Physiologic β-adrenergic activation of PKA during the sympathetic “fight-or-flight” response increases calcium influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. The second chapter of this dissertation describes our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Recent data confirm that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites on α1C or β2B subunits. Proximity proteomic labeling methods led us to other potential PKA targets near the CaV1.2 complex, including Rad, a calcium channel inhibitor that changes its position within the calcium channel neighborhood after β-adrenergic stimulation. With expression of α1C, β2B, and Rad in a heterologous expression system, we reconstituted forskolin-PKA regulation of CaV1.2. By mutating potential PKA phosphorylation sites on Rad, we identified specific residues that are critical for this mechanism to occur and validated Rad as the functional PKA target for regulation of CaV1.2. In the third chapter, we probe the contribution of both CaV1.2 α1C and β subunits in β-adrenergic regulation. Previous results have shown that binding between α1C and β subunits is required for adrenergic stimulation of the calcium channel in the heart. Using transgenic mouse models, we demonstrate that this phenomenon requires a rigid IS6-AID linker in the α1C subunit, as introduction of glycines in this region increased flexibility of the linker and abolished a response to adrenergic agonists even though α1C was able to bind to β. The fourth chapter examines the role of the auxiliary β subunit in β-adrenergic regulation of CaV1.2. Binding of Rad to the β subunit is also necessary for this mechanism to occur. Although the β2B isoform is the predominant subunit in the heart, we show that transgenic mice with β3 or β4 replacing β2B in the heart are viable and still have normal β-adrenergic regulation of CaV1.2, indicating that this mechanism is universal to other voltage gated calcium channels that bind to β subunits and RGK proteins. The fifth chapter verifies that Rad is the PKA target in the heart. Using a knock-in mouse model with four PKA phosphorylation sites mutated to alanine, we definitively show that phosphorylation of Rad is necessary for β-adrenergic regulation of CaV1.2 in the heart. We investigate the importance of Rad phosphorylation on many levels. First, we study Rad’s role in regulating the calcium channel. Second, we observe the effect phosphorylation of Rad has on the calcium transient using isolated cardiomyocytes. Third, we examine cardiovascular function in vivo using radiotelemetry and echocardiograms. Finally, we assess the “fight-or-flight” response in an animal model with exercise capacity testing. Together, these findings conclusively show that in the heart, phosphorylation of Rad is the essential mechanism for the sympathetic nervous system control of calcium influx in both atrial and ventricular cardiomyocytes. Additionally, Rad modulates both heart rate and contractility in vivo. In the sixth chapter, we explore the mechanism of Rad modulation of CaV1.2 in depth using a flow-cytometry Förster resonance energy transfer (FRET) two-hybrid assay. We closely examine the roles of phosphorylation sites on both Rad’s N-terminus and C-terminus. By creating phosphomimetic mutations on Rad, we uncover the importance of phosphorylating the C-terminus for release of Rad from both the membrane and the β subunit. Taken together, these findings elucidate the mechanism behind β-adrenergic regulation of CaV1.2 in the heart – a longstanding query for over forty years in the cardiovascular ion channel field. At baseline, Rad “tunes” the amount of calcium influx into the cell by inhibiting a population of channels as a functional reserve. Upon adrenergic stimulation, Rad is phosphorylated, lessening its interaction with the membrane, and releasing inhibition of the calcium channel. The enhanced local calcium influx allows for additional calcium release into the cytoplasm through ryanodine receptors leading to increased contractility of the heart, a notable characteristic of the evolutionary survival mechanism— “fight-or-flight.”

