TY - JOUR A1 - Kremer, Mark A1 - Biesenthal, Tobias A1 - Maczewsky, Lukas J. A1 - Heinrich, Matthias A1 - Thomale, Ronny A1 - Szameit, Alexander T1 - Demonstration of a two-dimensional PT-symmetric crystal JF - Nature Communications N2 - With the discovery of PT-symmetric quantum mechanics, it was shown that even non-Hermitian systems may exhibit entirely real eigenvalue spectra. This finding did not only change the perception of quantum mechanics itself, it also significantly influenced the field of photonics. By appropriately designing one-dimensional distributions of gain and loss, it was possible to experimentally verify some of the hallmark features of PT-symmetry using electromagnetic waves. Nevertheless, an experimental platform to study the impact of PT-symmetry in two spatial dimensions has so far remained elusive. We break new grounds by devising a two-dimensional PT-symmetric system based on photonic waveguide lattices with judiciously designed refractive index landscape and alternating loss. With this system at hand, we demonstrate a non-Hermitian two-dimensional topological phase transition that is closely linked to the emergence of topological mid-gap edge states. KW - micro-optics KW - optical materials and structures KW - topological matter Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230132 VL - 10 ER - TY - JOUR A1 - Kraus, Michael A1 - Grimm, Clemens A1 - Seibel, Jürgen T1 - Reversibility of a Point Mutation Induced Domain Shift: Expanding the Conformational Space of a Sucrose Phosphorylase JF - Scientific Reports N2 - Despite their popularity as enzyme engineering targets structural information about Sucrose Phosphorylases remains scarce. We recently clarified that the Q345F variant of Bifidobacterium adolescentis Sucrose Phosphorylase is able to accept large polyphenolic substrates like resveratrol via a domain shift. Here we present a crystal structure of this variant in a conformation suitable for the accommodation of the donor substrate sucrose in excellent agreement with the wild type structure. Remarkably, this conformation does not feature the previously observed domain shift which is therefore reversible and part of a dynamic process rather than a static phenomenon. This crystallographic snapshot completes our understanding of the catalytic cycle of this useful variant and will allow for a more rational design of further generations of Sucrose Phosphorylase variants. KW - biocatalysis KW - X-ray crystallography Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224845 VL - 8 ER - TY - JOUR A1 - Müller, Laura S. M. A1 - Cosentino, Raúl O. A1 - Förstner, Konrad U. A1 - Guizetti, Julien A1 - Wedel, Carolin A1 - Kaplan, Noam A1 - Janzen, Christian J. A1 - Arampatzi, Panagiota A1 - Vogel, Jörg A1 - Steinbiss, Sascha A1 - Otto, Thomas D. A1 - Saliba, Antoine-Emmanuel A1 - Sebra, Robert P. A1 - Siegel, T. Nicolai T1 - Genome organization and DNA accessibility control antigenic variation in trypanosomes JF - Nature N2 - Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation. KW - histone variants KW - genome architecture KW - single molecule real time (SMRT) KW - brucei genome KW - distance-dependent decay Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224265 VL - 563 ER - TY - JOUR A1 - Munz, Matthias A1 - Richter, Gesa M. A1 - Loos, Bruno G. A1 - Jepsen, Søren A1 - Divaris, Kimon A1 - Offenbacher, Steven A1 - Teumer, Alexander A1 - Holtfreter, Birte A1 - Kocher, Thomas A1 - Bruckmann, Corinna A1 - Jockel-Schneider, Yvonne A1 - Graetz, Christian A1 - Munoz, Loreto A1 - Bhandari, Anita A1 - Tennstedt, Stephanie A1 - Staufenbiel, Ingmar A1 - van der Velde, Nathalie A1 - Uitterlinden, André G. A1 - de Groot, Lisette C. P. G. M. A1 - Wellmann, Jürgen A1 - Berger, Klaus A1 - Krone, Bastian A1 - Hoffmann, Per A1 - Laudes, Matthias A1 - Lieb, Wolfgang A1 - Andre, Franke A1 - Dommisch, Henrik A1 - Erdmann, Jeanette A1 - Schaefer, Arne S. T1 - Genome-wide association meta-analysis of coronary artery disease and periodontitis reveals a novel shared risk locus JF - Scientific Reports N2 - Evidence for a shared genetic basis of association between coronary artery disease (CAD) and periodontitis (PD) exists. To explore the joint genetic basis, we performed a GWAS meta-analysis. In the discovery stage, we used a German aggressive periodontitis sample (AgP-Ger; 680 cases vs 3,973 controls) and the CARDIoGRAMplusC4D CAD meta-analysis dataset (60,801 cases vs 123,504 controls). Two SNPs at the known CAD risk loci ADAMTS7 (rs11634042) and VAMP8 (rs1561198) passed the pre-assigned selection criteria (PAgP-Ger < 0.05; PCAD < 5 × 10−8; concordant effect direction) and were replicated in an independent GWAS meta-analysis dataset of PD (4,415 cases vs 5,935 controls). SNP rs1561198 showed significant association (PD[Replication]: P = 0.008 OR = 1.09, 95% CI = [1.02–1.16]; PD [Discovery + Replication]: P = 0.0002, OR = 1.11, 95% CI = [1.05–1.17]). For the associated haplotype block, allele specific cis-effects on VAMP8 expression were reported. Our data adds to the shared genetic basis of CAD and PD and indicate that the observed association of the two disease conditions cannot be solely explained by shared environmental risk factors. We conclude that the molecular pathway shared by CAD and PD involves VAMP8 function, which has a role in membrane vesicular trafficking, and is manipulated by pathogens to corrupt host immune defense. KW - vesicle-associated membrane protein 8 (VAMP8) KW - ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS7) KW - shared genetic basis KW - genome-wide association studies (GWAS) KW - GWAS meta-analysis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231647 VL - 8 ER - TY - THES A1 - Karwen, Till T1 - Platelets promote insulin secretion of pancreatic β-cells T1 - Thrombozyten fördern die Insulinsekretion von pankreatischen β-Zellen N2 - The pancreas is the key organ for the maintenance of euglycemia. This is regulated in particular by α-cell-derived glucagon and β-cell-derived insulin, which are released in response to nutrient deficiency and elevated glucose levels, respectively. Although glucose is the main regulator of insulin secretion, it is significantly enhanced by various potentiators. Platelets are anucleate cell fragments in the bloodstream that are essential for hemostasis to prevent and stop bleeding events. Besides their classical role, platelets were implemented to be crucial for other physiological and pathophysiological processes, such as cancer progression, immune defense, and angiogenesis. Platelets from diabetic patients often present increased reactivity and basal activation. Interestingly, platelets store and release several substances that have been reported to potentiate insulin secretion by β-cells. For these reasons, the impact of platelets on β-cell functioning was investigated in this thesis. Here it was shown that both glucose and a β-cell-derived substance/s promote platelet activation and binding to collagen. Additionally, platelet adhesion specifically to the microvasculature of pancreatic islets was revealed, supporting the hypothesis of their influence on glucose homeostasis. Genetic or pharmacological ablation of platelet functioning and platelet depletion consistently resulted in reduced insulin secretion and associated glucose intolerance. Further, the platelet-derived lipid fraction was found to enhance glucose-stimulated insulin secretion, with 20-hydroxyeicosatetraenoic acid (20-HETE) and possibly also lyso-precursor of platelet-activating factor (lysoPAF) being identified as crucial factors. However, the acute platelet-stimulated insulin secretion was found to decline with age, as did the levels of platelet-derived 20-HETE. In addition to their direct stimulatory effect on insulin secretion, specific defects in platelet activation have also been shown to affect glucose homeostasis by potentially influencing islet vascular development. Taking together, the results of this thesis suggest a direct and indirect mechanism of platelets in the regulation of insulin secretion that ensures glucose homeostasis, especially in young individuals. N2 - Der Pankreas ist das Schlüsselorgan für die Aufrechterhaltung der Glukosehomöostase. Diese wird insbesondere durch das von α-Zellen stammende Glukagon und von β-Zellen stammende Insulin reguliert, die als Reaktion auf Nährstoffmangel beziehungsweise erhöhte Glukosespiegel freigesetzt werden. Obwohl Glukose der Hauptregulator der Insulinsekretion ist, wird sie durch verschiedene Potentiatoren erheblich gesteigert. Thrombozyten sind kernlose Zellfragmente im Blutkreislauf, die für die Hämostase unerlässlich sind. Neben ihrer klassischen Funktion sind sie auch an anderen physiologischen und pathophysiologischen Prozessen beteiligt, etwa an der Tumorentwicklung, der Immunabwehr und der Angiogenese. Thrombozyten von Diabetikern weisen häufig eine erhöhte Reaktivität und basale Aktivierung auf. Außerdem speichern und sekretieren sie Substanzen, von denen bekannt ist, dass sie die Insulinsekretion durch β-Zellen verstärken. Aus diesen Gründen wurde in dieser Arbeit der Einfluss von Thrombozyten auf die Funktion von β-Zellen untersucht. Es konnte gezeigt werden, dass sowohl Glukose als auch eine aus β-Zellen stammende Substanz/en die Thrombozytenaktivierung und die Bindung an Kollagen fördern. Darüber hinaus wurde eine spezifische Thrombozytenadhäsion an der Mikrovaskulatur der pankreatischen Inseln festgestellt, was die Hypothese ihres Einflusses auf die Glukosehomöostase unterstützt. Eine genetische oder pharmakologische Ablation der Thrombozytenfunktion sowie eine Depletion von Thrombozyten führten zu einer verminderten Insulinsekretion und einer damit verbundenen Glukoseintoleranz. Hierbei erwies sich die Lipidfraktion von Thrombozyten als essentieller Potentiator für die glukosestimulierte Insulinsekretion, wobei 20-Hydroxyeicosatetraensäure (20-HETE) und die Lyso-Vorstufe des Plättchen-Aktivierenden Faktors (LysoPAF) als entscheidende Faktoren identifiziert werden konnten. Weiterhin wurde festgestellt, dass sowohl der direkte stimulierende Effekt von Thrombozyten auf die Insulinsekretion, als auch deren 20-HETE Sekretion mit zunehmendem Alter abnimmt. Thrombozyten beeinflussten außerdem die Inselvaskularisierung, welche mutmaßlich zusätzlich zu Glukoseintoleranz führt. Insgesamt deuten die Ergebnisse dieser Arbeit auf einen direkten und indirekten Mechanismus der Thrombozyten bei der Regulierung der Insulinsekretion hin, der die Glukosehomöostase insbesondere bei jungen Menschen gewährleistet. KW - platelet KW - β cell KW - insulin KW - pancreas KW - diabetes KW - Thrombozyt KW - Insulinsekretion Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313933 ER - TY - THES A1 - Pätzel [geb. Ditter], Katharina Sabine T1 - Molekulare Charakterisierung eines Mitgliedes der TNF-Rezeptor-Superfamilie des Fuchsbandwurmes \(Echinococcus\) \(multilocularis\) T1 - Molecular characterization of a TNF-receptor-superfamily member of \(Echinococcus\) \(multilocularis\) N2 - Die alveoläre Echinokokkose (AE), die durch den Fuchsbandwurm Echinococcus multilocularis verursacht wird, ist eine seltene jedoch schwere und oft tödlich verlaufende Erkrankung. Aufgrund der späten Diagnosestellung sind kurative Behandlungsmethoden häufig nicht durchführbar und als einzige Behandlungsmöglichkeit bleibt eine lebenslange und nebenwirkungsreiche Therapie mit Benzimidazolen. Verbesserte Therapieoptionen durch die Entwicklung neuer Medikamente sind dringend notwendig. Hierfür kann es hilfreich sein die Biologie des Fuchsbandwurmes und die Kommunikationswege zwischen Parasit und Wirt zu verstehen. Bereits in vorherigen Arbeiten als auch in dieser Arbeit erwiesen sich evolutionsgeschichtlich konservierte Signalwege als Kommunikationsweg zwischen dem Fuchsbandwurm und seinem Wirt von zentraler Rolle. Die Entschlüsselung des Echinococcus-Genoms gab Hinweise darauf, dass ein Mitglied der Tumornekrosefaktor-Rezeptor-Superfamilie, jedoch kein endogener TNF α ähnlicher Ligand im Genom kodiert wird. Ein Mitglied der TNFR-Superfamilie des Fuchsbandwurmes (EmTNFR) wurde in dieser Arbeit als membranständiger Rezeptor mit einer intrazellulären Todesdomäne (DD) und hoher Ähnlichkeit zum humanen Typ 16 der TNF-Rezeptor-Superfamilie, auch 〖p75〗^NTR genannt, charakterisiert. Sowohl in bioinformatischen als auch in Sequenzanalysen wurden drei alternative Splicing-Formen von emtnfr (emtnfr, emtnfr-v2 und emtnfr-v3) nachgewiesen. emtnfr-v2 entsteht durch Alternatives Splicing und kodiert ein Protein, das keine intrazelluläre Todesdomäne besitzt. emtnfr-v3 verwendet einen alternativen Transkriptionstart und wird von den letzten 3 Exons von emtnfr kodiert. emtnfr-v3, kodiert ein Protein ohne extrazelluläre Region, aber mit intrazellulärer Todesdomäne. Ein löslicher TNF-Rezeptor konnte auf Proteinebene nicht nachgewiesen werden. Aufgrund von phylogenetischen Analysen und der Rezeptor-Struktur ist zu vermuten, dass EmTNFR ein p75NTR Homolog ist und damit der ursprünglichen Form der TNF-Rezeptoren entspricht. Mitglieder eines intrazellulären TNF-Signalweges wurden in bioinformatischen Analysen beim Fuchsbandwurm E. multilocularis identifiziert. Expressionsuntersuchungen zeigten sowohl in Trankriptomdaten als auch auf Proteinebene eine starke Expression von EmTNFR in Primärzellen und im Metazestoden (MZ), dem pathogenen Stadium für den Zwischenwirt. Echinococcus-Stammzellkulturen zeigten nach RNA-Interferenz-basiertem Knockdown des EmTNFR-kodierenden Gens deutliche Entwicklungsdefekte. Des Weiteren zeigten Echinococcus-Stammzellkulturen nach einer Behandlung mit TNF-α, einem potentiellen Liganden des TNF-Rezeptors und einem zentralen Zytokin in der Immunabwehr des Zwischenwirtes, Entwicklungsfortschritte, wie eine verbesserte Bildung von MZ aus Stammzellen. Zusätzlich wurde in whole-mount in situ Hybridisierungs-Versuchen eine ubiquitäre Expression von emtnfr in der Germinalschicht des MZ sowie eine Spezifität von emtnfr für den MZ, welcher ursächlich für die AE ist, nachgewiesen. Somit scheinen sowohl EmTNFR als auch TNF-α eine wichtige Funktion bei der Entwicklung und Etablierung des Fuchsbandwurmes während der frühen Phase der Infektion des Zwischenwirtes zu haben. TNF-α könnte ein weiterer Faktor für den ausgeprägten Organtropismus des Parasiten zur Leber sein, denn dort bestehen durch Kupfferzellen produzierte hohe lokale Konzentration von TNF-α. Zusammenfassend deuten die hier erarbeiteten Daten darauf hin, dass EmTNFR über die Bindung von Wirts-TNF-α bei der frühen Entwicklung des Echincoccus-Metazestoden eine Rolle spielt. N2 - Alveolar echinococcosis (AE), which is caused by the metacestode larval stage of the fox tapeworm Echinococcus multilocularis, is a rare but severe, often fatal disease. Due to late diagnosis and advanced spread of the infection curative therapy is often not possible and the only treatment option is benzimidazole chemotherapy, which often must be taken lifelong and has adverse side effects. Improvement of therapeutic options is thus urgently needed. To this end, a closer understanding of parasite biology and communication mechanisms between parasite and host are helpful. In this work, focus was laid on the possibility of host-parasite cross-communication involving an evolutionarily conserved signalling pathway. By mining the Echinococcus genome sequence, a gene encoding a member of the tumor necrosis-factor-receptor family (TNF-R), was identified. In this work, EmTNFR, a member of the TNF-R superfamily, of the fox tapeworm was identified as a membrane bound receptor with intracellular death domain and highest similarity to human TNFRSF 16, also called p75NTR. In in silico analysis and cDNA sequencing, 3 alternative splice forms of emtnfr (emtnfr-v1, -v2 and -v3) were found. emtnfr-v2 is the result of alternative splicing and encodes a protein lacking the intracellular death domain. emtnfr-v3 employs an alternative transcription start and is encoded by the last 3 exons of emtnfr. emtnfr-v3 encodes a protein without extracellular domain, but containing an intracellular death domain. A soluble TNF-receptor could not be found in proteomic analysis. Based on phylogenetic analysis and receptor structure, EmTNFR is thought to be a homolog of p75NTR, corresponding to the ancient form of TNF receptors. Members of an intracellular TNF signaling pathway were identified in bioinformatic analyses in the fox tapeworm E. multilocularis, indicating the presence of a full TNFR signalling pathway. Expression studies showed in transcriptome data and at protein level a strong expression of EmTNFR in primary cells and in the metacestode (MZ), the pathogenic stage for the intermediate host. Echinococcus stem cell cultures showed marked developmental defects after RNAi based knockdown of the EmTNFR-encoding gene. Furthermore, Echinococcus stem cell culture displayed accelerated developmental progress such as enhanced formation of MZ from stem cells after treatment with TNF-α, a potential ligand of the TNF receptor, and a central cytokine in the immune defense of the intermediate host. In addition, whole-mount in situ hybridization experiments demonstrated ubiquitous expression of emtnfr in the germinal layer of MZ and specificity of emtnfr for MZ, the causative agent of AE. Thus, both EmTNFR and TNF-α appear to have an important function in development and establishment of the fox tapeworm during the early phase of infection of the intermediate host. TNF-α could be an additional factor for the pronounced organ tropism of the parasite to the liver, caused by a high local concentration of TNF-α produced by Kupffer cells. In summary, the data generated in this work suggest that EmTNFR plays a role in the early development of Echinococcus metacestode via binding of host TNF-α. KW - Fuchsbandwurm KW - Wirt-Parasit-Beziehung KW - Parasit KW - Tumor-Nekrose-Faktor KW - Echinococcus multilocularis KW - TNF-Rezeptor KW - Wirt-Parasiten-Interaktion KW - Molekulare Charakterisierung Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369397 ER - TY - THES A1 - Zimmermann, Sebastian Andres T1 - Drug Monitoring of Kinase Inhibitors in the Context of Precision Medicine – Focus on Minimally Invasive Microsampling T1 - Arzneistoffspiegelmessung von Kinaseinhibitoren im Kontext der Präzisionsmedizin – Fokus auf minimalinvasives Mikrosampling N2 - The aim of the present work was to improve drug monitoring in patients with various diseases in the context of precision medicine. This was pursued through the development and validation of mass spectrometric methods for determining the