O peptídeo idêntico ao C-terminal da proteína S100A9 murina (pS100A9mH92-G110) inibe a hiperalgesia inflamatória induzida pela carragenina. Em adição, este peptídeo inibe a hiperalgesia inflamatória induzida por tripsina, uma serino protease capaz de ativar receptores ativados por protease do tipo 2 (PAR2). O objetivo inicial deste trabalho foi caracterizar a relação estrutura/ efeito do pS100A9m, a fim de determinar a menor seqüência peptídica dotada de atividade antinociceptiva. Ainda, como parte dos objetivos, neste trabalho foram investigados os mecanismos envolvidos no efeito antinociceptivo do pS100A9m e da menor seqüência ativa sobre a hiperalgesia induzida pela ativação de PAR2. Diferentes seqüências peptídicas homólogas ao pS100A9m foram sintetizadas e avaliadas em ratos submetidos ao modelo de hiperalgesia mecânica induzida por carragenina. Dentre todas as seqüências peptídicas investigadas, o peptídeo denominado AcE97-G102 foi determinado como a menor seqüência ativa com efeito semelhante ao pS100A9m. Com relação aos estudos sobre a ativação de PAR2, os resultados obtidos demonstraram que o pS100A9m bem como o AcE97-G102 inibem a hiperalgesia térmica e mecânica decorrentes da ativação de PAR2 (induzida por um peptídeo agonista deste receptor ? PAR2AP). A análise por imuno-histoquímica demonstrou que a ativação de PAR2 aumenta a expressão da proteína Egr-1 em neurônios nociceptivos, sendo o pS100A9m capaz de inibir este efeito. Em adição, ambos pS100A9m e AcE97-G102 inibiram o influxo de cálcio induzido por PAR2AP ou tripsina, em neurônios sensoriais do gânglio da raiz dorsal da medula espinhal (DRG). Por outro lado, nenhum dos peptídeos apresentou efeito sobre a mobilização de cálcio em células HEK-293, que naturalmente expressam PAR2, ou em células KNRK transfectadas com este tipo de receptor, sugerindo que o efeito tanto do pS100A9m quanto do AcE97-G102, sobre a ativação de PAR2, seja específico para neurônios sensoriais. O pS100A9m e o AcE97-G102 inibiram o influxo de cálcio nos neurônios DRG estimulados com bradicinina, capsaicina ou KCl. Ainda, o pS100A9m inibiu a liberação de substância P induzida por PAR2. Os resultados obtidos com o tratamento de neurônios DRG com tapsigaragina ou com ionóforo de cálcio sugerem um efeito direto do pS100A9m sobre os canais de cálcio. Desta forma, foi avaliada atividade do pS100A9m e do AcE97-G102 sobre culturas de células HEK-tsA transfectadas com canais de cálcio dependente de voltagem do tipo N ou do tipo L. Os resultados obtidos demonstraram que ambos peptídeos inibirem o influxo de cálcio em células transfectadas com receptores do tipo N. Em conjunto, os dados aqui obtidos demonstram que o efeito do C-terminal da proteína S100A9 murina sobre a nocicepção experimental é devido a uma inibição de canais de cálcio do tipo N, por uma ação direta em neurônios sensoriais. Ainda, a seqüência responsável por este efeito está localizada na porção E97-G102 do domínio C-terminal da proteína S100A9 murina. / Peptide identical to the C-terminus of S100A9 protein (mS100A9pH92-G110) inhibits inflammatory hyperalgesia induced by carrageenan and trypsin, a serine protease that activates protease-activated receptors 2 (PAR2). The aim of this work was to characterize the relationship between structure and function of mS100A9p in order to identify the shortest peptide sequence endowed with antinociceptive effect. Furthermore, the mechanisms involved on the antinociceptive effect of both mS100A9p and the shortest homologous sequence on PAR2-induced hyperalgesia were also evaluated. Different peptide sequences homologous to mS100A9p were synthesized and evaluated in rats