@phdthesis{Kramer2021, author = {Kramer, Sofia}, title = {Hemmung pathologischer kardialer Hypertrophie {\"u}ber das Dimer-Interface von ERK1/2}, doi = {10.25972/OPUS-23373}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-233739}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Die extrazellul{\"a}r Signal-regulierten Kinasen 1 und 2 (ERK1/2) spielen eine zentrale Rolle bei der Vermittlung kardialer Hypertrophie und dem Zell{\"u}berleben. Hypertrophe Stimuli aktivieren ERK1/2, triggern deren Dimerisierung und in der Folge die ERK188-Autophosphorylierung. Diese neu entdeckte Autophosphorylierung ist eine Voraussetzung f{\"u}r den nukle{\"a}ren Import von ERK1/2 und f{\"u}hrt zum Entstehen pathologischer kardialer Hypertrophie. Da das Dimer Interface von ERK eine m{\"o}gliche Zielstruktur darstellt, um selektiv die nukle{\"a}ren Signalwege von ERK zu unterbrechen, wurde untersucht, ob man mit Hemmung der ERK-Dimerisierung eine therapeutische M{\"o}glichkeit hat, um pathologische kardiale Hypertrophie zu verhindern. Dazu wurden verschiedene ERK2 Mutanten und Peptide generiert, um die ERK-Dimerisierung zu verhindern. Die Effekte dieser Konstrukte auf die ERK-Dimerisierung und den Kernimport wurden in verschiedenen Zelltypen mittels Fluoreszenzmikroskopie, Co-Immunopr{\"a}zipitationen und Duolink proximity ligation assays getestet. Es konnte gezeigt werden, dass die Peptide effektiv die ERK-ERK Interaktion nach Stimulation mit Phenylephrin und/oder Carbachol verhindern. Zus{\"a}tzlich reduzierten die Peptide ERKT188-Phosphorylierung und in der Folge den ERK-Import in den Nukleus und Kardiomyozytenhypertrophie. Normale ERK-Aktivierung wurde jedoch durch die Peptide nicht verhindert. Insgesamt konnte gezeigt werden, dass das ERK-Dimer Interface eine wertvolle Zielstruktur ist, mit dem man nukle{\"a}re ERK1/2 Signalwege selektiv unterbrechen und damit effektiv Kardiomyozytenwachstum reduzieren kann, ohne gleichzeitig das Zell{\"u}berleben zu gef{\"a}hrden.}, subject = {Dimerisierung}, language = {de} } @phdthesis{Classen2021, author = {Claßen, Alexandra}, title = {The ERK-cascade in the pathophysiology of cardiac hypertrophy}, doi = {10.25972/OPUS-22966}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229664}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {ERK1/2 are known key players in the pathophysiology of heart failure, but the members of the ERK cascade, in particular Raf1, can also protect the heart from cell death and ischemic injury. An additional autophosphorylation (ERK1 at Thr208, ERK2 at Thr188) empowers ERK1/2 translocation to the nucleus and phosphorylation of nuclear targets which take part in the development of cardiac hypertrophy. Thereby, targeting this additional phosphorylation is a promising pharmacological approach. In this thesis, an in silico model of ERK cascade in the cardiomyocyte is introduced. The model is a semi-quantitive model and its behavior was tested with different softwares (SQUAD and CellNetAnalyzer). Different phosphorylation states of ERK1/2 as well as different stimuli can be reproduced. The different types of stimuli include hypertrophic as well as non-hypertrophic stimuli. With the introduced in-silico model time courses and synergistic as well as antagonistic receptor stimuli combinations can be predicted. The simulated time courses were experimentally validated. SQUAD was mainly used to make predictions about time courses and thresholds, whereas CNA was used to analyze steady states and feedback loops. Furthermore, new targets of ERK1/2 which partially contribute, also in the formation of cardiac hypertrophy, were identified and the most promising of them were illuminated. Important further targets are Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 and SPIN90. Cardiomyocyte gene expression data sets were analyzed to verify involved components and to find further significantly altered genes after induced hypertrophy with TAC (transverse aortic constriction). Changes in the ultrastructure of the cardiomyocyte are the final result of induced hypertrophy.}, subject = {Herzhypertrophie}, language = {en} } @article{ChengOthmanStopperetal.2017, author = {Cheng, Cheng and Othman, Eman M. and Stopper, Helga and Edrada-Ebel, RuAngelie and Hentschel, Ute and Abdelmohsen, Usama Ramadan}, title = {Isolation of petrocidin A, a new cytotoxic cyclic dipeptide from the marine sponge-derived bacterium \(Streptomyces\) sp. SBT348}, series = {Marine Drugs}, volume = {15}, journal = {Marine Drugs}, number = {12}, doi = {10.3390/md15120383}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172644}, year = {2017}, abstract = {A new cyclic dipeptide, petrocidin A (\(\textbf{1}\)), along with three known compounds—2,3-dihydroxybenzoic acid (\(\textbf{2}\)), 2,3-dihydroxybenzamide (\(\textbf{3}\)), and maltol (\(\textbf{4}\))—were isolated from the solid culture of \(Streptomyces\) sp. SBT348. The strain \(Streptomyces\) sp. SBT348 had been prioritized in a strain collection of 64 sponge-associated actinomycetes based on its distinct metabolomic profile using liquid chromatography/high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR). The absolute configuration of all α-amino acids was determined by HPLC analysis after derivatization with Marfey's reagent and comparison with commercially available reference amino acids. Structure elucidation was pursued in the presented study by mass spectrometry and NMR spectral data. Petrocidin A (\(\textbf{1}\)) and 2,3-dihydroxybenzamide (\(\textbf{3}\)) exhibited significant cytotoxicity towards the human promyelocytic HL-60 and the human colon adenocarcinoma HT-29 cell lines. These results demonstrated the potential of sponge-associated actinomycetes for the discovery of novel and pharmacologically active natural products.}, language = {en} } @article{SegererHadamekZundleretal.2016, author = {Segerer, Gabriela and Hadamek, Kerstin and Zundler, Matthias and Fekete, Agnes and Seifried, Annegrit and Mueller, Martin J. and Koentgen, Frank and Gessler, Manfred and Jeanclos, Elisabeth and Gohla, Antje}, title = {An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation}, series = {Scientific Reports}, volume = {6}, journal = {Scientific Reports}, doi = {10.1038/srep35160}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181094}, year = {2016}, abstract = {Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive Pgp\(^{D34N}\) mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation.}, language = {en} } @article{MaurerHuppBischoffetal.2017, author = {Maurer, Jana and Hupp, Sabrina and Bischoff, Carolin and Foertsch, Christina and Mitchell, Timothy J. and Chakraborty, Trinad and Iliev, Asparouh I.}, title = {Distinct neurotoxicity profile of listeriolysin O from \(Listeria\) \(monocytogenes\)}, series = {Toxins}, volume = {9}, journal = {Toxins}, number = {1}, doi = {10.3390/toxins9010034}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172130}, year = {2017}, abstract = {Cholesterol-dependent cytolysins (CDCs) are protein toxins that originate from Gram-positive bacteria and contribute substantially to their pathogenicity. CDCs bind membrane cholesterol and build prepores and lytic pores. Some effects of the toxins are observed in non-lytic concentrations. Two pathogens, \(Streptococcus\) \(pneumoniae\) and \(Listeria\) \(monocytogenes\), cause fatal bacterial meningitis, and both produce toxins of the CDC family—pneumolysin and listeriolysin O, respectively. It has been demonstrated that pneumolysin produces dendritic varicosities (dendrite swellings) and dendritic spine collapse in the mouse neocortex, followed by synaptic loss and astrocyte cell shape remodeling without elevated cell death. We utilized primary glial cultures and acute mouse brain slices to examine the neuropathological effects of listeriolysin O and to compare it to pneumolysin with identical hemolytic activity. In cultures, listeriolysin O permeabilized cells slower than pneumolysin did but still initiated non-lytic astrocytic cell shape changes, just as pneumolysin did. In an acute brain slice culture system, listeriolysin O produced dendritic varicosities in an NMDA-dependent manner but failed to cause dendritic spine collapse and cortical astrocyte reorganization. Thus, listeriolysin O demonstrated slower cell permeabilization and milder glial cell remodeling ability than did pneumolysin and lacked dendritic spine collapse capacity but exhibited equivalent dendritic pathology.}, language = {en} } @article{SchoeneggeGallionPicardetal.2017, author = {Sch{\"o}negge, Anne-Marie and Gallion, Jonathan and Picard, Louis-Philippe and Wilkins, Angela D. and Le Gouill, Christian and Audet, Martin and Stallaert, Wayne and Lohse, Martin J. and Kimmel, Marek and Lichtarge, Olivier and Bouvier, Michel}, title = {Evolutionary action and structural basis of the allosteric switch controlling β\(_2\)AR functional selectivity}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, doi = {10.1038/s41467-017-02257-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172268}, year = {2017}, abstract = {Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the β\(_2\)-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or βarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity.}, language = {en} } @article{AdakuChilakaMally2020, author = {Adaku Chilaka, Cynthia and Mally, Angela}, title = {Mycotoxin Occurrence, Exposure and Health Implications in Infants and Young Children in Sub-Saharan Africa: A Review}, series = {Foods}, volume = {9}, journal = {Foods}, number = {11}, issn = {2304-8158}, doi = {10.3390/foods9111585}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-219250}, year = {2020}, abstract = {Infants and young children (IYC) remain the most vulnerable population group to environmental hazards worldwide, especially in economically developing regions such as sub-Saharan Africa (SSA). As a result, several governmental and non-governmental institutions including health, environmental and food safety networks and researchers have been proactive toward protecting this group. Mycotoxins, toxic secondary fungal metabolites, contribute largely to the health risks of this young population. In SSA, the scenario is worsened by socioeconomic status, poor agricultural and storage practices, and low level of awareness, as well as the non-establishment and lack of enforcement of regulatory limits in the region. Studies have revealed mycotoxin occurrence in breast milk and other weaning foods. Of concern is the early exposure of infants to mycotoxins through transplacental transfer and breast milk as a consequence of maternal exposure, which may result in adverse health effects. The current paper presents an overview of mycotoxin occurrence in foods intended for IYC in SSA. It discusses the imperative evidence of mycotoxin exposure of this population group in SSA, taking into account consumption data and the occurrence of mycotoxins in food, as well as biomonitoring approaches. Additionally, it discusses the health implications associated with IYC exposure to mycotoxins in SSA.}, language = {en} } @article{KlaunigDekantPlotzkeetal.2016, author = {Klaunig, James E. and Dekant, Wolfgang and Plotzke, Kathy and Scialli, Anthony R.}, title = {Biological relevance of decamethylcyclopentasiloxane (D5) induced rat uterine endometrial adenocarcinoma tumorigenesis: Mode of action and relevance to humans}, series = {Regulatory Toxicology and Pharmacology}, volume = {74}, journal = {Regulatory Toxicology and Pharmacology}, number = {Supplement}, doi = {10.1016/j.yrtph.2015.06.021}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-190952}, pages = {S44-S56}, year = {2016}, abstract = {Decamethylcyclopentasiloxane (D5) is a cyclic siloxane used in the production and formulation of consumer products with potential exposure to manufacturing workers, consumer, and the general public. Following a combined 2-year inhalation chronic bioassay performed in Fischer 344 (F344) rats, an increase in uterine endometrial adenocarcinomas was noted at the highest concentration to which animals were exposed. No other neoplasms were detected. In this study, a dose of 160 ppm produced an incidence of 8\% endometrial adenocarcinomas. Based on a number of experimental studies with D5, the current manuscript examines the biological relevance and possible modes of action for the uterine endometrial adenocarcinomas observed in the rat following chronic exposure to D5. Variable rates of spontaneous uterine endometrial adenocarcinomas have been reported for untreated F344 CrIBr rats. As such, we concluded that the slight increase in uterine endometrial adenocarcinomas observed in the D5 chronic bioassay might not be the result of D5 exposure but may be related to variability of the spontaneous tumor incidence in this strain of rat. However, if the uterine endometrial adenocarcinomas are related to D5-exposure, alteration in the estrous cycle in the aging F344 rat is the most likely mode of action. D5 is not genotoxic or estrogenic. The alteration in the estrous cycle is caused by a decrease in progesterone with an increase in the estrogen:progesterone ratio most likely induced by a decrease in prolactin concentration. Available data support that exposure to D5 influences prolactin concentration. Although the effects on prolactin concentrations in a number of experiments were not always consistent, the available data support the conclusion that D5 is acting via a dopamine receptor agonist-like mechanism to alter the pituitary control of the estrous cycle. In further support of this mode of action, studies in F344 aged animals showed that the effects of D5 on estrous cyclicity produced a response consistent with a dopamine-like effect and further suggest that D5 is accelerating the aging of the reproductive endocrine system in the F344 rat utilized in this study. This mode of action for uterine endometrial adenocarcinoma tumorigenesis is not relevant for humans.}, language = {en} } @article{BalasubramanianOthmanKampiketal.2017, author = {Balasubramanian, Srikkanth and Othman, Eman M. and Kampik, Daniel and Stopper, Helga and Hentschel, Ute and Ziebuhr, Wilma and Oelschlaeger, Tobias A. and Abdelmohsen, Usama R.}, title = {Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation}, series = {Frontiers in Microbiology}, volume = {8}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2017.00236}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171844}, year = {2017}, abstract = {Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections.}, language = {en} } @article{DekantKlaunig2016, author = {Dekant, Wolfgang and Klaunig, James E.}, title = {Toxicology of decamethylcyclopentasiloxane (D5)}, series = {Regulatory Toxicology and Pharmacology}, volume = {74}, journal = {Regulatory Toxicology and Pharmacology}, number = {Supplement}, doi = {10.1016/j.yrtph.2015.06.011}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-190914}, pages = {S67-S76}, year = {2016}, abstract = {Decamethylcyclopentasiloxane (D5) is a cyclic siloxane used in the formulation of consumer products as well as an industrial intermediate. A summary of the previous studies on the toxicology of D5 is provided. Toxicokinetic studies with D5 after dermal administration demonstrate a very low uptake of due to rapid evaporation. Following inhalation exposure, exhalation of unchanged D5 and excretion of metabolites with urine are major pathways for clearance in mammals. Due to this rapid clearance by exhalation, the potential for bioaccumulation of D5 is considered unlikely. The available toxicity data on D5 adequately cover the relevant endpoints regarding potential human health hazards. D5 was not DNA reactive or mutagenic in standard in vitro and in vivo test systems. D5 also did not induce developmental and reproductive toxicity in appropriately performed studies. In repeated studies in rats with subacute, subchronic and chronic inhalation exposure, mild effects on the respiratory tract typically seen after inhalation of irritating materials, increases in liver weight (28- and 90-day inhalation studies), and a small increase in the incidence of uterine adenocarcinoma (uterine tumor) in female rats (two-year inhalation chronic bioassay) were observed. The liver effects induced by D5 were consistent with D5 as a weak "phenobarbital-like" inducer of xenobiotic metabolizing enzymes and these effects are considered to be an adaptive response. Mechanistic studies to elucidate the mode-of-action for uterine tumor induction suggest an interaction of D5 with dopamine signal transduction pathways altering the pituitary control of the estrus cycle. The resulting estrogen imbalance may cause the small increase in uterine tumor incidence at the highest D5-exposure concentration over that seen in control rats. A genotoxic mechanism or a direct endocrine activity of D5 is not supported as a mode-of-action to account for the induction of uterine tumors by the available data.}, language = {en} } @phdthesis{Schihada2021, author = {Schihada, Hannes}, title = {Novel optical methods to monitor G-protein-coupled receptor activation in microtiter plates}, doi = {10.25972/OPUS-17541}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175415}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor's conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function.}, subject = {G-Protein gekoppelte Rezeptoren}, language = {en} } @phdthesis{PerpinaViciano2020, author = {Perpi{\~n}{\´a} Viciano, Cristina}, title = {Study of the activation mechanisms of the CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3)}, doi = {10.25972/OPUS-19237}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192371}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β-arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β-arrestin-dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different F{\"o}rster resonance energy transfer (FRET)-based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild-type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET-based receptor sensors in the field of chemokine receptors. Additional FRET-based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand-induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12-induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands.}, subject = {G protein-coupled receptors}, language = {en} } @phdthesis{Witzinger2020, author = {Witzinger, Linda}, title = {Rolle der Pyridoxal 5´-Phosphat Phosphatase PDXP im Vitamin B6-Metabolismus muriner Erythrozyten und Hippocampi}, doi = {10.25972/OPUS-21654}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216546}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Die Phosphatase PDXP (auch bekannt als Chronophin) geh{\"o}rt zur Familie der HAD Phosphatasen, einer ubiquit{\"a}r exprimierten Enzymklasse mit wichtigen physiologischen Funktionen. PDXP zeigt Phosphatase-Aktivit{\"a}t gegen{\"u}ber seinem Substrat Pyridoxal 5´-Phosphat (PLP), der aktivierten Form von Vitamin B6. PDXP-defiziente M{\"a}use (Knockout-M{\"a}use) weisen im Vergleich zu Wildtypen verdoppelte PLP-Konzentrationen in Erythrozyten sowie im Gesamthirn auf. Vermutlich kommt PDXP daher eine wichtige Funktion in Erythrozyten und im Hirn zu. Ziel dieser Arbeit war es, erste Einblicke in diese Funktion(en) von PDXP zu erlangen. Hierzu wurden HPLC-basierte Analysen der erythrozyt{\"a}ren PLP-Konzentrationen in Wildtyp- sowie PDXP-defizienten M{\"a}usen durchgef{\"u}hrt. Dabei ließen sich die rund doppelt so hohen erythrozyt{\"a}ren PLP-Level in den KO-M{\"a}usen best{\"a}tigen. Zudem ist es gelungen, eine Methode zur Messung der endogenen Phosphatase-Aktivit{\"a}t von PDXP in Erythrozytenlysaten zu etablieren. So konnte im Wildtyp anhand der Verringerung der PLP-Konzentrationen pro Zeiteinheit eine erythrozyt{\"a}re PDXP-Aktivit{\"a}t nachgewiesen werden. Dazu waren die Inkubation mit Pyridoxin, sowie die Anwendung eines Inhibitors der PDXK notwendig. Eine bis dato vermutete Funktion der PDXP, zur Mobilisation von erythrozyt{\"a}rem PLP w{\"a}hrend Fastenzeiten, konnte ausgeschlossen werden. So zeigte der Vergleich der erythrozyt{\"a}ren PLP-Konzentrationen aus gefasteten mit normal gef{\"u}tterten Tieren in beiden Genotypen exakt dieselbe prozentuale PLP-Verringerung. W{\"a}hrend Nahrungszufuhr ließ sich jedoch eine Funktion der Phosphatase PDXP als „Converter" von Pyridoxin zu Pyridoxal erkennen. Ausgehend von PN konnte im Wildtyp ({\"u}ber die Zwischenprodukte PNP und PLP) eine PDXP-abh{\"a}ngige Dephosphorylierung von PLP zu PL erfolgen. So wies der Wildtyp eine rund vierfach h{\"o}here PL-Produktion auf, verglichen mit der PDXP-defizienten Maus. Die Phosphatase PDXP erwies sich als essenziell f{\"u}r die erythrozyt{\"a}re Konversion von Pyridoxin zu Pyridoxal. Dadurch erreicht der Organismus eine metabolische Flexibilit{\"a}t, die ihn bis zu einem gewissen Grad unabh{\"a}ngig von der Nahrungsauswahl macht. Zudem k{\"o}nnen Zellen oder Organe, denen durch das Fehlen der PNPO, die Konversion zu PLP nicht m{\"o}glich ist, mit PL versorgt werden. Aus der hohen Reaktivit{\"a}t von PLP mit umliegenden Nucleophilen ergibt sich eine gewisse Problematik f{\"u}r die Zelle im Umgang mit freiem PLP. So liegt der Großteil des erythrozyt{\"a}ren PLPs gebunden an Proteine (vor allem H{\"a}moglobin) vor. Anhand von Filtern (MWCO, 3000) ließ sich zwischen der hier definiert als „freien" und der „gebundenen" Form von PLP differenzieren. So konnten erste Erkenntnisse zur Rolle von PDXP als Determinator freier PLP-Konzentrationen in Erythrozyten und insbesondere im Hippocampus erlangt werden. Im Hippocampus ergaben sich insgesamt deutlich h{\"o}here Konzentrationen an freiem PLP als in den Erythrozyten und es bestand zudem ein Unterschied zwischen den Genotypen. So wiesen die KO-M{\"a}use ~1/3 h{\"o}here freie PLP-Konzentrationen im Vergleich zu den Wildtypen auf. Schließlich konnte ein Effekt des Tieralters auf den PLP-Metabolismus festgestellt werden. Sowohl in den Erythrozyten als auch im Hippocampus ergaben sich alterskorrelierte {\"A}nderungen ihrer PLP-Konzentrationen. Zudem zeigten Western Blot Analysen altersbedingte Unterschiede ihrer Vitamin B6-Enzymexpressionen. So wiesen {\"a}ltere Wildtypen im Hippocampus eine f{\"u}nffach erh{\"o}hte PDXP-Expression verglichen mit j{\"u}ngeren Tieren auf. In den Erythrozytenlysaten hingegen zeigten {\"a}ltere Tiere beider Genotypen eine rund vierfach geringere PNPO-Expression gegen{\"u}ber j{\"u}ngeren Tieren. Die mit dem Alter eintretende physiologische Verringerung der erythrozyt{\"a}ren PNPO-Expression w{\"u}rde somit f{\"u}r den Organismus einen Verlust seiner metabolischen Flexibilit{\"a}t bedeuten, die mit der Konversion von PN zu PL einhergeht.}, subject = {Vitamin B6}, language = {de} } @phdthesis{Kircher2020, author = {Kircher, Malte Tim}, title = {Neuronale Genotoxizit{\"a}t von Angiotensin II}, doi = {10.25972/OPUS-21427}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214273}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {In recent decades, the acceptance has steadily increased that oxidative stress plays an important role in the development of chronic diseases, malignant neoplasia and the acceleration of the aging process. As one of the most common chronic diseases, hypertension is often associated with a misregulated renin-angiotensin-aldosterone system that causes chronic oxidative stress. Hypertension is a risk factor for neurological diseases such as vascular dementia (VaD) and many neurological disorders, including VaD, have an ROS-associated or inflammatory component in their etiology. Our group has already demonstrated AT-II-induced genotoxicity in kidney and myocardial cells and tissues. The aim of this dissertation was to investigate a possible association between AT-II and neurodegeneration that is triggered by neuronal genotoxicity of AT-II. First, we showed in two neuronal cell lines that AT-II causes dose-dependent genome damage. Subsequent experiments could attribute this toxicity to NOX-produced superoxide generated after AT-II binding to the AT1R. In addition, AT-II-induced depletion of the most important intracellular antioxidant - glutathione - was demonstrated. In vivo, we were able to show that AT1aR knockout mice after AT-II treatment showed significantly more genome damage in the subfornic organ (SFO) than wild-type mice. The SFO is one of the few structures in the brain with an interrupted blood-brain barrier, which makes it accessible and particularly sensitive to circulating AT-II. In the recent literature, these genome damages were also observed in kidney and heart tissues and prove an additional genotoxicity of AT-II independent of AT1aR and consequently independent of blood pressure. In summary, this work shows that increased AT-II levels in neuronal cells cause genome damage due to NOX-produced superoxide. It is hoped that these results will one day help to decipher the complete development of VaD.