@phdthesis{Horn2024, author = {Horn, Daniela}, title = {Kardiotoxizit{\"a}t von CTRPs und das Vorkommen der CTRP-Rezeptoren in Kardiomyozyten}, doi = {10.25972/OPUS-34902}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-349029}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Die C1q/tumor necrosis factor-related proteins (CTRPs) sind eine Ligandenfamilie aus sezernierten Plasmaproteinen, welche sich in ihrem Grundbauplan {\"a}hneln. Daten aus der Literatur deuten darauf hin, dass sie zum Teil positive Effekte auf den Stoffwechsel und das Herz-Kreislaufsystem besitzen und somit eine m{\"o}gliche therapeutische Zielstruktur darstellen. W{\"a}hrend f{\"u}r manche CTRPs bereits Rezeptoren identifiziert werden konnten, ist f{\"u}r andere immer noch nicht gekl{\"a}rt, an welche Rezeptoren sie binden oder {\"u}ber welche sie diese Wirkungen erzielen. Um die CTRPs zuk{\"u}nftig therapeutisch nutzen zu k{\"o}nnen, muss die Wirkung der CTRPs auf verschiedene Zellen weiter analysiert werden. Daf{\"u}r wurden in dieser Arbeit Zellen, auf die Expression bereits bekannter CTRP-Rezeptoren hin, untersucht. Des Weiteren wurden die durch CTRP2, CTRP3, CTRP4, CTRP9A, CTRP10, CTRP11, CTRP13 und CTRP14 induzierten {\"A}nderungen in der ATP- und Laktatproduktion als Surrogatparameter f{\"u}r Kardiotoxizit{\"a}t in den Kardiomyozytenzelllinien H9c2 und AC16 getestet, um potenziell kardiotoxische Wirkungen fr{\"u}hzeitig erkennen zu k{\"o}nnen. Es konnte gezeigt werden, dass die CTRPs sicher f{\"u}r Kardiomyozyten zu sein scheinen, was eine wichtige Grundlage f{\"u}r die therapeutische Nutzbarkeit darstellt.}, subject = {Herzmuskelzelle}, language = {de} } @article{EberlRebsHoppeetal.2024, author = {Eberl, Hanna and Rebs, Sabine and Hoppe, Stefanie and Sedaghat-Hamedani, Farbod and Kayvanpour, Elham and Meder, Benjamin and Streckfuss-B{\"o}meke, Katrin}, title = {Generation of an RBM20-mutation-associated left-ventricular non-compaction cardiomyopathy iPSC line (UMGi255-A) into a DCM genetic background to investigate monogenetic cardiomyopathies}, series = {Stem Cell Research}, volume = {74}, journal = {Stem Cell Research}, issn = {1873-5061}, doi = {10.1016/j.scr.2023.103290}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350565}, year = {2024}, abstract = {RBM20 mutations account for 3 \% of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient.}, language = {en} } @article{JanzWalzCirnuetal.2024, author = {Janz, Anna and Walz, Katharina and Cirnu, Alexandra and Surjanto, Jessica and Urlaub, Daniela and Leskien, Miriam and Kohlhaas, Michael and Nickel, Alexander and Brand, Theresa and Nose, Naoko and W{\"o}rsd{\"o}rfer, Philipp and Wagner, Nicole and Higuchi, Takahiro and Maack, Christoph and Dudek, Jan and Lorenz, Kristina and Klopocki, Eva and Erg{\"u}n, S{\"u}leyman and Duff, Henry J. and Gerull, Brenda}, title = {Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes}, series = {Molecular Metabolism}, volume = {79}, journal = {Molecular Metabolism}, issn = {2212-8778}, doi = {10.1016/j.molmet.2023.101859}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350393}, year = {2024}, abstract = {Highlights • Loss of DNAJC19's DnaJ domain disrupts cardiac mitochondrial structure, leading to abnormal cristae formation in iPSC-CMs. • Impaired mitochondrial structures lead to an increased mitochondrial respiration, ROS and an elevated membrane potential. • Mutant iPSC-CMs show sarcomere dysfunction and a trend to more arrhythmias, resembling DCMA-associated cardiomyopathy. Background Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. Methods We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca\(^{2+}\) kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tv\(_{HeLa}\)). Results Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca\(^{2+}\) concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to β-adrenergic stimulation. Conclusions Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca\(^{2+}\) kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.}, language = {en} } @unpublished{BrennerZinkWitzingeretal.2024, author = {Brenner, Marian and Zink, Christoph and Witzinger, Linda and Keller, Angelika and Hadamek, Kerstin and Bothe, Sebastian and Neuenschwander, Martin and Villmann, Carmen and von Kries, Jens Peter and Schindelin, Hermann and Jeanclos, Elisabeth and Gohla, Antje}, title = {7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase}, series = {eLife}, journal = {eLife}, doi = {10.7554/eLife.93094.2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350446}, year = {2024}, abstract = {Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.}, language = {en} } @phdthesis{Kaestner2023, author = {Kaestner, Alexandra Annika Nadine}, title = {Charakterisierung pharmakologischer Phosphoglykolatphosphatase-Inhibitoren}, doi = {10.25972/OPUS-27239}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-272394}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {In dieser Arbeit geht es um die Phosphoglykolatphosphatase (PGP), die als Phosphatase vom Haloazid Dehalogenase-Typ (HAD-Phosphatase) zu der ubiquit{\"a}r vorkommenden Superfamilie der HAD-Hydrolasen geh{\"o}rt. In der Literatur ist eine in vitro Phosphatase-Aktivit{\"a}t gegen{\"u}ber 2-Phospho-L-Laktat (2PL), 4-Phospho-D-Erythronat (4PE), Phosphoglykolat (PG) und Glycerol-3-Phosphat (G3P) beschrieben. 2PL und 4PE entstehen in Nebenreaktionen w{\"a}hrend der Glykolyse und hemmen bei Akkumulation die Glykolyse bzw. den Pentosephosphatweg. PG kann auch in einer Nebenreaktion w{\"a}hrend der Glykolyse oder im Rahmen der Reparatur von oxidativen DNA-Sch{\"a}den entstehen. G3P entsteht aus Dihydroxyacetonphosphat und bildet das Kohlenhydratger{\"u}st der Triacylglyceride (TAG). Zellul{\"a}re Studien konnten Hinweise auf die Regulierung des epidermalen wachstumsfaktor-(EGF-)induzierten Zytoskelettumbaus durch die PGP liefern und die Untersuchung von M{\"a}usen mit PGP-Inaktivierung zeigte einen Einfluss auf die Zellproliferation und embryonale Entwicklung. Die Regulation der PGP-Expression f{\"u}hrte zu Ver{\"a}nderungen im Kohlenhydrat- und Fettstoffwechsel. Die Untersuchung der PGP-Funktionen erfolgte bislang ausschließlich mit genetischen Ans{\"a}tzen. Aufgrund von m{\"o}glichen Kompensationsmechanismen und Off-Target-Effekten m{\"u}ssen genetische und pharmakologische Methoden als sich erg{\"a}nzende Ans{\"a}tze verstanden werden. Um die Funktionen der PGP besser zu verstehen, fokussiert sich die vorliegende Arbeit auf die gezielte pharmakologische PGP-Inhibition. In Vorarbeiten wurden 41.000 Molek{\"u}le gescreent und f{\"u}nf potentielle Inhibitoren identifiziert. Ziele dieser Arbeit waren zum einen die Implementierung der Inhibitor \# 1-Behandlung in der Zellkultur, zum anderen die Charakterisierung der PGP-Hemmung durch Inhibitor \# 48 und die Durchf{\"u}hrung erster Selektivit{\"a}tstestungen mit Inhibitor \# 48. Zusammenfassend kann festgehalten werden, dass Inhibitor \# 1 in der Lage ist, die endogene PGP in Zelllysaten der murinen spermatogonialen Zelllinie (GC1) zu hemmen. Unter bestimmten Bedingungen f{\"u}hrte die Inhibitor \# 1-Behandlung der GC1-Zellen zur Hemmung der PGP. Erste Analysen zellul{\"a}rer Inhibitoreffekte konnten eine Steigerung der TAG-Konzentration in behandelten GC1-Zellen nachweisen. Die PGP-Hemmung durch Inhibitor \# 48 wurde als unkompetitive Inhibition charakterisiert und es zeigten sich keine relevanten Inhibitoreffekte auf die HAD-Phosphatasen Magnesium-abh{\"a}ngige Phosphatase 1 (MDP1), Lysin-Histidin-Pyrophosphat-Phosphatase (LHPP) und Polynukleotidase 5'-Kinase/3'-Phosphatase (PnkP). Dagegen konnte eine Aktivit{\"a}tssteigerung von Phospho 2 beobachtet werden. Die vorliegende Arbeit liefert somit erste Erkenntnisse {\"u}ber die Anwendung des PGP-Inhibitors \# 1 in der Zellkultur und schafft die Grundlage f{\"u}r nachfolgende Untersuchungen mit Inhibitor \# 48. Weitere Experimente sind notwendig, die die Inhibitorbehandlung in der Zellkultur optimieren und die Selektivit{\"a}t weiter charakterisieren, um mithilfe der Inhibitoren neue Erkenntnisse {\"u}ber die physiologische und pathophysiologische Rolle der PGP gewinnen zu k{\"o}nnen.}, subject = {Phosphoglykolatphosphatase}, language = {de} } @phdthesis{Lotz2023, author = {Lotz, Arietta Lucia}, title = {Eine in-vitro-Untersuchung des Einflusses von Angiotensin II und Sulforaphan auf die Modulation des oxidativen Stresses anhand der NFκB- und Nrf 2-Aktivit{\"a}t in LLC-PK1 Zellen}, doi = {10.25972/OPUS-31057}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-310573}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Ausgangspunkt der Arbeit ist die klinische Beobachtung, dass Patienten mit arteriellem Hypertonus vermehrt Nierenerkrankungen entwickeln. Dabei zeigten sich in der Subgruppenanalyse vor allem erh{\"o}hte Inzidenzen der Niereninsuffizienz und der Nierenzellkarzinome. Als m{\"o}glicher Pathomechanismus steht das Renin-Angiotensin-Aldosteron-System (RAAS-System) im Vordergrund. Dabei wird postuliert, dass erh{\"o}hte Angiotensin II-Spiegel zu einem Missverh{\"a}ltnis zwischen den Oxidations- und Reduktionspartnern in der Zelle f{\"u}hren, wodurch sich das oxidative Potential der Zelle {\"a}ndert, und es vermehrt zur Bildung von Radikalen (ROS) kommt, die meist ungepaarte Elektronen in der Valenzschale oder instabile Verbindungen enthalten, wodurch sie besonders reaktionsfreudig mit Proteinen, Lipiden, Kohlenhydraten und auch der DNA interagieren. In der Folge kommt es zu DNA-Ver{\"a}nderungen in Form von Doppel- oder Einzelstrangbr{\"u}chen, DNA-Protein-Crosslinks, Basenmodifikationen und Basenverlusten, wodurch sich ein hohes mutagenes Potential ergibt. Dieser Ansatz zur Pathophysiologie best{\"a}tigte sich auch an den hier verwendeten porkinen Nierenzellmodell. Dabei zeigte sich nicht nur eine Ver{\"a}nderung der genomischen Stabilit{\"a}t nach Exposition gegen{\"u}ber erh{\"o}hten Angiotensin II-Spiegeln, sondern auch eine Ver{\"a}nderung der DNA in Abh{\"a}ngigkeit von der Expositionsdauer der Zellen. Als n{\"a}chster Schritt konnte die Modulation der Transkriptionsfaktoren Nrf 2 und NF-κB durch die Behandlung mit Angiotensin II und Sulforaphan nachgewiesen werden. Bei der Behandlung mit Sulforaphan ließ sich eine Nrf 2-Induktion nachweisen mit vermehrter Expression von antioxidativen und detoxifizierender Enzyme. Weiterhin zeigte sich im Rahmen der Behandlung erniedrigte NF-κB-Level. Bei der Modulation durch Angiotensin II stellte sich zun{\"a}chst ein signifikant erniedrigtes Level an Nrf 2 in den Zellen dar, das im Verlauf von 24 Stunden anstieg und konsekutiv ließ sich eine maximale Proteinexpression zwischen 24 und 48 Stunden messen. Weiterhin wiesen die Zellen, die mit Angiotensin II behandelt wurden, erh{\"o}hte NF-κB Mengen/Zelle auf. Zudem zeigte sich der Einfluss erh{\"o}hter Glucosekonzentrationen auf eine progrediente genomischen Instabilit{\"a}t, die Ver{\"a}nderung der Transkriptionsfaktoren mit erh{\"o}hter Nrf 2-Induktion und mit Deregulation des Transkriptionsfaktors NF-κB wurde durch die Behandlung mit Sulforaphan nachgewiesen. Aufgrund dieser Rolle in der Tumorgenese sind mittlerweile einige Bestandteile des NF-κB- und des Nrf 2-Signalweges und auch Nrf 2-Aktivatoren wie Sulforaphan wichtige Zielstrukturen f{\"u}r die Entwicklung neuer Medikamente und Therapieoptionen. Besonders zeigt sich hierbei die Wichtigkeit bei Diabetes induzierten kardiovaskul{\"a}ren Folgesch{\"a}den mit fr{\"u}hzeitiger medikament{\"o}ser Behandlung.}, subject = {Oxidativer Stress}, language = {de} } @phdthesis{Reiser2023, author = {Reiser, Pia}, title = {Das Adverse Outcome Pathway (AOP) - Konzept als Grundlage f{\"u}r die Entwicklung mechanistischer tierversuchsfreier Ans{\"a}tze: Eine Fallstudie {\"u}ber Nephrotoxizit{\"a}t initiiert durch rezeptorvermittelte Endozytose und lysosomalen Overload}, doi = {10.25972/OPUS-31804}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-318046}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Zur Verbesserung der Pr{\"u}fung und Risikobewertung der zunehmenden Menge von Chemikalien und Arzneimitteln, gilt es neue Alternativen in Form von in vitro Pr{\"u}fmethoden mit mechanistisch relevanten Endpunkten zu finden. Einen solchen Rahmen bietet das konzeptionelle Konstrukt des Adverse Outcome Pathway (AOP)- Konzepts. Es erzeugt auf der Basis bestehenden Wissens einen mechanistischen und kausalen Zusammenhang mit Hilfe von mehreren Schl{\"u}sselereignissen (Key Event [KE]) zwischen einem initierenden molekularen Ereignis (Molecular Initiating Event [MIE]) und einem adversen Effekt (Adverse Outcome [AO]) auf biologischer Ebene. Im Rahmen dieser Arbeit wurde der AOP „Rezeptorvermittelte Endozytose und lysosomaler Overload f{\"u}hren zu Nephrotoxizit{\"a}t" am Zellkulturmodell proximaler Nierentubuluszellen weiterentwickelt. Es wurden in vitro Assays f{\"u}r die Zelllinien RPTEC/TERT1 (Mensch) und NRK-52 E (Ratte) f{\"u}r jedes KE etabliert. In dem AOP wird die Initiierung der Sch{\"a}digung des Nierengewebes durch rezeptorvermittelte Endozytose der Substanzen (MIE) mit folgendem lysosomalem Overload (KE 1) und der lysosomalen Membranruptur (KE 2) beschrieben. Es kommt zur Zellsch{\"a}digung (KE 3) und endet mit einem Schaden auf Organebene (AO). F{\"u}r KE 1 erfolgte die Visualisierung des lysosomal-assoziierten Membranproteins (lysosomal-associated Membranprotein [LAMP]) und in KE 2 die Darstellung der Protease Cathepsin D (CTSD) mittels Immunfluoreszenz. F{\"u}r KE 3 wurden spezifische Toxizit{\"a}tsdaten der Testsubstanzen mit dem CellTiter-Glo® Lumineszenz-Zellviabilit{\"a}tstest generiert. Gew{\"a}hlte Stressoren f{\"u}r den AOP war die Gruppe der Polymyxin-Antibiotika (Polymyxin B, Colistin, Polymyxin B Nonapeptid), das Aminoglykosid Gentamicin, das Glykopeptid Vancomycin sowie Cadmiumchlorid. In Zusammenschau der Ergebnisse der drei KEs war die Rangfolge der Auswirkungen der drei Polymyxin-Derivate {\"u}ber alle KEs konsistent. Polymyxin B erwies sich als aktivste Substanz, w{\"a}hrend Polymyxin B Nonapeptid die geringsten Auswirkungen zeigte. Als Ausblick in weiterf{\"u}hrenden Analysen der Arbeitsgruppe konnten bei Cadmiumchlorid trotz einer signifikanten Zytotoxizit{\"a}t (KE 3) nur geringe Auswirkungen in der LAMPExpression (KE 1) aufgezeigt werden. Des Weiteren erfolgte die Erstellung von Response-Response-Analysen, um mittels vorgeschalteter Schl{\"u}sselereignisse nachfolgende Effekte vorhersagen zu k{\"o}nnen. Projektpartner der Universit{\"a}t Utrecht entwickelten dar{\"u}ber hinaus eine quantitative in vitro in vivo Extrapolation (QIVIVE) mittels eines physiologisch basierten pharmakokinetischen (PBPK) Modells.