<p>TRF1 is an essential shelterin protein that binds to double stranded telomeric DNA. TRF1 is best known for its role as a negative regulator of telomere length. Post-translational modifications, like phosphorylation, have been shown to regulate TRF1 function in cells. Mass spectrometric analysis revealed three potential TRF1 phosphorylation sites, threonine 271, serine 279 and threonine 305 <em>in vivo</em>. To analyze the function of these three potential phosphorylation sites, phosphomimic (aspartic acid, D) and non-phosphorylatable (alanine, A) mutations were made to be analyzed <em>in vitro</em>. Through <em>in vitro</em> gel shift assays, the phosphomimic mutation at threonine 271 mutant exhibits a DNA binding defect, whereas serine 279 and threonine 305 mutants have no binding defects. However, <em>in vivo</em> analysis needs to be conducted in order to determine whether this binding defect is authentic.</p> <p>PIN2 is a TRF1 isoform, which is identical in its protein sequence with the exception of 20 amino acids (residue 296-316 of TRF1). The second study looks to characterize any potential functional differences between PIN2 and TRF1. <em>In vitro</em>, CDK1 kinase assay was conducted on TRF1 and PIN2 mutants to assess whether there was a difference in phosphorylation. The kinase assay revealed that both PIN2 threonine 351 and TRF1 threonine 371 are both phosphorylated by CDK1. However, the net phosphorylation level of PIN2 by CDK1 is far lower than the net phosphorylation level of TRF1. An <em>in vitro</em> gel shift assay was also conducted to analyze the binding difference between TRF1 threonine 371 and PIN2 threonine 351 mutants. The DNA binding assay revealed that TRF1 threonine 371D mutant has a binding defect, whereas PIN2 threonine 351D mutant has no binding defect. However, this data needs to be verified to determine whether a PIN2 threonine 351D mutant behaves like a phosphomimic.</p> / Master of Science (MSc)

Kappa opioid receptor (KOPR) is involved in many physiological functions and pharmacological responses such as analgesia, anti-pruritic effect, sedation, motor incoordination and aversion (Simonin et al., 1998; Liu-Chen, 2004). The cellular mechanisms following activation of KOPR involve in part Gi/o protein-dependent pathways (Law et al., 2000). Following KOPR activation, the receptor is phosphorylated and arrestins are recruited. Arrestins mediate agonist-dependent KOPR desensitization, internalization and down-regulation (Liu-Chen, 2004). In recent years, arrestins were found to initiate arrestin-dependent downstream signaling. Thus, agonist-promoted KOPR phosphorylation plays a pivotal role in KOPR regulation and signaling. Previous studies from our lab showed that in Chinese hamster ovary (CHO) cells stably transfected with the human KOPR (hKOPR), U50,488H induced phosphorylation (Li et al., 2002a); however, sites of phosphorylation were not determined. Using LC-MS/MS, our lab recently identified four residues (S356, T357, T363 and S369) to be the sites of U50,488H-promoted phosphorylation in the mouse KOPR (mKOPR) stably expressed in N2A cells (Chen et al., 2016). Antibodies were generated against phosphopeptides and purified and three antibodies were found to have high specificity for the mKOPR phosphorylated at S356/T357, T363 and S369, respectively (Chen et al., 2016). Our lab previously showed that while U50,488H promoted robust hKOPR phosphorylation and internalization, etorphine induced little phosphorylation and internalization, although both were potent full agonists in enhancing [35S]GTPγS (Li et al., 2002a; Zhang et al., 2002; Li et al., 2003). Etorphine caused lower levels of KOPR phosphorylation at all the four residues than U50,488H by immunoblotting with the phospho-specific antibodies (Chen et al., 2016). Using the SILAC (stable isotope labeling by amino acids in cell culture) approach, we have found that compared to etorphine, U50,488H promoted higher levels of single phosphorylation at T363 and S369 and double phosphorylation at T363+S369 and T357+S369 as well as triple phosphorylation at S356+T357+S369 (Chen et al., 2016). These results indicate that an above-threshold phosphorylation is required for KOPR internalization. It has been reported that KOPR is involved in neuronal differentiation and neurogenesis. In the first chapter, I focused on whether there are differences in the mechanisms underlying neurite outgrowth induced by U50,488H and etorphine. In the chapter 2, mechanisms of KOPR phosphorylation were characterized in detail using phospho-specific KOPR antibodies. Protein kinase C was found, for the first time, to be involved in agonist-promoted KOPR phosphorylation. The roles of PKC in