Toxoplasma gondii ist ein obligat intrazellulärer protozoischer Parasit, der Toxoplasmose beim Menschen und bei Warmblütern verursacht. Die Signalübertragung durch zyklische Nukleotide ist entscheidend für das erfolgreiche intrazelluläre Überleben und die Vermehrung der Parasiten. Hier haben wir die physiologische und biochemische Bedeutung der wesentlichen Phosphodiesterasen (PDEs) in Toxoplasma gondii Tachyzoiten untersucht. Durch C-terminale Markierung von 18 PDEs konnten wir die Expression von 11 PDEs nachweisen. Die Immunogold-Färbung zeigte, dass TgPDE1, TgPDE2 und TgPDE9 im gesamten Parasitenkörper verteilt sind, einschließlich des inneren Membrankomplexes, des apikalen Pols, der Plasmamembran, des Zytosols, der dichten Granula und der Rhoptry, was auf eine räumliche Kontrolle der Signalübertragung innerhalb der Tachyzoiten hindeutet. Anschließend stellten wir fest, dass die meisten Enzyme berüchtigte dual-spezifische Phosphodiesterasen sind, wobei TgPDE2 anders als T.gondii cAMP-spezifisch ist, während T.gondii keine cGMP-spezifische Phosphodiesterase besitzt. Unsere enzymkinetischen Daten zeigen, dass TgPDE2 die höchste Affinität zu seinem Substrat aufweist, während die dualen PDEs (TgPDE1, TgPDE7 und TgPDE9) eine höhere Affinität zu cGMP als zu cAMP haben. Ein Screening der Hemmung gängiger PDE-Inhibitoren auf TgPDEs ergab, dass TgPDE1 das Ziel von BIPPO und Zaprinast ist. Darüber hinaus ergab die biologische Bedeutung, dass TgPDE1 und TgPDE2 einzeln für das Wachstum des Parasiten notwendig sind und ihr Verlust zum Tod des Parasiten führt, was auf ihre funktionelle Redundanz hindeutet. Darüber hinaus identifizierten wir Kinasen und Phosphatasen innerhalb der TgPDE1- und TgPDE2-Interaktome, die die enzymatische Aktivität über Protein-Protein-Interaktionen oder posttranslationale Modifikationen steuern könnten. Insgesamt unterstreichen unsere Erkenntnisse über die subzelluläre Lokalisierung, die katalytische Funktion, die medikamentöse Hemmung und die physiologische Bedeutung der wichtigsten Phosphodiesterasen die unvorhersehbare Plastizität und das therapeutische Potenzial der zyklischen Nukleotid-Signalübertragung in T. gondii. Der Datensatz der cAMP-bindenden Interaktoren, den wir in einem anderen Aspekt dieser Studie offengelegt haben, wird wertvolle Einblicke in die allgegenwärtige Natur der cAMP-vermittelten Signalübertragung in T. gondii Tachyzoiten liefern. / Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in human and warm-blood organisms. Cyclic nucleotide signaling is crucial for the successful intracellular survival and replication of the parasites. Here, we dissected the physiological and biochemical importance of the essential phosphodiesterases (PDEs) in Toxoplasma gondii tachyzoite. By C-terminal tagging of 18 PDEs, we detected the expression of 11 PDEs. Immunogold staining revealed that TgPDE1, TgPDE2 and TgPDE9 are distributed throughout the parasite body, including the inner membrane complex, the apical pole, the plasma membrane, the cytosol, dense granules, and rhoptry, suggesting the spatial control of signaling within tachyzoites. Subsequently, we identified that most enzymes are notorious dual-specific phosphodiesterases, and TgPDE2 is cAMP specific differently, whilst T.gondii lacks of cGMP specific phosphodiesterase. Our enzyme kinetic data demonstrated that the highest affinity to its substrate belongs to TgPDE2, while the dual PDEs (TgPDE1, TgPDE7 and TgPDE9) have higher affinity with cGMP than cAMP. Inhibition screening of commonly-used PDE inhibitors on TgPDEs, signifying TgPDE1 as the target of BIPPO and zaprinast. Furthermore, the biological significance revealed TgPDE1 and TgPDE2 are individually necessary for parasite growth, and their loss associatively results in parasite death, implying their functional redundancy. In addition, we identified kinases and phosphatases within the TgPDE1 and TgPDE2 interactomes, which may operate the enzymatic activity via protein-protein interactions or posttranslational modifications. Collectively, our findings on subcellular localization, catalytic function, drug inhibition, and physiological relevance of major phosphodiesterases highlight the unforeseeable plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii. The data set of cAMP-binding interactors, which we disclosed in another aspect of this study, will provide valuable insight into the pervasive nature of cAMP-mediated signaling in T. gondii tachyzoites.

