Intra- und extrazelluläre ß-Amyloid-Ablagerungen (Abeta) sind ein neuropathologisches Hauptmerkmal der Alzheimerschen Demenz (AD). Aktuelle Studien belegen, dass nicht Abeta-Plaques, sondern Abeta-Oligomere die Schädigung von Synapsen und Nervenzellen verursachen und dass ihre Konzentration gut mit der Schwere der kognitiven Dysfunktion korreliert. Allerdings sind Abeta-Peptide eine heterogene Gruppe schwer wasserlöslicher Peptide mit zahlreichen C- und N-terminalen Modifikationen. Dabei hängt die Tendenz von Abeta-Peptiden Oligomeren zu bilden, ihre proteolytische Resistenz und ihr neurotoxisches Potential maßgeblich von ihrer N-terminalen Struktur ab. Abeta-Peptide, die N-terminal einen Pyroglutamyl-Laktamring (pE-Abeta) aufweisen, machen einen Hauptbestandteil der Abeta-Last in den frühen Stadien der AD aus. Diese modifizierten Abeta-Peptide aggregieren schneller als unmodifiziertes Abeta, sind gegen Proteolyse geschützt und wirken als Aggregationskeim für andere Abeta-Spezies. Das Enzym Glutaminylzyklase (QC) katalysiert die n-terminale pE-Modifikation von Abeta in vitro und in vivo und wird in Neuronenpopulationen gefunden, für die ein starker Verlust von Synapsen und Neuronen im Zusammenhang mit der AD beschrieben wurde. Diese Arbeit stellt die schichtspezifische Verteilung von QC im temporalen Kortex und der hippocampalen Formation von Alzheimerpatienten und Kontrollen vergleichend dar und zeigt einen direkten Zusammenhang zwischen der Überexpression von QC und der Vulnerabilität betreffender Neuronenpopulationen auf. Darüber hinaus bestätigen die vorgestellten Ergebnisse die These, wonach QC und pE-Abeta das Potential haben, nach axonalem Transport eine Kaskade in efferenten Hirnregionen zu initiieren, an deren Ende der Verlust von Nervenzellen steht. Diese Erkenntnisse unterstützen das Interesse an QC als Gegenstand zukünftiger Grundlagenforschung und Wirkstoffentwicklungen für die Therapie der AD.:1. Einleitung 1 1.1. Fallbeispiel 1 1.2. Epidemiologie der Alzheimerschen Erkrankung 1-2 1.3. Derzeitige Pharmakotherapie 2 1.4. Neuropathologie der Alzheimerschen Demenz 3 1.5. Amyloidprozessierung 3-5 1.6. Das Enzym Glutaminylzyklase 7-8 1.7. Fragestellung 8-9 2. Material und Methoden 10 2.1. Humanes Hirngewebe von Alzheimer- und Kontrollfällen 10-11 2.2. Anfertigung von Gefrierschnitten 11 2.3. Kresylviolett-Färbung nach Nissl 12 2.4. Immunhistochemie 12-16 2.5. Vergleich von vier Anti-QC-Antikörpern 17-18 2.6. Zählmethodik 19-22 2.7. Verwendete Hard- und Software 20 3. Ergebnisse 23 3.1. Neuronendichten der untersuchten Hirnregionen in 23-24 Alzheimer- und Kontrollgehirnen 3.2. QC-Immunreaktivitat in Alzheimer- und Kontrollgehirnen 25 3.2.1. QC-Immunreaktivität im temporalen Kortex 25-26 3.2.2. QC-Immunreaktivität im entorhinalen Kortex 27-28 3.2.3. QC-Immunreaktivität im Subikulum und Ammonshorn 29-31 3.3. Stärke der QC-Immunreaktivität in Alzheimer- und Kontrollgehirnen 32 3.3.1. Stärke der QC-Immunreaktivität im temporalen Kortex 32-33 3.3.2. Stärke der QC-Immunreaktivität im entorhinalen Kortex 34-35 3.4. Schichtspezifische Verteilung der QC-Immunreaktivität im temporalen und entorhinalen Kortex in Alzheimer- und Kontrollgehirnen 36-38 3.5. QC-Immunreaktivität der Ammonshornregionen CA1 – CA4 39-40 4. Diskussion 41 4.1. Abeta-Spezies und QC in der AD 41-42 4.2. QC im temporalen Kortex 42-44 4.3. QC im entorhinalen Kortex 44-47 4.4. QC im Hippocampus 47-49 4.5. Regionale, schichtspezifische und neuronale Verteilung von QC 49-51 4.6. Ausblick 51-53 5. Zusammenfassung (Deutsch und Englisch) 54-55 6. Literaturverzeichnis 56-65 7. Anhang 66 7.1. Färbeprotokoll für Immunhistochemie 66 7.2. Erklärung über die eigenständige Abfassung der Arbeit 67 7.3. Lebenslauf 68 7.4. Danksagung 69 / Intra- and extracellular s-amyloid (Abeta) deposits are a major neuropathological hallmark of Alzheimer\''s disease (AD). Recent studies demonstrate that Abeta oligomers rather than Abeta plaques cause severe damage of synapses and nerve cells and in addition the concentration of Abeta oligomers correlates well with the severity of cognitive dysfunction. However, Abeta peptides are a heterogeneous group of poorly water-soluble peptides with various C- and N-terminal modifications. Biophysical properties of these peptides such as their propensity to form oligomers, their proteolytic resistance and their