behavioral effects induced by KOPR agonists in mice were examined. For the chapter 1, in Neuro2a mouse neuroblastoma cells stably transfected with the hKOPR (N2A-3HA-hKOPR), U50,488H robustly induced neurite outgrowth, but etorphine caused outgrowth to a much lower extent. G protein-dependent pathway was found to be involved in the actions of both agonists, but β-arrestin-dependent pathway was not. Inhibition of ERK1/2 phosphorylation decreased neurite outgrowth promoted by both agonists, indicating the roles of MAP kinase cascades in KOPR agonist-induced neuritogenesis. In contrast, β-arrestin2, 14-3-3ζ, GEC1 and Rap1 are not involved in U50,488H- or etorphine-promoted neurite outgrowth. Thus, the two agonists appear to share the same signaling pathways and the difference between two agonists is likely due to the lower efficacy of etorphine. For the chapter 2, U50,488H caused phosphorylation of the mKOPR at S356, T357, T363 and S369 in N2A cells stably transfected with FmK6H (FmK6H-N2A cells). NorBNI abolished U50,488H-induced KOPR phosphorylation at all four residues. GRKs (GRKs2, 3, 5 and 6) and PKCs were involved in U50,488H-mediated KOPR phosphorylation. In addition, PKC also participated in agonist-independent KOPR phosphorylation. This is the first time that PKC was shown to be involved in agonist-induced KOPR phosphorylation. We found that U50,488H caused KOPR phosphorylation at T363 and S369 in the mouse brain and PKC participated in phosphorylation of S369, but not T363, by using the PKC inhibitor chelerythrine (CHL). Thus, we further characterized effects of PKC inhibition on KOPR-mediated behaviors in CD1 mice. PKC was involved in KOPR-mediated sedation, motor incoordination and conditioned place aversion, but not analgesia and anti-scratching effect in mice. Studies in this thesis revealed the mechanisms of KOPR-mediated neurite outgrowth and KOPR-mediated phosphorylation and the involvement of PKC in KOPR-mediated pharmacological effects in vivo. These studies push the frontier of molecular pharmacology of the KOPR, which may be useful for development of KOPR agonists for therapeutic use. / Pharmacology

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24558号 / 薬科博第175号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, remains elusive. To investigate the impact of PTMs on chaperone function, we chose to characterize two sites of phosphorylation on the linker of HspA1, the stress inducible human Hsp70. To mimic these phosphoserines, we used aspartate as a phosphomimetic substitution for all experiments. Interdomain allostery ties together chaperone structure and function. Therefore, the impact of phosphorylation on interdomain allostery is probed using biophysical and biochemical techniques. Altogether, data suggest that phosphorylation of the linker and SBD destabilizes the chaperone, while shifting the population towards the docked state. This result alludes to a previously described region of the protein that uncouples domain docking from conformational changes in the substrate-binding domain. The cross-communication between these phosphorylation sites reveals a novel, synergistic effect on chaperone structure and function.

La division cellulaire constitue le principe fondamental de la vie et repose sur des changements architecturaux cellulaires spectaculaires. Plusieurs de ces changements sont dirigés par le remodelage précis de structures mécano-sensibles à base d'actine. De plus en plus d'évidences suggèrent une relation étroite entre le contrôle de qualité des protéines et la régulation spatiotemporelle de la dynamique des structures d'actine entre autres, par l'intermédiaire de mécanismes de séquestration ou de dégradation des protéines. Les petites protéines de choc thermique (HSPB) sont des chaperons moléculaires qui font partie intégrante du réseau de contrôle de qualité des protéines, lesquelles contribuent à l'homéostasie du protéome. Ces chaperons émergent comme des modulateurs des structures à base d'actine en conditions physiologiques et comme des protecteurs de l'intégrité de ces structures en conditions de stress. Selon le modèle prévalent, l'assemblage des HSPB en structures oligomériques dynamiques leur confère leur fonction dans la séquestration de composantes cellulaires pour prévenir une agrégation protéique non-spécifique. Néanmoins, leur mode d'action demeure encore élusif : le fait que certaines HSPB ne formeraient pas d'oligomères suggère un autre mécanisme d'action pour ces HSPB. C'est le cas de HSPB8, qui forme un complexe avec le co-chaperon moléculaire BAG3. Les prémices des travaux de cette thèse ont été la découverte d'un nouveau rôle pour ce complexe au cours de la mitose : BAG3, d'une manière dépendante