submitted to the carrageenan-induced mechanical hyperalgesia model. Among all evaluated sequences, the peptide AcE97-G102 was found to be the shortest sequence that showed an antinociceptive effect similar to that induced by mS100A9p. In regard to PAR2 activation, data obtained herein demonstrated that both mS100A9p and AcE97-G102 inhibit PAR2-induced mechanical and thermal hyperalgesia, induced by the selective agonist peptide ? PAR2AP. Imunohistochemical evaluation demonstrated that PAR2 activation increased Egr-1 protein expression on sensory neurons and mS100A9p inhibited this effect. In addition, both mS100A9p and AcE97-G102 inhibited PAR2- and trypsin-induced calcium influx in dorsal root ganglia neurons (DRG). On the other hand, no effect on the calcium influx of the peptides were observed on HEK-293 cells or KNRK-PAR2 transfected cells, suggesting that the effects of mS100A9p and AcE97-G102 on PAR2 activation are specific for sensory neurons. Both mS100A9p and AcE97-G102 inhibited DRG calcium flux when cells were stimulated with bradykinin, capsaicin or KCl. Also, mS100A9p inhibited PAR2-induced substance P release in DRG. Treatment of DRG with either thapsigargin or calcium ionophore suggest a direct effect of mS100A9p on calcium channels. To evaluate this hypothesis the effects of mS100A9p and AcE97-G102 were evaluated on N-type or L-type voltage-dependent calcium channel transfected HEK-tsA cells. Both peptides inhibited calcium influx of N-type transfected cells. In conclusion, data presented herein demonstrate that the C-terminus of murine S100A9 protein inhibits experimental nociception through a block of N-type voltage-dependent calcium channels, directly on sensory neurons. Also, the domain involved in this effect is localized on the sequence E97-G102 of the C-terminus of murine S100A9 protein.

O peptídeo idêntico ao C-terminal da proteína S100A9 murina (pS100A9mH92-G110) inibe a hiperalgesia inflamatória induzida pela carragenina. Em adição, este peptídeo inibe a hiperalgesia inflamatória induzida por tripsina, uma serino protease capaz de ativar receptores ativados por protease do tipo 2 (PAR2). O objetivo inicial deste trabalho foi caracterizar a relação estrutura/ efeito do pS100A9m, a fim de determinar a menor seqüência peptídica dotada de atividade antinociceptiva. Ainda, como parte dos objetivos, neste trabalho foram investigados os mecanismos envolvidos no efeito antinociceptivo do pS100A9m e da menor seqüência ativa sobre a hiperalgesia induzida pela ativação de PAR2. Diferentes seqüências peptídicas homólogas ao pS100A9m foram sintetizadas e avaliadas em ratos submetidos ao modelo de hiperalgesia mecânica induzida por carragenina. Dentre todas as seqüências peptídicas investigadas, o peptídeo denominado AcE97-G102 foi determinado como a menor seqüência ativa com efeito semelhante ao pS100A9m. Com relação aos estudos sobre a ativação de PAR2, os resultados obtidos demonstraram que o pS100A9m bem como o AcE97-G102 inibem a hiperalgesia térmica e mecânica decorrentes da ativação de PAR2 (induzida por um peptídeo agonista deste receptor ? PAR2AP). A análise por imuno-histoquímica demonstrou que a ativação de PAR2 aumenta a expressão da proteína Egr-1 em neurônios nociceptivos, sendo o pS100A9m capaz de inibir este efeito. Em adição, ambos pS100A9m e AcE97-G102 inibiram o influxo de cálcio induzido por PAR2AP ou tripsina, em neurônios sensoriais do gânglio da raiz dorsal da medula espinhal (DRG). Por outro lado, nenhum dos peptídeos apresentou efeito sobre a mobilização de cálcio em células HEK-293, que naturalmente expressam PAR2, ou em células KNRK transfectadas com este tipo de receptor, sugerindo