TY - JOUR A1 - Heydarian, Motaharehsadat A1 - Rühl, Eva A1 - Rawal, Ravisha A1 - Kozjak-Pavlovic, Vera T1 - Tissue models for Neisseria gonorrhoeae research — from 2D to 3D JF - Frontiers in Cellular and Infection Microbiology N2 - Neisseria gonorrhoeae is a human-specific pathogen that causes gonorrhea, the second most common sexually transmitted infection worldwide. Disease progression, drug discovery, and basic host-pathogen interactions are studied using different approaches, which rely on models ranging from 2D cell culture to complex 3D tissues and animals. In this review, we discuss the models used in N. gonorrhoeae research. We address both in vivo (animal) and in vitro cell culture models, discussing the pros and cons of each and outlining the recent advancements in the field of three-dimensional tissue models. From simple 2D monoculture to complex advanced 3D tissue models, we provide an overview of the relevant methodology and its application. Finally, we discuss future directions in the exciting field of 3D tissue models and how they can be applied for studying the interaction of N. gonorrhoeae with host cells under conditions closely resembling those found at the native sites of infection. KW - ex vivo KW - biomimetic tissue models KW - Neisseria gonorrhoeae KW - in vivo KW - in vitro Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263046 SN - 2235-2988 VL - 12 ER - TY - THES A1 - Zwettler, Fabian Ulrich T1 - Expansionsmikroskopie kombiniert mit hochauflösender Fluoreszenzmikroskopie T1 - Expansion Microscopy combined with Super-Resolution Fluorescence Microscopy N2 - Fluorescence microscopy is a form of light microscopy that has developed during the 20th century and is nowadays a standard tool in Molecular and Cell biology for studying the structure and function of biological molecules. High-resolution fluorescence microscopy techniques, such as dSTORM (direct Stochastic Optical Reconstruction Microscopy) allow the visualization of cellular structures at the nanometre scale (10−9 m). This has already made it possible to decipher the composition and function of various biopolymers, such as proteins, lipids and nucleic acids, up to the three-dimensional (3D) structure of entire organelles. In practice, however, it has been shown that these imaging methods and their further developments still face great challenges in order to achieve an effective resolution below ∼ 10 nm. This is mainly due to the nature of labelling biomolecules. For the detection of molecular structures, immunostaining is often performed as a standard method. Antibodies to which fluorescent molecules are coupled, recognize and bind specifcally and with high affnity to the molecular section of the target structure, also called epitope or antigen. The fluorescent molecules serve as reporter molecules which are imaged with the use of a fluorescence microscope. However, the size of these labels with a length of about 10-15 nm in the case of immunoglobulin G (IgG) antibodies, cause a detection of the fluorescent molecules shifted to the real position of the studied antigen. In dense regions where epitopes are located close to each other, steric hindrance between antibodies can also occur and leads to an insuffcient label density. Together with the shifted detection of fluorescent molecules, these factors can limit the achievable resolution of a microscopy technique. Expansion microscopy (ExM) is a recently developed technique that achieves a resolution improvement by physical expansion of an investigated object. Therefore, biological samples such as cultured cells, tissue sections, whole organs or isolated organelles are chemically anchored into a swellable polymer. By absorbing water, this so-called superabsorber increases its own volume and pulls the covalently bound biomolecules isotropically apart. Routinely, this method achieves a magnifcation of the sample by about four times its volume. But protocol variants have already been developed that result in higher expansion factors of up to 50-fold. Since the ExM technique includes in the frst instance only the sample treatment for anchoring and magnifcation of the sample, it can be combined with various standard methods of fluorescence microscopy. In theory, the resolution of the used imaging technique improves linearly with the expansion factor of the ExM treated sample. However, an insuffcient label density and the size of the antibodies can here again impair the effective achievable resolution. The combination of ExM with high-resolution fluorescence microscopy methods represents a promising strategy to increase the resolution of light microscopy. In this thesis, I will present several ExM variants I developed which show the combination of ExM with confocal microscopy, SIM (Structured Illumination Microscopy), STED (STimulated Emission Depletion) and dSTORM. I optimized existing ExM protocols and developed different expansion strategies, which allow the combination with the respective imaging technique. Thereby, I gained new structural insights of isolated centrioles from the green algae Chlamydomonas reinhardtii by combining ExM with STED and confocal microscopy. In another project, I combined 3D-SIM imaging with ExM and investigated the molecular structure of the so-called synaptonemal complex. This structure is formed during meiosis in eukaryotic cells and contributes to the exchange of genetic material between homologous chromosomes. Especially in combination with dSTORM, the ExM method showed its high potential to overcome the limitations of modern fluorescence microscopy techniques. In this project, I expanded microtubules in mammalian cells, a polymer of the cytoskeleton as well as isolated centrioles from C. reinhardtii. By labelling after expansion of the samples, I was able to signifcantly reduce the linkage error of the label and achieve an improved label density. In future, these advantages together with the single molecule sensitivity and high resolution obtained by the dSTORM method could pave the way for achieving molecular resolution in fluorescence microscopy N2 - Die Fluoreszenzmikroskopie ist eine Form der Lichtmikroskopie, die sich im Laufe des 20. Jahrhunderts entwickelt hat und heutzutage standardmäßig in der Molekular-und Zellbiologie zur Erforschung von Aufbau und Funktion biologischer Moleküle eingesetzt wird. Hochauflösende Verfahren der Fluoreszenzmikroskopie, wie die dSTORM (direct Stochastic Optical Reconstruction Microscopy) Technik, ermöglichen die Visualisierung zellulärer Strukturen im Nanometer-Größenbereich (10−9 m). Dadurch konnte bereits die Zusammensetzung und Funktion unterschiedlicher Biopolymere, wie die von Proteinen, Lipiden und Nukleinsäuren, bis hin zum dreidimensionalen (3D) Aufbau ganzer Organellen entschlüsselt werden. In der Praxis zeigt sich jedoch, dass diese Bildgebungsverfahren und ihre Weiterentwicklungen immer noch vor großen Herausforderungen stehen, bevor eine effektive Auflösung von unter ∼10 nm erreicht werden kann. Die größte Hürde stellt die Art und Weise der Markierung von Biomolekülen dar. Bei dieser wird zum Nachweis molekularer Strukturen häufig die sogenannte Immunfärbung als Standardmethode eingesetzt. Antikörper, welche mit Fluoreszenzmolekülen gekoppelt werden, erkennen und binden hierbei spezifisch und mit hoher Affinität den Molekülabschnitt der Zielstruktur, auch Epitop oder Antigen genannt. Die Fluoreszenzmoleküle dienen als Reportermoleküle, welche mit Hilfe eines Fluoreszenzmikroskops abgebildet werden. Die Größe der Antikörper, mit einer Länge von etwa 10-15 nm im Falle von Immunglobulin G (IgG) Antikörpern, bewirkt jedoch eine Detektion der fluoreszierenden Moleküle verschoben zur eigentlichen Lage des untersuchten Antigens. In Regionen mit räumlich dicht nebeneinander liegenden Epitopen kann es zusätzlich zur sterischen Hinderung zwischen den Antikörpern kommen. Dies führt zu einer unzureichenden Markierungsdichte und stellt - zusammen mit der verschobenen Detektion der Fluoreszenzmoleküle - eine Limitierung der zu erreichenden Auflösung dar. Die Expansionsmikroskopie (ExM) ist ein neu entwickeltes Verfahren, welches eine Auflösungsverbesserung durch die physikalische Expansion eines untersuchten Objekts erreicht. Hierbei werden biologische Proben, wie beispielsweise kultivierte Zellen, Gewebeschnitte, ganze Organe oder isolierte Organellen, chemisch in ein quellbares Polymer verankert. Durch Absorption von Wasser vergrößert dieser sogenannte Superabsorber sein eigenes Volumen und zieht während der räumlichen Expansion die kovalent gebundenen Biomoleküle isotrop auseinander. Standardmäßig wird durch dieses Verfahren eine Vergrößerung der Proben um etwa das vierfache Volumen erreicht, wobei bereits Protokollvarianten entwickelt wurden, die eine bis zu 50-fache Expansion erzielt haben. Da die ExM-Technik zunächst nur die Probenbehandlung zur Verankerung und Vergrößerung der Probe selbst beinhaltet, kann sie mit unterschiedlichen Standardmethoden der Fluoreszenzmikroskopie kombiniert werden. Dadurch verbessert sich die Auflösung des verwendeten Bildgebungsverfahrens theoretisch linear um den Faktor der Volumenvergrößerung der ExM behandelten Probe. Eine unzureichende Markierungsdichte und die Größe der verwendeten Antikörper können auch hier die effektiv erreichbare Auflösung beeinträchtigen. Die Kombination der ExM mit hochauflösenden Verfahren der Fluoreszenzmikroskopie stellt eine vielversprechende Strategie zur Erhöhung der bisher erreichbaren Auflösung in der Lichtmikroskopie dar. In dieser Arbeit werde ich mehrere von mir entwickelte ExM Varianten vorstellen, welche die Kombination von ExM mit konfokaler Mikroskopie, SIM (Structured Illumination Microscopy), STED (STimulated Emission Depletion) und dSTORM zeigen. Um die Verbindung mit dem jeweiligen Bildgebungsverfahren zu ermöglichen, optimierte ich bestehende ExM-Protokolle und entwickelte unterschiedliche Expansionsstrategien. Dadurch konnte ich neue strukturelle Erkenntnisse von isolierten Zentriolen aus der Grünalge Chlamydomonas reinhardtii durch die Verbindung von ExM mit STED und konfokaler Mikroskopie gewinnen. In einem weiteren Projekt kombinierte ich 3D-SIM mit ExM und untersuchte den molekularen Aufbau des sogenannten synaptonemalen Komplexes. Diese Struktur bildet sich in eukaryotischen Zellen während der Reifeteilung (Meiose) aus und trägt zum Austausch des genetischen Materials zwischen homologen Chromosomen bei. Vor allem in Verbindung mit dSTORM zeigte sich das hohe Potential der ExM-Methode, die bisherigen Limitierungen moderner Techniken der Fluoreszenzmikroskopie zu überwinden. In diesem Projekt expandierte ich Mikrotubuli in Säugetierzellen, ein Polymer des Zytoskeletts, sowie isolierte Zentriolen aus C. reinhardtii. Dadurch, dass die Markierung erst nach dem Expandieren der Proben erfolgte, gelang es, den Abstandsfehler der Markierung deutlich zu verringern und eine verbesserte Markierungsdichte zu erreichen. Diese Vorteile könnten in Verbindung mit der Einzelmolekülsensititvität und hohen Auflösung der dSTORM Methode Wegbereiter zur Erreichung