drug concentrations of kinase inhibitors and their clinical application. Besides conventional approaches to determine plasma level concentrations, the focus was also on alternative sampling techniques using volumetric absorptive microsampling (VAMS). A conventional LC-MS/MS method was developed for the determination of cabozantinib in human EDTA plasma and validated according to the guidelines of the European and United States drug authorities (EMA, FDA). The method met the required criteria for linearity, accuracy and precision, selectivity, sensitivity, and stability of the analyte. Validation was also performed for dilution integrity, matrix effect, recovery, and carry-over, with results also in accordance with the requirements. The importance of monitoring the exposure of cabozantinib was demonstrated by a clinical case report of a 34-year-old female patient with advanced adrenocortical carcinoma who also required hemodialysis due to chronic kidney failure. Expected cabozantinib plasma concentrations were simulated for this off-label use based on a population pharmacokinetic model. It was shown that the steady state trough levels were much lower than expected but could not be explained by hemodialysis. Considering the critical condition and potential drug-drug interaction with metyrapone, a substance the patient had taken among several others during the observation period, individual pharmacokinetics could consequently not be estimated without drug monitoring. In addition, a VAMS method for simultaneous determination of ten kinase inhibitors from capillary blood was developed. This microsampling technique was mainly characterized by the collection of a defined volume of blood, which could be dried and subsequently analyzed. The guidelines for bioanalytical method validation of the EMA and FDA were also used for this evaluation. As the nature of dried blood samples differs from liquid matrices, further parameters were investigated. These include the investigation of the hematocrit effect, process efficiency, and various stability conditions, for example at increased storage temperatures. The validation showed that the developed method is suitable to analyze dried matrix samples accurate, precise, and selective for all analytes. Apart from the stability tests, all acceptance criteria were met. The decreased stability of two analytes was probably due to the reproducible but reduced recovery. In vitro studies provided results on the VAMS-to-plasma correlation to predict the analyte distribution between both matrices, at least in an exploratory manner. It revealed a heterogeneous picture of analytes with different VAMS-to-plasma distributions. Furthermore, the analysis of 24 patient samples indicated the applicability of at-home VAMS. Both should be confirmed later as part of the clinical validation. The clinical investigation of the VAMS method pursued two objectives. On the one hand, the simultaneous collection of VAMS and serum samples should enable a conversion of the determined concentrations and, on the other hand, the feasibility of autonomous microsampling at home should be examined more closely. For the former, it could be shown that different conversion methods are suitable for converting VAMS concentrations into serum levels. The type of conversion was secondary for the prediction. However, the previously defined criteria could not be fulfilled for all five kinase inhibitors investigated. The framework conditions of the study led to increased variability, especially for analytes with short half-life. A low and varying hematocrit, caused by the underlying disease, also made prediction difficult for a specific patient collective. For the second objective, investigating the feasibility of VAMS, different aspects were considered. It could be shown that the majority of patients support home-based microsampling. The acceptance is likely to increase even further when microsampling is no longer part of a non-interventional study, but participation is accompanied by targeted monitoring and subsequent adjustment of the therapy. The fact that additional training increases understanding of the correct sampling procedure is also a source of confidence. Demonstrated stability during storage under real-life conditions underlines the practicality of this sampling technique. Taken together, mass spectrometric methods for both plasma and VAMS could be developed and validated, and their clinical application could be successfully demonstrated. The availability of simple bioanalytical methods to determine kinase inhibitor exposure could improve access to prospective studies and thus facilitate the implementation of routine therapeutic drug monitoring. N2 - Ziel der vorliegenden Arbeit war es, den Einsatz des Drug Monitorings bei Patienten mit verschiedenen Erkrankungen im Rahmen der Präzisionsmedizin zu erleichtern. Dies wurde durch die Entwicklung und Validierung massenspektrometrischer Methoden zur Bestimmung der Wirkstoffkonzentrationen von Kinaseinhibitoren und deren klinische Anwendung verfolgt. Dabei standen neben konventionellen Ansätzen zur Bestimmung der Plasmaspiegelkonzentration auch alternative Entnahmetechniken in Form von „Volumetric Absorptive Microsampling (VAMS)“ im Mittelpunkt. Eine solche konventionelle LC-MS/MS-Methode wurde zur Bestimmung von Cabozantinib in humanem EDTA-Plasma entwickelt und nach den Richtlinien der europäischen und US-amerikanischen Arzneimittelbehörden (EMA, FDA) validiert. Die Methode erfüllte die geforderten Kriterien bezüglich Linearität, Richtigkeit und Präzision, Selektivität, Sensitivität und Stabilität des Analyten. Die Validierung erfolgte auch hinsichtlich Verdünnungsintegrität, Matrixeffekt, Wiederfindung, und Analyt-Verschleppung, deren Ergebnisse ebenso in Übereinstimmung mit den Vorgaben standen. Die Bedeutung der Expositionsüberwachung von Cabozantinib wurde anhand eines klinischen Fallberichts einer 34-jährigen Patientin mit fortgeschrittenem Nebennierenrindenkarzinom, die aufgrund chronischen Nierenversagens zudem dialysepflichtig war, gezeigt. Die erwartbaren Cabozantinib-Plasmakonzentrationen wurden für diesen Off-Label-Use basierend auf einem populations-pharmakokinetischen Modell simuliert. Es zeigte sich, dass die Steady-State-Talspiegel wesentlich niedriger waren als angenommen, sich aber nicht durch die Hämodialyse erklären ließen. Unter Berücksichtigung des kritischen Gesundheitszustandes und möglicher Arzneimittelinteraktionen mit Metyrapon, einer Substanz, die die Patientin neben einigen anderen während des Beobachtungszeitraums eingenommen hatte, konnte die individuelle Pharmakokinetik folglich nicht ohne Arzneistoffspiegelmessung abgeschätzt werden. Darüber hinaus konnte eine VAMS-Methode zur simultanen Bestimmung von zehn Kinaseinhibitoren aus Kapillarblut entwickelt werden. Dieses Mikrosampling-Verfahren zeichnete sich vor allem durch die Entnahme eines definierten Blutvolumens aus, welches getrocknet und anschließend analysiert werden konnte. Auch hierbei wurden die Richtlinien für bioanalytische Methodenvalidierung der EMA und FDA zur Beurteilung herangezogen. Da sich die Beschaffenheit der Trockenblutproben von flüssigen Matrices unterscheidet, wurden weitere Einflussfaktoren untersucht. Dazu gehören die Untersuchung des Hämatokrit-Effekts, der Prozesseffizienz und verschiedener Stabilitätsbedingungen, zum Beispiel bei erhöhten Lagertemperaturen. Die Validierung zeigte, dass die entwickelte Methode in der Lage ist Trockenmatrixproben für alle Analyten richtig, präzise und selektiv zu messen. Von den Stabilitätsuntersuchungen abgesehen wurden alle Akzeptanzkriterien erfüllt. Die verminderte Stabilität von zwei Analyten ist vermutlich durch die zwar reproduzierbare, aber geringere Wiederfindungsrate zu begründen. In vitro Untersuchungen lieferten Ergebnisse über die VAMS-zu-Plasma-Korrelation, um die Analytverteilung zwischen beiden Matrizes zumindest exploratorisch vorherzusagen. Dabei zeigte sich ein heterogenes Bild von Analyten mit unterschiedlicher VAMS-zu-Plasma-Verteilung. Darüber hinaus zeigten die Messungen von 24 Patientenproben die Anwendbarkeit von VAMS im häuslichen Umfeld. Beides sollte später im Rahmen der klinischen Validierung bestätigt werden. Die klinische Untersuchung der VAMS-Methode verfolgte zweierlei Ziele. Zum einen sollte durch die zeitgleiche Entnahme von VAMS und Serumproben eine Umrechnung der ermittelten Konzentrationen ermöglicht und zum anderen die Durchführbarkeit der eigenständigen Mikroprobenentnahme zuhause genauer überprüft werden. Für Ersteres konnte gezeigt werden, dass verschiedene Umrechnungsmethoden geeignet sind, VAMS-Konzentrationen in Serumspiegel umzurechnen. Die Art der Umrechnung war für die Vorhersage zweitrangig. Allerdings konnten nicht für alle fünf untersuchten Kinaseinhibitoren die zuvor festgelegten Kriterien erfüllt werden. Die Rahmenbedingungen der Studie führten vor allem bei Analyten mit geringer Halbwertszeit zu einer erhöhten Variabilität. Ein geringer und schwankender Hämatokritwert, bedingt durch die zugrundeliegende Erkrankung, erschwerte zudem die Vorhersage bei einem bestimmten Patientenkollektiv. Für das zweite Ziel, die Durchführbarkeit von VAMS zu untersuchen, wurden verschiedene Aspekte betrachtet. Es konnte gezeigt werden, dass die Mehrheit der Patienten die häusliche Mikroprobenentnahme befürwortet. Die Akzeptanz dürfte sogar noch weiter steigen, wenn die Mikroprobenentnahme nicht mehr nur Teil einer nicht-interventionellen Studie ist, sondern die Teilnahme mit einer gezielten Überwachung und anschließenden Anpassung der Therapie einhergeht. Zuversichtlich stimmte auch die Tatsache, dass ein zusätzliches Training das Verständnis für die korrekte Durchführung erhöht. Dass sich die Stabilität auch bei der Lagerung unter Realbedingungen zeigen ließ, unterstreicht die Praxistauglichkeit dieser Sampling-Technik. Insgesamt konnten sowohl für Plasma als auch VAMS massenspektrometrische Methoden entwickelt, validiert und deren klinische Anwendung erfolgreich demonstriert werden. Die Verfügbarkeit von simplen bioanalytischen Methoden zur Bestimmung der Kinaseinhibitor-Exposition könnte den Zugang zu prospektiven Studien und damit die Einführung von routinemäßigem Therapeutischen Drug Monitoring erleichtern. KW - Arzneimittelüberwachung KW - Blutspiegel KW - Klinische Pharmazie KW - Kinaseinhibitor KW - Therapeutisches Drug Monitoring KW - TDM KW - Precision medicine KW - Volumetric absorptive microsampling KW - VAMS Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369550 ER - TY - THES A1 - de Sunda, Angela T1 - Effekte der Tiefenhirnstimulation bei Patienten mit idiopathischem Parkinson-Syndrom auf Symptome der Stimme und des Sprechens T1 - Effects of Deep Brain Stimulation of the subthalamic nucleus on symptoms of voice and speech in patients with Parkinson´s Disease N2 - Sprech- und Stimmstörungen sind häufige Symptome der Idiopathischen Parkinson Erkrankung (IPS), wobei bis zu 89% der Patienten im Verlauf der Krankheit unter einer Dysarthrie leiden. Die Tiefenhirnstimulation des Nucleus subthalamicus (STN-DBS) ist eine etablierte Behandlung für die motorischen Symptome des IPS (Allert et al., 2004). Während STN-DBS positive Effekte auf einige Teilfunktionsbereiche der Dysarthrie zu haben scheint, berichten die meisten Studien entweder über keine Verbesserung oder eine Verschlechterung der Sprech- und Stimmfunktionen nach Implantation der STN-DBS (Tsuboi et al., 2015; Wang et al., 2003; Wertheimer et al., 2014). Klinische Erfahrungswerte sowie Fallberichte und Studien lassen vermuten, dass diese sprachtherapeutisch relevanten Nebenwirkungen unabhängig von der therapeutischen Wirksamkeit der STN-DBS sind und daher als unerwünschte, aber nicht therapieimmanente Interferenzfaktoren anzusehen sind (Bouthour et al., 2018), die es genauer zu untersuchen gilt, da die Lebensqualität von IPS-Erkrankten als stark einschränkend wahrgenommen wird (Hariz et al., 2010). Eine aufwendige und methodisch fundierte Klassifizierung wurde von Tsuboi und Kollegen vorgenommen, die im Zusammenhang mit STN-DBS fünf Cluster von Sprech- und Stimmstörungen identifizierten (Tanaka et al., 2020; Tsuboi et al., 2015, 2017). Dazu zählten die Phänotypen „spastische Dysarthrie“, „Stottern“, „rigid-hypokinetischer Typ“, „behauchte Stimme“ und „gepresste Stimme“. Erste Hinweise lassen darauf schließen, dass die Nebenwirkungen von STN-DBS auf die Stimulation spezifischer Gehirnkreise zurückzuführen sein könnte (Fox et al., 2014). In dieser Arbeit wird eine retrospektive Studie mit STN-DBS stimulierten IPS Erkrankten vorgestellt, die sprachtherapeutisch relevante Sprech- und Stimmstörungen unter zwei Bedingungen bewertet (ein- und ausgeschaltete Stimulation) sowie eine prospektive Studie mit den beiden gleichen Bedingungen. Beide Studien haben das Ziel einer Replizierbarkeit der Ergebnisse von Tsuboi et al. (2015, 2017). Die zweite prospektive Studie bezieht außerdem konnektombasierte Daten ein. Die Ergebnisse beider Studien lassen quantitativ keine Signifikanzen hinsichtlich der o.g. dysarthrischen Phänotypen zu, quantitativ lassen sich jedoch deutliche Tendenzen ähnlich der Ausgangsstudie erkennen. Zudem wurden das Cluster „Stottern“ in der retrospektiven Studie als weiteres möglicherweise STN-DBS immantentes Cluster identifiziert. In der prospektiven Studie wurde ein Cluster hinzugefügt, da in den Beurteilungen zusätzlich die Symptomatik „hasty speech“ oder auch „hastiges Sprechen“ beobachtet wurde. N2 - Speech and voice disorders are common symptoms of Parkinson's disease (PD) with up to 89% of patients suffering from dysarthria during the course of the disease. Deep brain stimulation of the subthalamic nucleus (STN-DBS) is an established treatment for the motor symptoms of PD (Allert et al., 2004). While STN-DBS appears to have positive effects on some subfunctional areas of dysarthria, most studies report either no improvement or a deterioration in speech and voice functions after implantation of STN-DBS (Tsuboi et al., 2015; Wang et al., 2003; Wertheimer et al., 2014). Clinical experience as well as case reports and studies suggest that these side effects relevant to speech therapy are independent of the therapeutic effectiveness of STN-DBS and should therefore be regarded as undesirable but not therapy-immanent interference factors (Bouthour et al., 2018), which need to be investigated more closely. It is considered to be a serious problem, as the quality of life of PD sufferers is perceived to be severely limiting (Hariz et al., 2010). A complex and methodologically solid classification was carried out by Tsuboi and colleagues, who identified five clusters of speech and voice disorders in connection with STN-DBS (Tanaka et al., 2020; Tsuboi et al., 2015, 2017). These included the phenotypes "spastic dysarthria", "stuttering", "rigid-hypokinetic type", "breathy voice" and "pressed voice". Initial evidence suggests that the side effects of STN-DBS could be due to the stimulation of specific brain circuits (Fox et al., 2014). This paper presents a retrospective study with STN-DBS-stimulated PD patients that assesses speech therapy-relevant disorders under two conditions (stimulation on and off) as well as a prospective study with the same two conditions. Both studies aim to replicate the results of Tsuboi et al. (2015, 2017). The second prospective study also includes connectome-based data. The results of both studies do not show any quantitative significance with regard to the dysarthric phenotypes mentioned above, but quantitatively clear tendencies similar to the original study can be seen. In addition, the "stuttering" cluster was identified in the retrospective study as another cluster possibly inherent in STN-DBS. A cluster was added in the prospective study because the symptoms of "hasty speech" were also observed in the assessments. KW - Dysarthrie KW - Parkinson-Krankheit KW - Parkinson Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-363014 ER - TY - THES A1 - Aroko, Erick Onyango T1 - Trans-regulation of \(Trypanosoma\) \(brucei\) variant surface glycoprotein (VSG) mRNA and structural analysis of a \(Trypanosoma\) \(vivax\) VSG using X-ray crystallography T1 - Trans-regulierung der mRNA des variablen Oberflächenglykoprotein (VSG) von \(Trypanosoma\) \(brucei\) und strukturelle Analyse eines \(Trypanosoma\) \(vivax\) VSG mittels Kristallstrukturanalyse N2 - African trypanosomes are unicellular parasites that cause nagana and sleeping sickness in livestock and man, respectively. The major pathogens for the animal disease include Trypanosoma vivax, T. congolense, and T. brucei brucei, whereas T. b. gambiense and T. b. rhodesiense are responsible for human infections. Given that the bloodstream form (BSF) of African trypanosomes is exclusively extracellular, its cell surface forms a critical boundary with the host environment. The cell surface of the BSF African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). This surface protein acts as an impenetrable shield that protects the cells from host immune factors and is also involved in antibody clearance and antigenic variation, which collectively ensure that the parasite stays ahead of the host immune system. Gene expression in T. brucei is markedly different from other eukaryotes: most genes are transcribed as long polycistronic units, processed by trans-splicing a 39-nucleotide mini exon at the 5′ and polyadenylation at the 3′ ends of individual genes to generate the mature mRNA. Therefore, gene expression in T. brucei is regulated post-transcriptionally, mainly by the action of RNA binding proteins (RBPs) and conserved elements in the 3′ untranslated regions (UTR) of transcripts. The expression of VSGs is highly regulated, and only a single VSG gene is expressed at a time from one of the ~15 subtelomeric domains termed bloodstream expression sites (BES). When cells are engineered to simultaneously express two VSGs, the total VSG mRNA do not exceed the wild type amounts. This suggests that a robust VSG mRNA balancing mechanism exists in T. brucei. The present study uses inducible and constitutive expression of ectopic VSG genes to show that the endogenous VSG mRNA is regulated only if the second VSG is properly targeted to the ER. Additionally, the endogenous VSG mRNA response is triggered when high amounts of the GFP reporter with a VSG 3′UTR is targeted to the ER. Further evidence that non-VSG ER import signals can efficiently target VSGs to the ER is presented. This study suggests that a robust trans-regulation of the VSG mRNA is elicited at the ER through a feedback