que o efeito tanto do pS100A9m quanto do AcE97-G102, sobre a ativação de PAR2, seja específico para neurônios sensoriais. O pS100A9m e o AcE97-G102 inibiram o influxo de cálcio nos neurônios DRG estimulados com bradicinina, capsaicina ou KCl. Ainda, o pS100A9m inibiu a liberação de substância P induzida por PAR2. Os resultados obtidos com o tratamento de neurônios DRG com tapsigaragina ou com ionóforo de cálcio sugerem um efeito direto do pS100A9m sobre os canais de cálcio. Desta forma, foi avaliada atividade do pS100A9m e do AcE97-G102 sobre culturas de células HEK-tsA transfectadas com canais de cálcio dependente de voltagem do tipo N ou do tipo L. Os resultados obtidos demonstraram que ambos peptídeos inibirem o influxo de cálcio em células transfectadas com receptores do tipo N. Em conjunto, os dados aqui obtidos demonstram que o efeito do C-terminal da proteína S100A9 murina sobre a nocicepção experimental é devido a uma inibição de canais de cálcio do tipo N, por uma ação direta em neurônios sensoriais. Ainda, a seqüência responsável por este efeito está localizada na porção E97-G102 do domínio C-terminal da proteína S100A9 murina. / Peptide identical to the C-terminus of S100A9 protein (mS100A9pH92-G110) inhibits inflammatory hyperalgesia induced by carrageenan and trypsin, a serine protease that activates protease-activated receptors 2 (PAR2). The aim of this work was to characterize the relationship between structure and function of mS100A9p in order to identify the shortest peptide sequence endowed with antinociceptive effect. Furthermore, the mechanisms involved on the antinociceptive effect of both mS100A9p and the shortest homologous sequence on PAR2-induced hyperalgesia were also evaluated. Different peptide sequences homologous to mS100A9p were synthesized and evaluated in rats submitted to the carrageenan-induced mechanical hyperalgesia model. Among all evaluated sequences, the peptide AcE97-G102 was found to be the shortest sequence that showed an antinociceptive effect similar to that induced by mS100A9p. In regard to PAR2 activation, data obtained herein demonstrated that both mS100A9p and AcE97-G102 inhibit PAR2-induced mechanical and thermal hyperalgesia, induced by the selective agonist peptide ? PAR2AP. Imunohistochemical evaluation demonstrated that PAR2 activation increased Egr-1 protein expression on sensory neurons and mS100A9p inhibited this effect. In addition, both mS100A9p and AcE97-G102 inhibited PAR2- and trypsin-induced calcium influx in dorsal root ganglia neurons (DRG). On the other hand, no effect on the calcium influx of the peptides were observed on HEK-293 cells or KNRK-PAR2 transfected cells, suggesting that the effects of mS100A9p and AcE97-G102 on PAR2 activation are specific for sensory neurons. Both mS100A9p and AcE97-G102 inhibited DRG calcium flux when cells were stimulated with bradykinin, capsaicin or KCl. Also, mS100A9p inhibited PAR2-induced substance P release in DRG. Treatment of DRG with either thapsigargin or calcium ionophore suggest a direct effect of mS100A9p on calcium channels. To evaluate this hypothesis the effects of mS100A9p and AcE97-G102 were evaluated on N-type or L-type voltage-dependent calcium channel transfected HEK-tsA cells. Both peptides inhibited calcium influx of N-type transfected cells. In conclusion, data presented herein demonstrate that the C-terminus of murine S100A9 protein inhibits experimental nociception through a block of N-type voltage-dependent calcium channels, directly on sensory neurons. Also, the domain involved in this effect is localized on the sequence E97-G102 of the C-terminus of murine S100A9 protein.

