TY - JOUR A1 - Meral, Derya A1 - Provasi, Davide A1 - Prada-Gracia, Diego A1 - Möller, Jan A1 - Marino, Kristen A1 - Lohse, Martin J. A1 - Filizola, Marta T1 - Molecular details of dimerization kinetics reveal negligible populations of transient µ-opioid receptor homodimers at physiological concentrations JF - Scientific Reports N2 - Various experimental and computational techniques have been employed over the past decade to provide structural and thermodynamic insights into G Protein-Coupled Receptor (GPCR) dimerization. Here, we use multiple microsecond-long, coarse-grained, biased and unbiased molecular dynamics simulations (a total of ~4 milliseconds) combined with multi-ensemble Markov state models to elucidate the kinetics of homodimerization of a prototypic GPCR, the µ-opioid receptor (MOR), embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol lipid bilayer. Analysis of these computations identifies kinetically distinct macrostates comprising several different short-lived dimeric configurations of either inactive or activated MOR. Calculated kinetic rates and fractions of dimers at different MOR concentrations suggest a negligible population of MOR homodimers at physiological concentrations, which is supported by acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments. This study provides a rigorous, quantitative explanation for some conflicting experimental data on GPCR oligomerization. KW - computational biophysics KW - fluorescence resonance energy transfer Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223995 VL - 8 ER - TY - JOUR A1 - Joos, J. P. A1 - Saadatmand, A. R. A1 - Schnabel, C. A1 - Viktorinová, I. A1 - Brand, T. A1 - Kramer, M. A1 - Nattel, S. A1 - Dobrev, D. A1 - Tomancak, P. A1 - Backs, J. A1 - Kleinbongard, P. A1 - Heusch, G. A1 - Lorenz, K. A1 - Koch, E. A1 - Weber, S. A1 - El-Armouche, A. T1 - Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila JF - Scientific Reports N2 - Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure. KW - cardiac hypertrophy KW - epigenetics KW - heart failure Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323637 VL - 8 ER - TY - JOUR A1 - Hommers, L. G. A1 - Richter, J. A1 - Yang, Y. A1 - Raab, A. A1 - Baumann, C. A1 - Lang, K. A1 - Schiele, M. A. A1 - Weber, H. A1 - Wittmann, A. A1 - Wolf, C. A1 - Alpers, G. W. A1 - Arolt, V. A1 - Domschke, K. A1 - Fehm, L. A1 - Fydrich, T. A1 - Gerlach, A. A1 - Gloster, A. T. A1 - Hamm, A. O. A1 - Helbig-Lang, S. A1 - Kircher, T. A1 - Lang, T. A1 - Pané-Farré, C. A. A1 - Pauli, P. A1 - Pfleiderer, B. A1 - Reif, A. A1 - Romanos, M. A1 - Straube, B. A1 - Ströhle, A. A1 - Wittchen, H.-U. A1 - Frantz, S. A1 - Ertl, G. A1 - Lohse, M. J. A1 - Lueken, U. A1 - Deckert, J. T1 - A functional genetic variation of SLC6A2 repressor hsa-miR-579-3p upregulates sympathetic noradrenergic processes of fear and anxiety JF - Translational Psychiatry N2 - Increased sympathetic noradrenergic signaling is crucially involved in fear and anxiety as defensive states. MicroRNAs regulate dynamic gene expression during synaptic plasticity and genetic variation of microRNAs modulating noradrenaline transporter gene (SLC6A2) expression may thus lead to altered central and peripheral processing of fear and anxiety. In silico prediction of microRNA regulation of SLC6A2 was confirmed by luciferase reporter assays and identified hsa-miR-579-3p as a regulating microRNA. The minor (T)-allele of rs2910931 (MAFcases = 0.431, MAFcontrols = 0.368) upstream of MIR579 was associated with panic disorder in patients (pallelic = 0.004, ncases = 506, ncontrols = 506) and with higher trait anxiety in healthy individuals (pASI = 0.029, pACQ = 0.047, n = 3112). Compared to the major (A)-allele, increased promoter activity was observed in luciferase reporter assays in vitro suggesting