neurotoxic potential particularly depends on their N-terminal structure. Abeta-peptides that contain a pyroglutamyl-a-lactam ring at their N-Terminus (pE-Abeta) constitute a major component of the Abeta load in the early stages of AD. These modified Abeta-peptides aggregate faster than unmodified Abeta, are protected against proteolysis and act as aggregation seed for other Abeta-species. The enzyme glutaminyl cyclase (QC)catalyzes the cyclization of Abeta to pE-Abeta in vitro and in vivo and is found in neuronal populations for which a strong loss of synapses and neurons in the context of AD is described. This thesis presents the layer-specific distribution of QC in the temporal cortex and the hippocampal formation of Alzheimer\''s patients and controls, showing a direct correlation between the overexpression of QC and the vulnerability of respective neuronal populations. Moreover, the presented results confirm the hypothesis that QC and pE-Abeta have the potential to initiate a cascade leading to the loss of nerve cells due to axonal transport and release in efferent brain regions. These findings support the interest in QC as a subject of fundamental research and future drug developments for the treatment of AD.:1. Einleitung 1 1.1. Fallbeispiel 1 1.2. Epidemiologie der Alzheimerschen Erkrankung 1-2 1.3. Derzeitige Pharmakotherapie 2 1.4. Neuropathologie der Alzheimerschen Demenz 3 1.5. Amyloidprozessierung 3-5 1.6. Das Enzym Glutaminylzyklase 7-8 1.7. Fragestellung 8-9 2. Material und Methoden 10 2.1. Humanes Hirngewebe von Alzheimer- und Kontrollfällen 10-11 2.2. Anfertigung von Gefrierschnitten 11 2.3. Kresylviolett-Färbung nach Nissl 12 2.4. Immunhistochemie 12-16 2.5. Vergleich von vier Anti-QC-Antikörpern 17-18 2.6. Zählmethodik 19-22 2.7. Verwendete Hard- und Software 20 3. Ergebnisse 23 3.1. Neuronendichten der untersuchten Hirnregionen in 23-24 Alzheimer- und Kontrollgehirnen 3.2. QC-Immunreaktivitat in Alzheimer- und Kontrollgehirnen 25 3.2.1. QC-Immunreaktivität im temporalen Kortex 25-26 3.2.2. QC-Immunreaktivität im entorhinalen Kortex 27-28 3.2.3. QC-Immunreaktivität im Subikulum und Ammonshorn 29-31 3.3. Stärke der QC-Immunreaktivität in Alzheimer- und Kontrollgehirnen 32 3.3.1. Stärke der QC-Immunreaktivität im temporalen Kortex 32-33 3.3.2. Stärke der QC-Immunreaktivität im entorhinalen Kortex 34-35 3.4. Schichtspezifische Verteilung der QC-Immunreaktivität im temporalen und entorhinalen Kortex in Alzheimer- und Kontrollgehirnen 36-38 3.5. QC-Immunreaktivität der Ammonshornregionen CA1 – CA4 39-40 4. Diskussion 41 4.1. Abeta-Spezies und QC in der AD 41-42 4.2. QC im temporalen Kortex 42-44 4.3. QC im entorhinalen Kortex 44-47 4.4. QC im Hippocampus 47-49 4.5. Regionale, schichtspezifische und neuronale Verteilung von QC 49-51 4.6. Ausblick 51-53 5. Zusammenfassung (Deutsch und Englisch) 54-55 6. Literaturverzeichnis 56-65 7. Anhang 66 7.1. Färbeprotokoll für Immunhistochemie 66 7.2. Erklärung über die eigenständige Abfassung der Arbeit 67 7.3. Lebenslauf 68 7.4. Danksagung 69

The Gram-negative aerobic coccobacillus Bordetella pertussis is one of the few exclusively human pathogens and the main causative agent of the respiratory infectious disease called pertussis, or whooping cough. Despite global vaccination programs, pertussis remains an important public-health burden and still accounts for over 100,000 infant deaths and over a dozen of millions of whooping cough cases every year. Substantial effort is devoted to studies on the mechanisms of action of virulence factors of B. pertussis, but the biology of interactions of B. pertussis with its human host remains largely underexplored. Evolution, genetics and adaptation of B. pertussis to the complex environment of human nasopharynx and the mechanisms enabling B. pertussis to overcome host innate and adaptive mucosal immune defenses, remain poorly understood. In such situations, unbiased exploratory omics approaches represent valuable tools for uncovering of unknown aspects of host-pathogen interactions and open the path to detailed analysis of virulence-underlying processes by mechanistic studies. In this thesis, I am presenting the results of three omics projects on B. pertussis biology that involved high-throughput proteomics. In the inital phosphoprotemics project, we analyzed the kinase signaling pathways hijacked...

