TY - JOUR A1 - Engstler, Markus A1 - Beneke, Tom T1 - Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit JF - eLife N2 - CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries. KW - CRISPR/Cas9 KW - Leishmania KW - cytosine base editor (CBE) toolbox KW - gene editing KW - scalable functional genomic screening KW - LeishBASEedit Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350002 VL - 12 ER - TY - JOUR A1 - Salihoglu, Rana A1 - Srivastava, Mugdha A1 - Liang, Chunguang A1 - Schilling, Klaus A1 - Szalay, Aladar A1 - Bencurova, Elena A1 - Dandekar, Thomas T1 - PRO-Simat: Protein network simulation and design tool JF - Computational and Structural Biotechnology Journal N2 - PRO-Simat is a simulation tool for analysing protein interaction networks, their dynamic change and pathway engineering. It provides GO enrichment, KEGG pathway analyses, and network visualisation from an integrated database of more than 8 million protein-protein interactions across 32 model organisms and the human proteome. We integrated dynamical network simulation using the Jimena framework, which quickly and efficiently simulates Boolean genetic regulatory networks. It enables simulation outputs with in-depth analysis of the type, strength, duration and pathway of the protein interactions on the website. Furthermore, the user can efficiently edit and analyse the effect of network modifications and engineering experiments. In case studies, applications of PRO-Simat are demonstrated: (i) understanding mutually exclusive differentiation pathways in Bacillus subtilis, (ii) making Vaccinia virus oncolytic by switching on its viral replication mainly in cancer cells and triggering cancer cell apoptosis and (iii) optogenetic control of nucleotide processing protein networks to operate DNA storage. Multilevel communication between components is critical for efficient network switching, as demonstrated by a general census on prokaryotic and eukaryotic networks and comparing design with synthetic networks using PRO-Simat. The tool is available at https://prosimat.heinzelab.de/ as a web-based query server. KW - network simulation KW - protein analysis KW - signalling pathways KW - dynamic protein-protein interactions KW - optogenetics KW - oncolytic virus KW - DNA storage Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350034 SN - 2001-0370 VL - 21 ER - TY - JOUR A1 - Conrad, David A1 - Kehl, Alexandra A1 - Müller, Tobias A1 - Klopfleisch, Robert A1 - Aupperle-Lellbach, Heike T1 - Immunohistochemical and molecular genetic analysis of canine digital mast cell tumours JF - Animals N2 - Grading, immunohistochemistry and c-kit mutation status are criteria for assessing the prognosis and therapeutic options of canine cutaneous mast cell tumours (MCTs). As a subset, canine digital MCTs have rarely been explored in this context. Therefore, in this retrospective study, 68 paraffin-embedded canine digital MCTs were analysed, and histological grading was assessed according to Patnaik and Kiupel. The immunohistochemical markers KIT and Ki67 were used, as well as polymerase chain reaction (PCR) for mutational screening in c-kit exons 8, 9, 11 and 14. Patnaik grading resulted in 22.1% grade I, 67.6% grade II and 10.3% grade III tumours. Some 86.8% of the digital MCTs were Kiupel low-grade. Aberrant KIT staining patterns II and III were found in 58.8%, and a count of more than 23 Ki67-positive cells in 52.3% of the cases. Both parameters were significantly associated with an internal tandem duplication (ITD) in c-kit exon 11 (12.7%). French Bulldogs, which tend to form well-differentiated cutaneous MCTs, had a higher proportion of digital high-grade MCTs and ITD in c-kit exon 11 compared with mongrels. Due to its retrospective nature, this study did not allow for an analysis of survival data. Nevertheless, it may contribute to the targeted characterisation of digital MCTs. KW - dog KW - digit KW - toe KW - CD117 KW - Ki67 KW - KIT KW - grading KW - PCR KW - sequencing KW - c-kit Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319199 SN - 2076-2615 VL - 13 IS - 10 ER - TY - THES A1 - Nirchal, Naveen Kumar T1 - Mechanistische Regulierung des gastroösophagealen Übergangs und die Rolle der Retinsäure bei der Entwicklung des Barrett-Ösophagus T1 - Mechanistic regulation of gastroesophageal junction and role of retinoic acid in the development of Barrett's esophagus N2 - Der gastroösophageale Übergang (GEJ), der die Region abgrenzt, in der der distale Ösophagus auf die proximale Magenregion trifft, ist bekannt für die Entwicklung pathologischer Zustände, wie Metaplasie und Adenokarzinom des Ösophagus (EAC). Es ist wichtig, die Mechanismen der Entwicklungsstadien zu verstehen, die zu EAC führen, da die Inzidenzrate von EAC in den letzten 4 Jahrzehnten um das 7-fache gestiegen ist und die Gesamtüberlebensrate von 5 Jahren 18,4 % beträgt. In den meisten Fällenwird die Diagnose im fortgeschrittenen Stadium ohne vorherige Symptome erstellt. Der Hauptvorläufer für die Entwicklung von EAC ist eine prämaligne Vorstufe namens Barrett-Ösophagus (BE). BE ist der metaplastische Zustand, bei dem das mehrschichtige Plattenepithel des nativen Ösophagus durch ein spezialisiertes einschichtiges Säulenepithel ersetzt wird, das die molekularen Eigenschaften des Magen- sowie des Darmepithels aufweist. Zu den wichtigsten Risikofaktoren für die Entwicklung von BE gehören die chronische gastroösophageale Refluxkrankheit (GERD), eine veränderte Mikrobiota und veränderte Retinsäure-Signalwege (RA). Es ist unklar, welche Zelle der Ursprung für BE ist, da es keine eindeutigen Beweisen für den Prozess der BE-Initiation gibt. In dieser Arbeit habe ich untersucht, wie die GEJ-Homöostase in gesundem Gewebe durch stammzellregulatorische Morphogene aufrechterhalten wird, welche Rolle der Vitamin-A (RA-Signalübertragung) spieltund wie ihre Veränderung zur BE-Entwicklung beiträgt. Im ersten Teil meiner Dissertation habe ich anhand von Einzelmolekül-RNA in situ-Hybridisierung und Immunhistochemie eindeutig das Vorhandensein von zwei Arten von Epithelzellen nachweisen können, dem Plattenepithel in der Speiseröhre und dem Säulenepithel imMagenbereich des GEJ. Mittels Abstammungsanalysen im Mausmodell konnte ich zeigen, dass die Epithelzellen des Ösophagus und des Magens von zwei verschiedenen epithelialen Stammzelllinien imGEJ abstammen. Die Grenze zwischen Plattenepithel und Säulenepithelzellen im SCJ des GEJ wirddurch gegensätzliche Wnt-Mikroumgebungen streng reguliert. Plattenepithelstammzellen des Ösophagus werden durch das Wnt-hemmende Mikroumgebungssignal aufrechterhalten, während Magensäulenepithelzellen durch das Wnt-aktivierende Signal aus dem Stromakompartiment erhalten werden. Ich habe die in vivo Erhaltung der Epithelstammzellen des GEJ mit Hilfe eines in vitro Epithel-3D-Organoidkulturmodells rekonstruiert. Das Wachstum und die Vermehrung von Magensäulenepithel-Organoiden hängen von Wnt-Wachstumsfaktoren ab, während das Wachstum von Plattenepithel-Organoiden von Wnt-defizienten Kulturbedingungen abhängt. Darüber hinaus zeigte die Einzelzell-RNA-Sequenzanalyse (scRNA-seq) der aus Organoiden gewonnenenEpithelzellen, dass der nicht-kanonische Wnt/ planar cell polarity (PCP) Signalweg an der Regulierung der Plattenepithelzellen beteiligt ist. Im Gegensatz dazu werden säulenförmige Magenepithelzellen durch den kanonischen Wnt/beta-Catenin- und den nicht-kanonischen Wnt/Ca2+-Weg reguliert. Meine Daten zeigen, dass die SCJ-Epithelzellen, die am GEJ verschmelzen, durch entgegengesetzte stromale Wnt-Faktoren und unterschiedliche Wnt-Weg-Signalee in den Epithelzellen reguliert werden. Im zweiten Teil der Dissertation untersuchte ich die Rolle der bioaktiven Vitamin A Verbindung RA auf Ösophagus- und Magenepithelstammzellen. Die In-vitro-Behandlung von epithelialen Organoiden der Speiseröhre und des Magens mitRA oder seinem pharmakologischen Inhibitors BMS 493 zeigte, dass jeder Zelltyp unterschiedlich reguliert wurde. Ich beobachtete, dass eine verstärkte RA die Differenzierung von Stammzellen und den Verlust der Schichtung förderte, während die RA-Hemmung zu einer verstärkten Stammzellbildung und Regeneration im mehrschichtigen Epithel der Speiseröhre führte. Im Gegensatz zur Speiseröhre ist der RA-Signalweg in Magen-Organoiden aktiv, und die Hemmung von RA hat ein reduziertes Wachstum von Magen-Organoiden. Globale transkriptomische Daten und scRNA-seq-Daten zeigten, dass derRA-Signalweg einen Ruhephänotyp in den Ösophaguszellen induziert. Dagegen führt das Fehlen von RA in Magenepithelzellen zur Expression von Genen, die mit BE assoziiert sind. Daher isteine räumlich definierte Regulation der Wnt- und Retinsäure-Signalgebung amGEJ entscheidend für eine gesunde Homöostase, und ihre Störung führt zur Entwicklung von Krankheiten. N2 - Gastroesophageal junction (GEJ), demarcating the region where the distal esophagus meets with the proximal stomach region, is known for developing pathological conditions, including metaplasia and esophageal adenocarcinoma (EAC). It is essential to understand the mechanisms of developmental stages which lead to EAC since the incidence rate of EAC increased over 7-fold during the past four decades, and the overall five years survival rate is 18.4%. In most cases, patients are diagnosed in the advanced stage without prior symptoms. The main precursor for the development of EAC is a pre-malignant condition called Barrett's esophagus (BE). BE is the metaplastic condition where the multilayered squamous epithelium of the native esophagus is replaced by specialized single-layered columnar epithelium, which shows the molecular characteristics of the gastric as well as intestinal epithelium. The main risk factors for BE development include chronic gastro-esophageal acid reflux disease (GERD), altered microbiota, and altered retinoic acid signaling (RA). The cell of origin of BE is under debate due to a lack of clear evidence demonstrating the process of BE initiation. Here, I investigated how GEJ homeostasis is maintained in healthy tissue by stem cell regulatory morphogens, the role of vitamin A (RA signaling), and how its alteration contributes to BE development. In the first part of my thesis, I showed the presence of two types of epithelial cells, the squamous type in the esophagus and the columnar type in the stomach region in the GEJ, using single-molecule RNA in situ hybridization (smRNA-ISH) and immunohistochemistry. Employing lineage tracing in the mouse model, I have demonstrated that the esophageal epithelial and stomach epithelial cells derived from two distinct epithelial stem cell lineages in the GEJ. The border between squamous and columnar epithelial cells in the Squamo-columnar junction (SCJ) of GEJ is regulated by opposing Wnt microenvironments. The regeneration of stomach columnar epithelial stem cells is maintained by Wnt activating signal from the stromal compartment while squamous epithelial stem cells of the esophagus are maintained by the Wnt inhibitory signals. I recapitulated the in vivo GEJ epithelial stem cell maintenance by using in vitro epithelial 3D organoid culture model. The growth and propagation of stomach columnar epithelial organoids depend on Wnt growth factors, while squamous epithelial organoids' development needs Wnt-deficient culture conditions. Further, single-cell RNA sequence (scRNA-seq) analysis of organoid-derived epithelial cells revealed the non-canonical Wnt/ planar cell polarity (PCP) pathway involvement in regulating the squamous epithelial cells. In contrast, columnar stomach epithelial cells are regulated by the canonical Wnt/ beta-catenin and non-canonical Wnt/Ca2+ pathways. My data indicate that the SCJ epithelial cells that merge at the GEJ are regulated by opposing stromal Wnt factors and distinct Wnt pathway signaling in the epithelial cells. In the second part of the thesis, I investigated the role of Vitamin A-derived bioactive compound RA on esophageal and stomach epithelial stem cells. In vitro treatment of esophageal and stomach, epithelial organoids with RA or its pharmacological inhibitor BMS 493 revealed that each cell type was regulated distinctly. I observed that enhanced RA promoted esophageal stem cell differentiation and loss of stratification, while RA inhibition led to enhanced stemness and regeneration of the esophagus stratified epithelium. As opposed to the esophagus, RA signaling is active in the stomach organoids, and inhibition of RA reduces the growth of stomach organoids. Global transcriptomic data and scRNA-seq data revealed that RA signaling induces dormancy phenotype in the esophageal cells. In contrast, the absence of RA in stomach epithelial cells induces the expression of genes associated with BE. Thus, spatially defined regulation of Wnt and RA signaling at GEJ is critical for healthy homeostasis, and its perturbation leads to disease development. KW - Retinoesäure KW - Esophageal adenocarcinoma KW - Intestinal metaplasia KW - Epithelial lineage KW - Organoid KW - Retinoic acid KW - Gastroesophageal reflux KW - Endobrachyösophagus KW - Esophageal disease Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311556 ER - TY - THES A1 - Weisert, Nadine T1 - Characterization of telomere-associated proteins in \(Trypanosoma\) \(brucei\) T1 - Charakterisierung Telomer-assoziierter Proteine in \(Trypanosoma\) \(brucei\) N2 - The unicellular pathogen Trypanosoma brucei is the causative agent of African trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF. In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis. N2 - Der einzellige Pathogen Trypanosoma brucei ist der Erreger der afrikanischen Trypanosomiasis, eine endemische Krankheit vertreten in der Sub-Sahara Zone Afrikas. Trypanosoma brucei wechselt zwischen einem Säugerwirt und dem Insektenvektor, der Tsetse-Fliege. Der im Blutstrom des Säugers vorkommende, extrazelluläre Parasit ändert seinen Oberflächenmantel bestehend aus dem ˈvariablen Oberflächenproteinˈ (VSG) in periodischen Abständen, um der Immunantwort des Wirtes auszuweichen. Diese antigenetische Variation wird durch die monoallelische Expression einer einzelnen VSG-Variante, lokalisiert auf den ˈBlutstromform Expressionsseitenˈ (BES), zu einem bestimmten Zeitpunkt erreicht. Diese stadienspezifischen Oberflächenproteine werden während der Differenzierung der ˈBlutstromformˈ (BSF) zur ˈprozyklischen Formˈ (PCF) im Mitteldarm der Tsetse-Fliege stillgelegt und durch Prozykline ersetzt. Wegen der subtelomeren Lokalisation wird die VSG Transkription und Stilllegung teilweise durch Homologe des Säuger Telomerkomplexes TbTRF, TbTIF2 und TbRAP1 als auch durch Telomer-assoziierte Proteine (TelAPs) wie TelAP1 reguliert. Um Einblicke in die Transkriptionsregulation der VSG Gene zu erhalten, ist die Identifikation und Charakterisierung anderer Telomer-assoziierter Proteine von großem Interesse. In einer vorherigen Studie wurden zwei komplementäre biochemische Versuchsansätze verwendet, um weitere neue TelAPs zu identifizieren. Es wurde eine ko-Immunpräzipitation (co-IP) durchgeführt, um mögliche Interaktionspartner von TbTRF zu identifizieren, sowie eine Affinitätschromatographie unter Verwendung telomerischen Wiederholungseinheiten. Hierdurch wurde eine Liste von potenziellen Kandidaten generiert. Mit diesem Ansatz wurde TelAP1 als neue Komponente des Telomerkomplexes identifiziert, welches an der Kinetik der transkriptionellen BES-Stilllegung während der Differenzierung von BSF zu PCF beteiligt ist. Um weitere Einblicke in die Zusammensetzung des Telomerkomplexes zu erhalten, wurden zuvor angereicherte Proteine durch einen Screening-Prozess unter Verwendung von RNA-Interferenz charakterisiert. Nach der Depletion von 21 Proteinen wurden massenspektrometrische Analysen der VSG Expressionsprofiländerungen sowie allgemeine Veränderungen des Proteomenprofils analysiert. Mit dieser Methode können Erkenntnisse über die Funktion der jeweiligen Proteine und ihrer Beteiligung an der Regulierung der antigenetischen Variation von T. brucei gewonnen werden. Um die Interaktionen von Proteinen zu validieren, welche bei den Co-Immunpräzipitationen mit TbTRF und TelAP1 angereichert wurden, habe ich eine reziproke Affinitätschromatographie mit vier der vielversprechendsten Kandidaten durchgeführt (TelAP2, TelAP3, PPL2 und PolIE). Zusätzlich bestätigte ich die Co-lokalisation von zwei Kandidaten mit TbTRF via Immunfluoreszenzaufnahmen (TelAP2, TelAP3). TelAP3 ko-lokalisiert mit TbTRF und TelAP1 und interagiert potenziell mit TbTRF, TbTIF2, TelAP1 und TelAP2 als auch mit den zwei Transläsionspolymerasen PPL2 und PolIE in BSF. PPL2 und PolIE stehen in engem Kontakt zueinander und nehmen an den Telomerenden unterschiedliche Funktionen ein, da nur PolIE an der VSG Regulation beteiligt ist. TelAP2 wurde in einer vorherigen Publikation als Telomer-assoziiertes Protein durch partielle Co-Lokalisation mit TbTRF identifiziert und Zellen zeigen nach der Depletion von TelAP2 eine Derepression von zuvor stillgelegten VSGs. In dieser Studie zeige ich, dass TelAP2 mit den Telomer-bindenden Proteinen TbTRF und TbTIF2 sowie mit dem telomerassoziierten Protein TelAP1 in BSF interagiert und dass die Depletion von TelAP2 zu dem Verlust der Co-Lokalisation von TelAP1 mit TbTRF in BSF führt. Zusammenfassend zeigt diese Studie, dass die Charakterisierung potenzieller TelAPs dazu beiträgt, Einblicke in die Zusammensetzung des Telomerkomplexes und dessen Rolle bei der VSG-Regulation in Trypanosoma brucei zu gewinnen. Das Verständnis dieser Interaktionen könnte möglicherweise zu neuen therapeutischen Ansatzpunkten zur Bekämpfung der afrikanischen Trypanosomiasis führen. KW - Telomer KW - Trypanosoma brucei KW - telomere-associated protein Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-352732 ER - TY - JOUR A1 - Amatobi, Kelechi M. A1 - Ozbek-Unal, Ayten Gizem A1 - Schäbler, Stefan A1 - Deppisch, Peter A1 - Helfrich-Förster, Charlotte A1 - Mueller, Martin J. A1 - Wegener, Christian A1 - Fekete, Agnes T1 - The circadian clock is required for rhythmic lipid transport in Drosophila in interaction with diet and photic condition JF - Journal of Lipid Research N2 - Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality. KW - hemolymph lipids KW - lipidomics KW - circadian rhythm KW - feeding KW - locomotor activity KW - light-driven metabolism Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349961 VL - 64 IS - 10 ER - TY - JOUR A1 - Schuhmann, Antonia A1 - Scheiner, Ricarda T1 - A combination of the frequent fungicides boscalid and dimoxystrobin with the neonicotinoid acetamiprid in field-realistic concentrations does not affect sucrose responsiveness and learning behavior of honeybees JF - Ecotoxicology and Environmental Safety N2 - The increasing loss of pollinators over the last decades has become more and more evident. Intensive use of plant protection products is one key factor contributing to this decline. Especially the mixture of different plant protection products can pose an increased risk for pollinators as synergistic effects may occur. In this study we investigated the effect of the fungicide Cantus® Gold (boscalid/dimoxystrobin), the neonicotinoid insecticide Mospilan® (acetamiprid) and their mixture on honeybees. Since both plant protection products are frequently applied sequentially to the same plants (e.g. oilseed rape), their combination is a realistic scenario for honeybees. We investigated the mortality, the sucrose responsiveness and the differential olfactory learning performance of honeybees under controlled conditions in the laboratory to reduce environmental noise. Intact sucrose responsiveness and learning performance are of pivotal importance for the survival of individual honeybees as well as for the functioning of the entire colony. Treatment with two sublethal and field relevant concentrations of each plant protection product did not lead to any significant effects on these behaviors but affected the mortality rate. However, our study cannot exclude possible negative sublethal effects of these substances in higher concentrations. In addition, the honeybee seems to be quite robust when it comes to effects of plant protection products, while wild bees might be more sensitive. Highlights • Mix of SBI fungicides and neonicotinoids can lead to synergistic effects for bees. • Combination of non-SBI fungicide and neonicotinoid in field-realistic doses tested. • Synergistic effect on mortality of honeybees. • No effects on sucrose responsiveness and learning performance of honeybees. • Synergistic effects by other pesticide mixtures or on wild bees cannot be excluded. KW - Apis mellifera KW - non-SBI fungicide KW - insecticide KW - pesticide mixture KW - synergistic effect KW - sublethal effect Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350047 VL - 256 ER - TY - JOUR A1 - Coelho, Luis Pedro A1 - Alves, Renato A1 - Monteiro, Paulo A1 - Huerta-Cepas, Jaime A1 - Freitas, Ana Teresa A1 - Bork, Peer T1 - NG-meta-profiler: fast processing of metagenomes using NGLess, a domain-specific language JF - Microbiome N2 - Background Shotgun metagenomes contain a sample of all the genomic material in an environment, allowing for the characterization of a microbial community. In order to understand these communities, bioinformatics methods are crucial. A common first step in processing metagenomes is to compute abundance estimates of different taxonomic or functional groups from the raw sequencing data. Given the breadth of the field, computational solutions need to be flexible and extensible, enabling the combination of different tools into a