more effective MIR579 expression and SLC6A2 repression in vivo (p = 0.041). Healthy individuals carrying at least one (T)-allele showed a brain activation pattern suggesting increased defensive responding and sympathetic noradrenergic activation in midbrain and limbic areas during the extinction of conditioned fear. Panic disorder patients carrying two (T)-alleles showed elevated heart rates in an anxiety-provoking behavioral avoidance test (F(2, 270) = 5.47, p = 0.005). Fine-tuning of noradrenaline homeostasis by a MIR579 genetic variation modulated central and peripheral sympathetic noradrenergic activation during fear processing and anxiety. This study opens new perspectives on the role of microRNAs in the etiopathogenesis of anxiety disorders, particularly their cardiovascular symptoms and comorbidities. KW - clinical genetics KW - psychiatric disorders Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-322497 VL - 8 ER - TY - JOUR A1 - Rietjens, Ivonne M. C. M. A1 - Dussort, P. A1 - Günther, Helmut A1 - Hanlon, Paul A1 - Honda, Hiroshi A1 - Mally, Angela A1 - O'Hagan, Sue A1 - Scholz, Gabriele A1 - Seidel, Albrecht A1 - Swenberg, James A1 - Teeguarden, Justin A1 - Eisenbrand, Gerhard T1 - Exposure assessment of process-related contaminants in food by biomarker monitoring JF - Archives of Toxicology N2 - Exposure assessment is a fundamental part of the risk assessment paradigm, but can often present a number of challenges and uncertainties. This is especially the case for process contaminants formed during the processing, e.g. heating of food, since they are in part highly reactive and/or volatile, thus making exposure assessment by analysing contents in food unreliable. New approaches are therefore required to accurately assess consumer exposure and thus better inform the risk assessment. Such novel approaches may include the use of biomarkers, physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry, and/or duplicate diet studies. This review focuses on the state of the art with respect to the use of biomarkers of exposure for the process contaminants acrylamide, 3-MCPD esters, glycidyl esters, furan and acrolein. From the overview presented, it becomes clear that the field of assessing human exposure to process-related contaminants in food by biomarker monitoring is promising and strongly developing. The current state of the art as well as the existing data gaps and challenges for the future were defined. They include (1) using PBK modelling and duplicate diet studies to establish, preferably in humans, correlations between external exposure and biomarkers; (2) elucidation of the possible endogenous formation of the process-related contaminants and the resulting biomarker levels; (3) the influence of inter-individual variations and how to include that in the biomarker-based exposure predictions; (4) the correction for confounding factors; (5) the value of the different biomarkers in relation to exposure scenario's and risk assessment, and (6) the possibilities of novel methodologies. In spite of these challenges it can be concluded that biomarker-based exposure assessment provides a unique opportunity to more accurately assess consumer exposure to process-related contaminants in food and thus to better inform risk assessment. KW - Dietary process-related contaminants KW - Biomarkers KW - External exposure assessment KW - Physiologically based kinetic models KW - Risk assessment Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226268 VL - 92 IS - 1 ER - TY - JOUR A1 - Salinger, Tim A1 - Hu, Kai A1 - Liu, Dan A1 - Taleh, Scharoch A1 - Herrmann, Sebastian A1 - Oder, Daniel A1 - Gensler, Daniel A1 - Müntze, Jonas A1 - Ertl, Georg A1 - Lorenz, Kristina A1 - Frantz, Stefan A1 - Weidemann, Frank A1 - Nordbeck, Peter T1 - Association between