Lebewesen nutzen nukleotid-basierte sekundäre Botenstoffe um extra- und intrazelluläre Signale zur Induktion einer entsprechenden Zellantwort weiterzuleiten. Das zyklische Dinukleotid c-di-AMP steuert verschiedene physiologische Prozesse und ist für viele Bakterien unter bestimmten Bedingungen essentiell. Dieses Signalmolekül muss präzise reguliert werden, da seine Akkumulation oft toxisch ist. Diadenylatzyklasen mit einer DAC-Domäne synthetisieren c-di-AMP, welches von Phosphodiesterasen (PDE) mit DHH/DHHA1- oder HD-Domänen abgebaut wird. Streptomyceten sind im Boden lebende, Gram-positive Actinobakterien mit einem komplexen Lebenszyklus, während welchem sie vielzählige sekundäre Metabolite, inklusive Antibiotika, produzieren. Die Regulierung des zellulären c-di-AMP und seine Bedeutung in der Streptomyceten-Biologie waren zu Beginn dieser Studie weitgehend unbekannt. Zur c-di-AMP-Synthese nutzen Streptomyces die DAC DisA, besitzen aber keine der typischen PDEs sowie die meisten der bekannten c-di-AMP-bindenden Effektoren. Diese Arbeit zeigt, dass DisA die wichtigste c-di-AMP-Synthetase in Streptomyces venezuelae ist. AtaC wurde als eine PDE identifiziert, welche eine neue Klasse von c-di-AMP PDEs begründet. Während eine ataC-Deletion zu Störungen in Differenzierung und Wachstum in S. Venezuelae führt, führt die Inaktivierung von disA zur Sensitivität gegenüber erhöhten Konzentrationen von monovalenten Kationen im Medium. CpeA und CpeD wurden als erste c-di-AMP-bindende Proteine im Streptomyces Signalnetzwerk charakterisiert. Die entsprechenden Gene sind in Operons mit putativen Kation/Proton-Antiportern cpeB bzw. cpeE kodiert und die jeweiligen Genprodukte interagieren c-di-AMP-abhängig in vivo. Obwohl Deletion von cpe und Überexpression von cpeABC in S. venezuelae keine Phänotypen zeigten, verbesserte die CpeABC-Expression in Escherichia coli das Wachstum in Kalium-supplementierten Medien, was auf eine Funktion von cpe in der Regulation von Kalium hindeutet. / Nucleotide second messengers are used by all forms of life to transduce extra and intracellular signals and translate them into a physiological cell response. The cyclic dinucleotide c-di-AMP is a signaling molecule involved in diverse functions in bacterial physiology and is essential for many bacteria under certain growth conditions. However, this second messenger has to be tightly regulated since increased levels of c-di-AMP can be toxic. In many bacteria diadenylate cyclases with a conserved DAC domain synthesize c-di-AMP and phosphodiesterases (PDEs) with a DHH/DHHA1 or HD domain degrade it. Streptomyces spp. are soil-inhabiting gram-positive Actinobacteria characterized by a sophisticated developmental life cycle during which they produce various secondary metabolites, including antibiotics. The regulation and role of c-di-AMP is not well understood in Streptomyces biology. For c-di-AMP synthesis, streptomycetes utilize the DAC DisA but do not encode any canonical PDE and most of the known effector proteins for c-di-AMP signal transduction are absent. In this work, I demonstrated that DisA is the primary c-di-AMP synthetase in Streptomyces venezuelae and characterized AtaC as the founding member of a novel class of c-di-AMP-specific PDEs. In S. venezuelae, deletion of ataC interferes with development and growth, whereas disA inactivation affects bacterial survival under high ion osmotic stress conditions. Further, I identified CpeA and CpeD as the first c-di-AMP-binding proteins in Streptomyces. The respective genes are encoded in operons with the predicted cation/proton antiporters cpeB and cpeE, respectively, and the gene products interact in vivo in a c-di-AMP-dependent manner. Although neither cpe deletion nor overexpression of cpeABC produced a phenotype in S. venezuelae, expression of cpeABC in Escherichia coli improved growth in liquid media supplemented with potassium, suggesting that Cpe transporters are involved in potassium homeostasis.

Hintergrund: Die Herzinsuffizienz ist ein internistisches Krankheitsbild, welches weltweit eines der höchsten Morbiditäten und Mortalitäten aufweist. Trotz etablierter Behandlungsmethoden sterben mehr als die Hälfte der Patienten innerhalb der ersten fünf Jahren nach Diagnosestellung. Zur pharmakologischen Therapie gehören in erster Linie die Betablocker, welche durch kompetitive Hemmung am β- Adrenorezeptor die β- Signalkaskade und somit die sympathische Wirkung am Herzen reduzieren. Gleichzeitig wird durch die Hemmung die Anzahl an β- Adrenorezeptoren erhöht, wodurch die Rezeptorsensitivität insgesamt herabgesetzt wird. Zudem verhindern sie ein Remodeling der Ventrikel, führen zu einer verbesserten myokardialen Energie- und Calciumnutzung, sowie zu einer Reduzierung von kardialen Arrhythmien. Aufgrund ihres Nebenwirkungsspektrums - Hypotonie, Bradykardie und erektile Dysfunktion - tolerieren nur wenige Patienten eine wirksame Dosis des Medikamentes. Fragestellung: Die Phosphodiesterasen stellen einen