}, subject = {Nephrotoxizit{\"a}t}, language = {de} } @phdthesis{Ramge2023, author = {Ramge, Vanessa Magali}, title = {Untersuchung der Genotoxizit{\"a}t von Pyrrolizidinalkaloiden \(in\) \(vitro\) am Beispiel von Riddelliin und Lasiocarpin}, doi = {10.25972/OPUS-31979}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-319793}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {PA sind nat{\"u}rliche Pflanzeninhaltsstoffe, die wegen ihres genotoxischen Potentials bekannt sind. Nach Applikation mikromolarer Konzentrationen k{\"o}nnen bei in vitro Untersuchungen von Leberzellen chromosomale Sch{\"a}den detektiert werden. PA stehen im Verdacht nach Aufnahme bei Menschen hepatotoxische und kanzerogene Wirkungen nach sich zu ziehen. In dieser Studie wurden Lasiocarpin und Riddelliin an der humanen Leberkarzinomzelllinie Huh6 auf Genotoxizit{\"a}t getestet. Die ausgew{\"a}hlten Methoden waren der MK-Test, der alkalische und der FPG Comet Assay und die γ-H2AX-F{\"a}rbung. In den Vorversuchen mit BaP und CPA wurde gezeigt, dass die Zellen durch Prodrugs genotoxisch gesch{\"a}digt werden. Zusammenfassend kann gesagt werden, dass Riddelliin und Lasiocarpin im MK-Test eine dosisabh{\"a}ngige, genotoxische Wirkung auf die Huh6 Zellen haben. Der Einfluss von Lasiocarpin war im MK-Test im Vergleich zum Einfluss von Riddelliin bei geringerer Konzentration detektierbar. Nach einer simultanen Behandlung der Huh6 Zellen mit verschiedenen PA kann konkludiert werden, dass keine signifikante Erh{\"o}hung an DNA-Sch{\"a}den im Vergleich zu Behandlungen mit den Einzelsubstanzen festgestellt werden konnte, was m{\"o}glicherweise auf eine Ersch{\"o}pfung der metabolischen Kapazit{\"a}t der Zellen zur{\"u}ckzuf{\"u}hren ist. Insgesamt ist es den Ergebnissen zufolge wahrscheinlich, dass die Entstehung von Crosslinks durch Lasiocarpin und Riddelliin eher eine Rolle in der Genotoxizit{\"a}tsinduktion auf Huh6 Zellen spielen als oxidativer Stress. Doppelstrangbr{\"u}che konnten nicht als sicherer Induktor von Genotoxizit{\"a}t identifiziert werden. Die Besonderheiten der Stoffwechselwege einzelner PA und die Spezifizierung einzelner, f{\"u}r die Metabolisierung relevanter Enzyme sollte in Zukunft Gegenstand der Forschung sein, um die kumulativen Wirkungen von PA besser nachzuvollziehen und die f{\"u}r den Menschen entstehenden Risiken durch die Aufnahme von PA konkretisieren zu k{\"o}nnen.}, subject = {Pyrrolizidinalkaloide}, language = {de} } @phdthesis{Nemec2023, author = {Nemec, Katarina}, title = {Modulation of parathyroid hormone 1 receptor (PTH1R) signaling by receptor activity-modifying proteins (RAMPs)}, doi = {10.25972/OPUS-28858}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288588}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {The receptor activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that interact with several G protein-coupled receptors (GPCRs), the largest and pharmacologically most important family of cell surface receptors. RAMPs can regulate GPCR function in terms of ligand-binding, G-protein coupling, downstream signaling, trafficking, and recycling. The integrity of their interactions translates to many physiological functions or pathological conditions. Regardless of numerous reports on its essential importance for cell biology and pivotal role in (patho-)physiology, the molecular mechanism of how RAMPs modulate GPCR activation remained largely elusive. This work presents new insights that add to the common understanding of the allosteric regulation of receptor activation and will help interpret how accessory proteins - RAMPs - modulate activation dynamics and how this affects the fundamental aspects of cellular signaling. Using a prototypical class B GPCR, the parathyroid hormone 1 receptor (PTH1R) in the form of advanced genetically encoded optical biosensors, I examined RAMP's impact on the PTH1R activation and signaling in intact cells. A panel of single-cell FRET and confocal microscopy experiments as well canonical and non-canonical functional assays were performed to get a holistic picture of the signaling initiation and transduction of that clinically and therapeutically relevant GPCR. Finally, structural modeling was performed to add molecular mechanistic details to that novel art of modulation. I describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity and kinetics of cAMP accumulation. Additionally, RAMP2 increases PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R and modulates cytosolic ERK1/2 phosphorylation. Structural homology modeling shows that structural motifs governing GPCR-RAMP interaction originate in allosteric hotspots and rationalize functional modulation. Moreover, to interpret the broader role of RAMP's modulation in GPCRs pharmacology, different fluorescent tools to investigate RAMP's spatial organization were developed, and novel conformational biosensors for class B GPCRs were engineered. Lastly, a high throughput assay is proposed and prototyped to expand the repertoire of RAMPs or other membrane protein interactors. These data uncover the critical role of RAMPs in GPCR activation and signaling and set up a novel platform for studying GPCR modulation. Furthermore, these insights may provide a new venue for precise modulation of GPCR function and advanced drug design.}, subject = {G-Protein gekoppelter Rezeptor}, language = {en} } @phdthesis{Eppli2023, author = {Eppli, Nenad}, title = {Untersuchung des Einflusses der ERK1/2-Autophosphorylierung an Threonin 188 auf Mausherzen mittels transgener M{\"a}use mit ubiquit{\"a}rer {\"U}berexpression von ERK2\(^{T188D}\)}, doi = {10.25972/OPUS-21655}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216558}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Die ERK2Thr188-Autophosphoylierung stellt einen regulatorischen Signalweg dar, der infolge einer hypertrophen Stimulation die kardiale Hypertrophie beg{\"u}nstigt. Eine Hemmung dieser Phosphorylierung in Kardiomyozyten verhindert die Ausbildung der kardialen Hypertrophie ohne Beeinflussung der kardioprotektiven Funktionen von ERK1/2. Demgegen{\"u}ber f{\"u}hrt die dauerhafte Simulation zu einem gain-of-function-Ph{\"a}notypen mit ausgepr{\"a}gter Hypertophie, Fibrose und einer reduzierten Herzfunktion. In dieser Arbeit wurde die dauerhafte Simulation ERK2Thr188-Phosphorylierung (T188D) in einem Mausmodell mit ubiquit{\"a}rer Expression dieser Mutation untersucht. Dabei konnte gezeigt werden, dass sich nach Stimulation durch TAC in diesen Tieren ein etwas st{\"a}rkerer hypertropher Ph{\"a}notyp mit vergr{\"o}ßerten Kardiomyozyten, gesteigerter interstitieller Fibrosierung und reduzierter Herzfunktion ausbildet als in M{\"a}usen mit kardiomyozyten-spezifischer {\"U}berexpression diese Mutante. In Fibroblasten- und VSMC-Zelllinien wurde eine gesteigerte Proliferation der T188D-{\"u}berexprimierenden Zellen im Vergleich zu Kontrollen festgestellt. Somit scheint die ERK2Thr188-Phosphorylierung auch in kardialen Nicht-Myozyten einen maladaptiven Einfluss auf das Herz auszu{\"u}ben.}, subject = {Herzhypertrophie}, language = {de} } @phdthesis{Zink2023, author = {Zink, Christoph}, title = {Biochemische und strukturbiologische Charakterisierung der Inhibition der Pyridoxal 5´-Phosphat Phosphatase durch 7,8-Dihydroxyflavon}, doi = {10.25972/OPUS-25151}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251511}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Die Pyridoxal-5'-Phosphat Phosphatase (PDXP), auch bekannt als Chronophin (CIN), ist eine HAD-Phosphatase, die beim Menschen ubiquit{\"a}r exprimiert wird und eine entscheidende Rolle im zellul{\"a}ren Vitamin-B6-Metabolismus einnimmt. PDXP ist in der Lage Pyridoxal-5'-Phosphat (PLP), die co-enzymatisch aktive Form von Vitamin B6, zu dephosphorylieren. In-vivo Studien mit M{\"a}usen zeigten, dass die Abwesenheit von PDXP mit verbesserten kognitiven Leistungen und einem verringerten Wachstum von Hirntumoren assoziiert ist. Dies begr{\"u}ndet die gezielte Suche nach einem pharmakologischen Inhibitor f{\"u}r PDXP. Ein Hochdurchsatz-Screen legte nahe, dass 7,8-Dihydroxyflavon (7,8-DHF) hierf{\"u}r ein potenzieller Kandidat ist. Zahlreiche Studien beschreiben bereits vielf{\"a}ltige positive neurologische Effekte nach in-vivo Administration von 7,8-DHF, allerdings bleibt der genaue Wirkmechanismus umstritten und wird bis dato nicht mit PDXP in Zusammenhang gebracht. Ziel dieser Arbeit ist es, die Inhibition von PDXP durch 7,8-DHF n{\"a}her zu charakterisieren und damit einen Beitrag zur Beantwortung der Frage zu leisten, ob PDXP an den 7,8-DHF-induzierten Effekten beteiligt ist. Hierzu wurde der Effekt von 7,8-DHF auf die enzymatische Aktivit{\"a}t von rekombinant hergestelltem, gereinigtem PDXP in in-vitro Phosphatase-Assays charakterisiert. Um die Selektivit{\"a}t von 7,8-DHF gegen{\"u}ber PDXP zu untersuchen, wurden f{\"u}nf weitere HAD-Phosphatasen getestet. Unter den analysierten Phosphatasen zeigte einzig die dem PDXP nah verwandte Phosphoglykolat Phosphatase (PGP) eine geringer ausgepr{\"a}gte Sensitivit{\"a}t gegen 7,8-DHF. Ein Vergleich von 7,8-DHF mit sechs strukturell verwandten, hydroxylierten Flavonen zeigte, dass 7,8-DHF unter den getesteten Substanzen die h{\"o}chste Potenz und Effektivit{\"a}t aufwies. Außerdem wurde eine Co-Kristallisation von PDXP mit 7,8-DHF durchgef{\"u}hrt, deren Struktur bis zu einer Aufl{\"o}sung von 2,0 {\AA} verfeinert werden konnte. Die in der Kristallstruktur identifizierte Bindungsstelle von 7,8-DHF an PDXP wurde mittels verschiedener, neu generierter PDXP-Mutanten enzymkinetisch best{\"a}tigt. Zusammenfassend zeigen die hier beschriebenen Ergebnisse, dass 7,8-DHF ein direkter, selektiver und vorwiegend kompetitiver Inhibitor der PDXP-Aktivit{\"a}t ist, mit einer IC50 im submikromolaren Bereich. Die Ergebnisse dieser in-vitro Untersuchungen motivieren zu weiterer Forschung bez{\"u}glich der 7,8-DHF-vermittelten Inhibition der PDXP-Aktivit{\"a}t in Zellen, um die Frage beantworten zu k{\"o}nnen, ob PDXP auch in-vivo ein relevantes Target f{\"u}r 7,8-DHF darstellt.}, subject = {Pyridoxalphosphat}, language = {de} } @article{NwoghaAbtewRaveendranetal.2023, author = {Nwogha, Jeremiah S. and Abtew, Wosene G. and Raveendran, Muthurajan and Oselebe, Happiness O. and Obidiegwu, Jude E. and Chilaka, Cynthia A. and Amirtham, Damodarasamy D.}, title = {Role of non-structural sugar metabolism in regulating tuber dormancy in white yam (Dioscorea rotundata)}, series = {Agriculture}, volume = {13}, journal = {Agriculture}, number = {2}, issn = {2077-0472}, doi = {10.3390/agriculture13020343}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304486}, year = {2023}, abstract = {Changes in sugar composition occur continuously in plant tissues at different developmental stages. Tuber dormancy induction, stability, and breaking are very critical developmental transitions in yam crop production. Prolonged tuber dormancy after physiological maturity has constituted a great challenge in yam genetic improvement and productivity. In the present study, biochemical profiling of non-structural sugar in yam tubers during dormancy was performed to determine the role of non-structural sugar in yam tuber dormancy regulation. Two genotypes of the white yam species, one local genotype (Obiaoturugo) and one improved genotype (TDr1100873), were used for this study. Tubers were sampled at 42, 56, 87, 101, 115, and 143 days after physiological maturity (DAPM). Obiaoturugo exhibited a short dormant phenotype and sprouted at 101-DAPM, whereas TDr1100873 exhibited a long dormant phenotype and sprouted at 143-DAPM. Significant metabolic changes were observed in non-structural sugar parameters, dry matter, and moisture content in Obiaoturugo from 56-DAPM, whereas in TDr1100873, significant metabolic changes were observed from 101-DAPM. It was observed that the onset of these metabolic changes occurred at a point when the tubers of both genotypes exhibited a dry matter content of 60\%, indicating that a dry matter content of 60\% might be a critical threshold for white yam tuber sprouting. Non-reducing sugars increased by 9-10-fold during sprouting in both genotypes, which indicates their key role in tuber dormancy regulation in white yam. This result implicates that some key sugar metabolites can be targeted for dormancy manipulation of the yam crop.}, language = {en} } @article{HadiBankogluStopper2023, author = {Hadi, Naji Said Aboud and Bankoglu, Ezgi Eyluel and Stopper, Helga}, title = {Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells}, series = {Archives of Toxicology}, volume = {97}, journal = {Archives of Toxicology}, number = {1}, doi = {10.1007/s00204-022-03394-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-324708}, pages = {295-306}, year = {2023}, abstract = {Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells.}, language = {en} } @article{SchanbacherHermannsLorenzetal.2023, author = {Schanbacher, Constanze and Hermanns, Heike M. and Lorenz, Kristina and Wajant, Harald and Lang, Isabell}, title = {Complement 1q/tumor necrosis factor-related proteins (CTRPs): structure, receptors and signaling}, series = {Biomedicines}, volume = {11}, journal = {Biomedicines}, number = {2}, issn = {2227-9059}, doi = {10.3390/biomedicines11020559}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304136}, year = {2023}, abstract = {Adiponectin and the other 15 members of the complement 1q (C1q)/tumor necrosis factor (TNF)-related protein (CTRP) family are secreted proteins composed of an N-terminal variable domain followed by a stalk region and a characteristic C-terminal trimerizing globular C1q (gC1q) domain originally identified in the subunits of the complement protein C1q. We performed a basic PubMed literature search for articles mentioning the various CTRPs or their receptors in the abstract or title. In this narrative review, we briefly summarize the biology of CTRPs and focus then on the structure, receptors and major signaling pathways of CTRPs. Analyses of CTRP knockout mice and CTRP transgenic mice gave overwhelming evidence for the relevance of the anti-inflammatory and insulin-sensitizing effects of CTRPs in autoimmune diseases, obesity, atherosclerosis and cardiac dysfunction. CTRPs form homo- and heterotypic trimers and oligomers which can have different activities. The receptors of some CTRPs are unknown and some receptors are redundantly targeted by several CTRPs. The way in which CTRPs activate their receptors to trigger downstream signaling pathways is largely unknown. CTRPs and their receptors are considered as promising therapeutic targets but their translational usage is still hampered by the limited knowledge of CTRP redundancy and CTRP signal transduction.