Les parasites Theileria (Theileria. annulata and T. parva) sont des protozoaires intracellulaires qui font partie du phylum des Apicomplexa. Theileria infecte les leucocytes bovins et les transforment en cellules cancéreuses, induisant un genre de leucémie chez le bovin et conduisant à la mort de l’animal. Les cellules infectées par Theileria démontrent certaines caractéristiques de cellules cancéreuses telles qu’une importante capacité d’invasion et de migration cellulaire. Cependant, le traitement de cellules infectées avec une drogue Theiléricide spécifique (buparvaquone) permet l'élimination du parasite et la réversion du phénotype transformé. De plus, la virulence peut être atténuée par passages répétés sur culture cellulaire. La similitude entre les cellules transformées par Theileria et la leucémie humaine fait de Theileria un modèle très important permettant l’étude des mécanismes cellulaires induits par le parasite au cours de la transformation de la cellule hôte. Mon laboratoire d’accueil a publié une augmentation significative de TGF-β2 dans les cellules virulentes et a constaté que parmi les 1158 cibles de TGF-β, 68 gènes ont été reconnus d'avoir modifié leurs niveaux de transcription concomitante avec l'atténuation. Dans ce travail de thèse, nous avons étudié les voies de signalisations impliquées dans la régulation de l’adhésion et l’invasion des cellules infectées par Theileria. Nous nous sommes particulièrement intéressés à l’étude de la voie de signalisation TGF-β2 et ses effecteurs. Nos résultats montrent que l’activation de la voie de signalisation de TGF-β2 par Theileria entraîne une augmentation de l’invasion et de l’adhérence des cellules transformées par deux mécanismes différents, soit en activant la voie de signalisation PGE2/EP4/cAMP/PKA/EPAC/CREB, soit en stimulant la voie GRB2/PI3-K/AP-1. Les macrophages atténués infectés par Theileria sont plus stressés oxydativement ce qui diminue leur adhérence et leur invasion cellulaire. Ceci nous a amené à étudier en collaboration avec un autre doctorant (Mehdi Metheni) le rôle de TGF-β2 dans la régulation du stress oxydatif dans les macrophages infectés par Theileria. Nos données montrent que les niveaux élevés de TGF-β2 stimule l’expression de la catalase, une enzyme anti-oxydante qui convertit le H2O2 en H2O et la baisse de H2O2 favorise la virulence en augmentant l’invasion et l’adhésion des cellules infectées par Theileria (résultats supplémentaires). De plus, nous avons examiné le statut de stress oxydatif et le type de glycolyse utilisé par les cellules infectées par Theileria. Les cellules transformées par Theileria agissent comme des cellules cancéreuses, elles consomment énormément de glucose. La protéine BAD joue un rôle important dans l’apoptose ainsi que dans la voie de glycolyse. Son activité est régulée par phosphorylation en réponse à des facteurs de croissance et de survie. BAD peut être phosphorylée par la PKA sur le résidu sérine 155. Durant ma thèse, nous avons examiné le rôle de la phosphorylation de BAD par la PKA dans la régulation du métabolisme cellulaire des macrophages infectés par Theileria. Nos résultats montrent que l’abolition de la phosphorylation de BAD par la PKA dissocie le complexe mitochondrial formé entre BAD et HK2, ce qui induit l’ubiquitynation et la dégradation de HK2 par le protéasome. La baisse de HK2 stimule la voie de phosphorylation oxydative en faveur de l’effet Warburg dans les cellules infectées par Theileria. / Theileria parasites (Theileria. annulata and T. parva) are intracellular protozoa and members of the phylum Apicomplexa. Theileria parasites are the only eukaryotes that possess the property of being able to transform another eukaryote, their leukocyte host cells. Transformed leukocytes show many characteristics of tumour cells such as heightened invasive capacity; however the tumour-like phenotype can be totally reversed upon drug induced parasite death and attenuated by multiple in vitro passages. Such multiple-passaged attenuated lines are used as live vaccines against tropical theileriosis. The similarities in tumour hyper-invasiveness between Theileria-transformed leukcocytes and human lymphomas imply that observations on Theileria-induced leukocyte transformation have the potential to give generally applicable insights into the mechanisms underpinning tumour virulence. My host laboratory described higher TGF-β2 levels in virulent infected macrophages and following microarray analysis of virulent compared to attenuated macrophages found that among the 1158 TGF-β-targets, 68 genes had altered transcript levels concomitant with attenuation. In this study, we investigate the signalling pathways involved in the regulation of cellular adhesion and invasiveness of Theileria-infected cells. We were especially interested in the study of TGF-β2 signalling in Theileria-transformed virulent versus attenuated macrophages. My results indicate that following Theileria infection of macrophages, the TGF-β2 signalling pathway is activated and induces an increase in adhesion of virulent transformed macrophages through two different mechanisms: either by activating a PGE2 / EP4 / cAMP / PKA / EPAC / CREB signaling pathway, or by stimulating a GRB2 / PI3-K / AP-1 pathway. As attenuated macrophages display heightened oxidative stress, which underpins their loss of adhesion and invasiveness, in collaboration with another PhD student (Mehdi Metheni) we investigated the role of TGF-β2 in the regulation of the oxidative stress status of Theileria-infected macrophages. Our data show that high levels of TGF-β2 increase the expression of catalase, an anti-oxidant enzyme that converts H2O2 into H2O and the drop in H2O2 output results in regain of the virulence trait heightened adhesion of Theileria-transformed macrophages to fibronectin. Theileria-transformed macrophages display many features of cancer cells such as their consumption of larger quantities of glucose. The BCL-2 family protein BAD has an alternative function in glucose metabolism separate from its role in apoptosis. The activity of BAD is regulated by phosphorylation in response to growth/survival factors. BAD can be phosphorylated on Ser155 by PKA. So during my thesis studies I examined the role of PKA mediated phosphorylation of BAD in the regulation of the cellular metabolism of Theileria-transformed macrophages. My results showed that ablation of BAD S155 phosphorylation dissociates the mitochondrial complex of BAD and HK2 and cytosolic HK2 becomes ubiquitinated and degraded by the proteasome. Loss of HK2 switches the metabolism of Theileria-transformed leukocytes from Warburg-like to OXPHOS-like glycolysis.