von mehreren Regulatoren der β-Signalkaskade dar, indem sie deren Second Messenger cAMP hydrolysieren und die Sympathikusaktivierung drosseln. In vorangegangenen Studien im Menschen ist die PDE2 bei einer Herzinsuffizienz hochreguliert. Gleichzeitig bildet die PDE2 eine Verbindung zum NO – sGC – cGMP – Signalweg, da sie als einzige PDE von cGMP allosterisch aktiviert werden kann und infolgedessen vermehrt cAMP hydrolysiert. PDE2 ist ein entscheidendes Enzym im negativen cross talk von der cAMP und cGMP Signalkaskade. Diese Arbeit untersucht die Funktion der PDE2 in der chronischen Herzinsuffizienz. Inwieweit zeigen sich vermehrt Rhythmusstörungen bei einem Mausgenotyp, dessen PDE2 überexprimiert vorliegt, im Gegensatz zum Wildtyp? Wie reagiert die transgene Maus auf adrenergen Stress? Und abschließend, schützt eine überexprimierte PDE2 vor einer Angiotensin II- induzierten kardialen Hypertrophie? Methoden: Nach Genotypisierung der Mäuse mit Hilfe von PCR Testung der Schwanzspitzen konnte mittels transthorakaler Echokardiographie die Morphologie des Herzens (Durchmesser der Ventrikelwand und des Ventrikels selbst) festgestellt und somit die Herzleistung (Ejektionsfraktion, systolisches und diastolisches Volumen) und das Herzgewicht errechnet werden. Zur dauerhaften Ableitung der Herzaktivität wurden den Mäusen (n = 7) pectoral telemetrische Transmitter implantiert, um die Herzfrequenzvariabilität und Arrhythmien (Salven, Extrasystolen, ventrikuläre Tachykardien) abzuleiten. Zur Arrhythmieprovokation wurde den Mäusen (n = 4) Ivabradin und Isoproterenol intraperitoneal verabreicht. Zur Provokation einer Kardiohypertrophie erhielten die Wildtyp (WT, n = 9) und transgenen Mäuse (TG, n = 10) mittels einer im zerviko-thorakalen Rückenbereich implantierten osmotischen Minipumpe für 14 bzw. 28 Tage Angiotensin II. Die Herzparameter zur Messung einer Hypertrophie wurden mittels transthorakaler Echokardiographie alle 7 Tage für maximal 28 Tage erfasst. Zudem wurden die Herzgewichte nach Tötung der Mäuse mittels vorausgehender Isoflurannarkose und anschließendem Genickbruch durch sofortiges Wiegen der Organe erlangt. Verglichen wurden die Ergebnisse der für 14 und der für 28 Tage dem Angiotensin II ausgesetzten Tieren. Post mortem wurden die Herzen der Mäuse nach Trennung der Ventrikel von den Vorhöfen kryokonserviert und das Ventrikelgewebe zur proteinchemischen Analyse mechanisch aufgeschlossen. Der zentrifugierte Überstand wurde für die Proteinbestimmung nach Bradford zur Erlangung der Proteinkonzentration verwendet. Im Anschluss erfolgte zur Größenselektion der Proteine eine Gelelektrophorese mit anschließendem Western Blotting zum Nachweis der Proteine. Zur Auswertung und Vergleichbarkeit der Ergebnisse wurden die Proteinlevel auf das Kardiomyozyten-spezifische Calsequestrin normiert. Ergebnisse: Bei überexprimierter PDE2 bestand eine grundsätzlich niedrigere Herzfrequenz mit erhaltender chronotroper Adaptionsfähigkeit und kompensatorisch erhöhter Kontraktilität. Zudem war bei den TG- Mäuse die Herzfrequenzvariabilität höher als bei den WT. Es zeigte sich kein Anhalt für eine Beeinflussung des HCN-Kanals durch die erhöhte PDE2. Bei vermehrter Stimulation der β-adrenergen Rezeptoren bestand kein signifikanter Unterschied in der Zunahme der Herzfrequenz, jedoch präsentierten sich deutlich weniger ventrikuläre Extrasystolen und Arrhythmien bei den TG- Mäusen als bei den WT- Mäusen. Bei den durch stete Ang. II-Applikation hypertrophierten Herzen stellte sich über die Zeit eine Zunahme der Herzfrequenz bei sowohl den WT- als auch den TG- Mäusen dar, bei gleichzeitig aufrecht erhaltener linksventrikulärer Funktion. Es zeigte sich ebenfalls bei beiden eine noch bestehende β-adrenerge Rezeptorsensibilität, vor allem bezüglich der Ejektionsfraktion. Von PKA- bzw. CaMKII-abhängige Zielproteine wiesen bei beiden Phänotypen keine vermehrte Phosphorylierung auf. Schlussfolgerung: Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass die PDE2 als Cross Link zwischen der cGMP- und der cAMP-Signalkaskade bei Überexpression ähnlich wie ein Betablocker die Herzfrequenz reduziert ohne die Herzleistung zu beinträchtigen. Sie schützte vor Arrhythmien und zeigte bei einer Kardiohypertrophie dennoch eine bestehende β-adrenerge Rezeptorsensibilität. Ein Schutz vor einer kardialen Hypertrophie bei den PDE2- überexprimierten Mäusen konnte in dieser Arbeit nicht nachgewiesen werden. Klinische Daten zeigen eine Hochregulation der PDE2 bei herzinsuffizienten Patienten. Ob eine Überstimulation der PDE2 in vivo und somit eine Zunahme der cAMP- Hydrolyse tatsächlich kardioprotektiv in der terminalen Herzinsuffizienz ist - oder sogar davor - bedarf noch weiterer Forschung.