de son association avec HSPB8, facilite le remodelage drastique du cytosquelette d'actine requis pour le positionnement du fuseau mitotique et la ségrégation adéquate des chromosomes. L'objectif de cette thèse était d'identifier le mode de régulation de la fonction mitotique de BAG3-HSPB8 et de disséquer les mécanismes moléculaires impliqués qui facilitent le remodelage du cytosquelette d'actine mitotique. Les travaux de cette thèse apportent des évidences que la modulation des fonctions mitotiques de BAG3 est dépendante de sa phosphorylation par la kinase mitotique CDK1 sur des résidus spécifiques ; Thr285 et Ser386. Ces phosphorylations lui confèrent une activité différentielle sur l'arrondissement cellulaire versus le positionnement du fuseau mitotique. De plus, BAG3 serait phosphorylée dès la phase G2/M sur le résidu Ser195, ce qui modulerait son enrichissement en périphérie du noyau à la transition G2/M. Nos résultats suggèrent que ces phosphorylations seraient impliquées dans la modulation d'associations protéiques différentielles, selon les phases du cycle cellulaire. En outre, l'entrée des cellules en mitose est marquée par l'association de BAG3 avec des protéines du cytosquelette d'actine, telle que cortactine, ainsi qu'avec des acteurs du contrôle de qualité des protéines, notamment le récepteur autophagique p62/SQSTM1 et la déacétylase HDAC6. De manière cruciale, la phosphorylation et les associations protéiques mitotiques de BAG3 sont dépendantes de sa liaison à HSPB8. Nos résultats suggèrent un model selon lequel le complexe BAG3-HSPB8 régule l'assemblage de p62/SQSTM1 en corps supramoléculaires qui pourraient offrir une plateforme pour isoler et réguler l'assemblage de complexes protéiques impliqués dans le remodelage des structures d'actine mitotiques. Via ce mécanisme d'action, BAG3-HSPB8 limiterait la polymérisation de l'actine branchée dépendante d'Arp2/3, en modulant négativement l'activité déacétylase de HDAC6 sur son substrat cortactine, un processus qui faciliterait l'arrondissement mitotique. Ainsi, nos résultats mettent en avant un rôle central pour la phosphorylation de BAG3 dans la modulation de son action mitotique, en étroite collaboration avec ses partenaires HSPB8 et p62/SQSTM1. L'ensemble de nos données contribue ainsi à une meilleure compréhension des mécanismes moléculaires par lesquels le complexe chaperon BAG3-HSPB8 orchestre le remodelage dynamique des structures cellulaires mitotiques à base d'actine et facilite les changements de forme des cellules requis pour la progression mitotique. Ces travaux ont également permis l'identification de nouvelles cibles moléculaires du complexe chaperon, entre autres impliquées dans la dynamique du cytosquelette d'actine. Ces travaux offrent de nouvelles pistes d'investigations intéressantes concernant le développement de pathologies associées à une dérégulation du complexe BAG3-HSPB8, notamment dans la progression tumorale. / Cell division is the fundamental principle of life and is based on spectacular cellular architectural changes. Many of them are driven by the accurate remodeling of mechanosensitive actin-based structures. Growing evidence suggests a close relationship between protein quality control and the spatiotemporal regulation of actin remodeling, through mechanisms that would promote protein sequestration and/or degradation. Small heat shock proteins (HSPBs) are molecular chaperones that are an integral part of the protein quality control network, which contribute to maintain proteome homeostasis. They emerge as modulators of actin-based structures under physiological conditions and as guardians of the integrity of cytoskeletal structures under stress conditions. According to the prevailing model, the assembly of HSPBs into large oligomers confers them with the ability to sequester cellular components and prevent unspecific aggregation of damaged proteins. Nevertheless, their mode of action remains elusive: the observation that some HSPBs do not form oligomers suggests another mechanism of action for these HSPBs. This is the case for HSPB8, which forms a complex with the molecular co-chaperone BAG3. The working model of this thesis is based on the initial discovery in our laboratory of a new role for this complex during cell division: BAG3 facilitates the drastic remodeling of the actin cytoskeleton required for spindle positioning and proper segregation of chromosomes, in a manner that requires HSPB8. The aim of this thesis was to identify the mechanisms whereby such a function of the BAG3-HSPB8 chaperone complex is regulated, and to investigate how the complex can facilitate mitotic actin cytoskeleton remodeling. The work presented here provides evidence that