Comorbidities and Progression of Transvalvular Pressure Gradients in Patients with Moderate and Severe Aortic Valve Stenosis JF - Cardiology Research and Practice N2 - Background. Fast progression of the transaortic mean gradient (P-mean) is relevant for clinical decision making of valve replacement in patients with moderate and severe aortic stenosis (AS) patients. However, there is currently little knowledge regarding the determinants affecting progression of transvalvular gradient in AS patients. Methods. This monocentric retrospective study included consecutive patients presenting with at least two transthoracic echocardiography examinations covering a time interval of one year or more between April 2006 and February 2016 and diagnosed as moderate or severe aortic stenosis at the final echocardiographic examination. Laboratory parameters, medication, and prevalence of eight known cardiac comorbidities and risk factors (hypertension, diabetes, coronary heart disease, peripheral artery occlusive disease, cerebrovascular disease, renal dysfunction, body mass index >= 30 Kg/m(2), and history of smoking) were analyzed. Patients were divided into slow (P-mean < 5 mmHg/year) or fast (P-mean >= 5 mmHg/year) progression groups. Results. A total of 402 patients (mean age 78 +/- 9.4 years, 58% males) were included in the study. Mean follow-up duration was 3.4 +/- 1.9 years. The average number of cardiac comorbidities and risk factors was 3.1 +/- 1.6. Average number of cardiac comorbidities and risk factors was higher in patients in slow progression group than in fast progression group (3.3 +/- 1.5 vs 2.9 +/- 1.7; P = 0.036). Patients in slow progression group had more often coronary heart disease (49.2% vs 33.6%; P = 0.003) compared to patients in fast progression group. LDL-cholesterol values were lower in the slow progression group (100 +/- 32.6 mg/dl vs 110.8 +/- 36.6 mg/dl; P = 0.005). Conclusion. These findings suggest that disease progression of aortic valve stenosis is faster in patients with fewer cardiac comorbidities and risk factors, especially if they do not have coronary heart disease. Further prospective studies are warranted to investigate the outcome of patients with slow versus fast progression of transvalvular gradient with regards to comorbidities and risk factors. KW - Valvular heart-desease KW - Prognostic impact KW - Risk-factors KW - Chronic heart-failure KW - Prevalence KW - mild KW - statins KW - therapy KW - mortality Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227291 ER - TY - JOUR A1 - Schihada, Hannes A1 - Vandenabeele, Sylvie A1 - Zabel, Ulrike A1 - Frank, Monika A1 - Lohse, Martin J. A1 - Maiellaro, Isabella T1 - A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics JF - Communications Biology N2 - G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the alpha(2 Lambda)-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning beta(2)-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands. KW - Fluorescence resonance energy transfer KW - G protein-coupled receptors KW - High-throughput screening Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228592 VL - 1 IS - 105 ER - TY - JOUR A1 - Bankoglu, Ezgi Eyluel A1 - Arnold, Charlotte A1 - Hering, Ilona A1 - Hankir, Mohammed A1 - Seyfried, Florian A1 - Stopper, Helga T1 - Decreased chromosomal damage in lymphocytes of obese patients after bariatric surgery JF - Scientific Reports N2 - The number of bariatric surgeries being performed worldwide has markedly risen. While the improvement in obesity-associated comorbidities after bariatric surgery is well-established, very little is known about its impact on cancer risk. The peripheral lymphocyte micronucleus test is a widely used method for the monitoring of chromosomal damage levels in vivo, and micronucleus frequency