loop to keep the VSG transcripts in check and avoid overshooting the secretory pathway capacity. Further, it was shown that induction of expression of the T. vivax VSG ILDat1.2 in T. brucei causes a dual cell cycle arrest, with concomitant upregulation of the protein associated with differentiation (PAD1) expression. It could be shown that T. vivax VSG ILDat1.2 can only be sufficiently expressed in T. brucei after replacing its native GPI signal peptide with that of a T. brucei VSG. Taken together, these data indicate that inefficient VSG GPI anchoring and expression of low levels of the VSG protein can trigger differentiation from slender BSF to stumpy forms. However, a second T. vivax VSG, ILDat2.1, is not expressed in T. brucei even after similar modifications to its GPI signals. An X-ray crystallography approach was utilized to solve the N-terminal domain (NTD) structure of VSG ILDat1.2. This is first structure of a non-T. brucei VSG, and the first of a surface protein of T. vivax to be solved. VSG ILDat1.2 NTD maintains the three-helical bundle scaffold conserved in T. brucei surface proteins. However, it is likely that there are variations in the architecture of the membrane proximal region of the ILDat1.2 NTD and its CTD from T. brucei VSGs. The tractable T. brucei system is presented as a model that can be used to study surface proteins of related trypanosome species, thus creating avenues for further characterization of trypanosome surface coats. N2 - Afrikanische Trypanosomen sind einzellige Parasiten, die Nagana in Nutzvieh und die Schlafkrankheit im Menschen verursachen. Zu den Hauptverursachern der Tierkrankheit gehören Trypanosoma vivax, T. congolense und T. brucei brucei, während T. b. gambiense und T. b. rhodesiense für Infektionen im Menschen verantwortlich sind. Da die Blutstromform (BSF) der afrikanischen Trypanosomen rein extrazellulär vorkommt, bildet die Zelloberfläche eine kritische Grenzregion mit der Wirtsumgebung. Die Zelloberoberfläche der BSF afrikanischer Trypanosomen ist mit einem dichten Mantel an immunogenen variablen Oberflächenglykoproteinen (variant surface glycoprotein, VSG) umgeben. Dieses Oberflächenprotein dient als Barriere zum Schutz gegen Faktoren des Wirtsimmunsystems und spielt ebenfalls eine Rolle in Antikörper-Clearance und antigener Variation, welche gemeinsam dafür sorgen, dass der Parasit dem Wirtsimmunsystem stets einen Schritt voraus bleibt. Die Genexpression von T. brucei weist dezidierte Unterschiede im Vergleich zu anderen Eukaryoten auf: Die meisten Gene werden als lange polyzystronische Einheiten transkribiert, die durch trans-Splicing eines Miniexons aus 39 Nukleotiden am 5′ und Polyadenylierung am 3′ Ende der individuellen Gene prozessiert wird. Daher wird die Genexpression in T. brucei posttranskriptionell reguliert, zumeist durch RNA Bindeproteine (RBPs) und konservierte Elemente in der 3′ untranslatierten Region (UTR). Die Expression der VSGs ist stark reguliert, so wird zu einer gegebenen Zeit stets nur ein VSG Gen aus einer von ~15 Subtelomerregionen, die Blutstrom Expressionsorte (bloodstream expression sites, BES) genannt werden, exprimiert. Zellen, die gentechnisch manipuliert wurden um zwei VSGs zu exprimieren, produzieren die gleiche Menge an VSG mRNA wie Wildtyp Zellen. Dies deutet auf die Existenz eines robusten Mechanismus zur Regulierung der Gesamt-VSG mRNA Menge in T. brucei hin. Diese Arbeit verwendet induzierbare sowie konstitutive Expression eines ektopischen VSG Gens um zu zeigen, dass die endogene VSG mRNA nur reguliert wird, wenn das zweite VSG zum ER gelangt. Außerdem wird die endogene VSG mRNA Antwort auch ausgelöst, wenn hohe Mengen eines GFP Reporters, der eine VSG 3′UTR enthält, zum ER geleitet wird. Weiterhin, wird gezeigt, dass ER Importsignale anderer Proteine VSGs effizient zum ER dirigieren können. Das Ergebnis dieser Studie deutet darauf hin, dass eine Rückkopplungsschleife am ER eine robuste trans-Regulation der VSG mRNA auslöst, die die VSG Transkripte limitiert und somit eine Überlastung des sekretorischen Wegs verhindert. Weiterhin konnte gezeigt werden, dass es nach Induktion der Expression des T. vivax VSGs ILDat1.2 in T. brucei zu einem doppelten Zellzyklusarrest mit gleichzeitiger Hochregulation der Expression des protein associated with differentation (PAD1) kam und dass dieses T. vivax VSG nur nach Austausch des GPI Signalpeptids durch das eines T. brucei VSGs effizient exprimiert werden konnte. Zusammengenommen suggerieren diese Daten, dass eine ineffiziente GPI-Verankerung und wenig abundante Expression des VSGs die Differenzierung der sogenannten slender BSF zur sogenannten stumpy Form einleiten kann. Ein zweites T. vivax VSG, ILDat2.1, konnte hingegen auch nach Austausch des GPI Signals nicht in T. brucei exprimiert werden. Mit Hilfe der Röntgenstrukturanalyse wurde die Struktur der N-terminalen Domäne (NTD) des ILDat1.2 VSGs gelöst. Es handelt sich hierbei um die erste Proteinstruktur eines VSGs, welches nicht aus T. brucei stammt und die erste Struktur eines Oberflächenproteins von T. vivax. Das in T. brucei Oberflächenproteinen konservierte drei-Helix Grundgerüst ist auch in der NTD des ILDat1.2 VSGs enthalten. Die Architektur der Membranproximalen Gegend der IlDat1.2 NTD und CTD unterscheiden sich aber vermutlich von der der T. brucei VSGs. Das leicht handhabbare T. brucei System bietet somit ein geeignetes Modell um die Oberflächenproteine anderer afrikanischer Trypanosomen Spezies zu untersuchen und eröffnet neue Wege zur Charakterisierung ihrer Oberflächenmäntel. KW - Trypanosoma vivax KW - Trypanosoma brucei KW - Variant surface glycoprotein KW - messenger RNA KW - Regulation of expression KW - messenger RNA regulation KW - VSG structure Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241773 ER - TY - THES A1 - Reissland, Michaela T1 - USP10 is a \(de\) \(novo\) tumour-specific regulator of β-Catenin and contributes to cancer stem cell maintenance and tumour progression T1 - USP10 ist ein \(de\) \(novo\) tumorspezifischer Regulator von ß-Catenin und trägt zur Erhaltung von Krebsstammzellen und zur Tumorprogression bei N2 - Colorectal Cancer (CRC) is the third most common cancer in the US. The majority of CRC cases are due to deregulated WNT-signalling pathway. These alterations are mainly caused by mutations in the tumour suppressor gene APC or in CTNNB1, encoding the key effector protein of this pathway, β-Catenin. In canonical WNT-signalling, β-Catenin activates the transcription of several target genes, encoding for proteins involved in proliferation, such as MYC, JUN and NOTCH. Being such a critical regulator of these proto-oncogenes, the stability of β-Catenin is tightly regulated by the Ubiquitin-Proteasome System. Several E3 ligases that ubiquitylate and degrade β-Catenin have been described in the past, but the antagonists, the deubiquitylases, are still unknown. By performing an unbiased siRNA screen, the deubiquitylase USP10 was identified as a de novo positive regulator of β-Catenin stability in CRC derived cells. USP10 has previously been shown in the literature to regulate both mutant and wild type TP53 stability, to deubiquitylate NOTCH1 in endothelial cells and to be involved in the regulation of AMPKα signalling. Overall, however, its role in colorectal tumorigenesis remains controversial. By analysing publicly available protein and gene expression data from colorectal cancer patients, we have shown that USP10 is strongly upregulated or amplified upon transformation and that its expression correlates positively with CTNNB1 expression. In contrast, basal USP10 levels were found in non-transformed tissues, but surprisingly USP10 is upregulated in intestinal stem cells. Endogenous interaction studies in CRC-derived cell lines, with different extend of APCtruncation, revealed an APC-dependent mode of action for both proteins. Furthermore, by utilising CRISPR/Cas9, shRNA-mediated knock-down and overexpression of USP10, we could demonstrate a regulation of β-Catenin stability by USP10 in CRC cell lines. It is widely excepted that 2D cell culture systems do not reflect complexity, architecture and heterogeneity and are therefore not suitable to answer complex biological questions. To overcome this, we established the isolation, cultivation and genetically modification of murine intestinal organoids and utilised this system to study Usp10s role ex vivo. By performing RNA sequencing, dependent on different Usp10 levels, we were able to recapitulate the previous findings and demonstrated Usp10 as important regulator of β-dependent regulation of stem cell homeostasis. Since genetic depletion of USP10 resulted in down-regulation of β-Catenin-dependent transcription, therapeutic intervention of USP10 in colorectal cancer was also investigated. Commercial and newly developed inhibitors were tested for their efficacy against USP10, but failed to significantly inhibit USP10 activity in colorectal cancer cells. To validate the findings from this work also in vivo, development of a novel mouse model for colorectal cancer has begun. By combining CRISPR/Cas9 and classical genetic engineering with viral injection strategies, WT and genetically modified mice could be transformed and, at least in some animals, intestinal lesions were detectable at the microscopic level. The inhibition of USP10, which we could describe as a de novo tumour-specific regulator of β-Catenin, could become a new therapeutic strategy for colorectal cancer patients. N2 - Darmkrebs ist die dritthäufigste Krebsart in den USA. Die Mehrheit der Darmkrebsfälle sind auf einen deregulierten WNT-Signalweg zurückzuführen. Diese Veränderungen wer- den hauptsächlich durch Mutationen im Tumorsuppressor-Gen APC oder in CTNNB1 verursacht, welches für das zentrale Protein dieses Signalwegs, β-Catenin, kodiert. Beim kanonischen WNT-Signalweg aktiviert β-Catenin die Transkription mehrerer Gene, die für, an der Proliferation beteiligte Proteine wie MYC, JUN und NOTCH, kodieren. Da β-Catenin ein kritischer Regulator dieser proto-Onkogene ist, wird die Stabilität von β-Catenin durch das Ubiquitin-Proteasom-System streng reguliert. In der Vergangen- heit wurden mehrere E3-Ligasen beschrieben, die β-Catenin ubiquitylieren und abbauen, aber die Deubiquitylasen, sind grö𐀀tenteils noch unbekannt. Mit Hilfe eines unvoreingenommenen siRNA-Screens wurde die Deubiquitylase USP10 als de novo Regulator der β-Catenin-Stabilität in Darmkrebs-Zellen identifiziert. In der Literatur wurde bereits gezeigt, dass USP10 sowohl die Stabilität von mutiertem als auch von wild typ TP53 reguliert, NOTCH1 in Endothelzellen deubiquityliert und an der Regulation des AMPKα Signalwegs beteiligt ist. Insgesamt bleibt seine Rolle in der kolorektalen Tumorgenese aber bisher umstritten. Anhand der Analyse öffentlich zugänglicher Protein- und Genexpressionsdaten haben wir gezeigt, dass USP10 bei der Transformation stark hochreguliert oder amplifiziert wird und dass seine Expression positiv mit der von CTNNB1 korreliert. Im Gegensatz dazu wurden in nicht transformiertem Gewebe basale USP10-Spiegel gefunden, aber überraschenderweise ist USP10 in intestinalen Stammzellen hochreguliert. Endogene Interaktionsstudien in Darmkrebs-Zelllinien mit unterschiedlichem Ausma𐀀 an APC-Trunkierung zeigten eine APC-abhängige Interaktion für beide Proteine. Darüber hinaus konnten wir mit Hilfe von CRISPR/Cas9, shRNA-vermitteltem Knock-down und Überexpression von USP10 eine Regulation der β-Catenin-Stabilität durch USP10 in Darmkrebs-Zelllinien nachweisen. Es ist allgemein bekannt, dass 2D-Zellkultursysteme die Komplexität, Architektur und Heterogenität nicht widerspiegeln und daher nicht geeignet sind, um komplexe biologische Fragen zu beantworten. Um dies zu überwinden, haben wir die Isolierung, Kultivierung und genetische Veränderung von murinen Dar- morganoiden etabliert und dieses System genutzt, um die Rolle von Usp10 ex vivo zu untersuchen. Durch die Durchführung von RNA-Sequenzierungen in Abhängigkeit von unterschiedlichen Usp10-Spiegeln konnten wir die bisherigen Ergebnisse rekapitulieren und Usp10 als wichtigen Regulator der β-Catenin-abhängigen Regulation der Stammzell- homöostase nachweisen. Da die genetische Depletion von USP10 zu einer Herunterregulierung der β-Catenin- abhängigen Transkription führte, wurde auch die therapeutische Intervention von USP10 in Darmkrebs untersucht. Kommerzielle und neu entwickelte Inhibitoren wurden auf ihre Wirksamkeit gegen USP10 getestet, konnten jedoch die Aktivität von USP10 in Darmkrebs- Zellen nicht hemmen. Um die Erkenntnisse aus dieser Arbeit auch in vivo zu validieren, wurde mit der Entwicklung eines neuartigen Mausmodells für Darmkrebs begonnen. Durch die Kombination von CRISPR/Cas9 und klassischer Gentechnik mit viralen Injektionsstrategien konnten WT- und gentechnisch veränderte Mäuse trans- formiert werden und zumindest bei einigen Tieren waren Darmläsionen auf mikroskopis- cher Ebene nachweisbar. Die Inhibtierung von USP10, als de novo tumorspezifischer Regulator von β-Catenin, könnte eine neue therapeutische Strategie für Darmkrebs-Patienten werden. KW - Biomedizin KW - Biomedicine Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319579 ER - TY - THES A1 - Schulz, Daniel T1 - Development of Inhibitory Control in Kindergarten Children T1 - Entwicklung von Inhibitionskontrolle bei Kindern im Kindergartenalter N2 - This dissertation explores the development and assessment of inhibitory control – a crucial component of executive functions – in young children. Inhibitory control, defined as the ability to suppress inappropriate responses (Verbruggen & Logan, 2008), is essential for adaptable and goal-oriented behavior. The rapid and non-linear development of this cognitive function in early childhood presents unique challenges for accurate assessment. As children age, they often exhibit a ceiling effect in terms of response accuracy (Petersen et al., 2016), underscoring the need to consider response latency as well. Ideally, combining response latency with accuracy could yield a more precise measure of inhibitory control (e.g., Magnus et al., 2019), facilitating a detailed tracking of developmental changes in inhibitory control across a wider age spectrum. The three studies of this dissertation collectively aim to clarify the relationship between response accuracy, response latency, and inhibitory control across different stages of child development. Each study utilizes a computerized Pointing Stroop Task (Berger et al., 2000) to measure inhibitory control, examining the task's validity and the integration of dual metrics for a more comprehensive evaluation. The first study focuses on establishing the validity of using both response accuracy and latency as indicators of inhibitory control. Utilizing the framework of explanatory item-response modeling (De Boeck & Wilson, 2004), the study revealed how the task characteristics congruency and item position influence both the difficulty level and timing aspects in young children’s responses in the computerized Pointing Stroop task. Further, this study found that integrating response accuracy with latency, even in a basic manner, provides additional insights. Building upon these findings, the second study investigates the nuances of integrating response accuracy and latency, examining whether this approach can account for age-related differences in inhibitory control. It also explores whether response latencies may contain different information depending on the age and proficiency of the children. The study leverages novel and established methodological perspectives to integrate response accuracy and latency into a single metric, showing the potential applicability of different approaches for assessing inhibitory control development. The third study extends the investigation to a longitudinal perspective, exploring the dynamic relationship between response accuracy, latency, and inhibitory control over time. It assesses whether children who achieve high accuracy at an earlier age show faster improvement in response latency, suggesting a non-linear maturation pathway of inhibitory control. The study also examines if the predictive value of early response latency for later fluid intelligence is dependent on the response accuracy level. Together, these empirical studies contribute to a more robust understanding of the complex interaction between inhibitory control, response accuracy, and response latency, facilitating valid evaluations of cognitive capabilities in children. Moreover, the findings may have practical implications for designing educational strategies and clinical interventions that address the developmental trajectory of inhibitory control. The nuanced approach advocated in this dissertation suggests prioritizing accuracy in assessment and interventions during the early stages of children's cognitive development, gradually shifting the focus to response latency as children mature and secure their inhibitory control abilities. N2 - Die vorliegende Dissertation erforscht die Erfassung und Entwicklung von Inhibitionskontrolle bei jungen Kindern – einer zentralen Komponente der Exekutiven Funktionen. Inhibitionskontrolle, also die Fähigkeit, automatisierte aber unangemessene Reaktionen zu unterdrücken (Verbruggen & Logan, 2008), ist wesentlich für adaptives und zielgerichtetes Verhalten. Die schnelle und nichtlineare Entwicklung dieser kognitiven Funktion im frühen Kindesalter gestaltet eine präzise Messung herausfordernd. Mit zunehmendem Alter der Kinder zeigt sich häufig ein Deckeneffekt hinsichtlich der Antwortgenauigkeit (Petersen et al., 2016), was die Notwendigkeit hervorhebt, auch die Reaktionszeit in Betracht zu ziehen. Idealerweise könnte durch die Integration von Reaktionszeit und Antwortgenauigkeit ein Messwert berechnet werden (z.B. Magnus et al., 2019), welcher eine detaillierte Erfassung von Entwicklungsveränderungen der Inhibitionskontrolle über ein breiteres Altersspektrum hinweg ermöglicht. Die drei Studien dieser Dissertation zielen darauf ab, die Beziehung zwischen Antwortgenauigkeit, Reaktionszeit und Inhibitionskontrolle in verschiedenen Stadien der kindlichen Entwicklung zu untersuchen. Jede Studie nutzt eine computergestützte Inhibitionsaufgabe, den computerized Pointing-Stroop Task (cPST; Berger et al., 2000), um die Inhibitionskontrolle zu messen, wobei die Validität dieses Tests und die Integration von Antwortgenauigkeit und Reaktionszeit für eine umfassendere Bewertung untersucht werden. In der ersten Studie wird untersucht, ob sowohl Antwortgenauigkeit als auch Reaktionszeit valide