Ce travail de thèse s’intéresse à l’introduction de méthodologies électrochimiques dans la problématique de la caractérisation du transport de peptides pénétrants (CPPs) à travers des membranes phospholipidiques. Malgré leur charge électrique globalement positive, ces peptides sont en effet capables de traverser les bicouches lipidiques de cellules réelles ou artificielles (liposomes) et il n’existe pas à ce jour de mécanisme d’internalisation universellement admis. Dans ce contexte, nous avons dans un premier temps développé des méthodologies visant à détecter des peptides pénétrants marqués par des sondes rédox en optimisant les conditions de volume et de confinement à l’électrode. Parallèlement, nous avons tenté d’observer le passage transmembranaire de ces peptides en utilisant un dispositif dérivé du patch-clamp, dans lequel un morceau (patch)de membrane lipidique est excisé d’une vésicule géante et le suivi du passage assuré par une détection ampérométrique sur ultramicro-électrode à proximité de la membrane. La sensibilité de la technique ampérométrique et le flux de CPP s’avérant faibles, nous nous sommes tournés vers une stratégie associant fluorescence (pour la sensibilité) et commande électrochimique (pour l’extinction de la fluorescence). Dans la mesure où les phospholipides constituent la barrière dynamique à travers laquelle doivent passer les CPPs, nous avons d’abord étudié l’extinction électrochimique de phospholipides marqués par une sonde à la fois rédox et fluorescente, le NBD. Observée sur des vésicules géantes au microscope confocale, la réduction électrochimique a permis l’extinction sélective des phospholipides situés sur le feuillet externe de la vésicule, un résultat que ne permettent ni la réduction par des agents chimiques (dithionite), ni les techniques de « photobleaching ». Cette propriété a été confirmée cette fois-ci pour des CPPs marqués par le NBD qui ont été mis à incuber en présence de vésicules géantes et pour lesquels un essai préliminaire semble confirmer une extinction de fluorescence essentiellement pour les peptides associés au feuillet externe de la vésicule. / This PhD work was aimed at introducing electrochemical strategies in the general topic devoted to the characterization of the passage of cell penetrating peptides (CPPs) across phospholipidic membranes. Although positively charged, CPPs are prone to cross lipidic bilayers of real and artificial cells (liposomes) and there is no commonly admitted internalization mechanism so far. Therefore, we first developed electrochemical setups aimed at improving the amperometric detection of redox-taggedCPPs, through optimization of volume and confinement. Additionally, we have made attempts to use patch-clamp inspired setups to monitor the passage of CPPs across a membrane patched from a giant vesicle using ultramicro-electrodes in the close vicinity of the patched membrane. Since the amperometric technique displayed poor sensibility and the flux of CPP was too narrow, we changed our strategy for a methodology coupling fluorescence (for the sensitivity) and an electrochemical command (to achieve fluorescence extinction). Considering that phospholipids are forefront actors of CPP internalization, we first focused on the electrochemical quenching of phospholipids tagged with a probe displaying both redox and fluorescent properties (NBD). Observed on giant unilamellar vesicles (GUVs) with confocal microscopy, the electrochemical reduction of the NBD probe led to the selective extinction of the phospholipids located on the outer leaflet of the vesicle, a selectivity which is not observed using chemical quenchers such as dithionite or photobleaching methods. That property was extended to NBD-tagged CPPs, previously incubated with unlabeled Guvs and that preliminary experiment confirmed that the electrochemical extinction mostly concerned peptides associated to the outer leaflet of the liposome.

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Un canal chlorure de 10 pS de la membrane basolatérale des cellules intercalaires du canal connecteur présente des propriétés proches de celles de ClC-K2. Nos données de patch clamp montrent que son activité et le nombre de canaux actifs (N) augmentent avec (i) la dépolarisation membranaire (ii) la concentration en calcium externe et (iii) l’alcalinisation extra et intracellulaire. L’alcalinisation extracellulaire déplace la dépendance au voltage vers des potentiels négatifs, alors que l’alcalinisation intracellulaire augmente leur activité à des potentiels négatifs. Ces données suggèrent que le Ca2+ extracellulaire et le pH modulent l’activité des canaux ClC-K2 via une action sur la porte commune plutôt que sur les protopores des autres canaux ClC. Nous avons cherché à établir le rôle et l’identité moléculaire des canaux potassiques basolatéraux du tubule contourné proximal (PCT), peu connus. Nos résultats montrent la présence d’ARNm codant pour les sous-unités Kir4.2 et Kir5.1 dans le PCT et, par western blot et par immunohistochimie, leur expression sur la membrane basolatérale du PCT. Un canal potassique, étudié par patch-clamp, présente des propriétés semblables à celles des canaux hétérotétramériques Kir4.2/Kir5.1 sur la membrane basolatérale du PCT de souris : il présente une conductance de 47 pS, une rectification entrante induite par le Mg2+ intracellulaire, une dépendance de son activité au pHi et un blocage par le Ba2+ extracellulaire. Cependant, un phénotype rénal chez des souris invalidées pour le gène Kcnj15 codant pour Kir4.2 n’a pas pu être mis en évidence. / A 10 pS chloride channel at the basolateral side of connecting duct intercalated cells shares properties with the cloned ClC-K2 channel. Patch-clamp experiments show that its activity and the number of active channels increase with (i) membrane depolarization (ii) external calcium concentration and (iii) external and internal alkalinization. External alkalinization also shifts the voltage-dependence curve towards negative voltages while internal alkalinization flattens the voltage-dependence curve thereby raising channel activity at negative potentials. These data suggest that extracellular calcium and both extra and intracellular protons modulate ClC-K2 channels activity through an action on the common gate rather than on the protopores present in others ClC channels.The role and the molecular identity of basolateral potassium channels of the proximal convoluted tubule (PCT) are not very well known. RT-PCR results revealed the presence of mRNA encoding the Kir4.2 and Kir5.1 potassium channels subunits in mouse PCT tubular cells, and western blot and immunohistochemistry experiments showed that both proteins are expressed at the basolateral membrane of these cells. The most frequent channel observed by patch-clamp on the basolateral membrane of PCT presents a conductance of 47 pS, an inward rectification induced by intracellular Mg2+, an inhibition by extracellular Ba2+ and an activity dependent on intracellular pH. These electrophysiological properties are consistent with the presence of heteromeric Kir4.2/Kir5.1 channels in the basolateral membrane of mouse PCT. The study of mice knocked out for the Kir4.2-encoding gene Kcnj15 did not highlight a renal phenotype.