the modulation of BAG3 mitotic functions depends on its phosphorylation by the mitotic kinase CDK1 at specific residues, Thr285 and Ser386, which confers differential activity on cell rounding versus mitotic spindle positioning. Evidence also suggests that BAG3 would be phosphorylated earlier in the G2/M phase, at Ser195, which would modulate its perinuclear enrichment. Our results suggest that these phosphorylations could be involved in defining specific protein associations, in a cell-cycle dependent manner. In addition, we found that mitotic entry is marked by the stimulation of BAG3'sassociation with proteins that organize the actin cytoskeleton, such as cortactin, as well as with protein quality control actors, notable, the autophagic receptor p62/SQSTM1 and the deacetylase HDAC6. Critically, BAG3 phosphorylation and its associations with mitotic protein partners rely on its binding to HSPB8. The results suggests a model whereby the BAG3-HSPB8 complex would regulate the molecular assembly of p62/SQSTM1 into mitotic bodies that could provide a platform to sequester and facilitate protein complex assembly implicated in mitotic actin cytoskeleton remodeling. Via this mechanism, BAG3-HSPB8 could limit branched actin polymerization that depends on Arp2/3 activity, by down-modulating HDAC6 deacetylase activity towards its substrate cortactin, a process that would facilitate mitotic cell rounding. Thus, our results highlight a central role of BAG3 phosphorylation in the modulation of its mitotic action, in close relationship with its partners HSPB8 and p62. Altogether, our data contribute to a better understanding of the molecular mechanisms by which the BAG3-HSPB8 chaperone complex orchestrates the dynamic remodeling of mitotic cell structures and thereby, facilitates the cell shape changes required for mitotic progression. This study has also identified new molecular targets of the chaperone complex there are, among others, involved in the dynamics of the actin cytoskeleton. Thus, this work offers new avenues of investigation regarding the development of pathologies associated with a deregulation of the BAG3-HSPB8 complex, particularly in tumor progression.

Three distinct families of PTPs, the conventional (cPTPs), low molecular weight (LMW PTPs), and Cdc25 PTPs, have converged upon a common catalytic mechanism and active site sequence, mainly, the phosphate-binding loop encompassing the PTP signature motif (H/V)<b>C</b>(X)₅<b>R</b>(S/T) and an essential Asp residue on a surface loop. There is little sequence similarity among the three families of phosphatases. All known LMW PTP remove phosphoryl groups esterified to the hydroxyl amino acid: tyrosine, whereas all members of the Cdc25 family are dual-specificity protein phosphatases that dephosphorylate all the hydroxyl amino acids: tyrosine, serine and threonine. The cPTP family primarily functions as tyrosine phosphatases, but it also includes dual-specific members. ORFs encoding potential cPTPs have been identified in five archaeal species: <i>Methanobacterium thermoautotrophicum</i>, <i>Methanococcus jannaschii</i>, <i>Thermococcus kodakaraensis</i>, <i>Pyrococcus horikoshii</i>, and <i>S. solfataricus</i>. Only one has been partially characterized, <i>Tk</i>-PTP from <i>T. kodakaraensis</i>. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented. The genome of <i>S. solfataricus</i> encodes a single conventional protein tyrosine phosphatase, SsoPTP. SsoPTP is the smallest known archaeal PTP (18.3 kDa) with a primary amino acid sequence that conforms to the cPTP protein tyrosine phosphatase paradigm, HCX₅R(S/T). Relatively little is known about its mode of action " whether it follows the conventional PTP mechanism or employs a different route for catalysis " or its physiological role. ORF <i>sso2453</i> from the genome of <i>Sulfolobus solfataricus</i>, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in <i>E. coli</i> and purified by immobilized metal affinity chromatography. SsoPTP displayed the ability to dephosphorylate protein-bound phosphotyrosine as well as protein-bound phosphoserine/phosphothreonine. SsoPTP hydrolyzed both isomers of naphthyl phosphate, an indication of dual specificity. The four conserved residues within the presumed active site sequence: Asp⁶⁹, His⁹⁵, and Arg¹⁰², and the invariant Gln¹³⁹ residue were essential for catalysis, as it was predicted for the established members of the PTP family in both bacteria and eukaryotes. A substrate trapping protein variant, SsoPTP-C96S/D69A, was constructed to isolate possible SsoPTP substrates present in <i>S. solfataricus</i> cell lysates. Several potential substrates were isolated and identified by mass spectroscopy. / Ph. D.

