal highway speed the day after construction.

CARDIAC AND MUSCLE CHANNELOPATHIES: ROLES AND REGULATION OF VOLTAGE-GATED SODIUM CHANNELS

Abstract :The contraction of the heart or skeletal muscles is mainly due to the propagation, through excitable cells, of an electrical influx called action potential (AP). The AP results from the sequential opening of ion channels that generate inward or outward currents through the cell membrane. Among all the channels involved, the voltage-gated sodium channel is responsible for the rising phase of the action potential. Ten genes encode the different isoforms of these channels (from Nav1.1 to Nav1.9 and an atypical channel named NavX). Nav1.4 and Nav1.5 are the main skeletal muscle and cardiac sodium channels respectively. Their importance for muscle and heart function has been highlighted by the description of mutations in their encoding genes SCN4A and SCNSA. They lead respectively to neuromuscular disorders such as myotonia or paralysis (for Nav1.4), and to cardiac arrhythmias that can deteriorate into sudden cardiac death (for Nav1.5).The general aim of my PhD work has been to study diseases linked with channels dysfunction, also called channelopathies. In that purpose, I investigated the function and the regulation of the muscle and cardiac voltage-gated sodium channels. During the two first studies, I characterized the effects of two mutations affecting Nav1.4 and Nav1.5 function. I used the HEK293 model cells to express wild-type or mutant channels and then studied their biophysical properties with the patch-clamp technique, in whole cell configuration. We found that the SCN4A mutation produced complex alterations of the muscle sodium channel function, that could explain the myotonic phenotype described in patients carrying the mutation. In the second study, the index case was an heterozygous carrier of a SCNSA mutation that leads to a "loss of function" of the channel. The decreased sodium current measured with mutated Nay 1.5 channels, at physiological temperature, was a one of the factors that could explain the observed Brugada syndrome. The last project aimed at identifying a new potential protein interacting with the cardiac sodium channel. We found that the protein SAP97 binds the three last amino-acids of the C-terminus of Na,, 1.5. Our results also indicated that silencing the expression of SAP97 in HEK293 cells decreased the sodium current. Sodium channels lacking their three last residues also produced a reduced INa. These preliminary results suggest that SAP97 is implicated in the regulation of sodium channel. Whether this effect is direct or imply the action of an adaptor protein remains to be investigated. Moreover, our group has previously shown that Nav1.5 channels are localized to lateral membranes of cardiomyocytes by the dystrophin multiprotein complex (DMC). This suggests that sodium channels are distributed in, at least, two different pools: one targeted at lateral membranes by DMC and the other at intercalated discs by another protein such as SAP97.These studies reveal that cardiac and muscle diseases may result from ion channel mutations but also from regulatory proteins affecting their regulation.Résumé :La contraction des muscles et du coeur est principalement due à la propagation, à travers les cellules excitables, d'un stimulus électrique appelé potentiel d'action (PA). C'est l'ouverture séquentielle de plusieurs canaux ioniques transmembranaires, permettant l'entrée ou la sortie d'ions dans la cellule, qui est à l'origine de ce PA. Parmi tous les canaux ioniques impliqués dans ce processus, les canaux sodiques dépendant du voltage sont responsables de la première phase du potentiel d'action. Les différentes isoformes de ces canaux (de Nav1.1 à Nav1.9 et NavX) sont codées par dix gènes distincts. Nav1.4 et Nav1.5 sont les principaux variants exprimés respectivement dans le muscle et le coeur. Plusieurs mutations ont été décrites dans les gènes qui codent pour ces deux canaux: SCN4A (pour Nav1.4) et SCNSA (pour Nav1.5). Elles sont impliquées dans des pathologies neuromusculaires telles que des paralysies ou myotonies (SCN4A) ou des arythmies cardiaques pouvant conduire à la mort subite cardiaque (SCNSA).Mon travail de thèse a consisté à étudier les maladies liées aux dysfonctionnements de ces canaux, aussi appelées canalopathies. J'ai ainsi analysé la fonction et la régulation des canaux sodiques dépendant du voltage dans le muscle squelettique et le coeur. A travers les deux premières études, j'ai ainsi pu examiner les conséquences de deux mutations affectant respectivement les canaux Nav1.4 et Nav1.5. Les canaux sauvages ou mutants ont été exprimés dans des cellules HEK293 afin de caractériser leurs propriétés biophysiques par la technique du patch clamp en configuration cellule entière. Nous avons pu déterminer que la mutation trouvée dans le gène SCN4A engendrait des modifications importantes de la fonction du canal musculaire. Ces altérations fournissent des indications nous permettant d'expliquer certains aspects de la myotonie observée chez les membres de la famille étudiée. Le patient présenté dans la deuxième étude était hétérozygote pour la mutation identifiée dans le gène SCNSA. La perte de fonction des canaux Nav1.5 ainsi engendrée, a été observée lors d'analyses à températures physiologiques. Elle représente l'un des éléments pouvant potentiellement expliquer le syndrome de Brugada du patient. La dernière étude a consisté à identifier une nouvelle protéine impliquée dans la régulation du canal sodique cardiaque. Nos expériences ont démontré que les trois derniers acides aminés de la partie C-terminale de Nav1.5 pouvaient interagir avec la protéine SAP97. Lorsque que l'expression de la SAP97 est réduite dans les cellules HEK293, cela induit une baisse importante du courant sodique. De même, les canaux tronqués de leurs trois derniers acides aminés génèrent un flux ionique réduit. Ces résultats préliminaires suggèrent que SAP97 est peut-être impliquée dans la régulation du canal Na,,1.5. Des expériences complémentaires permettront de déterminer si ces deux protéines interagissent directement ou si une protéine adaptatrice est nécessaire. De plus, nous avons préalablement montré que les canaux Nav1.5 étaient localisés au niveau de la membrane latérale des cardiomyocytes par le complexe multiprotéique de la dystrophine (DMC). Ceci suggère que les canaux sodiques peuvent être distribués dans un minimum de deux pools, l'un ciblé aux membranes latérales pax le DMC et l'autre dirigé vers les disques intercalaires par des protéines telles que SAP97.L'ensemble de ces études met en évidence que certaines maladies musculaires et cardiaques peuvent être la conséquence directe de mutations de canaux ioniques, mais que l'action de protéines auxiliaires peut aussi affecter leur fonction.