Indikatoren für Inhibitionskontrolle in jungen Kindern darstellen. Unter Verwendung von explanatorischen Item-Response-Modellen zeigte die Studie, wie die Aufgabenmerkmale Kongruenz und Item-Position die Aufgabenschwierigkeit sowohl in Bezug auf Antwortgenauigkeit als auch Reaktionszeit im cPST beeinflussen. Darüber hinaus zeigten sich erste Hinweise, dass bereits eine rudimentäre Integration von Antwortgenauigkeit und Reaktionszeit zusätzliche Einsichten liefert. Aufbauend auf diesen Erkenntnissen untersucht die zweite Studie die Feinheiten der Integration von Antwortgenauigkeit und Reaktionszeit und prüft, ob moderne Methoden der Integration dieser beiden Metriken altersbedingte Unterschiede in der Inhibitionskontrolle berücksichtigen können. Sie erforscht auch, ob sich aus den Reaktionszeiten in Inhibitionsaufgaben, abhängig vom Alter und Können der Kinder, unterschiedliche Schlussfolgerungen ziehen lassen. Die Studie nutzt neue und etablierte methodische Ansätze, um Antwortgenauigkeit und Reaktionszeit zu einer Metrik zu integrieren und zeigt die potenzielle Anwendbarkeit verschiedener Ansätze zur Bewertung der Entwicklung der Inhibitionskontrolle. Die dritte Studie erweitert die Untersuchung auf eine Längsschnittperspektive und erforscht die dynamische Beziehung zwischen Antwortgenauigkeit, Reaktionszeit und Inhibitionskontrolle im Laufe der Entwicklung. Sie betrachtet, ob Kinder, die in jüngerem Alter eine hohe Genauigkeit erreichen, eine schnellere Verbesserung in der Reaktionszeit zeigen. Die Studie untersucht weiter, ob der prädiktive Wert von Reaktionszeit für zukünftige fluide Intelligenz in Abhängigkeit zu der Antwortgenauigkeit steht. Zusammen tragen diese empirischen Arbeiten zu einem tieferen Verständnis der komplexen Interaktion zwischen Inhibitionskontrolle, Antwortgenauigkeit und Reaktionszeit bei und erleichtern valide Bewertungen dieser kognitiven Fähigkeiten bei Kindern. Darüber hinaus könnten die Ergebnisse praktische Implikationen für die Gestaltung von Interventionen haben, die sich mit dem Entwicklungsverlauf der Inhibitionskontrolle befassen. Der in dieser Dissertation vertretene Ansatz legt nahe, Antwortgenauigkeit bei der Bewertung und Interventionen während der frühen Phasen der kognitiven Entwicklung von Kindern zu priorisieren und den Fokus allmählich auf die Reaktionszeit zu verlagern, sobald Kinder ihre Inhibitionskontrolle festigen und ausbauen. KW - Kognitive Entwicklung KW - Kognition KW - Psychologie KW - Executive Functions KW - Inhibitory Control KW - Inhibitionskontrolle KW - Linear-Mixed Models KW - Linear Gemischte Modelle KW - Cognition KW - Child Development KW - Kindliche Entwicklung KW - Exekutive Funktionen KW - Kinderentwicklung Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357152 ER - TY - THES A1 - Heimberger, Kevin T1 - Regulation pathways of c-MYC under glutamine-starving conditions in colon carcinoma cells T1 - Regulierungsmechanismen von c-MYC in Darmkrebszellen unter Glutaminmangelbedingungen N2 - Colon carcinomas (CRC) are statistically among the most fatal cancer types and hence one of the top reasons for premature mortality in the developed world. CRC cells are characterized by high proliferation rates caused by deregulation of gene transcription of proto-oncogenes and general chromosomal instability. On macroscopic level, CRC cells show a strongly altered nutrient and energy metabolism. This work presents research to understand general links between the metabolism and transcription alteration. Mainly focussing on glutamine dependency, shown in colon carcinoma cells and expression pathways of the pro-proliferation protein c-MYC. Previous studies showed that a depletion of glutamine in the cultivation medium of colon carcinoma cell lines caused a proliferation arrest and a strong decrease of overall c-MYC levels. Re-addition of glutamine quickly replenished c-MYC levels through an unknown mechanism. Several proteins altering this regulation mechanism were identified and proposed as possible starting point for further in detail studies to unveil the precise biochemical pathway controlling c-MYC translation repression and reactivation in a rapid manner. On a transcriptional level the formation of RNA:DNA hybrids, so called R-loops, was observed under glutamine depleted conditions. The introduction and overexpression of RNaseH1, a R-loop degrading enzyme, in combination with an ectopically expressed c-MYC variant, independent of cellular regulation mechanisms by deleting the regulatory 3’-UTR of the c-MYC gene, lead to a high rate of apoptotic cells in culture. Expression of a functionally inactive variant of RNaseH1 abolished this effect. This indicates a regulatory function of R-loops formed during glutamine starvation in the presence of c-MYC protein in a cell. Degradation of R-loops and high c-MYC levels in this stress condition had no imminent effect on the cell cycle progression is CRC cells but disturbed the nucleotide metabolism. Nucleotide triphosphates were strongly reduced in comparison to starving cells without R-loop degradation and proliferating cells. This study proposes a model of a terminal cycle of transcription termination, unregulated initiation and elongation of transcription leading to a depletion of energy resources of cells. This could finally lead to high apoptosis of the cells. Sequencing experiments to determine a coinciding of termination sites and R-loop formation sides failed so far but show a starting point for further studies in this essential survival mechanism involving R-loop formation and c-MYC downregulation. N2 - Darmkrebs gehört statistisch zu den Krebsarten mit den höchsten Sterblichkeitsraten und zählen somit zu den häufigsten Todesursachen der entwickelten Länder. Darmkrebszellen zeichnen sich durch chromosomale Instabilität und hohe Proliferationsraten aus, die durch eine Deregulierung der Expression verschiedener Proto-Onkogene zustande kommen. Generell besitzen diese Krebszellen einen stark veränderten Nährstoff- und Energiestoffwechsel im Vergleich zu gesunden somatischen Zellen. Diese Arbeit strebt ein besseres Verständnis der Verbindung zwischen dem Metabolismus und der Gen-Expression an. Das Hauptaugenmerk liegt hierbei auf dem Mechanismus der Expression des proliferationsfördernden Proteins c-MYC und der Abhängigkeit von Glutamin, die Darmkrebszellen charakterisiert. Frühere Studien haben gezeigt, dass der Entzug von Glutamin aus dem Kulturmedium von Darmkrebszelllinien eine Arretierung des Zellzyklus bewirkt sowie die Konzentration des Proteins c-MYC reduziert. Erneute Zugabe von Glutamin zum Medium stellt die MYC-Konzentration schnell wieder her. Die Hintergründe dieses Mechanismus sind bislang aber kaum verstanden. Einige Proteine wurden hier als potenzielle Kandidaten identifiziert, die einen Einfluss auf den biochemischen Prozess haben könnten, der die schnelle Wiederaufnahme der c-MYC Translation gewährleistet. Auf Translationsebene wurden RNA:DNA-Hybriden, sogenannte R-loops, gefunden, die sich unter anderem unter Glutamin-Mangelbedingungen im Genom bilden können. Ein gezielter Abbau dieser R-loops mithilfe des Enzyms RNaseH1, in Kombination mit der ektopischen Expression einer c-MYC-Variante, die unempfindlich gegenüber der zelleigenen Regulationsmechanismen ist, führte zu einer erhöhten Anzahl an apoptotischen Zellen in Kultur. Exprimiert man eine funktionell inaktive Variante der RNaseH1, statt der funktionellen, so kann dieser Apoptose-fördernde Prozess nicht beobachtet werden. Dies bestärkt die Hypothese, dass die R-loops, die sich während eines Glutamin-Mangels und hoher c-MYC-Konzentration bilden, eine regulatorische Funktion innehaben. Als Ursache für die Apoptose konnte ein Effekt der veränderten Expression auf das Fortschreiten des Zellzyklus ausgeschlossen werden. Jedoch zeigte sich eine Veränderung im Nukleotid-Metabolismus. Betroffene Zellen zeigten deutlich reduzierte Nuklotidtriphosphat-Konzentrationen im Vergleich zu Zellen unter Glutaminmangelbedingungen ohne R-loop-Abbau. In dieser Arbeit wurde ein Modell entwickelt, das einen sich selbst negativ verstärkenden Zyklus vorschlägt, der die Zellen zur Apoptose führt. Transkriptionstermination und eine unkontrollierte Initiation der Transkription im Wechsel führt zu einem Verbrauch der lebensnotwendigen Energieressourcen der Zellen. Sequenzierungsexperimente zur Lokalisierung der R-loops und Terminationsstellen sind bislang fehlgeschlagen, bieten jedoch Ansätze für künftige Forschung. KW - Myc KW - cMYC regulation KW - Colorectal Cancer KW - Glutamine KW - Translation regulation Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-363316 ER - TY - JOUR A1 - Morimoto, Yoshiro A1 - Shimada-Sugimoto, Mihoko A1 - Otowa, Takeshi A1 - Yoshida, Shintaro A1 - Kinoshita, Akira A1 - Mishima, Hiroyuki A1 - Yamaguchi, Naohiro A1 - Mori, Takatoshi A1 - Imamura, Akira A1 - Ozawa, Hiroki A1 - Kurotaki, Naohiro A1 - Ziegler, Christiane A1 - Domschke, Katharina A1 - Deckert, Jürgen A1 - Umekage, Tadashi A1 - Tochigi, Mamoru A1 - Kaiya, Hisanobu A1 - Okazaki, Yuji A1 - Tokunaga, Katsushi A1 - Sasaki, Tsukasa A1 - Yoshiura, Koh-ichiro A1 - Ono, Shinji T1 - Whole-exome sequencing and gene-based rare variant association tests suggest that PLA2G4E might be a risk gene for panic disorder JF - Translational Psychiatry N2 - Panic disorder (PD) is characterized by recurrent and unexpected panic attacks, subsequent anticipatory anxiety, and phobic avoidance. Recent epidemiological and genetic studies have revealed that genetic factors contribute to the pathogenesis of PD. We performed whole-exome sequencing on one Japanese family, including multiple patients with panic disorder, which identified seven rare protein-altering variants. We then screened these genes in a Japanese PD case–control group (384 sporadic PD patients and 571 controls), resulting in the detection of three novel single nucleotide variants as potential candidates for PD (chr15: 42631993, T>C in GANC; chr15: 42342861, G>T in PLA2G4E; chr20: 3641457, G>C in GFRA4). Statistical analyses of these three genes showed that PLA2G4E yielded the lowest p value in gene-based rare variant association tests by Efficient and Parallelizable Association Container Toolbox algorithms; however, the p value did not reach the significance threshold in the Japanese. Likewise, in a German case–control study (96 sporadic PD patients and 96 controls), PLA2G4E showed the lowest p value but again did not reach the significance threshold. In conclusion, we failed to find any significant variants or genes responsible for the development of PD. Nonetheless, our results still leave open the possibility that rare protein-altering variants in PLA2G4E contribute to the risk of PD, considering the function of this gene. KW - clinical genetics KW - medical genetics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224192 VL - 8 ER - TY - JOUR A1 - Schneider, Christian A1 - Glazov, Mikhail M. A1 - Korn, Tobias A1 - Höfling, Sven A1 - Urbaszek, Bernhard T1 - Two-dimensional semiconductors in the regime of strong light-matter coupling JF - Nature Communications N2 - The optical properties of transition metal dichalcogenide monolayers are widely dominated by excitons, Coulomb-bound electron–hole pairs. These quasi-particles exhibit giant oscillator strength and give rise to narrow-band, well-pronounced optical transitions, which can be brought into resonance with electromagnetic fields in microcavities and plasmonic nanostructures. Due to the atomic thinness and robustness of the monolayers, their integration in van der Waals heterostructures provides unique opportunities for engineering strong light-matter coupling. We review first results in this emerging field and outline future opportunities and challenges. KW - optical physics KW - two-dimensional materials Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231295 VL - 9 ER - TY - JOUR A1 - Selkrig, Joel A1 - Mohammad, Farhan A1 - Ng, Soon Hwee A1 - Chua, Jia Yi A1 - Tumkaya, Tayfun A1 - Ho, Joses A1 - Chiang, Yin Ning A1 - Rieger, Dirk A1 - Pettersson, Sven A1 - Helfrich-Förster, Charlotte A1 - Yew, Joanne Y. A1 - Claridge-Chang, Adam T1 - The Drosophila microbiome has a limited influence on sleep, activity, and courtship behaviors JF - Scientific Reports N2 - In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this dependency is present in other animal species. We proposed the hypothesis that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal’s survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved. KW - behavioural ecology KW - sleep Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235891 VL - 8 ER - TY - JOUR A1 - Nerreter, Thomas A1 - Letschert, Sebastian A1 - Götz, Ralph A1 - Doose, Sören A1 - Danhof, Sophia A1 - Einsele, Hermann A1 - Sauer, Markus A1 - Hudecek, Michael T1 - Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T JF - Nature Communications N2 - Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens. KW - cancer imaging KW - cancer immunotherapy KW - imaging Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232258 VL - 10 ER - TY - JOUR A1 - Odin, Per A1 - Chaudhuri, K. Ray A1 - Volkmann, Jens A1 - Antonini, Angelo A1 - Storch, Alexander A1 - Dietrichs, Espen A1 - Pirtošek, Zvezdan A1 - Henriksen, Tove A1 - Horne, Malcolm A1 - Devos, David A1 - Bergquist, Filip T1 - Viewpoint and practical recommendations from a movement disorder specialist panel on objective measurement in the clinical management of Parkinson’s disease JF - npj Parkinson's Disease N2 - Motor aspects of Parkinson’s disease, such as fluctuations and dyskinesia, can be reliably evaluated using a variety of “wearable” technologies, but practical guidance on objective measurement (OM) and the optimum use of these devices is lacking. Therefore, as a first step, a panel of movement disorder specialists met to provide guidance on how OM could be assessed and incorporated into clinical guidelines. A key aspect of the incorporation of OM into the management of Parkinson’s disease (PD) is defining cutoff values that separate “controlled” from “uncontrolled” symptoms that can be modified by therapy and that relate to an outcome that is relevant to the person with PD (such as quality of life). Defining cutoffs by consensus, which can be subsequently tested and refined, is the first step to optimizing OM in the management of PD. OM should be used by all clinicians that treat people with PD but the least experienced may find the most value, but this requires guidance from experts to allow non-experts to apply guidelines. While evidence is gained for devices that produce OM, expert opinion is needed to supplement the evidence base. KW - Parkinson's disease Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234435 VL - 4 ER - TY - JOUR A1 - Mussel, Patrick A1 - Hewig, Johannes T1 - A neural perspective on when and why trait greed comes at the expense of others JF - Scientific Reports N2 - Depending on the point of view, conceptions of greed range from being a desirable and inevitable feature of a well-regulated, well-balanced economy to the root of all evil - radix omnium malorum avaritia (Tim 6.10). Regarding the latter, it has been proposed that greedy individuals strive for obtaining desired goods at all costs. Here, we show that trait greed predicts selfish economic decisions that come at the expense of others in a resource dilemma. This effect was amplified when individuals strived for obtaining real money, as compared to points, and when their revenue was at the expense of another person, as compared to a computer. On the neural level, we show that individuals high, compared to low in trait greed showed a characteristic signature in the EEG, a reduced P3 effect to positive, compared to negative feedback, indicating that they may have a lack of sensitivity to adjust behavior according to positive and negative stimuli from the environment. Brain-behavior relations further confirmed this lack of sensitivity to behavior adjustment as a potential underlying neuro-cognitive mechanism which explains selfish and reckless behavior that may come at the expense of others. KW - human behaviour KW - psychology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231652 VL - 9 ER - TY - JOUR A1 - Schmitt, Martin A1 - Moras, Paolo A1 - Bihlmayer, Gustav A1 - Cotsakis, Ryan A1 - Vogt, Matthias A1 - Kemmer, Jeannette A1 - Belabbes, Abderrezak A1 - Sheverdyaeva, Polina M. A1 - Kundu, Asish K. A1 - Carbone, Carlo A1 - Blügel, Stefan A1 - Bode, Matthias T1 - Indirect chiral magnetic exchange through Dzyaloshinskii–Moriya-enhanced RKKY interactions in manganese oxide chains on Ir(100) JF - Nature Communications N2 - Localized electron spins can couple magnetically via the Ruderman–Kittel–Kasuya–Yosida interaction even if their wave functions lack direct overlap. Theory predicts that spin–orbit scattering leads to a Dzyaloshinskii–Moriya type enhancement of this indirect exchange interaction, giving rise to chiral exchange terms. Here we present a combined spin-polarized scanning tunneling microscopy, angle-resolved photoemission, and density functional theory study of MnO2 chains on Ir(100). Whereas we find antiferromagnetic Mn–Mn coupling along the chain, the inter-chain coupling across the non-magnetic Ir substrate turns out to be chiral with a 120° rotation between adjacent MnO2 chains. Calculations reveal that the Dzyaloshinskii–Moriya interaction results in spin spirals with a periodicity in agreement with experiment. Our findings confirm the existence of indirect chiral magnetic exchange, potentially giving rise to exotic phenomena, such as chiral spin-liquid states in spin ice systems or the emergence of new quasiparticles. KW - magnetic properties and materials KW - surfaces, interfaces and thin films Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230986 VL - 10 ER - TY - JOUR A1 - Schurr, Yvonne A1 - Spindler, Markus A1 - Kurz, Hendrikje A1 - Bender, Markus T1 - The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis JF - Scientific Reports N2 - Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice. KW - actin KW - microtubules Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234966 VL - 9 ER - TY - JOUR A1 - Scholz, S. L. A1 - Cosgarea, I. A1 - Süßkind, D. A1 - Murali, R. A1 - Möller, I. A1 - Reis, H. A1 - Leonardelli, S. A1 - Schilling, B. A1 - Schimming, T. A1 - Hadaschik, E. A1 - Franklin, C. A1 - Paschen, A. A1 - Sucker, A. A1 - Steuhl, K. P. A1 - Schadendorf, D. A1 - Westekemper, H. A1 - Griewank, K. G. T1 - NF1 mutations in conjunctival melanoma JF - British Journal of Cancer N2 - Background