Dopamine (DA) receptor supersensitivity refers to the phenomenon of an enhanced physiological, behavioral or biochemical response to a DA agonist. Literature related to ontogenetic aspects of this process was reviewed. Neonatal 6-hydroxydopamine (6-OHDA) destruction of rat brain DA neurons produces overt sensitization to D1 agonist-induced oral activity, overt sensitization of some D2 agonist-induced stereotyped behaviors and latent sensitization of D1 agonist-induced locomotor and some stereotyped behaviors. This last process is unmasked by repeated treatments with D1 (homologous "priming") or D2 (heterologous "priming") agonists. A serotonin (5-HT) neurotoxin (5,7-dihydroxytryptamine) and 5-HT2C receptor antagonist (mianserin) attenuate some enhanced behavioral effects of D1 agonists, indicating that 5-HT neurochemical systems influence D1 receptor sensitization. Unlike the relative absence of change in brain D1 receptor number, DA D2 receptor proliferation accompanies D2 sensitization in neonatal 6-OHDA-lesioned rats. Robust D2 receptor supersensitization can also be induced in intact rats by repeated treatments in ontogeny with the D2 agonist quinpirole. In these rats quinpirole treatments produce vertical jumping at 3-5 wk after birth and subsequent enhanced quinpirole-induced antinociception and yawning. The latter is thought to represent D3 receptor sensitization. Except for enhanced D1 agonist-induced expression of c-fos, there are no changes in the receptor or receptor-mediated processes which account for receptor sensitization. Adaptive mechanisms by multiple "in series" neurons with different neurotransmitters may account for the phenomenon known as receptor supersensitivity.

Rhodopsin-Guanylyl-Zyklasen (RGCs) gehören zur Familie der Enzymrhodopsine, welche sich durch eine Lichtregulation ihrer Enzymaktivität durch ein Rhodopsin (Rh) auszeichnen. Das membranständige Rh ist hierbei mit einer Guanylylzyklase (GC) verbunden, welches nach Lichtaktivierung des Rh GTP zu zyklisiertem GMP (cGMP) umsetzt. Der sekundäre Botenstoff cGMP sowie das verwandte cAMP spielen eine wichtige Rolle in einer Vielzahl von biologischen Prozessen. Die lichtgesteuerte Kontrolle dieser Botenstoffe bietet der Optogenetik somit eine Möglichkeit zur Erforschung ihrer Signalwege und könnte so den Weg zu medizinisch nutzbaren Erkenntnissen weisen. Im Rahmen dieser Arbeit wurde eine RGC, gefunden im Genom des Pilzes Catenaria anguillulae aus der Abteilung der Blastocladiomycota, spektroskopisch untersucht. Zu diesem Zweck wurde ein FTIR- und UV-Vis-spektroskopischer Messaufbau entwickelt, der eine parallele Aufzeichnung von UV-Vis- und FTIR-Spektren an derselben Proteinprobe erlaubt. Neben konventionellen Belichtungsmethoden, wurde ein durchstimmbarer Hochleistungspulslaser integriert, welcher den praktisch simultanen Umsatz der Proteinprobe erlaubt. Um auch früheste Prozesse spektroskopisch zu erfassen, wurde zusätzlich ein Hochdruck-Heliumkryostat integriert, der Messungen bis unterhalb des Siedepunkts von Helium ermöglicht (bis ~3 K). Nach der UV-Vis- und FTIR-spektroskopischen Charakterisierung der Photointermediate konnte ein Modell des Photozyklus abgeleitet werden, während Messungen an trunkierten Varianten eine aktive Rolle des N-Terminus in der Enzymregulation aufzeigten. Unter Verwendung eines photolabilen und nichtumsetzbaren GTP-Substrats konnte die Aktivität von RGC und freiem GC in Echtzeit spektroskopisch untersucht werden. Neben der Identifizierung des aktiven Zustands wurde entgegen bisheriger Annahmen gezeigt, dass GTP schon vor Lichtaktivierung an RGC bindet. Die Lichtregulation erfolgt demnach direkt über Modifikationen in der Bindetasche und nicht deren Zugänglichkeit. Ein Aktivierungsmechanismus wurde skizziert, der sowohl die hier vorgelegten Ergebnisse, als auch Ergebnisse vorhergehender Untersuchungen kombiniert. / Rhodopsin guanylyl cyclases (RGCs) belong to the family of enzymerhodopsins, which are characterized by light regulation of their enzyme activity by a rhodopsin (Rh). The embrane-bound Rh is linked to a type III guanylyl cyclase (GC). Upon light activation of Rh, inhibition of GC is abolished and conversion of GTP to cyclic GMP (cGMP) is initiated. The secondary messengers cGMP and the closely related cAMP play important roles in a variety of biological processes. Hence, light-controlled regulation of these messengers provides an opportunity to investigate their signaling pathways using optogenetics and could pave the way for medically useful findings. In this work, a RGC found in the genome of the fungus Catenaria anguillulae from the division of Blastocladiomycota was spectroscopically investigated. For this purpose, an FTIR and UV-Vis spectroscopic