Based on preliminary reports, an extended series of bolamphiphilic oligoester compounds with structural symmetry were synthesized and then tested using a planar bilayer experiment with the voltage-clamp technique. The main structures of these compounds are identical, consisting of a mono or tri-aromatic core, two octamethylene chains and two benzoyl headgroups which are all connected through ester linkages. The structural variance was provided by the four differently functionalized benzoyl headgroups. The synthetic methods of three to five steps were mainly adapted from the previously reported method.1 The methods successfully produced eight compounds with overall yields of 20 to 30%. The voltage-clamp data suggested voltage-dependent behaviors occur at low concentrations while Ohmic behaviors require at high concentrations. The activity at low potentials showed relatively erratic behavior but the channels frequently switched between opening and closing states. The activity at high potential lasted longer as the channel maintained a longer state of opening. The exponential voltage-dependent behaviors were observed at higher potential while the voltage-independent Ohmic behaviors occur at low potential. These channel behaviors are highly time-dependent as there is no control over the stability and the aggregation level for the compounds forming active channels in the membrane. In some cases the current-voltage responses appear to be asymmetrical between the positive and the negative potentials. Mechanisms consistent with the observations are proposed. / Graduate / 0485 / 0490 / yzong@uvic.ca

Based on preliminary reports, an extended series of bolamphiphilic oligoester compounds with structural symmetry were synthesized and then tested using a planar bilayer experiment with the voltage-clamp technique. The main structures of these compounds are identical, consisting of a mono or tri-aromatic core, two octamethylene chains and two benzoyl headgroups which are all connected through ester linkages. The structural variance was provided by the four differently functionalized benzoyl headgroups. The synthetic methods of three to five steps were mainly adapted from the previously reported method.1 The methods successfully produced eight compounds with overall yields of 20 to 30%. The voltage-clamp data suggested voltage-dependent behaviors occur at low concentrations while Ohmic behaviors require at high concentrations. The activity at low potentials showed relatively erratic behavior but the channels frequently switched between opening and closing states. The activity at high potential lasted longer as the channel maintained a longer state of opening. The exponential voltage-dependent behaviors were observed at higher potential while the voltage-independent Ohmic behaviors occur at low potential. These channel behaviors are highly time-dependent as there is no control over the stability and the aggregation level for the compounds forming active channels in the membrane. In some cases the current-voltage responses appear to be asymmetrical between the positive and the negative potentials. Mechanisms consistent with the observations are proposed. / Graduate / 0485 / 0490 / yzong@uvic.ca

No description available.

Dans le diabète de type 2 établi, l'hyperglycémie chronique, un taux élevé d'acides gras libres et l'inflammation induisent un stress oxydatif (SO) au niveau de la cellule beta. Le SO, qui apparaît dès le stade de pré-diabète, peut induire un dysfonctionnement précoce de cette cellule. Ainsi, la protection de la cellule β par des molécules anti-oxydantes pourrait ralentir la progression du pré-diabète au diabète.La quercétine, un flavonoïde, a présenté des propriétés antidiabétiques dans plusieurs études in vivo. Cependant, très peu de données traitent de son mécanisme d'action directement au niveau de la cellule beta. Dans ce contexte, nous avons étudié les effets de la quercétine au niveau de la cellule beta dans des conditions physiologiques et des conditions de SO.Nos résultats montrent qu'en présence de concentrations stimulantes de sécrétagogue, la quercétine potentialise la sécrétion d'insuline par un mécanisme impliquant l'augmentation de calcium intracellulaire et la potentialisation de ERK1/2 via l'activation des voies de la PKA et de la CaMK II. De plus, la quercétine protège la cellule beta du SO en sur-activant ERK1/2. Le resvératrol et la NAC, deux antioxydants de référence, sont inactifs dans ces conditions expérimentales.En absence de concentrations stimulantes de sécrétagogue, la quercétine induit une sécrétion d'insuline modérée en augmentant le calcium intracellulaire suite à une activation directe des CaV de type L. Dans ces conditions, l'activation de ERK1/2 induite par la quercétine, qui est indépendante de l'activation des voies de la PKA et de la CaMK II, ne serait pas impliquée dans le mécanisme sécrétoire. Nos résultats indiquent que le mécanisme d'action de la quercétine au niveau de la cellule β ne repose pas uniquement sur ses capacités anti-oxydantes mais fait intervenir des cibles pharmacologiques et la régulation de voies de signalisation intracellulaires. / In type 2 diabetes, chronic hyperglycaemia, elevated free fatty acids and inflammation induce oxidative stress (OS) in pancreatic β cell. SO, which appears at the stage of pre-diabetes, may induce early dysfunction of this cell. Thus, the β cell protection by antioxidant molecules could slow the progression of pre-diabetes to diabetes.Quercetin, a flavonoid, has shown antidiabetic properties in several in vivo studies. However, very few data address its mechanism of action directly at the β cell. In this context, we studied the effects of quercetin at the β cell under physiological conditions and conditions of OS.Our results show that in the presence of stimulating concentrations of secretagogue, quercetin potentiates insulin secretion by a mechanism involving increased intracellular calcium and potentiation of ERK1 / 2 via activation of the PKA and the CaMK II pathways. In addition, quercetin protects beta cell from OS via a suractivation of ERK1/2. Resveratrol and NAC, two antioxidants of reference are inactive under these experimental conditions.In the absence of stimulating concentration of secretagogue, quercetin induced moderate insulin secretion by increasing the intracellular calcium via a direct activation of L-type CaV Under these conditions, the activation of ERK1/2 induced by quercetin, which is independent of the activation pathways of PKA and CaMK II to, would not be involved in the secretory mechanism.Our results indicate that the mechanism of action of quercetin at the β cell not only based on its antioxidant capacity but involves pharmacological targets and the regulation of intracellular signaling pathways.