}, subject = {Angiotensin II}, language = {de} } @phdthesis{Schewe2020, author = {Schewe, Victoria Kristina}, title = {Mikrokernbildung in Mundschleimhautzellen von Patienten mit Kopf-Hals-Tumoren w{\"a}hrend Radio-/Radiochemotherapie}, doi = {10.25972/OPUS-21535}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-215354}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Das Ziel der vorliegenden Arbeit war die Mikrokernbildung in Mundschleimhautzellen von 35 Patienten mit Kopf-Hals-Tumoren w{\"a}hrend einer sechsw{\"o}chigen Radio-/Radiochemotherapie und sechs Wochen danach darzustellen. Die Ergebnisse der vorliegenden Arbeit zeigten, dass Patienten mit Kopf-Hals-Tumoren im Vergleich zu gesunden Probanden erh{\"o}hte Mikrokernraten aufwiesen. Ebenfalls konnte gezeigt werden, dass es zu einer vermehrten Bildung von Mikrokernen w{\"a}hrend einer sechsw{\"o}chigen Radio-/Radiochemotherapie kam. Nach Therapiebeendigung sanken die Werte nach drei bis sechs Wochen und lagen unter dem Ausgangswert, in dem Bereich von spontan entstehenden Mikrokernen. In Bezug auf die Tumorgr{\"o}ße konnte nur in der zweiten Woche ein signifikanter Unterschied in der Mikrokernrate zwischen T1- und T4-Stadium beobachtet werden. Es konnte keine Korrelation zwischen einer zus{\"a}tzlich verabreichten Chemotherapie, Grading des Tumors, Alter sowie Geschlecht der Patienten und einem Anstieg der Mikrokernrate festgestellt werden.}, subject = {Kleinkern}, language = {de} } @article{TanBabakVenkatesanetal.2019, author = {Tan, Aaron and Babak, Maria V. and Venkatesan, Gopalakrishnan and Lim, Clarissa and Klotz, Karl-Norbert and Herr, Deron Raymond and Cheong, Siew Lee and Federico, Stephanie and Spalluto, Giampiero and Ong, Wei-Yi and Chen, Yu Zong and Loo, Jason Siau Ee and Pastorin, Giorgia}, title = {Design, Synthesis and Evaluation of New Indolylpyrimidylpiperazines for Gastrointestinal Cancer Therapy}, series = {Molecules}, volume = {24}, journal = {Molecules}, number = {20}, issn = {1420-3049}, doi = {10.3390/molecules24203661}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193271}, pages = {3661}, year = {2019}, abstract = {Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors' ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3-6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30\% and EC50 of 2.89 ± 0.55 μM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.}, language = {en} } @phdthesis{Schaefer2020, author = {Sch{\"a}fer, Florian}, title = {Kontraktionsverhalten neonataler Rattenkardiomyozyten bei {\"U}berexpression phosphorylierungsdefizienter RKIP Mutanten S51/S52}, doi = {10.25972/OPUS-21374}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213747}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Durch Phosphorylierung inaktiviert GRK2 kardiale ß-Adrenorezeptoren und vermindert dadurch die Kontraktilit{\"a}t von neonatalen Rattenkardiomyozyten. Als nat{\"u}rlicher Inhibitor der GRK2 beeinflusst RKIP die Signalweiterleitung bei Stimulation von ß-Adrenorezeptoren. In dieser Arbeit konnte gezeigt werden, dass eine {\"U}berexpression von RKIP die Kontraktilit{\"a}t von neonatalen Rattenkardiomyozyten erh{\"o}ht. Es wurde die Anzahl auftretender Spontankontraktionen vor und nach Stimulation mit Isoproterenol erfasst sowie eine zeitliche Analyse der Calciumfreisetzung und -aufnahme nach elektrischer Stimulation durchgef{\"u}hrt. Im unstimulierten Zustand zeigten neonatale Rattenkardiomyozyten, die Wildtyp-RKIP {\"u}berexprimierten, verglichen mit der Kontrollgruppe, keine signifikanten Unterschiede hinsichtlich auftretender Spontankontraktionen. Nach Stimulation mit Isoproterenol zeigten neonatale Rattenkardiomyozyten, in denen Wildtyp-RKIP {\"u}berexprimiert wurde, eine signifikant h{\"o}here Anzahl auftretender Spontankontraktionen. Eine Analyse der Calciumtransienten zeigte bei RKIP-Wildtyp {\"u}berexprimierenden neonatalen Rattenkardiomyozyten eine erh{\"o}hte Calciumfreisetzung w{\"a}hrend der Systole sowie eine beschleunigte Calciumwiederaufnahme w{\"a}hrend der Diastole. Zudem wurden die RKIP-Mutanten RKIPS51V und RKIPS52V im Hinblick auf ihre Kontraktilit{\"a}t untersucht. Neonatale Rattenkardiomyozyten welche RKIPS51V und RKIPS52V {\"u}berexprimierten zeigten weder im Hinblick auf auftretende Spontankontraktionen noch bei der Analyse der Calciumtransienten signifikante Unterschiede zur Kontrollgruppe. Da auch eine Phosphorylierung an Aminos{\"a}ureposition 51 bzw. Aminos{\"a}ureposition 52 ohne direkte Auswirkung auf die Kontraktilit{\"a}t m{\"o}glich ist, wurde in einem in vitro Kinase Assay analysiert, ob neben der bekannten Phosphorylierungsstelle S153 eine weitere Phosphorylierung von RKIP durch PKA oder PKC erfolgt. Bei Einsatz der an Aminos{\"a}ureposition 153 phosphorylierungsdefizienten RKIP Mutante RKIPS153A konnte keine Phosphorylierung beobachtet werden. In der vorliegenden Arbeit konnte neben einer Phosphorylierung an S153 keine weitere Phosphorylierung von RKIP durch PKC oder PKA beobachtet werden.}, subject = {Herzinsuffizienz}, language = {de} } @phdthesis{Arnold2020, author = {Arnold, Charlotte Antonia}, title = {Reduktion des Genomschadens in peripheren Lymphozyten adip{\"o}ser Patienten nach bariatrischer Operation}, doi = {10.25972/OPUS-21174}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211748}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {{\"U}bergewicht, das als Volkskrankheit ein wachsendes globales Problem darstellt, ist mit mehreren folgenreichen Komorbidit{\"a}ten behaftet und die Assoziation der Erkrankung mit nachweisbarer Sch{\"a}digung des Erbguts durch oxidativen Stress ist mittlerweile unangefochten. In der vorliegenden Studie wurden periphere Lymphozyten stark bis morbid adip{\"o}ser Patienten mit Hilfe des Mikrokern-Assays untersucht und es konnte - begleitend zu der zu erwartenden BMI-Abnahme - eine signifikante Reduktion des Genomschadens durch Magenbypass bzw. Sleeve-Gastrektomie 12 Monate postoperativ detektiert werden. Daneben demonstrierte die Analyse zus{\"a}tzlich erhobener Patientendaten, die u. a. N{\"u}chternglucose, HbA1c, Blutdruck und Herzfrequenz sowie ein Blutbild der Patienten (inklusive CRP als Entz{\"u}ndungsmarker, Transaminasen, gGT sowie Lipidprofil) umfasste, eine deutliche Besserung vieler Parameter bis auf teilweise wieder physiologische Normwerte. All diese Ergebnisse st{\"u}tzen die These der metabolischen Wirksamkeit sowohl des Roux-en-Y-Magenbypass als auch der Sleeve- Gastrektomie. Ihre Bedeutung liegt nicht zuletzt in der angenommenen Reduktion des Krebserkrankungsrisikos, da jeweils ein Zusammenhang mit der Adipositas an sich, dem Diabetes und durch oxidativen Stress verursachter DNA-Sch{\"a}digung besteht. Mit Blick auf eine gegenw{\"a}rtig in der Forschung diskutierte potentielle therapeutische Anwendung von Antioxidantien zur Reduktion von Erbgutsch{\"a}digungen, die durch oxidativen Stress zustande kommen, wurden die Substanzen Tricetinidin, Curcumin und Resveratrol im Rahmen des Mikrokerntests an HL60- und NRK-Zellen untersucht, in denen mit der Mischung aus Insulin und Angiotensin II eine gentoxische Wirkung erzielt worden war.}, subject = {Fettsucht}, language = {de} } @article{ChristianSeierDrakopoulosetal.2020, author = {Christian, Gentzsch and Seier, Kerstin and Drakopoulos, Antonios and Jobin, Marie-Lise and Lanoisel{\´e}e, Yann and Koszegi, Zsombor and Maurel, Damien and Sounier, R{\´e}my and H{\"u}bner, Harald and Gmeiner, Peter and Granier, S{\´e}bastien and Calebiro, Davide and Decker, Michael}, title = {Selective and Wash-Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single-Molecule Imaging of μ-Opioid Receptor Dimerization}, series = {Angewandte Chemie International Edition}, volume = {59}, journal = {Angewandte Chemie International Edition}, number = {15}, doi = {10.1002/anie.201912683}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212398}, pages = {5958-5964}, year = {2020}, abstract = {μ-Opioid receptors (μ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of μ-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ-OR pharmacology at single-molecule level.}, language = {en} } @phdthesis{Koussemou2020, author = {Kouss{\´e}mou, Y{\´e}wa Bony Marthe}, title = {A\(_{2B}\) adenosine receptor signaling in MDA-MB-231 breast cancer cells: Mechanism of A\(_{2B}\)-mediated reduction of ERK1/2 phosphorylation}, doi = {10.25972/OPUS-20965}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-209655}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Recently, it was shown that MDA-MB-231 breast cancer cells express very high levels of the A2BAR as the sole adenosine receptor subtype, and stimulation of the A2BAR in MDA-MB-231 cells triggers an unusual inhibitory signal on ERK1/2 phosphorylation. The ERK1/2 pathway is reported to be associated with the control of growth, proliferation and differentiation of cells and as such might serve as a promising target for tumor treatment. The present study investigated signaling mechanisms involved in linking A2BAR to ERK1/2 phosphorylation in MDA-MB-231 cells. The A2BAR mediated reduction of ERK1/2 phosphorylation and of proliferation of MDA-MB-231 cell is in good agreement with previous results from (Dubey et al., 2005). These observations provide support to the hypothesis that activation of A2BAR could attenuate the growth of some types of cancer cell and argue against a stimulation of proliferation resulting from the activation of A2BAR as discussed by (Fernandez-Gallardo et al., 2016). AC activation by forskolin has recently been shown to enhance the activity of the chemotherapeutic agent doxorubicin in TNBC cells via a mechanism dependent on the PKA-mediated inhibition of ERK1/2 phosphorylation. Furthermore, forskolin also increased the sensitivity of MDA-MB-231 and MDA-MB-468 triple negative breast cancer cells to 5-fluorouracil and taxol (Illiano et al., 2018), and sustains the evidence of anticancer activity mediated by cAMP/PKA-mediated ERK1/2 inhibition. Similar to these studies, a reduced amount of pERK1/2 was also observed after stimulation of AC with FSK, application of cAMP-AM or inhibition of PDE-4. The inhibition of ERK1/2 phosphorylation was mimicked by UTP and abolished with the PLC inhibitor U73122 or by chelating intracellular Ca2+ with BAPTA-AM. These results point to an important role for both cAMP and Ca2+ signaling in the pathway leading to a decrease in ERK1/2 phosphorylation. This study encourages the idea that A2BAR could be used as target in cancer therapy. But A2BAR did not only stimulate signaling cascades associated with cell survival and proliferation reduction, but also key phases relevant in angiogenesis like Ca2+ mobilization (Kohn et al., 1995). Whereas the potency toward AC and Ca2+ are similar for the diverse agonists, the potency to promote ERK1/2 reduction is much higher. Interestingly, the proliferation of MDA-MB-231 cells is inhibited by low nanomolar agonist concentration which is inactive in Ca2+ mobilization. This means that it is certainly possible to reduce the proliferation without promoting angiogenesis. LUF6210 is particularly interesting when considering that it preferentially stimulates a reduction in ERK1/2 phosphorylation over Ca2+ and therefore may not promote angiogenesis. LUF6210 is therapeutically appealing as adjuvant in treatment of cancer. Given that stimulation of AC can activate a reduction of ERK1/2 phosphorylation and proliferation in cancer cells, agonist bias toward Gs-AC-PKA-mediated ERK1/2 inhibition represent a potential therapy of various malignancies. The fact that the reduction of ERK1/2 phosphorylation followed by reduced proliferation observed in MDA-MB-231 cells were mediated by the activation of the A2BAR illustrates the importance of this receptor subtype in cancer. A2BARs must be considered as a key factor in cancer treatment and deserve attention for the development of new therapeutic strategies.}, subject = {Adenosinrezeptor}, language = {en} } @article{DekantBridges2016, author = {Dekant, Wolfgang and Bridges, James}, title = {Assessment of reproductive and developmental effects of DINP, DnHP and DCHP using quantitative weight of evidence}, series = {Regulatory Toxicology and Pharmacology}, volume = {81}, journal = {Regulatory Toxicology and Pharmacology}, doi = {10.1016/j.yrtph.2016.09.032}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186750}, pages = {397-406}, year = {2016}, abstract = {Quantitative weight of evidence (QWoE) methodology utilizes detailed scoring sheets to assess the quality/reliability of each publication on toxicity of a chemical and gives numerical scores for quality and observed toxicity. This QWoE-methodology was applied to the reproductive toxicity data on diisononylphthalate (DINP), di-n-hexylphthalate (DnHP), and dicyclohexylphthalate (DCHP) to determine if the scientific evidence for adverse effects meets the requirements for classification as reproductive toxicants. The scores for DINP were compared to those when applying the methodology DCHP and DnHP that have harmonized classifications. Based on the quality/reliability scores, application of the QWoE shows that the three databases are of similar quality; but effect scores differ widely. Application of QWoE to DINP studies resulted in an overall score well below the benchmark required to trigger classification. For DCHP, the QWoE also results in low scores. The high scores from the application of the QWoE methodology to the toxicological data for DnHP represent clear evidence for adverse effects and justify a classification of DnHP as category 1B for both development and fertility. The conclusions on classification based on the QWoE are well supported using a narrative assessment of consistency and biological plausibility.}, language = {en} } @article{ProppingLorenzMicheletal.2016, author = {Propping, Stefan and Lorenz, Kristina and Michel, Martin C. and Wirth, Manfred P. and Ravens, Ursula}, title = {beta-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in beta 2-Adrenoceptor Knockout Mice}, series = {Frontiers in Pharmacology}, volume = {7}, journal = {Frontiers in Pharmacology}, number = {118}, doi = {10.3389/fphar.2016.00118}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165245}, year = {2016}, abstract = {Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug's affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation.}, language = {en} } @article{ArimanyNardiMinuesaPastorAngladaetal.2016, author = {Arimany-Nardi, Cristina and Minuesa, Gerard and Pastor-Anglada, Mar{\c{c}}al and Keller, Thorsten and Erkizia, Itziar and Koepsell, Hermann and Martinez-Picado, Javier}, title = {Role of Human Organic Cation Transporter 1 (hOCT1) Polymorphisms in Lamivudine (3TC) Uptake and Drug-Drug Interactions}, series = {Frontiers in Pharmacology}, volume = {7}, journal = {Frontiers in Pharmacology}, number = {175}, doi = {10.3389/fphar.2016.00175}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165236}, year = {2016}, abstract = {Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level.}, language = {en} } @phdthesis{Gruse2020, author = {Gruse, Tamara}, title = {Untersuchung der Rolle der ERK-Dimerisierung bei der ERK1/2- vermittelten Proliferation von Tumorzellen}, doi = {10.25972/OPUS-15984}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159847}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Bei vielen Erkrankungen wie z.B. Herzhypertrophie, Diabetes und Entz{\"u}ndungen spielt die Raf-MEK-ERK-Signalkaskade eine wichtige Rolle. ERK1/2 ist in vielen zellul{\"a}ren Prozessen, u.a. Proliferation, Differenzierung, Wachstum, Hypertrophie und Apoptose involviert. Auch in der Tumorentstehung besitzt dieser MAPK-Signalweg eine signifikante Funktion, da er bei ca. 50\% aller Krebsarten deutlich aktiviert ist. Ziel dieser Arbeit war es, die Rolle einer neu entdeckten Phosphorylierungsstelle an Threonin188 an ERK2 bei der Entstehung und der m{\"o}glichen Therapie von Tumoren zu erarbeiten. Daf{\"u}r wurde ein Myc-ERK2309-357-Peptid verwendet, das 2013 in der Arbeitsgruppe Lorenz entwickelt wurde. Myc-ERK2309-357 zeigte in bisher unver{\"o}ffentlichten Versuchen, dass es direkt an ERK2 bindet, eine Dimerisierung von ERK1/2 hemmen und eine vermehrte Lokalisation von ERK2 im Zellkern verhindern kann. Im Rahmen dieses Projekts konnten wir belegen, dass mit Hilfe des Myc-ERK2309-357-Peptids die Tumorzellproliferation von verschiedenen Krebszelllinien (Caco-2, SCC68, PC/1-1 und PC/13-1) um 60-80\% vermindert werden konnte. Des Weiteren konnten wir zeigen, dass Myc-ERK2309-357 keinen Einfluss auf die Phosphorylierung von ERK1/2 am TEY-Motiv besitzt. Die Aktivierung von ERK1/2 durch die Kinasen MEK1/2 wird somit nicht beeinflusst und die zytosolischen ERK-Funktionen, wie z.B. der anti-apoptotische Effekt, w{\"u}rden somit bestehen bleiben. Außerdem fanden wir heraus, dass Myc-ERK2309-357 im Vergleich zu den MEK-Inhibitoren U0126 und PD98059 und verglichen mit dem EGF-Rezeptor-Antik{\"o}rper Cetuximab die Proliferation signifikant besser hemmt.}, subject = {Dimerisierung}, language = {de} } @article{FathyFawzyHintzscheetal.2019, author = {Fathy, Moustafa and Fawzy, Michael Atef and Hintzsche, Henning and Nikaido, Toshio and Dandekar, Thomas and Othman, Eman M.}, title = {Eugenol exerts apoptotic effect and modulates the sensitivity of HeLa cells to cisplatin and radiation}, series = {Molecules}, volume = {24}, journal = {Molecules}, number = {21}, issn = {1420-3049}, doi = {10.3390/molecules24213979}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193227}, pages = {3979}, year = {2019}, abstract = {Eugenol is a phytochemical present in different plant products, e.g., clove oil. Traditionally, it is used against a number of different disorders and it was suggested to have anticancer activity. In this study, the activity of eugenol was evaluated in a human cervical cancer (HeLa) cell line and cell proliferation was examined after treatment with various concentrations of eugenol and different treatment durations. Cytotoxicity was tested using lactate dehydrogenase (LDH) enzyme leakage. In order to assess eugenol's potential to act synergistically with chemotherapy and radiotherapy, cell survival was calculated after eugenol treatment in combination with cisplatin and X-rays. To elucidate its mechanism of action, caspase-3 activity was analyzed and the expression of various genes and proteins was checked by RT-PCR and western blot analyses. Eugenol clearly decreased the proliferation rate and increased LDH release in a concentration- and time-dependent manner. It showed synergistic effects with cisplatin and X-rays. Eugenol increased caspase-3 activity and the expression of Bax, cytochrome c (Cyt-c), caspase-3, and caspase-9 and decreased the expression of B-cell lymphoma (Bcl)-2, cyclooxygenase-2 (Cox-2), and interleukin-1 beta (IL-1β) indicating that eugenol mainly induced cell death by apoptosis. In conclusion, eugenol showed antiproliferative and cytotoxic effects via apoptosis and also synergism with cisplatin and ionizing radiation in the human cervical cancer cell line.}, language = {en} } @phdthesis{Seier2020, author = {Seier, Kerstin}, title = {Investigation of dynamic processes of prototypical class A GPCRs by single-molecule microscopy}, doi = {10.25972/OPUS-19973}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199739}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {In this work, two projects were pursued. In the first project, I investigated two different subtypes of opioid receptors, which play a key role as target for analgesia. A set of subtype specific fluorescent ligands for μ opioid receptor (MOR) and δ opioid receptor (DOR) was characterised and used to gain insights into the diffusion behaviour of those receptors. It was shown that the novel ligands hold photophysical and pharmacological properties making them suitable for single-molecule microscopy. Applying them to wild-type receptors expressed in living cells revealed that both sub-types possess a heterogeneous diffusion behaviour. Further- more, the fluorescent ligands for the MOR were used to investigate homodomerisation, a highly debated topic. The results reveal that only ≈ 5 \% of the receptors are present as homodimers, and thus the majority is monomeric. G-protein coupled receptors (GPCRs) play a major role as drug targets. Accordingly, understanding the activation process is very important. For a long time GPCRs have been believed to be either active or inactive. In recent years several studies have shown, that the reality is more complex, involving more substates. [1, 2, 3, 4] In this work the α 2A AR was chosen to investigate the activation process on a single-molecule level, thus being able to distinguish also rare or short-lived events that are hidden in ensemble mea- surements. With this aim, the receptor was labelled intracellular with two fluorophores using supported membranes. Thus it was possible to acquire movies showing qualita- tively smFRET events. Unfortunately, the functionality of the used construct could not be demonstrated. To recover the functionality the CLIP-tag in the third intracellular loop was replaced successfully with an amber codon. This stop codon was used to insert an unnatural amino acid. Five different mutants were created and tested and the most promising candidate could be identified. First ensemble FRET measurements indicated that the functionality might be recovered but further improvements would be needed. Overall, I could show that single-molecule microscopy is a versatile tool to investigate the behaviour of typical class A GPCRs. I was able to show that MOR are mostly monomeric under physiological expression levels. Furthermore, I could establish intra- cellular labelling with supported membranes and acquire qualitative smFRET events.}, subject = {PhD thesis pharmacology}, language = {en} } @phdthesis{Maimari2020, author = {Maimari, Theopisti}, title = {The influence of N-terminal peptides of G-protein coupled receptor kinase (GRK) 2, 3 and 5 on β-adrenergic signaling}, doi = {10.25972/OPUS-19932}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199322}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {G protein coupled receptor kinases (GRK) phosphorylate and thereby desensitize G protein coupled receptors (GPCR) including β-adrenergic receptors (βAR), which are critical regulators of cardiac function. We identified the Raf kinase inhibitor protein (RKIP) as an endogenous inhibitor of GRK2 that leads to increased cardiac contractility via βAR activation. RKIP binds to the N-terminus (aa1-185) of GRK2, which is important for the GRK2/receptor interaction. Thereby it interferes with the GRK2/receptor interaction without interference with cytosolic GRK2 target activation. In this project, the RKIP/GRK interface was investigated to develop strategies that simulate the effects of RKIP on βAR. RKIP binding to different isoforms of GRK expressed in the heart was analyzed by protein interaction assays using full-length and N-termini of GRK2, GRK3 and GRK5: 1-53, 54-185 and 1-185. Co-immunoprecipitation (Co-IPs) and pull-down assays revealed that RKIP binds to the peptides of GRK2 and GRK3 but not to the ones of GRK5, which suggests the existence of several binding sites of RKIP within the N-termini of GRK2 and GRK3. To analyze whether the peptides of GRK2 and GRK3 are able to simulate the RKIP mediated interference of the GRK2/receptor interaction, we analyzed the β2-AR phosphorylation in the absence and presence of the peptides. Interestingly, N-termini (aa1-185) of GRK2 and GRK3 reduced β2AR phosphorylation to a comparable extent as RKIP. In line with reduced receptor phosphorylation, the peptides also reduced isoproterenol-stimulated receptor internalization as shown by [3H] CGP-12177 radioligand binding assay and fluorescence microscopy compared to control cells. Subsequently, these peptides increased downstream signaling of β2AR, i.e. the phosphorylation of the PKA substrate phosducin. In an attempt to elucidate the mechanism behind the observed effects, Co-IPs were performed in order to investigate whether the peptides bind directly to the β2-AR and block its phosphorylation by GRK2. Indeed, GRK2 1-185 and GRK3 1-185 could bind the receptor, suggesting that this way GRK2 is prevented from inhibiting the receptor. To investigate the physiological effect of GRK2 1-185, GRK3 1-185 and GRK5 1-185, their effect on neonatal mouse cardiomyocyte contractility and hypertrophy was analyzed. After long-term isoproterenol stimulation, in the presence of GRK2 1 185 and GRK3 1-185 the cross-sectional area of the cardiomyocytes showed no significant increase in comparison to the unstimulated control cells. In addition, upon isoproterenol stimulation, GRK2 1-185 and GRK3 1-185 increased the beat rate in cardiomyocytes, mimicking RKIP while the base impedance, an indicator of viability, remained stable. The N-termini (1-185) of GRK2 and GRK3 simulated RKIP's function and had a significant influence on β2AR phosphorylation, on its downstream signaling and internalization, could bind β2-AR, increased beat rate and did not significantly induce hypertrophy, suggesting that they may serve as a model for the generation of new and more specific targeting strategies for GRK mediated receptor regulation.