La protéine kinase AMPc-dépendante (PKA) joue un rôle crucial dans la régulation neurohormonale de la fonction cardiaque. L’activation aiguë de la PKA est bénéfique car elle conduit à une augmentation de la contraction cardiaque en phosphorylant les acteurs clés du couplage excitation-contraction. En revanche, son activation chronique est délétère et ces effets semblent faire intervenir la régulation de protéines nucléaires pouvant conduire au remodelage hypertrophique et à l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurée par des protéines d’ancrage (AKAPs), est importante pour la rapidité et la spécificité d’action des hormones mettant en jeu la voie de l’AMPc. Les niveaux d’AMPc sont régulés par l’activité des adénylate cyclases et des phosphodiestérases (PDEs), et l’état de phosphorylation des protéines cibles de la PKA dépend de l’activité des Ser/Thr phosphatases (PPs). Dans le cœur, les PDEs les plus importantes dégradant l’AMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une première partie de mon travail, j’ai mis au point, dans les cardiomyocytes de rats adultes, une mesure de l’activité de la PKA en temps réel dans les compartiments cytoplasmiques et nucléaires. J’ai utilisé pour cela des sondes de type AKAR (A-kinase activity reporters) basées sur le transfert d’énergie de fluorescence (FRET) et localisées spécifiquement dans le noyau ou dans le cytoplasme par des séquences d’adressage ou d’exclusion nucléaires. J’ai ainsi pu montrer qu’une stimulation maintenue des récepteurs β-adrénergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se développe lentement au niveau nucléaire que dans le cytoplasme. De ce fait, une stimulation brève des récepteurs β-adrénergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de l’étude, je me suis intéressée au rôle des PDE3 et PDE4 ainsi qu’à celui de PP1, PP2A et PP2B dans la régulation de l’activité PKA cytoplasmique et nucléaire, en réponse à une stimulation β-adrénergique. J’ai montré que la PDE4, mais pas la PDE3, régule l’activité de la PKA cytoplasmique et nucléaire. L’utilisation de souris invalidées pour les gènes Pde4b et Pde4d a révélé que l’isoforme PDE4B est prédominante pour la modulation de l’activité PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent à la régulation de l’activité PKA nucléaire. Finalement, j’ai montré que la PP1 et la PP2A, mais pas la PP2B, participent à la terminaison des réponses β-adrénergiques dans le cytoplasme, alors qu’au niveau nucléaire, la PP1 semble jouer un rôle majeur. En conclusion, ce travail a mis en évidence le rôle des phosphodiestérases et des phosphatases dans l’intégration différentielle des réponses PKA à une stimulation β-adrénergique dans le cytoplasme et le noyau de cardiomyocytes adultes. / The cAMP-dependent protein kinase (PKA) exerts short term beneficial effects on cardiac function by phosphorylating several key excitation-contraction coupling (ECC) proteins. However, its chronic activation is deleterious on the long term, and this may involve regulation of nuclear effectors ultimately leading to hypertrophic remodelling and heart failure. The subcellular localization of PKA, mediated by anchoring proteins (AKAPs), is important for the speed and specificity of hormones that activate the cAMP pathway. The levels of cAMP are regulated by adenylyl cyclase and phosphodiesterases (PDEs), and PKA activity is counterbalanced by Ser/Thr phosphatases (PPs). In heart, the most important PDEs that degrade cAMP belong to the PDE3 and PDE4 famillies, whereas the major cardiac PPs are PP1, PP2A and PP2B. In a first part, I developed, in adult rat cardiomyocytes, a technique to measure PKA activity in real time specifically in the cytoplasm and the nucleus. For this I used genetically-encoded fluorescence resonance energy transfer (FRET) sensors called AKAR (A-kinase activity reporters) that can be targeted specifically to the nucleus or the cytoplasm by nuclear localization or exclusion sequences, respectively. Using this approach, I showed that maintained β-adrenergic stimulation activates PKA more efficiently and more potently in the cytoplasm than in the nucleus, and that the kinetics of PKA activation was much slower in the nucleus than in the cytoplasm. Accordingly, a short β-adrenergic stimulation maximally activated PKA in the cytoplasm but marginally activated PKA in the nucleus. In a second part, I characterized the respective contribution of PDE3, PDE4, and PP1, PP2A and PP2B families in the regulation of cytoplasmic and nuclear PKA activity in response to β-adrenergic stimulation. PDE4, but not PDE3, regulates PKA activity in the cytoplasm and in the nucleus. The use of knock out mice for Pde4b and Pde4d genes revealed that PDE4B plays a predominant role to modulate β-AR stimulation of cytoplasmic PKA, whereas in the nucleus both PDE4B and PDE4D isoforms contribute. Finally, I showed that both PP1 and PP2A, but not PP2B, participate to the termination of β-adrenergic PKA responses in the cytoplasm, whereas PP1 appears to play a major role in the nuclei. In conclusion, this work highlights the role of phosphodiesterases and phosphatases in the differential integration of PKA responses to β-adrenergic stimulation in the cytoplasm and the nucleus of adult cardiomyocytes.