Rhythmische neuronale Aktivität spielt vermutlich eine wichtige Rolle in der Informationsverarbeitung im zentralen Nervensystem. Oszillationen neuronaler Netze sind heterogen, von der Hirnregion und ihrer Funktion abhängig und werden entsprechend ihrer Frequenz eingeteilt. Für ihre Entstehung sind über die Verschaltung der Neuronen und der synaptischen Übertragung hinaus insbesondere die Erregbarkeit und Oszillationseigenschaften einzelner Neurone von Bedeutung. Bestimmte Zellen der hippokampalen Formation wie zum Beispiel Sternzellen (SC) der Schicht II des Entorhinalkortex zeigen oszillatorische Aktivität und antworten verstärkt auf Stimuli einer bestimmten Frequenz – sie sind resonant. Beide Phänomene werden auf spezifische spannungsabhängige Leitfähigkeiten in der Membran zurückgeführt. Es stellte sich heraus, dass die Resonanzfrequenz von SCs durch das Muster der vorhandenen Leitfähigkeiten bestimmt wird und von der Position der Zelle entlang der dorso-ventralen Achse abhängt. Dieser Gradient ist bereits in frühen Entwicklungsstadien nachweisbar. Im Zuge der weiteren Entwicklung werden SCs weniger erregbar und der Bereich der Resonanzfrequenz dehnt sich nach dorsal aus. Pharmakologische Experimente ergaben, dass die Resonanz von SCs von HCN-Kanälen abhängt und von Kv7-Kanälen moduliert wird. Außerdem konnten zwei, bisher unbekannte Klassen von oszillatorischen Interneuronen beschrieben werden, deren Resonanz ebenfalls im Theta-Bereich liegt und auf ähnliche Leitfähigkeiten zurückgeführt werden kann. Weitere, auch CA1-Pyramidenzellen einschließende Experimente ergaben, dass HCN-Kanäle die allgemeine Voraussetzung für Resonanz zu sein scheinen während Kv7-Kanäle potente Modulatoren darstellen. Die pharmakologische Blockade dieser Kanäle unterbrach Netzwerkoszillation im Hippokampus. Dies unterstützt die These, dass bestimmte Leitfähigkeiten Neuronen Resonanzeigeschaften verleihen und somit wiederum Netzwerkoszillationen unterstützen. / Rhythmic neuronal activity is thought to be crucial for information processing in the brain. Neuronal network oscillations are heterogeneous, vary with brain region and type of information processed. They are classified according to their frequency content. Their generation relies on network circuitry, synaptic transmission and neuronal properties. Oscillatory behavior of individual cells has been particularly implicated. Different cell types within the hippocampal formation such as layer II stellate cells (SC) of the medial entorhinal cortex display oscillatory activity and are resonant, i.e., respond preferentially to stimuli of a given frequency. Voltage dependent ionic conductances have been suggested to give rise to these phenomena. It was found that resonance of SCs is defined by the composition of voltage-dependent channels embedded in their membrane and changes with their position along the dorsal-ventral axis. This gradient of SC properties develops during early postnatal life. During the transition to adulthood cells become less excitable and the range of resonance frequencies expands in the dorsal direction. Pharmacological experiments reveal the resonance of SCs to depend strongly on HCN-channels and to be modulated by Kv7-channels. Also, two previously unknown classes of oscillating interneurons were identified in the stratum radiatum of the CA1 region. These are targeted by neurons from the dentate gyrus, display frequency preferences in the theta range which relies on similar membrane conductances. Further experiments including CA1 pyramidal cells suggested HCN-channels to be the primary global requirement for resonance whereas Kv7-channels appear to be effective modulators. Pharmacological blockade of these channels disrupted ongoing network oscillations in the hippocampus. This supports the notion that specific ion channels support rhythmic activity of individual cells and in turn of entire networks.