}, language = {en} } @phdthesis{Soliman2022, author = {Soliman, Alexander}, title = {Einfluss des Gewichtsverlusts auf den oxidativen Stress und den DNS-Schaden in adip{\"o}sen Patient*innen nach bariatrischer Chirurgie}, doi = {10.25972/OPUS-25973}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259737}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Einfluss des Gewichtsverlusts auf den oxidativen Stress und den DNS-Schaden in adip{\"o}sen Patient*innen nach bariatrischer Chirurgie Adipositas ist eine Erkrankung, die durch ein erh{\"o}htes Krebsrisiko neben zahlreichen anderen Komorbidit{\"a}ten mit weitreichenden Folgen f{\"u}r die Gesundheit adip{\"o}ser Patient*innen einhergeht. In der Pathogenese der adipositas-assoziierten Krebsarten sind dabei ein erh{\"o}hter oxidativer Stress sowie die damit einhergehende Sch{\"a}digung der DNS maßgeblich beteiligt. Im Umkehrschluss wurde in der vorliegenden Arbeit der Einfluss eines durch bariatrische Chirurgie induzierten Gewichtsverlusts auf den oxidativen Stress und DNS-Schaden in adip{\"o}sen Patient*innen anhand von Blutproben pr{\"a}operativ sowie 6 und 12 Monate postoperativ untersucht. In einer Subpopulation der Patient*innen konnte eine tendenzielle Verringerung des DNS-Schadens anhand des Comet-Assays in peripheren Lymphozyten beobachtet werden. Im Hinblick auf den oxidativen Stress wurde im Plasma die Eisenreduktionsf{\"a}higkeit als Maß f{\"u}r antioxidative Kapazit{\"a}t sowie Malondialdehyd als Surrogatmarker f{\"u}r das Ausmaß an Lipidperoxidation bestimmt. Weiterhin wurde in Erythrozyten das Gesamtglutathion und oxidierte Glutathion bestimmt. Die oxidativen Stressparameter zeigten insgesamt nach einer initialen Zunahme im oxidativen Stress 6 Monate postoperativ eine r{\"u}ckl{\"a}ufige Tendenz im oxidativen Stress am Studienende. Somit geben die Beobachtungen dieser Arbeit Anlass zur Hoffnung, dass adip{\"o}se Patient*innen durch einen bariatrisch induzierten Gewichtsverlust von einer Verringerung des Krebsrisikos profitieren k{\"o}nnten.}, subject = {Magenchirurgie}, language = {de} } @phdthesis{Jarzina2022, author = {Jarzina, Sebastian Oskar}, title = {Assessment of systemic toxicity in vitro using the Adverse Outcome Pathway (AOP) concept: nephrotoxicity due to receptor-mediated endocytosis and lysosomal overload and inhibition of mtDNA polymerase-ɣ as case studies}, doi = {10.25972/OPUS-26484}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-264842}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {The US National Research Council (NRC) report "Toxicity Testing in the 21st Century: A Vision and a strategy (Tox21)", published in 2007, calls for a complete paradigm shift in tox-icity testing. A central aspect of the proposed strategy includes the transition from apical end-points in in vivo studies to more mechanistically based in vitro tests. To support and facilitate the transition and paradigm shift in toxicity testing, the Adverse Outcome Pathway (AOP) concept is widely recognized as a pragmatic tool. As case studies, the AOP concept was ap-plied in this work to develop AOPs for proximal tubule injuries initiated by Receptor-mediated endocytosis and lysosomal overload and Inhibition of mtDNA polymerase-. These AOPs were used as a mechanistic basis for the development of in vitro assays for each key event (KE). To experimentally support the developed in vitro assays, proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) were treated with model compounds. To measure the dis-turbance of lysosomal function in the AOP - Receptor-mediated endocytosis and lysosomal overload, polymyxin antibiotics (polymyxin B, colistin, polymyxin B nonapeptide) were used as model compounds. Altered expression of lysosomal associated membrane protein 1/2 (LAMP-1/2) (KE1) and cathepsin D release from lysosomes (KE2) were determined by im-munofluorescence, while cytotoxicity (KE3) was measured using the CellTiter-Glo® cell via-bility assay. Importantly, significant differences in polymyxin uptake and susceptibility be-tween cell lines were observed, underlining the importance of in vitro biokinetics to determine an appropriate in vitro point of departure (PoD) for risk assessment. Compared to the in vivo situation, distinct expression of relevant transporters such as megalin and cubilin on mRNA and protein level in the used cell lines (RPTEC/TERT1 and NRK-52E) could not be con-firmed, making integration of quantitative in vitro to in vivo extrapolations (QIVIVE) neces-sary. Integration of QIVIVE by project partners of the University of Utrecht showed an im-provement in the modelled biokinetic data for polymyxin B. To assess the first key event, (KE1) Depletion of mitochondrial DNA, in the AOP - Inhibition of mtDNA polymerase-, a RT-qPCR method was used to determine the mtDNA copy number in cells treated with mod-el compounds (adefovir, cidofovir, tenofovir, adefovir dipivoxil, tenofovir disoproxil fumarate). Mitochondrial toxicity (KE2) was measured by project partners using the high-content imaging technique and MitoTracker® whereas cytotoxicity (KE3) was determined by CellTiter-Glo® cell viability assay. In contrast to the mechanistic hypothesis underlying the AOP - Inhibition of mtDNA polymerase-, treatment with model compounds for 24 h resulted in an increase rather than a decrease in mtDNA copy number (KE1). Only minor effects on mitochondrial toxicity (KE2) and cytotoxicity (KE3) were observed. Treatment of RPT-EC/TERT1 cells for 14 days showed only a slight decrease in mtDNA copy number after treatment with adefovir dipivoxil and tenofovir disoproxil fumarate, underscoring some of the limitations of short-term in vitro systems. To obtain a first estimation for risk assessment based on in vitro data, potential points of departure (PoD) for each KE were calculated from the obtained in vitro data. The most common PoDs were calculated such as the effect concentra-tion at which 10 \% or 20_\% effect was measured (EC10, EC20), the highest no observed effect concentration (NOEC), the lowest observed effect concentration (LOEC), the benchmark 10 \% (lower / upper) concentrations (BMC10, BMCL10, BMCU10) and a modelled non-toxic con-centration (NtC). These PoDs were then compared with serum and tissue concentrations de-termined from in vivo studies after treatment with therapeutic / supratherapeutic doses of the respective drugs in order to obtain a first estimate of risk based on in vitro data. In addition, AOPs were used to test whether the quantitative key event relationships between key events allow prediction of downstream effects and effects on the adverse outcome (AO) based on measurements of an early key event. Predictions of cytotoxicity from the mathematical rela-tionships showed good concordance with measured cytotoxicity after treatment with colistin and polymyxin b nonapeptide. The work also revealed uncertainties and limitations of the ap-plied strategy, which have a significant impact on the prediction and on a risk assessment based on in vitro results.}, language = {en} } @article{JeanclosSchloetzerHadameketal.2022, author = {Jeanclos, Elisabeth and Schl{\"o}tzer, Jan and Hadamek, Kerstin and Yuan-Chen, Natalia and Alwahsh, Mohammad and Hollmann, Robert and Fratz, Stefanie and Yesilyurt-Gerhards, Dilan and Frankenbach, Tina and Engelmann, Daria and Keller, Angelika and Kaestner, Alexandra and Schmitz, Werner and Neuenschwander, Martin and Hergenr{\"o}der, Roland and Sotriffer, Christoph and von Kries, Jens Peter and Schindelin, Hermann and Gohla, Antje}, title = {Glycolytic flux control by drugging phosphoglycolate phosphatase}, series = {Nature Communications}, volume = {13}, journal = {Nature Communications}, number = {1}, doi = {10.1038/s41467-022-34228-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300928}, year = {2022}, abstract = {Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.}, language = {en} } @article{MallyJarzina2022, author = {Mally, Angela and Jarzina, Sebastian}, title = {Mapping adverse outcome pathways for kidney injury as a basis for the development of mechanism-based animal-sparing approaches to assessment of nephrotoxicity}, series = {Frontiers in Toxicology}, volume = {4}, journal = {Frontiers in Toxicology}, issn = {2673-3080}, doi = {10.3389/ftox.2022.863643}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284405}, year = {2022}, abstract = {In line with recent OECD activities on the use of AOPs in developing Integrated Approaches to Testing and Assessment (IATAs), it is expected that systematic mapping of AOPs leading to systemic toxicity may provide a mechanistic framework for the development and implementation of mechanism-based in vitro endpoints. These may form part of an integrated testing strategy to reduce the need for repeated dose toxicity studies. Focusing on kidney and in particular the proximal tubule epithelium as a key target site of chemical-induced injury, the overall aim of this work is to contribute to building a network of AOPs leading to nephrotoxicity. Current mechanistic understanding of kidney injury initiated by 1) inhibition of mitochondrial DNA polymerase γ (mtDNA Polγ), 2) receptor mediated endocytosis and lysosomal overload, and 3) covalent protein binding, which all present fairly well established, common mechanisms by which certain chemicals or drugs may cause nephrotoxicity, is presented and systematically captured in a formal description of AOPs in line with the OECD AOP development programme and in accordance with the harmonized terminology provided by the Collaborative Adverse Outcome Pathway Wiki. The relative level of confidence in the established AOPs is assessed based on evolved Bradford-Hill weight of evidence considerations of biological plausibility, essentiality and empirical support (temporal and dose-response concordance).}, language = {en} } @article{MaurerHartmannArgyriouetal.2022, author = {Maurer, Wiebke and Hartmann, Nico and Argyriou, Loukas and Sossalla, Samuel and Streckfuss-B{\"o}meke, Katrin}, title = {Generation of homozygous Na\(_{v}\)1.8 knock-out iPSC lines by CRISPR Cas9 genome editing to investigate a potential new antiarrhythmic strategy}, series = {Stem Cell Research}, volume = {60}, journal = {Stem Cell Research}, doi = {10.1016/j.scr.2022.102677}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300936}, year = {2022}, abstract = {The sodium channel Na\(_{v}\)1.8, encoded by SCN10A, is reported to contribute to arrhythmogenesis by inducing the late I\(_{Na}\) and thereby enhanced persistent Na\(^{+}\) current. However, its exact electrophysiological role in cardiomyocytes remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) with a homozygous SCN10A knock-out from a healthy iPSC line by CRISPR Cas9 genome editing. The edited iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro differentiation capacity. The iPSCs are able to differentiate into iPSC-cardiomyocytes, hence making it possible to investigate the role of Na\(_{v}\)1.8 in the heart.}, language = {en} } @phdthesis{BathePeters2022, author = {Bathe-Peters, Marc}, title = {Spectroscopic approaches for the localization and dynamics of β\(_1\)- and β\(_2\)-adrenergic receptors in cardiomyocytes}, doi = {10.25972/OPUS-25812}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-258126}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {In the heart the β\(_1\)-adrenergic receptor (AR) and the β\(_2\)-AR, two prototypical G protein-coupled receptors (GPCRs), are both activated by the same hormones, namely adrenaline and noradrenaline. Both receptors couple to stimulatory G\(_s\) proteins, mediate an increase in cyclic adenosine monophosphate (cAMP) and influence the contractility and frequency of the heart upon stimulation. However, activation of the β\(_1\)-AR, not the β\(_2\)-AR, lead to other additional effects, such as changes in gene transcription resulting in cardiac hypertrophy, leading to speculations on how distinct effects can arise from receptors coupled to the same downstream signaling pathway. In this thesis the question of whether this distinct behavior may originate from a differential localization of these two receptors in adult cardiomyocytes is addressed. Therefore, fluorescence spectroscopy tools are developed and implemented in order to elucidate the presence and dynamics of these endogenous receptors at the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. This allows the visualization of confined localization and diffusion of the β\(_2\)-AR to the T-tubular network at endogenous expression. In contrast, the β\(_1\)-AR is found diffusing at both the outer plasma membrane and the T-tubules. Upon overexpression of the β\(_2\)-AR in adult transgenic cardiomyocytes, the receptors experience a loss of this compartmentalization and are also found at the cell surface. These data suggest that distinct signaling and functional effects can be controlled by specific cell surface targeting of the receptor subtypes. The tools at the basis of this thesis work are a fluorescent adrenergic antagonist in combination of fluorescence fluctuation spectroscopy to monitor the localization and dynamics of the lowly expressed adrenergic receptors. Along the way to optimizing these approaches, I worked on combining widefield and confocal imaging in one setup, as well as implementing a stable autofocus mechanism using electrically tunable lenses.}, subject = {G-Protein gekoppelte Rezeptoren}, language = {en} } @article{LorenzRosner2022, author = {Lorenz, Kristina and Rosner, Marsha Rich}, title = {Harnessing RKIP to combat heart disease and cancer}, series = {Cancers}, volume = {14}, journal = {Cancers}, number = {4}, issn = {2072-6694}, doi = {10.3390/cancers14040867}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262185}, year = {2022}, abstract = {Cancer and heart disease are leading causes of morbidity and mortality worldwide. These diseases have common risk factors, common molecular signaling pathways that are central to their pathogenesis, and even some disease phenotypes that are interdependent. Thus, a detailed understanding of common regulators is critical for the development of new and synergistic therapeutic strategies. The Raf kinase inhibitory protein (RKIP) is a regulator of the cellular kinome that functions to maintain cellular robustness and prevent the progression of diseases including heart disease and cancer. Two of the key signaling pathways controlled by RKIP are the β-adrenergic receptor (βAR) signaling to protein kinase A (PKA), particularly in the heart, and the MAP kinase cascade Raf/MEK/ERK1/2 that regulates multiple diseases. The goal of this review is to discuss how we can leverage RKIP to suppress cancer without incurring deleterious effects on the heart. Specifically, we discuss: (1) How RKIP functions to either suppress or activate βAR (PKA) and ERK1/2 signaling; (2) How we can prevent cancer-promoting kinase signaling while at the same time avoiding cardiotoxicity.