Conjunctival melanoma is a potentially deadly eye tumour. Despite effective local therapies, tumour recurrence and metastasis remain frequent. The genetics of conjunctival melanomas remain incompletely understood. Methods A large cohort of 63 conjunctival melanomas was screened for gene mutations known to be important in other melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with patient prognosis. Results Frequent mutations in genes activating the MAP kinase pathway were identified. NF1 mutations were most frequent (n = 21, 33%). Recurrent activating mutations were also identified in BRAF (n = 16, 25%) and RAS genes (n = 12, 19%; 11 NRAS and 1 KRAS). Conclusions Similar to cutaneous melanomas, conjunctival melanomas can be grouped genetically into four groups: BRAF-mutated, RAS-mutated, NF1-mutated and triple wild-type melanomas. This genetic classification may be useful for assessment of therapeutic options for patients with metastatic conjunctival melanoma KW - cancer genetics KW - eye cancer Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233329 VL - 118 ER - TY - JOUR A1 - Steuer Costa, Wagner A1 - Van der Auwera, Petrus A1 - Glock, Caspar A1 - Liewald, Jana F. A1 - Bach, Maximilian A1 - Schüler, Christina A1 - Wabnig, Sebastian A1 - Oranth, Alexandra A1 - Masurat, Florentin A1 - Bringmann, Henrik A1 - Schoofs, Liliane A1 - Stelzer, Ernst H. K. A1 - Fischer, Sabine C. A1 - Gottschalk, Alexander T1 - A GABAergic and peptidergic sleep neuron as a locomotion stop neuron with compartmentalized Ca2+ dynamics JF - Nature Communications N2 - Animals must slow or halt locomotion to integrate sensory inputs or to change direction. In Caenorhabditis elegans, the GABAergic and peptidergic neuron RIS mediates developmentally timed quiescence. Here, we show RIS functions additionally as a locomotion stop neuron. RIS optogenetic stimulation caused acute and persistent inhibition of locomotion and pharyngeal pumping, phenotypes requiring FLP-11 neuropeptides and GABA. RIS photoactivation allows the animal to maintain its body posture by sustaining muscle tone, yet inactivating motor neuron oscillatory activity. During locomotion, RIS axonal Ca2+ signals revealed functional compartmentalization: Activity in the nerve ring process correlated with locomotion stop, while activity in a branch correlated with induced reversals. GABA was required to induce, and FLP-11 neuropeptides were required to sustain locomotion stop. RIS attenuates neuronal activity and inhibits movement, possibly enabling sensory integration and decision making, and exemplifies dual use of one cell across development in a compact nervous system. KW - cellular neuroscience KW - neural circuits Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223273 VL - 10 ER - TY - JOUR A1 - Carradec, Quentin A1 - Pelletier, Eric A1 - Da Silva, Corinne A1 - Alberti, Adriana A1 - Seeleuthner, Yoann A1 - Blanc-Mathieu, Romain A1 - Lima-Mendez, Gipsi A1 - Rocha, Fabio A1 - Tirichine, Leila A1 - Labadie, Karine A1 - Kirilovsky, Amos A1 - Bertrand, Alexis A1 - Engelen, Stefan A1 - Madoui, Mohammed-Amin A1 - Méheust, Raphaël A1 - Poulain, Julie A1 - Romac, Sarah A1 - Richter, Daniel J. A1 - Yoshikawa, Genki A1 - Dimier, Céline A1 - Kandels-Lewis, Stefanie A1 - Picheral, Marc A1 - Searson, Sarah A1 - Jaillon, Olivier A1 - Aury, Jean-Marc A1 - Karsenti, Eric A1 - Sullivan, Matthew B. A1 - Sunagawa, Shinichi A1 - Bork, Peer A1 - Not, Fabrice A1 - Hingamp, Pascal A1 - Raes, Jeroen A1 - Guidi, Lionel A1 - Ogata, Hiroyuki A1 - de Vargas, Colomban A1 - Iudicone, Daniele A1 - Bowler, Chris A1 - Wincker, Patrick T1 - A global ocean atlas of eukaryotic gene JF - Nature Communications N2 - While our knowledge about the roles of microbes and viruses in the ocean has increased tremendously due to recent advances in genomics and metagenomics, research on marine microbial eukaryotes and zooplankton has benefited much less from these new technologies because of their larger genomes, their enormous diversity, and largely unexplored physiologies. Here, we use a metatranscriptomics approach to capture expressed genes in open ocean Tara Oceans stations across four organismal size fractions. The individual sequence reads cluster into 116 million unigenes representing the largest reference collection of eukaryotic transcripts from any single biome. The catalog is used to unveil functions expressed by eukaryotic marine plankton, and to assess their functional biogeography. Almost half of the sequences have no similarity with known proteins, and a great number belong to new gene families with a restricted distribution in the ocean. Overall, the resource provides the foundations for exploring the roles of marine eukaryotes in ocean ecology and biogeochemistry. KW - genomics KW - marine biology KW - microbial ecology KW - water microbiology Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222250 VL - 9 ER - TY - JOUR A1 - Brunk, Michael A1 - Sputh, Sebastian A1 - Doose, Sören A1 - van de Linde, Sebastian A1 - Terpitz, Ulrich T1 - HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi JF - Scientific Reports N2 - The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains. KW - bioinformatics KW - cell growth KW - fungal biology KW - microscopy Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221691 VL - 8 ER - TY - JOUR A1 - China, Swarup A1 - Burrows, Susannah M. A1 - Wang, Bingbing A1 - Harder, Tristan H. A1 - Weis, Johannes A1 - Tanarhte, Meryem A1 - Rizzo, Luciana V. A1 - Brito, Joel A1 - Cirino, Glauber G. A1 - Ma, Po-Lun A1 - Cliff, John A1 - Artaxo, Paulo A1 - Gilles, Mary K. A1 - Laskin, Alexander T1 - Fungal spores as a source of sodium salt particles in the Amazon basin JF - Nature Communications N2 - In the Amazon basin, particles containing mixed sodium salts are routinely observed and are attributed to marine aerosols transported from the Atlantic Ocean. Using chemical imaging analysis, we show that, during the wet season, fungal spores emitted by the forest biosphere contribute at least 30% (by number) to sodium salt particles in the central Amazon basin. Hydration experiments indicate that sodium content in fungal spores governs their growth factors. Modeling results suggest that fungal spores account for ~69% (31–95%) of the total sodium mass during the wet season and that their fractional contribution increases during nighttime. Contrary to common assumptions that sodium-containing aerosols originate primarily from marine sources, our results suggest that locally-emitted fungal spores contribute substantially to the number and mass of coarse particles containing sodium. Hence, their role in cloud formation and contribution to salt cycles and the terrestrial ecosystem in the Amazon basin warrant further consideration. KW - atmospheric chemistry KW - biogeochemistry KW - environmental sciences Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222492 VL - 9 ER - TY - JOUR A1 - Bruchhagen, Christin A1 - Jarick, Marcel A1 - Mewis, Carolin A1 - Hertlein, Tobias A1 - Niemann, Silke A1 - Ohlsen, Knut A1 - Peters, Georg A1 - Planz, Oliver A1 - Ludwig, Stephan A1 - Ehrhardt, Christina T1 - Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound JF - Scientific Reports N2 - Influenza virus (IV) infections cause severe respiratory illnesses that can be complicated by bacterial super-infections. Previously, we identified the cellular Raf-MEK-ERK cascade as a promising antiviral target. Inhibitors of MEK, such as CI-1040, showed potent antiviral activity. However, it remained unclear if this inhibitor and its active form, ATR-002, might sensitize host cells to either IV or secondary bacterial infections. To address these questions, we studied the anti-pathogen activity of ATR-002 in comparison to CI-1040, particularly, its impact on Staphylococcus aureus (S. aureus), which is a major cause of IV super-infections. We analysed IV and S. aureus titres in vitro during super-infection in the presence and absence of the drugs and characterized the direct impact of ATR-002 on bacterial growth and phenotypic changes. Importantly, neither CI-1040 nor ATR-002 treatment led to increased bacterial titres during super-infection, indicating that the drug does not sensitize cells for bacterial infection. In contrast, we rather observed reduced bacterial titres in presence of ATR-002. Surprisingly, ATR-002 also led to reduced bacterial growth in suspension cultures, reduced stress- and antibiotic tolerance without resistance induction. Our data identified for the first time that a particular MEK-inhibitor metabolite exhibits direct antibacterial activity, which is likely due to interference with the bacterial PknB kinase/Stp phosphatase signalling system. KW - antimicrobials KW - pathogens Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221648 VL - 8 ER - TY - JOUR A1 - Castilho, Miguel A1 - Hochleitner, Gernot A1 - Wilson, Wouter A1 - van Rietbergen, Bert A1 - Dalton, Paul D. A1 - Groll, Jürgen A1 - Malda, Jos A1 - Ito, Keita T1 - Mechanical behavior of a soft hydrogel reinforced with three-dimensional printed microfibre scaffolds JF - Scientific Reports N2 - Reinforcing hydrogels with micro-fibre scaffolds obtained by a Melt-Electrospinning Writing (MEW) process has demonstrated great promise for developing tissue engineered (TE) constructs with mechanical properties compatible to native tissues. However, the mechanical performance and reinforcement mechanism of the micro-fibre reinforced hydrogels is not yet fully understood. In this study, FE models, implementing material properties measured experimentally, were used to explore the reinforcement mechanism of fibre-hydrogel composites. First, a continuum FE model based on idealized scaffold geometry was used to capture reinforcement effects related to the suppression of lateral gel expansion by the scaffold, while a second micro-FE model based on micro-CT images of the real construct geometry during compaction captured the effects of load transfer through the scaffold interconnections. Results demonstrate that the reinforcement mechanism at higher scaffold volume fractions was dominated by the load carrying-ability of the fibre scaffold interconnections, which was much higher than expected based on testing scaffolds alone because the hydrogel provides resistance against buckling of the scaffold. We propose that the theoretical understanding presented in this work will assist the design of more effective composite constructs with potential applications in a wide range of TE conditions. KW - biomedical engineering KW - biomedical materials KW - gels and hydrogels Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222280 VL - 8 ER - TY - JOUR A1 - Ciuchi, Sergio A1 - Di Sante, Domenico A1 - Dobrosavljević, Vladimir A1 - Fratini, Simone T1 - The origin of Mooij correlations in disordered metals JF - npj Quantum Materials N2 - Sufficiently disordered metals display systematic deviations from the behavior predicted by semi-classical Boltzmann transport theory. Here the scattering events from impurities or thermal excitations can no longer be considered as additive-independent processes, as asserted by Matthiessen’s rule following from this picture. In the intermediate region between the regime of good conduction and that of insulation, one typically finds a change of sign of the temperature coefficient of resistivity, even at elevated temperature spanning ambient conditions, a phenomenology that was first identified by Mooij in 1973. Traditional weak coupling approaches to identify relevant corrections to the Boltzmann picture focused on long-distance interference effects such as “weak localization”, which are especially important in low dimensions (1D and 2D) and close to the zero-temperature limit. Here we formulate a strong-coupling approach to tackle the interplay of strong disorder and lattice deformations (phonons) in bulk three-dimensional metals at high temperatures. We identify a polaronic mechanism of strong disorder renormalization, which describes how a lattice locally responds to the relevant impurity potential. This mechanism, which quantitatively captures the Mooij regime, is physically distinct and unrelated to Anderson localization, but realizes early seminal ideas of Anderson himself, concerning the interplay of disorder and lattice deformations. KW - electronic properties and materials KW - phase transitions and critical phenomena KW - theory and computation Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223148 VL - 3 ER - TY - JOUR A1 - Al-Zaben, Naim A1 - Medyukhina, Anna A1 - Dietrich, Stefanie A1 - Marolda, Alessandra A1 - Hünniger, Kerstin A1 - Kurzai, Oliver A1 - Figge, Marc Thilo T1 - Automated tracking of label-free cells with enhanced recognition of whole tracks JF - Scientific Reports N2 - Migration and interactions of immune cells are routinely studied by time-lapse microscopy of in vitro migration and confrontation assays. To objectively quantify the dynamic behavior of cells, software tools for automated cell tracking can be applied. However, many existing tracking algorithms recognize only rather short fragments of a whole cell track and rely on cell staining to enhance cell segmentation. While our previously developed segmentation approach enables tracking of label-free cells, it still suffers from frequently recognizing only short track fragments. In this study, we identify sources of track fragmentation and provide solutions to obtain longer cell tracks. This is achieved by improving the detection of low-contrast cells and by optimizing the value of the gap size parameter, which defines the number of missing cell positions between track fragments that is accepted for still connecting them into one track. We find that the enhanced track recognition increases the average length of cell tracks up to 2.2-fold. Recognizing cell tracks as a whole will enable studying and quantifying more complex patterns of cell behavior, e.g. switches in migration mode or dependence of the phagocytosis efficiency on the number and type of preceding interactions. Such quantitative analyses will improve our understanding of how immune cells interact and function in health and disease. KW - image processing KW - software Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221093 VL - 9 ER - TY - JOUR A1 - Dammert, Marcel A. A1 - Brägelmann, Johannes A1 - Olsen, Rachelle R. A1 - Böhm, Stefanie A1 - Monhasery, Niloufar A1 - Whitney, Christopher P. A1 - Chalishazar, Milind D. A1 - Tumbrink, Hannah L. A1 - Guthrie, Matthew R. A1 - Klein, Sebastian A1 - Ireland, Abbie S. A1 - Ryan, Jeremy A1 - Schmitt, Anna A1 - Marx, Annika A1 - Ozretić, Luka A1 - Castiglione, Roberta A1 - Lorenz, Carina A1 - Jachimowicz, Ron D. A1 - Wolf, Elmar A1 - Thomas, Roman K. A1 - Poirier, John T. A1 - Büttner, Reinhard A1 - Sen, Triparna A1 - Byers, Lauren A. A1 - Reinhardt, H. Christian A1 - Letai, Anthony A1 - Oliver, Trudy G. A1 - Sos, Martin L. T1 - MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer JF - Nature Communications N2 - MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients. KW - genetic engineering KW - oncogenes KW - small-cell lung cancer KW - targeted therapies Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223569 VL - 10 ER - TY - JOUR A1 - de Jong, Simone A1 - Diniz, Mateus Jose Abdalla A1 - Saloma, Andiara A1 - Gadelha, Ary A1 - Santoro, Marcos L. A1 - Ota, Vanessa K. A1 - Noto, Cristiano A1 - Curtis, Charles A1 - Newhouse, Stephen J. A1 - Patel, Hamel A1 - Hall, Lynsey S. A1 - O'Reilly, Paul F. A1 - Belangero, Sintia I. A1 - Bressan, Rodrigo A. A1 - Breen, Gerome T1 - Applying polygenic risk scoring for psychiatric disorders to a large family with bipolar disorder and major depressive disorder JF - Communications Biology N2 - Psychiatric disorders are thought to have a complex genetic pathology consisting of interplay of common and rare variation. Traditionally, pedigrees are used to shed light on the latter only, while here we discuss the application of polygenic risk scores to also highlight patterns of common genetic risk. We analyze polygenic risk scores for psychiatric disorders in a large pedigree (n ~ 260) in which 30% of family members suffer from major depressive disorder or bipolar disorder. Studying patterns of assortative mating and anticipation, it appears increased polygenic risk is contributed by affected individuals who married into the family, resulting in an increasing genetic risk over generations. This may explain the observation of anticipation in mood disorders, whereby onset is earlier and the severity increases over the generations of a family. Joint analyses of rare and common variation may be a powerful way to understand the familial genetics of psychiatric disorders. KW - bipolar disorder KW - depression KW - genetic association study KW - genetic linkage study Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223622 VL - 1 ER - TY - JOUR A1 - Dekker, Annelot M. A1 - Diekstra, Frank P. A1 - Pulit, Sara L. A1 - Tazelaar, Gijs H. P. A1 - van der Spek, Rick A. A1 - van Rheenen, Wouter A1 - van Eijk, Kristel R. A1 - Calvo, Andrea A1 - Brunetti, Maura A1 - Van Damme, Philip A1 - Robberecht, Wim A1 - Hardiman, Orla A1 - McLaughlin, Russell A1 - Chiò, Adriano A1 - Sendtner, Michael A1 - Ludolph, Albert C. A1 - Weishaupt, Jochen H. A1 - Pardina, Jesus S. Mora A1 - van den Berg, Leonard H. A1 - Veldink, Jan H. T1 - Exome array analysis of rare and low frequency variants in amyotrophic lateral sclerosis JF - Scientific Reports N2 - Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects 1 in ~350 individuals. Genetic association studies have established ALS as a multifactorial disease with heritability estimated at ~61%, and recent studies show a prominent role for rare variation in its genetic architecture. To identify rare variants associated with disease onset we performed exome array genotyping in 4,244 cases and 3,106 controls from European cohorts. In this largest exome-wide study of rare variants in ALS to date, we performed single-variant association testing, gene-based burden, and exome-wide individual set-unique burden (ISUB) testing to identify single or aggregated rare variation that modifies disease risk. In single-variant testing no variants reached exome-wide significance, likely due to limited statistical power. Gene-based burden testing of rare non-synonymous and loss-of-function variants showed NEK1 as the top associated gene. ISUB analysis did not show an increased exome-wide burden of deleterious variants in patients, possibly suggesting a more region-specific role for rare variation. Complete