Les canaux calciques dépendants du voltage (CCVD) sont impliqués dans de nombreux processus cellulaires tels que la libération de neurotransmetteurs, la contraction musculaire ou encore la régulation de l'expression génique. Les CCVD sont constitués d'une sous-unité canalaire (alpha1 ou Cav) par laquelle les ions Ca2+ entrent dans le milieu intracellulaire, associée à différentes sous-unités auxiliaires, alpha2delta, beta et gammaqui régulent leur fonction. Ma thèse a contribué à la mise en évidence d'une nouvelle voie de régulation du couplage excitation-transcription impliquant la sous-unité beta4 des CCVD. Dans ce cadre, nous nous sommes intéressés à la compréhension des déterminants de l'entrée de beta4 dans le noyau et aux mécanismes de régulation de l'expression génique par cette sous-unité des CCVD. Un modèle animal nous a été particulièrement utile, la souris léthargique (lh), déficiente pour la sous-unité beta4 et considérée comme un modèle d'étude de l'épilepsie-absences. Une translocation de beta4 du cytoplasme vers le noyau est observée au cours de la différenciation neuronale. Cette translocation est dépendante de l'intégrité structurale de beta4 et plus précisément de l'interaction de ses domaines SH3 (Src Homology 3) et GK (Guanylate Kinase). La translocation de beta4 au noyau nécessite son association avec un partenaire : la sous-unité régulatrice de la protéine phosphatase 2A (PP2A), B56delta. La dépolarisation membranaire permet un décrochage de beta4 du canal et son association à B56delta. beta4 migre donc vers le noyau sous forme de complexe avec B56delta/PP2A. Une étude transcriptomique réalisée pour comparer le profil d'expression dans le cervelet de souris lh par rapport aux souris wild-type a montré l'implication de beta4 dans la répression et l'activation d'un certain nombre de gènes. Particulièrement beta4 réprime fortement l'expression du gène qui code pour la tyrosine hydroxylase (TH). Dans le noyau, beta4 interagit avec un facteur de transcription, le récepteur aux hormones thyroïdiennes alpha(TRalpha). Cette association permet au complexe beta4/B56delta/PP2A de cibler la région promotrice du gène TH comme cela a été montré par des expériences d'immunoprécipitation de la chromatine (ChIP). Le complexe beta4/B56delta/PP2A est capable de s'associer aux histones et de déphosphoryler spécifiquement les histones H3 en Ser10 au niveau de la région promotrice du gène TH. Cette modification de la chromatine est corrélée avec le recrutement de Heterochromatin Protein 1 gamma (HP1gamma au niveau du promoteur du gène TH. HP1gamma est impliquée dans la formation d'hétérochromatine et pourrait expliquer la répression de l'expression du gène TH. Ainsi dans le cervelet de souris lh, l'absence de beta4 déclenche un dérèglement de cette voie de signalisation qui entraîne la sur-expression du géne TH. La mutation humaine R482X à l'origine de la délétion d'une partie du domaine C-terminal de beta4 et responsable d'une forme d'épilepsie juvénile myoclonique, perturbe la localisation nucléaire de beta4. En effet, le mutant beta1-481 incapable de s'associer à B56delta/PP2A et de migrer au noyau n'interagit pas avec les histones. La voie de signalisation permettant la régulation de l'expression génique par beta4 n'est donc plus assurée par le mutant. Ainsi, la fonction debeta4 ne se limite pas à son action cytoplasmique en tant que sous-unité auxiliaire des CCVD. En effet, ce travail montre combien dans le noyau,beta4, joue un rôle important dans la régulation de l'expression génique. / Voltage dependent calcium channels (VDCC) participate to various cellular processes such as neurotransmitters release, muscular contraction or gene expression regulation. VDCC are composed of a pore-forming subunit (alpha1 or Cav), that allows Ca2+ to enter the cells associated with auxiliary subunits, alpha2delta, beta and gamma. My thesis studies on a new signaling pathway in which the beta4 subunit of VDCC couples neuronal excitability to transcription. In particular it focuses on the understanding of the