La régulation de la télomérase s’effectue à de nombreux niveaux dont la transcription de la sous-Unité catalytique (hTERT). Les travaux du laboratoire effectués sur les cellules NB4, modèle de Leucémie Aiguë Promyélocytaire (LAP), ont montré que l'acide rétinoïque tout-Trans (ATRA) réprime la transcription de hTERT. Cette répression peut être associée à la différenciation (cas des cellules NB4) ou en être dissociée conduisant à la mort des cellules (cas des cellules NB4-LR1 résistantes à la maturation induite par l’ATRA). A partir de la lignée NB4-LR1 a été sélectionnée la lignée NB4-LR1SFD résistante à cette mort cellulaire du fait de la ré-Expression de hTERT même en présence d’ATRA. Cependant cette résistance à la répression de hTERT peut être levée par le co-Traitement ATRA et trioxide d’Arsenic (As2O3) qui conduit à la mort des cellules. Il s'agit donc d'une propriété nouvelle de cette lignée dont le mécanisme reste à élucider.Les résultats obtenus par le laboratoire suggèrent l'importance du statut de méthylation de l’ADN du promoteur de hTERT jusque là peu explorée qui pourrait rendre compte de la résistance à la répression de hTERT. Mon projet a pour objectif de valider cette hypothèse en tirant profit de la diversité des réponses biologiques (différenciation, prolifération, mort cellulaire et expression de hTERT) des variants cellulaires du modèle NB4. Une coopération entre le statut épigénétique (méthylation de l’ADN et modification des histones) du promoteur de hTERT et la fixation de facteurs activateurs et/ou répresseurs sera étudiée. Le statut de méthylation du promoteur de hTERT sur une région allant de -2500pb à +1000pb par rapport au site d’initiation de la transcription a été étudié par la technique de séquençage (Illumina) après traitement des cellules NB4-LR1SFD par l’ATRA seul ou en combinaison avec As2O3. Le résultat obtenu à ce jour montre une hypométhylation d’une région limitée du domaine distal (de -1300pb à -800pb) du promoteur de hTERT associée à la répression de hTERT dans les cellules traitées par la combinaison ATRA+ As2O3 par rapport aux traitements seuls par ATRA ou As2O3. Ceci renforce l’importance du statut de méthylation de cette région du promoteur dans la régulation de l’expression de hTERT. Ce co-Traitement induit également une diminution de l’expression protéique de cMyc et WT1, et aussi de l’ADN methyltransférase 1 (DNMT1) suggérant un rôle de cette enzyme dans le maintien de la méthylation de cette région du promoteur de hTERT. Dans le but d’évaluer le rôle de c-Myc dans la régulation de hTERT, nous avons montré qu’un analogue de l’AMPc, le 8-CPT-CAMP, induisait une dégradation (en partie protéasome dépendant) de la protéine c-Myc dès 6h de traitement dans la lignée résistante NB4-LR1SFD et non la lignée parentale NB4. La lignée NB4-LR1SFD est caractérisée par un déficit en sous unité régulatrice PKA RII. Spécifique knock-Down de PKA RII et l’utilisation d’agonistes et d’antagonistes spécifiques de PKAI a montré : 1) PKAI et PKAII ont des rôles différents sur la stabilité de la protéine c-Myc; 2) le rapport PKAI/PKAII déterminait la stabilité de c-Myc suite à l’activation de la signalisation PKA. Ces résultats suggèrent un rôle possible de PKA comme régulatrice de expression de hTERT via son implication dans le maintien de la stabilité de la protéine c-Myc. / The regulation of telomerase occurs at various levels, including the transcriptional regulation of hTERT. Previous results in our laboratory from acute promyolocytic leukemia cell model NB4, have shown that all-Trans retinoid acid (ATRA) repress the transcription of hTERT. This repression can be associated with differentiation (in the case of NB4 cells), or be dissociated with differentiation and triggers cellular death (the case of maturation resistant NB4-LR1 cells). Another variant NB4-LR1SFD cells were isolated from NB4-LR1 cells with continuous presence of ATRA and were resistant to the cellular death induced by ATRA. In fact, this resistance is related to the re-Expression of hTERT in presence of ATRA. However, this resistance can be overcome by combination of ATRA and AS2O3 and triggers cellular death.The results obtained in our laboratory suggested the importance of the DNA methylation status in the promoter region of hTERT and could be the one mechanism of the resistance to the repression of hTERT induced by ATRA. My project is by taking the diversity of biological response of the NB4 cells variants to validate the hypothesis. And the cooperation between epigenetic modifications and the binding of transcriptional factors will be equally studied.The DNA methylation status in the promoter region of hTERT from -2500bp to +1000bp has been analyzed with the sequencing technique (illumina) in NB4-LR1SFD treated by ATRA alone or in combination with AS2O3. The results showed a distal hypomethylated region from -1300bp to -800bp associated with the repression of hTERT by the co-Treatment of ATRA and AS2O3 compared with the treatment by ATRA or AS2O3 alone. This result strengthens the importance of methylation status in this region in the regulation of hTERT. The co-Treatment induces also a diminution in protein expression of cMyc, WT1 and DNA methyltransferase 1 (DNMT 1), suggesting this enzyme may play a role in the maintenance of methylation level in this region.In order to evaluate the role of cMyc in the regulation of hTERT, we have shown that an analog de cAMP, 8-CPT-CAMP, induces degradation (partly proteasome-Dependent) of c-Myc protein since 6h in NB4-LR1SFD cells but not in NB4 cells. NB4-LR1SFD cells are characterized by a defect of the PKA regulatory subunit II. Specific knockdown of PKA RII and utilizations of agonists and antagonists of PKA I have shown that: 1) PKA I and PKA II have distinct functional roles on the steady-State of c-Myc protein. 2) The ratio of PKA I/PKA II determines the stability of c-Myc protein with the activation of PKA signalization. These results suggest a possible role of PKA in the regulation of hTERT expression through its modulation on the stability of c-Myc.