einer molekularen Auflösung sein KW - Fluoreszenzmikroskopie KW - Expansionsmikroskopie KW - Einzelmolekül-Lokalisationsmikroskopie KW - Zentriolen KW - Synaptonemaler Komplex Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212362 ER - TY - THES A1 - Kehrberger, Sandra T1 - Effects of climate warming on the timing of flowering and emergence in a tritrophic relationship: plants - bees - parasitoids T1 - Auswirkungen der Klimaerwärmung auf die zeitliche Regulierung der Blüte und des Schlupfes in einer tritrophischen Beziehung: Pflanzen - Bienen - Parasitoide N2 - The right timing of phenological events is crucial for species fitness. Species should be highly synchronized with mutualists, but desynchronized with antagonists. With climate warming phenological events advance in many species. However, often species do not respond uniformly to warming temperatures. Species-specific responses to climate warming can lead to asynchrony or even temporal mismatch of interacting species. A temporal mismatch between mutualists, which benefit from each other, can have negative consequences for both interaction partners. For host-parasitoid interactions temporal asynchrony can benefit the host species, if it can temporally escape its parasitoid, with negative consequences for the parasitoid species, but benefit the parasitoid species if it increases synchrony with its host, which can negatively affect the host species. Knowledge about the drivers of phenology and the species-specific responses to these drivers are important to predict future effects of climate change on trophic interactions. In this dissertation I investigated how different drivers act on early flowering phenology and how climate warming affects the tritrophic relationship of two spring bees (Osmia cornuta & Osmia bicornis), an early spring plant (Pulsatilla vulgaris), which is one of the major food plants of the spring bees, and three main parasitoids of the spring bees (Cacoxenus indagator, Anthrax anthrax, Monodontomerus). In Chapter II I present a study in which I investigated how different drivers and their change over the season affect the reproductive success of an early spring plant. For that I recorded on eight calcareous grasslands around Würzburg, Germany the intra-seasonal changes in pollinator availability, number of co-flowering plants and weather conditions and studied how they affect flower visitation rates, floral longevity and seed set of the early spring plant P. vulgaris. I show that bee abundances and the number of hours, which allowed pollinator foraging, were low at the beginning of the season, but increased over time. However, flower visitation rates and estimated total number of bee visits were higher on early flowers of P. vulgaris than later flowers. Flower visitation rates were also positively related to seed set. Over time and with increasing competition for pollinators by increasing numbers of co-flowering plants flower visitation rates decreased. My data shows that a major driver for early flowering dates seems to be low interspecific competition for pollinators, but not low pollinator abundances and unfavourable weather conditions. Chapter III presents a study in which I investigated the effects of temperature on solitary bee emergence and on the flowering of their food plant and of co-flowering plants in the field. Therefore I placed bee cocoons of two spring bees (O. cornuta & O. bicornis) on eleven calcareous grasslands which differed in mean site temperature. On seven of these grasslands the early spring plant P. vulgaris occurred. I show that warmer temperatures advanced mean emergence in O. cornuta males. However, O. bicornis males and females of both species did not shift their emergence. Compared to the bees P. vulgaris advanced its flowering phenology more strongly with warmer temperatures. Co-flowering plants did not shift flowering onset. I suggest that with climate warming the first flowers of P. vulgaris face an increased risk of pollinator limitation whereas for bees a shift in floral resources may occur. In Chapter IV I present a study in which I investigated the effects of climate warming on host-parasitoid relationships. I studied how temperature and photoperiod affect emergence phenology in two spring bees (O. cornuta & O. bicornis) and three of their main parasitoids (C. indagator, A. anthrax, Monodontomerus). In a climate chamber experiment with a crossed design I exposed cocoons within nest cavities and cocoons outside of nest cavities to two different temperature regimes (long-term mean of Würzburg, Germany and long-term mean of Würzburg + 4 °C) and three photoperiods (Würzburg vs. Snåsa, Norway vs. constant darkness) and recorded the time of bee and parasitoid emergence. I show that warmer temperatures advanced emergence in all studied species, but bees advanced less strongly than parasitoids. Consequently, the time period between female bee emergence and parasitoid emergence decreased in the warm temperature treatment compared to the cold one. Photoperiod influenced the time of emergence only in cocoons outside of nest cavities (except O. bicornis male emergence). The data also shows that the effect of photoperiod compared to the effect of temperature on emergence phenology was much weaker. I suggest that with climate warming the synchrony of emergence phenologies of bees and their parasitoids will amplify. Therefore, parasitism rates in solitary bees might increase which can negatively affect reproductive success and population size. In this dissertation I show that for early flowering spring plants low interspecific competition for pollinators with co-flowering plants is a major driver of flowering phenology, whereas other drivers, like low pollinator abundances and unfavourable weather conditions are only of minor importance. With climate warming the strength of different drivers, which act on the timing of phenological events, can change, like temperature. I show that warmer temperatures advance early spring plant flowering more strongly than bee emergence and flowering phenology of later co-flowering plants. Furthermore, I show that warmer temperatures advance parasitoid emergence more strongly than bee emergence. Whereas temperature changes can lead to non-uniform temporal shifts, I demonstrate that geographic range shifts and with that altered photoperiods will not change emergence phenology in bees and their parasitoids. In the tritrophic system I investigated in this dissertation climate warming may negatively affect the reproductive success of the early spring plant and the spring bees but not of the parasitoids, which may even benefit from warming temperatures. N2 - Der richtige Zeitpunkt von phänologischen Ereignissen ist maßgeblich für das Überleben und die Fortpflanzung einer Art. Arten sollten eine möglichst hohe Synchronisation mit Mutualisten aufweisen, aber eine möglichst geringe mit Antagonisten. Die Klimaerwärmung führt dazu, dass sich bei vielen Arten phänologische Ereignisse verfrühen. Allerdings reagieren Arten unterschiedlich auf wärmere Temperaturen. Artspezifische Reaktionen auf die Klimaerwärmung können zu Asynchronität oder sogar zu zeitlicher Diskrepanz bei interagierenden Arten führen. Eine zeitliche Diskrepanz zwischen Mutualisten, die voneinander profitieren, kann sich negativ auf beide Interaktionspartner auswirken. Bei Wirt-Parasitoid Beziehungen kann der Wirt von einer zeitlichen Diskrepanz profitieren, wenn er seinem Parasitoid zeitlich entfliehen kann, was wiederum negative Folgen für den Parasitoid haben kann. Jedoch kann der Parasitoid profitieren, wenn er die Synchronisation mit seinem Wirt erhöhen kann, was wiederum den Wirt negativ beeinflussen kann. Das Wissen über die Treiber von phänologischen Ereignissen und die artspezifischen Reaktionen auf diese Treiber sind von Bedeutung um die Auswirkungen des Klimawandels auf trophische Beziehungen vorherzusagen. In meiner Doktorarbeit habe ich untersucht, wie verschiedene Treiber mit einer frühen Blüte zusammenhängen und wie der Klimawandel die tritrophische Beziehung von zwei Frühlingsbienen (Osmia cornuta & Osmia bicornis), einer Frühlingspflanzenart (Pulsatilla vulgaris), die eine der wichtigen Futterpflanzen der Bienen ist, und der drei Hauptparasitoiden der Frühlingsbienen (Cacoxenus indagator, Anthrax anthrax, Monodontomerus) beeinflusst. In Kapitel II präsentiere ich eine Studie, in der ich den Einfluss verschiedener Treiber und ihre saisonale Veränderung auf den Fortpflanzungserfolg einer Frühlingspflanzenart untersucht habe. Dazu habe ich auf acht Kalkmagerrasen bei Würzburg (Deutschland) die innersaisonalen Veränderungen der Bestäuberverfügbarkeit, der Anzahl an gleichzeitig blühenden Pflanzenarten und die Wetterbedingungen aufgezeichnet. Des Weiteren habe ich erforscht wie diese Faktoren die Blütenbesuchsrate, die Blütenlanglebigkeit und den Samenansatz der Frühlingspflanze P. vulgaris beeinflussen. Ich konnte zeigen, dass die Anzahl an Bienen und die Anzahl an Stunden, die ein Furagieren von Bestäubern ermöglicht hätten, am Anfang der Saison niedrig waren und mit der Zeit zunahmen. Jedoch war die Blütenbesuchsrate und die geschätzte Anzahl an Bienenbesuchen höher bei frühen als bei späten P. vulgaris Blüten. Die Blütenbesuchsrate wirkte sich auch positiv auf den Samenansatz aus. Die Blütenbesuchsrate nahm mit der Zeit und mit zunehmender Konkurrenz um Bestäuber durch eine zunehmende Anzahl an gleichzeitig blühenden Pflanzenarten ab. Meine Daten zeigen, dass ein Hauptreiber von frühen Blühzeitpunkten die geringe zwischenartliche Konkurrenz um Bestäuber ist, aber nicht die niedrige Bestäuberanzahl und ungünstige Wetterbedingungen. Kapitel III präsentiert eine Studie, in welcher ich die Auswirkungen der Temperatur auf den Schlupf von Solitärbienen und die Blüte ihrer Futterpflanzen und gleichzeitig blühenden Pflanzen im Freiland untersucht habe. Dafür habe ich Bienenkokons von zwei Frühlingsbienen (O. cornuta & O. bicornis) auf elf Kalkmagerrasen, die sich in der mittleren Flächentemperatur unterschieden, platziert. Auf sieben dieser Kalkmagerrasen kam die Frühlingspflanzenart P. vulgaris vor. Ich konnte zeigen, dass wärmere Temperaturen den mittleren Schlupf von O. cornuta Männchen verfrühen. Die Männchen von O. bicornis und die Weibchen beider Arten haben ihren Schlupfzeitpunkt jedoch nicht verschoben. Im Vergleich zu den Bienen verfrühte P. vulgaris seine Blühphänologie bei warmen Temperaturen stärker. Die gleichzeitig blühenden Pflanzenarten verschoben ihren Blühbeginn nicht. Die Daten zeigen, dass wärmere Temperaturen den Bienenschlupf weniger stark verfrühen als die Blüte ihrer Futterpflanze. Das lässt darauf schließen, dass mit dem Klimawandel die ersten Blüten von P. vulgaris ein erhöhtes Risiko haben nicht bestäubt zu werden, während die Bienen möglicherweise auf andere Blühressourcen ausweichen müssen. Kapitel IV beschreibt eine Studie, in welcher ich die Auswirkungen der Klimaerwärmung auf Wirt-Parasitoid Beziehungen untersucht habe. Dabei habe ich die Auswirkungen von Temperatur und Photoperiode auf die Schlupfphänologie