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis V Abkürzungsverzeichnis VI 1 Einleitung 1 1.1 Herzinsuffizienz 1 1.1.1 Epidemiologie 1 1.1.2 Ätiologie 2 1.1.3 Pathophysiologie 3 1.2 Behandlung der Herzinsuffizienz 4 1.2.1 Medikamentöse Behandlung 5 1.2.1.1 ACE-Hemmer und AT1-Antagonisten 5 1.2.1.2 Betablocker 5 1.2.1.3 Ivabradin 6 1.3 Wirkungen des vegetativen Nervensystems am Herzen 7 1.3.1 Physiologie der kardialen β-adrenergen Signalkaskade 7 1.3.2 Kompartimentierung in Kardiomyozyten 8 1.4 Physiologie der Phosphodiesterasen 9 1.4.1 Cyclisches Guanosinmonophosphat 9 1.4.2 Die Subtypen der Phosphodiesterasen 10 1.4.3 Die Interaktion von cGMP und cAMP 14 1.4.4 Die PDE2 in der Herzinsuffizienz 16 1.5 Vordaten 17 1.5.1 Überexpression von PDE2 im Mausmodell 17 1.6 Ziele dieser Arbeit 18 2 Material und Methoden 20 2.1 Herkunft und Gewinnung der Wildtyp- und transgenen Mäuse 20 2.1.1 Tierhaltung und Tötung 20 2.1.2 Genotypisierung der Mäuse 20 2.2 Echokardiographie zur Messung der kardiologischen Parameter 21 2.3 Einbau der telemetrischen Transmitter 24 2.4 Aufzeichnung des EKG 25 2.5 Einbau der osmotischen Minipumpen 25 2.6 Herzentnahme bei Mäusen 26 2.7 Proteinchemische Methode 26 2.7.1 Aufschluss des Herzgewebes zur proteinchemischen Analyse 26 2.7.2 Proteinbestimmung nach Bradford 27 2.7.3 SDS-PAGE 28 2.7.4 Transfer der Proteine auf Membranen (Westernblot) 29 2.7.5 Auftragen der Antikörper auf die Membranen 30 2.7.6 Aufnahmen 32 2.8 Statistische Auswertungen 32 3 Ergebnisse 33 3.1 Charakterisierung der Herzfunktion bei PDE2-Überexpression mittels EKG 33 3.1.1 Zirkadiane Messung der Herzfrequenz und Aktivität 33 3.1.2 Herzfrequenzvariabilität 35 3.1.3 Autonome Herzfunktion unter Ivabradin 36 3.1.4 Arrhythmieprovokation mit Isoproterenol 37 3.2 Die Rolle von PDE2 im Angiotensin II- induzierten kardialen Remodeling 41 3.2.1 Grundcharakterisierung der Herzfunktion bei niedriger PDE2- Überexpression mittels Echokardiographie 41 3.2.2 Auswirkung erhöhter PDE2-Spiegel nach chronischer Angiotensin II Applikation 43 3.3 Auswertung der Western Blots der PDE2A3-4808 Mäuse 49 4 Diskussion 54 4.1 Phosphodiesterase 2 reguliert die Herzfrequenz 54 4.2 Phosphodiesterase 2 schützt vor ventrikulären Arrhythmien 55 4.3 Phosphodiesterase 2 im kardialen Remodeling 56 4.4 Limitation der Überexpression durch zelluläre Kompartimentierung 58 4.5 Diskussion der Methodik und Bewertung der Ergebnisse 58 4.6 Ausblick: PDE2 als Downstream Target für Beta-Adrenorezeptor-Blockade? 59 5 Zusammenfassung/ Summary 61 5.1 Zusammenfassung 61 5.2 Summary 63 6 Anhang 66 6.1 Puffer 70 7 Literaturverzeichnis 72 / Background: Congestive heart failure is a medical condition, which has one of the highest morbidity and mortality rates worldwide. Despite having established treatments, more than half of the patients die within the five years after diagnosis. First-line pharmacological therapy includes beta-blockers. By competitive inhibition at the β-adrenoreceptor, they reduce the β-signal cascade and thus the sympathetic effect on the heart. Simultaneously, this inhibition increases the number of β-adrenoreceptors, which then reduces the sensitivity of the receptors. Additionally, beta-blockers prevent remodeling of the ventricles, lead to improved myocardial energy and calcium utilization, and reduce the risk of cardiac arrhythmias. Due to its side effects – such as hypotension, bradycardia, and erectile dysfunction – only few patients tolerate an effective dose of the drug. Hypothesis: Phosphodiesterases are one of several regulators of the β- signaling cascade. They hydrolyze their second messenger cAMP and reduce sympathetic activation. Previous studies in humans showed an upregulated PDE2 in heart failure. PDE2 is a special PDE, since it forms a connection between the β adrenergic and the NO – sGC – cGMP signaling pathway. It is the only PDE, which is activated by cGMP allosterically. As a result, PDE2 increasingly hydrolyzes cAMP. Therefore, PDE2 is a key enzyme in the negative cross talk of the cAMP and cGMP signaling cascade. This thesis investigates the function of PDE2 in chronic heart failure. Are there increased cardiac arrhythmias in mice, which have an overexpressed PDE2, in comparison to wild type mice? How do these mice react to adrenergic stress? And finally, does an overexpressed PDE2 protect against an Angiotensin II-induced cardiac hypertrophy? Methods: After genotyping the mice’s tail tips with the help of PCR tests, the morphology of the heart (diameter of the ventricular wall and the ventricle itself) could be determined with the help of transthoracic echocardiography. With this data, the cardiac output (ejection fraction, systolic and diastolic volume) and the heart weight were calculated. To record cardiac activity permanently, telemetric transmitters were implanted pectoral in the mice (n = 7), in order to gain heart rate variability and arrhythmia data (salvos, extrasystoles, ventricular tachycardias). To provoke the arrhythmia, mice (n = 4) were intraperitoneally administered ivabradine and isoproterenol. To provoke cardiac hypertrophy, the wild type (WT, n = 9) and transgenic mice (TG, n = 10) received angiotensin II using osmotic minipumps, implanted in the cervicothoracic back area for 14 