}, language = {en} } @article{SchanbacherBieberReindersetal.2022, author = {Schanbacher, Constanze and Bieber, Michael and Reinders, Yvonne and Cherpokova, Deya and Teichert, Christina and Nieswandt, Bernhard and Sickmann, Albert and Kleinschnitz, Christoph and Langhauser, Friederike and Lorenz, Kristina}, title = {ERK1/2 activity is critical for the outcome of ischemic stroke}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {2}, issn = {1422-0067}, doi = {10.3390/ijms23020706}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-283991}, year = {2022}, abstract = {Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood-brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.}, language = {en} } @phdthesis{İşbilir2022, author = {İ{\c{s}}bilir, Ali}, title = {Localization and Trafficking of CXCR4 and CXCR7}, doi = {10.25972/OPUS-24937}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-249378}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins, and are the master components that translate extracellular stimulus into intracellular signaling, which in turn modulates key physiological and pathophysiological processes. Research within the last three decades suggests that many GPCRs can form complexes with each other via mechanisms that are yet unexplored. Despite a number of functional evidence in favor of GPCR dimers and oligomers, the existence of such complexes remains controversial, as different methods suggest diverse quaternary organizations for individual receptors. Among various methods, high resolution fluorescence microscopy and imagebased fluorescence spectroscopy are state-of-the-art tools to quantify membrane protein oligomerization with high precision. This thesis work describes the use of single molecule fluorescence microscopy and implementation of two confocal microscopy based fluorescence fluctuation spectroscopy based methods for characterizing the quaternary organization of two class A GPCRs that are important clinical targets: the C-X-C type chemokine receptor 4 (CXCR4) and 7 (CXCR7), or recently named as the atypical chemokine receptor 3 (ACKR3). The first part of the results describe that CXCR4 protomers are mainly organized as monomeric entities that can form transient dimers at very low expression levels allowing single molecule resolution. The second part describes the establishment and use of spatial and temporal brightness methods that are based on fluorescence fluctuation spectroscopy. Results from this part suggests that ACKR3 forms clusters and surface localized monomers, while CXCR4 forms increasing amount of dimers as a function of receptor density in cells. Moreover, CXCR4 dimerization can be modulated by its ligands as well as receptor conformations in distinct manners. Further results suggest that antagonists of CXCR4 display distinct binding modes, and the binding mode influences the oligomerization and the basal activity of the receptor: While the ligands that bind to a "minor" subpocket suppress both dimerization and constitutive activity, ligands that bind to a distinct, "major" subpocket only act as neutral antagonists on the receptor, and do not modulate neither the quaternary organization nor the basal signaling of CXCR4. Together, these results link CXCR4 dimerization to its density and to its activity, which may represent a new strategy to target CXCR4.}, subject = {G-Protein gekoppelter Rezeptor}, language = {en} } @article{SpinaciLambertucciBuccionietal.2022, author = {Spinaci, Andrea and Lambertucci, Catia and Buccioni, Michela and Dal Ben, Diego and Graiff, Claudia and Barbalace, Maria Cristina and Hrelia, Silvana and Angeloni, Cristina and Tayebati, Seyed Khosrow and Ubaldi, Massimo and Masi, Alessio and Klotz, Karl-Norbert and Volpini, Rosaria and Marucci, Gabriella}, title = {A\(_{2A}\) adenosine receptor antagonists: are triazolotriazine and purine scaffolds interchangeable?}, series = {Molecules}, volume = {27}, journal = {Molecules}, number = {8}, issn = {1420-3049}, doi = {10.3390/molecules27082386}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270618}, year = {2022}, abstract = {The A\(_{2A}\) adenosine receptor (A\(_{2A}\)AR) is one of the four subtypes activated by nucleoside adenosine, and the molecules able to selectively counteract its action are attractive tools for neurodegenerative disorders. In order to find novel A\(_{2A}\)AR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A\(_{2A}\)AR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted.}, language = {en} } @article{JarzinaDiFioreEllingeretal.2022, author = {Jarzina, Sebastian and Di Fiore, Stefano and Ellinger, Bernhard and Reiser, Pia and Frank, Sabrina and Glaser, Markus and Wu, Jiaqing and Taverne, Femke J. and Kramer, Nynke I. and Mally, Angela}, title = {Application of the adverse outcome pathway concept to in vitro nephrotoxicity assessment: kidney injury due to receptor-mediated endocytosis and lysosomal overload as a case study}, series = {Frontiers in Toxicology}, volume = {4}, journal = {Frontiers in Toxicology}, issn = {2673-3080}, doi = {10.3389/ftox.2022.864441}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284796}, year = {2022}, abstract = {Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.}, language = {en} } @article{KlenkHommersLohse2022, author = {Klenk, Christoph and Hommers, Leif and Lohse, Martin J.}, title = {Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor}, series = {Frontiers in Endocrinology}, volume = {13}, journal = {Frontiers in Endocrinology}, issn = {1664-2392}, doi = {10.3389/fendo.2022.839351}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262055}, year = {2022}, abstract = {Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.}, language = {en} } @phdthesis{Soliman2022, author = {Soliman, Alexander}, title = {Einfluss des Gewichtsverlusts auf den oxidativen Stress und den DNS-Schaden in adip{\"o}sen Patient*innen nach bariatrischer Chirurgie}, doi = {10.25972/OPUS-27835}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-278354}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Adipositas ist eine Erkrankung, die durch ein erh{\"o}htes Krebsrisiko neben zahlreichen anderen Komorbidit{\"a}ten mit weitreichenden Folgen f{\"u}r die Gesundheit adip{\"o}ser Patient*innen einhergeht. In der Pathogenese der adipositas-assoziierten Krebsarten sind dabei ein erh{\"o}hter oxidativer Stress sowie die damit einhergehende Sch{\"a}digung der DNS maßgeblich beteiligt. Im Umkehrschluss wurde in der vorliegenden Arbeit der Einfluss eines durch bariatrische Chirurgie induzierten Gewichtsverlusts auf den oxidativen Stress und DNS-Schaden in adip{\"o}sen Patient*innen anhand von Blutproben pr{\"a}operativ sowie 6 und 12 Monate postoperativ untersucht. In einer Subpopulation der Patient*innen konnte eine tendenzielle Verringerung des DNS-Schadens anhand des Comet-Assays in peripheren Lymphozyten beobachtet werden. Im Hinblick auf den oxidativen Stress wurde im Plasma die Eisenreduktionsf{\"a}higkeit als Maß f{\"u}r die antioxidative Kapazit{\"a}t sowie Malondialdehyd als Surrogatmarker f{\"u}r das Ausmaß an Lipidperoxidation bestimmt. Weiterhin wurde in Erythrozyten das Gesamtglutathion und das oxidierte Glutathion bestimmt. Die oxidativen Stressparameter zeigten insgesamt nach einer initialen Zunahme im oxidativen Stress 6 Monate postoperativ eine r{\"u}ckl{\"a}ufige Tendenz im oxidativen Stress am Studienende. Somit geben die Beobachtungen dieser Arbeit Anlass zur Hoffnung, dass adip{\"o}se Patient*innen durch einen bariatrisch induzierten Gewichtsverlust von einer Verringerung des Krebsrisikos profitieren k{\"o}nnten.}, subject = {Magenchirurgie}, language = {de} } @phdthesis{Bertelsmann2022, author = {Bertelsmann, Dietmar}, title = {Analysis of the Frequency of Kidney Toxicity in Preclinical Safety Studies using the eTOX Database}, doi = {10.25972/OPUS-25710}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257104}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {This research aimed to obtain reliable data on the frequency of different types of renal toxicity findings in 28-day oral gavage studies in Wistar rats, their consistency across species and study duration, as well as the correlation between histopathological endpoints and routinely used clinical chemistry parameters indicative of kidney injury. Analysis of renal histopathological findings was carried out through extraction of information from the IMI eTOX database. Spontaneous renal histopathological findings in 28-day oral gavage studies in control Wistar rats and beagle dogs confirmed tubular basophilia and renal dilation as the most frequent incidental findings in controls, whereas necrosis and glomerulosclerosis were not identified at all or only rarely as a background lesion. Histopathological evidence of necrosis and glomerulosclerosis was associated with changes in clinical chemistry parameters in 28-day oral gavage Wistar rat studies. Necrosis was frequently accompanied by a statistically significant rise in serum creatinine and serum urea, whereas serum albumin was frequently found to decrease statistically significantly in treatment groups in which necrosis was recorded. In contrast to necrosis, glomerulosclerosis was not associated with statistically significant changes in serum creatinine and urea in any of the 28-day oral gavage Wistar rat treatment groups, but appears to be best reflected by a pattern of statistically significantly lowered serum albumin and serum protein together with a statistically significant increase in serum cholesterol. As might have been expected based on the high background incidences of tubular basophilia and dilation, no consistent changes in any of the clinical chemistry parameters were evident in animals in which renal lesions were con� fined to renal tubular basophilia or dilation. In summary, the routinely provided clinical chemistry parameters are rather insensitive - novel kidney biomarkers such as Cystatin C, β-trace protein and Kidney injury molecule 1 should further be evaluated and integrated into routine preclinical and clinical practice. However, evaluation of clinical chemistry data was limited by the lack of individual animal data. Even though an extensive amount of preclinical studies is accessible through the eTOX database, comparison of consistency across time was limited by the limited number of shorter- and longer term studies conducted with the compounds identified as causing renal histopathological changes within a 28- day study in rats. A high consistency across time for both treatment-related tubular basophilia and treatment-related dilation cannot be confirmed for either of the two effects as these two findings were both induced only rarely in studies over a different treatment-duration other than 28 days after administration of the compounds which provoked the respective effect in a 28-day study. For the finding of necrosis consistency across time was low with the exception of "AZ_GGA_200002321", in which renal papillary necrosis was identified consist� ently throughout different treatment durations (2, 4, 26, 104 weeks). No shorter and longer-term studies were available for the compounds identified as causing glomerulosclerosis within a 28-day study in rats. No consistent findings of the selected histopathological endpoints were identified in any of the corresponding 28-day oral gavage beagle dog studies after treatment with the identical compounds, which caused the respective ef� fect after 28-day treatment in rats. However, in the overwhelming majority of cases, beagle dogs were administered lower doses in these studies in compar� ison to the corresponding 28-day Wistar rat studies. Searching the eTOX database yielded no 28-day oral gavage studies in Wistar and Wistar Han rats in which accumulation of hyaline droplets, tubular atrophy or hyperplasia was recorded. Only one 28-day oral gavage Wistar rat study was identified with the histopathological result of neutrophilic inflammation. Consequently, evaluation of these four renal findings in relation to clinical chemistry parameters and consistency across time and species cannot be made. In summary, this work contributes knowledge through mining and evaluating the eTOX database on a variety of specific renal endpoints that frequently occur after administration of trial substances in 28-day oral gavage studies in Wistar rats in the field of preclinical toxicity with specific focus on their frequency relation to background findings, as well as consistency across time and species. Targeted statistical evaluation of in vivo data within joint research ventures such as the eTOX project, presents an enormous opportunity for an innovative future way of aiding preclinical research towards a more efficient research in the preclinical stage of drug development. This could be achieved through the aug� mentation of methodological strategies and possibly novel software tools in order to predict in vivo toxicology of new molecular entities by means of information that is already available before early stages of the drug development pipeline begin.}, language = {en} } @article{ChilakaObidiegwuChilakaetal.2022, author = {Chilaka, Cynthia Adaku and Obidiegwu, Jude Ejikeme and Chilaka, Augusta Chinenye and Atanda, Olusegun Oladimeji and Mally, Angela}, title = {Mycotoxin regulatory status in Africa: a decade of weak institutional efforts}, series = {Toxins}, volume = {14}, journal = {Toxins}, number = {7}, issn = {2072-6651}, doi = {10.3390/toxins14070442}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-278941}, year = {2022}, abstract = {Food safety problems are a major hindrance to achieving food security, trade, and healthy living in Africa. Fungi and their secondary metabolites, known as mycotoxins, represent an important concern in this regard. Attempts such as agricultural, storage, and processing practices, and creation of awareness to tackle the menace of fungi and mycotoxins have yielded measurable outcomes especially in developed countries, where there are comprehensive mycotoxin legislations and enforcement schemes. Conversely, most African countries do not have mycotoxin regulatory limits and even when available, are only applied for international trade. Factors such as food insecurity, public ignorance, climate change, poor infrastructure, poor research funding, incorrect prioritization of resources, and nonchalant attitudes that exist among governmental organisations and other stakeholders further complicate the situation. In the present review, we discuss the status of mycotoxin regulation in Africa, with emphasis on the impact of weak mycotoxin legislations and enforcement on African trade, agriculture, and health. Furthermore, we discuss the factors limiting the establishment and control of mycotoxins in the region.