Für ein tiefergehendes Verständnis von Entwicklung und Funktion der quergestreiften Muskulatur ist eine Betrachtung der am Aufbau der Myofibrillen, den kontraktilen Organellen, beteiligten Proteine essentiell.<br> Die vorliegende Arbeit beschäftigt sich mit Myomesin, einem Protein der sarkomeren M-Bande. Zunächst wurde die cDNA des humanen Myomesins vollständig kloniert, sequenziert und nachfolgend die komplette Größe der aminoterminalen Kopfdomäne bestimmt. Es konnte gezeigt werden, daß Myomesin in vitro mit den Domänen 1 und 12 an Myosin bindet. Die muskelspezifische Isoform der Kreatinkinase bindet an die Domänen 7 und 8.<br> Stimulations- und Inhibitionsexperimente belegen, daß Myomesin an Serin 618 in vivo durch die Proteinkinase A phosphoryliert wird und daß diese Phosphorylierung durch Aktivierung beta2-adrenerger Rezeptoren stimulierbar ist. In Muskelgewebeproben von Patienten, die an der Hypertrophen Kardiomyopathie, einer genetisch bedingten Herzmuskelkrankheit, erkrankt sind, konnte mit einem neu hergestellten phosphorylierungsabhängigen Antikörper eine Verminderung der Menge phosphorylierten Myomesins nachgewiesen werden. Mögliche Ursachen werden diskutiert.<br> Myomesin bildet Dimere, wie durch hefegenetische und biochemische Experimente gezeigt werden konnte. Die Dimerisierung von Myomesin könnte eine zentrale Rolle für den Einbau der Myosinfilamente in die naszierende Myofibrille haben. Anhand der gewonnenen Daten wurde ein verbessertes Modell der zentralen M-Bande erstellt. / A deep understanding of the development and function of the sarcomeric muscle depends on the careful study of proteins involved in the assembly of myofibrills, the contractile organelles in cross-striated muscle.<br> This thesis deals with the sarcomeric M-band protein myomesin. First, the complete cDNA of human myomesin was cloned, sequenced, and subsequently the size of the aminoterminal head domain of myomesin was determined. Myomesin binds to myosin in vitro via domains 1 and 12. Musclespecific creatine kinase is binding to the domains 7 and 8 of myomesin.<br> Stimulation and inhibition experiments revealed, that serin 618 in human myomesin is phosphorylated in vivo by protein kinase A and that this phosphorylation can be stimulated by activation of beta2-adrenergic receptors. In muscle tissue of patients showing symptoms of the hypertrophic cardiomyopathy, a cardiac disease caused by genetic defects, the amount of phosphorylated myomesin was lowered as detected by a phosphospecific antibody which was established new.<br> Myomesin dimerizes as shown by yeast two hybrid and biochemical experiments. Myomesin dimerization could be a central point in myofibrillogenesis, when myosin filaments were incorporated in nascent myofibrills. Taking all the data together, an improved model of the central M-band was developed.

Die vakuoläre Protonen-ATPase, kurz V-ATPase, ist ein multimerer Enzymkomplex, der in fast jeder eukaryotischen Zelle zu finden ist und den aktiven elektrogenen Transport von Protonen über Membranen katalysiert. Die Aktivität der V-ATPase ist essentiell für eine Vielzahl physiologischer Prozesse. Ein grundlegender Mechanismus zur Regulation der V-ATPase-Aktivität ist die reversible Dissoziation des Holoenzyms in den integralen VO-Komplex, der als Protonenkanal dient, und den cytosolischen V1-Komplex, der ATP hydrolysiert und somit den Protonentransport energetisiert. Die Untereinheit C, die im dissoziierten Zustand der V-ATPase als einzige Untereinheit isoliert im Cytoplasma vorliegt, scheint bei der Bildung des aktiven Holoenzyms eine Schlüsselrolle zu übernehmen. In den Speicheldrüsen der Schmeißfliege Calliphora vicina ist die V-ATPase an der Speichelsekretion beteiligt. In den sekretorischen Zellen wird die Bildung des V-ATPase-Holoenzyms in der apikalen Plasmamembran durch das Neurohormon Serotonin (5-HT) stimuliert. Der Effekt von 5-HT auf die V-ATPase wird intrazellulär durch die Proteinkinase A (PKA) vermittelt und hält nur für die Dauer der Stimulierung an. In der vorliegenden Arbeit wurde mittels Phosphoproteinfärbungen und 2D-Elektrophorese nachgewiesen, dass infolge einer Stimulierung der Drüsenzellen mit 5-HT die Untereinheit C der V-ATPase durch die PKA reversibel phosphoryliert wird. Die Phosphorylierung geht einher mit einer Umverteilung der Untereinheit C aus dem Cytoplasma zur apikalen Plasmamembran und der Bildung des aktiven Holoenzyms. Immuncytochemische Untersuchungen zeigten, dass die katalytische Untereinheit der PKA ebenfalls umverteilt wird und in stimulierten Zellen im Bereich der apikalen Plasmamembran konzentriert vorliegt. Um herauszufinden welche Proteinphosphatase der PKA entgegenwirkt, wurden luminale pH-Messungen durchgeführt und der Effekt von spezifischen Proteinphosphatase-Inhibitoren und veresterten Komplexbildnern zweiwertiger Kationen auf die V-ATPase-Aktivität untersucht. Diese Messungen führten zu der Schlussfolgerung, dass eine Proteinphosphatase des Typs 2C an der Inaktivierung der V-ATPase beteiligt ist. Mit weiteren Phosphoproteinfärbungen konnte gezeigt werden, dass die Dephosphorylierung der Untereinheit C ebenfalls durch eine Proteinphosphatase 2C katalysiert wird und dies vermutlich die Dissoziation des VO- und V1-Komplexes begünstigt. Darüber hinaus konnte durch luminale pH-Messungen und ergänzende biochemische Untersuchungen eine Calcineurin-vermittelte Modulation des cAMP/PKA-Signalweges durch den parallel aktivierten IP3/Ca2+-Signalweg und damit einhergehend eine Beeinflussung der V-ATPase-Aktivität durch den [Ca2+]-Spiegel nachgewiesen werden. / The vacuolar-type H+-ATPase (V-ATPase) is a multimeric enzyme that can be found in nearly every eukaryotic cell. It catalyses the active electrogenic transport of protons across membranes and is essential for a multitude of physiological processes. A fundamental mechanism to regulate V-ATPase activity is the reversible dissociation of the holoenzyme into an integral proton conducting VO-complex and a cytosolic V1-complex that hydrolyses ATP and thus energises proton translocation. Subunit C occurs isolated in the cytoplasm upon dissociation of the V-ATPase complexes and seems to be critical for the formation of active holoenzymes. In the salivary glands of the blowfly Calliphora vicina the V-ATPase is involved in fluid secretion. In secretory cells, formation of the V-ATPase holoenzyme is stimulated by the hormone serotonin (5-HT). The effect of 5-HT on V-ATPase activity is mediated by protein kinase A (PKA) and persists for the duration of the 5-HT stimulus. In this study, it was shown by phosphoprotein stainings and two-dimensional electrophoresis that subunit C of the V-ATPase becomes phosphorylated by PKA upon exposure of blowfly salivary glands to 5-HT. Parallel to the phosphorylation event, subunit C translocates from the cytoplasm to the apical plasma membrane for the assembly of active V-ATPase holoenzymes. Using immunofluorescence staining, it could be shown that PKA catalytic subunit translocates as well to the apical membrane upon 5-HT stimulation. To examine which protein phosphatase counteracts PKA, luminal pH-measurements were carried out. Based on the results with protein phosphatase inhibitors and esterified chelating agents of bivalent cations, it may be concluded that a protein phosphatase 2C is involved in the process leading to V-ATPase inactivation. Phosphoprotein stainings revealed that dephosphorylation of subunit C is likewise catalysed by a protein phosphatase 2C. Therefore the dephosphorylation of subunit C seems to promote dissociation of VO- and V1-complexes. Finally, luminal pH-measurements and supplemental biochemical experiments revealed a Ca2+/calcineurin-mediated modulation of the cAMP/PKA signalling cascade and an influence of intracellular calcium on the V-ATPase activity.