larger pipeline. Results We present NGLess and NG-meta-profiler. NGLess is a domain specific language for describing next-generation sequence processing pipelines. It was developed with the goal of enabling user-friendly computational reproducibility. It provides built-in support for many common operations on sequencing data and is extensible with external tools with configuration files. Using this framework, we developed NG-meta-profiler, a fast profiler for metagenomes which performs sequence preprocessing, mapping to bundled databases, filtering of the mapping results, and profiling (taxonomic and functional). It is significantly faster than either MOCAT2 or htseq-count and (as it builds on NGLess) its results are perfectly reproducible. Conclusions NG-meta-profiler is a high-performance solution for metagenomics processing built on NGLess. It can be used as-is to execute standard analyses or serve as the starting point for customization in a perfectly reproducible fashion. NGLess and NG-meta-profiler are open source software (under the liberal MIT license) and can be downloaded from https://ngless.embl.de or installed through bioconda. KW - metagenomics KW - next-generation sequencing KW - domain-specific language Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223161 VL - 7 IS - 84 ER - TY - JOUR A1 - Coelho, Luis Pedro A1 - Kultima, Jens Roat A1 - Costea, Paul Igor A1 - Fournier, Coralie A1 - Pan, Yuanlong A1 - Czarnecki-Maulden, Gail A1 - Hayward, Matthew Robert A1 - Forslund, Sofia K. A1 - Schmidt, Thomas Sebastian Benedikt A1 - Descombes, Patrick A1 - Jackson, Janet R. A1 - Li, Qinghong A1 - Bork, Peer T1 - Similarity of the dog and human gut microbiomes in gene content and response to diet JF - Microbiome N2 - Background Gut microbes influence their hosts in many ways, in particular by modulating the impact of diet. These effects have been studied most extensively in humans and mice. In this work, we used whole genome metagenomics to investigate the relationship between the gut metagenomes of dogs, humans, mice, and pigs. Results We present a dog gut microbiome gene catalog containing 1,247,405 genes (based on 129 metagenomes and a total of 1.9 terabasepairs of sequencing data). Based on this catalog and taxonomic abundance profiling, we show that the dog microbiome is closer to the human microbiome than the microbiome of either pigs or mice. To investigate this similarity in terms of response to dietary changes, we report on a randomized intervention with two diets (high-protein/low-carbohydrate vs. lower protein/higher carbohydrate). We show that diet has a large and reproducible effect on the dog microbiome, independent of breed or sex. Moreover, the responses were in agreement with those observed in previous human studies. Conclusions We conclude that findings in dogs may be predictive of human microbiome results. In particular, a novel finding is that overweight or obese dogs experience larger compositional shifts than lean dogs in response to a high-protein diet. KW - microbiome KW - diet KW - metagenomics KW - dog microbiome KW - human microbiome KW - mouse microbiome KW - pig microbiome Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223177 VL - 6 ER - TY - JOUR A1 - Dedukh, Dmitrij A1 - Da Cruz, Irene A1 - Kneitz, Susanne A1 - Marta, Anatolie A1 - Ormanns, Jenny A1 - Tichopád, Tomáš A1 - Lu, Yuan A1 - Alsheimer, Manfred A1 - Janko, Karel A1 - Schartl, Manfred T1 - Achiasmatic meiosis in the unisexual Amazon molly, Poecilia formosa JF - Chromosome Research N2 - Unisexual reproduction, which generates clonal offspring, is an alternative strategy to sexual breeding and occurs even in vertebrates. A wide range of non-sexual reproductive modes have been described, and one of the least understood questions is how such pathways emerged and how they mechanistically proceed. The Amazon molly, Poecilia formosa, needs sperm from males of related species to trigger the parthenogenetic development of diploid eggs. However, the mechanism, of how the unreduced female gametes are produced, remains unclear. Cytological analyses revealed that the chromosomes of primary oocytes initiate pachytene but do not proceed to bivalent formation and meiotic crossovers. Comparing ovary transcriptomes of P. formosa and its sexual parental species revealed expression levels of meiosis-specific genes deviating from P. mexicana but not from P. latipinna. Furthermore, several meiosis genes show biased expression towards one of the two alleles from the parental genomes. We infer from our data that in the Amazon molly diploid oocytes are generated by apomixis due to a failure in the synapsis of homologous chromosomes. The fact that this failure is not reflected in the differential expression of known meiosis genes suggests the underlying molecular mechanism may be dysregulation on the protein level or misexpression of a so far unknown meiosis gene, and/or hybrid dysgenesis because of compromised interaction of proteins from diverged genomes. KW - meiosis KW - parthenogenesis KW - synaptonemal complex KW - recombination KW - crossing-over KW - achiasmatic KW - transcriptome KW - oogenesis Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325128 VL - 30 IS - 4 ER - TY - JOUR A1 - Rössler, Wolfgang A1 - Grob, Robin A1 - Fleischmann, Pauline N. T1 - The role of learning-walk related multisensory experience in rewiring visual circuits in the desert ant brain JF - Journal of Comparative Physiology A N2 - Efficient spatial orientation in the natural environment is crucial for the survival of most animal species. Cataglyphis desert ants possess excellent navigational skills. After far-ranging foraging excursions, the ants return to their inconspicuous nest entrance using celestial and panoramic cues. This review focuses on the question about how naïve ants acquire the necessary spatial information and adjust their visual compass systems. Naïve ants perform structured learning walks during their transition from the dark nest interior to foraging under bright sunlight. During initial learning walks, the ants perform rotational movements with nest-directed views using the earth’s magnetic field as an earthbound compass reference. Experimental manipulations demonstrate that specific sky compass cues trigger structural neuronal plasticity in visual circuits to integration centers in the central complex and mushroom bodies. During learning walks, rotation of the sky-polarization pattern is required for an increase in volume and synaptic complexes in both integration centers. In contrast, passive light exposure triggers light-spectrum (especially UV light) dependent changes in synaptic complexes upstream of the central complex. We discuss a multisensory circuit model in the ant brain for pathways mediating structural neuroplasticity at different levels following passive light exposure and multisensory experience during the performance of learning walks. KW - central complex KW - mushroom body KW - multisensory navigation KW - visual memory KW - neuronal and synaptic plasticity Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325096 VL - 209 IS - 4 ER - TY - JOUR A1 - Fricke, Ute A1 - Redlich, Sarah A1 - Zhang, Jie A1 - Tobisch, Cynthia A1 - Rojas-Botero, Sandra A1 - Benjamin, Caryl S. A1 - Englmeier, Jana A1 - Ganuza, Cristina A1 - Riebl, Rebekka A1 - Uhler, Johannes A1 - Uphus, Lars A1 - Ewald, Jörg A1 - Kollmann, Johannes A1 - Steffan-Dewenter, Ingolf T1 - Plant richness, land use and temperature differently shape invertebrate leaf-chewing herbivory on plant functional groups JF - Oecologia N2 - Higher temperatures can increase metabolic rates and carbon demands of invertebrate herbivores, which may shift leaf-chewing herbivory among plant functional groups differing in C:N (carbon:nitrogen) ratios. Biotic factors influencing herbivore species richness may modulate these temperature effects. Yet, systematic studies comparing leaf-chewing herbivory among plant functional groups in different habitats and landscapes along temperature gradients are lacking. This study was conducted on 80 plots covering large gradients of temperature, plant richness and land use in Bavaria, Germany. We investigated proportional leaf area loss by chewing invertebrates (‘herbivory’) in three plant functional groups on open herbaceous vegetation. As potential drivers, we considered local mean temperature (range 8.4–18.8 °C), multi-annual mean temperature (range 6.5–10.0 °C), local plant richness (species and family level, ranges 10–51 species, 5–25 families), adjacent habitat type (forest, grassland, arable field, settlement), proportion of grassland and landscape diversity (0.2–3 km scale). We observed differential responses of leaf-chewing herbivory among plant functional groups in response to plant richness (family level only) and habitat type, but not to grassland proportion, landscape diversity and temperature—except for multi-annual mean temperature influencing herbivory on grassland plots. Three-way interactions of plant functional group, temperature and predictors of plant richness or land use did not substantially impact herbivory. We conclude that abiotic and biotic factors can assert different effects on leaf-chewing herbivory among plant functional groups. At present, effects of plant richness and habitat type outweigh effects of temperature and landscape-scale land use on herbivory among legumes, forbs and grasses. KW - climate KW - ecosystem function KW - land use KW - plant guilds KW - plant–insect interactions Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325079 VL - 199 IS - 2 ER - TY - JOUR A1 - Zupanc, Günther K. H. A1 - Rössler, Wolfgang T1 - Government funding of research beyond biomedicine: challenges and opportunities for neuroethology JF - Journal of Comparative Physiology A N2 - Curiosity-driven research is fundamental for neuroethology and depends crucially on governmental funding. Here, we highlight similarities and differences in funding of curiosity-driven research across countries by comparing two major funding agencies—the National Science Foundation (NSF) in the United States and the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). We interviewed representatives from each of the two agencies, focusing on general funding trends, levels of young investigator support, career-life balance, and international collaborations. While our analysis revealed a negative trend in NSF funding of biological research, including curiosity-driven research, German researchers in these areas have benefited from a robust positive trend in DFG funding. The main reason for the decrease in curiosity-driven research in the US is that the NSF has only partially been able to compensate for the funding gap resulting from the National Institutes of Health restricting their support to biomedical research using select model organisms. Notwithstanding some differences in funding programs, particularly those relevant for scientists in the postdoctoral phase, both the NSF and DFG clearly support curiosity-driven research. KW - German Research Foundation KW - Government research funding KW - National Science Foundation KW - Deutsche Forschungsgemeinschaft KW - neuroethology Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325113 VL - 208 IS - 3 ER - TY - JOUR A1 - Englmeier, Jana A1 - Mitesser, Oliver A1 - Benbow, M. Eric A1 - Hothorn, Torsten A1 - von Hoermann, Christian A1 - Benjamin, Caryl A1 - Fricke, Ute A1 - Ganuza, Cristina A1 - Haensel, Maria A1 - Redlich, Sarah A1 - Riebl, Rebekka A1 - Rojas Botero, Sandra A1 - Rummler, Thomas A1 - Steffan-Dewenter, Ingolf A1 - Stengel, Elisa A1 - Tobisch, Cynthia A1 - Uhler, Johannes A1 - Uphus, Lars A1 - Zhang, Jie A1 - Müller, Jörg T1 - Diverse effects of climate, land use, and insects on dung and carrion decomposition JF - Ecosystems N2 - Land-use intensification and climate change threaten ecosystem functions. A fundamental, yet often overlooked, function is decomposition of necromass. The direct and indirect anthropogenic effects on decomposition, however, are poorly understood. We measured decomposition of two contrasting types of necromass, rat carrion and bison dung, on 179 study sites in Central Europe across an elevational climate gradient of 168–1122 m a.s.l. and within both local and regional land uses. Local land-use types included forest, grassland, arable fields, and settlements and were embedded in three regional land-use types (near-natural, agricultural, and urban). The effects of insects on decomposition were quantified by experimental exclusion, while controlling for removal by vertebrates. We used generalized additive mixed models to evaluate dung weight loss and carrion decay rate along elevation and across regional and local land-use types. We observed a unimodal relationship of dung decomposition with elevation, where greatest weight loss occurred between 600 and 700 m, but no effects of local temperature, land use, or insects. In contrast to dung, carrion decomposition was continuously faster with both increasing elevation and local temperature. Carrion reached the final decomposition stage six days earlier when insect access was allowed, and this did not depend on land-use effect. Our experiment identified different major drivers of decomposition on each necromass form. The results show that dung and carrion decomposition are rather robust to local and regional land use, but future climate change and decline of insects could alter decomposition processes and the self-regulation of ecosystems. KW - decay KW - ecosystem function KW - global change KW - land-use intensification KW - necrobiome KW - urbanization Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325064 SN - 1432-9840 VL - 26 IS - 2 ER - TY - JOUR A1 - Shirakashi, Ryo A1 - Sisario, Dmitri A1 - Taban, Danush A1 - Korsa, Tessa A1 - Wanner, Sophia B. A1 - Neubauer, Julia A1 - Djuzenova, Cholpon S. A1 - Zimmermann, Heiko A1 - Sukhorukov, Vladimir L. T1 - Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes JF - Biomechanics and Modeling in Mechanobiology N2 - Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux. KW - electroporation KW - cell velocimetry KW - hemoglobin jet KW - non-muscle myosin KW - echinocytes KW - cytoskeleton Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325107 VL - 22 IS - 2 ER - TY - JOUR A1 - Moustafa, Moataz A. M. A1 - Fouad, Eman A. A1 - Ibrahim, Emad A1 - Erdei, Anna Laura A1 - Kárpáti, Zsolt A1 - Fónagy, Adrien T1 - The comparative toxicity, biochemical and physiological impacts of chlorantraniliprole and indoxacarb on Mamestra brassicae (Lepidoptera: Noctuidae) JF - Toxics N2 - Background: The cabbage moth, Mamestra brassicae, is a polyphagous pest that attacks several crops. Here, the sublethal and lethal effects of chlorantraniliprole and indoxacarb were investigated on the developmental stages, detoxification enzymes, reproductive activity, calling behavior, peripheral physiology, and pheromone titer of M. brasssicae. Methods: To assess pesticide effects, the second instar larvae were maintained for 24 h on a semi-artificial diet containing insecticides at their LC\(_{10}\), LC\(_{30}\), and LC\(_{50}\) concentrations. Results: M. brassicae was more susceptible to chlorantraniliprole (LC\(_{50}\) = 0.35 mg/L) than indoxacarb (LC\(_{50}\) = 1.71 mg/L). A significantly increased developmental time was observed with both insecticides at all tested concentrations but decreases in pupation rate, pupal weight, and emergence were limited to the LC50 concentration. Reductions in both the total number of eggs laid per female and the egg viability were observed with both insecticides at their LC\(_{30}\) and LC\(_{50}\) concentrations. Both female calling activity and the sex pheromone (Z11-hexadecenyl acetate and hexadecenyl acetate) titer were significantly reduced by chlorantraniliprole in LC\(_{50}\) concentration. Antennal responses of female antennae to benzaldehyde and 3-octanone were significantly weaker than controls after exposure to the indoxocarb LC\(_{50}\) concentration. Significant reductions in the enzymatic activity of glutathione S-transferases, mixed-function oxidases, and carboxylesterases were observed in response to both insecticides. KW - toxicity KW - sublethal effects KW - chlorantraniliprole KW - indoxacarb KW - Mamestra brassicae Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-303931 SN - 2305-6304 VL - 11 IS - 3 ER - TY - THES A1 - Pitsch, Maximilian Jonathan T1 - Zyklisches Adenosinmonophosphat (cAMP) als Äquivalent akkumulierter neuronaler Evidenz T1 - Cyclic adenosine monophosphate (cAMP) as an equivalent of accumulated neuronal evidence N2 - Die vier Crz-Neurone des ventralen Nervensystems von Drosophila melanogaster sammeln Evidenz, wann im Rahmen eines Paarungsakts zirka 6 Minuten vergangen sind. Diese Entscheidung ist für die männliche Fliege von Bedeutung, da das Männchen vor Ablauf dieser ~6 Minuten, welche den Zeitpunkt der Ejakulation darstellen, eher das eigene Leben opfern würde, als dass es die Paarung beenden würde. Nach Ablauf der ~6 Minuten fällt die Motivation des Männchens dagegen dramatisch ab. Im Rahmen der vorliegenden Arbeit wurde zunächst mittels optogenetischer neuronaler Inhibitionsprotokolle sowie Verhaltensanalysen das Phänomen der Evidenz-akkumulation in den Crz-Neuronen genauer charakterisiert. Dabei zeigte sich, dass die akkumulierte Evidenz auch während einer elektrischen Inhibition der Crz-Neurone persistierte. Dieses Ergebnis warf die Hypothese auf, dass das Äquivalent der akkumulierten Evidenz in den Crz-Neuronen biochemischer Natur sein könnte. Es wurde daraufhin ein Hochdurchsatzscreening-Verfahren entwickelt, mittels dessen 1388 genetische Manipulationen der Crz-Neurone durchgeführt und auf eine Änderung der Evidenzakkumulation getestet wurden. Nur ~30 genetische Manipulationen zeigten eine veränderte Evidenzakkumulation, wobei die meisten dieser Manipulationen den cAMP-Signalweg betrafen. Mittels der optogenetischen Photoadenylatzyklase bPAC, einer Reihe weiterer genetischer Manipulationen des cAMP-Signalwegs sowie der ex vivo Kalzium-Bildgebung und Fluoreszenzlebensdauer-Mikroskopie konnte bestätigt werden, dass cAMP das Äquivalent der in den Crz-Neuronen spannungsabhängig akkumulierten Evidenz darstellt, wobei die Kombination dieser Methoden nahelegte, dass der Schwellenwert der Evidenzakkumulation durch die cAMP-Bindungsaffinität der regulatorischen PKA-Untereinheiten festgelegt sein könnte. Mittels genetischer Mosaikexperimente sowie bildgebenden Verfahren konnte darüber hinaus gezeigt werden, dass innerhalb des Crz-Netzwerks eine positive Rückkopplungsschleife aus rekurrenter Aktivität sowie der cAMP-Akkumulation besteht, welche, sobald die cAMP-Spiegel den Schwellenwert erreichen, zu einem netzwerkweit synchronisierten massiven Kalziumeinstrom führt, was die Abgabe des Crz-Signals an nachgeschaltete Netzwerke triggert. Dieses Phänomen könnte ein Analogon des Aktionspotenzials auf Netzwerkebene sowie auf Intervallzeitskalen darstellen und wurde als „Eruption“ bezeichnet. Genetische, optogenetische sowie Bildgebungsexperimente konnten zeigen, dass die CaMKII derartige Eruptionen durch Niedrighalten der cAMP-Spiegel unterdrückt, was den Zeitmessmechanismus des ersten beschriebenen Intervallzeitmessers CaMKII offenlegt. N2 - The four Crz neurons of the ventral nervous system of Drosophila melanogaster collect evidence about when approximately 6 minutes have elapsed during a mating act. This decision is of importance for the male fly as the male would rather sacrifice his own life than terminate mating before the expiration of these ~6 minutes, which represent the time of ejaculation. After these ~6 minutes, however, the male's motivation drops dramatically. In this dissertation, optogenetic neuronal inhibition protocols as well as behavioral analyses were used to characterize the phenomenon of evidence accumulation in the Crz neurons in more detail. This showed that the accumulated evidence persisted during electrical inhibition of the Crz neurons. This result raised the hypothesis that the equivalent of accumulated evidence in the Crz neurons might be biochemical in nature. A high-throughput-screening-assay was developed using which 1388 genetic manipulations of the Crz neurons were performed and tested for a change in evidence accumulation. Only ~30 genetic manipulations showed altered evidence accumulation, with most of these manipulations involving the cAMP pathway. Using the optogenetic photoadenylyl cyclase bPAC, a number of other genetic manipulations of the cAMP pathway, as well as ex vivo calcium imaging and fluorescence lifetime microscopy techniques, it was confirmed that cAMP represents the equivalent of accumulated evidence in the Crz neurons, and the combination of these methods suggested that the evidence accumulation threshold may be set by the cAMP-binding affinity of regulatory PKA subunits. Using genetic mosaic experiments as well as imaging techniques, it was further shown that within the Crz network there is a positive feedback loop between the recurrent activity as well as the cAMP accumulation, which, once cAMP levels reach the threshold, leads to a network-wide synchronized massive calcium influx, triggering the delivery of the Crz signal to downstream networks. This phenomenon could represent an analog of the action potential at the network level as well as at interval time scales and has been termed an "eruption." Genetic, optogenetic as well as imaging experiments could show that CaMKII suppresses such eruptions by keeping cAMP