measurement setup was developed which supports parallel recording of UV-Vis and FTIR spectra of the same protein sample. In addition to conventional illumination methods, a tunable high-power pulse laser was integrated which allows virtually simultaneous turnover of the protein sample due to its pulse duration in the nanosecond and power in the megawatt range. To investigate the earliest molecular processes a high-pressure helium cryostat was integrated which allows measurements down to below the boiling point of helium (~3 K). Based upon the UV-Vis and FTIR spectroscopic characterization of the photointermediates, a model of the photocycle was derived, while experiments on truncated variants revealed an active role of the N-terminus in enzyme regulation. Using a photolabile and non-convertible GTP substrate, the activity of RGC and free GC could be investigated spectroscopically in real time. In addition to identifying the active state, it was shown, contrary to previous assumptions, that GTP binds to RGC even before light activation. Thus, light regulation occurs directly via modifications in the binding pocket rather than its accessibility. An activation mechanism was outlined that combines both the results presented here and results of previous studies.

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.

The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....

EN Heme containing gas sensor proteins play important role in bacterial physiology in regulating many processes such as cell differentiation, virulence, biofilm formation or intercellular communication. For their structure, typical modular architecture is characteristic where various sensor domains (usually at the N-terminus) regulate the activity of the catalytic or functional domains (usually at the C-terminus). In this dissertation thesis, we focused on three representatives from the group of oxygen sensing proteins, namely histidine kinase AfGcHK, diguanylate cyclase YddV, phosphodiesterase EcDOS and also on protein RR, which is the interaction partner of AfGcHK. The main aim of the thesis was to study intra-protein/inter-domain signal transduction in two representatives of heme sensor proteins with a globin fold of the sensor domain (AfGcHK, YddV) and in one representative with PAS fold of the sensor domain (EcDOS). Another objective was to describe inter-protein signal transduction in the two component signaling system AfGcHK-RR and structurally characterize these two interacting partners. Emphasis was also placed on the study of the interaction between model sensor domains and different signaling molecules and also on function of individual amino acids involved in the binding of these...

Die lichtaktivierte Kinase Phototropin aus Chlamydomonas reinhardtii, die photoaktivierte Adenylatcyclase (PAC) aus Euglena gracilis und das BLUF-Protein Slr1694 aus Synechocystis sp. PCC 6803 wurden in Hinblick auf die molekularen Details der primären photochemischen Prozesse sowie der Signalweiterleitung untersucht. Phototropin wurde mit Hilfe von Arginin aus Escherichia coli in Milligramm Mengen isoliert. Ohne Arginin wurde E. coli cAMP Rezeptorprotein assoziiert aufgefunden, welches eine hohe Homologie zu einer cAMP aktivierten Kinase aus C. reinhardtii besitzt. Volllängen Phototropin bildet wie einzelne LOV-Domänenkonstrukte ohne Kinasedomäne den Flavin-Triplettzustand und das kovalente Cysteinyl-Addukt. Der Zerfall des Signalzustandes ist in Anwesenheit von ATP beschleunigt und deutet auf Photorezeptor-Kinase Interaktion hin. Strukturelle Änderungen in der Kinasedomäne wurden durch FTIR-Differenzspektroskopie gezeigt. Über ELDOR-Spektroskopie wurde der Abstand der Photorezeptordomänen auf etwa 25 Angstrom bestimmt. Mutationen in Slr1694 an S28, N31 und W91 zeigten keine konservierten Einfluss auf die Dynamik des Signalzustands. Die Entfernung der Seitenkette von S28 führte zu einer 15 nm Rotverschiebung des Absorptionsspektrums aufgrund veränderter Wasserstoffbrückenkoordination des Kofaktors. Die Einführung von positiv geladenen Seitenketten an Stelle von N31 erhöhte die Kofaktorbindung von phosphorylierten Flavinen. Künstliche Kofaktoren wie Roseoflavin konnten in Slr1694 durch Koexpression eines prokaryotischen Flavintransporters erreicht werden. Die Rolle von M152 in PAC für die Signalweiterleitung wurde anhand der lichtaktivierten cAMP Synthese-Aktivität gezeigt. Durch ultraschnelle IR-Spektroskopie wurde die Beteiligung der Seitenketten von Y8 sowie Q50 bestätigt und eine genauere Beschreibung der Wasserstoffbrücken im langlebigen Signalzustand ermöglicht. / The light activated kinase Phototropin from Chlamydomonas reinhardtii, the photoactivated adenylylcyclase (PAC) from Euglena gracilis