Nuolat kintanti aplinka yra pagrindinis veiksnys, kontroliuojantis mikroorganizmų augimą ir vystymąsi. Evoliucijos eigoje organizmuose išsivystė signalinės sistemos, gebančios sujungti aplinkos signalus su ląstelės transkripcijos, transliacijos ir kt. procesais. Viena iš tokių universalių signalinių sistemų yra Ras/PKA signalinis kelias. Ši sistema leidžia mielių ląstelėms reaguoti į aplinkoje esančius maisto medžiagų šaltinius ir įvairius stresinius veiksnius. Vienas iš pagrindinių aplinkos signalų, įtakojančių ląstelių fiziologiją, yra aplinkos pH. Mielių ląstelėse į aplinkos pH reaguoja Rim101 signalinė sistema, kuri kartu su Ras/PKA reguliuoja tokius procesus kaip sporuliacija ir pseudohifinis augimas. Ekstraląstelinės terpės rūgštėjimas lemia viduląstelinį rūgštėjimą, o tai sukelia mielių ląstelėms stresą ir taip aktyvina Ras/PKA signalinį kelią. Streso poveikio pasekmė yra ląstelių augimo sulėtėjimas, gyvenimo trukmės trumpėjimas, augimo sustabdymas ar netgi žūtis. Pastaraisiais metais parodyta, kad būtent Ras/PKA signalinis kelias reguliuoja ląstelės senėjimo procesą kaip atsaką į aplinkos pH. Taigi mielių, kaip modelinio organizmo, panaudojimas žmogaus genų, reguliuojančių ląstelės senėjimą ir žūtį, tyrimuose vis plečiasi. Disertacinio darbo metu buvo įvertintas izogeninių mielių kamienų turinčių Ras/PKA signalinio kelio komponentų mutacijas augimas bei mitybinės terpės pH kitimas. Nustatyta, kad mutacijos Ras/PKA signalinio kelio komponentuose lemia skirtingą... [toliau žr. visą tekstą] / Constantly changing environment is the major factor controlling the growth and development of the microorganisms. For quick generation of the cell response, information about changes in the cell environment is rapidly transmitted to the inner molecules of the cell. During the course of evolution the cells have developed signaling systems that are able to combine extracellular signals with the inner processes, as transcription, translation etc. One of the universal signaling systems is Ras/PKA signal transduction pathway. This system helps cells recognize the nutrient sources present in the growth medium.Environmental pH is one of the main factors influencing the growth, physiology and differentiation of yeast. In Saccharomyces cerevisiae, response to pH is determined by the RIM101 pathway. Recently, it was shown, that Ras/PKA signal transduction pathway regulates cell aging as response to environmental pH. Many aging and apoptosis features are conserved between yeast and multicellular microorganisms, and this makes them perfect model organisms. Yeasts are also suitable also for acidosis related disease studies. Evaluation of the cell growth and medium acidification of isogenic strains containing mutation in the members of Ras/PKA signal transduction pathway was performed. Mutations in these genes cause changes in metabolic activity of the cell.Members of the Ras/PKA signal transduction pathway participate in regulation of cell viability and lifespan during the natural... [to full text]

Darbo tikslas: dviem metodais gauti azodažiklius iš aromatinių aminų diazonio druskų, nustatyti jų pKa reikšmes, įvertinti spektrines savybes bei pritaikyti kaip indikatorius. Darbo uždaviniai: Gauti azodažiklius iš metilanilinų ir 4-nitroanilino vykdant jų diazonio druskų hidrolizę arba jungiant su 1-naftoliu bei 2-naftoliu, apskaičiuoti gautų junginių išeigas, nustatyti jų pKa reikšmes, spektrines savybes, galimą kaip indikatorių pritaikymą. Metodai: Tyrimui atlikti naudojami du azodažiklių sintezės metodai. Apskaičiuotos abiem metodais gautų azodažiklių procentinės išeigos. ESC įvertintas junginių grynumas. Gautų azodažiklių pKa reikšmės bei pH ribos, kuriose keičiasi jų spalva, vertinamos atliekant potenciometrinį titravimą. Vandeniniai azodažiklių tirpalai vertinti spektrofotometriškai, matuojant tirpalų absorbcijos maksimumus ir optinius tankius. Rezultatai: Azodažikliai, atliekant aromatinių aminų diazonio druskų hidrolizę (hidrolizatas sudarytas iš 44 ml vandens ir 20 ml koncentruotos H2SO4), gaunami veikiant aminus koncentruota HCl, kai hidrolizato temperatūra pakeliama iki 104-110 oC. Atliekant tradicinę azodažiklių sintezę išeigos yra geros-labai geros (vidutinė išeiga yra 76,95 proc.), o vykdant diazonio druskų hidrolizę – vidutinės (junginiai gaunami su vidutine 41,44 proc. išeiga). Tradiciniu metodu gautų naftolinių azodažiklių pKa reikšmės yra 8,8-11,0 ribose, o fenolinių, gautų atliekant eksperimentinę sintezę, - 7,5 8,0. Spektrofotometrinis tyrimas parodė... [toliau žr. visą tekstą] / Aim of the work: synthesis of azo dyes from aromatic amines diazonium salts by two methods, determination of their pKa values, spectral features and applicability as indicators of produced azo dyes. Main objectives: to obtain azo dyes from methylanilines and 4-nitroaniline by hydrolysis of their diazonium salts or by binding with 1-naphtol and 2-naphtol, to calculate yields of obtained compounds, to determine their pKa values, spectral features and their possible application as indicators. Methods: Two methods of azo dyes synthesis are used to perform the assay. Percentage yields of synthesized compounds, obtained by both methods, are calculated. Purity of compounds is evaluated by HPLC. pKa values and range of pH where the colour changes of received compounds are evaluated by potentiometric titration. Aqueous solutions of azo dyes are assessed by measuring absorption peaks and optical densities with a spectrophotometer. Results: it is necessary to synthesise aromatic amines diazonium salts with conc. HCl and to perform hydrolysis in 104-110 oC temperature when hydrolysate consist of 44 ml water and 20 ml conc. H2SO4 in order to obtain azo dyes by experimental method. Yields of traditional method are good-very good (average yield is 76,95 proc.), yields of experimental method are moderate (average yield is 41,44 proc.). pKa values of naphtolic azo dyes which are obtained by traditional method are 8,8-11,0 and phenolic azo dyes – 7,5-8,0. Spectrophotometric studies showed that... [to full text]