}, language = {en} } @phdthesis{Huemmert2020, author = {H{\"u}mmert, Martin W.}, title = {Untersuchung einer monomeren Mutante der extrazellul{\"a}r regulierten Kinase 2 (ERK2) bei kardialer Hypertrophie}, doi = {10.25972/OPUS-15564}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155644}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Die Raf-MEK-ERK1/2-Kaskade spielt eine wichtige Rolle in der Vermittlung von kardialer Hypertrophie und Zell{\"u}berleben. Durch unsere Arbeitsgruppe konnte im Vorfeld gezeigt werden, dass die Dimerisierung von ERK2 eine Voraussetzung f{\"u}r dessen Autophosphorylierung an Thr188 darstellt, welche wiederum f{\"u}r die {\"U}bermittlung der hypertrophen Effekten von ERK1/2 erforderlich ist. Im Rahmen dieser Arbeit wurde daraus abgeleitet die Fragestellung untersucht, ob mit Verhinderung der ERK2-Dimerisierung eine n{\"u}tzliche Strategie zur Inhibition von Hypertrophie vorliegt und welchen Einfluss diese auf das Zell{\"u}berleben hat. Die Auswirkungen der Dimerisierungsdefizienz von ERK2 wurden in neonatalen Kardiomyozyten der Ratte und in transgenen M{\"a}usen mithilfe einer ERK2-Mutante untersucht, der einige Aminos{\"a}uren in der ERK-ERK-Interaktionsfl{\"a}che fehlen und daher keine Dimere bilden kann (ERK2Δ174-177). Eine {\"U}berexpression von ERK2Δ174-177 in neonatalen Kardiomyozyten verringerte signifikant die Antwort auf hypertrophe Stimuli (Phenylephrin, Endothelin 1). Im Anschluss daran wurden die Effekte der Dimerisierungsdefizienz von ERK2 in vivo an transgenen M{\"a}usen mit kardialer {\"U}berexpression von ERK2Δ174-177 erforscht. Diese M{\"a}use zeigten unter basalen Bedingungen keine Unterschiede gegen{\"u}ber Wildtyp-M{\"a}usen hinsichtlich Kardiomyozytengr{\"o}ße, Ventrikelwanddicke und kardialer Funktion. Unter chronischer Druckbelastung mittels TAC ließ sich hingegen ein signifikant vermindertes Ausmaß an Hypertrophie im Vergleich zu Wildtyp quantifizieren. Da der ERK1/2-Signalweg auch am {\"U}berleben von Kardiomyozyten beteiligt ist, wurde die Apoptose an histologischen Schnitten von Mausherzen analysiert. Interessanterweise fand sich bei Herzen, die das dimerisierungsdefiziente ERK2-Protein {\"u}berexprimierten, eine mit Wildtyp vergleichbare Anzahl TUNEL-positiver Zellen. Ein {\"a}hnliches Ergebnis konnte bei der Messung des Fibrosegrades an Sirius-Rot gef{\"a}rbten histologischen Schnitten beobachtet werden. Zuletzt wurden die Folgen der ERK2-Dimerisierungsdefizienz auf physiologische Hypertrophie mit einem Laufrad-Versuchsaufbau evaluiert. Transgene ERK2Δ174-177- und Wildtyp-M{\"a}use zeigten unter diesem physiologischen Stimulus keine Unterschiede im Hinblick auf die Zunahme an kardialer Hypertrophie. Da die Dimerisierungsdefizienz von ERK2 zu einer reduzierten pathologischen Hypertrophie, ohne negative Auswirkungen auf ERK1/2-vermittelte anti-apoptotische Effekte noch auf kardiale Funktion oder physiologische Hypertrophieprozesse f{\"u}hrt, stellt die Hemmung der ERK-Dimerisierung ein attraktives Ziel zur Therapie pathologischer Hypertrophie sowie potentiell auch anderer auf den ERK1/2-Signalweg basierenden Krankheiten dar.}, subject = {ERK2d4}, language = {de} } @article{LohseBockMaiellaroetal.2017, author = {Lohse, Christian and Bock, Andreas and Maiellaro, Isabella and Hannawacker, Annette and Schad, Lothar R. and Lohse, Martin J. and Bauer, Wolfgang R.}, title = {Experimental and mathematical analysis of cAMP nanodomains}, series = {PLoS ONE}, volume = {12}, journal = {PLoS ONE}, number = {4}, doi = {10.1371/journal.pone.0174856}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170972}, pages = {e0174856}, year = {2017}, abstract = {In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells.}, language = {en} } @article{ScholzGuanNieberleretal.2017, author = {Scholz, Nicole and Guan, Chonglin and Nieberler, Matthias and Grotmeyer, Alexander and Maiellaro, Isabella and Gao, Shiqiang and Beck, Sebastian and Pawlak, Matthias and Sauer, Markus and Asan, Esther and Rothemund, Sven and Winkler, Jana and Pr{\"o}mel, Simone and Nagel, Georg and Langenhan, Tobias and Kittel, Robert J}, title = {Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons}, series = {eLife}, volume = {6}, journal = {eLife}, number = {e28360}, doi = {10.7554/eLife.28360}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170520}, year = {2017}, abstract = {Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.}, language = {en} } @article{GodboleLygaLohseetal.2017, author = {Godbole, Amod and Lyga, Sandra and Lohse, Martin J. and Calebiro, Davide}, title = {Internalized TSH receptors en route to the TGN induce local G\(_{S}\)-protein signaling and gene transcription}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {443}, doi = {10.1038/s41467-017-00357-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170375}, year = {2017}, abstract = {A new paradigm of G-protein-coupled receptor (GPCR) signaling at intracellular sites has recently emerged, but the underlying mechanisms and functional consequences are insufficiently understood. Here, we show that upon internalization in thyroid cells, endogenous TSH receptors traffic retrogradely to the trans-Golgi network (TGN) and activate endogenous Gs-proteins in the retromer-coated compartment that brings them to the TGN. Receptor internalization is associated with a late cAMP/protein kinase A (PKA) response at the Golgi/TGN. Blocking receptor internalization, inhibiting PKA II/interfering with its Golgi/TGN localization, silencing retromer or disrupting Golgi/TGN organization all impair efficient TSH-dependent cAMP response element binding protein (CREB) phosphorylation. These results suggest that retrograde trafficking to the TGN induces local G\(_{S}\)-protein activation and cAMP/PKA signaling at a critical position near the nucleus, which appears required for efficient CREB phosphorylation and gene transcription. This provides a new mechanism to explain the functional consequences of GPCR signaling at intracellular sites and reveals a critical role for the TGN in GPCR signaling.}, language = {en} } @phdthesis{Zundler2019, author = {Zundler, Matthias}, title = {Einfluss der Phosphoglykolat-Phosphatase auf den Metabolismus von Signal-, Membran- und Speicherlipiden in murinen Embryonen und Lymphozyten}, doi = {10.25972/OPUS-16844}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168442}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Die Phosphoglykolat-Phosphatase PGP (fr{\"u}her auch als AUM bezeichnet) wurde in unserem Labor als Mitglied der HAD-Typ-Phosphatasen identifiziert. Die genetische Inaktivierung des Enzyms im gesamten Mausorganismus f{\"u}hrt ab E8.5 zu einer Wachstumsverz{\"o}gerung muriner Embryonen und bis E12.5 schließlich zu deren Tod. Im Gegensatz dazu sind M{\"a}use mit einer PGP-Inaktivierung in h{\"a}matopoetischen Zellen und im Endothel lebensf{\"a}hig und ph{\"a}notypisch unauff{\"a}llig. Neue Erkenntnisse schreiben dem Enzym neben einer Aktivit{\"a}t gegen{\"u}ber Phosphoglykolat auch Aktivit{\"a}ten gegen{\"u}ber Glycerin-3-phosphat (G3P), P-Erythronat und P-Lactat zu. Da diese Phosphatase-Aktivit{\"a}ten Auswirkungen auf den Lipidstoffwechsel nahelegen, wurde in der vorliegenden Arbeit mittels massenspektrometrischer Methoden der Einfluss der Phosphoglykolat-Phosphatase auf den Metabolismus von Signal-, Membran- und Speicherlipiden in murinen Embryonen und Lymphozyten untersucht. Nach Inaktivierung der PGP im gesamten Organismus wurden in E8.5-Embryonen erh{\"o}hte Diacylglycerin (DG)-, Triacylglycerin (TG)- und Sphingomyelin (SM)-Spiegel gemessen, w{\"a}hrend niedrigere Phosphatidylcholin (PC)-Level vorlagen. In PGP-inaktivierten Lymphozyten waren G3P-, DG-, TG-, PC- und SM-Level nicht ver{\"a}ndert. Daf{\"u}r kam es zu signifikanten Erh{\"o}hungen der Phosphatidylglycerol (PG*)- und Cardiolipin (CL)-Spiegel. Zusammenfassend konnte gezeigt werden, dass die PGP in unterschiedlichen Geweben differenzielle Effekte auf die Spiegel verschiedener Lipide hat. Dies deckt neue Funktionen der PGP f{\"u}r die Regulation des Lipidmetabolismus auf. Die vorliegende Arbeit stellt somit die Grundlage f{\"u}r weitere Untersuchungen {\"u}ber die genauen Ursachen und Folgen dieser Regulation dar und l{\"a}sst auf eine wichtige Rolle der PGP als metabolische Phosphatase im Organismus schließen.}, subject = {Phosphoglykolatphosphatase}, language = {de} } @phdthesis{Link2019, author = {Link, Samuel}, title = {Aldosteron-vermittelte oxidative Nierensch{\"a}digung - Einfluss der antioxidativen Abwehr}, doi = {10.25972/OPUS-18603}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186037}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Patienten mit erh{\"o}hten Aldosteronspiegeln zeigen eine gesteigerte Inzidenz f{\"u}r Malignome, insbesondere von Nierenzellkarzinomen. Das Ziel dieser Arbeit war es, die Aldosteron-vermittelte oxidative Nierensch{\"a}digung n{\"a}her zu analysieren sowie die auf Zellebene gezeigte Beeinflussung der antioxidativen Schutzmechanismen im lebenden Organismus nachzuweisen und m{\"o}gliche therapeutische Ansatzpunkte zu identifizieren. Dazu wurde ein Interventions-versuch {\"u}ber 28 Tage durchgef{\"u}hrt. Neben einer Aldosterongabe wurden folgende Interventionen verwendet: Spironolacton zur Blockade des Mineralkortikoid-Rezeptors (MR), Apocynin als Hemmstoff der NADPH-Oxidasen (Nox), L-NAME zur Blockade der NO-Synthasen (NOS), PDTC, einen Hemmstoff des Transkriptionsfaktors NF-kB sowie Sulforaphan, ein nat{\"u}rlicher Nrf2-Induktor. Eine weitere Gruppe erhielt Sulforaphan ohne additive Aldosterongabe. Die Nierensch{\"a}den wurden mittels histopathologischer Sch{\"a}digungsscores und der Anzahl an DNA-Doppelstrangbr{\"u}che analysiert. Die Beeinflussung der antioxidativen Abwehr wurde durch die Aktivierung des Transkriptionsfaktors Nrf2 und durch die Quantifizierung antioxidativer Enzyme bestimmt. Im Nierengewebe f{\"u}hrte Aldosteron zu einer Zunahme von oxidativem Stress. Histologisch zeigte sich ein Anstieg von glomerul{\"a}ren Sch{\"a}den. Auch kam es zu einer deutlichen Zunahme von Doppelstrangbr{\"u}chen der DNA. Des Weiteren konnten wir zeigen, dass Aldosteron auch in vivo zu einer Zunahme der Nrf2-Aktivit{\"a}t f{\"u}hrte, wobei sich dies auf Proteinebene nicht in einer (dauerhaften) Synthesesteigerung von antioxidativen Enzymen wiederspiegelte und keinen ausreichenden Schutz des Nierengewebes bot. F{\"u}r die Interventionsgruppen konnte keine signifikante Auswirkung auf das Vorliegen von oxidativem Stress gezeigt werden. Dies k{\"o}nnte an der Versuchsdauer bzw. an der gew{\"a}hlten Nachweismethode gelegen haben. Nichtsdestotrotz zeigte die Blockade der Nox durch Apocynin bzw. der NOS durch L-NAME eine effektive Reduktion der histologischen und genomischen Sch{\"a}den. Die L-NAME-Gruppe wies dabei die h{\"o}chsten Blutdruckwerte auf, diese waren auch zur Aldosterongruppe signifikant gesteigert. Die beobachteten Effekte waren folglich nicht durch den in der Aldosterongruppe erfolgten Blutdruckanstieg, sondern vielmehr durch den Anstieg von oxidativem Stress zu erkl{\"a}ren. Ebenfalls blieb die Nrf2-Aktivit{\"a}t bei der Gabe von Apocynin und L-NAME weitgehend auf Kontrollniveau, was daf{\"u}rspricht, dass der in der Aldosterongruppe messbare Nrf2-Anstieg am ehesten als Reaktion auf chronisch erh{\"o}hten oxidativen Stress erfolgte, welcher durch die Interventionen ausblieb. Die Blockade von NF-κB mittels PDTC f{\"u}hrte zu vergleichbaren Effekten wie Apocynin und L-NAME. Das deutet darauf hin, dass Aldosteron {\"u}ber die Aktivierung von NF-κB die vermehrte Synthese von pro-oxidativen Enzymen wie Nox und NOS anregt. Die Gabe von Spironolacton hatte den st{\"a}rksten protektiven Effekt, sowohl auf histologische Ver{\"a}nderungen als auch auf das Entstehen von DNA-Doppelstrangbr{\"u}chen, wobei die Nrf2-Aktivit{\"a}t in dieser Gruppe ebenfalls auf Kontrollniveau blieb. Die Aldosteroneffekte wurden folglich {\"u}ber den MR vermittelt. Eine additive Nrf2-Induktion mittels Sulforaphan konnte auch keinen (dauerhaften) Effekt auf die Synthese antioxidativer Enzyme zeigen. Dennoch zeigte diese Gruppe einen {\"a}hnlich effektiven Schutz vor den oxidativen Nierensch{\"a}den wie die Gabe von Spironolacton. Vieles spricht daf{\"u}r, dass die Wirkung von Sulforaphan dabei {\"u}ber seine Wirkung als direktes Antioxidans bzw. Radikalf{\"a}nger und nicht {\"u}ber den Nrf2-Weg zu erkl{\"a}ren ist. Aldosteron f{\"u}hrt in der Niere {\"u}ber oxidativen Stress zu glomerul{\"a}rer Fibrose und DNA-Sch{\"a}den. Das k{\"o}nnte eine Erkl{\"a}rung f{\"u}r die gesteigerte Inzidenz von Nierenzellkarzinomen in Patienten mit erh{\"o}hten Aldosteronspiegeln darstellen. Unsere Ergebnisse sprechen daf{\"u}r, dass Aldosteron {\"u}ber eine Signalkaskade {\"u}ber den MR zu einer Aktivierung von Nox und NOS f{\"u}hrt. Der Aktivierung des Transkriptionsfaktors NF-κB scheint dabei durch die Synthese pro-oxidativer Enzyme eine Art Verst{\"a}rker-Effekt zuzukommen. Als Reaktion auf den durch Aldosteron gesteigerten oxidativen Stress kommt es zu einer Aktivierung des antioxidativen Transkriptionsfaktors Nrf2, jedoch ohne dass dies zu einem ausreichenden Schutz des Nierengewebes f{\"u}hrt. M{\"o}gliche therapeutische Ansatzpunkte f{\"u}r einen Schutz vor den durch Aldosteron vermittelten oxidativen Nierensch{\"a}den scheinen eher innerhalb der Aldosteronsignalkaskade, insbesondere in der Blockade des MR, als in der antioxidativen Abwehr zu liegen.}, subject = {Aldosteron}, language = {de} } @article{SchuppStopperHeidland2016, author = {Schupp, Nicole and Stopper, Helga and Heidland, August}, title = {DNA Damage in Chronic Kidney Disease: Evaluation of Clinical Biomarkers}, series = {Oxidative Medicine and Cellular Longevity}, volume = {2016}, journal = {Oxidative Medicine and Cellular Longevity}, number = {3592042}, doi = {10.1155/2016/3592042}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-166569}, year = {2016}, abstract = {Patients with chronic kidney disease (CKD) exhibit an increased cancer risk compared to a healthy control population. To be able to estimate the cancer risk of the patients and to assess the impact of interventional therapies thereon, it is of particular interest to measure the patients' burden of genomic damage. Chromosomal abnormalities, reduced DNA repair, and DNA lesions were found indeed in cells of patients with CKD. Biomarkers for DNA damage measurable in easily accessible cells like peripheral blood lymphocytes are chromosomal aberrations, structural DNA lesions, and oxidatively modified DNA bases. In this review the most common methods quantifying the three parameters mentioned above, the cytokinesis-block micronucleus assay, the comet assay, and the quantification of 8-oxo-7,8-dihydro-2′-deoxyguanosine, are evaluated concerning the feasibility of the analysis and regarding the marker's potential to predict clinical outcomes.}, language = {en} } @phdthesis{Wilde2019, author = {Wilde, Sabrina}, title = {Einsatz von mechanistischen Biomarkern zur Charakterisierung und Bewertung von \(in\) \(vitro\) Genotoxinen}, doi = {10.25972/OPUS-18278}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182782}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Die verf{\"u}gbaren in vitro Genotoxizit{\"a}tstests weisen hinsichtlich ihrer Spezifit{\"a}t und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschr{\"a}nkungen auf. Um diese M{\"a}ngel zu {\"u}berwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Pr{\"u}fung auf Genotoxizit{\"a}t in der Arzneimittelentwicklung beitragen. 1. Etablierung und Bewertung einer neuen in vitro Genotoxizit{\"a}tsmethode (MultiFlow Methode) Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbr{\"u}che), phosphorylierten H3 (S10) (mitotische Zellen), nukle{\"a}ren Protein p53 (Genotoxizit{\"a}t) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und erg{\"a}nzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre F{\"a}higkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen" gegen{\"u}ber der „alten" Methode f{\"u}hrte zu einer verbesserten Sensitivit{\"a}t von 95\% gegen{\"u}ber 90\%, Spezifit{\"a}t von 90\% gegen{\"u}ber 72\% und einer MoA-Klassifizierungsrate von 85\% gegen{\"u}ber 45\% (Aneugen vs. Klastogen). 2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen Die Analyse 67 ausgew{\"a}hlter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen k{\"o}nnen. Die Kombination der h{\"o}chstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 erm{\"o}glichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivit{\"a}t, Spezifit{\"a}t und Pr{\"a}diktivit{\"a}t von 86\%, 83\% und 85\%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizit{\"a}t mit TP53I3, Klastogenit{\"a}t mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann. Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen f{\"u}r BubR1 (S670) und pH3 (S28) mit Aneugenit{\"a}t, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenit{\"a}t, p53 (K373) mit Genotoxizit{\"a}t und Nrf2 (S40) mit oxidativem Stress identifiziert. Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinver{\"a}nderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivit{\"a}ten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden k{\"o}nnen und daher eine bessere Risikobewertung von Wirkstoffkandidaten erm{\"o}glichen.}, subject = {Genotoxizit{\"a}t}, language = {de} } @article{BankogluArnoldHeringetal.2018, author = {Bankoglu, Ezgi Eyluel and Arnold, Charlotte and Hering, Ilona and Hankir, Mohammed and Seyfried, Florian and Stopper, Helga}, title = {Decreased chromosomal damage in lymphocytes of obese patients after bariatric surgery}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, number = {11195}, doi = {10.1038/s41598-018-29581-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177090}, year = {2018}, abstract = {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.}, language = {en} } @article{HintzscheMontagStopper2018, author = {Hintzsche, Henning and Montag, Gracia and Stopper, Helga}, title = {Induction of micronuclei by four cytostatic compounds in human hematopoietic stem cells and human lymphoblastoid TK6 cells}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, number = {3371}, doi = {10.1038/s41598-018-21680-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176210}, year = {2018}, abstract = {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.}, language = {en} } @phdthesis{Berisha2019, author = {Berisha, Filip}, title = {Molekulare Wirkmechanismen von Sulfonylharnstoffen: Direkte Epac-Aktivierung oder Hemmung der Phosphodiesterasen}, doi = {10.25972/OPUS-17653}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176535}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Diabetes mellitus ist die h{\"a}ufigste Stoffwechselerkrankung in Deutschland. Sulfonylharnstoffe (SH) stellen die {\"a}lteste und eine sehr prominente Gruppe in der oralen Therapie des Diabetes mellitus Typ II dar, die eine verst{\"a}rkte Insulinfreisetzung vorrangig durch die Hemmung eines ATP-sensitiven Kaliumkanals (K+ATPKanal) erreichen. Daneben konnten weitere Proteine identifiziert werden, die mit SH interagieren und zu deren Effekten beitragen. W{\"a}hrend bereits in fr{\"u}hen Arbeiten gezeigt werden konnte, dass SH Vertreter der Phosphodiesterasen (PDE)Familie in ihrer Funktion behindern k{\"o}nnen, wurde k{\"u}rzlich Epac2 (exchange protein directly activated by cAMP 2) als weiteres Zielprotein f{\"u}r SH angef{\"u}hrt. Insbesondere die F{\"a}higkeit von SH, direkt an Epac2 zu binden, wird in der Literatur kontrovers diskutiert und eine indirekte Aktivierung durch eine PDE-Hemmung und einen erh{\"o}hten cAMP-Spiegel als Mechanismus vermutet. Zur weiteren Untersuchung wurden in dieser Arbeit FRET-basierte Biosensoren verwendet, um die Wirkung von SH auf Epac und PDEs n{\"a}her zu untersuchen. Dabei konnte sowohl in einem photometrischen Ansatz als auch in lebenden Zellen, die einen Epac2-basierten Sensor enthalten, gezeigt werden, dass eine Aktivierung durch SH stattfindet. Da sowohl Epac2-camps, der von allen hier verwendeten Sensoren mit der h{\"o}chsten Sensitivit{\"a}t f{\"u}r cAMP, als auch CFP-Epac1δDEPYFP nicht auf SH reagieren, ist diese Aktivierung selektiv f{\"u}r die Isoform Epac2 und wird vorrangig nicht durch eine PDE-Hemmung verursacht. Die Verwendung weiterer Sensoren mit verschiedenen Varianten von Epac2 (verl{\"a}ngerte Version von Epac2-camps) zeigen mit zunehmender L{\"a}nge {\"u}ber die cAMP-Bindedom{\"a}ne hinaus eine beginnende Reaktion im Sinne einer instabilen FRET-Kurve (Epac2camps long) bzw. eine deutliche Aktivierung durch den SH (Epac2-camps superlong), wodurch eine direkte Aktivierung best{\"a}tigt wird, und suggerieren eine Bindestelle f{\"u}r SH, die sich von denen von cAMP unterschiedet und weiter eingeengt werden konnte (im n{\"a}heren Bereich von Q454 bzw. E460). Obwohl hierdurch eine direkte Aktivierung gezeigt werden konnte, ist die grunds{\"a}tzliche F{\"a}higkeit der SH, PDE zu beeinflussen, keineswegs gekl{\"a}rt. Daher wurden weitere Sensoren konstruiert bzw. verwendet, die basierend auf Epac1-camps und Epac2-camps verschiedene PDEs enthalten. Dabei konnte durch die Zugabe von SH eine deutliche Aktivierung des jeweiligen Sensors und somit eine PDEHemmung nachgewiesen werden. Dies konnte sowohl f{\"u}r PDE4A als auch f{\"u}r die in Inselzellen {\"u}berwiegend vorkommende PDE3B gezeigt werden. Dadurch ergeben sich einige (klinisch relevante) Implikationen. Zum einen stellt neben der direkten Epac-Aktivierung auch die direkte Hemmung der PDE einen wichtigen Mechanismus f{\"u}r die Sekretion von Insulin dar. Außerdem sind bei PDEHemmung und direkter Epac-Aktivierung außerhalb der Inselzellen auch Nebenwirkungen in anderen Organen zu erwarten wie z.B. die Entstehung lebensgef{\"a}hrlicher Rhythmusst{\"o}rungen in Herzmuskelzellen.}, subject = {Sulfonylharnstoffe}, language = {de} } @phdthesis{Segerer2019, author = {Segerer, Gabriela}, title = {Characterization of cell biological and physiological functions of the phosphoglycolate phosphatase AUM}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123847}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer. Still, most of the mammalian HAD phosphatases remain functionally uncharacterized. This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos. To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation. The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells. These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling. Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase. To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions. Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20\% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under hypoxic (~1\% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3'-phosphate (GADP). Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism. Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.}, subject = {Phosphoglykolatphosphatase}, language = {en} } @phdthesis{Curtaz2019, author = {Curtaz, Carolin Julia}, title = {\(In\) \(vitro\) Analysen der Wechselwirkung erh{\"o}hter Temperatur mit Zytostatika am Beispiel von Cisplatin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174543}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Neben der Chemotherapie ist heutzutage auch die Hyperthermie-Behandlung eine wichtige S{\"a}ule der antitumor{\"o}sen Therapie. W{\"a}hrend der sogenannten HIPEC Therapie (Hypertherme intraperitoneale Chemoperfusion) werden die beiden Arten der Therapieformen kombiniert und in der klinischen Praxis erfolgreich angewendet. Genauere Kenntnisse {\"u}ber die zu Grunde liegenden toxikologischen in-vitro Mechanismen k{\"o}nnten zu neuen M{\"o}glichkeiten in der klinischen Anwendung f{\"u}hren. In unserer Arbeit untersuchten wir verschiedenen Tumorzelllinien (HT29,CaCo-2,HCT116,HaCaT) in Kombination mit Cisplatin und Hyperthermie mit verschiedenen Methoden, wie zum Beispiel Mikrokerntest, Comet-Assay, Durchflusszytometrie, Vitalit{\"a}tstest und mikroskopischen Analysen. Unsere Ergebnisse f{\"u}hrten uns zu der Hypothese, dass Hyperthermie alleine zu einer sogenannte mitotic catastrophe f{\"u}hrt und zum Absterben der Tumorzellen. Im Gegensatz dazu zeigten Tumorzellen, welche mit Cisplatin alleine oder auch in Kombination mit Hyperthermie nicht in die Mitose eintreten und daher nicht durch Apoptose in den Zelltod gehen.