and the BLUF protein Slr1694 from Synechocystis sp. PCC 6803 were investigated concerning the molecular details of the primary photochemistry as well as signal transduction. Phototropin was isolated from Escherichia coli in mg amounts after solubilization with arginine. Without arginine E. coli cAMP receptor protein, which shows high homology to a cAMP activated kinase from C. reinhardtii, was copurified. Full length Phototropin shows similar photochemistry to LOV-domain containing proteins without the kinase including triplet and covalent cysteinyl adduct formation. Signaling state decay is accelerated in the presence of ATP and suggests photoreceptor-kinase interaction. FTIR spectroscopy showed light induced structural changes in the kinase domain. The distance of the photoreceptor domains of 25 Angstrom was determined by ELDOR spectroscopy. Mutation of the side chains of S28, N31 and W91 in Slr1694 showed no conserved influence on the dynamic of the signaling state. Removal of the hydroxyl group of S28 lead to a 15 nm red shift of the absorption spectrum as a result of altered hydrogen bond coordination of the cofactor. Introduction of positively charged side chains at the position of N31 strengthened the binding of phosphorylated flavins. An artificial flavin like roseoflavin was introduced in Slr1694 by coexpression of a bacterial flavin transporter. The essential role of M152 in PAC for signal transduction was shown by determination of light activated cAMP synthesis activity. Ultrafast IR spectroscopy confirmed the contribution of Y8 and Q50 in the photocycle and gave a more detailed description of the hydrogen bonding situation in the signaling state.

In this work, the influence of the second messenger cAMP on the functional plasticity of M-DC8+ dendritic cells (DC) was examined. The marker M-DC8 defines a population of native DC first described in blood. After their isolation, M-DC8+ DC acquire a mature CD83+ phenotype during a short culture ex vivo. After a challenge with LPS and IFN-g, M-DC8+ DC secrete large amounts of the proinflammatory cytokines IL-12(p70) and TNF-a surpassing by far other DC populations and monocytes. Due to their preferential induction of TH1-dominated T cell responses, M-DC8+ DC might play a role in the pathogenesis of inflammatory diseases. Different cAMP-elevating agents suppressed the proinflammatory cytokine production and enhanced the secretion of anti-inflammatory IL-10. Activity of phosphodiesterase (PDE) 4, the most important cAMP-hydrolysing enzyme in immune cells, was detected and RT-PCR revealed the expression of PDE4 subtypes 4A, 4B and 4D in M-DC8+ DC, whereas 4C was not detectable. The PDE4-specific inhibitors AWD12-281 and Roflumilast were then used to elevate cAMP concentrations. These substances have been proven to be efficient in anti-inflammatory therapies. In the presence of PDE4 inhibitors, the LPS/IFN-g-induced production of IL-12 and TNF-a was decreased by 90 % and 60 %, respectively, whereas the IL-10-release was doubled. These effects were only observed, if the PDE4 inhibitors where present from the beginning of the culture. The inhibition of the IL-12 secretion was reverted using an a-IL-10-receptor antibody. PDE4 inhibitor-treated M-DC8+ DC showed a reduced capacity to polarize TH1-cells, which was demonstrated analysing culture supernatants by ELISA and by single-cell analysis detecting intracellular IFN-g und IL-4. These results suggest that PDE4 inhibitors may not only be useful in the therapy of TH2-mediated diseases but also in TH1-dominated indications such as multiple sclerosis and Crohn´s disease. Despite the shift of the cytokine profile, the in vitro maturation of M-DC8+ DC was not affected by PDE4 inhibitors. The expression of CD83, CD80, CD86, MHC-molecules as well as CD54 and CD58, was assessed by FACS analysis. Correspondingly, in the presence of AWD12-281, M-DC8+ DC efficiently stimulated the proliferation of allogeneic CD4+CD45RA+ T-cells. In the second part of this study, the effects of an inhibition of cAMP-synthesis in M-DC8+ DC were analyzed. Two adenylyl cyclase (AC) inhibitors, 2,5-Dideoxyadenosine and SQ22536, clearly hampered the in vitro maturation of M-DC8+ DC. The expression of the DC maturation marker CD83 could be reconstituted using the stable cAMP-analogon 8-Br-cAMP. Measuring the intracellular cAMP concentration in M-DC8+ DC, initially low cAMP-levels were observed, but within 30 min the concentration raised and returned to original levels within 2 hrs. Blocking the cAMP synthesis by AC inhibitors, the LPS/IFN-g-induced production of IL-12, TNF-a and IL-10 was strongly reduced. Furthermore, it was demonstrated that M-DC8+ DC can only release IL-12 after a transient