zweier Frühlingsbienen (O. cornuta & O. bicornis) und drei ihrer Hauptparasitoide (C. indagator, A. anthrax, Monodontomerus) erforscht. In einem Klimakammerexperiment mit gekreuztem Design habe ich Kokons in Nesthöhlen und Kokons außerhalb von Nesthöhlen, zwei verschiedenen Temperaturregimen (Langzeitmittel von Würzburg, Deutschland und Langzeitmittel von Würzburg + 4 °C) und drei Photoperioden (Würzburg, Deutschland contra Snåsa, Norwegen contra Dauerdunkel) ausgesetzt und die Zeitpunkte des Bienen- und Parasitoidenschlupfes aufgezeichnet. Ich konnte zeigen, dass warme Temperaturen in allen untersuchten Arten den Schlupfzeitpunkt verfrühten, jedoch bei den Bienen weniger stark als bei den Parasitoiden. Eine Folge daraus ist, dass sich die Zeitspanne zwischen dem Schlupf der Bienenweibchen und dem Schlupf der Parasitoide im warmen Temperaturregime im Vergleich zum kalten verkürzte. Die Photoperiode hatte auf den Zeitpunkt des Schlupfes nur in Kokons außerhalb von Nisthöhlen einen Effekt (außer beim Schlupf von O. bicornis Männchen). Die Daten zeigen auch, dass der Effekt der Photoperiode auf die Schlupfphänologie im Vergleich zu dem Effekt der Temperatur viel schwächer war. Daraus schließe ich, dass sich im Zuge der Klimaerwärmung die Synchronisation der Schlupfphänologien von Bienen und ihren Parasitoiden verstärken wird. Eine Folge davon könnten erhöhte Parasitierungsraten bei Solitärbienen sein, welche den Reproduktionserfolg und die Populationsgröße negativ beeinflussen können. In dieser Doktorarbeit habe ich gezeigt, dass einer der Haupttreiber einer frühen Blüte bei Frühlingspflanzen geringe zwischenartliche Konkurrenz um Bestäuber mit später gleichzeitig blühenden Pflanzenarten ist, während andere Treiber, wie geringe Bestäuberabundanzen und ungünstige Wetterbedingungen nur von geringer Bedeutung sind. Im Zuge des Klimawandels könnte sich die Stärke verschiedener Treiber, die den Zeitpunkt von phänologischen Ereignissen beeinflussen, verändern. Ich konnte außerdem zeigen, dass wärmere Temperaturen die Blüte von frühen Frühlingspflanzen stärker verfrühen, als den Schlupf von Bienen und die Blüte von später gleichzeitig blühenden Pflanzenarten. Des Weiteren zeigte ich, dass wärmere Temperaturen den Schlupf von Parasitoiden stärker verfrühen als den Schlupf der Bienen. Ich konnte zeigen, dass während Temperaturveränderungen zu verschieden starken zeitlichen Verschiebungen führen können, Verschiebungen von geografischen Verbreitungsgebieten und damit geänderten Photoperioden die Schlupfphänologie von Bienen und ihren Parasitoiden wahrscheinlich nicht ändern werden. In dem tritrophischen System, das ich in dieser Doktorarbeit untersucht habe, könnte die Erwärmung des Klimas den Fortpflanzungserfolg der frühen Frühlingspflanze und der Frühlingsbienen negativ beeinflussen, aber wahrscheinlich nicht die der Parasitoide, die vielleicht sogar davon profitieren können. KW - Biene KW - Klimaänderung KW - Interaktion KW - Parasitoid KW - Pflanzen KW - Klimaerwärmung KW - Mutualismus KW - Antagonismus KW - Synchronisation KW - Phänologie Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213932 ER - TY - JOUR A1 - Paponov, Ivan A. A1 - Dindas , Julian A1 - Król , Elżbieta A1 - Friz, Tatyana A1 - Budnyk, Vadym A1 - Teale, William A1 - Paponov, Martina A1 - Hedrich , Rainer A1 - Palme, Klaus T1 - Auxin-Induced plasma membrane depolarization is regulated by Auxin transport and not by AUXIN BINDING PROTEIN1 JF - Frontiers in Plant Science N2 - Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling. KW - auxin KW - ABP1 KW - plasma membrane depolarization KW - AUX1 KW - endocytosis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195914 SN - 1664-462X VL - 9 ER - TY - THES A1 - Maistrenko, Oleksandr T1 - Pangenome analysis of bacteria and its application in metagenomics T1 - Bakterielle Pan-Genome und ihre Anwendungen in der Metagenomik N2 - The biosphere harbors a large quantity and diversity of microbial organisms that can thrive in all environments. Estimates of the total number of microbial species reach up to 1012, of which less than 15,000 have been characterized to date. It has been challenging to delineate phenotypically, evolutionary and ecologically meaningful lineages such as for example, species, subspecies and strains. Even within recognized species, gene content can vary considerably between sublineages (for example strains), a problem that can be addressed by analyzing pangenomes, defined as the non-redundant set of genes within a phylogenetic clade, as evolutionary units. Species considered to be ecologically and evolutionary coherent units, however to date it is still not fully understood what are primary habitats and ecological niches of many prokaryotic species and how environmental preferences drive their genomic diversity. Majority of comparative genomics studies focused on a single prokaryotic species in context of clinical relevance and ecology. With accumulation of sequencing data due to genomics and metagenomics, it is now possible to investigate trends across many species, which will facilitate understanding of pangenome evolution, species and subspecies delineation. The major aims of this thesis were 1) to annotate habitat preferences of prokaryotic species and strains; 2) investigate to what extent these environmental preferences drive genomic diversity of prokaryotes and to what extent phylogenetic constraints limit this diversification; 3) explore natural nucleotide identity thresholds to delineate species in bacteria in metagenomics gene catalogs; 4) explore species delineation for applications in subspecies and strain delineation in metagenomics. The first part of the thesis describes methods to infer environmental preferences of microbial species. This data is a prerequisite for the analyses performed in the second part of the thesis which explores how the structure of bacterial pangenomes is predetermined by past evolutionary history and how is it linked to environmental preferences of the species. The main finding in this subchapter that habitat preferences explained up to 49% of the variance for pangenome structure, compared to 18% by phylogenetic inertia. In general, this trend indicates that phylogenetic inertia does not limit evolution of pangenome size and diversity, but that convergent evolution may overcome phylogenetic constraints. In this project we show that core genome size is associated with higher environmental ubiquity of species. It is likely this is due to the fact that species need to have more versatile genomes and most necessary genes need to be present in majority of genomes of that species to be highly prevalent. Taken together these findings may be useful for future predictive analyses of ecological niches in newly discovered species. The third part of the thesis explores data-driven, operational species boundaries. I show that homologous genes from the same species from different genomes tend to share at least 95% of nucleotide identity, while different species within the same genus have lower nucleotide identity. This is in line with other studies showing that genome-wide natural species boundary might be in range of 90-95% of nucleotide identity. Finally, the fourth part of the thesis discusses how challenges in species delineation are relevant for the identification of meaningful within-species groups, followed by a discussion on how advancements in species delineation can be applied for classification of within-species genomic diversity in the age of metagenomics. N2 - Die Biosphäre beherbergt eine große Zahl verschiedener Mikroorganismen, die fast alle bekannten Lebensräume besiedeln können. Die Gesamtzahl mikrobieller Spezies liegt Schätzungen zu Folge bei bis zu 1012, von denen jedoch bis heute erst 15.000 beschrieben worden sind. Die Beschreibung von phänotypisch, evolutionsbiologisch und ökologisch kohärenten Spezies, Sub-Spezies oder Stämmen stellt Forscher vor konzeptionelle Herausforderungen. Selbst innerhalb anerkannter Spezies kann die Kombination einzelner Gene oft stark variieren. Diese Beobachtung ist die Grundlage der Analyse von Pan-Genomen. also der Konstellation originärer Gene innerhalb einer Abstammunsglinie, als evolutionsbiologische Einheiten. Spezies entsprechen prinzipiell ökologisch und evolutionär kohärenten Einheiten, jedoch sind die primären Habitate und ökologischen Nischen vieler prokaryotischer Spezies bis heute nur unzureichend beschrieben, insbesondere mit Blick auf den Einfluss ökologischer Präferenzen auf die Evolution von Genomen. Die Mehrheit vergleichender genomischer Studien untersucht einzelne prokaryotische Spezies mit Bezug auf deren klinische oder ökologische Relevanz. Aufgrund der wachsenden Verfügbarkeit genomischer Daten ist es nun jedoch möglich, vergleichende Studien über Speziesgrenzen hinweg durchzuführen, um allgemeine Prinzipien der Evolution von Pan-Genomen, Spezies und Sub-Spezies zu untersuchen. Die wesentlichen Ziele der vorliegenden Arbeit waren 1) die Annotation von Habitatpräferenzen prokaryotischer Spezies und Stämme; 2) die Quantifizierung des Einflusses von Umwelt und Evolutionsgeschichte (Phylogenie) auf die genomische Diversität von Prokaryoten; 3) die Bestimmung natürlicher Schwellenwerte der Genomsequenzähnlichkeit zwischen Spezies, auch anhand von Genkatalogen; 4) die Untersuchung der Abgrenzung zwischen Spezies, Sub-Spezies und Stämmen mithilfe metagenomischer Daten. Im ersten Teil der Arbeit werden Methoden zur Bestimmung ökologischer Präferenzen mikrobieller Spezies beschrieben. Die so gewonnenen Daten dienen in der Folge als Grundlage für die Quantifizierung von Umwelt- und evolutionsgeschichtlichen Einflüssen auf die Struktur und Evolution bakterieller Pan-Genome im zweiten Teil der Arbeit. Ein zentrales Ergebnis dieser Untersuchung war, dass bis zu 49% der strukturellen Varianz in Pan-Genomen durch Habitatpräferenzen erklärt werden kann, im Gegensatz zu lediglich 18% durch phylogenetische Trägheitseffekte. Dies zeigt, dass die Größe und Diversität von Pan-Genomen nicht phylogenetisch limitiert ist, insbesondere in Fällen von konvergenter Evolution. Große Kern-Genome sind ferner mit einer weiten ökologischen Verbreitung von Spezies assoziiert; eine mögliche Erklärung ist, dass weit verbreitete Spezies vielseitigere Genome mit mehr notwendigen Genen besitzen, die ein Überleben in vielfältigen Umgebungen ermöglichen. Die vorgelegte Arbeit kann weiterhin einen Beitrag zur Vorhersage ökologischer Profile neu beschriebener Spezies leisten. Im dritten Teil der Arbeit werden datenbezogene, operationelle Definition von Spezies-Grenzen untersucht. Es konnte gezeigt werden, dass Gene verschiedener Genome innerhalb derselben Spezies normalerweise mindestens 95% Ähnlichkeit der Nukleotidsequenz aufweisen, während die Ähnlichkeit zwischen Spezies desselben Genus geringer ausfällt. Dieser Wert liegt im Rahmen früherer Schätzungen. Der vierte Teil der Arbeit beschreibt abschließend die Herausforderungen bei der Bestimmung von evolutionären Linien innerhalb von Spezies und diskutiert anschließend, wie konzeptionelle