levels low, revealing the timing mechanism of CaMKII, the first described interval timer. KW - Evidenz KW - Drosophila KW - Biologische Uhr KW - cAMP KW - Intervallzeitmessung Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-351292 ER - TY - JOUR A1 - Römer, Daniela A1 - Aguilar, Gonzalo Pacheco A1 - Meyer, Annika A1 - Roces, Flavio T1 - Symbiont demand guides resource supply: leaf-cutting ants preferentially deliver their harvested fragments to undernourished fungus gardens JF - The Science of Nature N2 - Leaf-cutting ants are highly successful herbivores in the Neotropics. They forage large amounts of fresh plant material to nourish a symbiotic fungus that sustains the colony. It is unknown how workers organize the intra-nest distribution of resources, and whether they respond to increasing demands in some fungus gardens by adjusting the amount of delivered resources accordingly. In laboratory experiments, we analyzed the spatial distribution of collected leaf fragments among nest chambers in Acromyrmex ambiguus leaf-cutting ants, and how it changed when one of the fungus gardens experienced undernourishment. Plant fragments were evenly distributed among nest chambers when the fungal symbiont was well nourished. That pattern changed when one of the fungus gardens was undernourished and had a higher leaf demand, resulting in more leaf discs delivered to the undernourished fungus garden over at least 2 days after deprivation. Some ants bypassed nourished gardens to directly deliver their resource to the chamber with higher nutritional demand. We hypothesize that cues arising from that chamber might be used for orientation and/or that informed individuals, presumably stemming from the undernourished chamber, may preferentially orient to them. KW - insect-fungus symbiosis KW - nutrition KW - pheromone trail KW - local cues KW - decision-making KW - decentralized control Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325080 VL - 109 IS - 3 ER - TY - THES A1 - Bakari Soale, Majeed T1 - Regulation of the Variant Surface Glycoprotein (VSG) Expression and Characterisation of the Nucleolar DExD/H box Protein Hel66 in \(Trypanosoma\) \(brucei\) T1 - Regulation der Expression des variable Oberflächen- Glykoprotein (VSG) und Charakterisierung des nukleolären DExD/H box Protein Hel66 in \(Trypanosoma\) \(brucei\) N2 - The variant surface glycoprotein (VSG) of African trypanosomes plays an essential role in protecting the parasites from host immune factors. These trypanosomes undergo antigenic variation resulting in the expression of a single VSG isoform out of a repertoire of around 2000 genes. The molecular mechanism central to the expression and regulation of the VSG is however not fully understood. Gene expression in trypanosomes is unusual due to the absence of typical RNA polymerase II promoters and the polycistronic transcription of genes. The regulation of gene expression is therefore mainly post-transcriptional. Regulatory sequences, mostly present in the 3´ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 100 % conserved 16mer motif within the 3´ UTR has been shown to modulate the stability of VSG transcripts and hence their expression. As a stability-associated sequence element, the absence of nucleotide substitutions in the motif is however unusual. It was therefore hypothesised that the motif is involved in other essential roles/processes besides stability of the VSG transcripts. In this study, it was demonstrated that the 100 % conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. It was further shown that the intact motif in the active VSG 3´ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Crosstalk between the VSG and procyclin genes during differentiation to the insect vector stage is also unaffected in cells with a mutated 16mer motif. Ectopic overexpression of a second VSG however requires the intact motif to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The 16mer motif therefore plays a dual role in VSG in situ switching and stability of VSG transcripts. The additional role of the 16mer in the essential process of antigenic variation appears to be the driving force for the 100 % conservation of this RNA motif. A screen aimed at identifying candidate RNA-binding proteins interacting with the 16mer motif, led to the identification of a DExD/H box protein, Hel66. Although the protein did not appear to have a direct link to the 16mer regulation of VSG expression, the DExD/H family of proteins are important players in the process of ribosome biogenesis. This process is relatively understudied in trypanosomes and so this candidate was singled out for detailed characterisation, given that the 16mer story had reached a natural end point. Ribosome biogenesis is a major cellular process in eukaryotes involving ribosomal RNA, ribosomal proteins and several non-ribosomal trans-acting protein factors. The DExD/H box proteins are the most important trans-acting protein factors involved in the biosynthesis of ribosomes. Several DExD/H box proteins have been directly implicated in this process in yeast. In trypanosomes, very few of this family of proteins have been characterised and therefore little is known about the specific roles they play in RNA metabolism. Here, it was shown that Hel66 is involved in rRNA processing during ribosome biogenesis. Hel66 localises to the nucleolus and depleting the protein led to a severe growth defect. Loss of the protein also resulted in a reduced rate of global translation and accumulation of rRNA processing intermediates of both the small and large ribosomal subunits. Hel66 is therefore an essential nucleolar DExD/H protein involved in rRNA processing during ribosome biogenesis. As very few protein factors involved in the processing of rRNAs have been described in trypanosomes, this finding represents an important platform for future investigation of this topic. N2 - Das variable Oberflächen-Glykoprotein (“varaint surface glycoprotein“, VSG) der Afrikanischen Trypanosomen schützt den Parasiten vor Immunfaktoren des Wirtes. Trypanosomen beherrschen die antigene Variation und expremieren nur eine einzige VSG Isoform aus einem Repertoire von ungefähr 2000 Genen. Der molekulare Mechanismus der die Expression dieser VSG Gene reguliert ist nicht komplett bekannt. Die Genexpression ist in Trypanosomen sehr ungewöhnlich. Es gibt keine typischen Promotoren für RNA Polymerase II und Gene werden polycistronisch transkribiert. Daher ist die Regulation der Genexpression hauptsächlich posttranskriptional. Die Expression individueller mRNAs wird durch regulatorische Sequenzen reguliert, die sich häufig in den 3´ UTRs befinden. In den VSG Genen von T. brucei moduliert ein zu 100% konserviertes 16mer Motiv in der 3´ UTR die Stabilität der VSG Transkripte und damit deren Expression. Für eine Sequenz, die die Stabilität der mRNA reguliert, ist das Fehlen von Nukleotid Substitutionen sehr ungewöhnlich. Es wurde deshalb spekuliert, dass das 16mer Motiv neben der Stabilisierung des VSG Transkriptes noch an anderen essentiellen Prozessen beteiligt ist. In dieser Arbeit wurde gezeigt, dass die 100%ige Konservierung des 16mer Motives weder für das Überleben der Zellen, noch für den Erhalt der Expression des VSG Protein in funktioneller Menge notwendig ist. Außerdem wurde gezeigt dass das intakte Motiv in der 3´UTR des aktiven VSGs weder für das „VSG silencing“ während des VSG Austausches („switching“) noch für die Differenzierung von Blutbahnformen zu prozyklischen Formen benötigt wird. Auch die Interaktionen („crosstalk“), die während der Differenzierung zum Insekten Stadium zwischen den VSG und Prozyklin Genen stattfinden, sind in Zellen mit mutiertem 16mer Motiv noch funktionell. Die ektopische Überexpression eines zweiten VSGs benötigt allerdings das intakte Motiv, um das aktive VSG zu inaktivieren und auszutauschen: dies suggeriert eine Rolle des Motivs im transkriptionalen „VSG switching“. Das 16mer Motif spielt daher eine Doppelrolle bei der Regulation der Stabilität der VSG Transkripte und im VSG in situ „switching“. Letzteres, die Rolle im essentiellen Prozess der antigenen Variation, ist dabei offensichtlich die treibende Kraft hinter der 100%igen Konservierung des RNA Motives. Eine Suche nach möglichen RNA bindenden Proteinen, die mit dem 16mer interagieren, führte zur Identifikation des DExD/H box Proteins Hel66. Obwohl das Protein wohl nicht direkt an der Regulation der VSG Expression über das 16mer beteiligt ist, spielen Mitglieder der DexD/H Proteinfamilie eine wichtige Rolle in der Biogenese von Ribosomen. Dieser Prozess ist in Trypanosomen noch nicht komplett verstanden und daher wurde das Protein für eine nähere Analyse ausgewählt, auch weil die 16mer Story ohne weitere Kandidaten zu einem Ende gekommen war. Die Biogenese von Ribosomen ist ein wichtiger zellulärer Prozess in Eukaryoten und benötigt ribosomale RNA, ribosomale Proteine sowie einige nicht-ribosomale, trans-agierende Protein Faktoren. Proteine der DExD/H box Familie sind die wichtigsten trans- agierenden Proteinfaktoren, die an der Biogenese der Ribosomen beteiligt sind. In der Hefe sind mehrere DExD/H box Proteine bekannt, die eine direkte Rolle in diesem Prozess spielen. In Trypanosomen sind erst sehr wenige Proteine aus dieser Familie untersucht worden und es ist daher kaum bekannt, welche spezifische Rollen sie im RNA Metabolismus spielen. In dieser Arbeit wurde gezeigt, dass Hel66 an der rRNA Prozessierung während der Biogenese der Ribosomen beteiligt ist. Hel66 ist im Nukleolus lokalisiert und die Reduktion des Proteins durch RNAi führte zu einem schweren Wachstumsphänotyp. Reduktion von Hel66 führte auch zu einer globalen Reduktion der Translation sowie zur Akkumulation von Synthese- Zwischenstadien der rRNAs sowohl der kleinen und als auch der großen ribosomalen Untereinheit. Hel66 ist daher ein essentielles nukleoläres DExD/H Protein dass an der Prozessierung der rRNA während der Biogenese der Ribosomen beteiligt ist. Da bisher erst wenige Proteine bekannt sind, die in Trypanosomen an diesem Prozess beteiligt sind, sind diese Ergebnisse ein sehr wichtiger Ausgangspunkt für weitere Untersuchungen in der Zukunft. KW - Trypanosoma brucei KW - Genexpression KW - Variant Surface Glycoprotein KW - VSG KW - DExD/H box protein KW - Ribosome biogenesis KW - rRNA processing KW - Ribosome Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-258090 ER - TY - JOUR A1 - Maloukh, Lina A1 - Nazzal, Yousef A1 - Kumarappan, Alagappan A1 - Howari, Fares A1 - Ambika, Lakshmi Kesari A1 - Yahmadi, Rihab A1 - Sharma, Manish A1 - Iqbal, Jibran A1 - Al-Taani, Ahmed A. A1 - Salem, Imen Ben A1 - Xavier, Cijo M. A1 - Naseem, Muhamad T1 - Metagenomic analysis of the outdoor dust microbiomes: a case study from Abu Dhabi, UAE JF - Atmosphere N2 - Outdoor dust covers a shattered range of microbial agents from land over transportation, human microbial flora, which includes pathogen and commensals, and airborne from the environment. Dust aerosols are rich in bacterial communities that have a major impact on human health and living environments. In this study, outdoor samples from roadside barricades, safety walls, and fences (18 samples) were collected from Abu Dhabi, UAE and bacterial diversity was assessed through a 16S rRNA amplicon next generation sequencing approach. Clean data from HiSeq produced 1,099,892 total reads pairs for 18 samples. For all samples, taxonomic classifications were assigned to the OTUs (operational taxonomic units) representative sequence using the Ribosomal Database Project database. Analysis such as alpha diversity, beta diversity, differential species analysis, and species relative abundance were performed in the clustering of samples and a functional profile heat map was obtained from the OTUs by using bioinformatics tools. A total of 2814 OTUs were identified from those samples with a coverage of more than 99%. In the phylum, all 18 samples had most of the bacterial groups such as Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Twelve samples had Propionibacteria acnes and were mainly found in RD16 and RD3. Major bacteria species such as Propionibacteria acnes, Bacillus persicus, and Staphylococcus captis were found in all samples. Most of the samples had Streptococcus mitis, Staphylococcus capitis. and Nafulsella turpanensis and Enhydrobacter aerosaccus was part of the normal microbes of the skin. Salinimicrobium sp., Bacillus alkalisediminis, and Bacillus persicus are halophilic bacteria found in sediments. The heat map clustered the samples and species in vertical and horizontal classification, which represents the relationship between the samples and bacterial diversity. The heat map for the functional profile had high properties of amino acids, carbohydrate, and cofactor and vitamin metabolisms of all bacterial species from all samples. Taken together, our analyses are very relevant from the perspective of out-door air quality, airborne diseases, and epidemics, with broader implications for health safety and monitoring. KW - dust microbiomes KW - metagenomics KW - microbial diversity KW - pollution KW - GIS Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304391 SN - 2073-4433 VL - 14 IS - 2 ER - TY - THES A1 - Maier [verh. Hartmann], Carina Ramona T1 - Regulation of the Mevalonate Pathway by the Deubiquitinase USP28 in Squamous Cancer T1 - Regulation des Mevalonat Stoffwechselwegs durch die Deubiquitinase USP28 in Plattenepithelkarzinomen N2 - The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment. De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis. SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis. SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation. It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth. A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation. In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients. N2 - Die Reprogrammierung metabolischer Stoffwechselwege ist ein Kennzeichen von Krebs: Tumorzellen sind abhängig von der Versorgung mit Metaboliten und Bausteinen, um ihren wachsenden Bedarf als hoch proliferierende Zellen zu decken. Vor allem die de novo Stoffwechselsynthesewege sich hochreguliert, wenn die Zellen des wachsenden Tumors nicht mehr in der Lage sind, ihr erforderliches metabolisches Niveau mithilfe der Aufnahme aus der Umgebung zu erfüllen. De novo Synthesewege sind oft unter der Kontrolle von zentralen Transkriptionsfaktoren die die Genexpression von Enzymen, die im Syntheseprozess beteiligt sind, regulieren. Die vorherrschenden Regulatoren, für die de novo Fettsäuresynthese und der Cholesterogenese sind die Steroid-regulatorisches-Element-bindende Proteine (SREBPs). Während SREBP1 bevorzugt die Expression von Enzymen die an der Fettsäuresynthese beteiligt sind kontrolliert, reguliert SREBP2 die Transkription von Enzymen des Mevalonat Stoffwechselwegs, sowie Prozesse unterhalb, namentlich die Cholesterol-, Isoprenoid- und die die Synthese von Bausteinen für die Ubiquinonsynthese. Die Aktivität von SREBP ist streng reguliert auf verschiedenen Ebenen: Die post-translationale Modifikation mittels Ubiquitinierung reduziert die Stabilität von aktiven SREBPs. Das Anhängen von K48-verlinkten Ubiquitinketten markiert die Transkriptionsfaktoren für den proteasomalen Abbau. In Tumorzellen sind hohe Niveaus von aktiven SREBPs essentiell für die Induktion der entsprechenden metabolischen Stoffwechselwege. Die erhöhte Stabilität und Aktivität von SREBPs wurden im Rahmen dieser Arbeit untersucht. SREBPs werden von der E3-Ligase Fbw7 ubiquitiniert, was zur Proteolyse der Transkriptionsfaktoren führt. In dieser Arbeit wurde gezeigt, dass das entgegenwirkende Deubiquitinierungsenzym USP28 die Ubiquitinketten von SREBPs entfernt und deren proteasomalen Abbau verhindert. Diese Forschungsarbeit zeigt weiterhin, dass die Stabilisierung von SREBP2 durch USP28 eine wichtige Rolle im Kontext von Epithelkarzinomen spielt. Erhöhte USP28 Niveaus werden mit einem schlechten Überleben von Patienten in der Krebs-Untergruppe der Plattenepithelkarzinomen verbunden. Es konnte gezeigt werden, dass reduzierte USP28 Niveaus, in Zelllinien und in vivo, niedrigere SREBP2-Aktivität und eine Herunterregulierung des Mevalonat Stoffwechselwegs ergeben. Diese Manipulation führte zu reduzierter Proliferation und Tumorwachstum. Ein direkter Vergleich von Adenokarzinomen und Plattenepithelkarzinomen in Lungenkrebspatienten zeigte zudem eine Hochregulierung von USP28 ebenso wie SREBP2 und dessen Zielgenen. Der gezielte Einsatz von Inhibitoren gegen die USP28-SREBP2 regulatorische Achse in Plattenepithelzellen reduzierte die Lebensfähigkeit und Proliferation der Zellen. Abschließend berichtet diese Forschungsarbeit von der Bedeutung des durch die USP28-SREBP2 Achse regulierten Mevalonat Stoffwechselwegs bei der Tumorinitiation und dem Fortschreiten von Plattenepithelkarzinomen. Die kombinatorische Behandlung mit USP28- und Inhibitoren der HMGCR, dem Schlüsselenzym des Mevalonat Stoffwechselwegs, mithilfe von Statinen eröffnet die Möglichkeit für eine gezielte therapeutische Behandlung von Patienten mit Plattenepithelkarzinomen. KW - Ubiquitin KW - Metabolismus KW - Deubiquitination KW - Mevalonate Pathway KW - Cancer Metabolism KW - Lung squamous cancer cells Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-348740 ER - TY - JOUR A1 - Dhillon, Maninder Singh A1 - Kübert-Flock, Carina A1 - Dahms, Thorsten A1 - Rummler, Thomas A1 - Arnault, Joel A1 - Steffan-Dewenter, Ingolf A1 - Ullmann, Tobias T1 - Evaluation of MODIS, Landsat 8 and Sentinel-2 data for accurate crop yield predictions: a case study using STARFM NDVI in Bavaria, Germany JF - Remote Sensing N2 - The increasing availability and variety of global satellite products and the rapid development of new algorithms has provided great potential to generate a new level of data with different spatial, temporal, and spectral resolutions. However, the ability of these synthetic spatiotemporal datasets to accurately map and monitor our planet on a field or regional scale remains underexplored. This study aimed to support future research efforts in estimating crop yields by identifying the optimal spatial (10 m, 30 m, or 250 m) and temporal (8 or 16 days) resolutions on a regional scale. The current study explored and discussed the suitability of four different synthetic (Landsat (L)-MOD13Q1 (30 m, 8 and 16 days) and Sentinel-2 (S)-MOD13Q1 (10 m, 8 and 16 days)) and two real (MOD13Q1 (250 m, 8 and 16 days)) NDVI products combined separately to two widely used crop growth models (CGMs) (World Food Studies (WOFOST), and the semi-empiric Light Use Efficiency approach (LUE)) for winter wheat (WW) and oil seed rape (OSR) yield forecasts in Bavaria (70,550 km\(^2\)) for the year 2019. For WW and OSR, the synthetic products’ high spatial and temporal resolution resulted in higher yield accuracies using LUE and WOFOST. The observations of high temporal resolution (8-day) products of both S-MOD13Q1 and L-MOD13Q1 played a significant role in accurately measuring the yield of WW and OSR. For example, L- and S-MOD13Q1 resulted in an R\(^2\) = 0.82 and 0.85, RMSE = 5.46 and 5.01 dt/ha for WW, R\(^2\) = 0.89 and 0.82, and RMSE = 2.23 and 2.11 dt/ha for OSR using the LUE model, respectively. Similarly, for the 8- and 16-day products, the simple LUE model (R\(^2\) = 0.77 and relative RMSE (RRMSE) = 8.17%) required fewer input parameters to simulate crop yield and was highly accurate, reliable, and more precise than the complex WOFOST model (R\(^2\) = 0.66 and RRMSE = 11.35%) with higher input parameters. Conclusively, both S-MOD13Q1 and L-MOD13Q1, in combination with LUE, were more prominent for predicting crop yields on a regional scale than the 16-day products; however, L-MOD13Q1 was advantageous for generating and exploring the long-term yield time series due to the availability of Landsat data since 1982, with a maximum resolution of 30 m. In addition, this study recommended the further use of its findings for implementing and validating the long-term crop yield time series in different regions of the world. KW - MODIS KW - Sentinel-2 KW - Landsat 8 KW - sustainable agriculture KW - decision-making KW - winter wheat KW - oil seed rape KW - resolution Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311132 SN - 2072-4292 VL - 15 IS - 7 ER - TY - JOUR A1 - Dhillon, Maninder Singh A1 - Dahms, Thorsten A1 - Kübert-Flock, Carina A1 - Liepa, Adomas A1 - Rummler, Thomas A1 - Arnault, Joel A1 - Steffan-Dewenter, Ingolf A1 - Ullmann, Tobias T1 - Impact of STARFM on crop yield predictions: fusing MODIS with Landsat 5, 7, and 8 NDVIs in Bavaria Germany JF - Remote Sensing N2 - Rapid and accurate yield estimates at both field and regional levels remain the goal of sustainable agriculture and food security. Hereby, the identification of consistent and reliable methodologies providing accurate yield predictions is one of the hot topics in agricultural research. This study investigated the relationship of spatiotemporal fusion modelling using STRAFM on crop yield prediction for winter wheat (WW) and oil-seed rape (OSR) using a semi-empirical light use efficiency (LUE) model for the Free State of Bavaria (70,550 km\(^2\)), Germany, from 2001 to 2019. A synthetic normalised difference vegetation index (NDVI) time series was generated and validated by fusing the high spatial resolution (30 m, 16 days) Landsat 5 Thematic Mapper (TM) (2001 to 2012), Landsat 7 Enhanced Thematic Mapper Plus (ETM+) (2012), and Landsat 8 Operational Land Imager (OLI) (2013 to 2019) with the coarse resolution