Universität Stuttgart
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Item Open Access Biophysical investigations of the in vitro effects of shock waves and ultrasound(1993) Brümmer, Franz; Suhr, Dierk; Irmer, Ulrich; Bachleitner, Christoph; Hülser, Dieter F.To investigate the interactions of ultrasonic waves with biological tissues, we developed and standardized several in vitro models. Using these systems - artificial stones, human erythrocytes, L1210 mouse leukemia cells, multicellular spheroids, cavitation assay - we are able to elucidate the mechanisms of interaction as well as the cause of clinically observed side effects.Item Open Access Construction of a super-competent Bacillus subtilis 168 using the PmtlA-comKS inducible cassette(2015) Rahmer, Regine; Morabbi Heravi, Kambiz; Altenbuchner, JosefCompetence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible PmtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of PmtlA-comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly.Item Open Access Die Spaltung von Arylether-Bindungen durch initiale Dioxygenierung: Grundlage des bakteriellen Dioxinabbaus(1991) Engesser, Karl-Heinrich; Strubel, Volker; Kirchner, S.; Schestag, S.; Schulte, P.; Knackmuss, Hans-JoachimBei der Untersuchung des bakteriellen Abbaus von Arylether-Modellsubstraten wie 2-Alkoxybenzoat, Carboxybiphenylether und Dibenzofuran wurde ein grundlegender Mechanismus für die Spaltung von Aryletherbindungen aufgedeckt. Demnach bewirken Dioxygenase-Enzyme unter Einführung zweier Hydroxylgruppen die Überführung von Ether- in Hemiacetalbindungen. Diese instabilen Hemiacetale reagieren unter Rearomatisierung zu aliphatischen Alkoholen und/oder Phenolverbindungen ab. Enzyme dieses Typs sind auch in der Lage, Dioxine zu spalten.Item Open Access Ein maschinenbezogenes Qualitätssicherungssystem für die intensitätsmodulierte Strahlentherapie : Entwicklung, Einführung, Untersuchung und Vergleich mit einem patientenplanbezogenen Qualitätssicherungssystem(2013) Hummel, Daniel; Gromoll, Christian (PD Dr.-Ing. habil.)Die dosimetrische Verifikation jedes Patienten-Bestrahlungsplans ist aktuell das Standardverfahren zur Qualitätssicherung der IMRT. Aufgrund der Eigenschaften und Beschränkungen der dabei eingesetzten Strahlungsdetektoren, Verifikationsmethoden und Vergleichsverfahren ist jedoch nur eine begrenzte Dosisgenauigkeit und Ortsauflösung erreichbar. Die dafür festgelegten Toleranzen können häufig nicht an allen Messpunkten eingehalten werden. Zudem ist es bei steigender Anzahl an mit IMRT behandelten Patienten nicht mehr möglich, jeden einzelnen Plan messtechnisch zu verifizieren. In einem maschinenbezogenen Qualitätssicherungssystem kann auf die Messung jedes einzelnen Patientenplans verzichtet werden, wenn alle IMRT-relevanten Kennmerkmale separat konstanzgeprüft werden. Ein solches QS-System wurde für eine Klinik entwickelt, eingeführt und getestet. Mittels einer täglich durchgeführten Testsequenz und wenigen zusätzlichen Messungen können alle wichtigen Kennmerkmale in kleinen Prüfintervallen geprüft und engere Toleranzen eingehalten werden. Damit sind eine höhere Dosisgenauigkeit, Patientensicherheit und Behandlungsqualität erreichbar. Für große Patientenzahlen ist ein maschinenbezogenes QS-System zudem effizienter als ein patientenplanbezogenes.Item Open Access Patterns of autologous and nonautologous interactions between core nuclear egress complex (NEC) proteins of α-, β- and γ-herpesviruses(2020) Häge, Sigrun; Sonntag, Eric; Borst, Eva Maria; Tannig, Pierre; Seyler, Lisa; Bäuerle, Tobias; Bailer, Susanne M.; Lee, Chung-Pei; Müller, Regina; Wangen, Christina; Milbradt, Jens; Marschall, ManfredNuclear egress is a regulated process shared by α-, β- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein-Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.Item Open Access The use of the tip potential of glass microelectrodes in the determination of low cell membrane potentials(1973) Hülser, Dieter F.; Webb, Dennis J.The tip potential of Ling-Gerard glass microelectrodes changes upon insertion into cells and thus impedes the determination of the actual membrane potential. The lower the membrane potential of a cell, the larger will be the error due to this tip potential. However, as is demonstrated, a relationship exists between the tip potential of the electrode and the measured potential difference, which allows the determination of the membrane potential of a particular cell type by linear regression. This method showed that resting lymphocytes had no membrane potential, whereas for the slime mould Dictyostelitium discoideum a membrane potential of about -9 mV could be calculated.Item Open Access Modeling of biocatalytic reactions: a workflow for model calibration, selection, and validation