Entwicklungen in dieser Frage für die Klassifizierung und Quantifizierung von Diversität anhand metagenomischer Daten genutzt werden kann. KW - Pangenom KW - phylogenetische Trägheit KW - Lebensraum KW - Stammvielfalt KW - mikrobielle Ökologie und Evolution KW - pangenome KW - phylogenetic inertia KW - habitat KW - strain diversity KW - microbial ecology and evolution KW - metagenomics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214996 ER - TY - JOUR A1 - Rössler, Wolfgang A1 - Spaethe, Johannes A1 - Groh, Claudia T1 - Pitfalls of using confocal-microscopy based automated quantification of synaptic complexes in honeybee mushroom bodies (response to Peng and Yang 2016) JF - Scientific Reports N2 - A recent study by Peng and Yang in Scientific Reports using confocal-microscopy based automated quantification of anti-synapsin labeled microglomeruli in the mushroom bodies of honeybee brains reports potentially incorrect numbers of microglomerular densities. Whereas several previous studies using visually supervised or automated counts from confocal images and analyses of serial 3D electron-microscopy data reported consistent numbers of synaptic complexes per volume, Peng and Yang revealed extremely low numbers differing by a factor of 18 or more from those obtained in visually supervised counts, and by a factor 22–180 from numbers in two other studies using automated counts. This extreme discrepancy is especially disturbing as close comparison of raw confocal images of anti-synapsin labeled whole-mount brain preparations are highly similar across these studies. We conclude that these discrepancies may reside in potential misapplication of confocal imaging followed by erroneous use of automated image analysis software. Consequently, the reported microglomerular densities during maturation and after manipulation by insecticides require validation by application of appropriate confocal imaging methods and analyses tools that rely on skilled observers. We suggest several improvements towards more reliable or standardized automated or semi-automated synapse counts in whole mount preparations of insect brains. KW - confocal-microscopy based automated quantification KW - mushroom bodies KW - honeybees KW - brain Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170451 VL - 7 IS - 9786 ER - TY - JOUR A1 - Bowler, Diana E. A1 - Bjorkman, Anne D. A1 - Dornelas, Maria A1 - Myers‐Smith, Isla H. A1 - Navarro, Laetitia M. A1 - Niamir, Aidin A1 - Supp, Sarah R. A1 - Waldock, Conor A1 - Winter, Marten A1 - Vellend, Mark A1 - Blowes, Shane A. A1 - Böhning‐Gaese, Katrin A1 - Bruelheide, Helge A1 - Elahi, Robin A1 - Antão, Laura H. A1 - Hines, Jes A1 - Isbell, Forest A1 - Jones, Holly P. A1 - Magurran, Anne E. A1 - Cabral, Juliano Sarmento A1 - Bates, Amanda E. T1 - Mapping human pressures on biodiversity across the planet uncovers anthropogenic threat complexes JF - People and Nature N2 - Climate change and other anthropogenic drivers of biodiversity change are unequally distributed across the world. Overlap in the distributions of different drivers have important implications for biodiversity change attribution and the potential for interactive effects. However, the spatial relationships among different drivers and whether they differ between the terrestrial and marine realm has yet to be examined. We compiled global gridded datasets on climate change, land‐use, resource exploitation, pollution, alien species potential and human population density. We used multivariate statistics to examine the spatial relationships among the drivers and to characterize the typical combinations of drivers experienced by different regions of the world. We found stronger positive correlations among drivers in the terrestrial than in the marine realm, leading to areas with high intensities of multiple drivers on land. Climate change tended to be negatively correlated with other drivers in the terrestrial realm (e.g. in the tundra and boreal forest with high climate change but low human use and pollution), whereas the opposite was true in the marine realm (e.g. in the Indo‐Pacific with high climate change and high fishing). We show that different regions of the world can be defined by Anthropogenic Threat Complexes (ATCs), distinguished by different sets of drivers with varying intensities. We identify 11 ATCs that can be used to test hypotheses about patterns of biodiversity and ecosystem change, especially about the joint effects of multiple drivers. Our global analysis highlights the broad conservation priorities needed to mitigate the impacts of anthropogenic change, with different priorities emerging on land and in the ocean, and in different parts of the world. KW - Anthropocene KW - biodiversity threats KW - direct drivers KW - global change Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213634 VL - 2 IS - 2 SP - 380 EP - 394 ER - TY - JOUR A1 - Gupta, Shishir K. A1 - Srivastava, Mugdha A1 - Osmanoglu, Oezge A1 - Dandekar, Thomas T1 - Genome-wide inference of the Camponotus floridanus protein-protein interaction network using homologous mapping and interacting domain profile pairs JF - Scientific Reports N2 - Apart from some model organisms, the interactome of most organisms is largely unidentified. High-throughput experimental techniques to determine protein-protein interactions (PPIs) are resource intensive and highly susceptible to noise. Computational methods of PPI determination can accelerate biological discovery by identifying the most promising interacting pairs of proteins and by assessing the reliability of identified PPIs. Here we present a first in-depth study describing a global view of the ant Camponotus floridanus interactome. Although several ant genomes have been sequenced in the last eight years, studies exploring and investigating PPIs in ants are lacking. Our study attempts to fill this gap and the presented interactome will also serve as a template for determining PPIs in other ants in future. Our C. floridanus interactome covers 51,866 non-redundant PPIs among 6,274 proteins, including 20,544 interactions supported by domain-domain interactions (DDIs), 13,640 interactions supported by DDIs and subcellular localization, and 10,834 high confidence interactions mediated by 3,289 proteins. These interactions involve and cover 30.6% of the entire C. floridanus proteome. KW - interaction map KW - drosophila KW - identification KW - evolutionary KW - reliability KW - annotation KW - database KW - target KW - cycle Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229406 VL - 10 IS - 1 ER - TY - JOUR A1 - Schneider-Schaulies, Sibylle A1 - Schumacher, Fabian A1 - Wigger, Dominik A1 - Schöl, Marie A1 - Waghmare, Trushnal A1 - Schlegel, Jan A1 - Seibel, Jürgen A1 - Kleuser, Burkhard T1 - Sphingolipids: effectors and Achilles heals in viral infections? JF - Cells N2 - As viruses are obligatory intracellular parasites, any step during their life cycle strictly depends on successful interaction with their particular host cells. In particular, their interaction with cellular membranes is of crucial importance for most steps in the viral replication cycle. Such interactions are initiated by uptake of viral particles and subsequent trafficking to intracellular compartments to access their replication compartments which provide a spatially confined environment concentrating viral and cellular components, and subsequently, employ cellular membranes for assembly and exit of viral progeny. The ability of viruses to actively modulate lipid composition such as sphingolipids (SLs) is essential for successful completion of the viral life cycle. In addition to their structural and biophysical properties of cellular membranes, some sphingolipid (SL) species are bioactive and as such, take part in cellular signaling processes involved in regulating viral replication. It is especially due to the progress made in tools to study accumulation and dynamics of SLs, which visualize their compartmentalization and identify interaction partners at a cellular level, as well as the availability of genetic knockout systems, that the role of particular SL species in the viral replication process can be analyzed and, most importantly, be explored as targets for therapeutic intervention. KW - glycosphingolipids KW - ceramides KW - sphingosine 1-phosphate KW - sphingomyelinase KW - HIV KW - SARS-CoV-2 KW - measles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245151 SN - 2073-4409 VL - 10 IS - 9 ER - TY - JOUR A1 - Haertle, Larissa A1 - El Hajj, Nady A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Nanda, Indrajit A1 - Lehnen, Harald A1 - Haaf, Thomas T1 - Epigenetic signatures of gestational diabetes mellitus on cord blood methylation JF - Clinical Epigenetics N2 - Background: Intrauterine exposure to gestational diabetes mellitus (GDM) confers a lifelong increased risk for metabolic and other complex disorders to the offspring. GDM-induced epigenetic modifications modulating gene regulation and persisting into later life are generally assumed to mediate these elevated disease susceptibilities. To identify candidate genes for fetal programming, we compared genome-wide methylation patterns of fetal cord bloods (FCBs) from GDM and control pregnancies. Methods and results: Using Illumina’s 450K methylation arrays and following correction for multiple testing, 65 CpG sites (52 associated with genes) displayed significant methylation differences between GDM and control samples. Four candidate genes, ATP5A1, MFAP4, PRKCH, and SLC17A4, from our methylation screen and one, HIF3A, from the literature were validated by bisulfite pyrosequencing. The effects remained significant after adjustment for the confounding factors maternal BMI, gestational week, and fetal sex in a multivariate regression model. In general, GDM effects on FCB methylation were more pronounced in women with insulin-dependent GDM who had a more severe metabolic phenotype than women with dietetically treated GDM. Conclusions: Our study supports an association between maternal GDM and the epigenetic status of the exposed offspring. Consistent with a multifactorial disease model, the observed FCB methylation changes are of small effect size but affect multiple genes/loci. The identified genes are primary candidates for transmitting GDM effects to the next generation. They also may provide useful biomarkers for the diagnosis, prognosis, and treatment of adverse prenatal exposures. KW - fetal programming KW - insulin treatment KW - DNA methylation KW - fetal cord blood KW - gestational diabetes mellitus Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159459 VL - 9 IS - 28 ER - TY - JOUR A1 - Roth, Nicolas A1 - Doerfler, Inken A1 - Bässler, Claus A1 - Blaschke, Markus A1 - Bussler, Heinz A1 - Gossner, Martin M. A1 - Heideroth, Antje A1 - Thorn, Simon A1 - Weisser, Wolfgang W. A1 - Müller, Jörg T1 - Decadal effects of landscape-wide enrichment of dead wood on saproxylic organisms in beech forests of different historic management intensity JF - Diversity and Distributions N2 - Aim: European temperate