summary statistics are released publicly. This study did not implicate new risk loci, emphasizing the immediate need for future large-scale collaborations in ALS that will expand available sample sizes, increase genome coverage, and improve our ability to detect rare variants associated to ALS. KW - amyotrophic lateral sclerosis KW - genome-wide association studies Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223686 VL - 9 ER - TY - JOUR A1 - Diehl-Schmid, Janine A1 - Licata, Abigail A1 - Goldhardt, Oliver A1 - Förstl, Hans A1 - Yakushew, Igor A1 - Otto, Markus A1 - Anderl-Straub, Sarah A1 - Beer, Ambros A1 - Ludolph, Albert Christian A1 - Landwehrmeyer, Georg Bernhard A1 - Levin, Johannes A1 - Danek, Adrian A1 - Fliessbach, Klaus A1 - Spottke, Annika A1 - Fassbender, Klaus A1 - Lyros, Epameinondas A1 - Prudlo, Johannes A1 - Krause, Bernd Joachim A1 - Volk, Alexander A1 - Edbauer, Dieter A1 - Schroeter, Matthias Leopold A1 - Drzezga, Alexander A1 - Kornhuber, Johannes A1 - Lauer, Martin A1 - Grimmer, Timo T1 - FDG-PET underscores the key role of the thalamus in frontotemporal lobar degeneration caused by C9ORF72 mutations JF - Translational Psychiatry N2 - C9ORF72 mutations are the most common cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). MRI studies have investigated structural changes in C9ORF72-associated FTLD (C9FTLD) and provided first insights about a prominent involvement of the thalamus and the cerebellum. Our multicenter, 18F-fluorodeoxyglucose positron-emission tomography study of 22 mutation carriers with FTLD, 22 matched non-carriers with FTLD, and 23 cognitively healthy controls provided valuable insights into functional changes in C9FTLD: compared to non-carriers, mutation carriers showed a significant reduction of glucose metabolism in both thalami, underscoring the key role of the thalamus in C9FTLD. Thalamic metabolism did not correlate with disease severity, duration of disease, or the presence of psychotic symptoms. Against our expectations we could not demonstrate a cerebellar hypometabolism in carriers or non-carriers. Future imaging and neuropathological studies in large patient cohorts are required to further elucidate the central role of the thalamus in C9FTLD. KW - diagnostic markers KW - psychiatric disorders Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225308 VL - 9 ER - TY - JOUR A1 - Dietrich, Thomas A1 - Krug, Ralf A1 - Krastl, Gabriel A1 - Tomson, Philip L. T1 - Restoring the unrestorable! Developing coronal tooth tissue with a minimally invasive surgical extrusion technique JF - British Dental Journal N2 - Surgical extrusion is a recognised treatment option for teeth that have insufficient coronal tooth structure remaining due to deep caries, resorption or traumatic injury. However, the technique has not been widely adopted, arguably because extraction of a severely compromised tooth may be difficult to achieve in a gentle and predictable way. In this paper, we present our novel approach to surgical extrusion and subsequent management of teeth using a vertical extraction system (Benex), which has become the method of choice in the authors' practice for many teeth that would otherwise be deemed unrestorable. We describe the clinical procedure in detail and discuss the advantages and disadvantages compared to alternative approaches, including surgical crown lengthening and orthodontic extrusion. Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225333 VL - 226 ER - TY - JOUR A1 - Krah, Franz-Sebastian A1 - Büntgen, Ulf A1 - Schaefer, Hanno A1 - Müller, Jörg A1 - Andrew, Carrie A1 - Boddy, Lynne A1 - Diez, Jeffrey A1 - Egli, Simon A1 - Freckleton, Robert A1 - Gange, Alan C. A1 - Halvorsen, Rune A1 - Heegaard, Einar A1 - Heideroth, Antje A1 - Heibl, Christoph A1 - Heilmann-Clausen, Jacob A1 - Høiland, Klaus A1 - Kar, Ritwika A1 - Kauserud, Håvard A1 - Kirk, Paul M. A1 - Kuyper, Thomas W. A1 - Krisai-Greilhuber, Irmgard A1 - Norden, Jenni A1 - Papastefanou, Phillip A1 - Senn-Irlet, Beatrice A1 - Bässler, Claus T1 - European mushroom assemblages are darker in cold climates JF - Nature Communications N2 - Thermal melanism theory states that dark-colored ectotherm organisms are at an advantage at low temperature due to increased warming. This theory is generally supported for ectotherm animals, however, the function of colors in the fungal kingdom is largely unknown. Here, we test whether the color lightness of mushroom assemblages is related to climate using a dataset of 3.2 million observations of 3,054 species across Europe. Consistent with the thermal melanism theory, mushroom assemblages are significantly darker in areas with cold climates. We further show differences in color phenotype between fungal lifestyles and a lifestyle differentiated response to seasonality. These results indicate a more complex ecological role of mushroom colors and suggest functions beyond thermal adaption. Because fungi play a crucial role in terrestrial carbon and nutrient cycles, understanding the links between the thermal environment, functional coloration and species’ geographical distributions will be critical in predicting ecosystem responses to global warming. KW - evolutionary ecology KW - fungal ecology KW - fungal evolution KW - macroecology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224815 VL - 10 ER - TY - JOUR A1 - Hauer, Nadine N. A1 - Popp, Bernt A1 - Schoeller, Eva A1 - Schuhmann, Sarah A1 - Heath, Karen E. A1 - Hisado-Oliva, Alfonso A1 - Klinger, Patricia A1 - Kraus, Cornelia A1 - Trautmann, Udo A1 - Zenker, Martin A1 - Zweier, Christiane A1 - Wiesener, Antje A1 - Jamra, Rami Abou A1 - Kunstmann, Erdmute A1 - Wieczorek, Dagmar A1 - Uebe, Steffen A1 - Ferrazzi, Fulvia A1 - Büttner, Christian A1 - Ekici, Arif B. A1 - Rauch, Anita A1 - Sticht, Heinrich A1 - Dörr, Helmuth-Günther A1 - Reis, André A1 - Thiel, Christian T. T1 - Clinical relevance of systematic phenotyping and exome sequencing in patients with short stature JF - Genetics in Medicine N2 - Purpose Short stature is a common condition of great concern to patients and their families. Mostly genetic in origin, the underlying cause often remains elusive due to clinical and genetic heterogeneity. Methods We systematically phenotyped 565 patients where common nongenetic causes of short stature were excluded, selected 200 representative patients for whole-exome sequencing, and analyzed the identified variants for pathogenicity and the affected genes regarding their functional relevance for growth. Results By standard targeted diagnostic and phenotype assessment, we identified a known disease cause in only 13.6% of the 565 patients. Whole-exome sequencing in 200 patients identified additional mutations in known short-stature genes in 16.5% of these patients who manifested only part of the symptomatology. In 15.5% of the 200 patients our findings were of significant clinical relevance. Heterozygous carriers of recessive skeletal dysplasia alleles represented 3.5% of the cases. Conclusion A combined approach of systematic phenotyping, targeted genetic testing, and whole-exome sequencing allows the identification of the underlying cause of short stature in at least 33% of cases, enabling physicians to improve diagnosis, treatment, and genetic counseling. Exome sequencing significantly increases the diagnostic yield and consequently care in patients with short stature. KW - growth KW - phenotypic spectrum KW - short stature KW - skeletal dysplasia KW - whole-exome sequencing Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227888 VL - 20 ER - TY - JOUR A1 - Kreinberg, Sören A1 - Grbešić, Tomislav A1 - Strauß, Max A1 - Carmele, Alexander A1 - Emmerling, Monika A1 - Schneider, Christian A1 - Höfling, Sven A1 - Porte, Xavier A1 - Reitzenstein, Stephan T1 - Quantum-optical spectroscopy of a two-level system using an electrically driven micropillar laser as a resonant excitation source JF - Light: Science & Applications N2 - Two-level emitters are the main building blocks of photonic quantum technologies and are model systems for the exploration of quantum optics in the solid state. Most interesting is the strict resonant excitation of such emitters to control their occupation coherently and to generate close to ideal quantum light, which is of utmost importance for applications in photonic quantum technology. To date, the approaches and experiments in this field have been performed exclusively using bulky lasers, which hinders the application of resonantly driven two-level emitters in compact photonic quantum systems. Here we address this issue and present a concept for a compact resonantly driven single-photon source by performing quantum-optical spectroscopy of a two-level system using a compact high-β microlaser as the excitation source. The two-level system is based on a semiconductor quantum dot (QD), which is excited resonantly by a fiber-coupled electrically driven micropillar laser. We dress the excitonic state of the QD under continuous wave excitation, and trigger the emission of single photons with strong multi-photon suppression (g\(^{(2)}\)(0)=0.02) and high photon indistinguishability (V = 57±9%) via pulsed resonant excitation at 156 MHz. These results clearly demonstrate the high potential of our resonant excitation scheme, which can pave the way for compact electrically driven quantum light sources with excellent quantum properties to enable the implementation of advanced quantum communication protocols. KW - near-infrared spectroscopy KW - photonic devices KW - semiconductor lasers KW - single photons and quantum effects Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229802 VL - 7 ER - TY - JOUR A1 - Ludwig, Heinz A1 - Delforge, Michel A1 - Facon, Thierry A1 - Einsele, Hermann A1 - Gay, Francesca A1 - Moreau, Philippe A1 - Avet-Loiseau, Hervé A1 - Boccadoro, Mario A1 - Hajek, Roman A1 - Mohty, Mohamad A1 - Cavo, Michele A1 - Dimopoulos, Meletios A A1 - San-Miguel, Jesús F A1 - Terpos, Evangelos A1 - Zweegman, Sonja A1 - Garderet, Laurent A1 - Mateos, María-Victoria A1 - Cook, Gordon A1 - Leleu, Xavier A1 - Goldschmidt, Hartmut A1 - Jackson, Graham A1 - Kaiser, Martin A1 - Weisel, Katja A1 - van de Donk, Niels W. C. J. A1 - Waage, Anders A1 - Beksac, Meral A1 - Mellqvist, Ulf H. A1 - Engelhardt, Monika A1 - Caers, Jo A1 - Driessen, Christoph A1 - Bladé, Joan A1 - Sonneveld, Pieter T1 - Prevention and management of adverse events of novel agents in multiple myeloma: a consensus of the European Myeloma Network JF - Leukemia N2 - During the last few years, several new drugs have been introduced for treatment of patients with multiple myeloma, which have significantly improved the treatment outcome. All of these novel substances differ at least in part in their mode of action from similar drugs of the same drug class, or are representatives of new drug classes, and as such present with very specific side effect profiles. In this review, we summarize these adverse events, provide information on their prevention, and give practical guidance for monitoring of patients and for management of adverse events. KW - disease prevention KW - myeloma Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-237338 VL - 32 ER - TY - JOUR A1 - Milanese, Alessio A1 - Mende, Daniel R A1 - Paoli, Lucas A1 - Salazar, Guillem A1 - Ruscheweyh, Hans-Joachim A1 - Cuenca, Miguelangel A1 - Hingamp, Pascal A1 - Alves, Renato A1 - Costea, Paul I A1 - Coelho, Luis Pedro A1 - Schmidt, Thomas S. B. A1 - Almeida, Alexandre A1 - Mitchell, Alex L A1 - Finn, Robert D. A1 - Huerta-Cepas, Jaime A1 - Bork, Peer A1 - Zeller, Georg A1 - Sunagawa, Shinichi T1 - Microbial abundance, activity and population genomic profiling with mOTUs2 JF - Nature Communications N2 - Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites). KW - microbiome KW - software Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224089 VL - 10 ER - TY - THES A1 - Machwart, Khaled T1 - Modulatorischer Einfluss von Levosimendan bei dem Ischämie-Reperfusionsschaden auf die myokardiale Mitochondrienfunktion T1 - Impact of Levosimendan in the ischemia-reperfusion damage on the myocardial Mitochondrial function N2 - Die vorliegende Studie untersuchte den Effekt von Levosimendan auf die mitochondriale Funktionen im Herzmuskel, insbesondere im Zusammenhang mit dem Ischämie/Reperfusions-Schaden. Methoden: In der Studie wurde ein retrogrades Langendorff-Modell verwendet, um die Auswirkungen von Levosimendan, dem Ischämie/Reperfusions-Schaden sowie deren Kombination auf die mitochondrialen Funktionen im Herzmuskel zu untersuchen. Dazu wurden vier verschiedene Gruppen von Rattenherzen entsprechend den experimentellen Bedingungen perfundiert, und ihre Funktionen wurden analysiert. Ergebnisse: Der Ischämie/Reperfusions-Schaden beeinträchtigte die myokardiale Ventrikelfunktion. Zusätzlich wurde eine Hypopolarisation des mithochondrialen Membranpotentials in den mit Levosimendan oder Ischämie behandelten Gruppen festgestellt. Die ATP-Synthese in den Gruppen mit Levosimendan und Ischämie war reduziert. Schlussfolgerung: Levosimendan zeigt signifikante Einflüsse auf die Atmungsfunktion der mitochondrialen Komplexe IV und V sowie auf das Membranpotential. Diese Phänomene könnten einem mito-K+ ATP-abhängigen Mechanismus zugrunde liegen. Obwohl Levosimendan während des Ischämie/Reperfusionsschadens eine protektive Wirkung hinsichtlich einer Ca2+- Überlastung aufweist, bleibt der kumulative Einfluss der beeinträchtigten ATP-Generierung auf die gesamte Myokardfunktion zu klären. N2 - The present study investigated the effect of levosimendan on mitochondrial functions in the heart muscle, particularly in connection with ischemia/reperfusion injury. Methods: In the study, a retrograde Langendorff model was used to examine the effects of levosimendan, ischemia/reperfusion injury, and their combination on mitochondrial functions in the heart muscle. For this purpose, four different groups of rat hearts were perfused according to the experimental conditions, and their functions were analyzed. Results: Ischemia/reperfusion injury impaired myocardial ventricular function. Additionally, a hypopolarization of the mitochondrial membrane potential was observed in the groups treated with levosimendan or ischemia. ATP synthesis was reduced in the groups with levosimendan and ischemia. Conclusion: Levosimendan shows significant effects on the respiratory function of mitochondrial complexes IV and V, as well as on the membrane potential. These phenomena could be based on a mito-K+ ATP-dependent mechanism. Although levosimendan has a protective effect during ischemia/reperfusion injury regarding Ca2+ overload, the cumulative impact of impaired ATP generation on overall myocardial function remains to be clarified. KW - Ischämie KW - Ischämie Reperfusion Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-361021 ER - TY - THES A1 - Schaefer, Bastian T1 - Eigenschaften von synthetischen Bandersatzmaterialien zum MPFL-Ersatz - biomechanische in vitro Studie am porcinen Modell T1 - Properties of synthetic ligament replacement materials for MPFL replacement - biomechanical in vitro study on a porcine model N2 - Der MPFL-Ersatz ist ein gängiges Therapieverfahren zur Behandlung einer patellofemoralen Instabilität. Die Operationstechniken unterscheiden sich zumeist an der patellaren Fixationsmethode und der Auswahl der Transplantate. Biomechanische Studien, welche sich mit den Eigenschaften implantatfreier ossärer Fixationsmethoden beim MPFL-Ersatz unter Verwendung künstlicher Transplantate beschäftigen gibt es nach aktueller Recherche nicht. Ziel dieser Arbeit war es, die biomechanischen Eigenschaften zweier patellarer Bohrkanalfixationstechniken beim MPFL-Ersatz mit unterschiedlichem künstlichen Bandersatzmaterial zu ermitteln. Die Hypothese war, dass die biomechanischen Eigenschaften in Elongation, Steifigkeit, Primärstabilität und maximaler Ausreißkraft mit denen der bereits etablierten Verfahren und dem nativen MPFL vergleichbar sind. Hierzu wurden 80 porcine Kniescheiben randomisiert in 8 Gruppen aufgeteilt und getestet. In den Gruppen 1-4 wurden parallele, transpatellare Bohrkanäle mit Tapes der Breiten 2 mm, 3 mm, 4 mm und 5 mm getestet. In den Gruppen 5-8 wurden V-Kanal-Fixationsmethoden mit Bändern der Breite von 2 mm, 3 mm, 4 mm und 5 mm untersucht. Zusätzlich wurden die biomechanischen Grundeigenschaften der nativen Tapes ermittelt. Alle Tests durchliefen jeweils drei Messabschnitte. Hierbei fand zunächst eine Präkonditionierung mit 10 Zyklen zwischen 5 N und 20 N statt. Daraufhin folgte eine zyklische Belastung mit 1000 Zyklen zwischen 5 N und 50 N. Am Ende wurde eine maximale Kraftapplikation bis zum Versagen der Fixationskomplexe durchgeführt. Im Rahmen der Messungen wurden Elongation, Steifigkeit, Yield Load und Maximum Load bestimmt. Es konnten Unterschiede zwischen den beiden Fixationsmethoden und den verwendeten Tapes festgestellt werden. Alle acht Gruppen zeigten eine höhere Primärstabilität als das humane MPFL. Bezogen auf die biomechanischen Eigenschaften und den Versagensmechanismus konnte in dieser Studie ein Vorteil der parallelen transpatellaren Bohrkanäle gegenüber den V- Kanaltechniken festgestellt werden. Die Werte mit der höchsten maximalen Ausreißkraft wurden in Gruppe 3 (631,6 ± 83,1 N) und Gruppe 1 (592,9 ± 170,1 N) gemessen. Diese zeigten eine höhere Primärstabilität mit geringerer Elongation und Steifigkeit im Vergleich zu den in der aktuellen Literatur beschriebenen biomechanischen Studien, welche sich mit unterschiedlichen und teilweise bereits etablierten MPFL-Ersatzverfahren beschäftigten. Eine implantatfreie MPFL-Rekonstruktion mit transpatellaren parallelen Bohrkanälen unter Verwendung eines 2 mm Fiber Tapes (Fa. Arthrex) oder eines 4 mm Tapes (Fa. Topester) könnten dementsprechend eine gute Alternative zur operativen Therapie einer patellofemoralen Instabilität sein. N2 - MPFL reconstruction is a common surgical treatment for patellofemoral instability. The surgical techniques usually differ in patellar fixation methods and selection of grafts. According to current research, there are no biomechanical studies that deal with the physical properties of implant-free MPFL reconstructions with osseous fixation using artificial grafts. The aim of this study was to determine the biomechanical properties of two patellar drill hole techniques in MPFL reconstruction with the use of different artificial grafts. The hypothesis was that the biomechanical properties in elongation, stiffness and maximum load are comparable to native MPFL and procedures with autologous grafts. Therefore 80 porcine