Ocotea puberula (Lauraceae) is a brazilian native tree, known in the South regions as canela guaicá or canela amarela. Its phytochemical composition includes several alkaloids of aporphinic type, some of them with biological activities already reported. Fruits collected in Curitiba, state of Paraná, were submitted to extraction and fractioning processes, allowing the isolation of an alkaloid identified as dicentrine, present as the majoritarian substance. In the present work we investigated the antinociceptive potential of the organic fractions obtained from O. puberula fruits extract and dicentrine (DCTN) isolated from the chloroform fraction (CF). Both CF and DCTN, given by oral route, were able to reduce the nociception induced by formalin and acetic acid with similar ID50, suggesting that the CF effect is due to the presence of DCTN. Hexanic (HF) or ethyl acetate (EAF) fractions had no antinociceptive effect. DCTN also presented antinociceptive effects in chronic models such as inflammatory (induced by intraplantar injection of Freund s complete adjuvant) and neuropathic (induced by partial sciatic nerve ligation), being able to reduce mechanical and cold hypersensitivity,with no changes in the response to heat. When evaluated against the thermo-receptors activation, DCTN administered either systemically (by oral route) or locally (by intraplantar route) reduced the nociception induced by cinnamaldehyde, a TRPA1 activator, but did not alter the response induced by capsaicin, a TRPV1 activator. The antinociceptive effect of DCTN was not affected by the pretreatment with antagonists of opioid, adrenergic or cannabinoid receptors, and neither by the pretreatment with an inhibitor of serotonin synthesis, suggesting that these descending mechanisms of pain control are not involved in the DCTN mechanism of action. On the other hand, DCTN was able to prevent the hypersensitivity induced by cAMP/PKA pathway activators, forskolin and PGE2, and it also reduced PKA activation, demonstrated by western blotting analysis, suggesting that DCTN may act by interaction with this signaling pathway. On the other hand, DCTN presented few or none action on the hypersensitivity induced by bradikinin or PMA, respectively, suggesting that the PLC/PKC pathway is not involved in DCTN antinociceptive action. Additionally, DCTN did not cause any sedative effect, neither alterations on motor activity or body temperature, and although daily treatment caused a slight increase in liver relative weight, alterations on AST, ALT or γ-GT levels were not observed. Together, these results demonstrate that DCTN has an important antinociceptive effect in acute and chronic models of pain, mainly of inflammatory origin, and its mechanism of action seems to involve an interaction with TRPA1 channels and the cAMP/PKA signaling pathway. In this way, DCTN may be considered a potential candidate to further pre-clinical and even clinical investigations for development of analgesic drugs. / A Ocotea puberula (Lauraceae) é uma árvore nativa brasileira, conhecida nos estados da região sul por canela guaicá ou canela amarela, cuja composição fitoquímica inclui diversos alcaloides do tipo aporfínicos, alguns dos quais com atividades biológicas já demonstradas. Frutos coletados na região de Curitiba, Paraná, foram submetidos a processos de extração e fracionamento, permitindo isolar um alcaloide identificado como dicentrina, presente como componente majoritário. Neste trabalho, foi investigado o potencial antinociceptivo das frações orgânicas obtidas a partir do extrato dos frutos de O. puberula e da dicentrina (DCTN) isolada da fração clorofórmica (FC). Tanto a FC quanto a DCTN, administradas por via oral, foram capazes de reduzir a nocicepção induzida pela formalina e pelo ácido acético, com DI50 semelhantes, sugerindo que o efeito de FC seja devido à presença da DCTN. As frações hexânica (FH) e acetato de etila (FAE), por sua vez, não apresentaram efeito antinociceptivo. A DCTN também apresentou efeito antinociceptivo em modelos crônicos, tanto inflamatório (injeção intraplantar de Adjuvante Completo de Freund) quanto neuropático (ligadura parcial do nervo ciático), sendo capaz de reduzir a hipersensibilidade mecânica e ao frio, porém sem alterar a resposta ao calor. Quando avaliada frente à ativação de termo-receptores, a DCTN administrada tanto por via sistêmica (via oral) quanto local (via intraplantar) reduziu a nocicepção induzida pelo cinamaldeído, um ativador de canais TRPA1, mas não alterou a resposta induzida pela capsaicina, um ativador de canais TRPV1. O efeito antinociceptivo da DCTN não foi revertido pelo pré-tratamento com antagonistas de receptores opióides, adrenérgicos ou canabinóides e nem pelo prétratamento com um inibidor da síntese de serotonina, sugerindo que estes sistemas descendentes de controle da dor não estão envolvidos no mecanismo de ação da DCTN. Por outro lado, a DCTN foi capaz de prevenir a hipersensibilidade induzida pelos ativadores da via AMPc/PKA, forscolina e PGE2, e também foi capaz de reduzir a ativação da PKA, demonstrada por análise de western blotting, sugerindo que a DCTN possa agir por interação com essa via de sinalização. Por outro lado, a DCTN apresentou pouca ou nenhuma ação frente à hipersensibilidade mecânica induzida pela bradicinina ou pelo PMA, respectivamente, o que sugere que a via PLC/PKC não está envolvida no seu efeito antinociceptivo. A DCTN não causou efeitos sedativos, alteração motora ou alteração na temperatura corporal dos animais e, embora o tratamento diário durante 14 dias tenha aumentado o peso relativo do fígado, não foram observadas alterações nos níveis sanguíneos de AST, ALT ou γ-GT. Coletivamente, os dados deste trabalho demonstram que a DCTN apresenta um importante efeito antinociceptivo em modelos de dor agudos e crônicos, principalmente de cunho inflamatório, e seu mecanismo de ação parece envolver interação com canais TRPA1 e com a via de sinalização AMPc/PKA. Desta forma, a DCTN constitui-se como uma potencial candidata para a continuidade de estudos pré-clínicos aprofundados, e futuramente clínicos, visando o desenvolvimento de fármacos analgésicos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The medicinal plant Condalia buxifolia has been traditionally used to treat inflammatory processes. The present study aimed to evaluate the phytochemical profile and the analgesic effect of the methanolic extract from the root bark of Condalia buxifolia (MECb) in models of acute pain as well as to investigate