using Bayesian statistics(2019) Eisenkolb, Ina; Jensch, Antje; Eisenkolb, Kerstin; Kramer, Andrei; Buchholz, Patrick C. F.; Pleiss, Jürgen; Spiess, Antje; Radde, NicoleWe present a workflow for kinetic modeling of biocatalytic reactions which combines methods from Bayesian learning and uncertainty quantification for model calibration, model selection, evaluation, and model reduction in a consistent statistical frame-work. Our workflow is particularly tailored to sparse data settings in which a considerable variability of the parameters remains after the models have been adapted to available data, a ubiquitous problem in many real-world applications. Our workflow is exemplified on an enzyme-catalyzed two-substrate reaction mechanism describing the symmetric carboligation of 3,5-dimethoxy-benzaldehyde to (R)-3,3',5,5'-tetramethoxybenzoin catalyzed by benzaldehyde lyase from Pseudomonas fluorescens. Results indicate a substrate-dependent inactivation of enzyme, which is in accordance with other recent studies.Item Open Access A universal polyphosphate kinase : PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate(2020) Hildenbrand, Jennie C.; Teleki, Attila; Jendrossek, DieterPolyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP.Item Open Access Development of artificial single and double reading domains to analyze chromatin modification patterns(2018) Mauser, Rebekka; Jeltsch, Albert (Prof. Dr.)The unstructured N-terminal tails of histone proteins carry many different post-translational modifications (PTMs), like methylation, acetylation or phosphorylation. These PTMs can alter the chromatin structure, influence the interaction of adjacent nucleosomes and serve as specific binding sites for histone interacting domains. Currently, the investigation of histone tail PTMs is mainly based on antibodies, however concerns about the specificity of these antibodies and reproducibility of data arouse. Therefore, it was one aim of this thesis to develop alternative approaches to histone tail PTM antibodies. Previous studies already showed that histone modification interacting domains (HiMIDs) can replace histone tail antibodies in a highly effective manner. As part of this work, the TAF3 PHD domain was established as new H3K4me3 specific HiMID. In peptide array binding and Far-western blot assays, the domain showed a specific interaction with H3K4me3 modifications. Also in ChIP like experiments (CIDOP: Chromatin Interacting Domain Precipitation) coupled to qPCR and next generation sequencing, the domain showed a similar performance as validated H3K4me3 antibodies. With the proposal of the histone code hypothesis the question was raised if combinations of histone modifications carry specific biological functions. However, so far, the experimental analysis of the co-occurrence of histone modification on the same nucleosome in a genome-wide manner is a challenging task. For this reason, the main aim of this work was to develop double reading domains in which two histone reading domains are fused together with a flexible linker to achieve simultaneously readout of dual histone tail modifications in a single CIDOP experiment. To validate the concept, the Dnmt3a PWWP domain and the MPP8 Chromo domain were fused together and their specific recognitions of H3K36me2/3 and H3K9me3 histone tail modifications were analyzed. Biochemical investigations like peptide arrays, Far-western blot and western blot experiments showed that both domains specifically interact with their targets and preferentially interact with double modified chromatin. Additionally, the preferred interaction with double modified chromatin could be further verified with binding pocket mutants and methyl-lysine analogues. The newly generated double domain was used in chromatin precipitation experiments to identify genome regions where both modifications are present. The genome-wide distribution of the H3K36me2/3-H3K9me3 showed that this combination of histone marks represents a novel bivalent chromatin state, which is associated with weakly transcribed genes and is enriched for binding sites of ZNF274 and SetDB1. Also in this work, mixed peptide arrays were introduced as new screening method for the efficient analysis of double reading domains. The naturally occurring double reading domain of the BPTF protein was used to demonstrate the capability of this new screening tool. BPTF contains a PHD domain, which binds to H3K4me3 and a Bromo domain, which interacts with acetyl groups of the H4 tail. Synergistic binding to both peptides was shown using the newly developed mixed peptide arrays. Additionally, in the course of this work mixed peptide arrays were used to optimize several of the designed double reading domains. Furthermore, some other double reading