}, subject = {Hyperthermie}, language = {de} } @phdthesis{Kauk2018, author = {Kauk, Michael}, title = {Investigating the Molecular Mechanism of Receptor Activation at Muscarinic Receptors by Means of Pathway-Specific Dualsteric Ligands and Partial Agonists}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173729}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {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.}, subject = {G-Protein gekoppelte Rezeptoren}, language = {en} } @article{VoglLutzSchoenfelderetal.2015, author = {Vogl, Silvia and Lutz, Roman W. and Sch{\"o}nfelder, Gilbert and Lutz, Werner K.}, title = {CYP2C9 genotype vs. metabolic phenotype for individual drug dosing - a correlation analysis using flurbiprofen as probe drug}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0120403}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148783}, pages = {e0120403}, year = {2015}, abstract = {Currently, genotyping of patients for polymorphic enzymes responsible for metabolic elimination is considered a possibility to adjust drug dose levels. For a patient to profit from this procedure, the interindividual differences in drug metabolism within one genotype should be smaller than those between different genotypes. We studied a large cohort of healthy young adults (283 subjects), correlating their CYP2C9 genotype to a simple phenotyping metric, using flurbiprofen as probe drug. Genotyping was conducted for CYP2C9*1, *2, *3. The urinary metabolic ratio MR (concentration of CYP2C9-dependent metabolite divided by concentration of flurbiprofen) determined two hours after flurbiprofen (8.75 mg) administration served as phenotyping metric. Linear statistical models correlating genotype and phenotype provided highly significant allele-specific MR estimates of 0.596 for the wild type allele CYP2C9*1, 0.405 for CYP2C9*2 (68 \% of wild type), and 0.113 for CYP2C9*3 (19 \% of wild type). If these estimates were used for flurbiprofen dose adjustment, taking 100 \% for genotype *1/*1, an average reduction to 84 \%, 60 \%, 68 \%, 43 \%, and 19\% would result for genotype *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. Due to the large individual variation within genotypes with coefficients of variation >= 20\% and supposing the normal distribution, one in three individuals would be out of the average optimum dose by more than 20 \%, one in 20 would be 40\% off. Whether this problem also applies to other CYPs and other drugs has to be investigated case by case. Our data for the given example, however, puts the benefit of individual drug dosing to question, if it is exclusively based on genotype.}, language = {en} } @article{HoffmannSchmidtKeimetal.2011, author = {Hoffmann, Linda S and Schmidt, Peter M and Keim, Yvonne and Hoffmann, Carsten and Schmidt, Harald H H W and Stasch, Johannes-Peter}, title = {Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells}, series = {PLOS ONE}, volume = {6}, journal = {PLOS ONE}, number = {8}, doi = {10.1371/journal.pone.0023596}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139631}, pages = {e23596}, year = {2011}, abstract = {In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e. g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.}, language = {en} } @article{WernerWakabayashiBaueretal.2018, author = {Werner, Rudolf and Wakabayashi, Hiroshi and Bauer, Jochen and Sch{\"u}tz, Claudia and Zechmeister, Christina and Hayakawa, Nobuyuki and Javadi, Mehrbod S. and Lapa, Constantin and Jahns, Roland and Erg{\"u}n, S{\"u}leyman and Jahns, Valerie and Higuchi, Takahiro}, title = {Longitudinal \(^{18}\)F-FDG PET imaging in a Rat Model of Autoimmune Myocarditis}, series = {European Heart Journal Cardiovascular Imaging}, journal = {European Heart Journal Cardiovascular Imaging}, issn = {2047-2404}, doi = {10.1093/ehjci/jey119}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165601}, pages = {1-8}, year = {2018}, abstract = {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.}, subject = {Myokarditis}, language = {en} } @phdthesis{Raab2018, author = {Raab, Annette}, title = {The role of Rgs2 in animal models of affective disorders}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152550}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Anxiety and depressive disorders result from a complex interplay of genetic and environmental factors and are common mutual comorbidities. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety as well as rodent depression. Rgs2 negatively regulates G protein-coupled receptor (GPCR) signaling by acting as a GTPase accelerating protein towards the Gα subunit. The present study investigates, whether mice with a homozygous Rgs2 deletion (Rgs2-/-) show behavioral alterations as well as an increased susceptibility to stressful life events related to human anxiety and depressive disorders and tries to elucidate molecular underlying's of these changes. To this end, Rgs2-/- mice were characterized in an aversive-associative learning paradigm to evaluate learned fear as a model for the etiology of human anxiety disorders. Spatial learning and reward motivated spatial learning were evaluated to control for learning in non-aversive paradigms. Rgs2 deletion enhanced learning in all three paradigms, rendering increased learning upon deletion of Rgs2 not specific for aversive learning. These data support reports indicating increased long-term potentiation in Rgs2-/- mice and may predict treatment response to conditioning based behavior therapy in patients with polymorphisms associated with reduced RGS2 expression. Previous reports of increased innate anxiety were corroborated in three tests based on the approach-avoidance conflict. Interestingly, Rgs2-/- mice showed novelty-induced hypo-locomotion suggesting neophobia, which may translate to the clinical picture of agoraphobia in humans and reduced RGS2 expression in humans was associated with a higher incidence of panic disorder with agoraphobia. Depression-like behavior was more distinctive in female Rgs2-/- mice. Stress resilience, tested in an acute and a chronic stress paradigm, was also more distinctive in female Rgs2-/- mice, suggesting Rgs2 to contribute to sex specific effects of anxiety disorders and depression. Rgs2 deletion was associated with GPCR expression changes of the adrenergic, serotonergic, dopaminergic and neuropeptide Y systems in the brain and heart as well as reduced monoaminergic neurotransmitter levels. Furthermore, the expression of two stress-related microRNAs was increased upon Rgs2 deletion. The aversive-associative learning paradigm induced a dynamic Rgs2 expression change. The observed molecular changes may contribute to the anxious and depressed phenotype as well as promote altered stress reactivity, while reflecting an alter basal stress level and a disrupted sympathetic tone. Dynamic Rgs2 expression may mediate changes in GPCR signaling duration during memory formation. Taken together, Rgs2 deletion promotes increased anxiety-like and depression-like behavior, altered stress reactivity as well as increased cognitive function.}, subject = {Angst}, language = {en} } @phdthesis{MontaggebKukielka2018, author = {Montag [geb. Kukielka], Gracia Anna}, title = {Rolle des Differenzierungszustandes f{\"u}r die Empfindlichkeit von S{\"a}ugerzellen gegen{\"u}ber genotoxischen Agenzien}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164670}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {In der vorliegenden Arbeit wurden die Einfl{\"u}sse verschiedener genotoxischer Substanzen auf S{\"a}ugertierzellen untersucht. Da ein Organismus der Ontogenese unterliegt und sich Zellen aus Stamm- und Vorl{\"a}uferzellen entwickelt, gilt es diese urspr{\"u}nglichen Zellen vor {\"a}ußeren Einfl{\"u}ssen zu sch{\"u}tzen. Da bisher kaum Untersuchungen von Zellen in verschiedenen Differenzierungsstadien durchgef{\"u}hrt wurden, wurden unter Verwendung vieler unterschiedlicher biologischer Endpunkte Effekte auf die Vitalit{\"a}t, Proliferation, Mitose und Apoptose dieser Zellen untersucht. Zudem erfolgte eine Interpretation der Ausbildung von Mikrokernen, Entstehung von DNS-Sch{\"a}den und der zugrundeliegenden Reparaturmechanismen. So konnte mit Hilfe der Untersuchungen der h{\"a}matopoetischen Stammzellen und der TK6-Zellen postuliert werden, dass h{\"a}matopoetische Stammzellen weitestgehend weniger empfindlich gegen{\"u}ber Zytostatika (Doxorubicin, Vinblastin, Methylmethansulfonat und Mitomycin C) sind als die lymphoblastoide Zelllinie TK6, welche in der Entwicklungshierarchie den Stammzellen folgt. Die Bef{\"u}rchtung, dass der Mikrokerntest in immortalisierten TK6-Zellen als Grundlage f{\"u}r Genotoxizit{\"a}tsuntersuchungen nicht gen{\"u}gen w{\"u}rden, konnte mit Hilfe der Versuchsergebnisse dieser Arbeit widerlegt werden. Die Ergebnisse belegen, dass der Mikrokerntest in TK6-Zellen relevant ist, da TK6-Zellen empfindlicher auf genotoxische Agentien im Vergleich zu h{\"a}matopoetischen Stammzellen reagieren. Bei der Untersuchung der Leuk{\"a}miezelllinie HL-60 wurden die Effekte klassischer (Vinblastin, Vincristin, Vinflunin und Vinorelbin) mit neu synthetisierten Vinca-Alkaloiden (4-Chlorochablastin, 4-Chlorochacristin, 16a, 17b und 18a) verglichen. Vinca-Alkaloide werden sehr h{\"a}ufig mit Nebenwirkungen, wie Neuropathien assoziiert, welche w{\"a}hrend einer Chemotherapie oftmals zu Therapieabbr{\"u}chen durch die Patienten f{\"u}hren. Aus diesem Grund war es erstrebenswert, neuartige Vinca-Alkaloide zu entwickeln, welche weniger Nebenwirkungen aber zugleich eine {\"a}hnliche Wirksamkeit aufweisen. Obwohl die Potenz der neuen Substanzen niedriger war als bei Vinblastin, Vincristin und Vinorelbin, zeigte ein Teil eine {\"a}hnliche Wirkung wie das Vinca-Alkaloid Vinflunin auf die Krebszelllinie HL-60 auf. Die Ergebnisse diese Arbeit k{\"o}nnen als erste Indikation in vitro genommen werden, dass sich diese Substanzen in der Krebstherapie als wirksam erweisen k{\"o}nnten und nach weiteren Ergebnissen in vivo als therapeutische Alternativen in Betracht gezogen werden. Auch bei der vergleichenden Untersuchung von exponentiell wachsenden mit differenzierten Zelllinien konnten Unterschiede detektiert werden. Die Zelllinie HT-22, welche selbst keine Krebszelllinie ist, zeigte nach Differenzierung zu nicht exponentiell wachsenden Zellen eine erh{\"o}hte Empfindlichkeit gegen{\"u}ber dem Alkylanz Methylmethansulfonat, was auf einer verminderten Basenexzisionsreparatur beruhen k{\"o}nnte. Auch die differenzierte Form der Adenokarzinom-Zelllinie CaCo2 zeigte eine gesteigerte Sensitivit{\"a}t gegen{\"u}ber dem Topoisomerase II-Inhibitor Etoposid auf, wohingegen der unselektive Topoisomerase II-Hemmer Doxorubicin keinen Effekt aufwies. Um den Sachverhalt zu kl{\"a}ren ob die festgestellten Unterschiede auf das Enzym Topoisomerase II zur{\"u}ckzuf{\"u}hren oder zellartspezifisch waren, wurden weitere Analysen der Zelllinien HL-60 und deren differenzierten Zellart durchgef{\"u}hrt. Auch hier konnten signifikante Unterschiede bei der Einzelzellgelelektrophorese nach Behandlung mit Doxorubicin und Etoposid festgestellt werden. Neben den in dieser Arbeit nachgewiesenen Unterschieden bei der Reparatur zwischen den Zelltypen, k{\"o}nnten aber auch weitere Faktoren zu Varianzen f{\"u}hren und die Mutagenit{\"a}tsforschung beeinflussen. Folglich ist davon auszugehen, dass zuk{\"u}nftige Testungen bei der pharmakologischen Substanzentwicklung in verschiedenen Zellsystemen von N{\"o}ten sind, bevor neue Substanzen zugelassen werden. Alles in allem konnte die Komplexit{\"a}t der Ergebnisse zwischen Zellen der verschiedenen Differenzierungsstadien in dieser Arbeit aufgezeigt werden. Deswegen sollte auch bei weiteren Forschungsvorhaben insbesondere ein Augenmerk auf den Differenzierungszustand der zu untersuchenden Zellpopulation geworfen werden.}, subject = {S{\"a}ugetiere}, language = {de} } @article{MaiellaroLohseKitteetal.2016, author = {Maiellaro, Isabella and Lohse, Martin J. and Kitte, Robert J. and Calebiro, Davide}, title = {cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons}, series = {Cell Reports}, volume = {17}, journal = {Cell Reports}, number = {5}, doi = {10.1016/j.celrep.2016.09.090}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162324}, pages = {1238-1246}, year = {2016}, abstract = {The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons.}, language = {en} } @phdthesis{Cirkel2018, author = {Cirkel, Nanett Christin}, title = {Beeinflussung des oxidativen Stress-Status in einem Rattenmodell: Effekt von Selenmangel auf Niere und Leber}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163631}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Das Spurenelement Selen und Vitamin E reduzieren reaktive Sauerstoff Spezies (ROS). Bei Mangel dieser wichtigen Stoffe erh{\"o}ht sich die Konzentration an ROS und der oxidative Stress steigt. Unter erh{\"o}hten ROS entstehen vermehrt DNA-Sch{\"a}den und Lipidperoxidationen. Das ROS Wasserstoffperoxid wird zu Wasser {\"u}ber das Enzym Gluthationperxoidase reduziert. Dessen Aktivit{\"a}t steigert Selen um den Faktor 100-1.000. Das Aktivit{\"a}tsmaximum des Enzyms liegt bei einer t{\"a}glichen Selenaufnahme von 60-80 Mikrogramm/Tag. Dadurch wird die Menge an ROS reduziert und der oxidative Stress in der Zelle nimmt ab. Vitamin E fungiert als Radikalf{\"a}nger. Sein Derivat alpha- Tocopherol besitzt die h{\"o}chste antioxidative Wirkung und kann Lipidperoxidationen unterbrechen. Die vorliegende Arbeit untersucht Auswirkungen von oxidativem Stress, den ein Mangel von Selen und Vitamin E in der Nahrung bei 6 Monate und 12 Monate alten Tieren auf Leber und Niere verursacht. Der Nachweis von oxidativem Stress erfolgte {\"u}ber sogenannte Hitzeschockproteine HSP70 und H{\"a}moxygenase 1. HSP 70 wird auch unter physiologischen Bedingungen exprimiert. Es wirkt als Chaperon und ist u.a. f{\"u}r die korrekte Faltung und Stabilisierung von Proteinen zust{\"a}ndig. Die Versuche zeigten, dass im Alter in der Niere die HSP70 Konzentration ansteigt und die Zelle unter vermehrtem oxidativen Stress leidet. Entsprechende Literaturergebnisse wurden best{\"a}tigt. Die H{\"a}moxygenase 1 (HO-1) ist ein Schl{\"u}sselenzym, das vermehrt bei oxidativem Stress gebildet wird. Hoch reaktionsfreudige und freie Blutbestandteile katalysiert die H{\"a}moxygenase. Einen Abfall der HO- 1 Konzentration zeigten Untersuchungen von Leber und Niere bei Selen, - Vitamin E Mangel und h{\"o}herem Lebensalter. Gr{\"u}nde f{\"u}r die verminderte Expression sind noch wenig erforscht. Die vermehrte Anreicherung von Superoxidanionradikalen wurde in den Geweben von Leber und Niere {\"u}ber Dihydroethidium (DHE) F{\"a}rbung nachgewiesen. Die Hypothese wurde best{\"a}tigt, dass bei Selen, -Vitamin E Mangelnahrung und h{\"o}herem Alter vermehrter oxidativer Stress entsteht. Selenmangel beg{\"u}nstigt die Entstehung verschiedener Krankheiten, z.B. Krebs, koronale Herzerkrankung und vor allem die Keshan-Krankheit, die den Herzmuskel bef{\"a}llt. Selen nimmt positiven Einfluss auf K{\"o}rperfunktionen: Fertilit{\"a}t, embryonalen Entwicklung und Entwicklung eines Neugeborenen. Einige Fragen bleiben ungekl{\"a}rt: Welche physiologischen Entwicklungsprozesse f{\"o}rdert Selen? Nimmt Selen eine wichtige Funktion bei der Befruchtung der Eizelle ein? Wie beeinflusst Selen die Entwicklung des Gehirns? Dem Spurenelement Selen kommen offensichtlich neben seiner Bedeutung zur Minderung des oxidativen Stresses noch weitere wichtige Funktionen zu, die bisher wenig untersucht wurden.}, subject = {Oxidativer Stress}, language = {de} } @article{IlinKulmanovNersesyanetal.2015, author = {Ilin, Alexander and Kulmanov, Murat and Nersesyan, Armen and Stopper, Helga}, title = {Genotoxic activity of the new pharmaceutical FS-1 in Salmonella/microsome test and mouse lymphoma L5178Y cells}, series = {Journal of BUON}, volume = {20}, journal = {Journal of BUON}, number = {2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143769}, pages = {595-601}, year = {2015}, abstract = {Purpose: The purpose of this study was to determine possible genotoxic effects of a new very promising antibacterial/ antiviral drug FS-1. Methods: The drug was tested in TA98, TA100, TA102, TA 1535 and TA1537 strains of Salmonella (Ames test) with and without metabolic activation, and also in mouse lymphoma L5178Y cells by means of micronucleus and comet assays. In microbes the drug was tested at concentrations up to 500 \(\mu\)g/plate and in mouse lymphoma cells up to 2,000 \(\mu\)g/ml. Results: In both test-systems in all experiments completely negative results were obtained although FS-1 was tested at maximum tolerated doses. Conclusions: The drug is not genotoxic. This is advantageous because many antibacterial/antiviral drugs possess such activity.}, language = {en} } @inproceedings{WernerWakabayashiJahnsetal.2017, author = {Werner, Rudolf and Wakabayashi, Hiroshi and Jahns, Roland and Erg{\"u}n, S{\"u}leyman and Jahns, Valerie and Higuchi, Takahiro}, title = {PET-Guided Histological Characterization of Myocardial Infiltrating Cells in a Rat Model of Myocarditis}, series = {European Heart Journal - Cardiovascular Imaging}, volume = {18}, booktitle = {European Heart Journal - Cardiovascular Imaging}, number = {Supplement}, publisher = {Oxford University Press}, issn = {2047-2404}, doi = {10.1093/ehjci/jex071}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161127}, pages = {i1-i3}, year = {2017}, abstract = {No abstract available.}, subject = {Myokarditis}, language = {en} } @article{AwadOthmanStopper2017, author = {Awad, Eman and Othman, Eman M. and Stopper, Helga}, title = {Effects of resveratrol, lovastatin and the mTOR-inhibitor RAD-001 on insulin-induced genomic damage in vitro}, series = {Molecules}, volume = {22}, journal = {Molecules}, number = {12}, doi = {10.3390/molecules22122207}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159260}, pages = {2207}, year = {2017}, abstract = {Diabetes mellitus (DM) is one of the major current health problems due to lifestyle changes. Before diagnosis and in the early years of disease, insulin blood levels are elevated. However, insulin generates low levels of reactive oxygen species (ROS) which are integral to the regulation of a variety of intracellular signaling pathways, but excess levels of insulin may also lead to DNA oxidation and DNA damage. Three pharmaceutical compounds, resveratrol, lovastatin and the mTOR-inhibitor RAD-001, were investigated due to their known beneficial effects. They showed protective properties against genotoxic damage and significantly reduced ROS after in vitro treatment of cultured cells with insulin. Therefore, the selected pharmaceuticals may be attractive candidates to be considered for support of DM therapy.}, language = {en} } @article{WeigandRonchiRizkRabinetal.2017, author = {Weigand, Isabel and Ronchi, Cristina L. and Rizk-Rabin, Marthe and Dalmazi, Guido Di and Wild, Vanessa and Bathon, Kerstin and Rubin, Beatrice and Calebiro, Davide and Beuschlein, Felix and Bertherat, J{\´e}r{\^o}me and Fassnacht, Martin and Sbiera, Silviu}, title = {Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, number = {49}, doi = {10.1038/s41598-017-00125-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157952}, year = {2017}, abstract = {Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40\% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.}, language = {en} } @phdthesis{Berlin2017, author = {Berlin, Christopher}, title = {Die Untersuchung der kardialen Folgen einer ubiquit{\"a}ren Deletion von RKIP in M{\"a}usen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152882}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die Herzinsuffizienz, eine der h{\"a}ufigsten chronischen Krankheiten in der westlichen Welt, ist als Folge einer Myokardsch{\"a}digung durch eine verschlechterte Pumpfunktion des Herzens charakterisiert, die der K{\"o}rper durch verschiedene Kompensationsmechanismen zur Kontraktilit{\"a}tssteigerung auszugleichen versucht. Wichtiger Mechanismus hierf{\"u}r ist die Kontraktilit{\"a}ts- und Frequenzsteigerung {\"u}ber ß-adrenerge Rezeptorsignale, welche bei langfristiger Stimulation allerdings zu einer Abnahme der Funktionalit{\"a}t und Minderexpression eben dieses Rezeptorsystems, sowie der gleichzeitigen Verschlechterung der Herzinsuffizienz f{\"u}hrt. Interessanterweise wird parallel zur verminderten Rezeptorexpression bei Herzinsuffizienzpatienten eine Zunahme der GRK-Aktivit{\"a}t beobachtet. Diese Kinase ist in der Lage, ß-adrenerge GPCR-Signale durch Phosphorylierung des membranst{\"a}ndigen Rezeptors herunterzuregulieren. Durch einen PKC-abh{\"a}ngigen switch von Raf1 zu GRK2 konnte mit RKIP ein kardialer, endogener Inhibitor der GRK2 identifiziert werden. Es wurde in vitro und in vivo in M{\"a}usen mit myokardialer {\"U}berexpression von RKIP gezeigt, dass RKIP f{\"a}hig ist, die kontraktile Funktion von Herzmuskelzellen zu verbessern, negative kardiale Langzeitfolgen wie eine Verschlechterung der Insuffizienz, Remodeling-Prozesse wie Zunahme der Fibrosierung und eine gesteigerte Apoptoserate, sowie kardiale Rhythmusst{\"o}rungen protektiv zu beeinflussen. Um die endogene Rolle von RKIP weiter zu er{\"o}rtern, wurde in dieser Arbeit der Knockout von RKIP unter basalen Bedingungen, als auch nach transverser Aortenkonstriktion (TAC) untersucht. Zur Untersuchung physiologischer Parameter wie der Verk{\"u}rzungsfraktion, oder dem linksventrikul{\"a}rem diastolischen Durchmesser wurden echokardiographische Verfahren herangezogen. In diesen Untersuchungen zeigte sich nach dreiw{\"o}chiger TAC eine Verschlechterung der Pumpfunktion, sowie eine verst{\"a}rkte Dilatation des linken Ventrikels in RKIP-/--M{\"a}usen. Gest{\"u}tzt wurden diese Ergebnisse durch einen erh{\"o}hten pulmonalen Blutr{\"u}ckstau in RKIP-/--M{\"a}usen nach chronischer Druckbelastung. Zudem wurde an isolierten Kardiomyozyten die Kinetik von Kalzium als f{\"u}r die Kontraktion verantwortlichen Botenstoff durch intrazellul{\"a}re Fluoreszenz-Echtzeit-Messungen, sowie die Kontraktion und Relaxation auf Zell- und Sarkomerebene durch ein optisches Kamerasystem untersucht. Hier zeigte sich ohne den Einfluss β-adrenerger Stimulantien {\"a}quivalent zum basalen Ph{\"a}notyp dieser Tiere in RKIP-/--Kardiomyozyten keine Ver{\"a}nderung der Kalzium-Kinetik, sowie der Kontraktion und Relaxation auf Zell- und Sarkomerebene. Des Weiteren wurden mittels realtime PCR die Expressionslevels von Insuffizienzmarkern wie BNP und ANP, sowie von Kollagen 3 bestimmt. Der Grad der Fibrosierung wurde zus{\"a}tzlich durch Quantifizierung der fibrosierten Areale in histologischen Querschnitten untersucht. Apoptotische Ver{\"a}nderungen wurden mittels TUNEL-Assay auf histologischer Ebene bestimmt. In all diesen Untersuchungen zeigte sich ein fortgeschrittenes kardiales Remodeling in RKIP-/--M{\"a}usen nach TAC im Vergleich zu Wildtyptieren. Hand in Hand mit dem Bild einer fortgeschrittenen Herzinsuffizienz in RKIP-/--M{\"a}usen nach TAC konnte zudem in diesen Tieren eine gesteigerte Mortalit{\"a}t nach chronischer Hochdruckbelastung festgestellt werden. In Kombination mit den protektiven Eigenschaften einer kardialen RKIP-{\"U}berexpression, sowie dem positiven Effekt einer retroviralen RKIP-Transfektion sprechen diese Ergebnisse f{\"u}r RKIP als einen interessanten k{\"o}rpereigenen Angriffspunkt f{\"u}r die kontraktilit{\"a}tssteigernde Therapie der Herzinsuffizienz, den es in weiteren klinischen Studien zu untersuchen gilt.