or 28 days. The cardiac parameters for hypertrophy were measured using transthoracic echocardiography every 7 days, recorded for a maximum of 28 days. In addition, each heart was weighted immediately after killing the mice using isoflurane anesthesia and subsequent neck fracture. The results of the animals exposed to angiotensin II for 14 and 28 days were compared. After separating the ventricles from the atria, the hearts of the mice were cryopreserved, and the ventricular tissue was mechanically crushed for protein-chemical analysis. The centrifuged supernatant was used for Bradford protein determination to identify the protein concentration. Following this, a gel electrophoresis and Western blotting took place to detect and to determine the size of the proteins. To evaluate and to compare the results, protein levels were calibrated to the cardiomyocyte specific Calsequestrin. Results: Overexpression of PDE2 resulted in a fundamentally lower heart rate with preserved chronotropic adaptability and compensatory increased contractility. In addition, the TG mice showed a higher heart rate variability than the WT mice. There were no signs of influence on the HCN channel by the increased PDE2. With intensified stimulation of the β-adrenergic receptors, there was no significant difference in the increase in heart rate, but significantly fewer ventricular extrasystoles and arrhythmias in the TG mice than in the WT mice. Over time, the hypertrophic hearts, induced by Angiotensin II, showed an increased heart rate with preserved left ventricular function within both the WT and the TG mice. Nevertheless, both still showed an unspoiled sensitivity of the β-receptors, especially in regard of the ejection fraction. Both PKA- or CaMKII-dependent target proteins did not show an increased phosphorylation in neither of the phenotypes. Conclusion: As a summary, this thesis demonstrates that an overexpression of PDE2 as a cross link between the cGMP and cAMP signaling cascade reduces the heart rate similar to a beta blocker, without affecting the cardiac output. PDE2 prevented arrhythmias and still showed existing β-adrenergic receptor sensitivity in cardiac hypertrophy. This thesis could not show a protection against cardiac hypertrophy within the transgenic mice. Clinical data show upregulation of PDE2 in heart failure patients. Whether overstimulation of PDE2 in vivo and a consecutive increase in cAMP hydrolysis is actually cardioprotective in end-stage heart failure - or even before - requires further research.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis V Abkürzungsverzeichnis VI 1 Einleitung 1 1.1 Herzinsuffizienz 1 1.1.1 Epidemiologie 1 1.1.2 Ätiologie 2 1.1.3 Pathophysiologie 3 1.2 Behandlung der Herzinsuffizienz 4 1.2.1 Medikamentöse Behandlung 5 1.2.1.1 ACE-Hemmer und AT1-Antagonisten 5 1.2.1.2 Betablocker 5 1.2.1.3 Ivabradin 6 1.3 Wirkungen des vegetativen Nervensystems am Herzen 7 1.3.1 Physiologie der kardialen β-adrenergen Signalkaskade 7 1.3.2 Kompartimentierung in Kardiomyozyten 8 1.4 Physiologie der Phosphodiesterasen 9 1.4.1 Cyclisches Guanosinmonophosphat 9 1.4.2 Die Subtypen der Phosphodiesterasen 10 1.4.3 Die Interaktion von cGMP und cAMP 14 1.4.4 Die PDE2 in der Herzinsuffizienz 16 1.5 Vordaten 17 1.5.1 Überexpression von PDE2 im Mausmodell 17 1.6 Ziele dieser Arbeit 18 2 Material und Methoden 20 2.1 Herkunft und Gewinnung der Wildtyp- und transgenen Mäuse 20 2.1.1 Tierhaltung und Tötung 20 2.1.2 Genotypisierung der Mäuse 20 2.2 Echokardiographie zur Messung der kardiologischen Parameter 21 2.3 Einbau der telemetrischen Transmitter 24 2.4 Aufzeichnung des EKG 25 2.5 Einbau der osmotischen Minipumpen 25 2.6 Herzentnahme bei Mäusen 26 2.7 Proteinchemische Methode 26 2.7.1 Aufschluss des Herzgewebes zur proteinchemischen Analyse 26 2.7.2 Proteinbestimmung nach Bradford 27 2.7.3 SDS-PAGE 28 2.7.4 Transfer der Proteine auf Membranen (Westernblot) 29 2.7.5 Auftragen der Antikörper auf die Membranen 30 2.7.6 Aufnahmen 32 2.8 Statistische Auswertungen 32 3 Ergebnisse 33 3.1 Charakterisierung der Herzfunktion bei PDE2-Überexpression mittels EKG 33 3.1.1 Zirkadiane Messung der Herzfrequenz und Aktivität 33 3.1.2 Herzfrequenzvariabilität 35 3.1.3 Autonome Herzfunktion unter Ivabradin 36 3.1.4 Arrhythmieprovokation mit Isoproterenol 37 3.2 Die Rolle von PDE2 im Angiotensin II- induzierten kardialen Remodeling 41 3.2.1 Grundcharakterisierung der Herzfunktion bei niedriger PDE2- Überexpression mittels Echokardiographie 41 3.2.2 Auswirkung erhöhter PDE2-Spiegel nach chronischer Angiotensin II Applikation 43 3.3 Auswertung der Western Blots der PDE2A3-4808 Mäuse 49 4 Diskussion 54 4.1 Phosphodiesterase 2 reguliert die Herzfrequenz 54 4.2 Phosphodiesterase 2 schützt vor ventrikulären Arrhythmien 55 4.3 Phosphodiesterase 2 im kardialen Remodeling 56 4.4 Limitation der Überexpression durch zelluläre Kompartimentierung 58 4.5 Diskussion der Methodik und Bewertung der Ergebnisse 58 4.6 Ausblick: PDE2 als Downstream Target für Beta-Adrenorezeptor-Blockade? 59 5 Zusammenfassung/ Summary 61 5.1 Zusammenfassung 61 5.2 Summary 63 6 Anhang 66 6.1 Puffer 70 7 Literaturverzeichnis 72