elevation of cAMP, i.e. they acquire a "license". Such a regulation of the IL-12 production has not been described before. Protein kinase A is an important effector molecule of cAMP. Inhibiting its activity resulted in a reduced expression of the DC maturation marker CD83 and a lower cytokine production underlining the importance of cAMP-signalling for the activation of M-DC8+ DC. In conclusion, this study provides evidence for a new concept of the immune-regulatory function of cAMP. Here, cAMP is essentially involved in the initial activation and maturation of DC and enables them to secrete large amounts of IL-12 and TNF-a upon stimulation with a TLR ligand. Conversely, a long-term elevation of cAMP-concentrations inhibits the proinflammatory effector functions of M-DC8+ DC and can induce anti-inflammatory responses by enhancing the secretion of IL-10. / In dieser Arbeit wurde der Einfluss des second messengers cAMP auf die funktionelle Plastizität von M-DC8+ dendritischen Zellen (DC) untersucht. Der Oberflächenmarker M-DC8 definiert eine zunächst im Blut beschriebene Population nativer DC. Nach ihrer Isolation erlangen M-DC8+ DC während einer kurzen Kultur einen maturen CD83+ Phänotyp. Nach Stimulation mit LPS und IFN-g produzieren native M-DC8+ DC deutlich höhere Mengen der proinflammatorischen Zytokine IL-12(p70) und TNF-a als andere DC-Populationen oder Monozyten. Dies resultiert in einer Programmierung TH1-dominierter T-Zellantworten. M-DC8+ DC könnten daher an der Pathogenese entzündlicher Krankheiten beteiligt sein. Unterschiedliche cAMP-erhöhende Substanzen supprimierten die proinflammatorische Zytokinproduktion und verstärkten gleichzeitig die Sekretion des anti-inflammatorischen IL-10. In M-DC8+ DC konnte die Aktivität von Phosphodiesterase (PDE) 4, dem wichtigsten cAMP-hydrolysierenden Enzym in Immunzellen, nachgewiesen werden. Durch RT-PCR wurde die Expression der PDE4-Subtypen 4A, 4B und 4D gezeigt, nicht aber 4C. Zur Erhöhung der cAMP-Konzentration wurden dann die PDE4-spezifischen Inhibitoren AWD12-281 und Roflumilast eingesetzt, deren klinische Effizienz bei anti-inflammatorischen Therapien belegt ist. Auch diese Substanzen verringerten die LPS/IFN-g-induzierte Produktion von IL-12 und TNF-a durch M-DC8+ DC um 90 % bzw. 60 %, während die IL-10-Freisetzung etwa verdoppelt wurde. Diese starken Effekte konnten nur erzielt werden, wenn die PDE4-Inhibitoren von Beginn der Kultur an eingesetzt wurden. Die Hemmung der IL-12-Sekretion wurde in Gegenwart eines a-IL-10-Rezeptor-Antikörpers aufgehoben. Unter dem Einfluss von PDE4-Inhibitoren war die TH1-Programmierung durch M-DC8+ DC deutlich reduziert, was sowohl durch die Analyse der Zellüberstände mittels ELISA als auch auf Einzelzell-Ebene durch intrazelluläre Detektion von IFN-g und IL-4 nachgewiesen wurde. Diese Ergebnisse legen nahe, dass PDE4-Inhibitoren nicht nur für TH2-vermittelte Erkrankungen sondern auch für TH1-dominierte Indikationen wie Multiple Sklerose oder Morbus Crohn von Nutzen sein könnten. Trotz der starken Modulation des Zytokinprofils blieb die in vitro-Ausreifung M-DC8+ DC unbeeinflusst von PDE4-Inhibitoren. Untersucht wurde die Expression von CD83, CD80, CD86, MHC-Molekülen, CD54 und CD58 mittels FACS-Analyse. Entsprechend induzierten M-DC8+ DC auch in Anwesenheit von AWD12-281 die Proliferation allogener CD4+CD45RA+ T-Zellen. Im zweiten Teil der Arbeit wurde untersucht, wie sich die Blockade der cAMP-Synthese auf M-DC8+ DC auswirkt. Zwei Adenylatcyclase-Inhibitoren, 2,5-Dideoxyadenosine und SQ22536, hemmten die in vitro-Maturation von M-DC8+ DC deutlich. Die CD83-Expression wurde mit 8-Br-cAMP rekonstituiert. Messungen der intrazellulären cAMP-Konzentration in unbehandelten M-DC8+ DC zeigten initial niedrige cAMP-Spiegel, die innerhalb von 30 min anstiegen und nach 2 h wieder auf das Ausgangsniveau abfielen. Die LPS/IFN-g-induzierte Produktion von IL-12, TNF-a und IL-10 wurde durch AC-Inhibitoren deutlich vermindert. M-DC8+ DC erhalten nur nach einer transienten cAMP-Erhöhung die "Lizenz" IL-12 freizusetzen. Eine derartige Regulation der IL-12-Sekretion ist bisher nicht beschrieben. Eine Hemmung des cAMP-Effektormoleküls Proteinkinase A resultierte in der reduzierten Expression des DC-Maturationsmarkers CD83 und einer verringerten Zytokinproduktion. Dies unterstreicht die Bedeutung von cAMP für die Aktivierung M-DC8+ DC. Zusammenfassend gibt diese Arbeit am Beispiel nativer humaner DC Anhalt für ein neues Konzept der immunregulatorischen Funktion von cAMP. Hierbei ist cAMP wesentlich an der Ausreifung von M-DC8+ DC beteiligt, woraufhin diese große Mengen IL-12 und TNF-a sekretieren können. Dagegen wirkt eine langfristige cAMP-Erhöhung durch die Induktion von IL-10 anti-inflammatorisch.