forests have lost dead wood and the associated biodiversity owing to intensive management over centuries. Nowadays, some of these forests are being restored by enrichment with dead wood, but mostly only at stand scales. Here, we investigated effects of a seminal dead-wood enrichment strategy on saproxylic organisms at the landscape scale. Location: Temperate European beech forest in southern Germany. Methods: In a before-after control-impact design, we compared assemblages and gamma diversities of saproxylic organisms in strictly protected old-growth forest areas (reserves) and historically moderately and intensively managed forest areas before and a decade after starting a landscape-wide strategy of dead-wood enrichment. Results: Before enrichment with dead wood, the gamma diversity of saproxylic organisms in historically intensively managed forest stands was significantly lower than in reserves and historically moderately managed forest stands; this difference disappeared after 10 years of dead-wood enrichment. The species composition of beetles in forest stands of the three historical management intensities differed before the enrichment strategy, but a decade thereafter, the species compositions of previously intensively logged and forest reserve plots were similar. However, the differences in fungal species composition between historical management categories before and after 10 years of enrichment persisted. Main conclusions: Our results demonstrate that intentional enrichment of dead wood at the landscape scale is a powerful tool for rapidly restoring saproxylic beetle communities and for restoring wood-inhabiting fungal communities, which need longer than a decade for complete restoration. We propose that a strategy of area-wide active restoration combined with some permanent strict refuges is a promising means of promoting the biodiversity of age-long intensively managed Central European beech forests. KW - dead-wood enrichment KW - integrative management strategy KW - land sharing KW - lowland beech forests KW - saproxylic organisms Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227061 VL - 25 IS - 3 ER - TY - JOUR A1 - Leverkus, Alexandro B. A1 - Thorn, Simon A1 - Gustafsson, Lena A1 - Noss, Reed A1 - Müller, Jörg A1 - Pausas, Juli G. A1 - Lindenmayer, David B. T1 - Environmental policies to cope with novel disturbance regimes–steps to address a world scientists’ warning to humanity JF - Environmental Research Letters N2 - No abstract available. KW - global change KW - novel disturbance KW - regime shift KW - forest management KW - risk management Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254180 SN - 1748-9326 VL - 16 IS - 2 ER - TY - JOUR A1 - Colizzi, Francesca Sara A1 - Beer, Katharina A1 - Cuti, Paolo A1 - Deppisch, Peter A1 - Martínez Torres, David A1 - Yoshii, Taishi A1 - Helfrich-Förster, Charlotte T1 - Antibodies Against the Clock Proteins Period and Cryptochrome Reveal the Neuronal Organization of the Circadian Clock in the Pea Aphid JF - Frontiers in Physiology N2 - Circadian clocks prepare the organism to cyclic environmental changes in light, temperature, or food availability. Here, we characterized the master clock in the brain of a strongly photoperiodic insect, the aphid Acyrthosiphon pisum, immunohistochemically with antibodies against A. pisum Period (PER), Drosophila melanogaster Cryptochrome (CRY1), and crab Pigment-Dispersing Hormone (PDH). The latter antibody detects all so far known PDHs and PDFs (Pigment-Dispersing Factors), which play a dominant role in the circadian system of many arthropods. We found that, under long days, PER and CRY are expressed in a rhythmic manner in three regions of the brain: the dorsal and lateral protocerebrum and the lamina. No staining was detected with anti-PDH, suggesting that aphids lack PDF. All the CRY1-positive cells co-expressed PER and showed daily PER/CRY1 oscillations of high amplitude, while the PER oscillations of the CRY1-negative PER neurons were of considerable lower amplitude. The CRY1 oscillations were highly synchronous in all neurons, suggesting that aphid CRY1, similarly to Drosophila CRY1, is light sensitive and its oscillations are synchronized by light-dark cycles. Nevertheless, in contrast to Drosophila CRY1, aphid CRY1 was not degraded by light, but steadily increased during the day and decreased during the night. PER was always located in the nuclei of the clock neurons, while CRY was predominantly cytoplasmic and revealed the projections of the PER/CRY1-positive neurons. We traced the PER/CRY1-positive neurons through the aphid protocerebrum discovering striking similarities with the circadian clock of D. melanogaster: The CRY1 fibers innervate the dorsal and lateral protocerebrum and putatively connect the different PER-positive neurons with each other. They also run toward the pars intercerebralis, which controls hormone release via the neurohemal organ, the corpora cardiaca. In contrast to Drosophila, the CRY1-positive fibers additionally travel directly toward the corpora cardiaca and the close-by endocrine gland, corpora allata. This suggests a direct link between the circadian clock and the photoperiodic control of hormone release that can be studied in the future. KW - aphids KW - circadian clock KW - cryptochrome KW - period KW - hemiptera KW - insects KW - photoperiodism Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242909 SN - 1664-042X VL - 12 ER - TY - JOUR A1 - Geisinger, Adriana A1 - Rodríguez-Casuriaga, Rosana A1 - Benavente, Ricardo T1 - Transcriptomics of Meiosis in the Male Mouse JF - Frontiers in Cell and Developmental Biology N2 - Molecular studies of meiosis in mammals have been long relegated due to some intrinsic obstacles, namely the impossibility to reproduce the process in vitro, and the difficulty to obtain highly pure isolated cells of the different meiotic stages. In the recent years, some technical advances, from the improvement of flow cytometry sorting protocols to single-cell RNAseq, are enabling to profile the transcriptome and its fluctuations along the meiotic process. In this mini-review we will outline the diverse methodological approaches that have been employed, and some of the main findings that have started to arise from these studies. As for practical reasons most studies have been carried out in males, and mostly using mouse as a model, our focus will be on murine male meiosis, although also including specific comments about humans. Particularly, we will center on the controversy about gene expression during early meiotic prophase; the widespread existing gap between transcription and translation in meiotic cells; the expression patterns and potential roles of meiotic long non-coding RNAs; and the visualization of meiotic sex chromosome inactivation from the RNAseq perspective. KW - meiosis KW - transcriptomics KW - RNAseq KW - meiotic prophase KW - spermatogenesis KW - lncRNAs KW - MSCI KW - spermatogenic cell sorting Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231032 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Maier, Carina R. A1 - Fischer, Thomas A1 - Prieto-Garcia, Cristian A1 - Baluapuri, Apoorva A1 - Schwarz, Jessica A1 - Schmitz, Werner A1 - Garrido-Rodriguez, Martin A1 - Pahor, Nikolett A1 - Davies, Clare C. A1 - Bassermann, Florian A1 - Orian, Amir A1 - Wolf, Elmar A1 - Schulze, Almut A1 - Calzado, Marco A. A1 - Rosenfeldt, Mathias T. A1 - Diefenbacher, Markus E. T1 - Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease JF - Frontiers in Cell and Developmental Biology N2 - Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research. KW - non-small cell lung cancer KW - CRISPR-Cas9 KW - mouse model KW - lung cancer KW - MYC KW - JUN Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230949 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Yu, Yidong A1 - Wolf, Ann-Katrin A1 - Thusek, Sina A1 - Heinekamp, Thorsten A1 - Bromley, Michael A1 - Krappmann, Sven A1 - Terpitz, Ulrich A1 - Voigt, Kerstin A1 - Brakhage, Axel A. A1 - Beilhack, Andreas T1 - Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms JF - Journal of Fungi N2 - Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates. KW - fungal infection model KW - calcofluor white staining KW - Aspergillus KW - Lichtheimia KW - silkworm Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228855 SN - 2309-608X VL - 7 IS - 2 ER - TY - JOUR A1 - Sponsler, Douglas A1 - Kallnik, Katharina A1 - Requier, Fabrice A1 - Classen, Alice A1 - Maihoff, A. Fabienne A1 - Sieger, Johanna A1 - Steffan-Dewenter, Ingolf T1 - Floral preferences of mountain bumble bees are constrained by functional traits but flexible through elevation and season JF - Oikos N2 - Patterns of resource use by animals can clarify how ecological communities have assembled in the past, how they currently function and how they are likely to respond to future perturbations. Bumble bees (Hymentoptera: Bombus spp.) and their floral hosts provide a diverse yet tractable system in which to explore resource selection in the context of plant–pollinator networks. Under conditions of resource limitation, the ability of bumble bees species to coexist should depend on dietary niche overlap. In this study, we report patterns and dynamics of floral morphotype preferences in a mountain bumble bee community based on ~13 000 observations of bumble bee floral visits recorded along a 1400 m elevation gradient. We found that bumble bees are highly selective generalists, rarely visiting floral morphotypes at the rates predicted by their relative abundances. Preferences also differed markedly across bumble bee species, and these differences were well-explained by variation in bumble bee tongue length, generating patterns of preference similarity that should be expected to predict competition under conditions of resource limitation. Within species, though, morphotype preferences varied by elevation and season, possibly representing adaptive flexibility in response to the high elevational and seasonal turnover of mountain floral communities. Patterns of resource partitioning among bumble bee communities may determine which species can coexist under the altered distributions of bumble bees and their floral hosts caused by climate and land use change. KW - resource selection KW - coexistence KW - competition KW - foraging KW - niche KW - pollinator Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259653 VL - 2022 IS - 3 ER - TY - JOUR A1 - De Lira, Maria Nathalia A1 - Raman, Sudha Janaki A1 - Schulze, Almut A1 - Schneider-Schaulies, Sibylle A1 - Avota, Elita T1 - Neutral Sphingomyelinase-2 (NSM 2) Controls T Cell Metabolic Homeostasis and Reprogramming During Activation JF - Frontiers in Molecular Biosciences N2 - Neutral sphingomyelinase-2 (NSM2) is a member of a superfamily of enzymes responsible for conversion of sphingomyelin into phosphocholine and ceramide at the cytosolic leaflet of the plasma membrane. Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether hyper-reactivity of NSM2-deficient T cells is supported by a deregulated metabolic activity in these cells. Here, we demonstrate that ablation of NSM2 activity affects metabolism of the quiescent CD4\(^+\) T cells which accumulate ATP in mitochondria and increase basal glycolytic activity. This supports enhanced production of total ATP and metabolic switch early after TCR/CD28 stimulation. Most interestingly, increased metabolic activity in resting NSM2-deficient T cells does not support sustained response upon stimulation. While elevated under steady-state conditions in NSM2-deficient CD4\(^+\) T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 h of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4\(^+\) T cells yet fails to support sustained T cell responses upon antigenic stimulation. KW - neutral sphingomyelinase-2 KW - T cell receptor KW - Seahorse XF KW - oxidative phosphorylation KW - ATP-adenosine triphosphate KW - Mitochondria Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211311 SN - 2296-889X VL - 7 ER - TY - JOUR A1 - Pauls, Dennis A1 - Blechschmidt, Christine A1 - Frantzmann, Felix A1 - el Jundi, Basil A1 - Selcho, Mareike T1 - A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster JF - Scientific Reports N2 - The modulation of an animal’s behavior through external sensory stimuli, previous experience and its internal state is crucial to survive in a constantly changing environment. In most insects, octopamine (OA) and its precursor tyramine (TA) modulate a variety of physiological processes and behaviors by shifting the organism from a relaxed or dormant condition to a responsive, excited and alerted state. Even though OA/TA neurons of the central brain are described on single cell level in Drosophila melanogaster, the periphery was largely omitted from anatomical studies. Given that OA/TA is involved in behaviors like feeding, flying and locomotion, which highly depend on a variety of peripheral organs, it is necessary to study the peripheral connections of these neurons to get a complete picture of the OA/TA circuitry. We here describe the anatomy of this aminergic system in relation to peripheral tissues of the entire fly. OA/TA neurons arborize onto skeletal muscles all over the body and innervate reproductive organs, the heart, the corpora allata, and sensory organs in the antennae, legs, wings and halteres underlining their relevance in modulating complex behaviors. KW - neural circuits KW - peripheral nervous system KW - Drosophila melanogaster Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177412 VL - 8 IS - 15314 ER - TY - JOUR A1 - Hensgen, Ronja A1 - England, Laura A1 - Homberg, Uwe A1 - Pfeiffer, Keram T1 - Neuroarchitecture of the central complex in the brain of the honeybee: Neuronal cell types JF - Journal of Comparative Neurology N2 - The central complex (CX) in the insect brain is a higher order integration center that controls a number of behaviors, most prominently goal directed locomotion. The CX comprises the protocerebral bridge (PB), the upper division of the central body (CBU), the lower division of the central body (CBL), and the paired noduli (NO). Although spatial orientation has been extensively studied in honeybees at the behavioral level, most electrophysiological and anatomical analyses have been carried out in other insect species, leaving the morphology and physiology of neurons that constitute the CX in the honeybee mostly enigmatic. The goal of this study was to morphologically identify neuronal cell types of the CX in the honeybee Apis mellifera. By performing iontophoretic dye injections into the CX, we traced 16 subtypes of neuron that connect a subdivision of the CX with other regions in the bee's central brain, and eight subtypes that mainly interconnect different subdivisions of the CX. They establish extensive connections between the CX and the lateral complex, the superior protocerebrum and the posterior protocerebrum. Characterized neuron classes and subtypes are morphologically similar to those described in other insects, suggesting considerable conservation in the neural network relevant for orientation. KW - RRID: AB_2337244 KW - RRID: AB_2315425 KW - central complex KW - insect brain KW - neuroanatomy KW - sky compass KW - Apis mellifera Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215566 VL - 529 ER - TY - JOUR A1 - Kunz, Tobias C. A1 - Rühling, Marcel A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kozjak-Pavlovic, Vera A1 - Rudel, Thomas A1 - Fraunholz, Martin T1 - The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy JF - Frontiers in Cellular and Infection Microbiology N2 - Expansion Microscopy (ExM) is a novel tool improving the resolution of fluorescence microscopy by linking the sample into a hydrogel that gets physically expanded in water. Previously, we have used ExM to visualize the intracellular Gram-negative pathogens Chlamydia trachomatis, Simkania negevensis, and Neisseria gonorrhoeae. Gram-positive bacteria have a rigid and thick cell wall that impedes classic expansion strategies. Here we developed an approach, which included a series of enzymatic treatments resulting in isotropic 4× expansion of the Gram-positive pathogen Staphylococcus aureus. We further demonstrate the suitability of the technique for imaging of planktonic bacteria as well as endocytosed, intracellular bacteria at a spatial resolution of approximately 60 nm with conventional confocal laser scanning microscopy. KW - high-resolution imaging KW - endosomes KW - autophagosomes KW - host-pathogen interaction KW - expansion microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232292 SN - 2235-2988 VL - 11 ER - TY - THES A1 - Reinhard, Sebastian T1 - Improving Super-Resolution Microscopy Data Reconstruction and Evaluation by Developing Advanced Processing Algorithms and Artifcial Neuronal Networks T1 - Verbesserung von Datenrekonstruktion und -auswertung in der Super-Resolution Mikroskopie durch die Entwicklung von fortgeschrittenen Verarbeitungsalgorithmen und künstlichen neuronalen Netzen N2 - The fusion of methods from several disciplines is a crucial component of scientific development. Artificial Neural Networks, based on the principle of biological neuronal networks, demonstrate how nature provides the best templates for technological advancement. These innovations can then be employed to solve the remaining mysteries of biology, including, in particular, processes that take place on microscopic scales and can only be studied with sophisticated techniques. For instance, direct Stochastic Optical Reconstruction Microscopy combines tools from chemistry, physics, and computer science to visualize biological processes at the molecular level. One of the key components is the computer-aided reconstruction of super-resolved images. Improving the corresponding algorithms increases the quality of the generated data, providing further insights into our biology. It is important, however, to ensure that the heavily processed images are still a reflection of reality and do not originate in random artefacts. Expansion microscopy is expanding the sample by embedding it in a swellable hydrogel. The method can be combined with other super-resolution techniques to gain additional resolution. We tested this approach on microtubules, a well-known filamentous reference structure, to evaluate the performance of different protocols and labelling techniques. We developed LineProfiler an objective tool for data collection. Instead of collecting perpendicular profiles in small areas, the software gathers line profiles from filamentous structures of the entire image. This improves data quantity, quality and prevents a biased choice of the evaluated regions. On the basis of the collected data, we deployed theoretical models of the expected intensity distribution across the filaments. This led to the conclusion that post-expansion labelling significantly reduces the labelling error and thus, improves the data quality. The software was further used to determine the expansion factor and arrangement of synaptonemal complex data. Automated Simple Elastix uses state-of-the-art image alignment to compare pre- and post-expansion images. It corrects linear distortions occurring under isotropic expansion, calculates a structural expansion factor and highlights structural mismatches in a distortion map. We used the software to evaluate expanded fungi and NK cells. We found that the expansion factor differs for the two structures and is lower than the overall expansion of the hydrogel. Assessing the fluorescence lifetime of emitters used for direct Stochastic Optical Reconstruction Microscopy can reveal additional information about the molecular environment or distinguish dyes emitting with a similar wavelength. The corresponding measurements require a confocal scanning of the sample in combination with the fluorescent switching of the underlying emitters. This leads to non-linear, interrupted Point Spread Functions. The software ReCSAI targets this problem by combining the classical algorithm of compressed sensing with modern methods of artificial intelligence. We evaluated several different approaches to combine these components and found, that unrolling compressed sensing into the network architecture yields the best performance in terms of reconstruction speed and accuracy. In addition to a deep insight into the functioning and learning of artificial intelligence in combination with classical algorithms, we were able to reconstruct the described non-linearities with significantly improved resolution, in comparison to other state-of-the-art architectures. N2 - Für die Weiterentwicklung der Wissenschaft wird es immer wichtiger, Methoden aus verschiedenen Gebieten zu kombinieren. Die künstliche Intelligenz beruht beispielsweise auf dem Prinzip biologischer neuronaler Netze. Hier wird die Natur als Vorlage für unsere technische Entwicklung genutzt. Diese Innovationen können dazu eingesetzt werden, die verbliebenen Rätsel der Biologie zu lösen. Dazu gehören insbesondere Prozesse, die sich auf mikroskopischer Ebene abspielen und nur mit hochentwickelten Techniken untersucht werden können. Die direkte Stochastisch Optische Rekonstruktionsmikroskopie kombiniert Methoden der Chemie, Physik und Informatik, um biologische Prozesse auf molekularer Ebene sichtbar zu machen. Eine der Schlüsselkomponenten ist die computergestützte Rekonstruktion von hochaufgelösten Bildern. Die Verbesserung der zugrunde liegenden Algorithmen erhöht die Qualität der erzeugten Daten und ermöglicht weitere Einblicke in unsere Biologie. Es muss jedoch sichergestellt werden, dass die künstlich erstellten Bilder immer noch ein Abbild der Realität sind und nicht auf zufälligen Artefakten beruhen. Expansionsmikroskopie vergrößert die Probe durch Einbettung in ein Hydrogel. Die Methode kann mit anderen hochauflösenden Techniken kombiniert werden, um die Auflösung noch weiter zu verbessern. Dieser Ansatz wurde an Mikrotubuli, einer bekannten flamentösen Referenzstruktur, verwendet, um verschiedene Protokolle und Markierungstechniken zu testen. Mit LineProfiler wurde ein objektives Werkzeug zur Datenerfassung