positively correlates with cancer risk. Therefore, the aim of this study was to compare the micronucleus frequency before and after bariatric surgery in obese subjects. Peripheral blood mononuclear cells were collected from 45 obese subjects before and at two time-points after bariatric surgery (6 and 12 months) to assess spontaneous micronucleus frequency. Consistent with the increased cancer risk previously shown, bariatric surgery-induced weight loss led to a significant reduction in lymphocyte micronucleus frequency after 12 months. Interestingly, comorbidities such as type 2 diabetes mellitus and metabolic syndrome further seemed to have an impact on the lymphocyte micronucleus frequency. Our findings may indicate a successful reduction of cancer risk in patients following weight loss caused by bariatric surgery. KW - obesity KW - bariatric surgery KW - cancer risk Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177090 VL - 8 IS - 11195 ER - TY - JOUR A1 - Hintzsche, Henning A1 - Montag, Gracia A1 - Stopper, Helga T1 - Induction of micronuclei by four cytostatic compounds in human hematopoietic stem cells and human lymphoblastoid TK6 cells JF - Scientific Reports N2 - For mutagenicity testing, primary lymphocytes or mammalian cell lines are employed. However, the true target for carcinogenic action of mutagenic chemicals may be stem cells. Since hematopoietic cancers induced by chemical agents originate at the hematopoietic stem cell (HSC) stage and since one of the side effects of chemotherapeutic cancer treatment is the induction of secondary tumors, often leukemias, HSC may be a suitable cell system. We compared the sensitivity of HSC with the genotoxicity testing cell line TK6 for chromosomal mutations. HSC were less sensitive than TK6 cells for the genotoxic effects of the model genotoxins and chemotherapeutic agents doxorubicin, vinblastine, methyl methanesulfonate (MMS) and equally sensitive for mitomycin C (MMC). However, loss of viability after mitomycin C treatment was higher in HSC than in TK6 cells. Among the factors that may influence sensitivity for genomic damage, the generation or response to reactive oxygen species (ROS) and the effectiveness of DNA damage response can be discussed. Here we show that HSC can be used in a standard micronucleus test protocol for chromosomal mutations and that their sensitivity was not higher than that of a classical testing cell line. KW - apoptosis KW - haematopoietic stem cells KW - TK6 cells KW - micronuclei Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176210 VL - 8 IS - 3371 ER - TY - THES A1 - Kauk, Michael T1 - Investigating the Molecular Mechanism of Receptor Activation at Muscarinic Receptors by Means of Pathway-Specific Dualsteric Ligands and Partial Agonists T1 - Molekulare Grundlagen der Rezeptoraktivierung von muskarinergen Acetylcholin Rezeptoren durch dualstere Liganden und Partialagonisten N2 - G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology. Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored. In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 % could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful. N2 - G Protein gekoppelte Rezeptoren (GPCRs) bilden die größte Proteinfamilie, die im humanen Genom verschlüsselt ist. Sie sind nicht nur die Zielstruktur für eine Vielzahl von derzeit gebräuchlichen Medikamenten, sondern gehören auch zu den vielversprechendsten Therapieansätzen für die moderne Medikamentenentwicklung. Muskarinerge Acetylcholin Rezeptoren (mAChRs) gehören zu den aminergen Klasse A GPCRs und sind bereits heute von klinischer Relevanz. Die muskarinerge Rezeptorfamilie wird von fünf Subtypen gebildet, die sich besonders durch eine hohe Sequenzidentität in der endogenen Ligandenbindestelle (orthostere Bindestelle) auszeichnen. Aus diesem