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi, B. garinii, and B. afzelii. These spirochetes experience environmental fluctuations as they are passed between mammalian and Ixodes tick hosts throughout their enzootic cycle. Recent studies have suggested cyclic diguanylate (c-di-GMP), a ubiquitous secondary messenger, is a key modulator of B. burgdorferi adaptive responses and may play a significant role in cycle progression. In this study, we examined the impact of the sole diguanylate cyclase (Rrp1), c-di-GMP binding proteins (PlzA and PlzB), and HD-GYP-containing phosphodiesterase (PdeB) in disease establishment of both murine and Ixodes tick systems. Strains harboring targeted gene deletions or plasmid-based constitutive gene expression constructs were generated. Rrp1 was required for tick colonization, yet overexpression abolished murine disease, thus implicating the requirement of finely regulated c-di-GMP levels for enzootic cycle progression. Deletion of rrp1 disrupted translational motion and swarming patterns by causing extended cell runs, eliminating stops/flexes, and reducing swarming capabilities. This was attributed to a defect in N-acetyl-D-glucosamine (NAG) metabolism and chemotaxis. NAG is a major source of nutrition for B. burgdorferi within the tick environment; therefore this defect would impede spirochete migration towards feeding ticks, as well as pathogen uptake and survival within the Ixodes vector. In contrast, the downstream c-di-GMP effector, PlzA, was critical for murine disease but nonessential for survival within ticks nor functionally complemented by PlzB. Deletion of plzA altered strain motility and swarming similarly to the rrp1 deletion mutant, yet had a distinct phenotype with significantly slower translational motion and no affect on NAG chemotaxis and metabolism. This indicates B. burgdorferi could possess alternate c-di-GMP effectors or Rrp1 could be directly influencing these cellular processes. Uniquely, PdeB did not abolish murine infection via needle inoculation, but wasrequired for natural transmission from ticks. This defect was linked to the decreased tick colonization efficiency upon pdeB deletion. Together, these analyses indicate that c-di-GMP signaling is an important virulence mechanism of Borrelia burgdorferi and demonstrate the complexity of this signaling pathway in an arthropod-borne pathogen. The data presented here additionally provide significant new insight into the gene regulatory mechanisms of the Lyme disease spirochetes.

Cell damage represents a major pathomechanism in many diseases of high clinical interest, such as myocardial infarction (MI), where it plays an important role in ischemia-reperfusion (I/R) injury. Considerable progress has been made towards identifying physiological and pharmacological agents that play a key role in myocardial preconditioning against I/R injury and also elucidating the molecular changes leading to such protection.Second messengers in cellular signaling pathways, such as cGMP have been well implicated as key players in ischemic and pharmacological preconditioning (PC) of the heart. Phosphodiesterase type 5 (PDE-5) is an enzyme that specifically hydrolyzes cGMP thereby decreasing its tissue concentration. Sildenafil is a potent selective inhibitor of PDE-5 and therefore allows the accumulation of cGMP in several tissues shown to express PDE-5, including pulmonary and coronary arteries. We initially hypothesized that vasodilation induced by sildenafil may release several endogenous mediators including adenosine, bradykinin or nitric oxide (NO), that may trigger a signaling cascade leading to protection against I/R injury. Our results show that sildenafil, at a clinically relevant dose, induced powerful acute and delayed cardioprotection against I/R injury in an in vivo rabbit model via opening of mitoKATP channels. The acute cardioprotective effect of sildenafil was dependent on activation of protein kinase C in rabbits. Moreover, we observed that sildenafil induced delayed PC by NO produced through activation of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in the mouse heart. The expression of iNOS/eNOS was regulated by ERK phosphorylation and the delayed protection against I/R was blocked by PD98059, a selective ERK inhibitor. Furthermore, sildenafil-induced delayed protection was abolished in the intact heart as well as adult myocytes derived from adenosine A1 receptor knock-out mice suggesting an essential role of A1 receptor in protection. Taken together, these studies suggest that sildenafil is a powerful tool to reduce I/R injury in the animal models. Future clinical studies with relatively safe and effective PDE-5 inhibitors may have an enourmous impact on the use of these compounds in reducing I/R injury in the heart and other organs.