of MOD13Q1 (250 m, 16 days) from 2001 to 2019. Except for some temporal periods (i.e., 2001, 2002, and 2012), the study obtained an R\(^2\) of more than 0.65 and a RMSE of less than 0.11, which proves that the Landsat 8 OLI fused products are of higher accuracy than the Landsat 5 TM products. Moreover, the accuracies of the NDVI fusion data have been found to correlate with the total number of available Landsat scenes every year (N), with a correlation coefficient (R) of +0.83 (between R\(^2\) of yearly synthetic NDVIs and N) and −0.84 (between RMSEs and N). For crop yield prediction, the synthetic NDVI time series and climate elements (such as minimum temperature, maximum temperature, relative humidity, evaporation, transpiration, and solar radiation) are inputted to the LUE model, resulting in an average R\(^2\) of 0.75 (WW) and 0.73 (OSR), and RMSEs of 4.33 dt/ha and 2.19 dt/ha. The yield prediction results prove the consistency and stability of the LUE model for yield estimation. Using the LUE model, accurate crop yield predictions were obtained for WW (R\(^2\) = 0.88) and OSR (R\(^2\) = 0.74). Lastly, the study observed a high positive correlation of R = 0.81 and R = 0.77 between the yearly R\(^2\) of synthetic accuracy and modelled yield accuracy for WW and OSR, respectively. KW - MOD13Q1 KW - precision agriculture KW - fusion KW - sustainable agriculture KW - decision making KW - winter wheat KW - oil-seed rape KW - crop models Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311092 SN - 2072-4292 VL - 15 IS - 6 ER - TY - JOUR A1 - Aupperle-Lellbach, Heike A1 - Heidrich, Daniela A1 - Kehl, Alexandra A1 - Conrad, David A1 - Brockmann, Maria A1 - Törner, Katrin A1 - Beitzinger, Christoph A1 - Müller, Tobias T1 - KITLG copy number germline variations in schnauzer breeds and their relevance in digital squamous cell carcinoma in black giant schnauzers JF - Veterinary Sciences N2 - Copy number variations (CNVs) of the KITLG gene seem to be involved in the oncogenesis of digital squamous cell carcinoma (dSCC). The aims of this study were (1) to investigate KITLG CNV in giant (GS), standard (SS), and miniature (MS) schnauzers and (2) to compare KITLG CNV between black GS with and without dSCC. Blood samples from black GS (22 with and 17 without dSCC), black SS (18 with and 4 without dSSC; 5 unknown), and 50 MS (unknown dSSC status and coat colour) were analysed by digital droplet PCR. The results are that (1) most dogs had a copy number (CN) value > 4 (range 2.5–7.6) with no significant differences between GS, SS, and MS, and (2) the CN value in black GS with dSCC was significantly higher than in those without dSCC (p = 0.02). CN values > 5.8 indicate a significantly increased risk for dSCC, while CN values < 4.7 suggest a reduced risk for dSCC (grey area: 4.7–5.8). Diagnostic testing for KITLG CNV may sensitise owners to the individual risk of their black GS for dSCC. Further studies should investigate the relevance of KITLG CNV in SS and the protective effects in MS, who rarely suffer from dSCC. KW - tumour KW - toe KW - miniature schnauzer KW - standard schnauzer KW - CNV KW - ddPCR KW - breed predisposition Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-303913 SN - 2306-7381 VL - 10 IS - 2 ER - TY - JOUR A1 - Cerezo-Echevarria, Argiñe A1 - Kehl, Alexandra A1 - Beitzinger, Christoph A1 - Müller, Tobias A1 - Klopfleisch, Robert A1 - Aupperle-Lellbach, Heike T1 - Evaluating the histologic grade of digital squamous cell carcinomas in dogs and copy number variation of KIT Ligand — a correlation study JF - Veterinary Sciences N2 - Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC). This may potentially suggest an underlying genetic predisposition not yet completely elucidated. Some authors have suggested a potential correlation between the number of copies KIT Ligand (KITLG) and the predisposition of dogs to DSCC, containing a higher number of copies in those affected by the neoplasm. In this study, the aim was to evaluate a potential correlation between the number of copies of the KITLG and the histological grade of malignancy in dogs with DSCC. For this, 72 paraffin-embedded DSCCs with paired whole blood samples of 70 different dogs were included and grouped according to their haircoat color as follow: Group 0/unknown haircoat color (n = 11); Group 1.a/black non-Schnauzers (n = 15); group 1.b/black Schnauzers (n = 33); group 1.c/black and tan dogs (n = 7); group 2/tan animals (n = 4). The DSCCs were histologically graded. Additionally, KITLG Copy Number Variation (CNV) was determined by ddPCR. A significant correlation was observed between KITLG copy number and the histological grade and score value. This finding may suggest a possible factor for the development of canine DSCC, thus potentially having an impact on personalized veterinary oncological strategies and breeding programs. KW - canine KW - cancer KW - toe KW - grading KW - haircoat KW - color KW - genetics KW - gene Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304824 SN - 2306-7381 VL - 10 IS - 2 ER - TY - JOUR A1 - Dong, Meng A1 - Böpple, Kathrin A1 - Thiel, Julia A1 - Winkler, Bernd A1 - Liang, Chunguang A1 - Schueler, Julia A1 - Davies, Emma J. A1 - Barry, Simon T. A1 - Metsalu, Tauno A1 - Mürdter, Thomas E. A1 - Sauer, Georg A1 - Ott, German A1 - Schwab, Matthias A1 - Aulitzky, Walter E. T1 - Perfusion air culture of precision-cut tumor slices: an ex vivo system to evaluate individual drug response under controlled culture conditions JF - Cells N2 - Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses. KW - precision-cut tumor slices KW - perfusion culture KW - tumor microenvironment KW - ovarian tumor KW - individual drug responses KW - mouse xenografts KW - preclinical model KW - personalized medicine Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311030 SN - 2073-4409 VL - 12 IS - 5 ER - TY - JOUR A1 - de Paz, Víctor A1 - Asís, Josep D. A1 - Holzschuh, Andrea A1 - Baños-Picón, Laura T1 - Effects of traditional orchard abandonment and landscape context on the beneficial arthropod community in a Mediterranean agroecosystem JF - Insects N2 - Agricultural abandonment is one of the main land-use changes in Europe, and its consequences on biodiversity are context- and taxa-dependent. While several studies have worked on this topic, few have focused on traditional orchards, especially in different landscapes and under a Mediterranean climate. In this context, we aimed to determine the effects of almond orchard abandonment on the communities of three groups of beneficial arthropods and the role of the landscape context in modulating these effects. Between February and September 2019, four samplings were carried out in twelve almond orchards (three abandoned and three traditional (active orchards under traditional agricultural management) located in simple landscapes as well as three abandoned and three traditional in complex landscapes). Abandoned and traditional almond orchards harbor different arthropod communities and diversity metrics that are strongly conditioned by seasonality. Abandoned orchards can favor pollinators and natural enemies, providing alternative resources in simple landscapes. However, the role that abandoned orchards play in simple landscapes disappears as the percentage of semi-natural habitats in the landscape increases. Our results show that landscape simplification, through the loss of semi-natural habitats, has negative consequences on arthropod biodiversity, even in traditional farming landscapes with small fields and high crop diversity. KW - abandonment KW - traditional almond orchard KW - spider KW - parasitoid KW - bee KW - landscape complexity Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311190 SN - 2075-4450 VL - 14 IS - 3 ER - TY - JOUR A1 - Wersebeckmann, Vera A1 - Biegerl, Carolin A1 - Leyer, Ilona A1 - Mody, Karsten T1 - Orthopteran diversity in steep slope vineyards: the role of vineyard type and vegetation management JF - Insects N2 - The abandonment of traditional agricultural practices and subsequent succession are major threats to many open-adapted species and species-rich ecosystems. Viticulture on steep slopes has recently suffered from strong declines due to insufficient profitability, thus increasing the area of fallow land considerably. Changing cultivation systems from vertically oriented to modern vineyard terraces offers an opportunity to maintain management economically viable and thus reduces further abandonment. Hillside parallel terraces favor mechanization, and their embankments offer large undisturbed areas that could provide valuable habitats. We investigated the effects of vineyard abandonment, different vineyard management types (vertically oriented vs. terraced), and local parameters on Orthoptera diversity in 45 study sites along the Upper Middle Rhine Valley in Germany. Our results show that woody structures and vineyard abandonment reduced Orthoptera diversity at the local and landscape scale due to decreased habitat quality, especially for open-adapted species. In contrast, open inter-rows of actively managed vineyard types supported heat-adapted Caelifera species. On terrace embankments, extensive management and taller vegetation benefited Ensifera species, while short and mulched vegetation in vertically oriented vineyards favored the dominance of one single Caelifera species. Our results highlight the significance of maintaining viticultural management on steep slopes for the preservation of both open-adapted Orthoptera species and the cultural landscape. KW - abandonment KW - alternating management KW - biodiversity conservation KW - grasshopper KW - insect conservation KW - succession KW - traditional land use KW - vineyard terrace Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304891 SN - 2075-4450 VL - 14 IS - 1 ER - TY - JOUR A1 - Reibetanz, Joachim A1 - Kelm, Matthias A1 - Uttinger, Konstantin L. A1 - Reuter, Miriam A1 - Schlegel, Nicolas A1 - Hankir, Mohamed A1 - Wiegering, Verena A1 - Germer, Christoph-Thomas A1 - Fassnacht, Martin A1 - Lock, Johan Friso A1 - Wiegering, Armin T1 - Differences in morbidity and mortality between unilateral adrenalectomy for adrenal Cushing’s syndrome and bilateral adrenalectomy for therapy refractory extra-adrenal Cushing’s syndrome JF - Langenbeck’s Archives of Surgery N2 - Purpose In selected cases of severe Cushing’s syndrome due to uncontrolled ACTH secretion, bilateral adrenalectomy appears unavoidable. Compared with unilateral adrenalectomy (for adrenal Cushing’s syndrome), bilateral adrenalectomy has a perceived higher perioperative morbidity. The aim of the current study was to compare both interventions in endogenous Cushing’s syndrome regarding postoperative outcomes. Methods We report a single-center, retrospective cohort study comparing patients with hypercortisolism undergoing bilateral vs. unilateral adrenalectomy during 2008–2021. Patients with adrenal Cushing’s syndrome due to adenoma were compared with patients with ACTH-dependent Cushing’s syndrome (Cushing’s disease and ectopic ACTH production) focusing on postoperative morbidity and mortality as well as long-term survival. Results Of 83 patients with adrenalectomy for hypercortisolism (65.1% female, median age 53 years), the indication for adrenalectomy was due to adrenal Cushing’s syndrome in 60 patients (72.2%; 59 unilateral and one bilateral), and due to hypercortisolism caused by Cushing’s disease (n = 16) or non-pituitary uncontrolled ACTH secretion of unknown origin (n = 7) (27.7% of all adrenalectomies). Compared with unilateral adrenalectomy (n = 59), patients with bilateral adrenalectomy (n = 24) had a higher rate of severe complications (0% vs. 33%; p < 0.001) and delayed recovery (median: 10.2% vs. 79.2%; p < 0.001). Using the MTL30 marker, patients with bilateral adrenalectomy fared worse than patients after unilateral surgery (MTL30 positive: 7.2% vs. 25.0% p < 0.001). Postoperative mortality was increased in patients with bilateral adrenalectomy (0% vs. 8.3%; p = 0.081). Conclusion While unilateral adrenalectomy for adrenal Cushing’s syndrome represents a safe and definitive therapeutic option, bilateral adrenalectomy to control ACTH-dependent extra-adrenal Cushing’s syndrome or Cushing’s disease is a more complicated intervention with a mortality of nearly 10%. KW - Cushing KW - adrenal surgery KW - MTL30 KW - complication Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323947 VL - 407 IS - 6 ER - TY - JOUR A1 - Köhler, Franziska A1 - Reese, Lena A1 - Hendricks, Anne A1 - Kastner, Carolin A1 - Müller, Sophie A1 - Lock, Johan F. A1 - Germer, Christoph-Thomas A1 - Wiegering, Armin T1 - Low-grade mucinous neoplasms (LAMN) of the appendix in Germany between 2011 and 2018: a nationwide analysis based on data provided by the German Center for Cancer Registry Data (ZfKD) at the Robert Koch Institute (RKI) JF - Langenbeck’s Archives of Surgery N2 - Introduction Low-grade appendiceal mucinous neoplasms (LAMN) are semi-malignant tumors of the appendix which are incidentally found in up to 1% of appendectomy specimen. To this day, no valid descriptive analysis on LAMN is available for the German population. Methods Data of LAMN (ICD-10: D37.3) were collected from the population-based cancer registries in Germany, provided by the German Center for Cancer Registry Data (Zentrum für Krebsregisterdaten—ZfKD). Data was anonymized and included gender, age at diagnosis, tumor staging according to the TNM-classification, state of residence, information on the performed therapy, and survival data. Results A total of 612 cases were reported to the ZfKD between 2011 and 2018. A total of 63.07% were female and 36.93% were male. Great inhomogeneity in reporting cases was seen in the federal states of Germany including the fact that some federal states did not report any cases at all. Age distribution showed a mean age of 62.03 years (SD 16.15) at diagnosis. However, data on tumor stage was only available in 24.86% of cases (n = 152). A total of 49.34% of these patients presented with a T4-stage. Likewise, information regarding performed therapy was available in the minority of patients: 269 patients received surgery, 22 did not and for 312 cases no information was available. Twenty-four patients received chemotherapy, 188 did not, and for 400 cases, no information was available. Overall 5-year survival was estimated at 79.52%. Patients below the age of 55 years at time of diagnosis had a significantly higher 5-year survival rate compared to patients above the age of 55 years (85.77% vs. 73.27%). Discussion In this study, we observed an incidence of LAMN in 0.13% of all appendectomy specimen in 2018. It seems likely that not all cases were reported to the ZfKD; therefore, case numbers may be considered underestimated. Age and gender distribution goes in line with international studies with females being predominantly affected. Especially regarding tumor stage and therapy in depth information cannot be provided through the ZfKD-database. This data analysis emphasizes the need for further studies and the need for setting up a specialized registry for this unique tumor entity to develop guidelines for the appropriate treatment and follow-up. KW - LAMN KW - low-grade mucinous neoplasm KW - appendix KW - epidemiology KW - ZfKD KW - Germany Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323919 VL - 407 IS - 8 ER - TY - JOUR A1 - Kotlyar, Mischa J. A1 - Krebs, Markus A1 - Solimando, Antonio Giovanni A1 - Marquardt, André A1 - Burger, Maximilian A1 - Kübler, Hubert A1 - Bargou, Ralf A1 - Kneitz, Susanne A1 - Otto, Wolfgang A1 - Breyer, Johannes A1 - Vergho, Daniel C. A1 - Kneitz, Burkhard A1 - Kalogirou, Charis T1 - Critical evaluation of a microRNA-based risk classifier predicting cancer-specific survival in renal cell carcinoma with tumor thrombus of the inferior vena cava JF - Cancers N2 - (1) Background: Clear cell renal cell carcinoma extending into the inferior vena cava (ccRCC\(^{IVC}\)) represents a clinical high-risk setting. However, there is substantial heterogeneity within this patient subgroup regarding survival outcomes. Previously, members of our group developed a microRNA(miR)-based risk classifier — containing miR-21-5p, miR-126-3p and miR-221-3p expression — which significantly predicted the cancer-specific survival (CSS) of ccRCC\(^{IVC}\) patients. (2) Methods: Examining a single-center cohort of tumor tissue from n = 56 patients with ccRCC\(^{IVC}\), we measured the expression levels of miR-21, miR-126, and miR-221 using qRT-PCR. The prognostic impact of clinicopathological parameters and miR expression were investigated via single-variable and multivariable Cox regression. Referring to the previously established risk classifier, we performed Kaplan–Meier analyses for single miR expression levels and the combined risk classifier. Cut-off values and weights within the risk classifier were taken from the previous study. (3) Results: miR-21 and miR-126 expression were significantly associated with lymphonodal status at the time of surgery, the development of metastasis during follow-up, and cancer-related death. In Kaplan–Meier analyses, miR-21 and miR-126 significantly impacted CSS in our cohort. Moreover, applying the miR-based risk classifier significantly stratified ccRCC\(^{IVC}\) according to CSS. (4) Conclusions: In our retrospective analysis, we successfully validated the miR-based risk classifier within an independent ccRCC\(^{IVC}\) cohort. KW - kidney cancer KW - RCC KW - venous infiltration KW - biomarker KW - miR KW - risk stratification Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311040 SN - 2072-6694 VL - 15 IS - 7 ER - TY - INPR A1 - Dandekar, Thomas T1 - Protein folding and crystallization applied to qubit interactions and fundamental physics yields a modified inflation model for cosmology N2 - Protein folding achieves a clear solution structure in a huge parameter space (the so-called protein folding problem). Proteins fold in water, and get by this a highly ordered structure. Finally, inside a protein crystal for structure resolution, you have everywhere the same symmetries as there is everywhere the same unit cell. We apply this to qubit interactions to do fundamental physics: in a modified cosmology, we replace the big bang by a condensation event in an eternal all-encompassing ocean of free qubits. Interactions of qubits in the qubit ocean are quite rare but provide a nucleus or seed for a new universe (domain) as the qubits become decoherent and freeze-out into defined bit ensembles. Second, we replace inflation by a crystallization event triggered by the nucleus of interacting qubits to which rapidly more and more qubits attach (like in everyday crystal growth). The crystal unit cell guarantees same symmetries everywhere inside the crystal. The textbook inflation scenario to explain the same laws of nature in our domain is replaced by the unit cell of the crystal formed. Interacting qubits solidify, quantum entropy decreases (but increases in the ocean around). In a modified inflation scenario, the interacting qubits form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. Then standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements. We explain by cosmological crystallization instead of inflation: early creation of large-scale structure of voids and filaments, supercluster formation, galaxy formation, and the dominance of matter: the unit cell of our crystal universe has a matter handedness avoiding anti-matter. We prove initiation of qubit interactions can only be 1,2,4 or 8-dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. Crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness. The phase space of the crystal agrees with the standard model of the basic four forces for n quanta. It includes all possible ensemble combinations of their quantum states m, a total of n**m states. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients. However, in our four dimensions there is only one bit overlap to neighbor states left (almost solid, only below Planck quantum there is liquidity left). The E8 symmetry of heterotic string theory has six curled-up, small dimensions which help to keep the qubit crystal together and will never expand. Mathematics focusses on the Hurwitz proof applied to qubit interaction, a toy model of qubit interaction and repulsive forces of qubits. Vacuum energy gets appropriate low inside the crystal. We give first energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit decoherence / crystal formation. Scalar fields for color interaction/confinement and gravity are derived from the qubit-interaction field. KW - protein folding KW - crystallization KW - qubit interaction KW - decoherence KW - modified inflation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-346156 ER - TY - THES A1 - Meiser, Elisabeth T1 - Single-molecule dynamics at a bottleneck: a systematic study of the narrow escape problem in a disc T1 - Einzelmoleküldynamik an einem Engpass: Eine systematische Untersuchung des Narrow Escape Problems in einer Scheibe N2 - Diffusion facilitates numerous reactions within the biological context of a cell. It is remarkable how the cost-efficient random process of Brownian motion promotes fast reactions. From the narrow escape theory, it is possible to determine the mean first passage time of such processes based on their reaction space and diffusion coefficient. The narrow escape theory of Brownian particles is characterized by a confining domain with reflective boundaries and a small reaction site. In this thesis, the mean first passage time was systematically tested in a disc as a function of the escape opening size in vitro and in silico. For the in vitro experiments, a model system of patterned supported-lipid bilayers (SLB) was established. Such a model is prepared by a combined colloid metalization approach, where a gold scaffold on glass facilitates assembly of SLB patches of distinct sizes through vesicle fusion. The model setup was evaluated and found to match all necessary requirements to test the nar- row escape problem in vitro. In particular, the reflectivity of the boundaries, the unhindered, free diffusion of the tracer lipids, and the distinct area were assessed. Observed results of the mean first passage time agreed with the theory of the narrow escape problem. There was excellent agreement in both absolute values and across a range of small escape opening sizes. Additionally, I developed a straightforward method, a correction factor, to calculate the mean first passage time from incomplete experimental traces. By re-scaling the mean first passage time to the fraction of particles that escaped, I was able to overcome the lifetime limitations of fluorescent probes. Previously inaccessible measurements of the mean first passage time relying on fluorescent probes will be made possible through this approach. The in vitro experiments were complemented with various in silico experiments. The latter were based on random walk simulations in discs, mimicking the in vitro situation with its uncertainties. The lifetime of single particles was either set sufficiently long to allow all particles to escape, or was adjusted to meet the lifetime limitations observed in the in vitro experiments. A comparison of the mean first passage time from lifetime-unlimited particles to the corrected, lifetime-limited particles did support the use of the correction factor. In agreement with the narrow escape theory, it was experimentally found that the mean first passage time is independent of the start point of the particle within the domain. This is when the particle adheres to a minimum distance to the escape site. In general, the presented random walk simulations do accurately represent the in vitro experiments in this study. The required hardware for the establishment of an astigmatism-based 3D system was installed in the existing microscope. The first attempts to analyze the obtained 3D imaging data gave insight into the potential of the method to investigate molecule dynamics in living trypanosome cells. The full functionality will be realized with the ongoing improvement of image analysis outside of this thesis. N2 - Diffusion erleichtert zahlreiche Reaktionen im biologischen Kontext einer Zelle. Es ist bemerkenswert, wie der kosteneffiziente Zufallsprozess der Brownschen Bewegung schnelle Reaktionen fördert. Mit Hilfe der Narrow Escape Theorie kann die mittlere erste Durchgangszeit (mean first passage time) solcher Prozesse auf der Grundlage ihres Reaktionsraums und des Diffusionskoeffizienten bestimmt werden. Die Narrow Escape Theorie von Brownschen Teilchen wird durch einen begrenzten Bereich mit reflektierenden Grenzen und einen kleinen Reaktionsraum gekennzeichnet. In dieser Arbeit wurde die mittlere erste Durchgangszeit in einer Scheibe systematisch als Funktion der Größe der Fluchtöffnung in vitro und in silico untersucht. Fur die in vitro-Experimente wurde ein Modellsystem aus strukturierten, Glas gestützten Lipiddoppelschichten (SLB) erstellt. Dieses Modell wurde durch einen kombinierten Kolloid-Metallisierungsansatz hergestellt, bei dem eine strukturierte Goldschicht auf Glas die Bildung von SLB-Scheiben unterschiedlicher Größe durch die Vesikelfusion ermöglicht. Es wurde festgestellt, dass das Modell alle Anforderungen erfüllt, um das Narrow escape problem in vitro zu testen. Insbesondere wurde das Reflexionsvermögen der Grenzen, die ungehinderte, freie Diffusion der Lipide und die Präzision der Fläche bewertet. Die beobachteten Ergebnisse der mittleren ersten Durchgangszeit stimmen mit der NEP-Theorie ̈uberein. Es besteht eine hervorragende ̈Ubereinstimmung sowohl bei den absoluten Werten als auch systematisch ̈uber einen Bereich von kleinen Fluchtöffnungsgrößen. Außerdem zeige ich eine einfache Methode, einen Korrekturfaktor, zur Berechnung der mittleren ersten Durchgangszeit aus unvollstandigen Lipid Trajektorien des Experimentes. Indem ich die mittlere erste Durchgangszeit auf den Anteil der entkommenen Par- tikel skalierte, konnte ich die Einschränkungen der Lebensdauer von fluoreszenten Farbstoffen ausgleichen. Mit dieser Technik werden bisher unzugängliche Messungen der mittleren ersten Durchgangszeit auf der Grundlage von fluoreszenten Farbstoffen möglich. In einem umfassenden Ansatz wurden die in vitro-Experimente durch verschiedene in silico-Experimente ergänzt. Letztere basierten auf Random- Walk-Simulationen in Scheiben, die die in vitro-Situation mit ihren Unsicherheiten nachahmten. Die Lebensdauer einzelner Partikel wurde entweder so lang angesetzt, dass alle Partikel entkommen konnten, oder sie wurde so angepasst, dass sie den in den in vitro-Experimenten beobachteten Lebensdauerbeschränkungen entsprach. Ein Vergleich der mittleren ersten Durchgangszeit von unsterblichen Partikeln mit den korrigierten, lebensdauerbegrenzten Partikeln hat die Verwendung des Korrekturfaktors bestätigt. In ̈Ubereinstimmung mit der Narrow Escape Theorie wurde experimentell festgestellt, dass die mittlere erste Durchgangszeit unabhängig vom Startpunkt des Teilchens innerhalb der Domäne ist. Dies ist dann der Fall, wenn das Teilchen einen Mindestabstand zur Austrittsstelle einhält. Im Allgemeinen bilden die vorgestellten Random-Walk-Simulationen die in dieser Studie durchgeführten Experimente genau ab. Die erforderliche Hardware für die Einrichtung eines auf Astigmatismus basierenden 3D-Systems wurde in ein vorhandenes Mikroskop eingebaut. Erste Versuche, die gewonnenen 3D-Bilddaten zu analysieren, gaben einen Einblick in das Potenzial der Methode zur Untersuchung der Moleküldynamik im lebenden Trypanosom. Die volle Funktionalität wird mit der laufenden Verbesserung der Bildanalyse außerhalb dieser Arbeit realisiert werden. Ein Teil der Ergebnisse und Methoden dieser Dissertation wurde zur Veröffentlichung unter dem Titel ’Experiments in micro-patterned model membranes support the narrow escape theory’ eingereicht. KW - Freies Molekül KW - Kolloid KW - Bimolekulare Lipidschicht KW - Mikroskopie KW - Brownsche Bewegung KW - Narrow escape problem KW - mean first passage time KW - single-molecule localization microscopy KW - single particle tracking KW - Random-walk simulations Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319650 ER - TY - THES A1 - Reuter, Christian Steffen T1 - Development of a tissue-engineered primary human skin infection model to study the pathogenesis of tsetse fly-transmitted African trypanosomes in mammalian skin T1 - Entwicklung eines primären humanen Hautinfektionsmodells basierend auf Gewebezüchtung zur Erforschung der Pathogenese von Tsetsefliegen-übertragenen Afrikanischen Trypanosomen in der Säugetierhaut N2 - Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host. Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression. A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts. The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes. In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection. After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host. In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods. N2 - Zahlreiche Arthropoden wie Stechmücken, Zecken, Wanzen und Fliegen sind Überträger für krankheitserregende Parasiten, Bakterien und Viren. Hierzu gehört der einzellige Parasit Trypanosoma brucei (T. brucei), welcher durch Tsetsefliegen übertragen wird und die Afrikanische Trypanosomiasis bei Menschen und Tieren verursacht. Der Entwicklungszyklus des Parasiten in der Fliege ist komplex und endet in der Speicheldrüse mit der Differenzierung in das metazyklische Lebensstadium. Diese metazyklischen Formen werden durch den Biss der blutsaugenden Tsetsefliege in die dermale Hautschicht des Säugetierwirts injiziert. Die zellzyklusarretierten metazyklischen Formen werden in der Dermis aktiviert und der Widereintritt in den Zellzyklus sowie die Differenzierung zu proliferativen Trypanosomen eingeleitet. Anschließend breitet sich der Parasit systemisch im Säugetierwirt aus. Obwohl T. brucei bereits seit Jahrzehnten erforscht wird, ist nur sehr wenig über das frühe Infektionsgeschehen in der Haut bekannt. Der genaue Zeitpunkt und die Mechanismen, die der Differenzierung des Parasiten in der Haut zugrunde liegen, sind unbekannt. Ebenso wurden die proliferativen Trypanosomen in der Haut bisher nur unzureichend charakterisiert. Das Verständnis über die ersten Schritte einer Infektion ist jedoch von entscheidender Bedeutung für die Entwicklung von neuen Strategien, die die Krankheitsentstehung und deren Fortschreiten verhindern sollen. Ein großes Hindernis bei der Erforschung der humanen Afrikanischen Trypanosomiasis ist der Mangel an geeigneten Infektionsmodellen, die den Krankheitsverlauf authentisch nachbilden. Außerdem werden für die Erzeugung der infektiösen metazyklischen Parasiten Tsetsefliegen benötigt, die aufwändig zu züchten sind. Tiermodelle haben es ermöglicht - hauptsächlich Mäuse -, viele Erkenntnisse über die Pathogenese von Trypanosomen im Säugetierwirt zu erlangen. Allerdings wurden diese überwiegend durch Nadelinjektion in den Bauchraum oder die Kaudalvene infiziert, wodurch die Haut als erste Eintrittspforte umgangen wurde. Darüber hinaus lassen Tiermodelle nicht immer Rückschlüsse auf den Infektionsverlauf beim Menschen zu. Zusätzlich erschwert die geringe Anzahl von metazyklischen Parasiten, die von Tsetsefliegen injiziert werden, die Isolation, Nachweis und Untersuchung im tierischen Wirt. Das Ziel der vorliegenden Arbeit war es, ein rekonstruiertes menschliches Hautäquivalent zu entwickeln und als Infektionsmodell zu validieren, um die Entwicklung von natürlich übertragenen metazyklischen Parasiten von T. brucei in der Säugetierhaut zu untersuchen. Der erste Teil dieser Arbeit beschreibt die Entwicklung und Charakterisierung eines primären menschlichen Hautäquivalents mit verbesserten mechanischen Eigenschaften. Zu diesem Zweck wurde ein computergesteuertes Kompressionssystem entworfen und hergestellt. Dieses System ermöglichte die gleichzeitige Verbesserung der mechanischen Stabilität von zwölf kollagenbasierten dermalen Äquivalenten durch plastische Kompression, die mittels Rheologie evaluiert wurden. Die verbesserten dermalen Äquivalente dienten als Fundament für die Erzeugung der Hautäquivalente und reduzierten deren Kontraktion und Gewichtsverlust während der Gewebebildung. Dadurch wurde ein hohes Maß an Standardisierung und Reproduzierbarkeit erreicht. Die Hautäquivalente wurden durch immunhistochemische und histologische Techniken charakterisiert und bildeten wichtige anatomische, zelluläre und funktionelle Aspekte der nativen menschlichen Haut nach. Des Weiteren wurde die zelluläre Heterogenität durch Einzelzell-RNA-Sequenzierung untersucht. Mit dieser Technik wurde ein umfangreiches Spektrum an extrazellulären Matrix-assoziierten Genen identifiziert, die von verschiedenen Zellsubpopulationen in der künstlichen Haut exprimiert werden. Zusätzlich wurden experimentelle Bedingungen etabliert, damit Tsetsefliegen eingesetzt werden konnten, um die Hautäquivalente auf natürlichem Weg mit Trypanosomen zu infizieren. Im zweiten Teil dieser Arbeit wurde die Entwicklung der Trypanosomen in der künstlichen Haut im Detail untersucht. Dies umfasste die Etablierung von Methoden zur erfolgreichen Isolierung der Trypanosomen aus der Haut, um deren Proteinsyntheserate, Zellzyklus- und Stoffwechselstatus, sowie Morphologie und Transkriptom zu bestimmen. Zusätzlich wurden Mikroskopietechniken zur Untersuchung der Trypanosomenmotilität und migration in der Haut optimiert. Nach der Injektion in die künstliche Haut durch Tsetsefliegen wurden die metazyklischen Parasiten schnell aktiviert und etablierten innerhalb eines Tages eine proliferative Population. Dieser Entwicklungsprozess wurde begleitet von (I) einer Reaktivierung der Proteinsynthese, (II) einem Wiedereintritt in den Zellzyklus, (III) einer Veränderung der Morphologie und (IV) einer erhöhten Motilität. Des Weiteren wurden diese Beobachtungen mit potentiell zugrundeliegenden entwicklungsbiologischen Mechanismen in Verbindung gebracht, indem eine Einzelzell RNA-Sequenzierung der Trypanosomen zu fünf verschiedenen Zeitpunkten nach der Infektion durchgeführt wurde. Nach der ersten proliferativen Phase traten die Tsetse-übertragenen Trypanosomen in der Haut in ein reversibles Ruhestadium ein. Diese ruhenden Trypanosomen waren durch eine sehr langsame Zellteilung, einen stark reduzierten Stoffwechsel und ein Transkriptom gekennzeichnet, dass sich deutlich von dem der injizierten metazyklischen Formen und der ersten proliferativen Trypanosomen unterschied. Durch Nachahmung der Migration von der Haut in den Blutkreislauf konnte dieser Phänotyp reaktiviert werden und die Parasiten kehrten in einen aktiven, proliferierenden Zustand zurück. Unter Berücksichtigung, dass vorangegangene Forschungsarbeiten die Haut als anatomisches Reservoir für T. brucei während des Krankheitsverlaufs identifiziert haben, ist anzunehmen, dass das Ruheprogramm eine authentische Facette im Verhalten des Parasiten in einem infizierten Wirt darstellt. Zusammenfassend zeigt diese Arbeit, das primäre menschliche Hautäquivalente eine neue und vielversprechende Möglichkeit bieten, vektorübertragene Parasiten unter naturnahen Bedingungen als Alternative zu Tierversuchen zu untersuchen. Durch die Verwendung des natürlichen Infektionsweges - dem Biss einer infizierten Tsetsefliege -, konnten die frühen Prozesse einer Trypanosomen-Infektion mit noch nie dagewesener Detailtiefe nachvollzogen werden. Des Weiteren könnte der hier erbrachte Nachweis einer ruhenden, hautresidenten Trypanosomen-Population die Persistenz von T. brucei in der Haut von aparasitämischen und asymptomatischen Personen erklären. Dies könnte eine wichtige Rolle bei der Aufrechterhaltung einer Infektion über lange Zeiträume spielen. KW - Trypanosoma brucei KW - Tissue Engineering KW - Trypanosomiasis KW - 3D-Zellkultur KW - Transkriptomanalyse KW - developmental differentiation KW - skin equivalent KW - artificial human skin KW - single-cell RNA sequencing KW - quiescence Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251147 ER - TY - THES A1 - Gotthard, Hannes T1 - Targeting Colorectal Cancer Stem Cells with Hemibodies T1 - Eliminierung von Krebsstammzellen des kolorektalen Karzinoms mithilfe von Hemibodies N2 - The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration. N2 - In den letzten Jahrzenten wurde neben der klonalen Evolution ein weiteres Modell zur Krebsentstehung und dessen Heterogenität entwickelt: die Krebsstammzellhypothe-se. Diese Hypothese besagt, dass die Heterogenität eines Tumors durch asymmetri-sche Teilung von sogenannten Krebsstammzellen entsteht. Nur diese sind tumorigen und in der Lage Metastasen zu bilden. Außerdem werden Krebsstammzellen als re-sistent gegen konventionelle Therapien beschrieben, weshalb es nach einer anfängli-chen Tumorregression oft zu einem Rezidiv durch erneutes Auswachsen von zurück-bleibenden Krebsstammzellen kommt. Deshalb ist es von großem Interesse genau diese Population abzutöten, um eine erfolgreiche Therapie zu gewährleisten. In den letzten Jahren wurden zahlreiche Medikationen entwickelt, um Krebsstammzellen ge-zielt anzugreifen. Ein vielversprechender Ansatz ist hierbei die immuntherapeutische Adressierung mittels Antikörpern gegen Krebsstammzellmarkern. Einzelne Marker sind allerdings auch auf normalen Stammzellen und gesundem Gewebe exprimiert, weshalb Therapien, die auf mindestens zwei verschiedene Oberflächenproteine ab-zielen, erfolgsversprechender sind. In dieser Arbeit wurde ein neues T-Zell rekrutie-rendes Antikörperformat entwickelt, sogenannte Hemibodies. Hierbei handelt es sich um ein trispezifisches und trivalentes Format, bestehend aus jeweils zwei Fragmen-ten. Jedes Fragment besteht aus einer Bindedomäne gegen ein Krebsstammzellmar-ker und einer geteilten Bindedomäne gegen CD3. Durch Bindung beider Fragmente an einen Stammzellmarker kommt es zur Komplementierung der geteilten anti-CD3 Domäne und zur T-Zellrekrutierung. Der erste Teil der Arbeit befasst sich mit der bioin-formatischen Analyse von Einzelzell-RNA-Daten des kolorektalen Karzinoms (KRK) zur Identifizierung von potentiellen Krebsstammzellmarkern. Dabei konnten die Ober-flächenproteine CD24, CD133, CD166 und CEA und besonders deren Kombination als geeignete Zielstrukturen identifiziert werden. Die gegen oben genannte Antigene gerichteten Hemibodies zeigten in den Kombinationen CD133xCD24, CD133xCD166 und CD133xCEA auf doppelt positiven CHO-Zellen eine hohe Effektivität. Außerdem konnte die Spezifität durch ein Ausbleiben von Zelltod auf einzel-positiven CHO Zellen bewiesen werden. Die Kombinationen CD133xCD24 und CD133xCD166 konnten Effektivität und Spezifität auch auf etablierten Krebszellen zeigen. Die oben genann-ten Kombinationen waren in einem therapeutischen Fenster von ein bis zwei Logstu-fen funktional. Neben der Testung verschiedener Hemibody-Kombinationen konnten die bereits publizierten Hemibodies der ersten Generation in ein neues Format der zweiten Generation weiterentwickelt werden. Das neue Format zeigte eine verbesser-te Halbwertszeit, Stabilität und Produzierbarkeit. In zukünftigen Experimenten werden die in der Thesis benutzten Hemibodies auf Mikrotumoren getestet, um weitere Vari-ablen, die die Effektivität und Spezifität beeinflussen zu ermitteln. KW - Monoklonaler bispezifischer Antikörper KW - Antikörper KW - T-Lymphozyt KW - Immunreaktion KW - Dickdarmkrebs KW - Hemibody KW - Hemibodies KW - Colorectal Cancer KW - trispecific KW - T-cell engager KW - dual targeting KW - Bispecific T-cell engager KW - stem cells KW - Kolorektales Karzinom Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-303090 ER - TY - JOUR A1 - Brenner, Daniela A1 - Geiger, Nina A1 - Schlegel, Jan A1 - Diesendorf, Viktoria A1 - Kersting, Louise A1 - Fink, Julian A1 - Stelz, Linda A1 - Schneider-Schaulies, Sibylle A1 - Sauer, Markus A1 - Bodem, Jochen A1 - Seibel, Jürgen T1 - Azido-ceramides, a tool to analyse SARS-CoV-2 replication and inhibition — SARS-CoV-2 is inhibited by ceramides JF - International Journal of Molecular Sciences N2 - Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication. KW - ceramides KW - SARS-CoV-2 KW - azido-ceramides KW - sphingolipids Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313581 SN - 1422-0067 VL - 24 IS - 8 ER - TY - JOUR A1 - Henriksson, Sofia A1 - Calderón-Montaño, José Manuel A1 - Solvie, Daniel A1 - Warpman Berglund, Ulrika A1 - Helleday, Thomas T1 - Overexpressed c-Myc sensitizes cells to TH1579, a mitotic arrest and oxidative DNA damage inducer JF - Biomolecules N2 - Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation. KW - MTH1 KW - TH588 KW - TH1579 KW - c-Myc KW - replication stress KW - DNA damage KW - cell death KW - cancer Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297547 SN - 2218-273X VL - 12 IS - 12 ER - TY - JOUR A1 - Rohmer, Carina A1 - Dobritz, Ronja A1 - Tuncbilek-Dere, Dilek A1 - Lehmann, Esther A1 - Gerlach, David A1 - George, Shilpa Elizabeth A1 - Bae, Taeok A1 - Nieselt, Kay A1 - Wolz, Christiane T1 - Influence of Staphylococcus aureus strain background on Sa3int phage life cycle switches JF - Viruses N2 - Staphylococcus aureus asymptomatically colonizes the nasal cavity of mammals, but it is also a leading cause of life-threatening infections. Most human nasal isolates carry Sa3 phages, which integrate into the bacterial hlb gene encoding a sphingomyelinase. The virulence factor-encoding genes carried by the Sa3-phages are highly human-specific, and most animal strains are Sa3 negative. Thus, both insertion and excision of the prophage could potentially confer a fitness advantage to S. aureus. Here, we analyzed the phage life cycle of two Sa3 phages, Φ13 and ΦN315, in different phage-cured S. aureus strains. Based on phage transfer experiments, strains could be classified into low (8325-4, SH1000, and USA300c) and high (MW2c and Newman-c) transfer strains. High-transfer strains promoted the replication of phages, whereas phage adsorption, integration, excision, or recA transcription was not significantly different between strains. RNASeq analyses of replication-deficient lysogens revealed no strain-specific differences in the CI/Mor regulatory switch. However, lytic genes were significantly upregulated in the high transfer strain MW2c Φ13 compared to strain 8325-4 Φ13. By transcriptional start site prediction, new promoter regions within the lytic modules were identified, which are likely targeted by specific host factors. Such host-phage interaction probably accounts for the strain-specific differences in phage replication and transfer frequency. Thus, the genetic makeup of the host strains may determine the rate of phage mobilization, a feature that might impact the speed at which certain strains can achieve host adaptation. KW - phage KW - virulence KW - induction KW - gene regulation KW - Staphylococcus KW - hemolysin Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297209 SN - 1999-4915 VL - 14 IS - 11 ER - TY - THES A1 - Kohl, Patrick Laurenz T1 - The buzz beyond the beehive: population demography, parasite burden and limiting factors of wild-living honeybee colonies in Germany T1 - Das Summen fern des Bienenstocks: Populationsdemographie, Parasitenlast und limitierende Faktoren wildlebender Honigbienenvölker in Deutschland N2 - The western honeybee (Apis mellifera) is widely known as the honey producer and pollinator managed by beekeepers but neglected as a wild bee species. Central European honeybee populations have been anthropogenically disturbed since about 1850 through introgression and moderate artificial selection but have never been truly domesticated due to a lack of mating control. While their decline in the wild was historically attributed to the scarcity of nesting cavities, a contemporary view considers the invasion of the parasitic mite Varroa destructor in the 1970s as the major driver. However, there are no longitudinal population data available that could substantiate either claim. Based on the insight that introduced European honeybees form viable wild populations in eastern North America and reports on the occurrence of wild-living colonies from various European countries, we systematically studied the ecology of wild-living honeybees in Germany. First, we investigated whether wild-living honeybees colonising German forests form a self-sustaining population. Second, we asked how the parasite burden of wild-living colonies relates to that of managed colonies. And third, we explored whether the winter mortality of wild-living colonies is associated with parasite burden, nest depredation, or the lack of resources on the landscape scale. Between 2017 and 2021, we monitored listed trees with black woodpecker cavities for honeybees in the managed forests of three study regions (Swabian Alb, counties Coburg and Lichtenfels, county Weilheim-Schongau). Continuity of occupation was determined using microsatellite genetic markers. Wild-living colonies predictably colonised forests in summer, when about 10% of all cavities were occupied. The annual colony survival rate and colony lifespan (based on N=112 colonies) were 10.6% and 0.6 years, with 90% of colonies surviving summer (July–September), 16% surviving winter (September–April), and 72% surviving spring (April–July). The average maximum and minimum colony densities were 0.23 (July) and 0.02 (April) colonies per km^2. During the (re-)colonisation of forests in spring, swarms preferred cavities that had already been occupied by other honeybee colonies. We estimate the net reproductive rate of the population to be R0= 0.318, meaning that it is currently not self-sustaining but maintained by the annual immigration of swarms from managed hives. The wild-living colonies are feral in a behavioural sense. We compared the occurrence of 18 microparasites among feral colonies (N=64) and managed colonies (N=74) using qPCR. Samples were collected in four regions (the three regions mentioned above and the city of Munich) in July 2020; they consisted of 20 workers per colony captured at flight entrances. We distinguished five colony types representing differences in colony age and management histories. Besides strong regional variation, feral colonies consistently hosted fewer microparasite taxa (median: 5, range 1–8) than managed colonies (median: 6, range 4–9) and had different parasite communities. Microparasites that were notably less prevalent among feral colonies were Trypanosomatidae, Chronic bee paralysis virus, and Deformed wing viruses A and B. In the comparison of five colony types, parasite burden was lowest in newly founded feral colonies, intermediate in overwintered feral colonies and managed nucleus colonies, and highest in overwintered managed colonies and hived swarms. This suggests that the natural mode of colony reproduction by swarming, which creates pauses in brood production, and well-dispersed nests, which reduce horizontal transmission, explain the reduced parasite burden in feral compared to managed colonies. To explore the roles of three potential drivers of feral colony winter mortality, we combined colony observations gathered during the monitoring study with data on colony-level parasite burden, observations and experiments on nest depredation, and landscape analyses. There was no evidence for an effect of summertime parasite burden on subsequent winter mortality: colonies that died (N=57) did not have a higher parasite burden than colonies that survived (N=10). Camera traps (N=15) installed on cavity trees revealed that honeybee nests are visited by a range of vertebrate species throughout the winter at rates of up to 10 visits per week. Four woodpecker species, great tits, and pine martens acted as true nest depredators. The winter survival rate of colonies whose nest entrances were protected by screens of wire mesh (N=32) was 50% higher than that of colonies with unmanipulated entrances (N=40). Analyses of land cover maps revealed that the landscapes surrounding surviving colonies (N=19) contained on average 6.4 percentage points more resource-rich cropland than landscapes surrounding dying colonies (N=94). We estimate that tens of thousands of swarms escape from apiaries each year to occupy black woodpecker cavities and other hollow spaces in Germany and that feral colonies make up about 5% of the regional honeybee populations. They are unlikely to contribute disproportionately to the spread of bee diseases. Instead, by spatially complementing managed colonies, they contribute to the pollination of wild plants in forests. Honeybees occupying tree cavities likely have various effects on forest communities by acting as nest site competitors or prey, and by accumulating biomass in tree holes. Nest depredation (a consequence of a lack of well-protected nest sites) and food resource limitation seem to be more important than parasites in hampering feral colony survival. The outstanding question is how environmental and intrinsic factors interact in preventing population establishment. Nest boxes with movable frames could be used to better study the environmental drivers of feral colonies’ mortality. Pairs of wild (self-sustaining) and managed populations known to exist outside Europe could provide answers to whether modern apiculture creates honeybee populations maladapted to life in the wild. In Europe, large continuous forests might represent evolutionary refuges for wild honeybees. N2 - Die Honigbiene (Apis mellifera) ist als Nutztier weitbekannt, doch als Wildtier vernachlässigt. Seit etwa 1850 sind ihre Populationen in Mitteleuropa durch Introgression und moderate künstliche Selektion vom Menschen beeinflusst. Die Art wurde jedoch aufgrund fehlender Paarungskontolle nie wirklich domestiziert. Früher wurde der Rückgang wildlebender Honigbienen dem Verlust geeigneter Nistplätze zugeschrieben. Heute wird meist die Bienenmilbe Varroa destructor als Hauptursache angenommen. Es gibt allerdings keine Langzeitdaten, welche diese Annahmen stützen könnten. Basierend auf der Erkenntnis, dass eingeführte Honigbienen in Nordamerika stabile wilde Populationen bilden, und aufgrund von Berichten über das Vorkommen wildlebender Bienenvölker in verschiedenen Ländern Europas, widmeten wir uns dem systematischen Studium wildlebender Honigbienen in Deutschland. Zunächst untersuchten wir, ob waldbewohnende Bienenvölker eine selbsterhaltende Population bilden. Zweitens stellten wir die Frage, inwiefern sich wildlebende und imkerlich gehaltene Völker in ihrer Parasitenlast unterscheiden. Drittens testeten wir, ob Winterverluste wildlebender Bienenvölker mit Parasitendruck, Nestprädation oder mangelndem Nahrungsangebot auf Landschaftsebene in Verbindung stehen. In Wirtschaftswäldern dreier Untersuchungsgebiete (Schwäbische Alb, Landkreise Coburg und Lichtenfels, Landkreis Weilheim-Schongau) kontrollierten wir zwischen 2017 und 2021 bekannte Höhlenbäume des Schwarzspechts auf Besiedlung durch Honigbienen. Das Überleben einzelner Bienenvölker wurde zusätzlich mittels Analyse von Mikrosatelliten DNA überprüft. Nach verlässlichem Muster besiedelten Honigbienen jeden Sommer etwa 10% der Baumhöhlen. Die jährliche Überlebensrate und die Lebenserwartung der Völker (N=112) betrugen 10,6% und 0,6 Jahre, wobei 90% den Sommer (Juli–September), 16% den Winter (September–April) und 72% das Frühjahr (April–Juli) überlebten. Die durchschnittliche maximale (Juli) und minimale (April) Koloniedichte betrug 0,23 bzw. 0,02 Bienenvölker pro km^2. Während der (Wieder)Besiedlung von Wäldern im Frühjahr bevorzugten Bienenschwärme solche Baumhöhlen, welche zuvor schon von Bienen besiedelt worden waren. Die Nettoreproduktionsrate der wildlebenden Population wird auf R0= 0,318 geschätzt, was bedeutet, dass diese zurzeit nicht selbsterhaltend ist, sondern durch die jährliche Einwanderung von Bienenschwärmen aus der Imkerei aufrechterhalten wird. Wir untersuchten wildlebende (N=64) und imkerlich gehaltene Bienenvölker (N=74) auf den Befall mit 18 verschiedenen Mikroparasiten mittels qPCR. Die Proben stammten aus den drei oben genannten Gebieten sowie aus dem Stadtgebiet von München. Eine Probe bestand aus 20 Arbeiterinnen, welche am Flugloch gefangen wurden. Wir unterschieden fünf Kolonietypen aufgrund des Alters (jünger oder älter als ein Jahr) und der unmittelbaren Geschichte der Bewirtschaftung durch Imkerinnen und Imker. Abgesehen von regionalen Unterschieden in der Parasitenlast waren wildlebende Völker mit einer geringeren Anzahl Parasitentaxa befallen (Median: 5, Spanne: 1–8) als imkerlich gehaltene Völker (Median: 6, Spanne: 4–9) und wiesen eine veränderte Zusammensetzung von Parasiten auf. Seltener bei wildlebenden Bienenvölkern waren besonders Trypanosomatidae, das Chronische-Paralysevirus, sowie die Flügeldeformationsviren A und B. Im Vergleich der fünf Kolonietypen war die Parasitenlast bei neu gegründeten wildlebenden Völkern am geringsten, intermediär bei überwinterten wildlebenden Völkern und Brutablegern, und am höchsten bei überwinterten Wirtschaftsvölkern und bei durch Schwärme gegründeten imkerlich gehaltenen Völkern. Dies deutet darauf hin, dass das Schwärmen (Entstehung von Brutpausen) sowie die größere Distanz zwischen Nestern (Verminderung der horizontalen Krankheitsübertragung) die geringere Parasitenlast wildlebender Bienenvölker erklären. Wir kombinierten Beobachtungen zum Winterüberleben aus dem Monitoring mit Daten zur Parasitenlast, mit Beobachtungen und Experimenten zur Nestprädation und mit Landschaftsanalysen. Es ergab sich kein Hinweis auf einen Zusammenhang zwischen Parasitenlast im Sommer und anschließendem Überwinterungserfolg: Völker, welche den Winter nicht überlebten (N=57), hatten zuvor keine höhere Parasitenlast als solche, welche den Winter überlebten (N=10). Kamerafallen (N=15) offenbarten, dass Honigbienennester im Winter von einer Vielzahl von Vögeln und Säugern mit bis zu 10 Besuchen pro Woche heimgesucht werden. Vier Spechtarten, Kohlmeisen und Baummarder wurden als echte Nestplünderer identifiziert. Bienenvölker, deren Nesteingang mit Maschendraht geschützt war (N=32), hatten eine 50% höhere Winterüberlebensrate als Völker ohne Schutz (N=40). Die Analyse von Landnutzungskarten zeigte, dass sich Bienenvölker, welche den Winter überlebten (N=19), in Landschaften mit durchschnittlich 6,4% höherem Anteil von Ackerflächen befanden als solche, die den Winter nicht überlebten (N=94). Wir schätzen, dass in Deutschland jährlich zehntausende Schwärme von Bienenständen entfliehen, um sich in Spechthöhlen oder anderen Hohlräumen anzusiedeln. Der Anteil wildlebender Völker an der Gesamtbienenpopulation beträgt im Sommer etwa 5%. Sie spielen vermutlich eine untergeordnete Rolle bei der Verbreitung von Bienenkrankheiten. Durch die Ergänzung imkerlich gehaltener Völker in Waldgebieten tragen sie zur Bestäubung waldbewohnender Pflanzenarten bei. Die Besiedlung von Baumhöhlen sollte vielseitige Auswirkungen auf Lebensgemeinschaften im Wald haben: Bienenvölker konkurrieren um Nistplätze, sind reiche Beute im Winter und akkumulieren organisches Material. Nestprädation (eine Folge des Mangels an sicheren Nisthöhlen) und Ressourcenlimitierung spielen offenbar derzeit eine größere Rolle als Parasiten bei der Erklärung von Winterverlusten. Eine offene Frage ist, inwiefern Umwelt und genetische Dispositionen die Etablierung wilder Honigbienenpopulationen verhindern. Künstliche Nistkästen könnten genutzt werden, um die Rolle von Umweltfaktoren genauer zu untersuchen. Populationen wilder Honigbienen außerhalb Europas könnten Erkenntnisse dazu liefern, inwiefern sich die moderne Imkerei auf die Anpassungen der Honigbienen als Wildtier auswirkt. In Europa könnten große zusammenhängende Waldgebiete als evolutionäre Refugien für wilde Honigbienen dienen. KW - Biene KW - Insektensterben KW - Wald KW - Spechte KW - Imkerei KW - wild honey bees KW - swarming KW - tree cavity KW - monitoring KW - bee diseases KW - Wilde Honigbienen KW - Bienenschwarm KW - Baumhöhle KW - Monitoring KW - Bienenkrankheiten KW - Nisthöhle Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-330327 ER - TY - JOUR A1 - Rackevei, Antonia S. A1 - Borges, Alyssa A1 - Engstler, Markus A1 - Dandekar, Thomas A1 - Wolf, Matthias T1 - About the analysis of 18S rDNA sequence data from trypanosomes in barcoding and phylogenetics: tracing a continuation error occurring in the literature JF - Biology N2 - The variable regions (V1–V9) of the 18S rDNA are routinely used in barcoding and phylogenetics. In handling these data for trypanosomes, we have noticed a misunderstanding that has apparently taken a life of its own in the literature over the years. In particular, in recent years, when studying the phylogenetic relationship of trypanosomes, the use of V7/V8 was systematically established. However, considering the current numbering system for all other organisms (including other Euglenozoa), V7/V8 was never used. In Maia da Silva et al. [Parasitology 2004, 129, 549–561], V7/V8 was promoted for the first time for trypanosome phylogenetics, and since then, more than 70 publications have replicated this nomenclature and even discussed the benefits of the use of this region in comparison to V4. However, the primers used to amplify the variable region of trypanosomes have actually amplified V4 (concerning the current 18S rDNA numbering system). KW - RNA secondary structure KW - variable regions KW - V1–V9 KW - V4 KW - V7/V8 KW - Trypanosoma Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297562 SN - 2079-7737 VL - 11 IS - 11 ER - TY - JOUR A1 - Brosch, Philippa K. A1 - Korsa, Tessa A1 - Taban, Danush A1 - Eiring, Patrick A1 - Hildebrand, Sascha A1 - Neubauer, Julia A1 - Zimmermann, Heiko A1 - Sauer, Markus A1 - Shirakashi, Ryo A1 - Djuzenova, Cholpon S. A1 - Sisario, Dmitri A1 - Sukhorukov, Vladimir L. T1 - Glucose and inositol transporters, SLC5A1 and SLC5A3, in glioblastoma cell migration JF - Cancers N2 - (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment. KW - volume regulation KW - transportome KW - phlorizin Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297498 SN - 2072-6694 VL - 14 IS - 23 ER - TY - THES A1 - Bergmann Borges, Alyssa T1 - The endo-lysosomal system of \(Trypanosoma\) \(brucei\): insights from a protist cell model T1 - Das Endo-lysosomale System von \(Trypanosoma\) \(brucei\): Erkenntnisse aus einem Protisten-Zellmodell N2 - Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain. The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis. Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group’s macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera. N2 - Die meisten Studien in der Zellbiologie konzentrieren sich in erster Linie auf Modelle aus der Opisthokont-Gruppe der Eukaryonten. Die Opisthokonten umfassen jedoch nicht die gesamte Vielfalt der Eukaryonten. Daher ist es notwendig, den Forschungsschwerpunkt auf andere Organismen auszuweiten, um ein umfassendes Verständnis grundlegender zellulärer Prozesse zu erlangen, die im gesamten Lebensbaum vorkommen. In diesem Sinne stellt Trypanosoma brucei, ein einzelliger Eukaryote, eine brauchbare Alternative dar. Die gemeinsamen Anstrengungen bei der Genomsequenzierung und der Markierung von Proteinen in den letzten zwei Jahrzehnten haben unser Wissen über diesen Organismus erheblich erweitert und wertvolle Instrumente für eine detailliertere Analyse dieses Parasiten bereitgestellt. Dennoch bleiben noch zahlreiche Fragen offen. Das Überleben von T. brucei im Säugetierwirt ist eng mit dem endo-lysosomalen System verknüpft, das eine entscheidende Rolle beim Recycling von Oberflächenglykoproteinen, der Antikörper-Clearance und der Homöostase der Plasmamembran spielt. Die Dynamik der Verdoppelung des endo-lysosomalen Systems während der Vermehrung von T. brucei und seine mögliche Beziehung zum Wachstum der Plasmamembran sind jedoch noch wenig bekannt. In dieser Arbeit wird daher das endo-lysosomale System von T. brucei im Kontext des Zellzyklus untersucht, um Erkenntnisse über das Wachstum der Zelloberfläche, die Verdopplung der Endosomen und die Clathrin-Rekrutierung zu gewinnen. Darüber hinaus wird in der Studie die Ferritin-Endozytose erneut untersucht, um quantitative Daten über die Beteiligung der TbRab-Proteine (TbRab5A, TbRab7 und TbRab11) und der verschiedenen endosomalen Subpopulationen (frühe, späte bzw. Recycling-Endosomen) am Transport dieses Flüssigphasenmarkers zu erhalten. Bemerkenswert ist, dass diese Subpopulationen zwar als unterschiedliche Kompartimente fungieren, aber verschiedene TbRabs in derselben Region oder Struktur gefunden werden können, was auf eine mögliche physische Verbindung zwischen den endosomalen Subpopulationen hindeutet. Die potenzielle physikalische Verbindung von Endosomen wird im Zusammenhang mit dem Zellzyklus weiter erforscht, und schließlich werden auch die Verdopplung und die morphologische Plastizität des Lysosoms untersucht. Insgesamt bieten diese Ergebnisse Einblicke in die Dynamik des Plasmamembranwachstums und die koordinierte Verdopplung des endo-lysosomalen Systems während der Proliferation von T. brucei. Die frühe Verdoppelung der Endosomen deutet auf ihre mögliche Beteiligung am Plasmamembranwachstum hin, während die späte Verdoppelung der Lysosomen auf eine geringere Rolle in diesem Prozess hindeutet. Die Rekrutierung von Clathrin- und TbRab-GTPasen an der Stelle der Endosomenbildung unterstützt die Annahme, dass das neu gebildete endosomale System während der Zellteilung aktiv ist, und deutet folglich auf seine potenzielle Rolle bei der Homöostase der Plasmamembran hin. In Anbetracht der enormen Vielfalt innerhalb der Gattung Trypanosoma, die etwa 500 beschriebene Arten umfasst, wurde die Makroevolution der Gruppe anhand der kombinierten Informationen der 18S rRNA-Gensequenz und Struktur untersucht. Die Sequenz-Struktur-Analyse von T. brucei und anderen 42 Trypanosomen-Arten wurde im Zusammenhang mit der Vielfalt der Trypanosomatida, der Ordnung, in die Trypanosomen eingeordnet werden, durchgeführt. Eine zusätzliche Analyse, die sich auf Trypanosoma konzentrierte, hob Schlüsselaspekte der Makroevolution dieser Gruppe hervor. Um diese Aspekte weiter zu erforschen, wurden zusätzliche Trypanosomenarten einbezogen und die Veränderungen in der Topologie des Trypanosoma-Baums analysiert. Die Sequenz-Struktur-Phylogenie bestätigte die unabhängige Evolutionsgeschichte der humanen Krankheitserreger T. brucei und Trypanosoma cruzi, während sie gleichzeitig Einblicke in die Evolution der aquatischen Klade, die Paraphylie von Gruppen und die Klassifizierung der Arten in Untergattungen lieferte. KW - 18S rRNA KW - Endocytose KW - Zellzyklus KW - Phylogenie KW - Endocytosis KW - Cell cycle KW - Trypanosoma KW - Phylogeny KW - Sequence-Structure KW - Endosomes KW - Lysosome Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-329248 ER - TY - THES A1 - Schilcher, Felix T1 - Regulation of the nurse-forager transition in honeybees (\(Apis\) \(mellifera\)) T1 - Regulation des Ammen–Sammlerinnen-Übergangs in Honigbienen (\(Apis\) \(mellifera\)) N2 - Honeybees are among the few animals that rely on eusociality to survive. While the task of queen and drones is only reproduction, all other tasks are accomplished by sterile female worker bees. Different tasks are mostly divided by worker bees of different ages (temporal polyethism). Young honeybees perform tasks inside the hive like cleaning and nursing. Older honeybees work at the periphery of the nest and fulfill tasks like guarding the hive entrance. The oldest honeybees eventually leave the hive to forage for resources until they die. However, uncontrollable circumstances might force the colony to adapt or perish. For example, the introduced Varroa destructor mite or the deformed wing virus might erase a lot of in-hive bees. On the other hand, environmental events might kill a lot of foragers, leaving the colony with no new food intake. Therefore, adaptability of task allocation must be a priority for a honeybee colony. In my dissertation, I employed a wide range of behavioral, molecular biological and analytical techniques to unravel the underlying molecular and physiological mechanisms of the honeybee division of labor, especially in conjunction with honeybee malnourishment. The genes AmOARα1, AmTAR1, Amfor and vitellogenin have long been implied to be important for the transition from in-hive tasks to foraging. I have studied in detail expression of all of these genes during the transition from nursing to foraging to understand how their expression patterns change during this important phase of life. My focus lay on gene expression in the honeybee brain and