Ion channels play a critical role in maintaining homeostasis by moving various ions in and out of cells. The Na+-K+-2Cl- or NKCC1 ion channel is involved in the regulation of Na+, K+, and Cl- across cell membranes, and plays a key role in many forms of cellular physiology. In the cochlea, NKCC1 is involved in endolymph production and maintenance of the endocochlear potential. Our hypothesis is that blocking NKCC1 channels should directly impact auditory sensitivity causing hearing loss. Our lab has also shown that the hormone aldosterone (ALD) can upregulate NKCC1 protein expression in vitro and in vivo. In the present investigation, we use electrophysiology and molecular biology techniques to study the biophysical mechanisms underlying the action of ALD in vitro on NKCC1 in the SH-SY5Y cell line. Our initial protein expression studies using RT-PCR found that proteins specific to NKCC1channels were present in SH-SY5Y neuronal cells. Whole cell currents measured using patch clamp methodology, were used to analyze the effects of various compounds on NKCC1 in the SH-SY5Y cell line. Control data were collected under perfusion of extracellular solution (ECS), then ECS containing 10µM bumetanide was applied, and, finally a washout condition completed the experiment. Similar experiments were conducted using ALD, and we observed an increase in K+ currents when bumetanide as well as when ALD was applied. This is the first report that indicates that ALD can directly regulate K+ channels in SH-SY5Y cells.

Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

Neurosteroids are an important group of substances that affect communication between neurons. They act as allosteric modulators of membrane receptors for neurotransmitters. One of the most important systems influenced by neurosteroids are NMDA receptors; however, a binding site(s) for their inhibition by steroids have not been found yet. This work is focused on the synthesis of fluorescently labeled photoaffinity probe, which may help explain the structure and location of binding site(s) and simplify the development of new neuroprotectives. A structural analogue of the endogenous neurosteroid, (20S)-20-Azido-5β-pregnan- 3α-yl N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-L-glutamyl 1-ester (8), was prepared. The structure of compound 8 includes photolabile azido group, as well as covalently bounded fluorescent NBD group. In addition, a photoaffinity probe with a modified steroid skeleton - pyridinium 17aα-azido-17α-methyl-17a-homo-5β-androstan-3α-yl 3-sulfate (29) - was synthesized. The ability of compound 8 and 29 to inhibit activated NMDA receptor has been verified for recombinant NR1-1a/NR2B receptors expressed in HEK293 cells using a patch-clamp technique. Additionally, the IC50 values of compounds 8 and 29 have been calculated. (In Czech) Key words: neuroactive steroid, NMDA receptor, photoffinity...

Ischemic injury leeds to sequence of pathophysiological events, which are accompanied by a release of growth factors and morphogens that significantly affect cell proliferation, migration and also their differentiation. Following ischemia, besides enhanced neurogenesis and gliogenesis in subventricular zone of the lateral ventricles and gyrus dentatus of the hippocampus, neurogenesis/gliogenesis also occurs in non-neurogenic regions, such as cortex or striatum. Recently, the attention was turned to a new glial cell type, termed polydendrocytes or NG2 glia. Under physiological conditions, these cells are able to divide and differentiate into mature oligodendrocytes due to they have often been equated with oligodendrocyte precursor cells. Based on recent reports, polydendrocytes are also able to generate protoplasmic astrocytes (Zhu et al., 2008) and neurons in vitro (Belachew et al., 2003), however their ability to differentiate into astrocytes or neurons under physiological or pathological conditions is still highly debated. Therefore, we have investigated the effect of different growth factors and morphogens, specifically brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and a morphogen sonic hedgehog (Shh), on...

N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...

Kainate receptors belong to the family of glutamate receptors, which include NMDA, AMPA and δ receptors. Glutamate receptors are widely found in the brain and therefore they are very dynamically investigated, especially from view of pharmacology, because there is great potential for finding new and more specific modulators which could be used in the treatment of neurodegenerative diseases. The aim of this work was to extend the knowledge about the influence of neurosteroids on homomeric kainate receptors (GluK1, GluK2, GluK3) in which is the study of modulation by neurosteroids still at the beginning. We have investigated interactions of homomeric kainate receptors with selected neurosteroids (pregnenolone sulfate, pregnanolone sulfate, dehydroepiandrosterone, dehydroepiandrosterone sulfate) by using patch clamp method in the configuration of whole-cell recording and also by using microfluorometry. We have found out that the biggest modulating effect on homomeric kainate receptors is caused by pregnenolone sulfate, which inhibits glutamate responses of these receptors. Keywords kainate receptor, glutamate, neurosteroids, steroids, patch-clamp technique

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