Evaluating aerodynamic noise from aircraft engines is a design stage process, so that it conform to regulations at airports. Aerodynamic noise is also a principal source of structural vibration and internal noise in short/vertical take off and landing and rocket launches. Acoustic loads may be critical for the proper functioning of electronic and mechanical components. It is imperative to have tools with capability to predict noise generation from turbulent flows. Understanding the mechanism of noise generation is essential in identifying methods for noise reduction. Lighthill (1952) and Lighthill (1954) provided the first explanation for the mechanism of aerodynamic noise generation and a procedure to estimate the radiated sound field. Many such procedures, known as acoustic analogies are used for estimating the radiated sound field in terms of the turbulent fluid flow properties. In these methods, the governing equations of the fluid flow are rearranged into two parts, the acoustic sources and the propagation terms. The noise source terms and propagation terms are different in different approaches. A good description of the turbulent flow field and the noise sources is required to understand the mechanism of noise generation. Computational aeroacoustics (CAA) tools are used to calculate the radiated far field noise. The inputs to the CAA tools are results from CFD simulations which provide details of the turbulent flow field and noise sources. Reynolds-Averaged Navier Stokes (RANS) solutions can be used as inputs to CAA tools which require only time-averaged mean quantities. The output of such tools will also be mean quantities. While complete unsteady turbulent flow details can be obtained from Direct Numerical Simulation (DNS), the computation is limited to low or moderate Reynolds number flows. Large eddy simulations (LES) provide accurate description for the dynamics of a range of large scales. Most of the kinetic energy in a turbulent flow is accounted by the large-scale structures. It is also the large-scale structures which accounts for the maximum contribution towards the radiated sound field. The results from LES can be used as an input to a suitable CAA tool to calculate the sound field. Numerical prediction of turbulent flow field, the acoustic sources and the radiated sound field is at the focus of this study. LES based on explicit filtering method is used for the simulations. The method uses a low-pass compact filter to account for the sub-grid scale effects. A one-parameter fourth-order compact filter scheme from Lele (1992) is used for this purpose. LES has been carried out for four different flow situations: (i) round jet (ii) plane jet (iii) impinging round jet and (iv) impinging plane jet. LES has been used to calculate the unsteady flow evolution of these cases and the Lighthill’s acoustic sources. A compact difference scheme proposed by Hixon & Turkel (1998) which involves only bi-diagonal matrices are used for evaluating spatial derivatives. The scheme provides similar spectral resolution as standard tridiagonal compact schemes for the first spatial derivatives. The scheme is computationally less intensive as it involves only bi-diagonal matrices. Also, the scheme employs only a two-point stencil. To calculate the radiated sound field, the Helmholtz equation is solved using the Green’s function approach, in the form of the Kirchhoff-Helmholtz integral. The integral is performed over a surface which is present entirely in the linear region and covers the volume where acoustic sources are present. The time series data of pressure and the normal component of the pressure gradient on the surface are obtained from the CFD results. The Fourier transforms of the time series of pressure and pressure gradient are then calculated and are used as input for the Kirchhoff-Helmholtz integral. The flow evolution for free jets is characterised by the growth of the instability waves in the shear layer which then rolls up into large vortices. These large vortical structures then break down into smaller ones in a cascade which are convected downstream with the flow. The rms values of the Lighthill’s acoustic sources showed that the sources are located mainly at regions immediately downstream of jet break down. This corresponds to the large scale structures at break down. The radiated sound field from free jets contains two components of noise from the large scales and from the small scales. The large structures are the dominant source for the radiated sound field. The contribution from the large structures is directional, mainly at small angles to the downstream direction. To account for the difference in jet core length, the far field SPL are calculated at points suitably shifted based on the jet core length. The peak value for the radiated sound field occurs between 30°and 35°as reported in literature. Convection of acoustic sources causes the radiated sound field to be altered due to Doppler effect. Lighthills sources along the shear layer were examined in the form of (x, t) plots and phase velocity pattern in (ω, k) plots to analyse for their convective speeds. These revealed that there is no unique convective speeds for the acoustic sources. The median convective velocity Uc of the acoustic sources in the shear layer is proportional to the jet velocity Uj at the center of the nozzle as Uc ≈ 