}, language = {en} } @article{TolstikAliGuoetal.2022, author = {Tolstik, Elen and Ali, Nairveen and Guo, Shuxia and Ebersbach, Paul and M{\"o}llmann, Dorothe and Arias-Loza, Paula and Dierks, Johann and Schuler, Irina and Freier, Erik and Debus, J{\"o}rg and Baba, Hideo A. and Nordbeck, Peter and Bocklitz, Thomas and Lorenz, Kristina}, title = {CARS imaging advances early diagnosis of cardiac manifestation of Fabry disease}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {10}, issn = {1422-0067}, doi = {10.3390/ijms23105345}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284427}, year = {2022}, abstract = {Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient's prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90-96\%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD.}, language = {en} } @article{GmachBathePetersTeluguetal.2022, author = {Gmach, Philipp and Bathe-Peters, Marc and Telugu, Narasimha and Miller, Duncan C. and Annibale, Paolo}, title = {Fluorescence spectroscopy of low-level endogenous β-adrenergic receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {18}, issn = {1422-0067}, doi = {10.3390/ijms231810405}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288277}, year = {2022}, abstract = {The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β\(_1\)- and β\(_2\)-adrenergic receptors (β\(_{1/2}\)-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs.}, language = {en} } @article{DjelićBorozanDimitrijevićSrećkovićetal.2022, author = {Djelić, Ninoslav and Borozan, Sunčica and Dimitrijević-Srećković, Vesna and Pajović, Nevena and Mirilović, Milorad and Stopper, Helga and Stanimirović, Zoran}, title = {Oxidative stress and DNA damage in peripheral blood mononuclear cells from normal, obese, prediabetic and diabetic persons exposed to thyroid hormone in vitro}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {16}, issn = {1422-0067}, doi = {10.3390/ijms23169072}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285988}, year = {2022}, abstract = {Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM.}, language = {en} } @article{SedaghatHamedaniRebsKayvanpouretal.2022, author = {Sedaghat-Hamedani, Farbod and Rebs, Sabine and Kayvanpour, Elham and Zhu, Chenchen and Amr, Ali and M{\"u}ller, Marion and Haas, Jan and Wu, Jingyan and Steinmetz, Lars M. and Ehlermann, Philipp and Streckfuss-B{\"o}meke, Katrin and Frey, Norbert and Meder, Benjamin}, title = {Genotype complements the phenotype: identification of the pathogenicity of an LMNA splice variant by nanopore long-read sequencing in a large DCM family}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {20}, issn = {1422-0067}, doi = {10.3390/ijms232012230}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290415}, year = {2022}, abstract = {Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20-40\% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.}, language = {en} } @article{BittnerBoonDelbancoetal.2022, author = {Bittner, Nataly and Boon, Andy and Delbanco, Evert H. and Walter, Christof and Mally, Angela}, title = {Assessment of aromatic amides in printed food contact materials: analysis of potential cleavage to primary aromatic amines during simulated passage through the gastrointestinal tract}, series = {Archives of Toxicology}, volume = {96}, journal = {Archives of Toxicology}, number = {5}, doi = {10.1007/s00204-022-03254-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-324697}, pages = {1423-1435}, year = {2022}, abstract = {Recent analyses conducted by German official food control reported detection of the aromatic amides N-(2,4-dimethylphenyl)acetamide (NDPA), N-acetoacetyl-m-xylidine (NAAX) and 3-hydroxy-2-naphthanilide (Naphthol AS) in cold water extracts from certain food contact materials made from paper or cardboard, including paper straws, paper napkins, and cupcake liners. Because aromatic amides may be cleaved to potentially genotoxic primary amines upon oral intake, these findings raise concern that transfer of NDPA, NAAX and Naphthol AS from food contact materials into food may present a risk to human health. The aim of the present work was to assess the stability of NDPA, NAAX and Naphthol AS and potential cleavage to 2,4-dimethylaniline (2,4-DMA) and aniline during simulated passage through the gastrointestinal tract using static in vitro digestion models. Using the digestion model established by the National Institute for Public Health and the Environment (RIVM, Bilthoven, NL) and a protocol recommended by the European Food Safety Authority, potential hydrolysis of the aromatic amides to the respective aromatic amines was assessed by LC-MS/MS following incubation of the aromatic amides with digestive fluid simulants. Time-dependent hydrolysis of NDPA and NAAX resulting in formation of the primary aromatic amine 2,4-DMA was consistently observed in both models. The highest rate of cleavage of NDPA and NAAX was recorded following 4 h incubation with 0.07 M HCl as gastric-juice simulant, and amounted to 0.21\% and 0.053\%, respectively. Incubation of Naphthol AS with digestive fluid simulants did not give rise to an increase in the concentration of aniline above the background that resulted from the presence of aniline as an impurity of the test compound. Considering the lack of evidence for aniline formation from Naphthol AS and the extremely low rate of hydrolysis of the amide bonds of NDPA and NAAX during simulated passage through the gastrointestinal tract that gives rise to only very minor amounts of the potentially mutagenic and/or carcinogenic aromatic amine 2,4-DMA, risk assessment based on assumption of 100\% cleavage to the primary aromatic amines would appear to overestimate health risks related to the presence of aromatic amides in food contact materials.}, language = {en} } @phdthesis{Konrad2021, author = {Konrad, Charlotte}, title = {Biochemische Charakterisierung von cAMP-Gradienten - Einfluss von Phosphodiesterasen}, doi = {10.25972/OPUS-20572}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205728}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Cyclisches Adenosinmonophosphat ist ein ubiquit{\"a}rer zweiter Botenstoff zahlreicher Signalwege im menschlichen K{\"o}rper. Auf eine Vielzahl verschiedenster extrazellul{\"a}rer Signale folgt jedoch eine Erh{\"o}hung desselben intrazellul{\"a}ren Botenstoffs - cAMP. Nichtsdestotrotz schafft es die Zelle, Signalspezifit{\"a}t aufrecht zu erhalten. Ein anerkanntes, wenn auch bisher unverstandenes Modell, um dieses zu erm{\"o}glichen, ist das Prinzip der Kompartimentierung. Die Zelle besitzt demnach Areale verschieden hoher cAMP-Konzentrationen, welche lokal begrenzt einzelne Signalkaskaden beeinflussen und somit eine differenzierte Signal{\"u}bertragung erm{\"o}glichen. Eine m{\"o}gliche Ursache f{\"u}r die Ausbildung solcher Bereiche geringerer cAMP- Konzentrationen (hier als Dom{\"a}nen bezeichnet), ist die hydrolytische Aktivit{\"a}t von Phosphodiesterasen (PDEs), welche als einzige Enzyme die F{\"a}higkeiten besitzen, cAMP zu degradieren. In dieser Arbeit wird der Einfluss der cAMP-Hydrolyse verschiedener PDEs auf die Gr{\"o}ße dieser Dom{\"a}nen evaluiert und mit denen der PDE4A1 verglichen, welche bereits durch unsere Arbeitsgruppe aufgrund ihrer Gr{\"o}ße als Nanodom{\"a}nen definiert wurden. Der Fokus wird dabei auf den Einfluss von kinetischen Eigenschaften der Phosphodiesterasen gelegt. So werden eine PDE mit hoher Umsatzgeschwindigkeit (PDE2A3) und eine PDE mit hoher Substrataffinit{\"a}t (PDE8A1) verglichen. Mithilfe sogenannter Linker, Abstandshaltern definierter L{\"a}nge, werden zus{\"a}tzlich die Nanodom{\"a}nen ausgemessen, um einen direkten Zusammenhang zwischen Gr{\"o}ße und kinetischer Eigenschaft anzugeben. Die Zusammenschau der Ergebnisse zeigt, dass die maximale Umsatzgeschwindigkeit der Phosphodiesterasen direkt mit der Gr{\"o}ße der Nanodom{\"a}nen korreliert. Durch den unmittelbaren Vergleich der gesamten PDE mit ihrer katalytischen Dom{\"a}ne wird zus{\"a}tzlich der Einfluss von regulatorischen Dom{\"a}nen evaluiert. Es wird gezeigt, dass diese cAMP-Gradienten modulieren k{\"o}nnen. Bei der PDE2A3 geschieht die Modulation u.a. durch Stimulation mit cGMP, welche h{\"o}chstwahrscheinlich dosisabh{\"a}ngig ist und somit graduell verl{\"a}uft. Hiermit pr{\"a}sentieren sich die Dom{\"a}nen als dynamische Bereiche, d.h. sie k{\"o}nnen in ihrer Auspr{\"a}gung reguliert werden. In dieser Arbeit wird die Hypothese best{\"a}tigt, dass Phosphodiesterasen eine wichtige Rolle in der Kompartimentierung von cAMP spielen, die Gruppe jedoch inhomogener ist, als bislang angenommen. Die Gradienten-Bildung l{\"a}sst sich nicht bei jeder Phosphodiesterase darstellen (PDE8A1). Einige Phosphodiesterasen (PDE2A3) jedoch bilden Kompartimente, die durch externe Stimuli in ihrer Gr{\"o}ße reguliert werden k{\"o}nnen. Die Arbeit legt den Grundstein zur breiteren Charakterisierung des spezifischen Einflusses weiterer PDEs auf cAMP-Kompartimentierung, welches nicht nur das Verst{\"a}ndnis der Kompartimentierungs-Strategien voranbringt, sondern auch essentiell f{\"u}r das Verst{\"a}ndnis der Pathophysiologie zahlreicher Krankheitsbilder, aber auch f{\"u}r das Verst{\"a}ndnis bereits angewandter aber auch potentiell neuer Medikamente ist.}, subject = {Cyclo-AMP}, language = {de} } @phdthesis{Kreutzmann2021, author = {Kreutzmann, Moritz Paul}, title = {Untersuchung von Markern f{\"u}r oxidativen Stress und DNA-Sch{\"a}den bei arterieller Hypertonie}, doi = {10.25972/OPUS-24338}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-243380}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Patienten mit arterieller Hypertonie haben ein erh{\"o}htes Risiko eine Tumorerkrankung, insbesondere Nierenzellkarzinome, zu entwickeln. Die arterielle Hypertonie ist {\"u}ber die Entstehung von oxidativem Stress mit der Entwicklung von DNA-Sch{\"a}den verkn{\"u}pft, wobei ein hochreguliertes Renin-Angiotensin-Aldosteron-System (RAAS) eine entscheidende Rolle einnimmt. Das Ziel dieser Arbeit war es zum einen Hypertoniker (HypAll) und gesunde Kontrollen und zum anderen gut (HypGut) und schlecht (HypSch) eingestellte Hypertoniker unter Ber{\"u}cksichtigung der eingenommenen Antihypertensiva bez{\"u}glich ihrer Level an oxidativem Stress und DNA-Sch{\"a}den zu vergleichen. Zus{\"a}tzlich erfolgte im Rahmen einer L{\"a}ngsschnittanalyse der intraindividuelle Vergleich unter den Hypertonikern. Hierf{\"u}r erfolgte die Bestimmung von SHp, D-ROM und 3-Nitrotyrosin als Marker f{\"u}r oxidativen Stress im Plasma, von 8-oxodG, 15-F2t-Isoprostan und Malondialdehyd als Marker f{\"u}r oxidativen Stress im Urin und von γ-H2AX und Mikrokernen als Marker f{\"u}r DNA-Sch{\"a}den in Lymphozyten. Dabei konnte ein erh{\"o}hter oxidativer Stress in der HypAll-Gruppe verglichen zu den Kontrollen anhand aller Marker f{\"u}r oxidativen Stress mit Ausnahme von Malondialdehyd festgestellt werden. Nach Altersadjustierung zeigte sich dieser Gruppenunterschied nur noch f{\"u}r die Proteinstressmarker SHp und 3-Nitrotyrosin signifikant. Bez{\"u}glich der Marker f{\"u}r DNA-Sch{\"a}den ergab sich kein Unterschied zwischen HypAll und Kontrollen. Ebenso zeigte sich kein signifikanter Unterschied in den Leveln f{\"u}r oxidativen Stress und DNA-Sch{\"a}den zwischen der HypGut- und HypSch-Gruppe. Zuletzt konnte im Rahmen der L{\"a}ngsschnittstudie ein positiver Zusammenhang zwischen der Entwicklung des Blutdrucks und des oxidativen Stresses anhand der Ver{\"a}nderung von D-ROM und des systolischen Blutdrucks beobachtet werden. Die teils nicht-signifikanten und teils mangelnden Unterschiede zwischen HypAll und Kontrollen sowie zwischen HypGut und HypSch sind am ehesten durch das besondere Patientengut, welches sich auch grundlegend von dem anderer vergleichbarer Studien unterscheidet, erkl{\"a}rbar. Die Patienten mit therapieresistenter Hypertonie (TRH) zeichnen sich durch eine langj{\"a}hrige Einnahme zahlreicher Antihypertensiva aus. Diese, insbesondere die RAAS-wirksamen, besitzen eine {\"u}ber die reine Blutdrucksenkung hinausgehende antioxidative und antigenotoxische Wirkung, welche vermutlich zu einer Angleichung der Level f{\"u}r oxidativen Stress und DNA-Sch{\"a}den gef{\"u}hrt hat. Um die Dynamik der Biomarker und den Einfluss der Antihypertensiva auf oxidativen Stress und DNA-Sch{\"a}den besser zu verstehen, sind weitere Studien {\"u}ber einen l{\"a}ngeren Beobachtungszeitraum sowie mit zus{\"a}tzlich therapienaiven Hypertonikern sinnvoll. Die weitere Erforschung von Biomarkern, um sie im klinischen Alltag zur Verbesserung der Patientenbehandlung einsetzen zu k{\"o}nnen, ist notwendig.