�}, subject = {RKIP}, language = {de} } @phdthesis{Arnaudov2017, author = {Arnaudov, Theresa Irina}, title = {Anthocyane - Modulation oxidativen Stresses in vivo und in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152593}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die menschliche Nahrung enth{\"a}lt antioxidative Stoffe, die den Menschen m{\"o}glicherweise vor oxidativem Stress und seinen Konsequenzen sch{\"u}tzen k{\"o}nnen. Im Fokus der vorliegenden Arbeit standen Anthocyane, die als vielversprechende antioxidative Pflanzenstoffe in unterschiedlichen Obst- und Gem{\"u}sesorten zu finden sind. Im ersten Teil der Arbeit wurden in einem HT-29-Zellkulturmodell die zwei wichtigsten Vertreter der Anthocyanidine, Delphinidin und Cyanidin, untersucht. Es galt zu pr{\"u}fen, ob beide Pflanzenstoffe in geringen Konzentrationen in humanen Zellen antioxidativ wirken und oxidativen Genomschaden verhindern k{\"o}nnen. Im Comet-Assay reduzierten sowohl Delphinidin (ab 3,2 µM) als auch Cyanidin (ab 1 µM) signifikant die durch 100 µM Wasserstoffperoxid induzierten DNA-Sch{\"a}den in den HT-29-Zellen. Im Comet-Assays mit FPG-Enzym wurde deutlich, dass eine Pr{\"a}inkubation mit Cyanidin wirksam die Oxidation der DNA-Basen verringert. Die Auswirkungen auf den Glutathionspiegel wurden mit Hilfe des Glutathion-Recycling-Assays nach Tietze untersucht. Die Pr{\"a}inkubation mit Cyanidin f{\"u}hrte hierbei zu keinen signifikanten Ver{\"a}nderungen. Um die Auswirkungen der Anthocyanidine auf die intrazellul{\"a}re ROS-Produktion zu beobachten, wurde der fluoreszierenden Farbstoffs DHE verwendet. Sowohl Delphinidin (10 und 15 µM) als auch Cyanidin (10 und 20 µM) senkten signifikant die durch 25 µM Antimycin A angeregte ROS-Produktion. Im zweiten Teil der Arbeit wurde ein anthocyanreicher roter Fruchtsaft in einer 10-w{\"o}chigen Interventionsstudie am Menschen getestet. Hieran nahmen sowohl 19 Fibromyalgiepatienten als auch 10 gesunde Probanden teil. Es sollte die Hypothese gepr{\"u}ft werden, dass die konzentrierte und andauernde Einnahme des Saftes messbar oxidative Stressparameter im Blut ver{\"a}ndert. Außerdem sollten m{\"o}gliche Unterschiede im oxidativen Stresslevel zwischen Patienten und gesunden Probanden aufgedeckt werden. Nach jeder Studienphase erfolgte eine Befragung nach klinischen Symptomen und die Abgabe einer Urin- und Blutprobe in der Schmerzambulanz der Uniklinik W{\"u}rzburg (2 Wochen Einwaschphase, 4 Wochen Fruchtsaftphase mit je 750 ml Saft t{\"a}glich, 4 Wochen Auswaschphase). Das ROS-Level wurde mit 2 Methoden in den mononukle{\"a}ren Blutzellen untersucht: In der photometrischen NBT-Messung konnten keine signifikanten Unterschiede zwischen den Gruppen oder Zeitpunkten beobachtet werden. Bei der durchflusszytometrischen Messung mit Hilfe des fluoreszierenden DCF-Farbstoffes lag das ROS-Level der Patientengruppe vor Fruchtsafteinnahme signifikant h{\"o}her als das der Kontrollgruppe. Zur Messung der antioxidativen Kapazit{\"a}t wurde die Eisen-Reduktionsf{\"a}higkeit (FRAP) im Plasma untersucht. In der Patientengruppe zeigte sich eine Steigerung der antioxidativen Kapazit{\"a}t nach Einnahme des Fruchtsaftes. Die Unterschiede zwischen den beiden Gruppen waren gering. Sowohl das Gesamtglutathion als auch die oxidierte und reduzierte Form wurden in den Erythrozyten der Probanden mit dem Glutathion-Recycling-Assay gemessen. Nach der Fruchtsafteinnahme stieg die Konzentration des Gesamtglutathions in der Patientengruppe an. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass Delphinidin und Cyanidin auch in geringen Konzentrationen (1µM - 20µM) einen antioxidativer Effekt in HT-29-Zellen haben und vor oxidativem DNA-Schaden sch{\"u}tzen k{\"o}nnen. Die Ergebnisse der Interventionsstudie unterschieden sich teilweise in den einzelnen Endpunkten. Es war nicht m{\"o}glich, den Fibromyalgiepatienten ein h{\"o}heres oxidatives Stresslevel nachzuweisen. Ein Grund f{\"u}r die geringeren Effekte des Fruchtsaftes k{\"o}nnte in der eher geringen Bioverf{\"u}gbarkeit der Anthocyane liegen. Außerdem k{\"o}nnte die Heterogenit{\"a}t der Fibromyalgieerkrankung genauso wie andere endogene oder exogene Faktoren wie etwa Alter oder Medikamenteneinnahme die teilweise großen interindividuellen Schwankungen der Messergebnisse hinsichtlich der oxidativen Stressparameter bedingen. Klinisch profitierten einige der Fibromyalgiepatienten von der Fruchtsafteinnahme insbesondere hinsichtlich der Reizdarmsymptomatik. Dieses Volksleiden k{\"o}nnte ein interessanter Ansatzpunkt f{\"u}r Folgeuntersuchungen mit einem anthocyanreichen Produkt sein.}, subject = {Oxidativer Stress}, language = {de} } @phdthesis{Basali2017, author = {Basali, Timo}, title = {Untersuchung der Nierensch{\"a}digung durch Aldosteron am Rattenmodell {\"u}ber die Quantifizierung von Sch{\"a}digungsmarkern mittels Real-Time PCR-Technik}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151311}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Die Breite der Wirkungen von Aldosteron auf Nierenzellen wurde lange Zeit untersch{\"a}tzt. Inzwischen zeigte sich ein nicht unerheblicher Anteil des Hyperaldosteronismus an arterieller Hypertonie und ebenso mehren sich die Hinweise auf damit assoziierter erh{\"o}hter Inzidenz f{\"u}r maligne Entartung von Nierengewebe. In dieser Arbeit wurde der Effekt von Hyperaldosteronismus auf Nierenzellen von Ratten in vivo untersucht. Mittels real time quantitative PCR wurden die relative Expressionsver{\"a}nderungen der mRNA von validierten Nierensch{\"a}digungsmarkern im Hyperaldosteronismusmodell kontrolliert beobachtet und statistisch ausgewertet. Anders als im analog durchgef{\"u}hrten Vorversuch mit DOCA an der Stelle von Aldosteron, ließ sich gr{\"o}ßtenteils kein {\"u}ber der nat{\"u}rlichen Streuung der Daten liegender, signifikanter Effekt der Nierensch{\"a}digung durch {\"u}berh{\"o}hte Aldosteronspiegel nachweisen. Hierf{\"u}r kommen vielf{\"a}ltige Gr{\"u}nde in Frage. Neben der technischen Variabilit{\"a}t, der Beschaffenheit der internen Kontrolle, potentiell vorhandenen Inhibitoren und der Qualit{\"a}t der mRNA, konnten eine Reihe von weiteren Gr{\"u}nden als Ursache f{\"u}r die Diskrepanz zu den Ergebnissen der mit DOCA behandelten Tiere ausgeschlossen werden. Neben der theoretischen M{\"o}glichkeit inter-methodischer Differenzen und sich daraus ergebender Variationen, sowie der noch weiter zu untersuchenden Rolle des Glukokortikoidrezeptors durch dessen variable gleichzeitige Aktivierung, ist die Interpretation im Sinne eines zu gering ausgepr{\"a}gten Sch{\"a}digungseffektes durch den Hyperaldosteronismus f{\"u}r den gew{\"a}hlten Stichprobenumfang naheliegend. Hiermit stimmt auch die Tatsache {\"u}berein, dass der Effekt der Behandlung mit Aldosteron im Vergleich zur Behandlung mit DOCA von vorne herein deutlich geringer ausfallend erwartet wurde.}, subject = {Aldosteron}, language = {de} } @phdthesis{Zimnol2017, author = {Zimnol, Anna}, title = {Relevance of angiotensin II type 1a receptor and NADPH oxidase for the formation of angiotensin II-mediated DNA damage}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137469}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Das Renin-Angiotensin-Aldosteron-System (RAAS) reguliert den Blutdruck sowie den Elektrolyt- und Wasserhaushalt. Das aktive Peptid, Angiotensin II (AngII), f{\"u}hrt dabei zur Vasokonstriktion und in h{\"o}heren Konzentrationen zu Bluthochdruck. Hypertensive Patienten haben ein erh{\"o}htes Risiko an Krebs zu erkranken, vor allem an Nierenkrebs. Wir konnten bereits in vivo zeigen, dass AngII in der Lage ist, den Blutdruck zu steigern und dosisabh{\"a}ngig zu DNA-Sch{\"a}den {\"u}ber den Angiotensin II Typ 1-Rezeptor (AT1R) f{\"u}hrt. Ein stimuliertes RAAS kann ferner {\"u}ber die Aktivierung der NADPH-Oxidase, einer Hauptquelle der Generierung reaktiver Sauerstoffspezies (ROS) in der Zelle, zu oxidativem Stress f{\"u}hren. Zielsetzung dieser Arbeit war es zum einen, mit Hilfe von AT1a-Rezeptor-defizienten M{\"a}usen in vivo zu pr{\"u}fen, ob die Bildung von ROS, sowie die Bildung von DNA-Sch{\"a}den in der Niere und im Herzen unabh{\"a}ngig von einem erh{\"o}hten Blutdruck auftreten. Zum anderen sollte, ebenfalls in vivo, untersucht werden, ob eine oder beide von zwei untersuchten Isoformen der NADPH-Oxidase (Nox) f{\"u}r die Ausl{\"o}sung oxidativen Stresses in der Niere verantwortlich ist. Zun{\"a}chst wurden f{\"u}r den Versuch zur {\"U}berpr{\"u}fung der Abh{\"a}ngigkeit AngII-induzierter DNA-Sch{\"a}den vom Blutdruck m{\"a}nnliche C57BL/6-M{\"a}use und AT1a-Knockout (KO)-M{\"a}use mit osmotischen Minipumpen ausgestattet, die AngII in einer Konzentrationen von 600 ng/kg min {\"u}ber einen Zeitraum von 28 Tagen abgaben. Zus{\"a}tzlich wurde eine Gruppe von AngII-behandelten Wildtyp (WT)-M{\"a}usen mit dem AT1-Rezeptor-Blocker Candesartan (Cand) behandelt. W{\"a}hrend des Versuchszeitraumes fanden regelm{\"a}ßige, nicht-invasive Blutdruckmessungen an den wachen M{\"a}usen statt. In WT-M{\"a}usen induzierte AngII Bluthochdruck, verursachte erh{\"o}hte Albumin-Level im Urin und f{\"u}hrte zur Bildung von ROS in Niere und im Herzen. Außerdem traten in dieser Gruppe DNA-Sch{\"a}den in Form von Einzel- und Doppelstrangbr{\"u}chen auf. All diese Reaktionen auf AngII konnten jedoch durch gleichzeitige Behandlung mit Cand verhindert werden. AT1a-KO-M{\"a}use hatten, verglichen mit WT-Kontrollm{\"a}usen, einen signifikant niedrigeren Blutdruck und normale Albumin-Level im Urin. In AT1a-KO-M{\"a}usen, die mit AngII behandelt wurden, konnte kein Anstieg des systolischen Blutdrucks sowie kein Einfluss auf die Nierenfunktion gefunden werden. Jedoch f{\"u}hrte AngII in dieser Gruppe zu einer Steigerung von ROS in der Niere und im Herzen. Zus{\"a}tzlich wurden genomische Sch{\"a}den, vor allem in Form von Doppelstrangbr{\"u}chen signifikant in dieser Gruppe induziert. Auch wenn AT1a-KO-Tiere, unabh{\"a}ngig von einer AngII-Infusion, keine eingeschr{\"a}nkte Nierenfunktion zeigten, so wiesen sie erhebliche histopathologische Sch{\"a}den im Hinblick auf die Glomeruli und das Tubulussystem auf. Diese Art von Sch{\"a}den deuten auf eine besondere Bedeutung des AT1aR im Hinblick auf die embryonale Entwicklung der Niere hin. Zusammenfassend beweisen die Ergebnisse dieses Experiments eindeutig, dass eine AngII-induzierte ROS-Produktion und die Induktion von DNA-Sch{\"a}den unabh{\"a}ngig von einem erh{\"o}hten Blutdruck auftreten. Da in der AngII-behandelten AT1a-KO-Gruppe eine signifikant h{\"o}here Expression des AT1b-Rezeptors zu finden war und die Blockade von beiden Rezeptorsubtypen mit Cand zu einer Verhinderung der sch{\"a}dlichen Effekte durch AngII f{\"u}hrte, scheint der AT1bR im Falle einer AT1aR-Defizienz f{\"u}r die Entstehung der Sch{\"a}den zust{\"a}ndig zu sein. Ziel des zweiten Experimentes war es, den Beitrag der Nox2 und Nox4 zum oxidativen DNA-Schaden in vivo zu untersuchen. Hierf{\"u}r wurden m{\"a}nnliche C57BL/6-M{\"a}use und Nox2- oder Nox4-defiziente M{\"a}use mit osmotischen Minipumpen ausgestattet, die AngII in einer Konzentration von 600 ng/kg min {\"u}ber einen Zeitraum von 28 Tagen abgaben. Im WT-Stamm und in beiden Nox-defizienten St{\"a}mmen induzierte AngII Bluthochdruck, verursachte erh{\"o}hte Albumin-Level im Urin und f{\"u}hrte zur Bildung von ROS in der Niere. Außerdem waren in allen AngII-behandelten Gruppen genomische Sch{\"a}den, vor allem in Form von Doppelstrangbr{\"u}chen, erh{\"o}ht. Auch in Abwesenheit von AngII wiesen Nox2- und Nox4-defiziente M{\"a}use mehr Doppelstrangbr{\"u}che im Vergleich zu WT-Kontrollm{\"a}usen auf. Interessanterweise kompensieren allerdings weder Nox2 noch Nox4 das Fehlen der jeweils anderen Isoform auf RNA-Basis. Aufgrund dieser Ergebnisse schließen wir, dass bislang keine Isoform alleine f{\"u}r die Generierung von oxidativen DNA-Sch{\"a}den in der Niere verantwortlich gemacht werden kann und dass eine Beteiligung einer weiteren Nox-Isoform sehr wahrscheinlich ist. M{\"o}glicherweise k{\"o}nnten aber auch andere ROS-generierende Enzyme, wie Xanthinoxidase oder Stickoxidsynthase involviert sein. Da genomische Sch{\"a}den in Nieren von Nox2- und Nox4-defizienten M{\"a}usen in Abwesenheit von AngII gegen{\"u}ber den Sch{\"a}den in WT-Kontrollm{\"a}usen erh{\"o}ht waren, k{\"o}nnten die beiden Isoformen auch eine sch{\"u}tzende Funktion im Bereich von Nierenkrankheiten {\"u}bernehmen. Da dies aber bislang nur f{\"u}r Nox4 beschrieben ist, ist es wahrscheinlicher, dass das Fehlen von einer der beiden Isoformen eher einen Einfluss auf die Embryonalentwicklung hat. Um dies jedoch abschließend zu kl{\"a}ren w{\"a}re es sinnvoll mit induzierbaren Knockout-Modellen zu arbeiten, bei denen m{\"o}gliche entwicklungsbedingte Effekte minimiert werden k{\"o}nnen.}, subject = {Angiotensin II}, language = {de} } @article{VandenbergChahoudHeindeletal.2012, author = {Vandenberg, Laura N. and Chahoud, Ibrahim and Heindel, Jerrold J. and Padmanabhan, Vasantha and Paumgartten, Francisco J. R. and Sch{\"o}nfelder, Gilbert}, title = {Urinary, Circulating, and Tissue Biomonitoring Studies Indicate Widespread Exposure to Bisphenol A}, series = {Ci{\^e}ncia \& Sa{\´u}de Coletiva}, volume = {17}, journal = {Ci{\^e}ncia \& Sa{\´u}de Coletiva}, number = {2}, doi = {10.1289/ehp.0901716}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134332}, pages = {407-434}, year = {2012}, abstract = {Bisphenol A (BPA) is one of the highest-volume chemicals produced worldwide, and human exposure to BPA is thought to be ubiquitous. Thus, there are concerns that the amount of BPA to which humans are exposed may cause adverse health effects. We examined many possibilities for why biomonitoring and toxicokinetic studies could come to seemingly conflicting conclusions. More than 80 published human biomonitoring studies that measured BPA concentrations in human tissues, urine, blood, and other fluids, along with two toxicokinetic studies of human BPA metabolism were examined. Unconjugated BPA was routinely detected in blood (in the nanograms per milliliter range), and conjugated BPA was routinely detected in the vast majority of urine samples (also in the nanograms per milliliter range). In stark contrast, toxicokinetic studies proposed that humans are not internally exposed to BPA. Available data from biomonitoring studies clearly indicate that the general population is exposed to BPA and is at risk from internal exposure to unconjugated BPA. The two toxicokinetic studies that suggested human BPA exposure is negligible have significant deficiencies, are directly contradicted by hypothesis-driven studies, and are therefore not reliable for risk assessment purposes.}, language = {en} } @article{OthmanNaseemAwadetal.2016, author = {Othman, Eman M. and Naseem, Muhammed and Awad, Eman and Dandekar, Thomas and Stopper, Helga}, title = {The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0168386}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147983}, pages = {e0168386}, year = {2016}, abstract = {Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.}, language = {en} } @phdthesis{Kestler2017, author = {Kestler, Christian}, title = {Untersuchungen {\"u}ber die Dimerisierung der HAD-Phosphatase Chronophin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149777}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Phosphatasen der HAD (haloacid dehalogenase)-Familie sind weit verbreitet in allen Dom{\"a}nen des Lebens und erf{\"u}llen die verschiedensten zellul{\"a}ren Aufgaben, beispielsweise in Metabolismus und Zellregulation. Die HAD-Phosphatase Chronophin zeigt Phosphataseaktivit{\"a}t unter anderem gegen{\"u}ber Pyridoxal-5'-Phosphat (PLP), einem essentiellen Kofaktor vieler biochemischer Prozesse, und Phosphocofilin, einem Regulator des Aktinzytoskeletts. Chronophin dimerisiert {\"u}ber die Interaktion zweier identischer Untereinheiten zu einem Homodimer. Ziel dieser Arbeit war, die Rolle dieser Dimerisierung, eines bei HAD-Phosphatasen weit verbreiteten Oligomerisierungszustandes, n{\"a}her zu untersuchen. Hierzu wurde die Dimerisierung erfolgreich durch den Austausch der Aminos{\"a}uren Alanin 194 und 195 zu Lysinen (Mutation A194K/A195K) gest{\"o}rt. Der Nachweis einer konstitutiv monomeren Chronophin-Mutante mittels Gr{\"o}ßenausschlusschromatographie, Rasterkraftmikroskopie, analytischer Ultra¬zentrifugation und Zellexperimenten wurde schließlich {\"u}ber die Struktur¬aufl{\"o}sung mittels R{\"o}ntgenstrukturanalyse best{\"a}tigt. Aktivit{\"a}tsmessungen der monomeren Mutante gegen{\"u}ber dem Substrat PLP zeigten eine deutliche Verminderung der Phosphataseaktivit{\"a}t. Die R{\"o}ntgenstrukturanalyse von Chronophin A194K/A195K im Vergleich mit Wildtyp-Chronophin enth{\"u}llte einen Mechanismus, wie die sogenannte Substratspezifit{\"a}tsschleife, die f{\"u}r die korrekte Positionierung des PLP sorgt, im Homodimer des Wildtyps durch Interaktionen mit dem zweiten Protomer stabilisiert wird. Diese Stabilisierung fehlt bei der monomeren Mutante und {\"a}ußert sich in einer ver{\"a}nderten Stellung der Substratspezifit{\"a}tsschliefe. Der Strukturvergleich von Chronophin mit weiteren HAD-Phosphatasen der selben strukturellen Untergruppe vom C2a-Typ l{\"a}sst eine allgemeine G{\"u}ltigkeit der hier beschriebenen allosterischen Kontrolle von Substratspezifit{\"a}t {\"u}ber Homodimerisierung bei HAD-Phosphatasen vermuten und k{\"o}nnte so neue Ansatzpunkte f{\"u}r m{\"o}glicherweise auch therapeutisch nutzbare Aktivit{\"a}tshemmungen liefern.}, subject = {Dimerisierung}, language = {de} } @phdthesis{Messerer2017, author = {Messerer, Regina}, title = {Synthesis of Dualsteric Ligands for Muscarinic Acetylcholine Receptors and Cholinesterase Inhibitors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149007}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {The study is dealing with the synthesis and pharmacological investigation of newly designed dualsteric ligands of muscarinic acetylcholine receptors belonging to the superfamily of G protein-coupled receptors. Such bipharmacophoric ligands combine the advantages of the orthosteric binding site (high-affinity) and of the topographically distinct allosteric binding site (subtype-selectivity) resulting in compounds with reduced side effects. This opens the way to a new therapeutic approach in the treatment of e.g. chronic pain, drug withdrawal, Parkinson`s and Alzheimer`s disease. Furthermore, the newly synthesized dualsteric compounds were pharmacologically investigated in order to get a better understanding of the activation and signaling processes in muscarinic acetylcholine receptors, especially with regard to partial agonism. The development of the "dynamic ligand binding" concept offers new perspectives for ligand binding and signaling at G protein-coupled receptors. GPCRs are no longer considered as simple on/off switches. Dualsteric ligands can bind in a dualsteric pose, reflecting an active receptor state as well as in a purely allosteric binding pose, characterized by an inactive receptor state resulting in partial agonism. The degree of partial agonism depends on the ratio of active versus inactive receptor populations. On this basis, orthosteric/orthosteric hybrid ligands consisting of the antagonist atropine and scopolamine, respectively, as well as of the agonist iperoxo and isoxazole, respectively, linked via different alkyl chain length were synthesized in order to investigate partial agonism (Figure 1). Figure 1: Structures of the synthesized iperoxo/isoxazole-atropine/scopolamine-hybrids. Furthermore, different sets of quaternary and tertiary homodimers consisting either of two iperoxo or two acetylcholine units were synthesized in order to study their extent on partial agonism (Figure 2). The two agonists were connected by varying alkyl chain length. Binding studies on CHO-hM2 cells of the quaternary compounds revealed that dimerization of the agonist results in a loss of potency. The iperoxo-dimers reached higher maximum effects on the Gi- as well as on the Gs pathway in comparison to the acetylcholine-dimers. Besides the choice of the orthosteric building block (potency of the agonist), the alkyl chain length is also crucial for the degree of partial agonism. Figure 2: Structures of the synthesized quat./tert. iperoxo/acetylcholine-homodimers. Quinolone-based hybrids connected to the superagonist iperoxo and to the endogenous ligand acetylcholine, respectively, linked through an alkyl chain of different length were synthesized in order to develop further partial agonists (Figure 3). FRET studies confirmed M1 subtype-selectivity as well as linker dependent receptor response. The greatest positive FRET signal was observed with quinolone-C6-iper resulting from a positive cooperativity between the two separated moieties, alloster and orthoster. However, the corresponding hybrids with a longer linker led to an inverse FRET signal indicating a different binding mode, e.g. purely allosteric, in contrast to the shorter linked hybrids. Furthermore, the flexible alkyl spacer was replaced by a rigidified linker resulting in the hybrid quinolone-rigid-iperoxo (Figure 3). FRET studies on the M1 receptor showed reduced FRET kinetics, resulting from interactions between the bulky linker and the aromatic lid, located between the orthosteric and allosteric binding site. A bitopic binding mode of the rigidified hybrid is presumed. For further clarity, mutational studies are necessary. Figure 3: M1-selective hybrid compounds. Another aim of this work was the design and synthesis of new hybrid compounds, acting as agonists at the M1 and M2 receptor and as inhibitors for AChE and BChE in the context of M. Alzheimer. Several sets of hybrid compounds consisting of different pharmacophoric units (catalytic active site: phthalimide, naphthalimide, tacrine; peripheric anionic site: iperoxo, isoxazole) linked through a polymethylene chain of varying length were synthesized. Tac-C10-iper (Figure 4), consisting of tacrine and the superagonist iperoxo linked by a C10 polymethylene spacer, was found to have excellent anticholinesterase activity for both AChE (pIC50 = 9.81) and BChE (pIC50 = 8.75). Docking experiments provided a structural model to rationalize the inhibitory power towards AChE. Additionally, the tacrine related hybrids showed affinity to the M1 and M2 receptor. Such compounds, addressing more than one molecular target are favorable for multifactorial diseases such as Alzheimer. Figure 4: Structure of the most active compound regarding anticholinesterase activity. In summary, the choice of the pharmacophoric units, their connecting point as well as the nature, length, and flexibility of the linker play an important role for the activity of designed bivalent ligands. A shorter linker length cannot bridge both binding sites simultaneously in contrast to longer linker chains. On the other hand, too long linker chains can result in unwanted steric interactions. Further investigations with respect to structural variations of hybrid compounds, with or without quaternary ammonium groups, are necessary in the light of drug development.}, subject = {Cholinesteraseinhibitor}, language = {en} } @article{BankogluTschoppSchmittetal.2016, author = {Bankoglu, Ezgi Eyluel and Tschopp, Oliver and Schmitt, Johannes and Burkard, Philipp and Jahn, Daniel and Geier, Andreas and Stopper, Helga}, title = {Role of PTEN in Oxidative Stress and DNA Damage in the Liver of Whole-Body Pten Haplodeficient Mice}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {11}, doi = {10.1371/journal.pone.0166956}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146970}, pages = {e0166956}, year = {2016}, abstract = {Type 2 diabetes (T2DM) and obesity are frequently associated with non-alcoholic fatty liver disease (NAFLD) and with an elevated cancer incidence. The molecular mechanisms of carcinogenesis in this context are only partially understood. High blood insulin levels are typical in early T2DM and excessive insulin can cause elevated reactive oxygen species (ROS) production and genomic instability. ROS are important for various cellular functions in signaling and host defense. However, elevated ROS formation is thought to be involved in cancer induction. In the molecular events from insulin receptor binding to genomic damage, some signaling steps have been identified, pointing at the PI3K/AKT pathway. For further elucidation Phosphatase and Tensin homolog (Pten), a tumour suppressor phosphatase that plays a role in insulin signaling by negative regulation of PI3K/AKT and its downstream targets, was investigated here. Dihydroethidium (DHE) staining was used to detect ROS formation in immortalized human hepatocytes. Comet assay and micronucleus test were performed to investigate genomic damage in vitro. In liver samples, DHE staining and western blot detection of HSP70 and HO-1 were performed to evaluate oxidative stress response. DNA double strand breaks (DSBs) were detected by immunohistostaining. Inhibition of PTEN with the pharmacologic inhibitor VO-OHpic resulted in increased ROS production and genomic damage in a liver cell line. Knockdown of Pten in a mouse model yielded increased oxidative stress levels, detected by ROS levels and expression of the two stress-proteins HSP70 and HO-1 and elevated genomic damage in the liver, which was significant in mice fed with a high fat diet. We conclude that PTEN is involved in oxidative stress and genomic damage induction in vitro and that this may also explain the in vivo observations. This further supports the hypothesis that the PI3K/AKT pathway is responsible for damaging effects of high levels of insulin.