the possible adjacente mechanism of its effect in mice. The phytochemical analysis of the extract showed the presence of large amounts of alkaloids. The MECb administered intragastrically 1 hour before the experiments (100 and 300 mg/kg, ig), mimicking the route used in humans (oral), promoted reduction of the nociceptive inflammatory pain (2nd phase) and paw oedema induced by intraplantar (i.pl.) injection of formalin, and its antiedematogenic and analgesic effect lasted until 4 and 6 h after MECb pretreatment. MECb (10-300) also prevented nociception induced by intraperitoneal injection of acetic acid. Furthermore, MECb (100 mg/kg, i.g.) prevented paw oedema and nociception induced by i.pl. injection of capsaicin, acidified saline, and nociception glutamate-induced by i.pl. injection. Centrally, MECb increased the latency to nociceptive stimulation in the hot plate model. Moreover, the opioid system is involved in the mechanism of action of MECb, since its effect (at the dose of 300 mg/kg, i.g.) was reversed by naloxone (an antagonist of opioid receptor). Acute (prolonged) treatment with MECb (100 mg/kg, i.g.) reduced the mechanical and thermal (heat) hyperalgesia caused by plantar incision (PI), a model of postoperative pain; and prevented the increase of the concentration of inflammatory cytokines [interleukin-1 β (IL-1 β), tumor necrosis factor-α (TNF-α)] and neurotrophin [nerve growth factor (NGF)]. Additionally, MECb was able to prevent nociception induced by PLC/PKC and cAMP/PKA pathway activators, PMA, forskolin and PGE2. Furthermore, MECb was also able to reduce PKA activation, demonstrated by western blotting analysis, suggesting that the MECb can act by interaction with this signaling pathway. Intrathecal treatment with capsaicin (for desensitization of C fibers) does not alter the mechanical hyperalgesia induced by IP and ablation did not influence the analgesic effect of MECb. Finally, MECb caused no sedative effects or alterations on motor activity of the animals in the open field test and the prolonged treatment did not promote macroscopic changes in important organs (liver, heart, spleen, kidneys and lungs), or changes in hematological and biochemical parameters (glucose, urea, creatinine, uric acid, AST, ALT, GGT, total cholesterol, HDL and LDL), and did not change the profile of food and water intake. Taken together, the results show that the antinociceptive effect of MECb is due, in part, by activation of the opioid system and inhibition of glutamatergic system, pro-inflammatory cytokines and TRPV1 and ASICs channels, as well as the PLC/PKC and/or cAMP/PKA signaling-dependent inhibition. Thus, the results of this study support that Condalia buxifolia has potential effect on pain control, and the continuity of extensive preclinical and clinical studies for the development of an analgesic phytoterapic. / A planta medicinal Condalia buxifolia tem sido tradicionalmente utilizada para o tratamento de processos inflamatórios. O presente estudo tem como objetivo avaliar o perfil fitoquímico e o efeito antinociceptivo do extrato metanólico da casca da raiz de Condalia buxifolia (EBMCb) em modelos de dor aguda, bem como investigar o possível mecanismo adjacente a este efeito em camundongos. A análise fitoquímica do extrato evidenciou a presença de grande quantidade de alcaloides. O EBMCb administrado 1 hora antes dos experimentos por via intragástrica (100 e 300 mg/kg, i.g.), reproduzindo a via utilizada em seres humanos (oral), promoveu redução da nocicepção de origem inflamatória (2ª fase) induzida pela injeção intraplantar (i.pl.) de formalina e do edema de pata, sendo que seu efeito antiedematogênico e antinociceptivo perdurou por até 4 e 6 h após a sua administração. O EBMCb (10-300) também preveniu a nocicepção induzida pela injeção intraperitoneal de ácido acético. Ademais, o EBMCb (100 mg/kg, i.g.) preveniu a nocicepção e edema de pata induzido pela injeção i.pl. de capsaicina, salina ácida e a nocicepção induzida pela injeção i.pl. de glutamato. Centralmente, o EBMCb atuou aumentando a latência ao estimulo nociceptivo no modelo de placa quente. Aliado a este resultado foi demonstrado que o sistema opióide está envolvido no mecanismo de ação do EBMCb, uma vez que seu efeito (na dose de 300 mg/kg, i.g.) foi revertido pela naloxona (antagonista de receptores opióides). O tratamento agudo (prolongado) com o EBMCb (100 mg/kg, i.g.) preveniu a hiperalgesia mecânica e térmica (calor) causada pela incisão plantar (IP), um modelo de dor pós-operatória; bem como preveniu o aumento das concentrações de citocinas inflamatórias [interleucina 1-β (IL-1 β), fator de necrose tumoral-α (TNF- α)] e do fator de crescimento do nervo (NGF). Adicionalmente, o EBMCb foi capaz de prevenir a nocicepção induzida pelos ativadores da via PLC/PKC e AMPc/PKA, como o PMA e a forscolina e PGE2. Além disso, o EBMCb também foi capaz de reduzir a ativação da PKA, demonstrada por análise do conteúdo da proteína fosforilada, sugerindo que o EBMCb possa agir por interação com essa via de sinalização. Também foi demonstrado que o tratamento intratecal com capsaicina (para dessensibilização das fibras C), não alterou a hiperalgesia mecânica induzida pela IP e que a ablação não influenciou no efeito antinociceptivo do EBMCb. Finalmente, o EBMCb não causou efeitos sedativos ou alteração na atividade locomotora dos animais no teste do campo aberto e o tratamento prolongado não promoveu alterações macroscópicas nos órgãos vitais (fígado, coração, rins e pulmões), nem alterações no hemograma e de parâmetros de bioquímica sanguínea (glicemia, ureia, creatinina, ácido úrico, AST, ALT, GGT, colesterol total, HDL e LDL), bem como não mudou o perfil de ingesta de água e ração. Tomados em conjunto, os resultados demonstram que o efeito antinociceptivo do EBMCb se deve, em parte, pela ativação do sistema opióide e inibição do sistema glutamatérgico, de citocinas pró-inflamatórias e de canais TRPV1 e ASICs, bem como da inibição da sinalização dependente da PLC/PKC e/ou AMPc/PKA. Assim, os resultados do presente estudo evidenciam o potencial efeito de Condalia buxifolia no controle da dor, bem como a continuidade de estudos pré-clínicos aprofundados, e futuramente clínicos, visando o desenvolvimento de um fitoterápico analgésico.