}, subject = {Oxidativer Stress}, language = {de} } @phdthesis{Reimann2021, author = {Reimann, Hauke}, title = {Schicksal von Mikrokernen bzw. mikrokernhaltigen Zellen und Bedeutung von Mikrokernen als Biomarker}, doi = {10.25972/OPUS-24010}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240109}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Mikrokerne sind als wichtiger Biomarker in der Gentoxizit{\"a}tsforschung seit langer Zeit etabliert und ihre Bildung ist mechanistisch gut verstanden, wohingegen das Mikrokernschicksal und die genaue Funktion von Mikrokernen in der Kanzerogenese unzureichend erforscht sind. Um das Schicksal von Mikrokernen und mikrokernhaltigen Zellen {\"u}ber einen l{\"a}ngeren Zeitraum zu untersuchen, wurden HeLa-Zellen, die mit einem GFP-markierten Histon H2B transfiziert worden sind, mittels Lebendzellmikroskopie nach Behandlung mit verschiedenen gentoxischen Agenzien f{\"u}r 96 h untersucht. Parameter wie die Mitose- oder Zelltodrate wurden dabei ebenso wie das Schicksal der Mikrokerne dokumentiert. W{\"a}hrend Persistenz und Reinkorporation von Mikrokernen h{\"a}ufig beobachtet wurden, waren Degradation und Auswurf von Mikrokernen selten bis gar nicht zu sehen. Auch konnte ein Teil der mikrokernhaltigen Zellen {\"u}ber mehrere Zellteilungen persistieren und proliferieren, wodurch die in Mikrokernen manifestierte chromosomale Instabilit{\"a}t unver{\"a}ndert bleiben kann. Ein eindeutiger Substanzeinfluss auf das Mikrokernschicksal konnte nicht ausgemacht werden. Extrusion sollte weiterhin durch Behandlung mit Hydroxyurea oder Cytochalasin B in Kombination mit gentoxischer Behandlung induziert werden, es wurde jedoch kein Effekt auf die Extrusionsrate beobachtet. Degradation wurde mittels γH2AX-Antik{\"o}rperf{\"a}rbung und transduziertem dsRed-markierten Autophagiemarker LC3B in HeLa-H2B-GFP-Zellen untersucht. Trotz erh{\"o}hter DNA-Degradation in Mikrokernen wurde nur selten eine Ko-Lokalisierung mit LC3B beobachtet. Daf{\"u}r gab es in HeLa-H2B-GFP-Zellen, die zus{\"a}tzlich mit dsRed markierten Kernmembranmarker Lamin B1 transduziert worden sind, Anzeichen f{\"u}r eine eingeschr{\"a}nkte Mikrokernmembranintegrit{\"a}t. Weiterhin wurden Zytokinese-Block Mikrokerntests nach Behandlung mit Thebain mit und ohne metabolische Aktivierung sowie Celecoxib und Celecoxibderivaten durchgef{\"u}hrt. Hierbei wurde nach Thebainbehandlung nur ohne metabolische Aktivierung und bei Anwesenheit von Zytotoxizit{\"a}t mehr Mikrokerne gefunden, w{\"a}hrend nach Behandlung mit Celecoxib und Celecoxibderivaten kein Anstieg beobachtet wurde. Zus{\"a}tzlich wurde der Einfluss durch neurodegenerative Ver{\"a}nderungen auf Mundschleimhautzellen in zwei großen Kohorten untersucht, wobei keine Effekte auf die H{\"a}ufigkeit von Mikrokernen oder mikrokernhaltigen Zellen zugeordnet werden konnten, w{\"a}hrend es teilweise bei Parametern, die auf Zytotoxizit{\"a}t hindeuten, zu Ver{\"a}nderungen kam. Es konnte insgesamt gezeigt werden, dass Mikrokerne und mikrokernhaltige Zellen zus{\"a}tzlich zu ihrer Funktion als Biomarker {\"u}ber wenigstens mehrere Zellteilungen bestehen bleiben k{\"o}nnen. Auf diese Weise k{\"o}nnen sie z. B. {\"u}ber Chromothripsis zu einer beschleunigten Kanzerogenese f{\"u}hren, was zu einer schlechten Prognose f{\"u}r Krebspatienten f{\"u}hren kann.}, subject = {Kleinkern}, language = {de} } @article{DekantLangerLuppetal.2021, author = {Dekant, Raphael and Langer, Michael and Lupp, Maria and Adaku Chilaka, Cynthia and Mally, Angela}, title = {In vitro and in vivo analysis of ochratoxin A-derived glucuronides and mercapturic acids as biomarkers of exposure}, series = {Toxins}, volume = {13}, journal = {Toxins}, number = {8}, issn = {2072-6651}, doi = {10.3390/toxins13080587}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-245146}, year = {2021}, abstract = {Ochratoxin A (OTA) is a widespread food contaminant, with exposure estimated to range from 0.64 to 17.79 ng/kg body weight (bw) for average consumers and from 2.40 to 51.69 ng/kg bw per day for high consumers. Current exposure estimates are, however, associated with considerable uncertainty. While biomarker-based approaches may contribute to improved exposure assessment, there is yet insufficient data on urinary metabolites of OTA and their relation to external dose to allow reliable estimates of daily intake. This study was designed to assess potential species differences in phase II biotransformation in vitro and to establish a correlation between urinary OTA-derived glucuronides and mercapturic acids and external exposure in rats in vivo. In vitro analyses of OTA metabolism using the liver S9 of rats, humans, rabbits and minipigs confirmed formation of an OTA glucuronide but provided no evidence for the formation of OTA-derived mercapturic acids to support their use as biomarkers. Similarly, OTA-derived mercapturic acids were not detected in urine of rats repeatedly dosed with OTA, while indirect analysis using enzymatic hydrolysis of the urine samples prior to LC-MS/MS established a linear relationship between urinary glucuronide excretion and OTA exposure. These results support OTA-derived glucuronides but not mercapturic acids as metabolites suitable for biomonitoring.}, language = {en} } @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} } @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{Schott2021, author = {Schott, Lea Marie}, title = {In vitro Untersuchung zur Genotoxizit{\"a}t ausgew{\"a}hlter Pyrrolizidinalkaloide}, doi = {10.25972/OPUS-24171}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-241716}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Pyrrolizidinalkaloide (PA) sind sekund{\"a}re Pflanzenstoffe, welche {\"u}ber Nahrungsmittel in den menschlichen Organismus gelangen k{\"o}nnen. Zahlreiche Studien belegen, dass PA in der Leber verstoffwechselt und dabei in aktive genotoxische Metabolite umgewandelt werden. Diese verursachen vor allem in der Leber zellul{\"a}re Sch{\"a}den, was sich klinisch in Form einer hepatischen ven{\"o}sen okklusiven Leberkrankheit, aber auch in der Entstehung von Tumoren zeigt. Die vorliegende Arbeit testet das genotoxische Potential der drei PA Lasiocarpin, Senecionin und Seneciphyllin anhand der Leberzelllinie Huh6 mit Hilfe des Mikrokerntests. Dar{\"u}ber hinaus wird die Wirkung von Lasiocarpin auf den intrazellul{\"a}ren Glutathion-Gehalt, die Superoxidproduktion und das mitochondriale Membranpotential analysiert. Zudem werden sowohl der eventuell negative Einfluss einer Glutathion Depletion, als auch die m{\"o}glicherweise sch{\"u}tzenden Effekte des pflanzlichen Antioxidans Delphinidin in Bezug auf die Genotoxizit{\"a}t von Lasiocarpin untersucht. Es konnte gezeigt werden, dass alle drei ausgew{\"a}hlten PA einen signifikanten Anstieg der Mikrokernfrequenz bewirken.Unsere Messungen zeigten f{\"u}r Lasiocarpin eine dezente Reduktion des Glutathion Gehalts. Dagegen f{\"u}hrte eine Glutathion-Depletion in den Huh6 Zellen zu keiner Steigerung der Genotoxizit{\"a}t von Lasiocarpin. In Kombination mit dem Antioxidans Delphinidin zeigte sich f{\"u}r Lasiocarpin eine signifikante Reduktion der Mikrokernfrequenz. Abschließend ist anzumerken, dass in Zukunft vor allem die Wechselwirkung der PA untereinander und mit anderen (Pflanzen-)bestandteilen f{\"u}r eine verbesserte Risikoabsch{\"a}tzung der PA-Exposition untersucht werden sollte.}, subject = {Pyrrolizidinalkaloide}, language = {de} } @phdthesis{Anton2021, author = {Anton, Selma}, title = {Characterization of cAMP nanodomains surrounding the human Glucagon-like peptide 1 receptor using FRET-based reporters}, doi = {10.25972/OPUS-19069}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-190695}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Cyclic adenosine monophosphate (cAMP), the ubiquitous second messenger produced upon stimulation of GPCRs which couple to the stimulatory GS protein, orchestrates an array of physiological processes including cardiac function, neuronal plasticity, immune responses, cellular proliferation and apoptosis. By interacting with various effector proteins, among others protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), it triggers signaling cascades for the cellular response. Although the functional outcomes of GSPCR-activation are very diverse depending on the extracellular stimulus, they are all mediated exclusively by this single second messenger. Thus, the question arises how specificity in such responses may be attained. A hypothesis to explain signaling specificity is that cellular signaling architecture, and thus precise operation of cAMP in space and time would appear to be essential to achieve signaling specificity. Compartments with elevated cAMP levels would allow specific signal relay from receptors to effectors within a micro- or nanometer range, setting the molecular basis for signaling specificity. Although the paradigm of signaling compartmentation gains continuous recognition and is thoroughly being investigated, the molecular composition of such compartments and how they are maintained remains to be elucidated. In addition, such compartments would require very restricted diffusion of cAMP, but all direct measurements have indicated that it can diffuse in cells almost freely. In this work, we present the identification and characterize of a cAMP signaling compartment at a GSPCR. We created a F{\"o}rster resonance energy transfer (FRET)-based receptor-sensor conjugate, allowing us to study cAMP dynamics in direct vicinity of the human glucagone-like peptide 1 receptor (hGLP1R). Additional targeting of analogous sensors to the plasma membrane and the cytosol enables assessment of cAMP dynamics in different subcellular regions. We compare both basal and stimulated cAMP levels and study cAMP crosstalk of different receptors. With the design of novel receptor nanorulers up to 60nm in length, which allow mapping cAMP levels in nanometer distance from the hGLP1R, we identify a cAMP nanodomain surrounding it. Further, we show that phosphodiesterases (PDEs), the only enzymes known to degrade cAMP, are decisive in constraining cAMP diffusion into the cytosol thereby maintaining a cAMP gradient. Following the discovery of this nanodomain, we sought to investigate whether downstream effectors such as PKA are present and active within the domain, additionally studying the role of A-kinase anchoring proteins (AKAPs) in targeting PKA to the receptor compartment. We demonstrate that GLP1-produced cAMP signals translate into local nanodomain-restricted PKA phosphorylation and determine that AKAP-tethering is essential for nanodomain PKA. Taken together, our results provide evidence for the existence of a dynamic, receptor associated cAMP nanodomain and give prospect for which key proteins are likely to be involved in its formation. These conditions would allow cAMP to exert its function in a spatially and temporally restricted manner, setting the basis for a cell to achieve signaling specificity. Understanding the molecular mechanism of cAMP signaling would allow modulation and thus regulation of GPCR signaling, taking advantage of it for pharmacological treatment.}, language = {en} } @article{WagnerSadekDybkovaetal.2021, author = {Wagner, Michael and Sadek, Mirna S. and Dybkova, Nataliya and Mason, Fleur E. and Klehr, Johann and Firneburg, Rebecca and Cachorro, Eleder and Richter, Kurt and Klapproth, Erik and Kuenzel, Stephan R. and Lorenz, Kristina and Heijman, Jordi and Dobrev, Dobromir and El-Armouche, Ali and Sossalla, Samuel and K{\"a}mmerer, Susanne}, title = {Cellular mechanisms of the anti-arrhythmic effect of cardiac PDE2 overexpression}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {9}, issn = {1422-0067}, doi = {10.3390/ijms22094816}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285888}, year = {2021}, abstract = {Background: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to β-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. Methods: Cellular arrhythmia, ion currents, and Ca\(^{2+}\)-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates. Results: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in I\(_{CaL}\) seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in I\(_{NaL}\) and Ca\(^{2+}\)-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. Conclusion: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.}, language = {en} } @article{SedaghatHamedaniRebsElBattrawyetal.2021, author = {Sedaghat-Hamedani, Farbod and Rebs, Sabine and El-Battrawy, Ibrahim and Chasan, Safak and Krause, Tobias and Haas, Jan and Zhong, Rujia and Liao, Zhenxing and Xu, Qiang and Zhou, Xiaobo and Akin, Ibrahim and Zitron, Edgar and Frey, Norbert and Streckfuss-B{\"o}meke, Katrin and Kayvanpour, Elham}, title = {Identification of SCN5a p.C335R variant in a large family with dilated cardiomyopathy and conduction disease}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {23}, issn = {1422-0067}, doi = {10.3390/ijms222312990}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284442}, year = {2021}, abstract = {Introduction: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. Methods: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. Results: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na\(^+\) channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. Conclusion: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.}, 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} } @article{BauerMallyLiedtke2021, author = {Bauer, Benedikt and Mally, Angela and Liedtke, Daniel}, title = {Zebrafish embryos and larvae as alternative animal models for toxicity testing}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {24}, issn = {1422-0067}, doi = {10.3390/ijms222413417}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284225}, year = {2021}, abstract = {Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.}, language = {en} } @article{BankogluSchueleStopper2021, author = {Bankoglu, Ezgi Eyluel and Schuele, Carolin and Stopper, Helga}, title = {Cell survival after DNA damage in the comet assay}, series = {Archives of Toxicology}, volume = {95}, journal = {Archives of Toxicology}, number = {12}, doi = {10.1007/s00204-021-03164-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265339}, pages = {3803-3813}, year = {2021}, abstract = {The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H\(_{2}\)O\(_{2}\)) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30\% DNA in tail caused the death of more than 50\% of the cells, with etoposide causing slightly more cell death than H\(_{2}\)O\(_{2}\) or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20\% DNA in tail, survival data for the cells are provided.}, language = {en} } @phdthesis{Kodandaraman2021, author = {Kodandaraman, Geema}, title = {Influence of insulin-induced oxidative stress in genotoxicity and disease}, doi = {10.25972/OPUS-24200}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242005}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Hormones are essential components in the body and their imbalance leads to pathological consequences. T2DM, insulin resistance and obesity are the most commonly occurring lifestyle diseases in the past decade. Also, an increased cancer incidence has been strongly associated with obese and T2DM patients. Therefore, our aim was to study the influence of high insulin levels in accumulating DNA damage in in vitro models and patients, through the induction of oxidative stress. The primary goal of this study was to analyze the genotoxicity induced by the combined action of two endogenous hormones (insulin and adrenaline) with in vitro models, through the induction of micronuclei and to see if they cause an additive increase in genomic damage. This is important for multifactorial diseases having high levels of more than one hormone, such as metabolic syndrome and conditions with multiple pathologies (e.g., T2DM along with high stress levels). Furthermore, the combination of insulin and the pharmacological inhibition of the tumor suppressor gene: PTEN, was to be tested in in vitro models for their genotoxic effect and oxidative stress inducing potential. As the tumor suppressor gene: PTEN is downregulated in PTEN associated syndromes and when presented along with T2DM and insulin resistance, this may increase the potential to accumulate genomic damage. The consequences of insulin action were to be further elucidated by following GFP-expressing cells in live cell-imaging to observe the ability of insulin, to induce micronuclei and replicative stress. Finally, the detrimental potential of high insulin levels in obese patients with hyperinsulinemia and pre-diabetes was to be studied by analyzing markers of oxidative stress and genomic damage. In summary, the intention of this work was to understand the effects of high insulin levels in in vitro and in patients to understand its relevance for the development of genomic instability and thus an elevated cancer risk.