fat body. I found an increase in the AmOARα1 and the Amforα mRNA expression with the transition from in-hive tasks to foraging and a decrease in expression of the other genes in both tissues. Interestingly, I found the opposite pattern of the AmOARα1 and AmTAR1 mRNA expression in the honeybee fat body during orientation flights. Furthermore, I closely observed juvenile hormone titers and triglyceride levels during this crucial time. Juvenile hormone titers increased with the transition from in-hive tasks to foraging and triglyceride levels decreased. Furthermore, in-hive bees and foragers also differ on a behavioral and physiological level. For example, foragers are more responsive towards light and sucrose. I proposed that modulation via biogenic amines, especially via octopamine and tyramine, can increase or decrease the responsiveness of honeybees. For that purpose, in-hive bees and foragers were injected with both biogenic amines and the receptor response was quantified 1 using electroretinography. In addition, I studied the behavioral response of the bees to light using a phototaxis assay. Injecting octopamine increased the receptor response and tyramine decreased it. Also, both groups of honeybees showed an increased phototactic response when injected with octopamine and a decreased response when injected with tyramine, independent of locomotion. Additionally, nutrition has long been implied to be a driver for division of labor. Undernourished honeybees are known to speed up their transition to foragers, possibly to cope with the missing resources. Furthermore, larval undernourishment has also been implied to speed up the transition from in-hive bees to foragers, due to increasing levels of juvenile hormone titers in adult honeybees after larval starvation. Therefore, I reared honeybees in-vitro to compare the hatched adult bees of starved and overfed larvae to bees reared under the standard in-vitro rearing diet. However, first I had to investigate whether the in-vitro rearing method affects adult honeybees. I showed effects of in-vitro rearing on behavior, with in-vitro reared honeybees foraging earlier and for a shorter time than hive reared honeybees. Yet, nursing behavior was unaffected. Afterwards, I investigated the effects of different larval diets on adult honeybee workers. I found no effects of malnourishment on behavioral or physiological factors besides a difference in weight. Honeybee weight increased with increasing amounts of larval food, but the effect seemed to vanish after a week. These results show the complexity and adaptability of the honeybee division of labor. They show the importance of the biogenic amines octopamine and tyramine and of the corresponding receptors AmOARα1 and AmTAR1 in modulating the transition from inhive bees to foragers. Furthermore, they show that in-vitro rearing has no effects on nursing behavior, but that it speeds up the transition from nursing to foraging, showing strong similarities to effects of larval pollen undernourishment. However, larval malnourishment showed almost no effects on honeybee task allocation or physiology. It seems that larval malnourishment can be easily compensated during the early lifetime of adult honeybees. N2 - Honigbienen gehören zu den wenigen Spezies, die in eusozialen Gemeinschaften leben. Die eierlegende Königin und die männlichen Drohnen dienen nur der Fortpflanzung. Alle anderen Arbeiten von den sterilen Arbeiterinnen ausgeführt werden. Die Arbeitsteilung wird meistens anhand des Alters der Bienen organisiert. Junge Arbeiterinnen bleiben im Inneren der Kolonie und führen beispielsweise Putzarbeiten und Ammentätigkeiten aus. Mit zunehmendem Alter verlagern sich ihre Tätigkeiten immer mehr in Richtung des Nestausgangs wo sie, unteranderem als Wächterbienen, den Stockeingang bewachen. Die ältesten Honigbienen verlassen das Nest, um Honig, Pollen, Wasser oder Propolis zu sammeln, bis sie am Ende sterben. Allerdings können unvorhersehbare Ereignisse dazu führen, dass sich die Kolonie anpassen muss, um nicht unterzugehen. Krankheiten wie der Flügeldeformationsvirus oder die, durch den Menschen eingeführte, Varroa destructor Milbe können auf einen Schlag eine große Zahl an Bienen auslöschen. Des Weiteren können beispielsweise starke Unwetter dafür sorgen, dass etliche Sammlerinnen auf ihrem Sammelflug sterben und die Kolonie ohne neuen Nektar oder Pollen zurückgelassen wird. Es liegt auf der Hand, dass eine starre Arbeitsverteilung nicht ausreicht, um solchen Umständen entgegenzuwirken und, dass eine gewisse Flexibilität notwendig ist. In meiner Dissertation habe ich eine weitreichende Anzahl an verhaltensbiologischen und molekularbiologischen Techniken verwendet, um die molekularen und physiologischen Mechanismen der Arbeitsteilung bei Honigbienen aufzuklären, vor allem im Bezug auf den Übergang von Ammenbienen zu Sammlerinnen. Es ist seit langer Zeit bekannt, dass die Gene AmOARα1, AmTAR1, Amfor und Vitellogenin beim Übergang von Ammenbienen zu Sammlerinnen von zentraler Bedeutung sind. Deshalb habe ich die Expression dieser Gene, sowohl im Gehirn als auch im Fettkörper, in genau diesem Zusammenhang betrachtet und die unterschiedlichen Veränderungen der Expressionsmuster während dieser wichtigen Phase im Leben einer Honigbiene analysiert. Ich konnte zeigen, dass sowohl die mRNA Expression des AmOARα1 und des Amforα beim Übergang von Ammenbienen zu Sammlerinnen anstieg, während die Expression der anderen Kandidatengene im gleichen Zeitraum sowohl im Gehirn als auch im Fettkörper abfiel. Interessanterweise zeigten die Expressionsmuster des AmOARα1 und des Am3 TAR1, während der Orientierungsflüge, genau in die entgegengesetzte Richtung. Zusätzlich habe ich mir bei denselben Bienen auch den Juvenilhormongehalt in der Hämolymphe und die Menge an Triglyceriden im Fettkörper angeschaut. Der Juvenilhormongehalt nahm schlagartig zu, als die Bienen mit dem Sammeln begannen. Die Menge an Triglyceriden nahm allerdings von Ammenbienen, über Bienen während des Orientierungsfluges zu Sammlerinnen konstant ab. Des Weiteren war bereits bekannt, dass sich Ammenbienen und Sammlerinnen nicht nur auf genetischer, sondern auch auf verhaltensbiologischer und physiologischer Ebene voneinander unterscheiden. Zum Beispiel sind Sammlerinnen empfindlicher für Licht und Saccharose. Ich stellte die Hypothese auf, dass die Empfindlichkeit von Honigbienen für solche Schwellen durch biogene Amine, insbesondere Oktopamin und Tyramin, moduliert werden kann. Oktopamin sollte die Empfindlichkeit von Bienen erhöhen, wohingegen Tyramin diese verringern sollte. Hierfür injizierte ich Stockbienen und Sammlerinnen beide biogenen Amine und analysierte die Rezeptorantwort mit einem Elektroretinogramm (ERG) und die Lichtempfindlichkeit in einer Phototaxisarena. Oktopamininjektion führte dazu, dass die Rezeptorantwort im ERG erhöht wurde und dass beide Gruppen eine erhöhte Lichtempfindlichkeit aufwiesen. Tyramin hatte in beiden Experimenten genau den gegenteiligen Effekt. Allerdings kann der Ammen-Sammlerinnen-Übergang nicht nur durch biogene Amine moduliert werden, auch die Ernährung hat einen großen Einfluss. Zum Beispiel fangen unterernährte Honigbienen eher an zu sammeln als satte Honigbienen. Des Weiteren sollte auch die larvale Unterernährung bereits einen Einfluss auf die spätere Arbeitsteilung haben, da man bei Arbeiterinnen, die im Larvenstadium bereits unterernährt waren, eine erhöhte Menge an Juvenilhormon festgestellt hatte. Dies sieht man auch beim Übergang von Ammenbienen zu Sammlerinnen. Deshalb nutzte ich eine Methode zur artifiziellen Aufzucht von Honigbienen, um die Standarddiät, die diese normalerweise erhalten, zu variieren. Allerdings musste ich zuerst den Effekt der in-vitro Aufzucht auf im Stock aufgezogene Honigbienen untersuchen. Ich konnte zeigen, dass die artifizielle Aufzucht das Sammelverhalten erwachsener Honigbienen signifikant beeinflusste, während das Ammenverhalten der in-vitro aufgezogenen Bienen nicht beeinflusst wurde. Artifiziell aufgezogene Honigbienen begannen, im Vergleich zu normalen Bienen, früher zu sammeln und sammelten für eine kürzere Zeit. Danach zog ich unterernährte, normal ernährte und überfütterte Honigbienen in-vitro 4 auf. Ich fand Unterschiede im Gewicht zwischen den Behandlungsgruppen. Unterernährte Bienen waren die leichtesten und überfütterte Bienen wogen am meisten. Dieser Unterschied verschwand aber über die Zeit. Des Weiteren konnte ich keinen Einfluss der Ernährung auf das Ammenverhalten oder das Sammelverhalten zeigen. Dieser Ergebnisse zeigen sowohl die Komplexität als auch das Anpassungsvermögen der Arbeitsteilung von Honigbienen. Sie zeigen, dass sowohl die beiden biogenen Amine Oktopamin und Tyramin, als auch die dazugehörigen Rezeptoren AmOARα1 und AmTAR1 bei der Modulation des Ammen-Sammlerinnen-Übergangs eine große Rolle spielen. Des Weiteren zeigen die Ergebnisse des Vergleichs von artifiziell und im Stock aufgezogenen Bienen, starke Gemeinsamkeiten zu einer larvalen Unterernährung mit Pollen. Jedoch scheint eine allgemeine larvale Unterernährung kaum einen Effekt auf den AmmenSammlerinnen-Übergang zu haben. Diese scheint während der ersten Lebenstage von Honigbienen relativ leicht kompensiert werden zu können. KW - Biene KW - juvenile hormone KW - nurse bee KW - forager KW - division of labor KW - malnourishment KW - diet KW - bee KW - honeybee Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-289352 ER - TY - THES A1 - Münch, Luca T1 - Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus T1 - The role of transposable elements in malignant melanoma development in the Xiphophorus fish model N2 - Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus. N2 - The term “transposable elements” (TEs) is based on their ability to change their genomic position. Through insertions, deletions or chromosomal aberrations, their genomic mobility can cause genetic instability. The extent to which they further exert regulatory influence on cellular functions is the subject of current research, as is the resulting question of their overall biological significance. To further pursue these questions the continuation of experimental research is indispensable. In this regard, the Xiphophorus- melanoma-model represents one of the oldest animal models for the study of malignant melanoma. Thanks to its clearly defined genetic background, it is excellently suited for research into melanoma, which continues to be the most lethal of all known forms of skin cancer. The work presented here investigated the role of transposable elements in malignant melanomagenesis of Xiphophorus. KW - Transposon KW - Platy KW - Melanom KW - Überexpression KW - Schwertkärpfling KW - Expression KW - expression KW - Xiphophorus KW - xiphophorus Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-289228 ER - TY - JOUR A1 - Mehmood, Rashid A1 - Alsaleh, Alanoud A1 - Want, Muzamil Y. A1 - Ahmad, Ijaz A1 - Siraj, Sami A1 - Ishtiaq, Muhammad A1 - Alshehri, Faizah A. A1 - Naseem, Muhammad A1 - Yasuhara, Noriko T1 - Integrative molecular analysis of DNA methylation dynamics unveils molecules with prognostic potential in breast cancer JF - BioMedInformatics N2 - DNA methylation acts as a major epigenetic modification in mammals, characterized by the transfer of a methyl group to a cytosine. DNA methylation plays a pivotal role in regulating normal development, and misregulation in cells leads to an abnormal phenotype as is seen in several cancers. Any mutations or expression anomalies of genes encoding regulators of DNA methylation may lead to abnormal expression of critical molecules. A comprehensive genomic study encompassing all the genes related to DNA methylation regulation in relation to breast cancer is lacking. We used genomic and transcriptomic datasets from the Cancer Genome Atlas (TGCA) Pan-Cancer Atlas, Genotype-Tissue Expression (GTEx) and microarray platforms and conducted in silico analysis of all the genes related to DNA methylation with respect to writing, reading and erasing this epigenetic mark. Analysis of mutations was conducted using cBioportal, while Xena and KMPlot were utilized for expression changes and patient survival, respectively. Our study identified multiple mutations in the genes encoding regulators of DNA methylation. The expression profiling of these showed significant differences between normal and disease tissues. Moreover, deregulated expression of some of the genes, namely DNMT3B, MBD1, MBD6, BAZ2B, ZBTB38, KLF4, TET2 and TDG, was correlated with patient prognosis. The current study, to our best knowledge, is the first to provide a comprehensive molecular and genetic profile of DNA methylation machinery genes in breast cancer and identifies DNA methylation machinery as an important determinant of the disease progression. The findings of this study will advance our understanding of the etiology of the disease and may serve to identify alternative targets for novel therapeutic strategies in cancer. KW - DNA methylation KW - epigenetic modification KW - breast cancer KW - genomics KW - in silico analysis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-321171 SN - 2673-7426 VL - 3 IS - 2 SP - 434 EP - 445 ER - TY - JOUR A1 - Han, Chao A1 - Ren, Pengxuan A1 - Mamtimin, Medina A1 - Kruk, Linus A1 - Sarukhanyan, Edita A1 - Li, Chenyu A1 - Anders, Hans-Joachim A1 - Dandekar, Thomas A1 - Krueger, Irena A1 - Elvers, Margitta A1 - Goebel, Silvia A1 - Adler, Kristin A1 - Münch, Götz A1 - Gudermann, Thomas A1 - Braun, Attila A1 - Mammadova-Bach, Elmina T1 - Minimal collagen-binding epitope of glycoprotein VI in human and mouse platelets JF - Biomedicines N2 - Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix. KW - GPVI KW - collagen KW - blood platelets KW - thrombosis KW - anti-thrombotic therapies Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304148 SN - 2227-9059 VL - 11 IS - 2 ER - TY - JOUR A1 - Hutin, Stephanie A1 - Ling, Wai Li A1 - Tarbouriech, Nicolas A1 - Schoehn, Guy A1 - Grimm, Clemens A1 - Fischer, Utz A1 - Burmeister, Wim P. T1 - The vaccinia virus DNA helicase structure from combined single-particle cryo-electron microscopy and AlphaFold2 prediction JF - Viruses N2 - Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements. KW - DNA replication KW - helicase KW - Pfam domain KW - poxvirus KW - cryo-electron microscopy KW - structure prediction KW - SF3 helicase KW - orthopoxvirus KW - DNA helicase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290523 SN - 1999-4915 VL - 14 IS - 10 ER - TY - THES A1 - Lippert, Juliane T1 - Die molekulargenetische Charakterisierung von Nebennierenrindenkarzinomen als Schritt in Richtung personalisierter Medizin T1 - Molecular Characterisation of Adrenocortical Carcinomas as a Step towards Personalized Medicine N2 - Nebennierenrindenkarzinome (NNR-Ca; engl. adrenocortical carcinoma (ACC)) zählen zu den sehr seltenen Tumorentitäten. Die Prognose für die Patient*innen ist insgesamt eher schlecht, kann aber, im Einzelnen betrachtet, sehr heterogen sein. Eine zuverlässige Prognose anhand klinischer und histopathologischer Marker – wie dem Tumorstadium bei Diagnose, dem Resektionsstatus und dem Proliferationsindex Ki-67 –, die routinemäßig erhoben werden, ist nicht für alle Erkrankten möglich. Außerdem wird deren Behandlung dadurch erschwert, dass Therapeutika fehlen, von denen ein Großteil der Patient*innen profitiert. Umfassende Multi-Omics-Studien aus den letzten Jahren halfen nicht nur das Wissen über Pathomechanismen in NNR-Cas zu erweitern, es konnte auch gezeigt werden, dass sich Patient*innen anhand molekularer Marker in Subgruppen mit jeweils unterschiedlicher Prognose einteilen lassen. Mit molekulargenetischen Untersuchungen wurden außerdem potentielle neue Therapieziele gefunden. Diese Erkenntnisse finden bisher jedoch keine oder kaum Anwendung, da die Analysen den zeitlichen und finanziellen Rahmen, der für den routinemäßigen Einsatz im Klinikalltag zu erfüllen wäre, deutlich überschreiten. Ziel dieser Arbeit war es, eine Strategie zur verbesserten Patientenversorgung der NNR-CaPatient*innen zu etablieren. Dafür sollte geklärt werden, ob ausgewählte molekulare prognostische Marker mit Methoden, die theoretisch einfach in den Klinikalltag zu implementieren wären, gefunden werden können. Außerdem sollte nach prädiktiven Markern gesucht werden, die helfen, NNR-Ca-Patient*innen zielgerichtet zu therapieren. Statt exom- oder genomweite Analysen durchzuführen wurden gezielt krebs- beziehungsweise NNR-Ca-assoziierte Gene mittels NGS (Next-Generation Sequencing) oder SangerSequenzierung (zusammen 161 Gene) und Pyrosequenzierung (4 Gene) auf somatische Veränderungen hin untersucht. Die Analysen wurden an DNA (Desoxyribonukleinsäure) durchgeführt, die aus FFPE (mit Formalin fixiert und in Paraffin eingebettet)-Gewebe isoliert worden war, welches standardmäßig nach Tumoroperationen in Pathologien für Untersuchungen zur Verfügung steht. Durch Analyse der Sequenzierergebnisse von insgesamt 157 Patient*innen aus einem retrospektiven (107 Patient*innen) und einem prospektiven Studienteil (50 Patient*innen) konnten in NNR-Cas bereits beschriebene Veränderungen von Genen und Signalwegen sowie Methylierungsunterschiede gefunden werden. Anhand der Sequenzierdaten der retrospektiven Studie wurden molekulare prognostische Marker (Anzahl an proteinverändernden Varianten pro Tumorprobe, Veränderungen im P53/Rb- und/oder dem Wnt/ß-Catenin-Signalweg und dem Methylierungsstatus von CpG-Inseln von vier 2 Tumorsuppressorgenen (GSTP1, PAX5, PAX6 und PYCARD)) definiert und für jeden einzelnen Marker ein signifikanter Zusammenhang zur Länge des progressionsfreien Überlebens (PFS) der Patient*innen gefunden. Durch die Kombination der molekularen Marker mit den klinischen und histopathologischen Markern war es zudem möglich, einen COMBI-Score zu bilden, der, verglichen mit den klinischen und histopathologischen Markern, eine spezifischere und sensitivere Aussage darüber erlaubt, ob Patient*innen innerhalb von 2 Jahren ein Fortschreiten der Tumorerkrankung erfahren. Mit Hilfe der Sequenzierdaten wurden in beiden Kohorten außerdem Veränderungen gefunden, die als prädiktive Marker zum Einsatz von zielgerichteten Therapien vewendet werden könnten. Als vielversprechendstes Therapieziel wurde – bei 46 Tumoren in der retrospektiven und 7 Tumoren in der prospektiven Studie – CDK4 identifiziert. CDK4/CDK6-Inhibitoren sind für die Behandlung von fortgeschrittenem und metastasiertem Brustkrebs von der Lebensmittel- überwachungs- und Arzneimittelbehörde (FDA; engl. Food and Drug Administration) zugelassene Therapeutika und bei anderen soliden Tumoren Gegenstand von Studien. Im Rahmen der Arbeit konnten außerdem von 12 Patient*innen jeweils zwei Tumoren molekulargenetisch untersucht und die Ergebnisse verglichen werden. Die Analyse zeigte, dass der Methylierungsstatus – im Vergleich zu Veränderungen in der DNA-Sequenz – der stabilere prognostische Marker ist. Mit dieser Arbeit wurde gezeigt, dass molekulare prognostische und prädiktive Marker für den Einsatz zielgerichteter Therapien mit Methoden identifiziert werden können, die sich im klinischen Alltag bei der Behandlung von NNR-Ca-Patient*innen implementieren lassen. Um einen allgemein anerkannten Leitfaden zu etablieren, fehlen allerdings noch die Ergebnisse weiterer – vor allem prospektiver – Studien zur Validierung der hier präsentierten Ergebnisse. Die gewonnenen Erkenntnisse sind jedoch als wichtiger Schritt in Richtung personalisierter Medizin bei Nebennierenrindenkarzinomen anzusehen. N2 - Adrenocortical carcinomas (ACC) are among the very rare tumor entities. Altogether prognosis for the patients is poor, though regarding individuals the outcome can be heterogenous. Prognostic stratification on the basis of clinical and histopathological markers – for example tumor stage at diagnosis, resection status and proliferation index Ki-67 – is not reliable for all patients. This fact and the lack off effective pharmacological therapies, makes the patient care challenging. In the last years comprehensive multi omics studies helped to increase the knowledge about pathogenetic mechanisms in ACC. With those data, scientists were also able to identify molecular markers useful to distinguish subgroups of patients with distinct clinical outcome. With molecular analysis also new potential drug targets for targeted therapies were identified. Till now these findings have not been transferred into the clinical routine care of ACC patients, mostly due to the time consuming and expensive methods required for the multi omics studies. The aim of this study was to establish a strategy for improved patient care of ACC patients. We chose methods theoretically applicable in a clinical routine workflow to analyze selected prognostic molecular markers, already correlated to outcome. Moreover it was searched for predictive markers for targeted therapy of ACC patients. Instead of comprehensive analysis a targeted approach via NGS (Next Generation Sequencing) or Sanger Sequencing (161 genes in total) and pyrosequencing (4 genes ) was conducted to find somatic variants in genes associated with cancer in general or particularly with ACC. For the analysis, DNA (deoxyribonucleic acid) was isolated from FFPE (formalin fixad and paraffin embedded) tissue which is routinely prepared and available in pathological institutions after tumor resections. Sequencing results of 157 patients in total, gained from a retrospective part of the study (107 patients) and a prospective part (50 patients), were in accordance to already published