Grund ist es mit den herkömmlich verwendeten Medikamenten nicht möglich, einen ganz bestimmten Subtyp zu therapieren, ohne auch andere Subtypen zu beeinflussen und so unerwünschte Nebenwirkungen zu erhalten. Eine Möglichkeit Ligandenbindung, dynamische Rezeptoraktivierung oder Signalweiterleitung von GPCRs nach pharmakologischen Gesichtspunkten zu charakterisieren, stellt der Floreszenz Resonanz Energietransfer (FRET) dar. Mit Hilfe dieser Methode kann über kleine Entfernungen (<10 nm) die relative Orientierung von zwei Fluorophoren mit überlappenden Spektralbereichen mit hoher zeitlicher Auflösung verfolgt werden. Integriert man das Fluorophorpaar mit Hilfe gentechnischer Methoden in die Molekülstruktur des Rezeptors, kann man dessen Konformationsänderung bzw. Aktivierung infolge einer Ligandenbindung aufzeichnen. Dualstere Liganden sind eine Substanzklasse von hohem zukünftigen klinischen Potential und zeichnen sich durch die Verknüpfung mehrerer pharmakologisch aktiver Untereinheiten aus. Der orthostere Molekülteil interagiert mit der endogenen Ligandenbindestelle und der allostere Molekülteil interagiert mit einem zweiten Rezeptorabschnitt, der häufig in den extrazellulären Schlaufen des Rezeptors zu finden ist. Diese allosteren Bindestellen zeichnet sich durch eine vergleichsweise geringe Sequenzidentität aus, weswegen allostere Modulatoren auch selektiv an Subtypen binden können. Aufgrund des Aufbaus können dualstere Liganden auf vielfältige Weise mit dem Rezeptor interagieren und dieser Bindemechanismus wurde als dynamische Ligandenbindung beschrieben. Zum einen können beide Molekülteile gleichzeitig mit dem Rezeptor interagieren und ihn aktivieren (dualsterer Bindemodus) und zum anderen findet man einen rein allosteren Bindemodus, der den Rezeptor nicht aktiviert. Der orthostere Molekülteil ist vor allem für die Rezeptoraktivierung zuständig, die sich durch eine hohe Affinität auszeichnet und der allostere Molekülteil kann selektive Rezeptorinteraktionen vermitteln. Da dualstere Moleküle immer Eigenschaften beider Untereinheiten besitzen, werden dualstere Liganden als sehr vielversprechend erachtet, zukünftig subtypselektive Medikamente darzustellen. In dieser Arbeit wurde der erste Rezeptor FRET Sensor für den muskarinergen Subtyp 1 (M1) beschrieben und es konnte gezeigt werden, dass sich dieser Rezeptorsensor in seiner physiologischen Funktion nicht von dem wild Typ unterscheidet. Des Weiteren können mit Hilfe dieses Sensors liganden- und konzentrationsabhängige Rezeptorantworten aufgezeichnet werden. Der M1-I3N-CFP wurde dazu genutzt verschiedene Reihen dualsterer Liganden zu charakterisieren und auf ihre aktivierenden Eigenschaften bezüglich des M1 zu testen. Es wurde gezeigt, dass die Kombination aus dem synthetischen und hochpotenten Agonisten Iperoxo als Orthoster und dem in der Literatur als M1 selektiven positiven allosteren Modulator beschriebenen BQCA als Alloster sehr vielversprechend ist. Es konnte gezeigt werden, dass die rezeptoraktivierenden Eigenschaften sowohl von orthosteren wie auch von dualsteren Liganden stark von der Linkerlänge abhängig sind. Für dualstere Liganden konnte so ein glockenförmiger Zusammenhang zwischen Linkerlänge und Rezeptoraktivierung herausgearbeitet werden. Des Weiteren wurde gezeigt, dass bestimmte Hybride, die den M1 aktivieren, an anderen Subtypen keine Effekte hervorrufen und somit als subtypselektiv beschrieben werden können. Im Anschluss wurde mit Hilfe des gewonnenen Wissens über Iperoxo/BQCA Hybride, das Moleküldesign der dualsteren Liganden weiterentwickelt. So wurden in dieser Arbeit die ersten photo-schaltbaren bzw. photo-dimmbaren dualsteren Liganden beschrieben und charakterisiert. Des Weiteren wurde in dieser Arbeit die herkömmliche Charakterisierung von dualsteren Liganden weiterentwickelt. Es