}, subject = {Insulin}, language = {en} } @article{BankogluStippGerberetal.2021, author = {Bankoglu, Ezgi Eyluel and Stipp, Franzisca and Gerber, Johanna and Seyfried, Florian and Heidland, August and Bahner, Udo and Stopper, Helga}, title = {Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay}, series = {Archives of Toxicology}, volume = {95}, journal = {Archives of Toxicology}, number = {5}, doi = {10.1007/s00204-021-03012-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265326}, pages = {1831-1841}, year = {2021}, abstract = {The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.}, language = {en} } @article{WeigandRonchiVanselowetal.2021, author = {Weigand, Isabel and Ronchi, Cristina L. and Vanselow, Jens T. and Bathon, Kerstin and Lenz, Kerstin and Herterich, Sabine and Schlosser, Andreas and Kroiss, Matthias and Fassnacht, Martin and Calebiro, Davide and Sbiera, Silviu}, title = {PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation}, series = {Science Advances}, volume = {7}, journal = {Science Advances}, number = {8}, doi = {10.1126/sciadv.abd4176}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270445}, year = {2021}, abstract = {Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing's syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing's syndrome.}, language = {en} } @article{GuthHueserRothetal.2021, author = {Guth, Sabine and H{\"u}ser, Stephanie and Roth, Angelika and Degen, Gisela and Diel, Patrick and Edlund, Karolina and Eisenbrand, Gerhard and Engel, Karl-Heinz and Epe, Bernd and Grune, Tilman and Heinz, Volker and Henle, Thomas and Humpf, Hans-Ulrich and J{\"a}ger, Henry and Joost, Hans-Georg and Kulling, Sabine E. and Lampen, Alfonso and Mally, Angela and Marchan, Rosemarie and Marko, Doris and M{\"u}hle, Eva and Nitsche, Michael A. and R{\"o}hrdanz, Elke and Stadler, Richard and van Thriel, Christoph and Vieths, Stefan and Vogel, Rudi F. and Wascher, Edmund and Watzl, Carsten and N{\"o}thlings, Ute and Hengstler, Jan G.}, title = {Contribution to the ongoing discussion on fluoride toxicity}, series = {Archives of Toxicology}, volume = {95}, journal = {Archives of Toxicology}, number = {7}, issn = {0340-5761}, doi = {10.1007/s00204-021-03072-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-307161}, pages = {2571-2587}, year = {2021}, abstract = {Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries.}, language = {en} } @article{BarileBerryBlaauboeretal.2021, author = {Barile, Frank A. and Berry, Colin and Blaauboer, Bas and Boobis, Alan and Bolt, Herrmann M. and Borgert, Christopher and Dekant, Wolfgang and Dietrich, Daniel and Domingo, Jose L. and Galli, Corrado L. and Gori, Gio Batta and Greim, Helmut and Hengstler, Jan G. and Heslop-Harrison, Pat and Kacew, Sam and Marquardt, Hans and Mally, Angela and Pelkonen, Olavi and Savolainen, Kai and Testai, Emanuela and Tsatsakis, Aristides and Vermeulen, Nico P.}, title = {The EU chemicals strategy for sustainability: in support of the BfR position}, series = {Archives of Toxicology}, volume = {95}, journal = {Archives of Toxicology}, number = {9}, issn = {0340-5761}, doi = {10.1007/s00204-021-03125-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-307154}, pages = {3133-3136}, year = {2021}, abstract = {The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.}, language = {en} } @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} } @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{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{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{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} } @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} } @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{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} } @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} } @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} } @article{FreyGassenmaierHofmannetal.2020, author = {Frey, Anna and Gassenmaier, Tobias and Hofmann, Ulrich and Schmitt, Dominik and Fette, Georg and Marx, Almuth and Heterich, Sabine and Boivin-Jahns, Val{\´e}rie and Ertl, Georg and Bley, Thorsten and Frantz, Stefan and Jahns, Roland and St{\"o}rk, Stefan}, title = {Coagulation factor XIII activity predicts left ventricular remodelling after acute myocardial infarction}, series = {ESC Heart Failure}, volume = {7}, journal = {ESC Heart Failure}, number = {5}, doi = {10.1002/ehf2.12774}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236013}, pages = {2354-2364}, year = {2020}, abstract = {Aims Acute myocardial infarction (MI) is the major cause of chronic heart failure. The activity of blood coagulation factor XIII (FXIIIa) plays an important role in rodents as a healing factor after MI, whereas its role in healing and remodelling processes in humans remains unclear. We prospectively evaluated the relevance of FXIIIa after acute MI as a potential early prognostic marker for adequate healing. Methods and results This monocentric prospective cohort study investigated cardiac remodelling in patients with ST-elevation MI and followed them up for 1 year. Serum FXIIIa was serially assessed during the first 9 days after MI and after 2, 6, and 12 months. Cardiac magnetic resonance imaging was performed within 4 days after MI (Scan 1), after 7 to 9 days (Scan 2), and after 12 months (Scan 3). The FXIII valine-to-leucine (V34L) single-nucleotide polymorphism rs5985 was genotyped. One hundred forty-six patients were investigated (mean age 58 ± 11 years, 13\% women). Median FXIIIa was 118 \% (quartiles, 102-132\%) and dropped to a trough on the second day after MI: 109\%(98-109\%; P < 0.001). FXIIIa recovered slowly over time, reaching the baseline level after 2 to 6 months and surpassed baseline levels only after 12 months: 124 \% (110-142\%). The development of FXIIIa after MI was independent of the genotype. FXIIIa on Day 2 was strongly and inversely associated with the relative size of MI in Scan 1 (Spearman's ρ = -0.31; P = 0.01) and Scan 3 (ρ = -0.39; P < 0.01) and positively associated with left ventricular ejection fraction: ρ = 0.32 (P < 0.01) and ρ = 0.24 (P = 0.04), respectively. Conclusions FXIII activity after MI is highly dynamic, exhibiting a significant decline in the early healing period, with reconstitution 6 months later. Depressed FXIIIa early after MI predicted a greater size of MI and lower left ventricular ejection fraction after 1 year. The clinical relevance of these findings awaits to be tested in a randomized trial.}, language = {en} } @article{PaisdziorDimitriouSchoepeetal.2020, author = {Paisdzior, Sarah and Dimitriou, Ioanna Maria and Sch{\"o}pe, Paul Curtis and Annibale, Paolo and Scheerer, Patrick and Krude, Heiko and Lohse, Martin J. and Biebermann, Heike and K{\"u}hnen, Peter}, title = {Differential signaling profiles of MC4R mutations with three different ligands}, series = {International Journal of Molecular Sciences}, volume = {21}, journal = {International Journal of Molecular Sciences}, number = {4}, issn = {1422-0067}, doi = {10.3390/ijms21041224}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285108}, year = {2020}, abstract = {The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin-melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via G\(_S\)-dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a G\(_S\) loss-of-function (S127L) and a G\(_S\) gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and β-arrestin2 recruitment, using the two endogenous agonists, α- and β-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the G\(_{q/11}\) pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations.}, language = {en} } @article{ObidiegwuLyonsChilaka2020, author = {Obidiegwu, Jude E. and Lyons, Jessica B. and Chilaka, Cynthia A.}, title = {The Dioscorea genus (yam) — an appraisal of nutritional and therapeutic potentials}, series = {Foods}, volume = {9}, journal = {Foods}, number = {9}, issn = {2304-8158}, doi = {10.3390/foods9091304}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213102}, year = {2020}, abstract = {The quest for a food secure and safe world has led to continuous effort toward improvements of global food and health systems. While the developed countries seem to have these systems stabilized, some parts of the world still face enormous challenges. Yam (Dioscorea species) is an orphan crop, widely distributed globally; and has contributed enormously to food security especially in sub-Saharan Africa because of its role in providing nutritional benefits and income. Additionally, yam has non-nutritional components called bioactive compounds, which offer numerous health benefits ranging from prevention to treatment of degenerative diseases. Pharmaceutical application of diosgenin and dioscorin, among other compounds isolated from yam, has shown more prospects recently. Despite the benefits embedded in yam, reports on the nutritional and therapeutic potentials of yam have been fragmented and the diversity within the genus has led to much confusion. An overview of the nutritional and health importance of yam will harness the crop to meet its potential towards combating hunger and malnutrition, while improving global health. This review makes a conscious attempt to provide an overview regarding the nutritional, bioactive compositions and therapeutic potentials of yam diversity. Insights on how to increase its utilization for a greater impact are elucidated.}, language = {en} } @article{VazquezRodriguezVilarKachleretal.2020, author = {Vazquez-Rodriguez, Saleta and Vilar, Santiago and Kachler, Sonja and Klotz, Karl-Norbert and Uriarte, Eugenio and Borges, Fernanda and Matos, Maria Jo{\~a}o}, title = {Adenosine receptor ligands: coumarin-chalcone hybrids as modulating agents on the activity of hARs}, series = {Molecules}, volume = {25}, journal = {Molecules}, number = {18}, issn = {1420-3049}, doi = {10.3390/molecules25184306}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213165}, year = {2020}, abstract = {Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin-chalcone hybrids were synthesized (compounds 1-8) and screened in radioligand binding (hA\(_1\), hA\(_{2A}\) and hA\(_3\)) and adenylyl cyclase (hA\(_{2B}\)) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin-chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA\(_1\) or hA\(_3\) subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA\(_1\), while the methoxy counterparts were selective for the hA\(_3\). The most potent hA\(_1\) ligand was compound 7 (K\(_i\) = 17.7 µM), whereas compound 4 was the most potent ligand for hA\(_3\) (K\(_i\) = 2.49 µM). In addition, docking studies with hA\(_1\) and hA\(_3\) homology models were established to analyze the structure-function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.}, language = {en} } @article{ReimannStopperPolaketal.2020, author = {Reimann, Hauke and Stopper, Helga and Polak, Thomas and Lauer, Martin and Herrmann, Martin J. and Deckert, J{\"u}rgen and Hintzsche, Henning}, title = {Micronucleus frequency in buccal mucosa cells of patients with neurodegenerative diseases}, series = {Scientific Reports}, volume = {10}, journal = {Scientific Reports}, doi = {10.1038/s41598-020-78832-y}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231430}, year = {2020}, abstract = {Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer's disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome.}, language = {en} } @article{ReimannStopperHintzsche2020, author = {Reimann, Hauke and Stopper, Helga and Hintzsche, Henning}, title = {Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells}, series = {Archives of Toxicology}, volume = {94}, journal = {Archives of Toxicology}, issn = {0340-5761}, doi = {10.1007/s00204-020-02840-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235039}, pages = {3553-3561}, year = {2020}, abstract = {Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.}, 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} } @article{JeanclosKnoblochHoffmannetal.2020, author = {Jeanclos, Elisabeth and Knobloch, Gunnar and Hoffmann, Axel and Fedorchenko, Oleg and Odersky, Andrea and Lamprecht, Anna-Karina and Schindelin, Hermann and Gohla, Antje}, title = {Ca\(^{2+}\) functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin}, series = {FEBS Letters}, volume = {594}, journal = {FEBS Letters}, number = {13}, doi = {10.1002/1873-3468.13795}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217963}, pages = {2099 -- 2115}, year = {2020}, abstract = {Pyridoxal 5′-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ-aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)- and integrin-binding protein 1 (CIB1) as a PDXP interactor by yeast two-hybrid screening and find a calmodulin (CaM)-binding motif that overlaps with the PDXP-CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP-CIB1 complex may functionally differ from the PDXP-Ca\(^{2+}\)-CaM complex.}, 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{WinkelbeinerWandtEbertetal.2020, author = {Winkelbeiner, Nicola and Wandt, Viktoria K. and Ebert, Franziska and Lossow, Kristina and Bankoglu, Ezgi E. and Martin, Maximilian and Mangerich, Aswin and Stopper, Helga and Bornhorst, Julia and Kipp, Anna P. and Schwerdtle, Tanja}, title = {A multi-endpoint approach to base excision repair incision activity augmented by PARylation and DNA damage levels in mice: impact of sex and age}, series = {International Journal of Molecular Sciences}, volume = {21}, journal = {International Journal of Molecular Sciences}, number = {18}, issn = {1422-0067}, doi = {10.3390/ijms21186600}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285706}, year = {2020}, abstract = {Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.