entwickelt. Anstatt Linienprofle in kleinen Bereichen zu erfassen, wertet die Software das gesamte Bild aus. Dies verbessert die Datenmenge und Datenqualität und verhindert eine voreingenommene Auswahl der ausgewerteten Regionen. Auf Grundlage der gesammelten Daten wurden theoretische Modelle für die erwartete Intensitätsverteilung über die Filamente erstellt. Daraus konnte geschlossen werden, dass die Markierung nach der Expansion den Markierungsfehler erheblich reduziert und somit die Qualität der Daten verbessert. Die Software wurde außerdem zur Bestimmung des Expansionsfaktors und der Anordnung der Daten des synaptonemalen Komplexes verwendet. Automated Simple Elastix verwendet modernste Bildregistrierung, um Bilder vor und nach der Expansion zu vergleichen. Lineare Verzerrungen, die bei isotroper Expansion auftreten, werden korrigiert. Der strukturelle Expansionsfaktor wird berechnet und strukturelle Unstimmigkeiten werden in einer Verzerrungskarte hervorgehoben. Die Software wurde zur Bewertung expandierter Pilze und NK-Zellen eingesetzt. Dabei wurde festgestellt, dass der Expansionsfaktor für die beiden Strukturen unterschiedlich ist und unter der Gesamtexpansion des Hydrogels liegt. Die Auswertung der Fluoreszenzlebensdauer von Emittern, die für die direkte Stochastische Optische Rekonstruktionsmikroskopie eingesetzt werden, kann zusätzliche Informationen über die molekulare Umgebung liefern oder Farbstoffe unterscheiden, die VI eine ähnliche Lichtwellenlänge emittieren. Die entsprechenden Messungen erfordern eine konfokale Abtastung der Probe in Kombination mit dem fluoreszenten Schalten der zugrunde liegenden Emitter. Dies führt zu nichtlinearen, unterbrochenen Punktspreizfunktionen. Die Software ReCSAI löst dieses Problem, indem sie den klassischen Algorithmus des Compressed Sensing mit modernen Methoden der künstlichen Intelligenz kombiniert. Es wurden verschiedene Ansätze zur Kombination der Komponenten ausgewertet und festgestellt, dass die Integration von Compressed Sensing in die Netzwerkarchitektur die beste Performance in Bezug auf Rekonstruktionsgeschwindigkeit und -genauigkeit bringt. Neben einem tiefen Einblick in die Funktionsweise und das Lernen von künstlicher Intelligenz in Kombination mit klassischen Algorithmen konnten die beschriebenen Nichtlinearitäten mit einer deutlich verbesserten Auflösung im Vergleich zu anderen modernen Architekturen rekonstruiert werden. KW - Mikroskopie KW - Künstliche Intelligenz KW - Datenanalyse KW - Bildverarbeitung KW - Compressed Sensing KW - Lifetime Imaging KW - dSTORM Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-316959 ER - TY - JOUR A1 - Mall, David A1 - Larsen, Ashley E. A1 - Martin, Emily A. T1 - Investigating the (mis)match between natural pest control knowledge and the intensity of pesticide use JF - Insects N2 - Transforming modern agriculture towards both higher yields and greater sustainability is critical for preserving biodiversity in an increasingly populous and variable world. However, the intensity of agricultural practices varies strongly between crop systems. Given limited research capacity, it is crucial to focus efforts to increase sustainability in the crop systems that need it most. In this study, we investigate the match (or mismatch) between the intensity of pesticide use and the availability of knowledge on the ecosystem service of natural pest control across various crop systems. Using a systematic literature search on pest control and publicly available pesticide data, we find that pest control literature is not more abundant in crops where insecticide input per hectare is highest. Instead, pest control literature is most abundant, with the highest number of studies published, in crops with comparatively low insecticide input per hectare but with high world harvested area. These results suggest that a major increase of interest in agroecological research towards crops with high insecticide input, particularly cotton and horticultural crops such as citrus and high value-added vegetables, would help meet knowledge needs for a timely ecointensification of agriculture. KW - ecological intensification KW - insecticides KW - agroecology KW - agricultural intensity KW - biological pest control KW - crop KW - study system Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158977 VL - 9 IS - 1 ER - TY - BOOK A1 - Halder, Partho T1 - Identification and characterization of synaptic proteins of Drosophila melanogaster using monoclonal antibodies of the Wuerzburg Hybridoma Library T1 - Identifikation und Charakterisierung von synaptischen Proteinen von Drosophila melanogaster mit Hilfe von monoklonalen Antikörpern der Würzburger Hybridoma-Bibliothek N2 - For a large fraction of the proteins expressed in the human brain only the primary structure is known from the genome project. Proteins conserved in evolution can be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and characterized with the aim to identify the target antigen. The mAb ab52 was found to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15 (epidermal growth factor receptor pathway substrate clone 15) to be a strong candidate. Another mAb from the library, aa2, was already found to recognize EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and 2D electrophoretic separations revealed similar patterns, hence indicating that both antigens could represent the same protein. Finally absence of the wild-type signal in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52 antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for applications like immunoprecipitation (IP). It has already been submitted to the Developmental Studies Hybridoma Bank (DSHB) to be easily available for the entire research community. The mAb na21 was also found to be an IgM. It recognizes a membrane associated antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to biochemically purify the endogenously expressed protein from the tissue, gave 99 promising results but could not be completed due to lack of time. Thus biochemical purification of the protein seems possible in order to facilitate its identification by mass spectrometry. Several other mAbs were studied for their staining pattern on cryosections and whole mounts of Drosophila brains. However, many of these mAbs stained very few structures in the brain, which indicated that only a very limited amount of protein would be available as starting material. Because these antibodies did not produce signals on Western blots, which made it impossible to enrich the antigens by electrophoretic methods, we did not attempt their purification. However, the specific localization of these proteins makes them highly interesting and calls for their further characterization, as they may play a highly specialized role in the development and/or function of the neural circuits they are present in. The purification and identification of such low expression proteins would need novel methods of enrichment of the stained structures. N2 - Für einen Großteil der Proteine, die im menschlichen Gehirn exprimiert werden, ist lediglich die Primärstruktur aus dem Genomprojekt bekannt. Proteine, die in der Evolution konserviert wurden, können in genetischen Modellsystemen wie Drosophila untersucht werden. In dieser Doktorarbeit werden monoklonale Antikörper (mAk) aus der Würzburger Hybridoma Bibliothek produziert und charakterisiert, mit dem Ziel, die erkannten Proteine zu identifizieren. Der mAk ab52 wurde als IgM typisiert, das auf Western Blots ein zytosolisches Protein von Mr ~110 kDa erkennt. Das Antigen wurde durch zwei-dimensionale Gelelektrophorese (2DE) als einzelner Fleck aufgelöst. Massenspektrometrische Analyse dieses Flecks identifizierte dass EPS-15 (epidermal growth factor receptor pathway substrate clone 15) als viel versprechenden Kandidaten. Da für einen anderen mAk aus der Bibliothek, aa2, bereits bekannt war, dass er EPS-15 erkennt, wurden die Western-Blot-Signale der beiden Antikörper nach 1D und 2D Trennungen von Kopfhomogenat verglichen. Die Ähnlichkeit der beiden Muster deuteten darauf hin, dass beide Antigene dasselbe Protein erkennen. Das Fehlen des Wildtyp-Signals in homozygoten Eps15 Mutanten in einem Western Blot mit mAk ab52 bestätigten schließlich, dass EPS-15 das Antigen zu mAk ab52 darstellt. Demnach erkennen beide mAk, aa2 und ab52, das Drosophila Homolog zu EPS- 15. Da mAk aa2 ein IgG ist, dürfte er für Anwendungen wie Immunpräzipitation (IP) besser geeignet sein. Er wurde daher bereits bei der Developmental Studies Hybridoma Bank (DSHB) eingereicht, um ihn der ganzen Forschergemeinde leicht zugänglich zu machen. Der mAk na21 wurde ebenfalls als IgM typisiert. Er erkennt ein Membran assoziiertes Antigen von Mr ~10 kDa auf Western Blots. Aufgrund der Membranassoziierung des Proteins war es nicht möglich, es in 2DE aufzulösen und 101 da es sich um ein IgM handelt, war eine Anreicherung des Antigens mittels IP nicht erfolgreich. Vorversuche zur biochemischen Reinigung des endogenen Proteins aus Gewebe waren Erfolg versprechend, konnten aber aus Zeitmangel nicht abgeschlossen werden. Daher erscheint eine biochemische Reinigung des Proteins für eine Identifikation durch Massenspektrometrie möglich. Eine Reihe weiterer mAk wurden hinsichtlich ihrer Färbemuster auf Gefrierschnitten und in Ganzpräparaten von Drosophila Gehirnen untersucht. Allerdings färbten viele dieser mAk sehr wenige Strukturen im Gehirn, so dass nur eine sehr begrenzte Menge an Protein als Startmaterial verfügbar wäre. Da diese Antikörper keine Signale auf Western Blots produzierten und daher eine Anreicherung des Antigens durch elektrophoretische Methoden ausschlossen, wurde keine Reinigung versucht. Andererseits macht die spezifische Lokalisation dieser Proteine sie hoch interessant für eine weitere Charakterisierung, da sie eine besonders spezialisierte Rolle in der Entwicklung oder für die Funktion von neuralen Schaltkreisen, in denen sie vorkommen, spielen könnten. Die Reinigung und Identifikation solcher Proteine mit niedrigem Expressionsniveau würde neue Methoden der Anreicherung der gefärbten Strukturen erfordern. KW - synaptic proteins KW - Taufliege KW - Synapse KW - Proteine KW - Monoklonaler Antikörper KW - synaptische Proteine KW - monoklonale Antikörper KW - Drosophila melanogaster KW - monoclonal antibodies Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270205 N1 - ursprüngliche Originalausgabe der Dissertation erschienen am 19.01.2012 unter: https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-67325 ER - TY - THES A1 - Roth, Nicolas Mériadec Max André T1 - Temporal development of communities with a focus on insects, in time series of one to four decades T1 - Entwicklung von Artengemeinschaften in der Zeit mit einem Fokus auf Insekten in Zeitreihen von einer bis vier Dekaden N2 - Changes and development are fundamental principles in biocenoses and can affect a multitude of ecological processes. In insect communities phenological and density changes, changes in species richness and community composition, as well as interactions between those changes, are the