beta4 nuclear translocation determinants and the mechanisms of gene expression regulation by this VDCC subunit. beta4 translocation from the cytoplasm to the nucleus is observed during neuronal differentiation. This translocation depends on beta4 structural integrity and more precisely on interaction between the beta4 SH3 (Src Homology 3) and GK (Guanylate Kinase) domains. beta4 nuclear translocation is conditioned by its association with a partner: the regulatory subunit of phosphatase protein 2A (PP2A), B56delta. Membrane depolarization induces beta4 channel uncoupling and association with B56delta. Thus, beta4 migrates to the nucleus in complex with B56delta/PP2A. A study on gene expression generated by microarray was carried on to compare profiles of gene expression in lethargic (lh) mice, considered as spontaneous beta4 KO with wild-type (WT) mice cerebellum. This study proved the influence ofbeta4 on the repression and the activation of certain genes. Particularly, beta4 strongly represses tyrosine hydroxylase (TH) gene expression. In the nucleus, beta4 interacts with a transcription factor: the thyroid hormone receptor alpha (TR alpha). This association allows beta4/B56delta/PP2A complex to target the TH gene promoter as shown by chromatin immunoprecipitation (ChIP) experiments. This complex is also able to associate itself with histones and dephosphorylate Ser10 histone H3 in the TH gene promoter. This chromatin modification is correlated with HP1 gamma (Heterochromatin Protein 1 gamma) recruitment in the TH gene promoter. HP1 gamma is known to promote heterochromatin formation that could explain the TH gene repression by beta4. Thus, in lh mice cerebellum, the absence of beta4 triggers a complete disorganization of this signaling pathway that results in the up-regulation of the TH gene. R482X, the human mutation inducing the C-terminus domain deletion of beta4 and responsible for a form of juvenile myoclonic epilepsy prevents beta4 nuclear localization. In fact, the mutant beta1-481 unable to associate with B56delta/PP2A and to migrate to the nucleus does not interact with HP1gamma and histones. The signaling pathway allowing gene regulation by beta4 is prevented by the mutant. Thus, beta4 does not play a confined role in the cytoplasm as CCVD auxiliary subunit but also functions in the nucleus as a gene expression regulator.

Les érythrocytes sont un modèle pour l'étude du transport des ions, des nutriments et de divers solutés au travers de la membrane cellulaire. L'identité moléculaire, la régulation et rôle physiologique des canaux ioniques sont pas clairement établis malgré leur implication évidente dans des processus physiologiques comme la sénescence ou pathologiques comme la drépanocytose ou le paludisme. Le présent travail de thèse a fait appel à la technique du 'patch-clamp' et à diverses méthodes biochimiques pour démontrer que: 1/ La diversité des courants anioniques enregistrés au travers de la membrane de l'érythrocyte humain sain, ou infecté par P. falciparum, correspond à différents états d'activité d'un type unique de canal 'maxi-anionique' comportant des niveaux de conductance, des modes d'activation et des propriétés pharmacologiques variables selon les conditions physico-chimiques. 2/ L'identité moléculaire de ce canal anionique est de type 'voltage dependent anion channel (VDAC)'. Il est l'une des trois composantes d'un récepteur 'peripheral-type benzodiazepine receptor (PBR)' présent dans la membrane érythrocytaire. 3/ Le canal VDAC, généralement peu actif correspond, lorsqu'il est activé, à la nouvelle voie de perméation 'new permeability pathway' décrite dans la membrane de l'érythrocyte infecté par P. falciparum. L'activation résulte alors en partie de l'insertion dans la membrane érythrocytaire de protéines plasmodiales de type 'Ring-infected Erythrocyte Surface Antigen (RESA). Ce travail contribue à l'élucidation de la nature exacte des canaux ioniques présents dans la membrane érythrocytaire et avance une hypothèse unificatrice quant au rôle joué par ces canaux.

Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded organisms. Systemic dissemination of T. gondii in the organism can cause life-threatening infection that manifests as Toxoplasma encephalitis in immune-compromised patients. In addition, mounting evidence from epidemiological studies indicates a link between chronic Toxoplasma infection and mental disorders. To better understand the pathogenesis of toxoplasmosis, basic knowledge on the host-parasite interactions and the dissemination mechanisms are essential. Previous findings have established that, upon infection with T. gondii, dendritic cells (DCs) and microglia exhibit enhanced migration, which was termed the hypermigratory phenotype. As a result of this enhanced migration, DCs and microglia are used as vehicle cells for dissemination (‘Trojan horse’) which potentiates dissemination of T. gondii in mice. However, the precise mechanisms behind the hypermigratory phenotype remained unknown. In this thesis, we characterized host-parasite interactions upon infection with T. gondii and investigated the basic mechanisms behind the hypermigratory phenotype of T. gondii-infected DCs and microglia. In paper I, we observed that upon infection with T. gondii, DCs underwent rapid morphological changes such as loss of adhesiveness and podosomes, with integrin redistribution. These rapid morphological changes were linked to hypermotility and were induced by active invasion of T. gondii within minutes. T. gondii-infected DCs exhibited up-regulation of the C-C chemokine receptor CCR7 and chemotaxis towards the CCR7 chemotactic cue, CCL19. In paper II, we developed a 3-dimensional migration assay in a collagen matrix, which allowed us to characterize the hypermigratory phenotype in a more in vivo-like environment. The migration of T. gondii-infected DCs exhibited features consistent with integrin-independent amoeboid type of migration. T. gondii-induced hypermigration of DCs was further potentiated in the presence of CCL19 in a 3D migration assay. In paper III, we identified a parasite effector molecule, a Tg14-3-3 protein derived from parasite secretory organelles. Tg14-3-3 was sufficient to induce the hypermigratory phenotype. Transfection with Tg14-3-3-containing fractions or recombinant Tg14-3-3 protein induced the hypermigratory phenotype in primary DCs and in a microglial cell line. In addition, Tg14-3-3 localized in the parasitophorous vacuolar space and host 14-3-3 proteins were rapidly recruited around the parasitophorous vacuole. In paper IV, we found that mouse DCs dominantly express the L-type voltage-dependent calcium channel, Cav1.3. Cav1.3 was linked to the GABAergic signaling-induced hypermigratory phenotype. Pharmacological inhibition of Cav1.3 and knockdown of Cav1.3 abolished the hypermigratory phenotype in T. gondii infected DCs. Blockade of voltage-dependent calcium channels reduced the dissemination of T. gondii in a mouse model. In paper V, we showed that microglia, resident immune cells in the brain, also exhibited rapid morphological changes and hypermotility upon infection with T. gondii. However, an alternative GABA synthesis pathway was shown to be involved in the hypermigratory phenotype in microglia. In summary, this thesis describes novel host-parasite interactions, including host cell migratory responses and key molecular mechanisms that mediate the hypermigratory phenotype. The findings define a novel motility-related signaling axis in DCs. Thus, T. gondii employs GABAergic non-canonical pathways to hijack host cell migration and facilitate dissemination. We believe that these findings represent a significant step forward towards a better understanding of the pathogenesis of T. gondii infection. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>