data concerning somatic variants in genes and signaling pathways and differences in the methylation patterns of particular genes. Molecular prognostic markers (number of protein changing variants per tumor sample, variants in P53/Rb- and/or Wnt/ß-Catenin signaling pathway and methylation pattern of CpG islands of four tumor suppressor genes (GSTP1, PAX5, PAX6 und PYCARD)) were defined with the data of the retrospective study. A significant prognostic role for progression free survival (PFS) was found for all of them. With the COMBI-Score – a combination of the molecular prognostic markers and the clinical and histopathological prognostic markers – it was possible to even better predict the progress of the disease within two years. Moreover variants reported to be predictive markers for the use of targeted therapies were identified in both cohorts. Most promising drug target seems to be CDK4 which was found to be amplified in 46 and 7 tumors in the retrospective and prospective study, respectively. CDK4/CDK6 inhibitors are drugs already approved by the Food and Drug Administration (FDA) for the treatment of advanced or metastatic breast cancer and under investigation in other solid tumors. Within this study it was also possible to compare molecular data from 12 tumor pairs, what means two tumors gained from one patient. It seems as if the methylation pattern is a more consistent prognostic marker than the changes detected on DNA sequence level. In conclusion, we demonstrated that molecular prognostic markers and predictive markers for targeted therapy can be identified using methods easily applicable in a clinical routine workflow for patients with ACC. Before implementing our strategy into a guideline that is commonly approved, further prospective studies are needed for the validation of the presented results. However our strategy can be regarded as an important step towards personalized medicine in adrenocortical carcinoma. KW - Nebennierentumor KW - Nebenniererindenkarzinom KW - molekulargenetische Charakterisierung KW - personalisierte Medizin Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-247172 ER - TY - THES A1 - Schmalz, Fabian Dominik T1 - Processing of behaviorally relevant stimuli at different levels in the bee brain T1 - Die Verarbeitung verhaltensrelevanter Stimuli auf unterschiedlichen Ebenen im Bienengehirn N2 - The behavior of honeybees and bumblebees relies on a constant sensory integration of abiotic or biotic stimuli. As eusocial insects, a sophisticated intraspecific communication as well as the processing of multisensory cues during foraging is of utter importance. To tackle the arising challenges, both honeybees and bumblebees have evolved a sophisticated olfactory and visual processing system. In both organisms, olfactory reception starts at the antennae, where olfactory sensilla cover the antennal surface in a sex-specific manner. These sensilla house olfactory receptor neurons (ORN) that express olfactory receptors. ORNs send their axons via four tracts to the antennal lobe (AL), the prime olfactory processing center in the bee brain. Here, ORNs specifically innervate spheroidal structures, so-called glomeruli, in which they form synapses with local interneurons and projection neurons (PN). PNs subsequently project the olfactory information via two distinct tracts, the medial and the lateral antennal-lobe tract, to the mushroom body (MB), the main center of sensory integration and memory formation. In the honeybee calyx, the sensory input region of the MB, PNs synapse on Kenyon cells (KC), the principal neuron type of the MB. Olfactory PNs mainly innervate the lip and basal ring layer of the calyx. In addition, the basal ring receives input from visual PNs, making it the first site of integration of visual and olfactory information. Visual PNs, carrying sensory information from the optic lobes, send their terminals not only to the to the basal ring compartment but also to the collar of the calyx. Receiving olfactory or visual input, KCs send their axons along the MB peduncle and terminate in the main output regions of the MB, the medial and the vertical lobe (VL) in a layer-specific manner. In the MB lobes, KCs synapse onto mushroom body output neurons (MBON). In so far barely understood processes, multimodal information is integrated by the MBONs and then relayed further into the protocerebral lobes, the contralateral brain hemisphere, or the central brain among others. This dissertation comprises a dichotomous structure that (i) aims to gain more insight into the olfactory processing in bumblebees and (ii) sets out to broaden our understanding of visual processing in honeybee MBONs. The first manuscript examines the olfactory processing of Bombus terrestris and specifically investigates sex-specific differences. We used behavioral (absolute conditioning) and electrophysiological approaches to elaborate the processing of ecologically relevant odors (components of plant odors and pheromones) at three distinct levels, in the periphery, in the AL and during olfactory conditioning. We found both sexes to form robust memories after absolute conditioning and to generalize towards the carbon chain length of the presented odors. On the contrary, electroantennographic (EAG) activity showed distinct stimulus and sex-specific activity, e.g. reduced activity towards citronellol in drones. Interestingly, extracellular multi-unit recordings in the AL confirmed stimulus and sex-specific differences in olfactory processing, but did not reflect the differences previously found in the EAG. Here, farnesol and 2,3-dihydrofarnesol, components of sex-specific pheromones, show a distinct representation, especially in workers, corroborating the results of a previous study. This explicitly different representation suggests that the peripheral stimulus representation is an imperfect indication for neuronal representation in high-order neuropils and ecological importance of a specific odor. The second manuscript investigates MBONs in honeybees to gain more insights into visual processing in the VL. Honeybee MBONs can be categorized into visually responsive, olfactory responsive and multimodal. To clarify which visual features are represented at this high-order integration center, we used extracellular multi-unit recordings in combination with visual and olfactory stimulation. We show for the first time that information about brightness and wavelength is preserved in the VL. Furthermore, we defined three specific classes of visual MBONs that distinctly encode the intensity, identity or simply the onset of a stimulus. The identity-subgroup exhibits a specific tuning towards UV light. These results support the view of the MB as the center of multimodal integration that categorizes sensory input and subsequently channels this information into specific MBON populations. Finally, I discuss differences between the peripheral representations of stimuli and their distinct processing in high-order neuropils. The unique activity of farnesol in manuscript 1 or the representation of UV light in manuscript 2 suggest that the peripheral representation of a stimulus is insufficient as a sole indicator for its neural activity in subsequent neuropils or its putative behavioral importance. In addition, I discuss the influence of hard-wired concepts or plasticity induced changes in the sensory pathways on the processing of such key stimuli in the peripheral reception as well as in high-order centers like the AL or the MB. The MB as the center of multisensory integration has been broadly examined for its olfactory processing capabilities and receives increasing interest about its visual coding properties. To further unravel its role of sensory integration and to include neglected modalities, future studies need to combine additional approaches and gain more insights on the multimodal aspects in both the input and output region. N2 - Honigbienen und Hummeln sind aufgrund ihrer Lebensweise auf die ständige Verarbeitung sensorischer Eindrücke abiotischen und biotischen Ursprungs angewiesen. Als eusoziale Insekten ist hierbei für beide Arten die Wahrnehmung innerartlicher Kommunikation wie auch die Verarbeitung multisensorischer Einflüsse während der Nahrungssuche von essenzieller Bedeutung. Um die daraus resultierenden vielfältigen Herausforderungen erfolgreich bewältigen zu können, verfügen Honigbienen und Hummeln über eine fortschrittliche Verarbeitung olfaktorischer und visueller Reize. In beiden Arten beginnt die Geruchsrezeption an den Antennen, welche geschlechtsspezifisch von zahlreichen olfaktorischen Sensillen besetzt sind. Diese beinhalten olfaktorische Rezeptorneurone (ORN), in welchen die Expression der Geruchsrezeptoren stattfindet. Axone der ORNs laufen dabei gebündelt über vier verschiedene Trakte in den Antennallobus (AL), das erste olfaktorische Verarbeitungszentrum im Bienengehirn. Im AL verschalten ORNs mit lokalen Interneuronen und Projektionsneuronen (PN) in kugelförmigen Strukturen, den sogenannten Glomeruli. PNs leiten die olfaktorische Information daraufhin über zwei charakteristische Trakte, den medialen und lateralen Antennallobustrakt, in den Pilzkörper (MB), das Verarbeitungszentrum für die Integration sensorischer Eindrücke und Gedächtnisbildung. Im Calyx der Honigbiene, der sensorischen Eingangsregion des MB, bilden die Endköpfchen der PNs synaptische Verbindungen mit Kenyonzellen (KC), den primären Nervenzellen im MB. Die Innervation des Calyx durch die PNs ist dabei spezifisch in drei verschiedenen Zonen organisiert, nämlich in Lippe, Hals und basalen Ring. Während die Lippe vornehmlich olfaktorische Information von PNs aus dem AL erhält, wird der basale Ring zusätzlich auch von visuellen PNs, welche Informationen aus dem optischen Lobus einbringen, angesteuert. Der basale Ring der Honigbiene wird dabei Ort der ersten räumlichen Integration visuellen und olfaktorischen Eingangs. Wiederum ähnlich zum unimodalen Eingang der Lippe, bezieht auch der Hals des Calyx grundsätzlich nur sensorischen Eingang einer Modalität, nämlich visuelle Information von PNs aus dem optischen Lobus. KCs verschalten im weiteren Verlauf die olfaktorischen und visuellen Informationen an Pilzkörperausgangsneurone (MBON). In einem bisher kaum erforschten Vorgang wird diese multimodale Information dabei verarbeitet und dann mithilfe der MBONs in verschiedene Bereiche des Gehirns geleitet, z.B. in die protocerebralen Loben, die kontralaterale Gehirnhemisphäre oder das Zentralgehirn. Diese Dissertation ist zweigeteilt und behandelt zuerst (i) die geschlechtsspezifische Verarbeitung olfaktorischer Reize in Hummeln und bespricht im zweiten Teil (ii) neue Einblicke in die neuronale Weiterverarbeitung visueller Reize durch MBONs in der Honigbiene. Manuskript 1 untersucht die Abläufe der Geruchsverarbeitung von Bombus terrestris und beschreibt geschlechtsspezifische Unterschiede. Hierbei wurden sowohl verhaltensbasierte als auch elektrophysiologische Methoden genutzt um die Wahrnehmung ökologisch relevanter Duftstoffe (Komponenten unterschiedlicher Pflanzendüfte oder Pheromone) auf drei verschiedene Weisen zu untersuchen, nämlich in der Peripherie, im AL und mittels olfaktorischer Konditionierung. Wir fanden in beiden Geschlechtern eine robuste Gedächtnisbildung nach absoluter Konditionierung und eine ausgeprägte Generalisierung anhand der Kohlenstoffkettenlänge der präsentierten Duftstoffe. Anders stellten sich die Ergebnisse der elektroantennographischen (EAG) Untersuchungen dar. Hier zeigten sowohl Drohnen als auch Arbeiterinnen neuronale Aktivität mit spezifischen Unterschieden zwischen den Stimuli, aber auch zwischen den Geschlechtern auf, z.B. löste die Applikation von Citronellol eine deutliche verringerte Reaktion in der EAG Aktivität der Drohnen aus. Interessanterweise zeigten auch extrazelluläre Ableitungen im AL stimulus- und geschlechtsspezifische Unterschiede, jedoch in unterschiedlicher Konstellation als in den EAG-Experimenten. Besonders Farnesol und 2,3-Dihydrofarnesol wiesen vor allem bei Arbeiterinnen eine deutliche Repräsentation in der neuronalen Aktivität auf; ein Alleinstellungsmerkmal welches für Farnesol bereits in einer früheren Studie beschrieben wurde. Diese explizit unterschiedliche neuronale Darstellung von Farnesol und 2,3-Dihydrofarnesol in der Peripherie und im AL führt zu der Annahme, dass die rezeptive Darstellung eines Stimulus in der Peripherie keine zuverlässigen Rückschlüsse über die neuronale Repräsentation in höheren Zentren oder die ökologische Relevanz zulässt. Im zweiten Manuskript stehen MBONs der Honigbiene im Fokus, um mehr Einblicke in die visuelle Verarbeitung im VL zu erlangen. Bisher können MBONs in folgende Klassen unterteilt werden: Visuelle, olfaktorische und multimodale MBONs, welche sensitiv für beide Modalitäten sind. Kern dieser Arbeit ist, mittels extrazellulärer Ableitungen festzustellen, welche zusätzlichen Aspekte eines visuellen Stimulus in diesem zentralen Verarbeitungszentrum repräsentiert sind. Dabei konnte zum ersten Mal gezeigt werden, dass Informationen über die Wellenlänge und die Intensität des Lichtstimulus im VL erhalten sind. Im weiteren Verlauf konnte eine Spezifizierung der bisherigen Kategorisierung visueller und multimodaler MBONs in drei weitere Untergruppen vollzogen werden: MBONs die spezifisch die Intensität, die Identität und dein Eingang eines Stimulus kodieren. Des Weiteren zeigte vor allem die Gruppe der Identitäts-MBONs eine bemerkenswerte Kategorisierung von UV-Licht. Diese neuen Erkenntnisse bestätigen die Ansicht, dass der MB, als Zentrum für sensorische Integration, eine Kategorisierung der verarbeiteten Eindrücke vornimmt und diese daraufhin auf die MBONs verschalten wird. Abschließend diskutiere ich Unterschiede in der peripheren Repräsentation von Stimuli und ihrer späteren neuronalen Verarbeitung. Hier zeige ich, die Aktivität von Farnesol in MS1 und UV-Licht MS2 als Beispiel nehmend, dass die periphere Repräsentation eines Stimulus keine sicheren Schlussfolgerungen über die nachfolgend induzierte neurale Aktivität oder die verhaltensrelevante Bedeutung zulässt. Im weiteren Verlauf werden dabei die Einflüsse konservierter Strukturen und plastischer Änderungen auf die Abläufe der sensorischen Peripherie oder der höheren Verarbeitungszentren, wie dem AL oder dem MB gezeigt. Obwohl der MB, das Zentrum für multimodale Integration und Gedächtnis, hinsichtlich seiner Rolle in der Geruchswahrnehmung ausgiebig erforscht ist, gibt es bezüglich der visuellen Verarbeitung oder dem Einfluss anderer Modalitäten noch ungeklärte Abläufe und Fragen. Wenngleich auch hier die Kenntnis speziell über die visuelle Verarbeitung im MB stetig zunimmt, sollten zukünftige Arbeiten mithilfe weiterer Methoden den MB Eingang und Ausgang explizit auf den Einfluss weiterer Modalitäten untersuchen, um so ein umfassenderes Bild über die Abläufe multimodaler Integration zu erhalten. KW - Biene KW - Elektrophysiologie KW - bee KW - electrophysiology KW - olfaction KW - vision KW - multi-unit recording KW - Olfaktorik KW - Sehen KW - Multi-Unit Aufnahmen Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288824 ER - TY - THES A1 - Geis, Maria T1 - Identifizierung von Zielmolekülen und Herstellung zweigeteilter trivalenter T-Zell-aktivierender Antikörperderivate zur immuntherapeutischen Behandlung von Multiplen Myelom T1 - Target identification and generation of trivalent T-cell activating antibody derivatives for multiple myeloma immunotherapy N2 - T-Zell-aktivierende Formate, wie BiTE (bispecific T-cell engagers) Antikörper und CAR T Zellen haben in den vergangen Jahren die Therapiemöglichkeiten für Tumorpatienten erweitert. Diese Therapeutika verknüpfen T-Zellen mit malignen Zellen über je ein spezifisches Oberflächenmolekül und initiieren, über eine T-Zell-vermittelte Immunantwort, die Lyse der Tumorzelle. Tumorspezifische Antigene sind jedoch selten. Häufig werden Proteine adressiert, die neben den Tumorzellen auch auf gesunden Zellen exprimiert werden. Die Folgen sind toxische Effekte abseits der Tumorzellen auf Antigen-positiven gesunden Zellen (on target/off tumor), welche nicht nur die Dosis des Therapeutikums und dessen Effektivität limitieren, sondern zu geringen bis letalen Begleiterscheinungen führen können. Der Bedarf an effektiven Therapieformen mit geringen Nebenwirkungen ist folglich immer noch sehr hoch. Diese Lücke soll durch ein neues Antikörperformat, sogenannten Hemibodies, geschlossen werden. Hemibodies sind eine neue Klasse von T-Zell-aktivierenden Antikörpern, die sich gegen eine Antigenkombination und nicht einzelne Antigene auf Tumorzellen richten. Sie bestehen aus zwei komplementären Molekülen mit je einer Antigen-bindenden Sequenz, die entweder mit der leichten (VL) oder der schweren (VH) Kette eines T-Zell-aktivierenden anti CD3 Antikörpers fusioniert ist. Nur wenn beide Hemibody-Fragmente gleichzeitig in unmittelbarer Nähe an ihr jeweiliges Antigenepitop auf der Tumorzelle binden, komplementieren die beiden Antikörperkonstrukte über das geteilte anti-CD3 und bilden einen trivalenten T Zell aktivierenden Komplex aus. Diese funktionale Einheit rekrutiert T-Zellen zur Tumorzelle und induzierte die T-Zell-vermittelte Lyse der malignen Zelle. Im Rahmen der vorliegenden Arbeit wurden geeignete Antigenkombinationen identifiziert und die erste effektive und spezifische Hemibody-basierte Immuntherapie gegen das Multiple Myelom (MM), ohne Nebenwirkungen auf Antigen-einfach-positiven gesunden Zellen, entwickelt. Basierend auf einer umfangreichen Analyse von Kandidaten-Antigenen wurden Kombinationen aus bekannten MM Zielmolekülen, wie BCMA, CD38, CD138, CD229 und SLAMF7, und für das MM unbekannte Oberflächenmolekülen, wie CHRM5 und LAX1, untersucht. Gegen die vielversprechendsten Antigene wurden Hemibodies entwickelt und produziert. Im Zusammenhang mit Analysen zur Produzierbarkeit sowie biochemischen und funktionalen Charakterisierungen, konnte aus 75 initialen Hemibody-Kombinationen drei Kombinationen mit geeigneten Eigenschaften identifiziert werden. Die Bindung von zwei Hemibody-Partnern auf der Oberfläche der MM Zelle führte zur Ausbildung eines trivalenten T-Zell-rekrutierenden Komplexes. Dieser initiierte nachfolgend über eine T-Zell-vermittelte Immunantwort die spezifische Lyse der malignen Zellen, ohne die Viabilität von Antigen-einfach-positiven gesunden Körper- oder Effektor-Zellen zu beeinflussen. Zusätzlich führte eine Hemibody-Therapie in vivo in einem NOD SCID MM-Mausmodel innerhalb von 7 Tagen zur kompletten Remission der MM Zellen. Diese Daten zeigten Hemibodies als ein neues, sehr vielversprechendes Antikörperformat für eine effektive und tumorspezifische Immuntherapie mit potentiell geringen Nebenwirkungen. N2 - T-cell activating therapies such as BiTEs (bispecific T-cell engagers) and CAR-T-cells have broadened the treatment options for cancer patients in the past years. These therapeutics induce a T-cell mediated immune response by linking T-cells with malignant cells by a specific target on the tumor cell. Tumor-specific antigens are rare and often antigens expressed on malignant and healthy tissues are addressed. Consequently, dosage and efficacy are limited by on-target/off-tumor toxicities, which can cause severe side effects. Efficient therapies with no side effects are still needed. To overcome these limitations and fill the gap of existing cancer immunotherapies, our novel strategy, coined hemibodies, targets an aberrant antigen signature uniquely expressed on tumor cells. Hemibodies are a new class of T-cell engaging antibodies consisting of two complementing molecules. Each hemibody molecule can bind one specific target on a tumor cell using a scFv fused to either the variable heavy (VH) or light (VL) chain domain of a T-cell activating anti-CD3 antibody. When both hemibodies simultaneously bind their specific target, the VL- and the VH-domain reconstitute and form a functional anti-CD3 domain, enabling T-cell recruitment for tumor cell lysis. This way, hemibodies form a trivalent protein complex only on tumor cells for safe cancer immunotherapy. The following work presents target combinations and the first hemibody-based immunotherapy for a precise multiple myeloma (MM) treatment, without side effects, on target-single-positiv cells. Besides combinations of known and often reported MM targets like CD138, CD38, BCMA and SLAMF7, new targets including CHRM5 and LAX1 are described. Moreover, three hemibody combinations out of 75 promising target combinations were identified that displayed favorable production and purification data as well as biochemical and functional characteristics. We demonstrated that hemibodies are able to recognize and bind MM cells on their specific targets and form a functional trivalent T-cell activating complex for tumor cell lysis. In contrast to BiTE antibodies, hemibody-fragments alone and in combination had no/low effects on the viability of target-single positive cells or on T-cells in the absence of tumor cells. Only in the presence of MM cells, hemibodies recruit T-cells to the tumor site and induce tumor specific lysis. In addition, human T-lymphocytes rejected MM cells after treatment with a hemibody combination for seven days in a murine NOD SCID model. In aggregate, the data reported here identified hemibodies as a promising therapeutic protein format for effective and safe cancer immunotherapy. KW - zweigeteilte trivalente T-Zell-aktivierende Antikörperderivate KW - Hemibodies Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186906 ER -