}, language = {en} } @article{MambrettiKistnerMayeretal.2016, author = {Mambretti, Egle M. and Kistner, Katrin and Mayer, Stefanie and Massotte, Dominique and Kieffer, Brigitte L. and Hoffmann, Carsten and Reeh, Peter W. and Brack, Alexander and Asan, Esther and Rittner, Heike L.}, title = {Functional and structural characterization of axonal opioid receptors as targets for analgesia}, series = {Molecular Pain}, journal = {Molecular Pain}, number = {12}, doi = {10.1177/1744806916628734}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145917}, pages = {1-17}, year = {2016}, abstract = {Background Opioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. µ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in na{\"i}ve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function. Results Comparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOP-mcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [D-Ala\(^2\), N-MePhe\(^4\), Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in na{\"i}ve Wistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using β-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl lead to a recruitment of β-arrestin-2 to the membrane followed by a β-arrestin-2 reappearance in the cytosol and MOP internalization. Pretreatment with hypertonic saline prevented MOP internalization. Conclusion MOPs are present and functional in the axonal membrane from na{\"i}ve animals. Hypertonic saline acutely decreases ligand-induced internalization of MOP and thereby might improve MOP function. Further studies should explore potential clinical applications of opioids together with enhancers for regional analgesia.}, language = {en} } @phdthesis{Glaeser2017, author = {Gl{\"a}ser, Katharina}, title = {Einfluss hochfrequenter Felder des Mobilfunks auf das blutbildende System in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145733}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Elektromagnetische Felder (EMF) sind in der Umwelt des Menschen allgegenw{\"a}rtig. Unter Verwendung unterschiedlicher Frequenzen bilden sie die Grundlage zahlreicher Technologien und begegnen uns im Alltag in einer Vielzahl von Anwendungen. Eine sehr wichtige Anwendung von EMF ist die mobile Kommunikation. Die hierf{\"u}r verwendeten Frequenzen liegen im hochfrequenten Bereich und variieren mit dem Mobilfunkstandard. Weit verbreitet ist die GSM- und UMTS-Modulation der zweiten (2G) und dritten Generation (3G). Zum neuesten Mobilfunkstandard z{\"a}hlt LTE (4G). Aus statistischen Daten geht hervor, dass derzeit weltweit mehr als sieben Milliarden Mobilfunk-Endger{\"a}te existieren. Die weitverbreitete und stetig ansteigende Verwendung dieser Technologien verdeutlicht, dass viele Menschen, darunter auch zunehmend Kinder und Jugendliche, regelm{\"a}ßig einer Exposition gegen{\"u}ber EMF ausgesetzt sind. Die wichtigste Expositionsquelle stellt dabei das Mobiltelefon dar, da sich in diesem Szenario die Quelle sehr nah am menschlichen K{\"o}rper befindet. In der Vergangenheit wurden zahlreiche in-vitro- und in-vivo-Untersuchungen sowie epidemiologische Studien durchgef{\"u}hrt, um potentielle, nicht-thermische Effekte von Mobilfunkstrahlung auf biologische Systeme beurteilen zu k{\"o}nnen. Ein vollst{\"a}ndiger Konsens konnte auf der Basis der erhaltenen Ergebnisse jedoch nicht erzielt werden, sodass weiterhin Bedenken zum sch{\"a}dlichen Potential dieser nichtionisierenden Strahlung bestehen. Insbesondere wurden Fragestellungen zu Langzeiteffekten sowie zu Effekten, die speziell bei Kindern eine besondere Rolle spielen, bisher nicht ausreichend adressiert. Kinder k{\"o}nnen empfindlicher auf Umwelteinfl{\"u}sse reagieren und sind im Vergleich zu Erwachsenen teilweise h{\"o}her gegen{\"u}ber EMF exponiert. Dies gilt vor allem f{\"u}r Kopfregionen, in denen sich das aktive, f{\"u}r die H{\"a}matopoese verantwortliche Knochenmark befindet. Vor diesem Hintergrund war es das Ziel der vorliegenden Arbeit, den Einfluss von Mobilfunkstrahlung auf das humane blutbildende System zu untersuchen. Im Fokus standen dabei humane h{\"a}matopoetische Stammzellen, die mit Frequenzen der Mobilfunkstandards GSM (900 MHz), UMTS (1.950 MHz) und LTE (2.535 MHz) jeweils {\"u}ber einen kurzen (4 h) und einen langen (20 h) Zeitraum und mit unterschiedlichen Intensit{\"a}ten (0 W/kg, 0,5 W/kg, 1 W/kg, 2 W/kg und 4 W/kg) exponiert wurden. Vergleichende Experimente erfolgten mit Zellen der Promyelozyten-Zelllinie HL-60. M{\"o}gliche Effekte wurden mit den Endpunkten Apoptose, oxidativer Stress, Zellzyklus, DNA-Schaden und -Reparatur sowie Differenzierung und Epigenetik in Form von Histonacetylierung bewertet. In keinem der genannten Endpunkte konnten klare Effekte durch Mobilfunkstrahlung ausgemacht werden, weder f{\"u}r die h{\"a}matopoetischen Stammzellen, noch f{\"u}r die Zelllinie HL-60. Die einzige Ver{\"a}nderung wurde bei der Quantifizierung von DNA-Sch{\"a}den beobachtet. Hier zeigte sich nach der Kurzzeitexposition der Stammzellen mit der Modulation GSM eine kleine, aber statistisch signifikante Abnahme der DNA-Sch{\"a}den verglichen mit der Scheinexposition. Diese Beobachtung ließ sich in weiteren Replikaten jedoch nicht reproduzieren und wurde daher als nicht biologisch relevant eingestuft. Insgesamt konnte mit dieser Arbeit gezeigt werden, dass durch Mobilfunkstrahlung mit Frequenzen der verbreiteten Modulationen GSM, UMTS und LTE sowie SAR-Werten, die unterhalb und oberhalb des empfohlenen Sicherheitsstandards liegen und typischerweise bei Handytelefonaten auftreten, keine Effekte in Zellen des blutbildenden Systems unter den gegebenen Versuchsbedingungen induziert wurden. Ein besonderer Fokus lag hierbei auf der Reproduzierbarkeit der Ergebnisse. Weiterhin wurden zum ersten Mal humane h{\"a}matopoetische Stammzellen f{\"u}r derartige Untersuchungen eingesetzt. Dies hat insofern eine besondere Bedeutung, als h{\"a}matopoetische Stammzellen aufgrund ihrer multipotenten Eigenschaften eine breitere Analyse mit Hinblick auf die Kanzerogenese und auf das Immunsystem erm{\"o}glichen. Um {\"u}ber die Mobilfunk-Untersuchungen hinaus die h{\"a}matopoetischen Stammzellen besser charakterisieren zu k{\"o}nnen, sowie die Sensitivit{\"a}t von Blutzellen mit unterschiedlichem Differenzierungsstatus zu analysieren, wurden sie anderen Zellen des blutbildenden Systems (undifferenzierte und differenzierte HL-60-Zellen und TK6-Zellen) gegen{\"u}bergestellt. Eine Behandlung der verschiedenen Zelltypen mit mutagenen Substanzen zeigte, dass sich die h{\"a}matopoetischen Stammzellen in den meisten der untersuchten Endpunkte von den Zelllinien unterschieden. Deutliche Abweichungen zeigten sich beim oxidativen Stress, der DNA-Reparatur und der Histonacetylierung; kein Unterschied konnte dagegen bei den DNA-Sch{\"a}den beobachtet werden. Eine erste Interpretation der erhaltenen Ergebnisse ist auf der Grundlage der unterschiedlichen Eigenschaften von Zellen mit abweichendem Differenzierungsstatus m{\"o}glich. Um jedoch eine eindeutige Aussage treffen zu k{\"o}nnen, m{\"u}ssten noch weitere Untersuchungen durchgef{\"u}hrt werden.}, subject = {Mobilfunk}, language = {de} } @phdthesis{Schmid2016, author = {Schmid, Evelyn}, title = {Effekte des Raf Kinase Inhibitor Proteins (RKIP) auf β-adrenerge Signalwege, Herzfunktion und die Entwicklung der Herzinsuffizienz}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142486}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Das Raf kinase inhibitor protein (RKIP) ist ein Kinaseregulator, der im Herzen eine Pr{\"a}ferenz f{\"u}r die G-Protein-gekoppelte Rezeptorkinase 2 (GRK2) zeigt. Die Regulation erfolgt durch direkte Interaktion beider Proteine, wird durch eine PKC-Phosphorylierung an Serin 153 des RKIP induziert und inhibiert die GRK2-vermittelte Phosphorylierung von G-Protein-gekoppelten Rezeptoren (GPCR). Die GRK2 desensitiviert GPCR und eine Hemmung der GRK2-Aktivit{\"a}t wirkt sich so positiv auf die Ansprechbarkeit von GPCR aus. Die \textbeta-adrenergen Rezeptoren (\textbeta AR) sind im Herzen maßgeblich an der Regulation der kardialen Kontraktilit{\"a}t beteiligt. Erste Zusammenh{\"a}nge zwischen der RKIP-Expression und der kontraktilen Antwort von Kardiomyozyten wurden bereits in einer fr{\"u}heren Arbeit untersucht und best{\"a}tigt. Sie begr{\"u}nden die Fragestellung nach Effekten einer verst{\"a}rkten RKIP-Expression auf \textbeta-adrenerge Rezeptorsignale, Herzfunktion und die Entwicklung der Herzinsuffizienz. Im Rahmen dieses Projektes konnten die Effekte des RKIP auf \textbeta-adrenerge Signalwege detaillierter beschrieben werden. Dabei erwies sich die inhibitorische Funktion auf die GRK2 als rezeptorspezifisch ohne Einfluss auf zytosolische Angriffspunkte der GRK2 zu nehmen. Verst{\"a}rkte \textbeta-adrenerge Signale zeigten sich in neonatalen Kardiomyozyten an Hand der erh{\"o}hten cAMP-Level, PKA-Aktivit{\"a}t, sowie Kontraktionsrate und Relaxationsgeschwindigkeit nach \textbeta-adrenerger Stimulation. Im Einklang damit konnte eine erh{\"o}hte PKA- und CaMKII-Aktivit{\"a}t und eine positive Inotropie in transgenen Tieren, mit herzspezifischer {\"U}berexpression von RKIP, beobachtet werden. Durch Messung des Calcium-\textit{Cyclings} in Kardiomyozyten konnte der Ph{\"a}notyp auf eine verbesserte R{\"u}ckf{\"u}hrung des Calciums, einer daraus resultierenden erh{\"o}hten Calciumbeladung des sarkoplasmatischen Retikulums und einem gesteigerten systolischen Calciumspiegel, zur{\"u}ckgef{\"u}hrt werden. Die Untersuchung der Phosphorylierung von Calciumkan{\"a}len, L-Typ-Calciumkanal und Ryanodin-Rezeptor 2, die den einw{\"a}rtsgerichteten Calciumstrom vermitteln konnte ihre Beteiligung an der positiv inotropen Wirkung ausschließen. Neben dem kontraktilen Ph{\"a}notyp konnten zus{\"a}tzliche protektive Effekte beobachtet werden. In Modellen, die eine chronische \textbeta-adrenerge Stimulation imitieren, bzw. eine Nachlasterh{\"o}hung induzieren konnte eine Verringerung der interstitiellen Fibrose und der damit assoziierten Marker, gezeigt werden. Mit Hilfe von \textit{in vivo} EKG-Messungen konnte die Neigung zur Ausbildung von Arrhythmien untersucht werden. Auch im Hinblick auf die Anzahl der Extrasystolen waren RKIP-transgene Tiere gesch{\"u}tzt. Infolge der Untersuchung der Ph{\"a}notypen in Deletionshintergr{\"u}nden der einzelnen \textbeta AR-Subtypen (\textbeta\textsubscript{1}AR, \textbeta\textsubscript{2}AR) konnte die positive Inotropie mit den spezifischen Signalwegen des \textbeta\textsubscript{1}AR assoziiert und die protektiven Effekte gegen{\"u}ber den Umbauprozessen und der Arrhythmieneigung dem \textbeta\textsubscript{2}-adrenergen Signalen zugeschrieben werden. Zus{\"a}tzlich best{\"a}tigt sich eine besondere Rolle der G\textalpha\textsubscript{i}-Kopplung des \textbeta\textsubscript{2}AR, durch die er einen hemmenden Einfluss auf die \textbeta\textsubscript{1}AR-Singale nehmen kann. Die Untersuchung einiger Marker, die eine physiologische von einer pathologischen Hypertrophie unterscheiden, konnte das in den RKIP-transgenen M{\"a}usen auftretende Wachstum der Kardiomyozyten als kompensatorische und physiologische Hypertrophie charakterisieren. Zusammengenommen weisen diese Ergebnisse auf eine ausgeglichene Aktivierung der beiden Rezeptoren hin, die sich gegenseitig regulieren und durch die Inhibition der GRK2 in ihrer Anregbarkeit erhalten bleiben. Mittels einer AAV9-vermittelten Gentherapie konnte das therapeutische Potential dieses Prinzips weiter best{\"a}tigt werden, da es die prominentesten Ver{\"a}nderungen w{\"a}hrend der Herzinsuffizienzentwicklung, wie die Verschlechterung der linksventrikul{\"a}ren Funktion, die Dilatation des linken Ventrikels, die Ausbildung von Lungen{\"o}demen und interstitieller Fibrose sowie die Expression von Herzinsuffizienz-assoziierten Genen, verhindern konnte. Auch konnten die Auswirkungen der Deletion des RKIP, die sich durch eine beschleunigte und gravierendere Herzinsuffizienzentwicklung auszeichnet, durch Reexpression von RKIP verhindert werden. Diese Arbeit kann somit zeigen, dass das RKIP eine ausgeglichene Verst{\"a}rkung von \textbeta-adrenergen Signalwegen verursacht, die positiv inotrop und gleichzeitig protektiv wirkt. Dieses Wirkprinzip k{\"o}nnte ferner eine Strategie zur Erh{\"o}hung der Kontraktilit{\"a}t in der Herzinsuffizienz darstellen, die entgegen etablierter Theorien auf der Stimulation beider \textbeta AR basiert.}, subject = {Herzinsuffizienz}, language = {de} } @phdthesis{Kahlert2016, author = {Kahlert, Katrin}, title = {Der Einfluss von RKIP auf die Progression von Herzinsuffizienz}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139191}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {GRK2 vermittelt {\"u}ber die Phosphorylierung und Inaktivierung kardialer β1-Rezeptoren eine verminderte kardiale Kontraktilit{\"a}t. RKIP als GRK2-Inhibitor spielt eine Rolle in der GPCR-Signalgebung. Die {\"U}berexpression des Proteins f{\"u}hrt zu einer verbesserten Herzfunktion. Dieser Effekt wird m{\"o}glicherweise {\"u}ber die GRK2-Inhibition vermittelt und er{\"o}ffnet die Diskussion {\"u}ber weitere durch RKIP vermittelte protektive Effekte im Herzen. In dieser Arbeit konnte ich zeigen, dass die kardiale RKIP-Expression in M{\"a}usen und humanem Herzgewebe bei Herzinsuffizienz gesteigert ist. Zudem beschrieb ich den protektiven Effekt einer gesteigerten Expression von RKIP in murinen Herzen im Hinblick auf die Auspr{\"a}gung typischer struktureller und morphologischer Zeichen von Herzinsuffizienz. Echokardiographische Untersuchungen zeigten, dass RKIP die Herzfunktion positiv beeinflusst. RKIP-tg-M{\"a}use wiesen eine gesteigerte Verk{\"u}rzungsfraktion und einen dauerhaft hyperkontraktilen Ph{\"a}notyp auf. Trotz fehlenden Einflusses auf die kardiale Hypertrophie bewirkte die chronische linksventrikul{\"a}re Druckbelastung durch TAC in RKIP-tg-M{\"a}usen eine geringere kardiale Dilatation und den Erhalt einer st{\"a}rkeren Kontraktilit{\"a}t als in Wildtyp-M{\"a}usen. Die Ligation der Aorta transversa bewirkte bei Wildtyp-M{\"a}usen zudem strukturelle und molekulare Ver{\"a}nderungen, die typisch f{\"u}r einen herzinsuffizienten Ph{\"a}notyp sind. Der Anteil fibrotischen Gewebes und die Apoptose im Herzen nahmen zu. Strukturelle Ver{\"a}nderungen des Herzgewebes sind ein Korrelat f{\"u}r ein herzinsuffizientes Herz. RKIP-tg-M{\"a}use zeigten diese Ver{\"a}nderungen in einem deutlich geringeren Ausmaß und weisen auf eine protektive Wirkung einer kardialen RKIP-{\"U}berexpression hin. Interessanterweise war die mRNA-Expression der Fibrosemarker CTGF und TGFß nach chronischer linksventrikul{\"a}rer Druckbelastung sowohl bei Wildtyp-M{\"a}usen als auch bei RKIP-transgenen M{\"a}usen erh{\"o}ht. Die Ursache f{\"u}r das Fehlen eines signifikanten Unterschiedes k{\"o}nnte sein, dass diese Marker nicht spezifisch f{\"u}r die kardiale Fibrosierung sind, sondern deren Expression auch mit der kardialen Hypertrophie zusammenh{\"a}ngt. Eine weitere Beobachtung war der Anstieg der mRNA-Expression der Herzinsuffizienz-Marker BNP und ANF nach chronischer Druckbelastung in Wildtyp- und RKIP-tg-M{\"a}usen. Die Ergebnisse best{\"a}tigten, dass BNP spezifischer f{\"u}r die durch chronische linksventrikul{\"a}re Druckerh{\"o}hung verursachte Herzinsuffizienz zu sein scheint. Ich beobachtete eine gesteigerte RKIP-Expression bei Herzinsuffizienz und kardialer Hypertrophie. Herzbiopsien herzinsuffizienter und an Aortenstenose erkrankter Patienten wiesen im Vergleich zu Kontrollen eine erh{\"o}hte RKIP-Proteinexpression auf. Auch C57BL/6J-M{\"a}use wiesen nach chronischer linksventrikul{\"a}rer Druckbelastung eine gesteigerte kardiale RKIP-Expression im Vergleich zu Kontrollen auf. Die Hochregulation der RKIP-Expression k{\"o}nnte als protektiver feedback-Mechanismus interpretiert werden. Resultat dieser Arbeit ist, dass RKIP eine protektive Wirkung bei der Progression von durch chronische linksventrikul{\"a}re Druckbelastung induzierte Herzinsuffizienz hat, am ehesten durch seine Funktion als GRK2-Inhibitor und seine Rolle bei der GPCR-Signalgebung.}, subject = {Herzinsuffizienz}, language = {de} } @phdthesis{Spiegel2015, author = {Spiegel, Silvana}, title = {Mikrokernfrequenzanalyse unter dem Einfluss von Methylphenidat und chronischem Stress bei adultem ADHS}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139387}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Einleitung: Methylphenidat (MPH) als Medikament der ersten Wahl bei Patienten mit einem Aufmerksamkeitsdefizit- /Hyperaktivit{\"a}tssyndrom (ADHS) ist f{\"u}r die Therapie von Kindern aber auch von Erwachsenen weit verbreitet. Weil es immer noch Sicherheitsbedenken gegen dieses Medikament gibt, wurde in der vorliegenden Studie untersucht, ob die Langzeiteinnahme von MPH unsch{\"a}dlich hinsichtlich eines zytogenetischen Effektes ist. Ein weiteres Ziel war die Beurteilung von chronischer psychosozialer Stressbelastung von Patienten im Vergleich zu Kontrollprobanden und zu beurteilen ob die Medikation einen Einfluss auf die H{\"o}he des Stresses hat. Nicht zuletzt war das dritte Ziel der Studie zu untersuchen, ob Stress selbst zu zytogenetischen Sch{\"a}den f{\"u}hrt. Material und Methoden: Lymphozyten von 72 (42 ADHS- und 28 gesunde Kontrollprobanden) geschlechts- und altersgematchte Probanden im Alter von 18-28 Jahren, wurden aus ven{\"o}sem Blut f{\"u}r den Mikronukleusassay isoliert. Hauptendpunkt der Studie war die Mikrokernanzahl in binukle{\"a}ren Zellen. Die psychosoziale Stressbelastung der letzten drei Monate wurde mit dem Trier Inventar zum chronischen Stress (TICS) gemessen. Zus{\"a}tzlich wurden Speichelproben f{\"u}r eine Cortisolmessung gesammelt. Ergebnisse: Ein Einfluss der MPH-Einnahme auf die Mikrokernfrequenz konnte nicht gefunden. ADHS-Patienten wiesen eine signifikant h{\"o}here Stressbelastung im Vergleich zu den Kontrollprobanden auf. Ein signifikanter positiver Einfluss auf das chronische Stresserleben unter MPH-Einnahme konnte bei Einnahme von mehr als 1 Jahr beobachtet werden. Die Stressbelastung der ADHS-Patienten und Kontrollprobanden zeigte keine Korrelation zu zytogenetischen Endpunkten. Eine kleine Untergruppe, ADHS-Patienten mit Komorbidit{\"a}t Depression, zeigte jedoch signifikante erh{\"o}hte Mikrokernfrequenzanzahlen unter stark erh{\"o}htem chronischen Stress. Aussichten: Aus unserer Sicht kann MPH auch in der Langzeittherapie sicher hinsichtlich eines Krebsrisikos in gewichts- und symptomadaptierter Dosis eingesetzt werden. Weitere Studien sind n{\"o}tig um das Krebsrisiko bei chronischer erh{\"o}hter Stressbelastung abzusch{\"a}tzen.}, subject = {ADHS}, language = {de} } @article{FedericoRedentiSturleseetal.2015, author = {Federico, Stephanie and Redenti, Sara and Sturlese, Mattia and Ciancetta, Antonella and Kachler, Sonja and Klotz, Karl-Norbert and Cacciari, Barbara and Moro, Stefano and Spalluto, Giampiero}, title = {The Influence of the 1-(3-Trifluoromethyl-Benzyl)-1H-Pyrazole-4-yl Moiety on the Adenosine Receptors Affinity Profile of Pyrazolo[4,3-e][1,2,4]Triazolo[1,5-c]Pyrimidine Derivatives}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0143504}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137133}, pages = {e0143504}, year = {2015}, abstract = {A new series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) derivatives has been developed in order to explore their affinity and selectivity profile at the four adenosine receptor subtypes. In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A\(_{2B}\) adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl) benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6(3H, 7H)-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA\(_{2B}\) AR but they displayed affinity at the hA\(_3\) AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA\(_3\) AR K\(_i\) = 11 nM) and selectivity (A\(_1\)/A\(_3\) and A\(_{2A}\)/A\(_3\) > 9090; A\(_{2B}\)/A\(_3\) > 909) at the hA\(_3\) AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs.}, language = {en} } @article{KlotzMentrupRegensburgeretal.2012, author = {Klotz, Barbara and Mentrup, Birgit and Regensburger, Martina and Zeck, Sabine and Schneidereit, Jutta and Schupp, Nicole and Linden, Christian and Merz, Cornelia and Ebert, Regina and Jakob, Franz}, title = {1,25-Dihydroxyvitamin D3 Treatment Delays Cellular Aging in Human Mesenchymal Stem Cells while Maintaining Their Multipotent Capacity}, series = {PLoS ONE}, volume = {7}, journal = {PLoS ONE}, number = {1}, doi = {10.1371/journal.pone.0029959}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133392}, pages = {e29959}, year = {2012}, abstract = {1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, beta-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence-and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to beta-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.}, language = {en} } @phdthesis{Leyh2015, author = {Leyh, Annekathrin}, title = {In vitro Untersuchungen zur Genotoxizit{\"a}t von Insulin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131986}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Insulin ist ein essentielles Hormon im menschlichen K{\"o}rper, welches f{\"u}r die Senkung der Blutglukosekonzentration, die Bildung von Energiespeichern und das Zellwachstum verantwortlich ist. Eine mit der Fehlregulation der Insulinproduktion einhergehenden Krankheit ist der Diabetes mellitus. F{\"u}r diese Arbeit spielt der Typ 2 dieser Erkrankung eine wichtige Rolle. Es entwickelt sich bei Patienten mit diesem Typ des Diabetes mellitus langsam eine Insulinresistenz, die zun{\"a}chst durch eine kompensatorische {\"U}berproduktion von Insulin charakterisiert ist. Dieser Zustand der Hyperinsulin{\"a}mie kann Jahre bis Jahrzehnte andauern, ehe es zu einem Versagen der ß-Zellen des Pankreas und somit zu einer Hypoinsulin{\"a}mie kommt. In dieser Arbeit war es Ziel herauszufinden, ob diese lange Zeit herrschende Hyperinsulin{\"a}mie einen Einfluss auf die menschliche DNA hat. Die Genotoxizit{\"a}t von hohen Insulinkonzentrationen wurde in Hep-G2 Zellen, HT29 Zellen, sowie prim{\"a}ren humanen peripheren Lymphozyten mithilfe des Comet Assays und des Mikrokerntests nachgewiesen. Oxidativer Stress bzw. dessen Reduzierung durch Antioxidantien und Inhibitoren wurde in HT29 Zellen mithilfe der DHE-F{\"a}rbung detektiert. Diese Arbeit belegt dass sich Insulin sch{\"a}digend auf das menschliche Genom in vitro auswirken kann. Eine besondere Relevanz haben die durchgef{\"u}hrten Experimente mit prim{\"a}ren menschlichen Lymphozyten. Denn bei ihnen handelt es sich um Zellen, die im Gegensatz zu der auch genutzten humanen Leberkarzinomzelllinie Hep-G2 und der humanen Kolonkarzinomzelllinie HT29 nicht transformiert sind. Eine weitere wesentliche Erkenntnis dieser Arbeit ist, dass schon pathophysiologisch vorliegende Insulinkonzentrationen in der Lage sind Genomsch{\"a}digungen in vitro zu induzieren. HT29 Zellen zeigten bei Kurzzeitbehandlung mit nur 1nM Insulin eine signifikante Erh{\"o}hung der DNA-Sch{\"a}digung. Bei Langzeitexposition von 6 Tagen konnten schon 0,5nM signifikante DNA-Sch{\"a}den hervorrufen. Diese durch Insulin hervorgerufenen Sch{\"a}den k{\"o}nnten, falls sie so auch in vivo entstehen, bei Versagen von Reparaturmechanismen zur Entstehung von Mutationen und sich daraus entwickelnden Karzinomen beitragen. Aus diesem Grund war ein weiteres Ziel dieser Arbeit herauszufinden, ob bestimmte Antioxidantien oder Inhibitoren in der Lage sind die Insulin-induzierten Genomsch{\"a}digungen zu verringern. Hierf{\"u}r wurde Tempol, Apocynin, Plumbagin, VAS2870, Rotenone, PPP, HNMPA-(AM)3 und Wortmannin genutzt. Tats{\"a}chlich sind diese Substanzen in der Lage die durch Insulin hervorgerufene Sch{\"a}digung zu reduzieren. Die positiven Ergebnisse dieser Arbeit k{\"o}nnten einen ersten Hinweis auf eine m{\"o}gliche pharmakologische Intervention bei Hyperinsulin{\"a}mie mit dem Ziel der Senkung des erh{\"o}hten Krebsrisikos geben. Eine wichtige Erkenntnis aus den Ergebnissen meiner Arbeit ist, dass die Reduzierung des oxidativen Stresses eine Reduzierung der Genomsch{\"a}digung bewirkt. Die genutzten Substanzen Apocynin, Tempol, VAS2870 und Rotenone bewirkten in HT29 Zellen eine signifikante Reduzierung des durch Insulin ausgel{\"o}sten oxidativen Stresses. Um aber genauere Aussagen {\"u}ber M{\"o}glichkeiten der Therapie bei Hyperinsulin{\"a}mie zu treffen, sollten Folgestudien auch in vivo folgen, welche die in dieser Arbeit beschriebenen Effekte best{\"a}tigen.