Plus de 25 millions de personnes à travers le monde sont atteintes de différentes formes de démences, la plus répandue étant la maladie d’Alzheimer (MA). Cette maladie neurodégénérative est caractérisée par la présence de plaques de peptides amyloïde-β et d’enchevêtrements neurofibrillaires formés d’agrégats toxiques de la protéine Tau. Ces agrégats sont dus à l’hyperphosphorylation de cette protéine associée aux microtubules, déstabilisant son interaction avec ceux-ci et participant à la dégénérescence neuronale. Pour étudier la protéine Tau, certains laboratoires utilisent des modèles animaux, et les méthodes de préparation des échantillons biologiques prélevés varient d’un laboratoire à l’autre, affectant potentiellement son observation. La MA est la plus connue des Tauopathies, qui est un groupe de maladies exprimant une forme pathologique de la protéine Tau. Il existe aussi des Tauopathies dites secondaires, où la pathologie Tau apparaît dans les stades tardifs de la maladie, comme la maladie de Huntington (MH) où il existe une pathologie Tau qui est cependant mal caractérisée chez l’humain. Dans ce contexte, nos hypothèses sont qu’une meilleure caractérisation immunohistochimique et biochimique de la protéine Tau est nécessaire puisque les méthodes présentement utilisées pour sa caractérisation ne sont pas optimales et que la MH est effectivement une Tauopathie secondaire chez l’humain due à l’hyperphosphorylation de Tau et une déficience de l’épissage alternatif aux stades avancés de la maladie. Nos objectifs étaient d’optimiser les méthodes de préparation pour l’étude de Tau par immunohistochimie dans des modèles murins et d’analyser les niveaux de phosphorylation et l’épissage alternatif de Tau dans les cerveaux post-mortem d’individus atteints de la MH. Les résultats obtenus suggèrent qu’une fixation au Bouin, sans perfusion et à 4°C semble être la meilleure méthode pour l’observation de Tau par immunohistochimie. L’hyperphosphorylation et les défauts d’épissage de Tau observés dans la MH permettent de définir cette pathologie comme une Tauopathie secondaire. / More than 25 million people worldwide are affected by different forms of dementia, the most known being Alzheimer’s disease (AD). This neurodegenerative disorder is characterized by the presence of β-amyloid plaques and neurofibrillary tangles formed by toxic aggregates of Tau protein. These aggregates are due to the hyperphosphorylation of this microtubule-associated protein, which destabilize its interaction with them and could participate to neuronal deterioration. To study Tau protein, many laboratories use animal models, and preparation methods of biological samples vary between each laboratory, which could potentially have effects on its observation. AD is the most common Tauopathy, a group of diseases expressing a pathological form of Tau protein. There are also secondary Tauopathies, where Tau pathology appears in late stages of the disease, such as Huntington’s disease (HD), with a Tau pathology that is not well characterized in humans. In this context, our hypotheses are that a better immunohistochemical and biochemical characterization of Tau protein is needed since methods currently used for its characterization are not optimal and that HD is indeed a secondary Tauopathy in human due to hyperphosphorylation of Tau and deficiency of splicing in advanced stage of the disease. Our objectives were to compare different preparation and fixation methods for the study of Tau protein by immunohistochemistry in mice models, and also to analyze phosphorylation and splicing levels of Tau protein in post-mortem brains of individuals with HD. Our results suggest that a fixation with Bouin, without perfusion and at 4°C would be the best method for the observation of Tau protein by immunohistochemistry, and that hyperphosphorylation and splicing impairments observed in individuals with HD allow to define this pathology as a secondary Tauopathy in humans.

No / We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.

Le facteur de transcription GATA4 est un puissant régulateur du développement cardiaque, gonadique et digestif. Bien qu'un grand nombre de ses gènes cibles soient connus, les mécanismes contrôlant son expression et son activité demeurent incertains. GATA4 est phosphorylé par la PKA en réponse à l'AMPc en serine 261 et des MAPK en serine 105. L'objectif de mon projet de maîtrise est donc de définir l'importance de ces sites de phosphorylations et plus particulièrement par les MAPK sur l'activité transcriptionnelle de GATA4 sur certains promoteurs cibles. Les résultats de transfection transitoire au phosphate de calcium montrent que la stimulation par les MAPK potentialise l'activité transcriptionnelle de GATA4 sur les promoteurs Star, Inha et Cypl9. Cette stimulation potentialise également la coopération de GATA4 avec ses partenaires transcriptionnels comme LRH1 et SF1 sur le promoteur Inha. Ceci a permits de mettre en évidence l'importance des MAPK dans la régulation de l'activité transcriptionnelle de GATA4 sur certains promoteurs cibles.