Recent studies have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor, sildenafil citrate, induces a powerful cardioprotective effect against ischemia-reperfusion (I/R) injury in rabbit and mouse hearts. However, the effect of this drug in inducing long-term protection against I/R injury remains unknown. The goal of this study was to identify the duration of the protective window of sildenafil citrate as well as vardenafil, a more potent PDE-5 inhibitor. Rabbits were treated with sildenafil (0.7 mg/kg, iv), vardenafil (0.143 mg/kg), or an equivalent volume of saline. After 24 hrs, 48 hrs, 96 hrs, or 7 days of sildenafil treatment, the hearts were subjected to I/R. In the vardenafil groups, the hearts were subjected to I/R at 24 hrs and 7 days after administration of the drug. To evaluate the role of nitric oxide (NO) in cardioprotection, a non-selective blocker of nitric oxide synthase, L-NAME (15 mg/kg, iv) was administered 10 minutes prior to I/R. The results show significant reductions in infarct size in hearts treated with sildenafil and vardenafil as compared to the corresponding saline controls at all time points. The protective effects of sildenafil and vardenafil were abrogated in animals treated with L-NAME. L-NAME had no effect on infarct size in saline treated control rabbits. These data suggest that both sildenafil and vardenafil induce a long-term protective effect against myocardial infarction which is mediated via a NO-dependent pathway. These studies are important in exploiting the clinical potential of PDE-5 inhibitors in terms of protection against ischemia/reperfusion injury in patients with coronary artery disease.

4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin

O segundo mensageiro cGMP está envolvido em diversas funções celulares incluindo a visão em mamíferos. Embora trabalhos anteriores mostrassem variações nos níveis de cGMP durante o ciclo de vida de Blastocladiela emersonii e evidências da existência de enzimas específicas envolvidas na sua síntese (guanilato ciclase) e degradação (cGMP fosfodiesterase), nenhum genoma de fungo publicado até o momento mostrou a existência de genes codificando estas enzimas. Este fato é atribuído por evolucionistas à completa perda de motilidade dos fungos em geral, já que cGMP está primordialmente associado a células com cílios. Blastocladiomicetos, como Blastocladiella, apresentam células móveis em pelo menos um estágio do seu ciclo de vida, o que poderia explicar a existência dessa via nesses fungos. Uma investigação no banco de ESTs de B. emersonii revelou a existência de cDNAs codificando parte de prováveis guanilato ciclases (BeGC1, BeGC2 e BeGC3) e uma possível cGMP fosfodiesterase (BePDE). Assim, este trabalho buscou confirmar a existência destas enzimas e caracterizar a sinalização por cGMP em B. emersonii. A proteína recombinante selvagem correspondente ao domínio catalítico de BePDE mostrou atividade de degradação sobre cGMP e a mutação E389A foi capaz de alterar a especificidade por cGMP. Com o sequênciamento do genoma de B. emersonii obteve-se as sequências completas das guanilato ciclases. Em BeGC2 não foi possível identificar o ligante responsável por sua ativação. Em BeGC3, a presença de um domínio Heme-Pas sugeriu sua ativação por óxido nítrico. A presença de um domínio rodopsina em BeGC1 sugeriu sua ativação por luz. Experimentos de microscopia por imunofluorescência localizaram BeGC1 no \"eyespot\", BeGC2 no capacete nuclear e BeGC3 no citoplasma de zoósporos de B. emersonii. Verificamos também que zoósporos realizam fototaxia em direção à luz verde e que a adição de hidroxilamina, inibidor de rodopsina, ou do inibidor de guanilato ciclase LY83583 tem efeito negativo na fototaxia, bem como impede o aumento dos níveis de cGMP observado em zoósporos expostos à luz verde. O bloqueio da síntese de retinal por Norflurazon também inibiu a fototaxia sendo esta restaurada quando adicionamos retinalA1. Estes dados, juntamente com o fato de o domínio rodopsina de BeGC1 ser a única rodopsina presente no genoma, indicam que BeGC1 é responsável pela fototaxia nos zoósporos de B. emersonii. O genoma do fungo apresenta ainda um possível canal de potássio ativado por cGMP (BeCNG1) localizado na membrana plasmática de zoósporos, similar ao canal regulado por cGMP envolvido na visão em humanos. Ensaios de microfluorimetria também evidenciaram a presença de um canal ativado por cGMP relacionado com o influxo de potássio e a motilidade dos zoósporos. Um modelo para a via de sinalização da fototaxia em B.emersonii foi proposto e comparado com a sinalização presente na visão de mamíferos, destacando a existência de cGMP e rodopsina em ambos os processos e sugerindo uma possível origem comum. Portanto, os resultados obtidos suportam a existência da sinalização por cGMP em B. emersonii, além de indicar o papel dessa sinalização na fototaxia dos zoósporos, sendo esta a primeira via de sinalização por cGMP caracterizada em fungos. / The second messenger cyclic GMP is involved in a wide array of cellular processes including vision in mammals. Although previous studies demonstrated changes in cGMP levels during the life cycle of Blastocladiela emersonii and evidences of specific enzymes involved in its synthesis (guanylyl cyclase) and hydrolysis (cGMP-phosphodiesterase), no fungal genome published so far shows the presence of genes encoding these enzymes. Evolutionists attribute the absence of cGMP signaling pathways in higher fungi to the sedentary life style of these organisms, since cGMP is primarily associated with ciliated cells. However, blastocladiomycetes like Blastocladiella, have motile cells in at least one stage of their life cycle, which could explain the existence of this