}, subject = {Insulin}, language = {de} } @article{ChenGassnerBoerneretal.2012, author = {Chen, Wen and Gaßner, Birgit and B{\"o}rner, Sebastian and Nikolaev, Viacheslav O. and Schlegel, Nicolas and Waschke, Jens and Steinbronn, Nadine and Strasser, Ruth and Kuhn, Michaela}, title = {Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway}, series = {Cardiovascular Research}, volume = {93}, journal = {Cardiovascular Research}, number = {1}, doi = {10.1093/cvr/cvr279}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126562}, pages = {141-151}, year = {2012}, abstract = {Aims Cardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP. Methods and results Intravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1-/- mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains. Conclusion ANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.}, language = {en} } @article{WippelMaurerFortschetal.2013, author = {Wippel, Carolin and Maurer, Jana and Fortsch, Christina and Hupp, Sabrina and Bohl, Alexandra and Ma, Jiangtao and Mitchell, Timothy J. and Bunkowski, Stephanie and Br{\"u}ck, Wolfgang and Nau, Roland and Iliev, Asparouh I.}, title = {Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage}, series = {PLoS Pathogens}, volume = {9}, journal = {PLoS Pathogens}, number = {6}, doi = {10.1371/journal.ppat.1003380}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130462}, pages = {e1003380}, year = {2013}, abstract = {Abstract Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage. Author Summary Bacterial meningitis is one of the most devastating brain diseases. Among the bacteria that cause meningitis, Streptococcus pneumoniae is the most common. Meningitis predominantly affects children, especially in the Third World, and most of them do not survive. Those that do survive often suffer permanent brain damage and hearing problems. The exact morphological substrates of brain damage in Streptococcus pneumoniae meningitis remain largely unknown. In our experiments, we found that the brain cortex of patients with meningitis demonstrated a loss of synapses (the contact points among neurons, responsible for the processes of learning and memory), and we identified the major pneumococcal neurotoxin pneumolysin as a sufficient cause of this loss. The effect was not direct but was mediated by the brain neurotransmitter glutamate, which was released upon toxin binding by one of the non-neuronal cell types of the brain - the astrocytes. Pneumolysin initiated calcium influx in astrocytes and subsequent glutamate release. Glutamate damaged the synapses via NMDA-receptors - a mechanism similar to the damage occurring in brain ischemia. Thus, we show that synaptic loss is present in pneumococcal meningitis, and we identify the toxic bacterial protein pneumolysin as the major factor in this process. These findings alter our understanding of bacterial meningitis and establish new therapeutic strategies for this fatal disease.}, language = {en} } @article{HuppFoertschWippeletal.2013, author = {Hupp, Sabrina and F{\"o}rtsch, Christina and Wippel, Carolin and Ma, Jiangtao and Mitchell, Timothy J. and Iliev, Asparouh I.}, title = {Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin}, series = {Journal of Molecular Biology}, volume = {425}, journal = {Journal of Molecular Biology}, number = {3}, doi = {10.1016/j.jmb.2012.11.034}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132297}, pages = {636-646}, year = {2013}, abstract = {The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by F{\"o}rster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170-190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought.}, language = {en} } @article{CapraBusnelliPerennaetal.2013, author = {Capra, Val{\´e}rie and Busnelli, Marta and Perenna, Alessandro and Ambrosio, Manuela and Accomazzo, Maria Rosa and Gal{\´e}s, Celine and Chini, Bice and Rovati, G. Enrico}, title = {Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0060475}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131013}, pages = {e60475}, year = {2013}, abstract = {The intrahelical salt bridge between \(E/D^{3.49}\) and \(R^{3.50}\) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of \(E^{3.49/6.30}\) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.}, language = {en} } @article{LohseKlotzUkenaetal.1984, author = {Lohse, M. J. and Klotz, K.-N. and Ukena, D. and Schwabe, U.}, title = {Characterization of \([^3H]\)Phenobarbital Binding to Rat Brain Membranes}, series = {Neuroscience Letters}, volume = {52}, journal = {Neuroscience Letters}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127894}, pages = {97-101}, year = {1984}, abstract = {The binding of \([^3H]\)phenobarbital to rat brain membranes was studied in order to determine its characteristics and specificity. The binding reaction was rapid and occurred at sites of low affinity. \((K_d = 700 μM)\) and very high density \((B_{max} = 2.7 nmoll/mg protein)\). It was unaffected by temperature changes from O°C to 95°C and was maximal at pH 5. Detergents in low concentrations markedly decreased the binding, apparently without solubilizing the binding sites. It is concluded that the binding of \([^3H]\) phenobarbital is a rather non-specific interaction with the plasma membrane.}, language = {en} } @article{LohseKlotzSalzeretal.1988, author = {Lohse, Martin J. and Klotz, Karl-Norbert and Salzer, Manfred J. and Schwabe, Ulrich}, title = {Adenosine regulates the \(Ca^{2+} \) sensitivity of mast cell mediator release : (histamine secretion/inositol phosphates/calcium)}, series = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {85}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127883}, pages = {8875-8879}, year = {1988}, abstract = {Mast cells release histamine and other mediators of allergy in response to stimulation of their IgE receptors. This release is generally thought to be mediated by an elevation of cytosolic \(Ca^{2+}\). Recent evidence suggests that there might be factors that modulate the coupling between \(Ca^{2+}\) levels and mediator release. The present report identifies adenosine as one such modulator. Adenosine and several of its metabolically stable analogues were shown to enhance histamine release from rat peritoneal mast cells in response to stimuli such as concanavalin A. Metabolizing endogenous adenosine with adenosine deaminase dampened the response to stimuli, whereas trapping endogenous adenosine inside mast cells with nucleoside-transport inhibitors markedly enhanced stimulated histamine release. The metabolically stable adenosine analogue 5' -(N-ethylcarboxamido)adenosine (NECA) did not affect the initial steps in the sequence from IgE-receptor activation to mediator release, which are generation of inositol trisphosphate and increase of cytosolic \(Ca^{2+}\). However, NECA did enhance the release induced in ATP-permeabilized cells by exogenous \(Ca^{2+}\), but it had no effect on the release induced by phorbol esters. These data suggest that adenosine sensitizes mediator release by a mechanism regulating stimulus-secretion coupling at a step distal to receptor activation and second-messenger generation.}, language = {en} } @article{OttLohseKlotzetal.1982, author = {Ott, Ilka and Lohse, Martin J. and Klotz, Karl-Norbert and Vogt-Moykopf, Ingolf and Schwabe, Ulrich}, title = {Effects of Adenosine on Histamine Release from Human Lung Fragments}, series = {International Archives of Allergy and Immunology}, volume = {98}, journal = {International Archives of Allergy and Immunology}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127877}, pages = {50-56}, year = {1982}, abstract = {The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A 23187 orwith concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl] methyloxy]-phenyl]-1,3-dipropylaxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioninosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence ofXAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a si te antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.}, language = {en} } @article{RychlikHumpfMarkoetal.2014, author = {Rychlik, Michael and Humpf, Hans-Ulrich and Marko, Doris and D{\"a}nicke, Sven and Mally, Angela and Berthiller, Franz and Klaffke, Horst and Lorenz, Nicole}, title = {Proposal of a comprehensive definition of modified and other forms of mycotoxins including "masked" mycotoxins}, series = {Mycotoxin Research}, volume = {30}, journal = {Mycotoxin Research}, number = {4}, doi = {10.1007/s12550-014-0203-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121240}, pages = {197-205}, year = {2014}, abstract = {As the term "masked mycotoxins" encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, "modified mycotoxins" into "biologically modified" and "chemically modified" with all variations of metabolites of the former and dividing the latter into "thermally formed" and "non-thermally formed" ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term "modified mycotoxins" should be used in the future and the term "masked mycotoxins" to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.}, language = {en} } @phdthesis{ZinkgebSondergeld2015, author = {Zink [geb. Sondergeld], Thomas Gerd}, title = {Der Cofilin-Signalweg im Glioblastoma multiforme - Ursachen f{\"u}r den Verlust von Chronophin und Einfluss von LIM-Kinase-Inhibitoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127065}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Das invasive Potential maligner Gliome beeinflusst maßgeblich die schlechte Prognose dieser Tumorentit{\"a}t. Migration und Invasion von Tumorzellen werden entscheidend durch die Cofilin-vermittelte Umstrukturierung des Aktin-Zytoskeletts gepr{\"a}gt, die durch die Aktivit{\"a}t antagonistischer Cofilin-Kinasen und -Phosphatasen reguliert wird. Im Rahmen der vorliegenden Arbeit konnte ein progressiver Expressionsverlust der Cofilin-Phosphatase Chronophin mit ansteigendem Malignit{\"a}tsgrad astrozyt{\"a}rer Gliome aufgezeigt werden, der mit einer Zunahme der Phosphorylierung von Cofilin einhergeht. In den entsprechenden Gewebeproben gelang gleichzeitig der Nachweis einer gesteigerten Expression der Cofilin-Kinase LIMK-2. Genetische und epigenetische Analysen des Chronophin-Locus konnten eine Hypermethylierung im Bereich der Promotorregion der Phosphatase identifizieren, die m{\"o}glicherweise dem Verlust von Chronophin in Glioblastom-Gewebeproben zugrunde liegt. In Glioblastom-Zelllinien, die unterschiedliche Expressionsmuster von Chronophin aufwiesen, konnten hingegen keine molekularen Alterationen festgestellt werden. Untersuchungen des Einflusses von ROCK- und LIMK-Inhibitoren auf Glioblastomzellen konnten ausgepr{\"a}gte Ver{\"a}nderungen der Zellmorphologie dokumentieren, wobei erstmals die Induktion eines stellate cell-Ph{\"a}notyps unter Einfluss des LIMK-Inhibitors BMS-5 beschrieben wird. W{\"a}hrend ROCK- und LIMK-Inhibitoren keinen Einfluss auf die 2D-Motilit{\"a}t der Tumorzellen hatten, wiesen die Glioblastomzellen in Abh{\"a}ngigkeit ihrer basalen Cofilin-Aktivit{\"a}t eine verst{\"a}rkte bzw. verminderte 3D-Invasivit{\"a}t auf. Die Erkenntnisse dieser Arbeit unterstreichen die Bedeutung des Cofilin-Signalweges f{\"u}r die Migration und Invasion von Gliomzellen, zeigen neue Angriffspunkte in der Therapie maligner Gliome auf und warnen zugleich vor einem unkritischen Einsatz neuer Wirkstoffe.}, subject = {Cofilin}, language = {de} } @phdthesis{Stumpf2015, author = {Stumpf, Anette D.}, title = {Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125469}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements. For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future.}, subject = {Adenosinrezeptor}, language = {en} } @article{BoivinBeyersdorfPalmetal.2015, author = {Boivin, Val{\´e}rie and Beyersdorf, Niklas and Palm, Dieter and Nikolaev, Viacheslav O. and Schlipp, Angela and M{\"u}ller, Justus and Schmidt, Doris and Kocoski, Vladimir and Kerkau, Thomas and H{\"u}nig, Thomas and Ertl, Georg and Lohse, Martin J. and Jahns, Roland}, title = {Novel Receptor-Derived Cyclopeptides to Treat Heart Failure Caused by \(Anti-β_1-Adrenoceptor\) Antibodies in a Human-Analogous Rat Model}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {2}, doi = {10.1371/journal.pone.0117589}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126028}, pages = {e0117589}, year = {2015}, abstract = {Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the \(β_1\) adrenergic receptor \((β_1EC2)\). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195-225) every 4 weeks; n = 38/114 rats were control-injected with 0.9\% NaCl. Intravenous application of a novel cyclic peptide mimicking \(β_1EC2\) (\(β_1EC2-CP\), 1.0 mg/kg every 4 weeks) or administration of the \(β_1-blocker\) bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received \(β_1EC2-CP/bisoprolol\) co-treatment. We found that \(β_1EC2-CP\) prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, \(β_1EC2-CP\) mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free \(anti-β_1EC2-antibodies\) and by targeting \(β_1EC2\)-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful \(anti-β_1EC2-antibodies\) and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to \(β_1\)-blockade represents a promising new therapeutic option in immune-mediated heart failure.}, language = {en} } @phdthesis{Bellwon2015, author = {Bellwon, Patricia}, title = {Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122693}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program "Predict-IV"; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of "omics" technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling. This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day. Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors. Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling. Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions. Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo. Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound's distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.}, subject = {Zellkultur}, language = {en} } @article{CalebiroMaiellaro2014, author = {Calebiro, Davide and Maiellaro, Isabella}, title = {cAMP signaling microdomains and their observation by optical methods}, series = {Frontiers in Cellular Neuroscience}, volume = {8}, journal = {Frontiers in Cellular Neuroscience}, issn = {1662-5102}, doi = {10.3389/fncel.2014.00350}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118252}, pages = {350}, year = {2014}, abstract = {The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains.}, language = {en} } @article{BrandAmannMandeletal.2014, author = {Brand, Susanne and Amann, Kerstin and Mandel, Philipp and Zimnol, Anna and Schupp, Nicole}, title = {Oxidative DNA Damage in Kidneys and Heart of Hypertensive Mice Is Prevented by Blocking Angiotensin II and Aldosterone Receptors}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {12}, issn = {1932-6203}, doi = {10.1371/journal.pone.0115715}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118011}, pages = {e115715}, year = {2014}, abstract = {INTRODUCTION: Recently, we could show that angiotensin II, the reactive peptide of the blood pressure-regulating renin-angiotensin-aldosterone-system, causes the formation of reactive oxygen species and DNA damage in kidneys and hearts of hypertensive mice. To further investigate on the one hand the mechanism of DNA damage caused by angiotensin II, and on the other hand possible intervention strategies against end-organ damage, the effects of substances interfering with the renin-angiotensin-aldosterone-system on angiotensin II-induced genomic damage were studied. METHODS: In C57BL/6-mice, hypertension was induced by infusion of 600 ng/kg • min angiotensin II. The animals were additionally treated with the angiotensin II type 1 receptor blocker candesartan, the mineralocorticoid receptor blocker eplerenone and the antioxidant tempol. DNA damage and the activation of transcription factors were studied by immunohistochemistry and protein expression analysis. RESULTS: Administration of angiotensin II led to a significant increase of blood pressure, decreased only by candesartan. In kidneys and hearts of angiotensin II-treated animals, significant oxidative stress could be detected (1.5-fold over control). The redox-sensitive transcription factors Nrf2 and NF-κB were activated in the kidney by angiotensin II-treatment (4- and 3-fold over control, respectively) and reduced by all interventions. In kidneys and hearts an increase of DNA damage (3- and 2-fold over control, respectively) and of DNA repair (3-fold over control) was found. These effects were ameliorated by all interventions in both organs. Consistently, candesartan and tempol were more effective than eplerenone. CONCLUSION: Angiotensin II-induced DNA damage is caused by angiotensin II type 1 receptor-mediated formation of oxidative stress in vivo. The angiotensin II-mediated physiological increase of aldosterone adds to the DNA-damaging effects. Blocking angiotensin II and mineralocorticoid receptors therefore has beneficial effects on end-organ damage independent of blood pressure normalization.}, language = {en} } @article{AgarwalYangRiceetal.2014, author = {Agarwal, Shailesh R. and Yang, Pei-Chi and Rice, Monica and Singer, Cherie A. and Nikolaev, Viacheslav O. and Lohse, Martin J. and Clancy, Colleen E. and Harvey, Robert D.}, title = {Role of Membrane Microdomains in Compartmentation of cAMP Signaling}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {4}, issn = {1932-6203}, doi = {10.1371/journal.pone.0095835}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116673}, pages = {e95835}, year = {2014}, abstract = {Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.}, language = {en} } @phdthesis{Riese2014, author = {Riese, Thorsten}, title = {Analysen zur potentiellen Gentoxizit{\"a}t von Terahertzstrahlung in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116469}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Im Rahmen dieser Arbeit wurden immortalisierte humane Keratinozyten (HaCaT-Zelllinie) {\"u}ber eine Dauer von 24 Stunden mit Terahertzstrahlung der Frequenz 0,106 THz und einer Leistungsflussdichte von 2 mW/cm² behandelt und anschließend mittels „cytokinesis-block micronucleus cytome assay" ausgewertet. Ferner wurden Proben der gleichen Zelllinie {\"u}ber einen Zeitraum von 24 Stunden Temperaturen zwischen 37 °C und 42 °C ausgesetzt und zum einen auf Hinweise f{\"u}r Gentoxizit{\"a}t mit Hilfe des Mikrokerntests untersucht und zum anderen mittels Western Blot die Expressionsrate von Hitzeschockproteinen der 70 kDa Familie bestimmt. Die der Terahertzstrahlung ausgesetzten Zellen zeigten gegen{\"u}ber den Kontrollen keine signifikanten Ver{\"a}nderungen der Marker f{\"u}r chromosomale Aberrationen oder der Zellteilung. Die der erh{\"o}hten Temperatur ausgesetzten Zellen zeigten einen signifikanten Anstieg der Mikrokernraten und weiterer Marker f{\"u}r Gentoxizit{\"a}t sowie eine damit korrelierende Zunahme der synthetisierten Proteinmenge der Hsp70 Proteine im Rahmen der Hitzeschockreaktion.}, subject = {Terahertzbereich}, language = {de} } @phdthesis{Ruebeling2014, author = {R{\"u}beling, Karsten}, title = {Identifizierung und pharmakologische Charakterisierung von Adenosinrezeptoren in humanen Melanomzelllinien}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115137}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Adenosin steuert seine physiologische Funktionen {\"u}ber die vier G-Protein gekoppelten Adenosinrezeptoren A1, A2A, A2B und A3 und kann enormen Einfluss auf die Zellphysiologie und das Immunsystem haben. Hohe Adenosinkonzentrationen in Tumorgeweben haben das Interesse vieler Forschungsgruppen geweckt und den Nachweis von AR und dessen tumorf{\"o}rdernde sowie -hemmende Wirkung auf diverse Krebszellen nach sich gezogen. Melanome sind f{\"u}r 90\% der letal ausgehenden Hautkrebsformen verantwortlich, da bereits kleinste Formen metastasieren k{\"o}nnen und vielmals eine rein chirurgische Therapie keine Heilung verspricht. Bei ung{\"u}nstiger Prognose schr{\"a}nken adjuvante und systemische Therapieerg{\"a}nzungen die Lebensqualit{\"a}t erheblich ein. Auf der Suche nach gezielteren und schonenderen Therapieans{\"a}tzen gilt es, die Rolle des Adenosins im Tumorgeschehen weiter aufzukl{\"a}ren, was den Nachweis und die Charakterisierung von AR und deren rezeptorspezifisch-gekoppelte Effekte voraussetzt. Daher sollten in der hier vorliegenden Arbeit die f{\"u}nf humanen Melanomzelllinien A375, Brown, MV3, SK MEL23 und MEL2A auf die Expression von AR untersucht werden. Zu Beginn wurden die Zellen mit Hilfe von Bindungsexperimenten unter Einsatz radioaktiv markierter Liganden ([3H]HEMADO, [3H]CCPA, [3H]NECA und [3H]ZM 241385) auf das Vorliegen der vier AR-Subtypen untersucht. A1- und A3-AR wurden nach Bindungsstudien mit [3H]CCPA bzw. [3H]HEMADO ausgeschlossen und nicht weiter untersucht. Durch Bindungsstudien mit [3H]ZM 241385 konnten in den Zellen Brown, MV3, SK-MEL23 und MEL2A geringe Mengen von A2B-Rezeptoren detektiert werden. Es folgten funktionelle Untersuchungen zur Stimulation der AC mit den Liganden NECA und CGS 21680 unter besonderem Blick auf die Differenzierung zwischen einer A2A- oder A2B vermittelten Stimulation. Ein NECA vermittelter Anstieg des c[32P]AMP im AC-Versuch best{\"a}tigte die Anwesenheit von A2B-Rezeptoren in den Zellen A375, Brown, MV3 und SK-MEL23. Durch CGS 21680 kam es hingegen zu keiner AC-Stimulation, was eine A2A-AR-Beteiligung ausschloss. Zur Best{\"a}tigung einer A2B gekoppelten AC-Aktivit{\"a}t wurde zum Abschluss der Versuche die Wirkung von drei selektiven Antagonisten (DPCPX, SCH 58261 und MRE 3008 F20) auf ein NECA induziertes cAMP-Signal {\"u}berpr{\"u}ft. Mit den drei Antagonisten konnte an den Zelllinien A375, Brown und MV3 die f{\"u}r A2B-AR zu erwartende Wirkung nachgewiesen und damit die Expression von A2B-AR in diesen Zellen best{\"a}tigt werden. Die gewonnenen Daten k{\"o}nnen als Ausgangspunkt weiterf{\"u}hrender Untersuchungen genutzt werden, um die Rolle von AR in Krebszellen besser zu verstehen. Sie k{\"o}nnten als Grundlage f{\"u}r die Suche nach potenziellen Angriffspunkten f{\"u}r neue Therapieans{\"a}tze dienen und so einen Beitrag f{\"u}r Fortschritte in der Behandlung von Tumoren leisten.}, subject = {Adenosin}, language = {de} } @phdthesis{Mandel2014, author = {Mandel, Philipp}, title = {Entstehung von oxidativen Stressmarkern in DNA und RNA nach der Behandlung mit den Hormonen Angiotensin II und Aldosteron in vitro und in vivo : Vergleich von drei Analysemethoden zum Nachweis von 8-Oxo-2'-desoxyguanosin in LLC-PK1-Zellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111190}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The detection of oxidative stress markers has gained increasing importancy in the early investigation of diseases like diabetes, cancer or hypertension. 8 oxo 2' deoxyguanosine (8-oxodG) is the main marker, which is used for the intracellular detection of oxidative stress levels. However, the oxidative stress markers 8 oxoguanine (8-oxoGua), a product of the DNA base excision repair and 8 oxoguanosine (8-oxoGuo), a marker for oxidative damaged RNA have received less attention up to now. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation processes of the blood pressure system. During hypertension angiotensin II (Ang II) and aldosterone (Aldo) are released in high concentrations over a longer period leading to non-physiological effects of the RAAS hormones. Subsequently, an increase of the intracellular oxidative stress level in kidney cells can be measured. The aim of this thesis is the in vitro and in vivo detection of the oxidative damage in DNA and RNA by measuring oxidative stress markers, especially 8-oxodG which is triggered by Ang II and Aldo. In vitro experiments were carried out in LLC-PK1, a cell line originated from porcine kidney cells. It could been shown that Ang II and Aldo led to a dose-dependent increase of DNA damage in the cells. A time-dependent increase was detected for the first 30 minutes of the treatment. For the rest of the experimental set up (4 h) the level of detected DNA damage remained constant. The FPG comet assay and the immunocytochemical staining showed a significant increase of 8-oxodG in the cells, whereas the HPLC-MS/MS measurement only detected a small increase of 8-oxodG in the DNA. The FPG enzyme, which recognises also other oxidized purines besides 8-oxodG, which led to an overestimation of 8-oxodG in the comet assay. Also, the 8 oxodG antibody, which was used in the immunocytochemical analysis, detected higher amounts of 8-oxodG most likely due to its side reactions with other oxidized DNA structures. One of the main advantages of the last mentioned methods is the direct measurement in damaged cells, whereas the HPLC-MS/MS requires an isolation of the DNA. During this isolation process the oxidative stress markers can be oxidized and the detection can become imprecise. The main purpose of the in vivo experiments was the detection of the oxidative stress marker 8-oxoGua, 8-oxodG and 8-oxoGuo in the urine of test animals. The treatment of C57BL/6 mice and Sprague Dawley (SD) rats with the RAAS hormones led to an increase of the blood pressure, higher DNA damage due to oxidative stress as well as an increased excretion rate of oxidative stress markers. The inhibition of the angiotensin II type 1- or mineralocorticoid receptor and a mutation of the AT1a gene could show, that the DNA damage is independent from the hypertension. In addition, it was shown that the NOX4 is not alone responsible for the oxidative stress. Other NADPH oxidases must contribute to the induction of oxidative stress inside the cell. Moreover, the activation of the Nrf2 pathway has an influence on the effect of Aldo in SD rats. The excretion rate of the oxidative stress markers in the 20 h urine of the treated animals showed how the equilibrium between the DNA repair and the oxidative stress level was changing over time. The measurement of 8-oxoGuo became more and more popular, because up to the fact that 80 \% of the DNA is translated into RNA. Overall, the detection of 8-oxodG and 8-oxoGuo is feasible for monitoring the disease or the healing process, because the measurement is non-invasive. The detection of 8-oxodG and 8-oxoGuo in nucleic acids is a first step into the field of basic research methods, because it reveals a snapshot of the nucleic acid damage in the cell at a specific time point. Usually, there will be an overestimation of the oxidative stress marker resulting from the analytical method. Although, it is possible to detect an underestimation of oxidative stress markers in tissue samples if not all cell types are damaged equally. Therefore, a primary goal should be the detection of a stable oxidation product of guanine to insure a reliable detection strategy and for a better understanding of the equilibrium of DNA oxidation and repair.}, subject = {Oxidativer Stress}, language = {de} } @phdthesis{Makiol2014, author = {Makiol, Christian}, title = {Die Rolle der durch NADPH-Oxidasen produzierten reaktiven Sauerstoffspezies (ROS) in der Pathophysiologie von Hypertonie und Alterung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109901}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Reaktive Sauerstoffradikale spielen eine große Rolle bei der Entstehung von Karzinomen, im Alterungsprozess und bei kardiovaskul{\"a}ren Erkrankungen (Valko et al., 2007). Solche Sauerstoffradikale k{\"o}nnen unter anderem durch die NADPH-Oxidase gebildet werden (Finkel and Holbrook, 2000). Ziel der Arbeit war es, die Rolle von ROS im Alterungsprozess und bei Bluthochdruck aufzuzeigen. Daf{\"u}r wurden zwei Tiermodelle, eines mit einer geringen ROS-Konzentration und eines mit einer hohen ROS-Konzentration, verwendet. Zum einen handelt es sich um ein p47-Knockout-Mausmodell f{\"u}r die geringere ROS-Konzentration, im Vergleich zu alten und jungen Wildtyp-Tieren. F{\"u}r die Rolle von ROS bei der Alterung wurden junge mit alten Wildtyp-Tieren verglichen. Um die Rolle der NOX2 in den ROS-Spiegeln der Organe zu ermitteln wurden gleichaltrige Wildtyp- mit p47-Knockout-Tieren verglichen. Zum anderen nutzten wir ein ren2-Tiermodell f{\"u}r die hohe ROS-Konzentration. Hierbei handelt es sich um ein Bluthochdruckmodell, in dem der Blutdruck mit Medikamenten normalisiert werden kann. Die Tiere wurden daher mit Ramipril (ACE-Inhibitor), Apocynin (NADPH-Oxidase-Inhibitor) und Tempol (Radikalf{\"a}nger) behandelt. Als Maß f{\"u}r die ROS-Konzentration wurden die Superoxidionenspiegel mithilfe einer Dihydroethidium-F{\"a}rbung ermittelt. Die DNA-Doppelstrangbr{\"u}che wurden mit einer γH2AX-Antik{\"o}rper-F{\"a}rbung nachgewiesen und die Expression von Sirtuin1 und Hsp70, welche als Anti-Aging Proteine bekannt sind, mittels Western Blot bestimmt.}, subject = {Reaktive Sauerstoffspezies}, language = {de} } @phdthesis{Mueller2014, author = {M{\"u}ller, Markus}, title = {Effekte kardioprotektiver Zyklopeptide auf Funktion und Morphometrie des Herzens im Rattenmodell der dilatativen Immunkardiomyopathie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101935}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Kardiomyopathien sind Erkrankungen des Herzmuskels, die mit einer kardialen Funktionsst{\"o}rung einhergehen. Formen, die ohne erkennbare Ursache zu einer progredienten Dilatation und Reduktion der Kontraktilit{\"a}t des linken Ventrikels f{\"u}hren, werden als idiopathische dilatative Kardiomyopathie (DCM) bezeichnet. Sie ist der Hauptgrund f{\"u}r schwere Herzinsuffizienz und die damit assoziierten Einschr{\"a}nkungen der Lebensqualit{\"a}t bei jungen Erwachsenen. Neben der beeintr{\"a}chtigten kardialen Funktion weisen diese Patienten oftmals auch Ver{\"a}nderungen im Bereich der humoralen und zellul{\"a}ren Immunit{\"a}t auf. Ein Teil der Patienten entwickelt Autoantik{\"o}rper, die sich gegen den kardialen β1-adrenergen Rezeptor richten und ihn {\"a}hnlich wie der nat{\"u}rliche Ligand Adrenalin aktivieren. Hieraus resultiert eine chronische {\"U}berstimulation des Rezeptors, die {\"u}ber eine initiale Hypertrophie dann zu einer eingeschr{\"a}nkten Pumpfunktion f{\"u}hrt. Nachdem sich die Therapie der Antik{\"o}rper-vermittelten Immunkardiomyopathie bisher auf die Behandlung der Herzinsuffizienz und die Kontrolle der Herz-insuffizienzsymptome beschr{\"a}nkt, k{\"o}nnten β1-ECII-homologe Peptide als Antik{\"o}rper-F{\"a}nger bei Antik{\"o}rper-positiven Patienten nun einen kausalen Therapieansatz darstellen. In diesem Zusammenhang wurden ein aus 25 Aminos{\"a}uren bestehendes zyklisches Peptid, eine aus 18 Aminos{\"a}uren bestehende Zyklopeptid-Mutante und ihre jeweiligen linearen {\"A}quivalente im Rattenmodell auf Antik{\"o}rper-neutralisierende Effekte und potentielle therapeutische Wirksamkeit getestet. Das Rattenmodell ist hierf{\"u}r besonders geeignet, da die Aminos{\"a}uresequenz der funktionell wichtigen zweiten extrazellul{\"a}ren Dom{\"a}ne des β1-adrenergen Rezeptors (β1-ECII) bei Mensch und Ratte absolut identisch ist. Auf immunologischer Ebene konnte der Titer der krankheitsinduzierenden β1-ECII-Antik{\"o}rper bereits nach der ersten Applikation zyklischer Peptide relevant gesenkt werden und nahm im weiteren Verlauf der Behandlung kontinuierlich ab. Nach Zyklopeptidgabe kam es am Herzen zu einer Reduktion des linksventrikul{\"a}ren Durchmessers und zu einer fast vollst{\"a}ndigen Normalisierung der anatomischen Proportionen. Auf die Morphologie der Myozyten selbst und auch den Kollagengehalt des Gewebes hatte die Zyklopeptidtherapie keinen wesentlichen Einfluss. Die funktionellen Eigenschaften des Herzens ließen sich durch die Neutralisation stimulatorischer β1-ECII-Antik{\"o}rper mittels intraven{\"o}ser Zyklopeptidapplikation deutlich verbessern: Die Verk{\"u}rzungsfraktion des linken Ventrikels und der Herzindex als Parameter f{\"u}r die kardiale Leistungsf{\"a}higkeit konnten durch die Behandlung wieder weitgehend normalisiert werden. Diese im Tiermodell erzielten Ergebnisse lassen einen therapeutischen Effekt der Zyklopeptide vermuten. Der Ansatz einer spezifisch gegen Antik{\"o}rper gerichteten Therapie zur Behandlung von Patienten mit β1-Antik{\"o}rper-positiver Herzinsuffizienz erscheint daher vielversprechend.}, subject = {Dilatative Kardiomyopathie}, language = {de} } @phdthesis{Sumski2014, author = {Sumski, Anna Magdalena}, title = {Adenosinrezeptoren auf Zervix-, Uterus- und Mammakarzinomzellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-99332}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Adenosinrezeptoren werden auf nahezu allen K{\"o}rperzellen exprimiert und {\"u}bernehmen dort vielf{\"a}ltige und wichtige Funktionen. Auch auf diversen Tumorzelllinien konnten bereits Adenosinrezeptoren nachgewiesen und - je nach Subtyp - mit Pro- oder Anti-tumor-Effekten in Zusammenhang gebracht werden. In dieser Arbeit wurden Geb{\"a}rmutterhalskrebszellen sowie endometriale und triple-negative Brustkrebszellen auf Expression und m{\"o}gliche Funktionen von Adenosinrezep-toren untersucht. Da spezifische Antik{\"o}rper bis heute nicht verf{\"u}gbar sind, wurde ein pharmakologischer Ansatz mit subtypspezifischen Agonisten und Antagonisten gew{\"a}hlt. In Radioliganden-Bindungsassays, konnte nachgewiesen werden, dass sich auf der Zer-vixkarzinom-Zelllinie SiHa und der Brustkrebs-Zelllinie HCC1806 Adenosinrezeptoren des Subtyps A1 befinden. Die endometrialen Krebszelllinien Ishikawa und HEC-1-A exprimieren Rezeptoren vom Subtyp A1 und A2A. A3-Adenosinrezeptoren wurden auf keiner der untersuchten Zelllinien gefunden. Der Nachweis von A2B-Rezeptoren kann mit dem Radioliganden-Bindungsassay nicht erbracht werden, da bislang kein Radioligand bekannt ist, der eine ausreichende Affini-t{\"a}t besitzt, um diesen Subtyp zweifelsfrei nachweisen zu k{\"o}nnen. Obwohl die Mehrheit der untersuchten Zelllinien Adenosinrezeptoren exprimiert, konnte ein signifikanter Effekt auf die Adenylatcyclase bei Stimulation der auf den Zellen vorhandenen Adenosinrezeptoren nur bei den HEC-1-A-Zellen festgestellt werden. Auch auf funktionelle A2B-Rezeptoren fand sich im Adenylatcyclaseassy kein Hinweis. Im durchgef{\"u}hrten Kristallviolettassay zeigte sich ein proapoptotischer Effekt auf Ishi-kawa- und HEC-1-A-Zellen bei hohen Adenosin-Konzentrationen (100 µM). Die im BrdU-Assay gemessene Proliferationsrate hingegen {\"a}nderte sich nach Vorbehandlung mit Adenosin nicht. Das metabolisch stabilere NECA (in Kombination mit ADA) hatte im Kristallviolettassay einen st{\"a}rkeren Einfluss auf die Apoptoserate der jeweiligen Zelllinie als Adenosin und auch im BrdU-Assay sank die Menge an inkorporiertem BrdU. Ein Synergismus zwischen Stimulation von Adenosinrezeptoren und diversen Todesliganden bzw. Chemotherapeutika konnte nicht nachgewiesen werden. Freies extrazellul{\"a}res Adenosin kann auch aus dem Abbau von ATP generiert werden, wenn Zellen die Ektonukleotidasen CD39 und CD73 exprimieren. Aufgrund der im-munsuppressiven Wirkung von Adenosin k{\"o}nnen diese Enzyme T-Zell- und NK-Zellantworten im Mikromilieu von Tumoren hemmen. Die durchflusszytometrische Analyse von HEC-1-A- und Ishikawa-Zellen zeigte zwar, dass die Expression von CD39 und CD73 nach Stimulation der Adenosinrezeptoren unver{\"a}ndert blieb. Die Ex-pression von Enzymen, l{\"a}sst aber vermuten, dass die Zellen in vivo von Adenosin profi-tieren k{\"o}nnten. Angesichts der in vitro Daten, die allenfalls einen wachstumshemmen-den Effekt von Adenosin zeigten, k{\"o}nnte die vorrangige Wirkung von Adenosin im Tumormikromilieu tats{\"a}chlich auf der Inhibition von Immunantworten beruhen. M{\"o}g-licherweise w{\"u}rden die Rezeptoren dann in erster Linie als Sensoren dienen. Weitere Forschungsarbeit wird helfen, die Rolle der Adenosinrezeptoren im Tumorge-schehen vollst{\"a}ndig zu verstehen und m{\"o}glicherweise f{\"u}r die Krebstherapie nutzbar zu machen.}, subject = {Adenosinrezeptor}, language = {de} } @article{EpeHaeringRamaiahetal.1993, author = {Epe, Bernd and H{\"a}ring, Martin and Ramaiah, Danaboyina and Stopper, Helga and Abou-Elzahab, Mohamed M. and Adam, Waldemar and Saha-M{\"o}ller, Chantu R.}, title = {DNA damage induced by furocoumarin hydroperoxides plus UV (360 nm)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86870}, year = {1993}, abstract = {Wben irradiated at 360 nm, furocoumarins with a hydroperoxide group in a side chain effciently give rise to a type of DNA damage that can best be explained by a photoinduced generation of hydroxyl radicals from the excited pbotosensitizers. The observed DNA damage profiles, i.e. the ratios of single-strand breaks, sites of base loss (AP sites) and base modifications sensitive to fonnamidopyrimidine-DNA glycosylase (FPG protein) and endonuclease m, are similar to the DNA damage profile produced by hydroxyl radicals generated by lonizing radiation or by xanthine and xanthine oxidase in the presence of Fe(III)-EDTA. No such damage is observed with the corresponding furocoumarin alcohols or in the absence of near-UV radiation. The damage caused by the photo-excited hydroperoxides is not influenced by superoxide dismutase (SOD) or catalase or by D2O as solvent. The presence of t-butanol, however, reduces both the formation of single-strand breaks and of base odifications sensitive to FPG protein. The cytotoxicity caused by one of the hydroperoxides in L5178Y mome lymphoma cells is found to be dependent on the near-UV irradiation and to be much higher than that of the corresponding alcohol. Therefore the new type of photoinduced damage occurs inside cells. Intercalating photosensitizers with an attached hydroperoxide group might represent a novel and versatile class of DNA damaging agents, e.g. for phototherapy.}, subject = {DNS-Sch{\"a}digung}, language = {en} } @article{ZimmermannStopper1986, author = {Zimmermann, U. and Stopper, Helga}, title = {Elektrofusion und Elektropermeabilisierung von Zellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86865}, year = {1986}, abstract = {No abstract available.}, subject = {Elektrofusion}, language = {de} } @article{JanevskiChohStopperetal.1993, author = {Janevski, J. and Choh, V. and Stopper, Helga and Schiffmann, D. and De Boni, U.}, title = {Diethylstilbestrol alters the morphology and calcium levels of growth cones of PC12 cells in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86858}, year = {1993}, abstract = {Diethylstilbestrol (DES) is a synthetic estrogen with carcinogenic properties. DES is known to alter cytoskeletal components, including the organization of actin stress fibres in C6 rat glioma cells. ln a test of the hypothesis that DES disrupts actin Filaments of growth cones in neuron-like cells, DES-induced changes in filopodial lengths were quantified in rat pheochromocytoma (PC12) cells in vitro. DES significantly altered growth cone morphology, with collapse of growth cone filopodia and neurite retraction invariably occurring at a concentration of 10 MikroM. At 5 MikroM DES, transient reductions in total filopodiallengths occurred. At DES concentrations of 0.1 nM and 1 nM, reductions in total filopodiallengths occurred in a fraction of growth cones. Evidence exists which shows that growth cone activity and morphology are intimately linked to Ieveis of intracellular, free calcium and that DES increases such levels. Measurements of free intracellular calcium levels by fluorescence microscopy, at times concurrent with the DES-induced reduction in total filopodial lengths, showed that calcium levels were indeed significantly increased by 10 MirkoM DES. Labelling of filamentaus actin (f-actin) with FITC-phalloidin showed that the f-actin distribution in growth cones exposed to DES could not be differentiated from the distribution found in spontaneously retracting growth cones. Tagether with evidence which showed that growth cone motility was not affected, the results are taken to indicate that DES, rather than acting directly on the cytoskeleton, exerts its effects indirectly, by a calcium-induced destabilization of actin filaments in the growth cone.}, subject = {Calcium}, language = {en} } @inproceedings{SchwinnRethwilmEsersetal.1990, author = {Schwinn, Andreas and Rethwilm, Axel and Esers, Stefan and Borisch, Bettina and ter Meulen, Volker}, title = {Interaction of HIV-1 and HHV-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86415}, year = {1990}, abstract = {No abstract available.}, subject = {HIV}, language = {en} } @incollection{ShephardMeierLutz1991, author = {Shephard, S. E. and Meier, I. and Lutz, Werner K.}, title = {Alkylating potency of nitrosated amino acids and peptides}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86320}, publisher = {Universit{\"a}t W{\"u}rzburg}, year = {1991}, abstract = {Tbe alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, 1)rr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, l)T-'I)T, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present durlog the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artiticial sweetener aspartame); only Met under these conditions bad a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Metproduces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the tirst-order reaction rate for nitrite. A decrease in nitrite concentration from the millimolar concentrations ofthe in-vitro assay to the micromolar concentrations in the stomach reduces the reaction rate by a factor of 1000 for the side-chain nitrosation, whereas a million-fold reduction will be observed for nitrosation of the amino group.}, subject = {Aminos{\"a}uren}, language = {en} } @article{LutzSchlatter1993, author = {Lutz, Werner K. and Schlatter, Josef}, title = {The relative importance of mutagens and carcinogens in the diet.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86311}, year = {1993}, abstract = {Known mutagens and carcinogens in the dict were compiled and the risk of cancer was estimated on the basis of average exposure Ievels in Switzerland and carcinogenic potencies from rodent bioassays. The analysis showed that, except for a1cohol, the sum of all known dietary carcinogens could only explain a few percent of the cancer deaths attributed by epidemiologists to dietary factors. The discrepancy was explained by a "carcinogenicity" of excess macronutrients. This hypothesis was based on an evaluation of dietary restriction experiments in rats and mice, where a dramatic reducing effect on spontaneaus tumour formation was seen. From these experiments, a "carcinogenic potency" was deduced for food in excess (TD50 approximately 16 g/kg per day). Ovemutrition in Switzerland was converted into excess food intake and the cancer risk estimated on the basis ofthe TD50 value. The resulting risk of60,000 cases per one million lives wou1d aJlow to explain by overnutrition almost all "diet-related" cancer deaths in humans.}, subject = {Medizin}, language = {en} } @incollection{CantoreggiGuptaLutz1993, author = {Cantoreggi, S. and Gupta, R. C. and Lutz, Werner K.}, title = {An improved 32P-postlabelling assay for detection and quantitation of styrene 7,8-oxide-DNA adducts}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86305}, publisher = {Universit{\"a}t W{\"u}rzburg}, year = {1993}, abstract = {Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P 1-enriched adducts were 32P-labelled and purified by high-salt ( 4.0 M ammonium formate, pH 6.1} C1s reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H20 (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected. The efficiency of the kinase reaction depended on the ATP concentration. Use of standard labelling conditions (['Y· 32P]ATP, <3000 Ci/mmol; <2 Mikromol) resulted in poor ( 4-7\%) adduct recovery. An ATP concentration of 40 Mikromol, however, increased the labeJling efficiency by a factor of 5-8 (35-55\% based on 3H-SO labelied DNA). The results indicate that the new separation technique is suitable for the relatively polar SO-DNA adducts and that high labelling efficiency can be achieved.}, subject = {Medizin}, language = {en} } @article{MeierGrossKlotzetal.1989, author = {Meier, Friedegund and Gross, Eva and Klotz, Karl-Norbert and Ruzicka, Thomas}, title = {Leukotriene B4 receptors on neutrophils in patients with psoriasis and atopic exzema}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86265}, year = {1989}, abstract = {Polymorphonuclear leukocyte (PMNL) infiltration is an important characteristic in psoriatic lesions. Elevated concentrations of the chemoattractant eicosanoid leukotriene B4 (L TB4) are present in psoriatic skin. Its chemotactic activity is mediated via high affinity receptors on PMNL. The goal of our work was to ascertain whether PMNL infiltration in psoriasis can be accounted for by functional abnormalities of the circulating PMNL due to alterations in the LTB4 receptor density or affinity (or both). No significant difference was found between patients with psoriasis, healthy controls and patients with another inflammatory dermatosis (atopic eczema) with regard to the binding parameters of LTB4 receptors on PMNL. Our findings suggest that PMNL accumulation in psoriatic skin may be the result of an excess of cutaneous hemoattractant rather than the increased readiness of psoriatic PMNL to migrate towards L TB4 due to altered LTB4 receptor density or affinity.}, subject = {Dermatologie}, language = {en} } @incollection{LohseKlotzSchwabeetal.1988, author = {Lohse, M. J. and Klotz, K.-N. and Schwabe, U. and Christalli, G. and Vittori, S. and Grifantini, M.}, title = {Pharmacology and Biochemistry of Adenosine Receptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86251}, publisher = {Universit{\"a}t W{\"u}rzburg}, year = {1988}, abstract = {Adenosine modulates a variety of physiological functions via membrane-bound receptors. These receptors couple via G proteins to adenylate cyclase and K+channels. The A1 subtype mediates an inhibition of adenylate cyclase and an opening of K+-channels, and the A2 subtype a Stimulation of adenylate cyclase. Both subtypes have been characterized by radioligand binding. This has facilitated the development of agonists and antagonists with more than 1000-fold A1 selectivity. A1-selective photoaffinity labels have been used for the biochemical characterization of A1 receptors and the study of their coupling to adenylate cyclase. Such selective ligands allow the analysis of the involvement of adenosine receptors in physiological functions. Selective interference with adenosine receptors provides new pharmacological tools and eventually new therapeutic approaches to a number of pathophysiological states.}, subject = {Adenosinrezeptor}, language = {en} } @incollection{ShephardHegiLutz1987, author = {Shephard, S. E. and Hegi, M. E. and Lutz, Werner K.}, title = {In-vitro assays to detect alkylating and mutagenic activities of dietary components nitrosated in situ}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86194}, publisher = {Universit{\"a}t W{\"u}rzburg}, year = {1987}, abstract = {Nitrosation of dietary components has been combined with the 4-(para-nitrobenzyl)pyridine (NBP) colorimetric test for screening alkylating agents and with the Ames test for the detection of mutagenic activity. This allowed the investigation of short-hved nitrosation products of dietary components which generate electrophilic degradation products requiring no metabolic activation (natural amino acids and some derivatives, ureas, guanidines, primary alkyl and aryl amines). In a first system, precursor, nitrous acid and NBP were present simultaneously. All amino acids tested, except glutamic acid and glutamine, gave positive results. The reactivities spanned more than three orders of magnitude, with the aromatic amino acids and methionine the most active; two primary amines, tryptamine and histamine, were also strongly reactive. All guanidines tested, except the amino acid arginine, gave negative results. A second system consisted of two phases: NBP was added only after destruction of residual nitrite and adjustment of the pH to neutrality. This system was useful for the study of ureas, which are stable in acid but not in neutral media. The range of responses covered more than two orders of magnitude. Most amino acids and primary amines also gave positive results, but could be assessed only after analysing the kinetics of the competing reactions and choosing appropriate reaction times. In a third system, Salmonella typhimurium strain TA1OO replaced NBP. Representatives of the class of amino acids, ureas, the primary amine tryptamine, and aniline became higbly mutagenic upon nitrosation. Methylguanidine was only weakly mutagenic under the present assay conditions. The results indicate that further studies with unstable nitrosation products of dietary components are required to understand more thoroughly the role of endogenous nitrosation in gastric cancer.}, subject = {Medizin}, language = {en} }