konnte zum ersten Mal gezeigt werden, dass es möglich ist, die Aktivierung eines Rezeptors durch einen dualsteren Liganden nachzustellen, indem die einzelnen Fragmente des ursprünglichen Liganden gleichzeitig appliziert werden. Diese auf Fragmenten basierende Charakterisierung ist die erste Anwendung dieser Art und birgt großes Potential für die zukünftige Suche nach neuen Wirkstoffen. Neben der Untersuchung von pharmakologischen Schwerpunkten wurde auch die Weiterentwicklung der Rezeptor FRET Methodik beschrieben. Die herkömmliche Anwendung der Rezeptor FRET Sensoren geschieht auf Objektträgern aus Quarzglas. In dieser Arbeit wurde diese Anwendung dahingehend weiterentwickelt, dass die Objektträger mit einer 20 nm dicken Goldschicht beschichtet wurden, um den Einfluss von Plasmonoberflächen auf physiologisch relevante FRET Messungen zu untersuchen. Es konnte gezeigt werden, dass mit Hilfe der Goldbeschichtung und in Abhängigkeit des Versuchsaufbaus die Energietransfereffizienz um bis zu 60 % gesteigert werden konnte. Diese Entdeckung zeigt Potential zukünftig die FRET-Reichweite zu erhöhen und so bisher nicht charakterisierbare Sachverhalte aufklären zu können. KW - G-Protein gekoppelte Rezeptoren KW - Muscarinrezeptor KW - Dualsteric Ligands KW - Partial Agonists KW - Dualstere Liganden KW - Partialagonismus Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173729 N1 - Online-Version enthält nicht den Appendix (Volltexte der Originalveröffentlichungen der Zeitschriftenaufsätze) ER - TY - JOUR A1 - Werner, Rudolf A1 - Wakabayashi, Hiroshi A1 - Bauer, Jochen A1 - Schütz, Claudia A1 - Zechmeister, Christina A1 - Hayakawa, Nobuyuki A1 - Javadi, Mehrbod S. A1 - Lapa, Constantin A1 - Jahns, Roland A1 - Ergün, Süleyman A1 - Jahns, Valerie A1 - Higuchi, Takahiro T1 - Longitudinal \(^{18}\)F-FDG PET imaging in a Rat Model of Autoimmune Myocarditis JF - European Heart Journal Cardiovascular Imaging N2 - Aims: Although mortality rate is very high, diagnosis of acute myocarditis remains challenging with conventional tests. We aimed to elucidate the potential role of longitudinal 2-Deoxy-2-\(^{18}\)F-fluoro-D-glucose (\(^{18}\)F-FDG) positron emission tomography (PET) inflammation monitoring in a rat model of experimental autoimmune myocarditis. Methods and results: Autoimmune myocarditis was induced in Lewis rats by immunizing with porcine cardiac myosin emulsified in complete Freund’s adjuvant. Time course of disease was assessed by longitudinal \(^{18}\)F-FDG PET imaging. A correlative analysis between in- and ex vivo \(^{18}\)F-FDG signalling and macrophage infiltration using CD68 staining was conducted. Finally, immunohistochemistry analysis of the cell-adhesion markers CD34 and CD44 was performed at different disease stages determined by longitudinal \(^{18}\)F-FDG PET imaging. After immunization, myocarditis rats revealed a temporal increase in 18F-FDG uptake (peaked at week 3), which was followed by a rapid decline thereafter. Localization of CD68 positive cells was well correlated with in vivo \(^{18}\)F-FDG PET signalling (R\(^2\) = 0.92) as well as with ex vivo 18F-FDG autoradiography (R\(^2\) = 0.9, P < 0.001, respectively). CD44 positivity was primarily observed at tissue samples obtained at acute phase (i.e. at peak 18F-FDG uptake), while CD34-positive staining areas were predominantly identified in samples harvested at both sub-acute and chronic phases (i.e. at \(^{18}\)F-FDG decrease). Conclusion: \(^{18}\)F-FDG PET imaging can provide non-invasive serial monitoring of cardiac inflammation in a rat model of acute myocarditis. KW - positron emission tomography KW - Myokarditis KW - myocarditis KW - inflammation KW - 18F-FDG KW - PET KW - personalized treatment Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165601 SN - 2047-2404 ER -