pathway in these primitive fungi. Inspection of B. emersonii EST data bank, revealed cDNAs encoding part of three putative guanylyl cyclases (BeGC1, BeGC2 e BeGC3) and one possible cGMP phosphodiesterase (BePDE). Thus, the purpose of this study was to confirm the existence of these enzymes and characterize the cGMP signaling pathway in this model. The recombinant protein containing the wild type catalytic domain of BePDE presented activity towards hydrolysis of cGMP and the E389A mutation of this domain changed the cGMP specificity of this enzyme. The complete nucleotide sequence of the guanylyl cyclases were obtained by sequencing of B. emersonii genome. In BeGC2 we were unable identify the ligand responsible for its activation, but in BeGC3, the presence of a Heme-Pas domain suggested its activation by nitric oxide. The presence of a rhodopsin domain in BeGC1 suggested its activation by light. Immunofluorescence microscopy localized BeGC1 in the \"eyespot\" structure, BeGC2 in the nuclear cap and BeGC3 in the cytoplasm of zoospores of B. emersonii. We found that Blastocladiella zoospores performed phototaxis toward green light and photobleaching of rhodopsin function using hydroxylamine prevented both phototaxis and the increased cGMP levels observed when zoospores were exposed to green light. The same effect was observed using the guanylyl cyclase inhibitor LY83583. Inhibition of retinal synthesis using Norflurazon prevented the phototaxis response, which could be restored by zoospore complementation with retinalA1. The BeGC1 gene is the only rhodopsin found in the draft assembly of B. emersonii genome, which indicates that BeGC1 is responsible for phototaxis observed in zoospores. We also found in the genome a possible cGMP-activated potassium channel (BeCNG1), localized in the plasma membrane of the zoospores, which is similar to the cGMP-activated channel involved in human vision. In addition, microfluorimetry assays revealed the presence of a cGMP-activated potassium channel involved in potassium influx and zoospore motility. The signaling model of B. emersonii phototaxis was proposed and compared with the mammalian vision system, with cGMP and rhodopsin acting in both signaling pathways, suggesting a common origin. Altogether our data indicate that Blastocladiella emersonii has a cGMP signaling system involved in phototaxis, being the first cGMP signaling pathway characterized in fungi.

Thesis advisor: Charles S. Hoffman / In this thesis, I describe the development of a screening platform for detecting PDE8A inhibitors using the cAMP-dependent glucose sensing pathway of the fission yeast Schizosaccharomyces pombe, which led us to discover several PDE8A selective inhibitors. In this system, the only PDE of the fission yeast is replaced with mammalian PDE8A1 in strains that have been engineered such that PDE inhibition is required to allow cell growth. Using this system, I screened 56 compounds obtained from PDE4 and PDE7 high throughput screens (HTSs) and identified a PDE4-PDE8 dual specificity inhibitor. Using this as a positive control, I developed a robust high-throughput screen (HTS) for PDE8A inhibitors and screened 240,267 compounds at the Harvard Medical School ICCB Screening Facility. Approximately 0.2 % of the screened compounds were potential PDE8A inhibitors with 0.03% displaying significant potency. Secondary assays of 367 of the most effective compounds against strains expressing PDE8A (both full length and catalytic domain), PDE4A and PDE7A or PDE7B led to the selection of structurally diverse compounds for further testing. To profile the selectivity of twenty-eight of these compounds, dose response assays were conducted using 16 yeast strains that express different PDE isoforms (representing all PDE families with the exception of the PDE6 family). These assays identified compounds with different patterns of inhibition, including structurally-distinct PDE8A-specific inhibitors. By evaluating the effects of these compounds for steroid production in mouse Leydig cells, biologically active compounds that can elevate steroid production were identified. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

In order to evaluate the suitability of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) as a potential drug target for an anti-parasite therapy, we are studying its role in the bloodstream form of Trypanosoma brucei brucei, the eukaryotic parasite that causes African Sleeping Sickness. Eukaryotic TDP1 removes covalently trapped topoisomerase IB and other adducts from the 3’ end of the DNA at DNA strand breaks. Covalent topoisomerase IB stalling is caused by endogenous DNA damage and by anti-cancer drugs such as camptothecin (CPT). A potential approach could be to use TDP1 inhibitors synergistically with CPT in a combined anti-parasite therapy. T. brucei TDP1 knock out cells are hypersensitive to CPT and accumulate in the late S phase of the cell cycle upon treatment with the drug. The CPT hypersensitivity of the TDP1-/- cells can be fully rescued through ectopic expression of wild type TDP1. The catalytic activity of TDP1 is required for complementation of the CPT sensitivity since overexpression of a catalytically inactive mutant form of TDP1 further sensitises TDP1-/- cells to CPT. In this context, expression of the mutant H358N, which shows reduced activity, also increases sensitivity of TDP1-/- cells to the drug. Surprisingly, expressing TDP1 carrying an analogous mutation to the one that causes SCAN1, a human neurodegenerative disease, does not sensitise TDP1-/- cells further. With this unique set of mutant TDP1 proteins in a TDP1-/- background we hope to answer questions concerning TDP1 function that have so far been elusive.