domains were generated in this work, like PWWP-ATRX, MPP8 Chromo domain-L-double Tudor and CBX7 Chromo domain-L-MPP8 Chromo domain and analyzed for specific dual readout. Also double reading domains with dual specificity for DNA methylation and histone marks were generated. The firstly used methyl-DNA binding domain of the MBD2 protein showed a strong binding, dominating the effect of the HiMIDs. Therefore, the weaker but still specific methyl-DNA binding domain of the MBD1 protein was used. First experiments with this new fusion constructs showed a simultaneously interaction with chromatin which is associated with DNA methylation and histone PTMs. In summary, the studies with double reading domains showed that with this novel method precipitation of double modified chromatin is possible and that the genome-wide investigation of newly studied bivalent chromatin states is feasible. Therefore, this novel approach makes it possible to analyze many different combinations of histone modifications, investigate their influence on chromatin and gain a deeper understanding of the biological role behind histone tail modification patterns.Item Open Access Molekulare Mechanismen der pro-apoptotischen Kooperation zwischen TNF-R1 und Nicht-Todesrezeptoren der TNF-Rezeptorfamilie(2002) Fotin-Mleczek, Mariola; Scheurich, Peter (Prof. Dr.)Ziel der vorliegenden Arbeit war es, die molekularen Mechanismen der apoptotischen Kooperation zwischen dem Todesrezeptor TNF-R1 und den Nicht-Todesrezeptoren der TNF-Rezeptorfamilie, insbesondere dem TNF-R2, aufzuklären. Es wurde hier gezeigt, dass die Art der zellulären Antwort, die vom TNF-R1 ausgeht, stark von der Dauer der TNF-Stimulation abhängen kann. So reicht ein TNF-Puls aus um den Transkriptionsfaktor NF-kB zu aktivieren, ist aber nicht hinreichend, um die apoptotische Zellmaschinerie in Gang zu setzen. Die Ko- und Vorstimulation des TNF-R2 konnten die TNF-vermittelte Apoptose-Induktion erhöhen und beschleunigen. Die Verstärkung der Apoptose korrelierte dabei mit der Zeitkinetik der TNF-R2-induzierten TRAF2-Depletion. Eine TNF-R2-Stimulation von 6 Stunden führte zu einer fast vollständigen Depletion von TRAF2 aus dem Zytoplasma, verstärkte den TNF-R1-vermittelten Zelltod dramatisch und inhibierte gleichzeitig die TNF-R1-induzierte NF-kB-Aktivierung zu 90%. Wurden die beiden TNF-Rezeptoren gleichzeitig aktiviert, so konnte noch immer eine beträchtliche Verstärkung der TNF-R1-induzierter Apoptose beobachtet werden, während die TNF-R1-vermittelte NF-kB-Aktivierung unbeeinflusst blieb. Mit Hilfe von der konfokalen Laserscanning-Mikroskopie konnte nachgewiesen werden, dass die Stimulation der Nicht-Todesrezeptoren CD40 und TNF-R2 zur TRAF2-abhängigen Rekrutierung der anti-apoptotischen Moleküle cIAP1 und cIAP2 an die Rezeptoren führte. Dies zeigt, dass der TNF-R1 und die Nicht-Todesrezeptoren der TNF-Rezeptorfamilie nicht nur um TRAF2 sondern auch um die mit TRAF2 assoziierten Schutzproteine kompetieren können. Diese Kompetition bildet den Kern des Modells zur apoptotischen Kooperation der beiden TNF-Rezeptoren. Demnach kann die Stimulation des TNF-R2 die TNF-R1-induzierte Apoptose auf zwei Ebenen verstärken: über die Inhibierung der NF-kB-abhängigen Induktion von Schutzproteinen und durch die Inhibition ihrer Schutzwirkung auf der post-transkriptionellen Ebene durch Kompetition. In beiden Fällen wird die Ausbildung eines Caspase-8-aktivierenden TNF-R1-Signalkomplexes ermöglicht. In vivo werden die Rezeptoren der TNF-Rezeptorfamilie auch durch membranständige Liganden aktiviert. Daher wurden im zweiten Abschnitt dieser Arbeit die Interaktionen zwischen TNF-R2, CD40 und ihren membranständigen Liganden analysiert. Unter Anwendung der Laser-Scanning-Mikroskopie und von Kokulturen konnte demonstriert werden, dass in Zell-Zell-Kontakten die Rezeptoren der TNF-Rezeptorfamilie mit ihren membranständigen Liganden Superaggregate bilden. Diese Aggregate entstehen innerhalb von 30 Minuten nach dem Zusammentreffen zweier Zellen, von denen eine den Rezeptor und die andere den membranständigen Ligand exprimiert. Die Rezeptor-Ligand-Superaggregate bleiben über mehrere Stunden stabil, wobei sich ihre Form und Lage stets verändern. Im Weiterem wurde beobachtet, dass die Rezeptor-Ligand-Superaggregate signalfähige Rezeptor-Komplexe enthalten. So wurde z.B. TRAF2 in die TNF-R2/memTNF-Aggregate rekrutiert. Wurde der CD40 mit einem löslichen CD40-Ligand stimuliert, so wurde die Internalisierung und möglicherweise der Abbau des Rezeptors beobachtet, nicht jedoch nach der Stimulation mit dem membranständigen Ligand. Dies deutet darauf hin, dass sich die Qualität der Bindung zwischen dem Rezeptor und seinem membranständigen Liganden wesentlich von der Interaktion des Rezeptors mit den löslichen Reagenzien unterscheidet.