}, language = {en} } @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} } @article{NaseemOthmanFathyetal.2020, author = {Naseem, Muhammad and Othman, Eman M. and Fathy, Moustafa and Iqbal, Jibran and Howari, Fares M. and AlRemeithi, Fatima A. and Kodandaraman, Geema and Stopper, Helga and Bencurova, Elena and Vlachakis, Dimitrios and Dandekar, Thomas}, title = {Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems}, series = {Scientific Reports}, volume = {10}, journal = {Scientific Reports}, doi = {10.1038/s41598-020-70253-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231317}, year = {2020}, abstract = {Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.}, language = {en} } @article{WoelfelSaetteleZechmeisteretal.2020, author = {W{\"o}lfel, Angela and S{\"a}ttele, Mathias and Zechmeister, Christina and Nikolaev, Viacheslov O. and Lohse, Martin J. and Boege, Fritz and Jahns, Roland and Boivin-Jahns, Val{\´e}rie}, title = {Unmasking features of the auto-epitope essential for β\(_1\)-adrenoceptor activation by autoantibodies in chronic heart failure}, series = {ESC Heart Failure}, volume = {7}, journal = {ESC Heart Failure}, number = {4}, doi = {10.1002/ehf2.12747}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235974}, pages = {1830-1841}, year = {2020}, abstract = {Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β\(_1\)-adrenoceptor (β1ECII) that is targeted by stimulating β\(_1\)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β\(_1\)EC\(_{II}\) loop (compared with the amino acids constituting the ECII loop of the β\(_2\)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β1ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β\(_1\)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β\(_1\)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β\(_1\)EC\(_{II}\) loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK\(^{211-214}\) motif and (ii) the intra-loop disulfide bond C\(^{209}\)↔C\(^{215}\). Of note, aberrant intra-loop disulfide bond C\(^{209}\)↔C\(^{216}\) almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β\(_1\)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β\(_1\)EC\(_{II}\) loop bearing the NDPK\(^{211-214}\) motif and the C\(^{209}\)↔C\(^{215}\) bridge while lacking cysteine C216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β\(_1\)-autoantibodypositive CHF.}, 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{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} } @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{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} } @article{BreitenbachLorenzDandekar2019, author = {Breitenbach, Tim and Lorenz, Kristina and Dandekar, Thomas}, title = {How to steer and control ERK and the ERK signaling cascade exemplified by looking at cardiac insufficiency}, series = {International Journal of Molecular Sciences}, volume = {20}, journal = {International Journal of Molecular Sciences}, number = {9}, issn = {1422-0067}, doi = {10.3390/ijms20092179}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285164}, year = {2019}, abstract = {Mathematical optimization framework allows the identification of certain nodes within a signaling network. In this work, we analyzed the complex extracellular-signal-regulated kinase 1 and 2 (ERK1/2) cascade in cardiomyocytes using the framework to find efficient adjustment screws for this cascade that is important for cardiomyocyte survival and maladaptive heart muscle growth. We modeled optimal pharmacological intervention points that are beneficial for the heart, but avoid the occurrence of a maladaptive ERK1/2 modification, the autophosphorylation of ERK at threonine 188 (ERK\(^{Thr188}\) phosphorylation), which causes cardiac hypertrophy. For this purpose, a network of a cardiomyocyte that was fitted to experimental data was equipped with external stimuli that model the pharmacological intervention points. Specifically, two situations were considered. In the first one, the cardiomyocyte was driven to a desired expression level with different treatment strategies. These strategies were quantified with respect to beneficial effects and maleficent side effects and then which one is the best treatment strategy was evaluated. In the second situation, it was shown how to model constitutively activated pathways and how to identify drug targets to obtain a desired activity level that is associated with a healthy state and in contrast to the maleficent expression pattern caused by the constitutively activated pathway. An implementation of the algorithms used for the calculations is also presented in this paper, which simplifies the application of the presented framework for drug targeting, optimal drug combinations and the systematic and automatic search for pharmacological intervention points. The codes were designed such that they can be combined with any mathematical model given by ordinary differential equations.}, 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{JochmannElkenaniMohamedetal.2019, author = {Jochmann, Svenja and Elkenani, Manar and Mohamed, Belal A. and Buchholz, Eric and Lbik, Dawid and Binder, Lutz and Lorenz, Kristina and Shah, Ajay M. and Hasenfuß, Gerd and Toischer, Karl and Schnelle, Moritz}, title = {Assessing the role of extracellular signal-regulated kinases 1 and 2 in volume overload-induced cardiac remodelling}, series = {ESC Heart Failure}, volume = {6}, journal = {ESC Heart Failure}, number = {5}, doi = {10.1002/ehf2.12497}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212735}, pages = {1015 -- 1026}, year = {2019}, abstract = {Aims Volume overload (VO) and pressure overload (PO) induce differential cardiac remodelling responses including distinct signalling pathways. Extracellular signal-regulated kinases 1 and 2 (ERK1/2), key signalling components in the mitogen-activated protein kinase (MAPK) pathways, modulate cardiac remodelling during pressure overload (PO). This study aimed to assess their role in VO-induced cardiac remodelling as this was unknown. Methods and results Aortocaval fistula (Shunt) surgery was performed in mice to induce cardiac VO. Two weeks of Shunt caused a significant reduction of cardiac ERK1/2 activation in wild type (WT) mice as indicated by decreased phosphorylation of the TEY (Thr-Glu-Tyr) motif (-28\% as compared with Sham controls, P < 0.05). Phosphorylation of other MAPKs was unaffected. For further assessment, transgenic mice with cardiomyocyte-specific ERK2 overexpression (ERK2tg) were studied. At baseline, cardiac ERK1/2 phosphorylation in ERK2tg mice remained unchanged compared with WT littermates, and no overt cardiac phenotype was observed; however, cardiac expression of the atrial natriuretic peptide was increased on messenger RNA (3.6-fold, P < 0.05) and protein level (3.1-fold, P < 0.05). Following Shunt, left ventricular dilation and hypertrophy were similar in ERK2tg mice and WT littermates. Left ventricular function was maintained, and changes in gene expression indicated reactivation of the foetal gene program in both genotypes. No differences in cardiac fibrosis and kinase activation was found amongst all experimental groups, whereas apoptosis was similarly increased through Shunt in ERK2tg and WT mice. Conclusions VO-induced eccentric hypertrophy is associated with reduced cardiac ERK1/2 activation in vivo. Cardiomyocyte-specific overexpression of ERK2, however, does not alter cardiac remodelling during VO. Future studies need to define the pathophysiological relevance of decreased ERK1/2 signalling during VO.}, language = {en} } @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} } @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{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{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} } @article{SchihadaVandenabeeleZabeletal.2018, author = {Schihada, Hannes and Vandenabeele, Sylvie and Zabel, Ulrike and Frank, Monika and Lohse, Martin J. and Maiellaro, Isabella}, title = {A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics}, series = {Communications Biology}, volume = {1}, journal = {Communications Biology}, number = {105}, doi = {10.1038/s42003-018-0072-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228592}, pages = {1-8}, year = {2018}, abstract = {G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the alpha(2 Lambda)-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning beta(2)-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands.}, language = {en} } @article{RietjensDussortGuentheretal.2018, author = {Rietjens, Ivonne M. C. M. and Dussort, P. and G{\"u}nther, Helmut and Hanlon, Paul and Honda, Hiroshi and Mally, Angela and O'Hagan, Sue and Scholz, Gabriele and Seidel, Albrecht and Swenberg, James and Teeguarden, Justin and Eisenbrand, Gerhard}, title = {Exposure assessment of process-related contaminants in food by biomarker monitoring}, series = {Archives of Toxicology}, volume = {92}, journal = {Archives of Toxicology}, number = {1}, doi = {10.1007/s00204-017-2143-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226268}, pages = {15-40}, year = {2018}, abstract = {Exposure assessment is a fundamental part of the risk assessment paradigm, but can often present a number of challenges and uncertainties. This is especially the case for process contaminants formed during the processing, e.g. heating of food, since they are in part highly reactive and/or volatile, thus making exposure assessment by analysing contents in food unreliable. New approaches are therefore required to accurately assess consumer exposure and thus better inform the risk assessment. Such novel approaches may include the use of biomarkers, physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry, and/or duplicate diet studies. This review focuses on the state of the art with respect to the use of biomarkers of exposure for the process contaminants acrylamide, 3-MCPD esters, glycidyl esters, furan and acrolein. From the overview presented, it becomes clear that the field of assessing human exposure to process-related contaminants in food by biomarker monitoring is promising and strongly developing. The current state of the art as well as the existing data gaps and challenges for the future were defined. They include (1) using PBK modelling and duplicate diet studies to establish, preferably in humans, correlations between external exposure and biomarkers; (2) elucidation of the possible endogenous formation of the process-related contaminants and the resulting biomarker levels; (3) the influence of inter-individual variations and how to include that in the biomarker-based exposure predictions; (4) the correction for confounding factors; (5) the value of the different biomarkers in relation to exposure scenario's and risk assessment, and (6) the possibilities of novel methodologies. In spite of these challenges it can be concluded that biomarker-based exposure assessment provides a unique opportunity to more accurately assess consumer exposure to process-related contaminants in food and thus to better inform risk assessment.}, language = {en} } @article{SalingerHuLiuetal.2018, author = {Salinger, Tim and Hu, Kai and Liu, Dan and Taleh, Scharoch and Herrmann, Sebastian and Oder, Daniel and Gensler, Daniel and M{\"u}ntze, Jonas and Ertl, Georg and Lorenz, Kristina and Frantz, Stefan and Weidemann, Frank and Nordbeck, Peter}, title = {Association between Comorbidities and Progression of Transvalvular Pressure Gradients in Patients with Moderate and Severe Aortic Valve Stenosis}, series = {Cardiology Research and Practice}, journal = {Cardiology Research and Practice}, doi = {10.1155/2018/3713897}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227291}, pages = {3713897, 1-7}, year = {2018}, abstract = {Background. Fast progression of the transaortic mean gradient (P-mean) is relevant for clinical decision making of valve replacement in patients with moderate and severe aortic stenosis (AS) patients. However, there is currently little knowledge regarding the determinants affecting progression of transvalvular gradient in AS patients. Methods. This monocentric retrospective study included consecutive patients presenting with at least two transthoracic echocardiography examinations covering a time interval of one year or more between April 2006 and February 2016 and diagnosed as moderate or severe aortic stenosis at the final echocardiographic examination. Laboratory parameters, medication, and prevalence of eight known cardiac comorbidities and risk factors (hypertension, diabetes, coronary heart disease, peripheral artery occlusive disease, cerebrovascular disease, renal dysfunction, body mass index >= 30 Kg/m(2), and history of smoking) were analyzed. Patients were divided into slow (P-mean < 5 mmHg/year) or fast (P-mean >= 5 mmHg/year) progression groups. Results. A total of 402 patients (mean age 78 +/- 9.4 years, 58\% males) were included in the study. Mean follow-up duration was 3.4 +/- 1.9 years. The average number of cardiac comorbidities and risk factors was 3.1 +/- 1.6. Average number of cardiac comorbidities and risk factors was higher in patients in slow progression group than in fast progression group (3.3 +/- 1.5 vs 2.9 +/- 1.7; P = 0.036). Patients in slow progression group had more often coronary heart disease (49.2\% vs 33.6\%; P = 0.003) compared to patients in fast progression group. LDL-cholesterol values were lower in the slow progression group (100 +/- 32.6 mg/dl vs 110.8 +/- 36.6 mg/dl; P = 0.005). Conclusion. These findings suggest that disease progression of aortic valve stenosis is faster in patients with fewer cardiac comorbidities and risk factors, especially if they do not have coronary heart disease. Further prospective studies are warranted to investigate the outcome of patients with slow versus fast progression of transvalvular gradient with regards to comorbidities and risk factors.}, 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{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} } @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{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} } @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} } @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} } @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} } @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} }