patellae were randomly divided into 8 groups. Group 1-4 tested, parallel, transpatellar tunnels with tapes measuring 2 mm, 3 mm, 4 mm and 5 mm. In group 5-8 a bone bridge method (V-channel) was used testing tapes of 2 mm, 3 mm, 4 mm and 5 mm. In addition, the basic physical properties of the native tapes were determined. The specimens were preconditioned with 10 cycles between 5 N and 20 N before they underwent cyclic load with 1000 cycles between 5 N and 50 N. In the end, the maximum load to failure was tested. Elongation, stiffness, yield load, maximum load and failure mode were determined. Differences could be found between the two fixation methods and the tapes used. All eight groups showed higher primary stability than human MPFL. An advantage of the parallel transpatellar tunnels over the bone bridge technique was found in this study. The results with the highest maximum load were found in group 4 (631.6 ± 83.1 N) and group 2 (592.9 ± 170.1 N). These showed a higher maximum load with lower elongation and stiffness compared to other biomechanical studies described in the current literature, which dealt with different MPFL reconstructions with autologous tendon grafts. Therefore an implant-free MPFL reconstruction with transpatellar parallel tunnels using a 2 mm fiber tape (Arthrex) or a 4 mm tape (Topester) could be a good alternative for surgical treatment of patellofemoral instability. KW - Patellaluxation KW - Patellar instability KW - MPFL reconstruction KW - FiberTape KW - Nonresorbable suture tape KW - Osseuous fixation KW - Patellainstabilität KW - MPFL Ersatz KW - FiberTape KW - Synthetisches Bandersatzmaterial KW - Knöcherne Fixation KW - Kniescheibenverrenkung KW - Synthetischer Bandersatz Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-361396 ER - TY - THES A1 - Schwebs, Marie T1 - Structure and dynamics of the plasma membrane: a single-molecule study in \(Trypanosoma\) \(brucei\) T1 - Die Struktur und Dynamik der Plasmamembran: eine Einzelmolekülstudie in \(Trypanosoma\) \(brucei\) N2 - The unicellular, flagellated parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and nagana in livestock. In the last decades, it has become an established eukaryotic model organism in the field of biology, as well as in the interdisciplinary field of biophysics. For instance, the dense variant surface glycoprotein (VSG) coat offers the possibility to study the dynamics of GPI-anchored proteins in the plasma membrane of living cells. The fluidity of the VSG coat is not only an interesting object of study for its own sake, but is critically important for the survival of the parasite in the mammalian host. In order to maintain the integrity of the coat, the entire VSG coat is recycled within a few minutes. This is surprisingly fast for a purely diffusive process with the flagellar pocket (FP) as the sole site for endo- and exocytosis. Previous studies characterising VSG dynamics using FRAP reported diffusion coefficients that were not sufficient to to enable fast turnover based on passive VSG randomisation on the trypanosome surface. In this thesis, live-cell single-molecule fluorescence microscopy (SMFM) was employed to elucidate whether VSG diffusion coefficients were priorly underestimated or whether directed forces could be involved to bias VSGs towards the entrance of the FP. Embedding the highly motile trypanosomes in thermo-stable hydrogels facilitated the investigation of VSG dynamics on living trypanosomes at the mammalian host's temperature of 37°C. To allow for a spatial correlation of the VSG dynamics to the FP entrance, a cell line was employed harbouring a fluorescently labelled structure as a reference. Sequential two-colour SMFM was then established to allow for recording and registration of the dynamic and static single-molecule information. In order to characterise VSG dynamics, an algorithm to obtain reliable information from short trajectories was adapted (shortTrAn). It allowed for the quantification of the local dynamics in two distinct scenarios: diffusion and directed motion. The adaptation of the algorithm to the VSG data sets required the introduction of an additional projection filter. The algorithm was further extended to take into account the localisation errors inherent to single-particle tracking. The results of the quantification of diffusion and directed motion were presented in maps of the trypanosome surface, including an outline generated from a super-resolved static structure as a reference. Information on diffusion was displayed in one map, an ellipse plot. The colour code represented the local diffusion coefficient, while the shape of the ellipses provided an indication of the diffusion behaviour (aniso- or isotropic diffusion). The eccentricity of the ellipses was used to quantify deviations from isotropic diffusion. Information on directed motion was shown in three maps: A velocity map, representing the amplitude of the local velocities in a colour code. A quiver plot, illustrating the orientation of directed motion, and a third map which indicated the relative standard error of the local velocities colour-coded. Finally, a guideline based on random walk simulations was used to identify which of the two motion scenarios dominated locally. Application of the guideline to the VSG dynamics analysed by shortTrAn yielded supermaps that showed the locally dominant motion mode colour-coded. I found that VSG dynamics are dominated by diffusion, but several times faster than previously determined. The diffusion behaviour was additionally characterised by spatial heterogeneity. Moreover, isolated regions exhibiting the characteristics of round and elongated traps were observed on the cell surface. Additionally, VSG dynamics were studied with respect to the entrance of the FP. VSG dynamics in this region displayed similar characteristics compared to the remainder of the cell surface and forces biasing VSGs into the FP were not found. Furthermore, I investigated a potential interference of the attachment of the cytoskeleton to the plasma membrane with the dynamics of VSGs which are anchored to the outer leaflet of the membrane. Preliminary experiments were conducted on osmotically swollen trypanosomes and trypanosomes depleted for a microtubule-associated protein anchoring the subpellicular microtubule cytoskeleton to the plasma membrane. The measurements revealed a trend that detachment of the cytoskeleton could be associated with a reduction in the VSG diffusion coefficient and a loss of elongated traps. The latter could be an indication that these isolated regions were caused by underlying structures associated with the cytoskeleton. The measurements on cells with an intact cytoskeleton were complemented by random walk simulations of VSG dynamics with the newly determined diffusion coefficient on long time scales not accessible in experiments. Simulations showed that passive VSG randomisation is fast enough to allow for a turnover of the full VSG coat within a few minutes. According to an estimate based on the known rate of endocytosis and the newly determined VSG diffusion coefficient, the majority of exocytosed VSGs could escape from the FP to the cell surface without being immediately re-endocytosed. N2 - Der einzellige, begeißelte Parasit Trypanosoma brucei ist der Erreger der humanen Afrikanischen Schlafkrankheit und Nagana bei Nutztieren. In den vergangenen Jahrzehnten hat er sich sowohl in der Biologie als auch im interdisziplinären Bereich der Biophysik als eukaryotischer Modellorganismus etabliert. So bietet der dichte variant surface glycoprotein (VSG) Mantel beispielsweise die Möglichkeit, die Dynamik von GPI-verankerten Proteinen in der Plasmamembran von lebenden Zellen zu untersuchen. Die Fluidität des VSG-Mantels ist nicht nur um ihrer selbst Willen ein interessantes Studienobjekt, sondern auch von entscheidender Bedeutung für das Überleben des Parasiten im Säugetierwirt. Damit die Integrität des Mantels erhalten bleibt, wird der gesamte VSG Mantel kontinuierlich innerhalb weniger Minuten ausgetauscht. Dies ist erstaunlich schnell für einen rein diffusiven Prozess, bei welchem die Geißeltasche (GT) der einzige Ort für Endo- und Exozytose ist. Bisherige Studien zur Charakterisierung der VSG Dynamik mit FRAP ermittelten Diffusionskoeffizienten, welche nicht ausreichten, um einen schnellen Austausch durch eine passive Randomisierung der VSG auf der Trypanosomenoberfläche zu ermöglichen. In dieser Arbeit wurde die Einzelmolekül-Fluoreszenzmikroskopie (EMFM) an lebenden Zellen eingesetzt, um herauszufinden, ob die VSG Diffusionskoeffizienten zuvor unterschätzt wurden oder ob gerichtete Kräfte beteiligt sein könnten, um VSGs zum Eingang der GT zu leiten. Die Einbettung der hochmotilen Trypanosomen in thermostabilen Hydrogelen erlaubte die Analyse der VSG Dynamik auf lebenden Trypanosomen bei einer Temperatur des Säugetierwirts von 37°C. Um eine räumliche Korrelation der VSG Dynamik mit dem Eingang zur GT zu ermöglichen, wurde eine Zelllinie verwendet, die eine fluoreszenzmarkierte Struktur als Referenz besaß. Anschließend wurde die sequenzielle EMFM in zwei Farben etabliert, um sowohl die Aufzeichnung als auch die Registrierung der dynamischen und statischen Einzelmolekülinformationen zu gewährleisten. Um die VSG Dynamik zu charakterisieren, wurde ein Algorithmus zur Gewinnung von zuverlässigen Informationen aus kurzen Trajektorien adaptiert (shortTrAn). Dieser ließ die Quantifizierung der lokalen Dynamik anhand zweier unterschiedlicher Szenarien zu: Diffusion und gerichtete Bewegung. Die Anpassung des Algorithmus an die VSG Datensätze erforderte die Einführung eines zusätzlichen Projektionsfilters. Darüber hinaus wurde der Algorithmus erweitert, um die Lokalisierungsfehler zu berücksichtigen, die bei der Verfolgung von Einzelpartikeln unvermeidbar auftreten. Anschließend wurden die Ergebnisse der Quantifizierung von Diffusion und gerichteter Bewegung in Karten präsentiert, die die Trypanosomenoberfläche abbildeten, einschließlich eines Umrisses, der als Referenz aus einer hochaufgelösten statischen Struktur generiert wurde. Die Informationen zur Diffusion wurden in einer Karte, einem Ellipsenplot, dargestellt. Dabei repräsentierte eine Farbkodierung die lokalen Diffusionskoeffizienten, während die Form der Ellipsen einen Hinweis auf das Diffusionsverhalten (aniso- oder isotrope Diffusion) gab. Die Exzentrizität der Ellipsen wurde hierbei genutzt, um die Abweichung von isotroper Diffusion zu quantifizieren. Die Informationen zur gerichteten Bewegung wurden in drei Karten wiedergegeben: Eine Karte für die Geschwindigkeit zeigte die Amplitude der lokalen Geschwindigkeiten farbkodiert. Ein Köcherplot veranschaulichte die Richtung der Geschwindigkeit und eine dritte Karte zeigte den relativen Standardfehler der lokalen Geschwindigkeiten farblich kodiert an. Abschließend wurde ein auf Random-Walk-Simulationen basierender Leitfaden herangezogen, um zu entscheiden, welches der beiden Szenarien lokal dominierte. Die Anwendung des Leitfadens auf die mit shortTrAn analysierte VSG Dynamik ergab Übersichtskarten, in denen der lokal dominierende Bewegungsmodus farblich kodiert war. Ich konnte zeigen, dass die VSG Dynamik von der Diffusion dominiert wird. Jedoch war diese um ein Vielfaches schneller als bisher angenommen. Das Diffusionsverhalten war zudem durch eine räumliche Heterogenität charakterisiert. Des Weiteren wurden auf der Zelloberfläche isolierte Regionen beobachtet, die die Eigenschaften von runden und länglichen Fallen aufwiesen. Zusätzlich wurde die VSG Dynamik in Bezug auf den Eingang der GT untersucht. Die VSG Dynamik in dieser Region wies ähnliche Kennwerte auf wie die restliche Zelloberfläche, und es konnten keine Kräfte festgestellt werden, welche die VSGs in die GT dirigieren. Des Weiteren habe ich den potenziellen Einfluss der Verankerung des Zytoskeletts an der Plasmamembran auf die Dynamik der VSGs untersucht, die in der äußeren Membranschicht verankert sind. Hierzu wurden vorläufige Experimente auf osmotisch geschwollenen Trypanosomen und Trypanosomen durchgeführt, denen ein Mikrotubuli assoziiertes Protein fehlte, welches das subpellikuläre Mikrotubuli-Zytoskelett an der Plasmamembran verankert. Bei den Messungen wurde ein Trend festgestellt, wonach die Ablösung des Zytoskeletts mit einer Verringerung des VSG Diffusionskoeffizienten und dem Verlust der länglichen Fallen korrelieren könnte. Letzteres könnte ein Hinweis darauf sein, dass diese isolierten Regionen durch darunter liegende, mit dem Zytoskelett verbundene Strukturen verursacht wurden. Die Messungen auf Zellen mit intaktem Zytoskelett wurden durch Random-Walk-Simulationen von VSG Trajektorien mit dem neu ermittelten Diffusionskoeffizienten auf langen, experimentell nicht zugänglichen Zeitskalen ergänzt. Die Simulationen zeigten, dass die passive Randomisierung der VSGs schnell genug ist, um einen Austausch des gesamten VSG Mantels innerhalb weniger Minuten zu ermöglichen. Einer Schätzung zufolge, die auf der bekannten Endozytoserate und dem neu ermittelten VSG Diffusionskoeffizienten basierte, könnte der Großteil der exozytierten VSGs aus der GT zur Zelloberfläche gelangen, ohne unmittelbar wieder endozytiert zu werden. KW - Trypanosoma brucei KW - Einzelmolekülmikroskopie KW - Membranproteine KW - Diffusionskoeffizient KW - Single-molecule fluorescence microscopy KW - Single-molecule tracking KW - Variant surface glycoprotein KW - GPI-anchored protein KW - Diffusion coefficient KW - Zellskelett KW - Zytoskelett Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275699 ER - TY - THES A1 - Iosip, Anda-Larisa T1 - Molecular Mechanosensing Mechanisms of the Carnivorous Plant \(Dionaea\) \(muscipula\) T1 - Molekulare Mechanismen der Mechanoperzeption in der fleischfressenden Pflanze \(Dionaea\) \(muscipula\) N2 - Plants are able to sense mechanical forces in order to defend themselves against predators, for instance by synthesizing repellent compounds. Very few plants evolved extremely sensitive tactile abilities that allow them to perceive, interpret and respond by rapid movement in the milliseconds range. One such rarity is the charismatic Venus flytrap (Dionaea muscipula) - a carnivorous plant which relies on its spectacular active trapping strategy to catch its prey. The snapping traps are equipped with touch-specialised trigger hairs, that upon bending elicit an action potential (AP). This electrical signal originates within the trigger hairs’ mechanosensory cells and further propagates throughout the whole trap, alerting the plant of potential prey. Two APs triggered within thirty seconds will set off the trap and more than five APs will initiate the green stomach formation for prey decomposition and nutrient uptake. Neither the molecular components of the plant’s AP nor the Venus flytrap’s fast closure mechanism have been fully elucidated yet. Therefore, the general objective of this study is to expound on the molecular basis of touch perception: from AP initiation to trap closure and finally to stomach formation. The typical electrical signal in plants lasts for minutes and its shape is determined by the intensity of the mechanical force applied. In contrast, the Venus flytrap’s one-second AP is of all-or-nothing type, similar in shape to the animal AP. In order to gain more insight into the molecular components that give rise to the Venus flytrap’s emblematic AP, the transcriptomic landscape of its unique mechanotransducer - the trigger hair – was compared to the rest of the non-specialised tissues and organs. Additionally, the transcriptome of the electrically excitable fully-developed adult trap was compared to non-excitable juvenile traps that are unable to produce sharp APs. Together, the two strategies helped with the identification of electrogenic channels and pumps for each step of the AP as follows: (1) the most specific to the trigger hair was the mechanosensitive channel DmMSL10, making up the best candidate for the initial AP depolarization phase, (2) the K+ outward rectifier DmSKOR could be responsible for repolarisation, (3) further, the proton pump DmAHA4, might kick in during repolarisation and go on with hyperpolarisation and (4) the hyperpolarization- and acid-activated K+ inward rectifier KDM1 might contribute to the re-establishment of electrochemical gradient and the resting potential. Responsible for the AP-associated Ca2+ wave and electrical signal propagation, the glutamate-like receptor DmGLR3.6 was also enriched in the trigger hairs. Together, these findings suggest that the reuse of genes involved in electrical signalling in ordinary plants can give rise to the Venus flytrap’s trademark AP. The Venus flytrap has been cultivated ever since its discovery, generating more than one hundred cultivars over the years. Among them, indistinguishable from a normal Venus flytrap at first sight, the ’ERROR’ cultivar exhibits a peculiar behaviour: it is unable to snap its traps upon two APs. Nevertheless, it is still able to elicit normal APs. To get a better understanding of the key molecular mechanisms and pathways that are essential for a successful trap closure, the ’ERROR’ mutant was compared to the functional wild type. Timelapse photography led to the observation that the ’ERROR’ mutants were able to leisurely half close their traps when repeated mechanostimulation was applied (10 minutes after 20 APs, 0.03 Hz). As a result of touch or wounding in non-carnivorous plants, jasmonic acid (JA) is synthesized, alerting the plants of potential predators. Curiously, the JA levels were reduced upon mechanostimulation and completely impaired upon wounding in the ’ERROR’ mutant. In search of genes accountable for the ’ERROR’ mutant’s defects, the transcriptomes of the two phenotypes were compared before and after mechanostimulation (1h after 10 APs, 0.01 Hz). The overall dampened response of the mutant compared to the wild type, was reflected at transcriptomic level as well. Only about 50% of wild type’s upregulated genes after touch stimulation were differentially expressed in ’ERROR’ and they manifested only half of the wild type’s expression amplitude. Among unresponsive functional categories of genes in ’ERROR’ phenotype, there were: cell wall integrity surveilling system, auxin biosynthesis and stress-related transcription factors from the ethylene-responsive AP2/ERF and C2H2-ZF families. Deregulated Ca2+-decoding as well as redox-related elements together with JA-pathway components might also contribute to the malfunctioning of the ’ERROR’ mutant. As the mutant does not undergo full stomach