Identification of Some Additional Loss Components in High-Power Low-Voltage Permanent Magnet Generators

Permanent magnet generators (PMG) represent the cutting edge technology in modern wind mills. The efficiency remains high (over 90%) at partial loads. To improve the machine efficiency even further, every aspect of machine losses has to be analyzed. Additional losses are often given as a certain percentage without providing any detailed information about the actual calculation process; meanwhile, there are many design-dependent losses that have an effect on the total amount of additional losses and that have to be taken into consideration. Additional losses are most often eddy current losses in different parts of the machine. These losses are usually difficult to calculate in the design process. In this doctoral thesis, some additional losses are identified and modeled. Further, suggestions on how to minimize the losses are given. Iron losses can differ significantly between the measured no-load values and the loss values under load. In addition, with embedded magnet rotors, the quadrature-axis armature reaction adds losses to the stator iron by manipulating the harmonic content of the flux. It was, therefore, re-evaluated that in salient pole machines, to minimize the losses and the loss difference between the no-load and load operation, the flux density has to be kept below 1.5 T in the stator yoke, which is the traditional guideline for machine designers. Eddy current losses may occur in the end-winding area and in the support structure of the machine, that is, in the finger plate and the clamping ring. With construction steel, these losses account for 0.08% of the input power of the machine. These losses can be reduced almost to zero by using nonmagnetic stainless steel. In addition, the machine housing may be subjected to eddy current losses if the flux density exceeds 1.5 T in the stator yoke. Winding losses can rise rapidly when high frequencies and 10–15 mm high conductors are used. In general, minimizing the winding losses is simple. For example, it can be done by dividing the conductor into transposed subconductors. However, this comes with the expense of an increase in the DC resistance. In the doctoral thesis, a new method is presented to minimize the winding losses by applying a litz wire with noninsulated strands. The construction is the same as in a normal litz wire but the insulation between the subconductors has been left out. The idea is that the connection is kept weak to prevent harmful eddy currents from flowing. Moreover, the analytical solution for calculating the AC resistance factor of the litz-wire is supplemented by including an end-winding resistance in the analytical solution. A simple measurement device is developed to measure the AC resistance in the windings. In the case of a litz-wire with originally noninsulated strands, vacuum pressure impregnation (VPI) is used to insulate the subconductors. In one of the two cases studied, the VPI affected the AC resistance factor, but in the other case, it did not have any effect. However, more research is needed to determine the effect of the VPI on litz-wire with noninsulated strands. An empirical model is developed to calculate the AC resistance factor of a single-layer formwound winding. The model includes the end-winding length and the number of strands and turns. The end winding includes the circulating current (eddy currents that are traveling through the whole winding between parallel strands) and the main current. The end-winding length also affects the total AC resistance factor.