0.6Uj. Simulations of the round jet at Mach number 0.9 were used for validating the LES approach. Five different cases of the round jet were used to understand the effect of Reynolds number and inflow perturbation on the flow, acoustic sources and the radiated sound field. Simulations were carried out for an Euler and LES at Reynolds number 3600 and 88000 at two different inflow perturbations. The LES results for the mean flow field, turbulence profiles and SPL directivity were compared with DNS of Freund (2001) and experimental data available in literature. The LES results showed that an increase in inflow forcing and higher Reynolds number caused the jet core length to reduce. The turbulent energy spectra showed that the energy content in smaller scale is higher for higher Reynolds number. LES of plane jets were carried out for two different cases, one with a co-flow and one without co-flow. LES of plane jets were carried out to understand the effect of co-flow on the sound field. The plane jets were of Mach number 0.5 and Reynolds number of 3000 based on center-line velocity excess at the nozzle. This is similar to the DNS by Stanley et al. (2002). It was identified that the co-flow leads to a reduction in turbulence levels. This was also corroborated by the turbulent energy spectrum plots. The far field radiation for the case without co-flow is higher over all angles. The contribution from the low frequencies is directional, mainly towards the downstream direction. The range of dominant convective velocities of the acoustic sources were different along shear layers and center-line. The plane jet results were also used to bring out a qualitative comparison of flow and the radiation characteristics with round jets. For the round jet, the center-line velocity decays linearly with the stream-wise distance. In the plane jet case, it is the square of the center-line velocity excess which decays linearly with the stream-wise distance. The turbulence levels at any section scales with the center-line stream-wise velocity. The decay of turbulence level is slower for the plane jet and hence the acoustic sources are present for longer distance along the downstream direction. Subsonic impinging jets are composed of four regions, the jet core, the fully developed jet, the impingement zone and the wall jet. The presence of the second region (fully developed free jet) depends on the distance of the wall from the nozzle and the length of the jet core. In impinging jets, reflection from the wall and the wall jet are additional sources of noise compared to the free jets. The results are analysed for the contribution of the different regions of the flow towards the radiated sound field. LES simulations of impinging round jets and impinging plane jet were carried out for this purpose. In addition, the results have been compared with equivalent free jets. The directivity plots showed that the SPL levels are significantly higher for the impinging jets at all angles. For free jets, a typical time scale for the acoustic sources is the ratio of the nozzle size to the jet velocity. This is ro/Uj for round jets and h/Uj for plane jets. For impinging jets, the non-dimensionlised rms of Lighthill’s source indicates that the time scale for acoustic sources is the ratio of the height of the nozzle from the wall to the jet velocity be L/Uj. LES of impinging round jets was carried out for two cases with different inflow perturbations. The jets were at Reynolds number of 88000 and Mach number of 0.9, same as the free jet cases. The impingement wall was at a distance L = 24ro from the nozzle exit. For impinging round jets, the SPL levels are found to be higher than the equivalent free jets. From the SPL levels and radiated noise spectra it was shown that the contribution from the large scale structures and its reflection from the wall is directional and at small angles to the wall normal. The difference in the range of angles where the radiation from the large scale structures were observed shows the significance of refraction of sound waves inside the flow. The rms values of the Lighthill’s sources indicate two dominant regions for the sources, just downstream of jet breakdown and in the impingement zone. The LES of impinging plane jet was done for a jet of Mach number 0.5 and Reynolds number of 6000. The impingement wall was at a distance L = 10h from the nozzle exit. The radiated sound field appears to emanate from this impingement zone. The directivity and the spectrum plots of the far field SPL indicate that there is no preferred direction of radiation from the impingement zone. The Lighthill’s sources are concentrated mainly in the impingement zone. The rms values of the sources indicate that the peak values occur in the impingement zone. The results from the different flow situations demonstrates the capability of LES with explicit filtering method in predicting the turbulent flow and radiated noise field. The method is robust and has been successfully used for moderate Reynolds number and an Euler simulation. An important feature is that LES can be used to identify acoustic sources and its convective speeds. It has been shown that the Lighthill source calculations, the calculated sound field and the observed radiation patterns agree well. An explanation for these based on the different turbulent flow structures has also been provided.