formation after mechanical treatment, these missing processes represent key milestones that might mediate growth-defence trade-offs under JA signalling. This confirms the idea that carnivory has evolved by recycling the already available molecular machineries of the ubiquitous plant immune system. To better understand the mutant’s defect in the trap snapping mechanism, the ground states (unstimulated traps) of the two phenotypes were compared. In this case, many cell wall-related genes (e.g. expansins) were downregulated in the ’ERROR’ mutant. For the first time, these data point to the importance of a special cell wall architecture of the trap, that might confer the mechanical properties needed for a functional buckling system - which amplifies the speed of the trap closure. This study provides candidate channels for each of the AP phases that give rise to and shape the sharp Venus flytrap-specific AP. It further underlines the possible contribution of the cell wall architecture to the metastable ready-to-snap configuration of the trap before stimulation - which might be crucial for the buckling-dependent snapping. And finally, it highlights molecular milestones linked to defence responses that ensure trap morphing into a green stomach after mechanostimulation. Altogether, these processes prove to be interdependent and essential for a successful carnivorous lifestyle. N2 - Pflanzen sind in der Lage, mechanische Einflüsse zu spüren, um sich gegen Fressfeinde zu verteidigen, indem sie zum Beispiel abweisende Verbindungen synthetisieren. Nur sehr wenige Pflanzen haben extrem sensible taktile Fähigkeiten entwickelt, die es ihnen ermöglichen, schnelle Bewegungen im Millisekundenbereich wahrzunehmen, zu interpretieren und darauf zu reagieren. Eine solche Rarität ist die charismatische Venusfliegenfalle (Dionaea muscipula) - eine fleischfressende Pflanze, die sich auf ihre spektakuläre aktive Fallenstrategie verlässt, um ihre Beute zu fangen. Die Schnappfallen sind mit berührungssensitiven Auslösehaaren ausgestattet, die beim Biegen ein Aktionspotenzial (AP) auslösen. Dieses elektrische Signal entsteht in den mechanosensorischen Zellen der Auslösehaare und breitet sich in der gesamten Falle aus, wodurch die Pflanze auf potenzielle Beute aufmerksam gemacht wird. Zwei APs, die innerhalb von dreißig Sekunden ausgelöst werden, lösen die Falle aus, und mehr als fünf APs leiten die Bildung des grünen Magens ein, der die Beute zersetzt und die Nährstoffe aufnimmt. Weder die molekularen Komponenten des AP der Pflanze noch der Schnellverschlussmechanismus der Venusfliegenfalle sind bisher vollständig geklärt. Daher besteht das allgemeine Ziel dieser Studie darin, die molekularen Grundlagen der Berührungswahrnehmung zu erforschen: von der Initiierung des AP bis zum Schließen der Falle und schließlich zur Magenbildung. Das typische elektrische Signal in Pflanzen dauert Minuten und seine Form wird durch die Intensität der angewandten mechanischen Kraft bestimmt. Im Gegensatz dazu ist das einsekündige AP der Venusfliegenfalle vom Alles-oder-Nichts-Typ und ähnelt in seiner Form dem tierischen AP. Um mehr Einblick in die molekularen Komponenten zu erhalten, die das emblematische AP der Venusfliegenfalle hervorbringen, wurde das Transkriptom ihres einzigartigen Mechanosensors - des Triggerhaars - mit den übrigen nicht spezialisierten Geweben und Organen verglichen. Darüber hinaus wurde das Transkriptom der elektrisch erregbaren, voll entwickelten adulten Falle mit nicht erregbaren juvenilen Fallen verglichen, die keine scharfen APs erzeugen können. Beide Strategien zusammen halfen bei der Identifizierung von elektrogenen Kanälen und Pumpen für jeden Schritt des AP: (1) Am spezifischsten für die Triggerhaare war der mechanosensitive Kanal DmMSL10, der der beste Kandidat für die anfängliche AP-Depolarisationsphase war, (2) der K+-Auswärtsgleichrichter DmSKOR könnte für die Repolarisation verantwortlich sein, (3) ferner, die H+-Pumpe DmAHA4, könnte während der Repolarisation einsetzen und mit der Hyperpolarisation fortfahren und (4) der durch Hyperpolarisation und Säure aktivierte K+-Einwärtsgleichrichter KDM1 könnte zur Wiederherstellung des elektrochemischen Gradienten und des Ruhepotentials beitragen. Der möglicherweise für die AP-assoziierte Ca2+-Welle und die elektrische Signalausbreitung verantwortliche Glutamatrezeptor DmGLR3.6 war ebenfalls in den Triggerhaaren angereichert. Zusammengenommen deuten diese Ergebnisse darauf hin, dass die Wiederverwendung von Genen, die an der elektrischen Signalübertragung in gewöhnlichen Pflanzen beteiligt sind, zu dem für die Venusfliegenfalle typischen AP führen kann. Die Venusfliegenfalle wird seit ihrer Entdeckung kultiviert und hat im Laufe der Jahre mehr als hundert Kultivare hervorgebracht. Die Sorte "ERROR", die auf den ersten Blick nicht von einer normalen Venusfliegenfalle zu unterscheiden ist, weist ein besonderes Verhalten auf: Sie ist nicht in der Lage, ihre Fallen nach dem Auslösen von 2 APs zu schließen. Dennoch ist sie in der Lage, normale APs auszulösen. Um ein besseres Verständnis der molekularen Schlüsselmechanismen und -wege zu erhalten, die für ein erfolgreiches Schließen der Fallen notwendig sind, wurde die "ERROR"-Mutante mit dem funktionalen Wildtyp verglichen. Zeitrafferaufnahmen führten zu der Beobachtung, dass die ’ERROR’-Mutanten in der Lage waren, ihre Fallen bei wiederholter mechanischer Stimulation (10 Minuten nach 20 APs, 0,03 Hz) sehr langsam etwa zur Hälfte zu schließen. Bei nicht karnivoren Pflanzen wird infolge von Berührungen oder Verletzungen Jasmonsäure (JA) synthetisiert, die die Pflanzen vor potenziellen Fressfeinden warnt. Merkwürdigerweise waren die JA-Spiegel bei mechanischer Stimulation reduziert und bei Verwundung in der "ERROR"-Mutante im Gegensatz zum WT überhaupt nicht erhöht. Auf der Suche nach Genen, die für die Defekte der "ERROR"-Mutante verantwortlich sind, wurden die Transkriptome der beiden Phänotypen vor und nach der Mechanostimulation (1 Stunde nach 10 APs, 0,01 Hz) verglichen. Die insgesamt gedämpfte Reaktion der Mutante im Vergleich zum Wildtyp spiegelte sich auch auf transkriptomischer Ebene wider. Nur etwa 50 % der nach Berührungsstimulation hochregulierten Gene des Wildtyps wurden in "ERROR" unterschiedlich exprimiert, und sie wiesen nur die Hälfte der Expressionsamplitude des Wildtyps auf. Zu den nicht reagierenden funktionellen Genkategorien gehörten: das System zur Überwachung der Zellwandintegrität, die Auxin-Biosynthese und stressbezogene Transkriptionsfaktoren aus den auf Ethylen reagierenden AP2/ERF- und C2H2-ZF-Familien. Deregulierte Ca2+-decodierende sowie redoxbezogene Elemente könnten zusammen mit Komponenten des JA-Signalwegs ebenfalls zur Fehlfunktion der "ERROR"-Mutante beitragen. Da die Mutante nach mechanischer Behandlung keine vollständige Magenbildung durchläuft, stellen diese fehlenden Prozesse wichtige Meilensteine dar, die bei der JA-Signalübertragung einen Kompromiss zwischen Wachstum und Verteidigung vermitteln könnten. Dies bestätigt die Idee, dass sich Karnivorie durch die Wiederverwertung bereits vorhandener Signalwege und -komponenten entwickelt hat. Um den Defekt der Mutante im Fallenschnappmechanismus besser zu verstehen, wurden die Grundzustände (unstimulierte Fallen) der beiden Phänotypen verglichen. In diesem Fall waren viele zellwandbezogene Gene (z. B. Expansine) in der "ERROR"-Mutante herunterreguliert. Diese Daten weisen zum ersten Mal auf die Bedeutung einer speziellen Zellwandarchitektur der Falle hin, die möglicherweise die mechanischen Eigenschaften für ein Umklappen der Fallenhälften verleiht, was wiederum die Geschwindigkeit des Fallenschlusses erhöht. Diese Studie liefert Kandidatenkanäle für jede der AP-Phasen, die das scharfe Venusfliegenfallen-spezifische AP hervorbringen und formen. Sie unterstreicht außerdem den möglichen Beitrag der Zellwandarchitektur zur metastabilen, schnappbereiten Konfiguration der Falle vor der Stimulation - die für das durch das Umklappen der Fallenhälften bedingte Zuschnappen der Falle entscheidend sein könnte. Und schließlich werden molekulare Meilensteine hervorgehoben, die mit Abwehrreaktionen verbunden sind und dafür sorgen, dass sich die Falle nach mechanischer Stimulation in einen grünen Magen verwandelt. Insgesamt erweisen sich diese Prozesse als voneinander abhängig und wesentlich für eine erfolgreiche fleischfressende Lebens-weise. KW - carnivorous plants KW - action potential KW - trap closure KW - jasmonic acid KW - mechanosensation KW - touch KW - molecular pathways KW - wounding KW - defence mechanisms KW - transcriptomics KW - Venusfliegenfalle KW - Dionaea muscipula Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-287649 ER - TY - JOUR A1 - Prusty, Bhupesh K. A1 - Gulve, Nitish A1 - Govind, Sheila A1 - Krueger, Gerhard R. F. A1 - Feichtinger, Julia A1 - Larcombe, Lee A1 - Aspinall, Richard A1 - Ablashi, Dharam V. A1 - Toro, Carla T. T1 - Active HHV-6 Infection of Cerebellar Purkinje Cells in Mood Disorders JF - Frontiers in Microbiology N2 - Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD. KW - HHV-6 KW - bipolar disorder KW - schizophrenia KW - major depressive disorder KW - Purkinje cells Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369222 VL - 9 ER - TY - JOUR A1 - Ticha, Olga A1 - Moos, Lukas A1 - Wajant, Harald A1 - Bekeredjian-Ding, Isabelle T1 - Expression of Tumor Necrosis Factor Receptor 2 Characterizes TLR9-Driven Formation of Interleukin-10-Producing B Cells JF - Frontiers in Immunology N2 - B cell-derived interleukin-10 (IL-10) production has been described as a hallmark for regulatory function in B lymphocytes. However, there is an ongoing debate on the origin of IL-10-secreting B cells and lack of specific surface markers has turned into an important obstacle for studying human B regulatory cells. In this study, we propose that tumor necrosis factor receptor 2 (TNFR2) expression can be used for enrichment of IL-10-secreting B cells. Our data confirm that IL-10 production can be induced by TLR9 stimulation with CpG ODN and that IL-10 secretion accompanies differentiation of peripheral blood B cells into plasma blasts. We further show that CpG ODN stimulation induces TNFR2 expression, which correlates with IL-10 secretion and terminal differentiation. Indeed, flow cytometric sorting of TNFR2+ B cells revealed that TNFR2+ and TNFR2− fractions correspond to IL-10+ and IL-10− fractions, respectively. Furthermore, CpG-induced TNFR2+ B cells were predominantly found in the IgM+ CD27+ B cell subset and spontaneously released immunoglobulin. Finally, our data corroborate the functional impact of TNFR2 by demonstrating that stimulation with a TNFR2 agonist significantly augments IL-10 and IL-6 production in B cells. Altogether, our data highlight a new role for TNFR2 in IL-10-secreting human B lymphocytes along with the potential to exploit this finding for sorting and isolation of this currently ill-defined B cell subset. KW - human KW - B cells KW - interleukin-10 KW - tumor necrosis factor receptor 2 KW - TLR 9 KW - Breg Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241323 VL - 8 ER - TY - JOUR A1 - Klotz, Peter A1 - Higgins, Paul G. A1 - Schaubmar, Andreas R. A1 - Failing, Klaus A1 - Leidner, Ursula A1 - Seifert, Harald A1 - Scheufen, Sandra A1 - Semmler, Torsten A1 - Ewers, Christa T1 - Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany JF - Frontiers in Microbiology N2 - Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans. KW - ESKAPE KW - Acinetobacter baumannii KW - antimicrobial susceptibility KW - MLST KW - cattle KW - epidemiology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325927 VL - 10 ER - TY - JOUR A1 - Schroeter, Matthias L. A1 - Pawelke, Sarah A1 - Bisenius, Sandrine A1 - Kynast, Jana A1 - Schuemberg, Katharina A1 - Polyakova, Maryna A1 - Anderl-Straub, Sarah A1 - Danek, Adrian A1 - Fassbender, Klaus A1 - Jahn, Holger A1 - Jessen, Frank A1 - Kornhuber, Johannes A1 - Lauer, Martin A1 - Prudlo, Johannes A1 - Schneider, Anja A1 - Uttner, Ingo A1 - Thöne-Otto, Angelika A1 - Otto, Markus A1 - Diehl-Schmid, Janine T1 - A Modified Reading the Mind in the Eyes Test Predicts Behavioral Variant Frontotemporal Dementia Better Than Executive Function Tests JF - Frontiers in Aging Neuroscience N2 - Behavioral variant frontotemporal dementia (bvFTD) is characterized by deep alterations in behavior and personality. Although revised diagnostic criteria agree for executive dysfunction as most characteristic, impairments in social cognition are also suggested. The study aimed at identifying those neuropsychological and behavioral parameters best discriminating between bvFTD and healthy controls. Eighty six patients were diagnosed with possible or probable bvFTD according to Rascovsky et al. (2011) and compared with 43 healthy age-matched controls. Neuropsychological performance was assessed with a modified Reading the Mind in the Eyes Test (RMET), Stroop task, Trail Making Test (TMT), Hamasch-Five-Point Test (H5PT), and semantic and phonemic verbal fluency tasks. Behavior was assessed with the Apathy Evaluation Scale, Frontal Systems Behavioral Scale, and Bayer Activities of Daily Living Scale. Each test’s discriminatory power was investigated by Receiver Operating Characteristic curves calculating the area under the curve (AUC). bvFTD patients performed significantly worse than healthy controls in all neuropsychological tests. Discriminatory power (AUC) was highest in behavioral questionnaires, high in verbal fluency tasks and the RMET, and lower in executive function tests such as the Stroop task, TMT and H5PT. As fluency tasks depend on several cognitive functions, not only executive functions, results suggest that the RMET discriminated better between bvFTD and control subjects than other executive tests. Social cognition should be incorporated into diagnostic criteria for bvFTD in the future, such as in the International Classification of Diseases (ICD)-11, as already suggested in the Diagnostic and Statistical Manual for Mental Disorders (DSM)-5. KW - behavioral variant frontotemporal dementia KW - diagnostic criteria KW - executive function KW - social cognition KW - theory of mind Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234254 VL - 10 ER - TY - JOUR A1 - Vaahtoranta, Enni A1 - Lenhart, Jan A1 - Suggate, Sebastian A1 - Lenhard, Wolfgang T1 - Interactive Elaborative Storytelling: Engaging Children as Storytellers to Foster Vocabulary JF - Frontiers in Psychology N2 - Positive effects of shared reading for children’s language development are boosted by including instruction of word meanings and by increasing interactivity. The effects of engaging children as storytellers on vocabulary development have been less well studied. We developed an approach termed Interactive Elaborative Storytelling (IES), which employs both word-learning techniques and children’s storytelling in a shared-reading setting. To systematically investigate potential benefits of children as storytellers, we contrasted this approach to two experimental groups, an Elaborative Storytelling group employing word-learning techniques but no storytelling by children and a Read-Aloud group, excluding any additional techniques. The study was a 3 × 2 pre-posttest randomized design with 126 preschoolers spanning 1 week. Measured outcomes were receptive and expressive target vocabulary, story memory, and children’s behavior during story sessions. All three experimental groups made comparable gains on target words from pre- to posttest and there was no difference between groups in story memory. However, in the Elaborative Storytelling group, children were the least restless. Findings are discussed in terms of their contribution to optimizing shared reading as a method of fostering language. KW - storytelling KW - shared reading KW - language intervention KW - preschool KW - language development Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232136 VL - 10 ER - TY - JOUR A1 - Lang, Isabell A1 - Füllsack, Simone A1 - Wajant, Harald T1 - Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies JF - Frontiers in Immunology N2 - Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF. KW - binding studies KW - Gaussia princeps luciferase fusion protein KW - progranulin KW - tumor necrosis factor KW - tumor necrosis factor receptor-1 KW - tumor necrosis factor receptor-2 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236373 VL - 9 ER - TY - JOUR A1 - Weiss, Esther A1 - Ziegler, Sabrina A1 - Fliesser, Mirjam A1 - Schmitt, Anna-Lena A1 - Hünniger, Kerstin A1 - Kurzai, Oliver A1 - Morton, Charles-Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - First Insights in NK—DC Cross-Talk and the Importance of Soluble Factors During Infection With Aspergillus fumigatus JF - Frontiers in Cellular and Infection Microbiology N2 - Invasive aspergillosis (IA) is an infectious disease caused by the fungal pathogen Aspergillus fumigatus that mainly affects immunocompromised hosts. To investigate immune cell cross-talk during infection with A. fumigatus, we co-cultured natural killer (NK) cells and dendritic cells (DC) after stimulation with whole fungal structures, components of the fungal cell wall, fungal lysate or ligands for distinct fungal receptors. Both cell types showed activation after stimulation with fungal components and were able to transfer activation signals to the counterpart not stimulated cell type. Interestingly, DCs recognized a broader spectrum of fungal components and thereby initiated NK cell activation when those did not recognize fungal structures. These experiments highlighted the supportive function of DCs in NK cell activation. Furthermore, we focused on soluble DC mediated NK cell activation and showed that DCs stimulated with the TLR2/Dectin-1 ligand zymosan could maximally stimulate the expression of CD69 on NK cells. Thus, we investigated the influence of both receptors for zymosan, Dectin-1 and TLR2, which are highly expressed on DCs but show only minimal expression on NK cells. Specific focus was laid on the question whether Dectin-1 or TLR2 signaling in DCs is important for the secretion of soluble factors leading to NK cell activation. Our results show that Dectin-1 and TLR2 are negligible for NK cell activation. We conclude that besides Dectin-1 and TLR2 other receptors on DCs are able to compensate for the missing signal. KW - natural killer cells KW - dendritic cells KW - NK-DC cross-talk KW - Aspergillus fumigatus KW - soluble factors KW - innate immunity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233565 VL - 8 ER -