Dans le cœur, l’activation aiguë de la voie AMPc/PKA via la stimulation des récepteurs β-adrénergiques (β-ARs) permet de réguler la contraction cardiaque alors que l’activation chronique de cette voie est délétère, car elle est source de survenue d’arythmies cardiaques et de remodelage hypertrophique du cœur. Au niveau des cardiomyocytes, Il existe principalement deux sous-types de récepteurs β-ARs ; β1- et β2-ARs, qui exercent des effets différents sur la fonction cardiaque.Dans une première partie de ma thèse, je me suis intéressé à l’étude du rôle des récepteurs β1- et β2-ARs dans la régulation différentielle de l’activité PKA cytoplasmique et nucléaire. J’ai ainsi pu montrer que contrairement aux récepteurs β1-ARs qui ont la capacité d’activer la PKA au niveau du cytoplasme et aux noyaux, les récepteurs β2-ARs activent la PKA uniquement au niveau du cytoplasme, et ce indépendamment de la capacité des récepteurs β2-ARs à induire une augmentation des niveaux d’AMPc dans les noyaux. En accord avec ces résultats, les récepteurs β1- mais pas β2-ARs activent le facteur pro-apoptotique régulé par la PKA, ICER.Dans une seconde partie de ma thèse, je me suis intéressé aux différents mécanismes responsables de l’incapacité des récepteurs β2-ARs à activer la PKA au niveau des noyaux. Mes résultats soulignent le rôle de la localisation des récepteurs β2-ARs au niveau des cavéoles, leurs couplage aux protéines Gi, leurs désensibilisation par la GRK2 ainsi que la dégradation de l’AMPc généré par ces récepteurs par la PDE3 et 4 dans la régulation de la signalisation PKA cytoplasmique et pointent vers la PDE4 comme un régulateur central permettant de limiter l’activation de la PKA holoenzyme responsable des réponses PKA nucléaires. Mes résultats montrent également que la mAKAP est un élément clé dans la transduction de la signalisation PKA nucléaire induite par les récepteurs β2-ARs et à un moindre degré, les récepteurs β1-ARs. Dans la dernière partie de ma thèse, j’ai étudié le remodelage de la signalisation PKA nucléaire induite par les récepteurs β1- et β2-ARs au cours de l’insuffisance cardiaque. J’ai ainsi pu montrer qu’en plus de la diminution de la signalisation PKA nucléaire induite par les récepteurs β1-ARs, il existe une signalisation PKA nucléaire de novo induite par les récepteurs β2-ARs dans les cardiomyocytes de rat adulte insuffisants.En conclusion, ce travail a mis à jour une nouvelle différence entre les récepteurs β1- et β2-ARs dans la signalisation PKA au niveau des noyaux des cardiomyocytes de rat adultes, et souligne le rôle important de la PDE4 et de la mAKAP dans la régulation de la signalisation PKA nucléaire induite par les récepteurs β2-ARs. / In the heart, acute activation of the cAMP/PKA pathway upon stimulation of β-adrenoceptors (β-ARs), plays a fundamental role in the regulation of cardiac function, whereas chronic activation of this pathway is deleterious, as it is responsible for cardiac arrhythmias and hypertrophic remodeling of the heart. In cardiac myocytes, there are mainly two subtypes of β-ARs: β1- and β2-ARs, which exert different effects on cardiac function.In the first part of my thesis, my work was focused on understanding the role of β1- and β2-ARs in the differential regulation of cytoplasmic and nuclear PKA activity. Hence, I have showed that unlike β1-ARs which have the capacity to induce the activation of PKA in the cytoplasm and the nucleus, β2-ARs induce the activation of PKA only in the cytoplasmic compartment, regardless of their ability to induce an increase in cAMP in the nuclei. Consistently, β1- but not β2-ARs were able to induce the activation of the pro-apoptotic factor regulated by PKA, ICER.The second aim of my thesis was to decipher the different mechanisms involved in the inability of β2-ARs to activate PKA in the nucleus. I concentrated my efforts on investigating the role of the localization of β2-ARs in caveolae, their coupling to Gi proteins, their desensitization by GRK2 as well as the hydrolysis of cAMP by PDE3 and 4 in the regulation of β2-AR-induced cytoplasmic PKA activity. My results point to PDE4 as a central regulator which limits the activation of the PKA holoenzyme pool involved in the nuclear PKA responses. My results also show that mAKAP is a key component of nuclear PKA signaling induced by β2-ARs and to a lesser extent by β1-ARs. In the last part of my thesis, I have studied the remodeling of nuclear PKA signaling induced by β1- and β2-ARs that occurs during heart failure. I showed that, besides a decrease in β1-AR-induced nuclear PKA signaling, there is a de novo β2-AR-induced nuclear PKA signaling in cardiomyocytes from rat with heart failure.In conclusion, this work uncovers a new difference in PKA signaling between β1- and β2-ARs at the nuclear compartment of adult rat cardiomyocytes and underlines the importance of PDE4 and mAKAP in the regulation of β2-AR-induced nuclear PKA signaling.