most important macro processes. However, climate change and other factors like habitat degradation and loss alter these processes leading to shifts and general biodiversity declines. Even though knowledge about insect decline in central Europe increased during the last decades, there are significant knowledge gaps about the development of insect communities in certain habitats and taxa. For example, insect communities in small lentic as well as in forested habitats are under-sampled and reported to be less endangered than communities in other habitats. Furthermore, the changes within habitats and taxa are additionally influenced by certain traits, like host or feeding specialization. To disentangle these influences and to increase the knowledge about the general long-term development of insect communities, comprehensive long-term monitoring studies are needed. In addition, long-term effects of conservation strategies should also be evaluated on large time scales in order to be able to decide on a scientific base which strategies are effective in promoting possibly declining taxa. Hence, this thesis also tackles the effects of an integrative conservation strategy on wood dependent beetle and fungi, beside the development of water beetle and macro moth communities over multiple decades. In Chapter 2 I present a study on the development of water beetle communities (Dytiscidae, Haliplidae, Noteridae) in 33 water bodies in Southern Germany from 1991 to 2018. Time-standardized capture per waterbody was used during three periods: between 1991 and 1995, 2007 and 2008, and 2017 and 2018. Results showed annual declines in both species number (ca. -1%) and abundance (ca. -2%). In addition, community composition shifted over time in part due to changing pH values. Hence, the recorded changes during the 28-year study period partly reflect natural succession processes. However, since also moor-related beetle species decreased significantly, it is likely that water beetles in southern Germany are also threatened by non-successional factors, including desiccation, increased nitrogen input and/or mineralization, as well as the loss of specific habitats. The results suggest, that in small to midsize lentic waterbodies, current development should aim for constant creation of new water bodies and protection of moor waterbodies in order to protect water beetle communities on a landscape scale. In Chapter 3 I present an analysis of the development of nocturnal macro moth species richness, abundance and biomass over four decades in forests of southern Germany. Two local scale data sets featuring a coppiced oak forest as well as an oak high forest were analysed separately from a regional data set representing all forest types in the temperate zone of Central Europe. At the regional scale species richness, abundance and biomass showed annual declines of ca. 1 %, 1.3 % and 1.4 %, respectively. These declines were more pronounced in plant host specialists and in dark coloured species. In contrast, species richness increased by ca. 1.5 % annually in the coppiced forest, while no significant trends were found in the high forest. In contrast to past assumptions, insect decline apparently affects also hyper diverse insect groups in forests. Since host specialists and dark coloured species were affected more heavily by the decline than other groups, habitat loss and climate change seem to be potential drivers of the observed trends. However, the positive development of species richness in the coppiced oak forest indicates that maintaining complex and diverse forest ecosystems through active management might compensate for negative trends in biodiversity. Chapter 4 features a study specifically aiming to investigate the long-term effect of deadwood enrichment as an integrative conservation strategy on saproxylic beetles and fungi in a central European beech forest at a landscape scale. A before–after control–impact design, was used to compare assemblages and gamma diversities of saproxylic organisms (beetles and fungi) in strictly protected old-growth forest areas (reserves) and previously moderately and intensively managed forest areas. Forests were sampled one year before and a decade after starting a landscape-wide strategy of dead-wood enrichment. Ten years after the start of the dead-wood enrichment, neither gamma diversities of saproxylic organisms nor species composition of beetles did reflect the previous management types anymore. However, fungal species composition still mirrored the previous management gradient. The results demonstrated that intentional enrichment of dead wood at the landscape scale can effectively restore communities of saproxylic organisms and may thus be a suitable strategy in addition to permanent strict reserves in order to protect wood dependent organisms in Europe. In this thesis I showed, that in contrast to what was assumed and partly reported so far, also water beetles in lentic water bodies and macro moths in forests decreased in species richness, abundance and biomass during the last three to four decades. In line with earlier studies, especially dark coloured species and specialists decreased more than light-coloured species and generalists. The reasons for these declines could partly be attributed to natural processes and pollution and possibly to climate change. However, further studies, especially experimental ones, will be needed to achieve a better understanding of the reasons for insect decline. Furthermore, analyses of time series data should be interpreted cautiously especially if the number of sampling years is smaller than ten years. In addition, validation techniques such as left- and right- censoring and cross validation should be used in order to proof the robustness of the analyses. However, the lack of knowledge, we are still facing today, should not prevent scientists and practitioners from applying conservation measures. In order to prove the effectiveness of such measures, long-term monitoring is crucial. Such control of success is essential for evidence based and thus adapted conservation strategies of threatened organisms. N2 - Veränderungen und Entwicklung sind grundlegende Prinzipien in Biozönosen und können eine Vielzahl von ökologischen Prozessen beeinflussen. In Insektengemeinschaften stellen Veränderungen in der Phänologie und Dichte, Veränderungen des Artenreichtums und der Artenzusammensetzung sowie die Wechselwirkungen zwischen diesen, die wichtigsten Makroprozesse dar. Klimawandel und andere Faktoren wie der Verlust von Lebensräumen oder deren Qualitätsverschlechterung beeinflussen diese Prozesse jedoch und führen zu Veränderungen und allgemeinen Rückgängen der Biodiversität. Auch wenn die Erkenntnisse zum „Insektensterben“ in Mitteleuropa in den letzten Jahrzehnten zugenommen haben, gibt es erhebliche Wissenslücken über die Entwicklung von Insektengemeinschaften in bestimmten Lebensräumen und Taxa. Beispielsweise ist die Entwicklung von Insektengemeinschaften in kleinen, stehenden Gewässern und in Wäldern wenig erforscht. Darüber hinaus werden die Veränderungen innerhalb von Habitaten und Taxa zusätzlich durch bestimmte Merkmale, wie Wirts- oder Nahrungsspezialisierung, beeinflusst. Um diese verschiedenen Einflüsse auseinanderhalten zu können und das Wissen über die allgemeine Langzeitentwicklung von Insektengemeinschaften zu vergrößern, sind umfassende Langzeitstudien erforderlich. Darüber hinaus sollten auch die langfristigen Auswirkungen von Naturschutzstrategien über lange Zeiträume evaluiert werden, um auf wissenschaftlicher Grundlage entscheiden zu können, welche Strategien zur Förderung bedrohter Taxa wirksam sind. Daher befasst sich diese Arbeit neben der Entwicklung von Wasserkäfer- und Großschmetterlingsgemeinschaften über mehrere Jahrzehnte auch mit den Auswirkungen einer integrativen Naturschutzmaßnahme auf xylobionte Käfer und Pilze. In Kapitel 2 stelle ich eine Studie über die Entwicklung von Wasserkäfergemeinschaften (Dytiscidae, Haliplidae, Noteridae) in 33 Gewässern Süddeutschlands von 1991 bis 2018 vor. Die zeitstandardisierte Erfassung pro Wasserkörper erfolgte in drei Zeiträumen: zwischen 1991 und 1995, 2007 und 2008 sowie 2017 und 2018. Die Ergebnisse zeigten einen jährlichen Rückgang sowohl der Artenzahl (ca. -1%) als auch der Abundanz (ca. -2%). Darüber hinaus verschob sich die Artenzusammensetzung im Laufe der Zeit zum Teil aufgrund sich ändernder pH-Werte. Daraus lässt sich schlussfolgern, dass die erfassten Veränderungen während des 28- jährigen Untersuchungszeitraums teilweise natürliche Sukzessionsprozesse widerspiegeln. Da aber auch an moorige Gewässer gebundene Käferarten deutlich abgenommen haben, ist es wahrscheinlich, dass die Wasserkäfer Süddeutschlands auch durch Faktoren wie Austrocknung, erhöhten Stickstoffeintrag und/oder Mineralisierung sowie durch den Verlust spezifischer Lebensräume bedroht sind. Aufgrund dieser Entwicklungen ist es empfehlenswert, auf Landschaftsebene auf die ständige Schaffung neuer Gewässer und den besonderen Schutz von Moorgewässern zu setzen, um Wasserkäfergemeinschaften erfolgreich schützen zu können. In Kapitel 3 präsentiere ich eine Analyse der Diversitäts-, Abundanz- und Biomassenentwicklung von nachtaktiven Großschmetterlingen über vier Jahrzehnte in Wäldern Süddeutschlands. Neben einem bayernweiten Datensatz, der alle typischen Waldtypen der gemäßigten Zone Mitteleuropas beinhaltet, wurden zwei lokale, besonders regelmäßig besammelte Gebiete getrennt analysiert. In diesen Gebieten werden die Eichenwälder als Hoch- bzw. als Mittelwald bewirtschaftet. Bayernweit wiesen Artenreichtum, Abundanz und Biomasse jährliche Rückgänge von ca. 1 %, 1,3 % bzw. 1,4 % auf. Diese Rückgänge waren bei Wirtspflanzenspezialisten und bei dunkel gefärbten Arten besonders stark ausgeprägt. Im Gegensatz dazu nahm der Artenreichtum im Mittelwald jährlich um ca. 1,5 % zu, während im Hochwald keine signifikanten Trends festgestellt werden konnten. Im Gegensatz zu früheren Annahmen betrifft der Insektenrückgang offenbar auch hyperdiverse Insektengruppen im Wald. Da Wirtspflanzenspezialisten und dunkel gefärbte Arten vom Rückgang stärker betroffen waren als andere, scheinen Lebensraumverlust und Klimawandel potentielle Treiber der beobachteten Trends zu sein. Die positive Entwicklung des Artenreichtums im Mittelwald zeigt jedoch, dass der Erhalt komplexer und vielfältiger Waldökosysteme durch aktives Management, negative Biodiversitätstrends zum Teil kompensieren könnte. Kapitel 4 enthält eine Studie, die die Langzeitwirkung von Totholzanreicherung als integrative Naturschutzmaßnahme auf xylobionte Käfer und Pilze in einem mitteleuropäischen Buchenwald auf der Landschaftsebene untersucht. Dabei wurde die Gamma-Diversität und die Artenzusammensetzung dieser beiden Gruppen anhand einer Vorher-Nachher Untersuchung mit Kontrollflächen (Naturwaldreservate) untersucht. Die bewirtschafteten Flächen wurden weiterhin in zuvor mäßig und intensiv bewirtschaftete Flächen eingeteilt. Die Wälder wurden ein Jahr vor und ein Jahrzehnt nach Beginn einer Totholzanreicherungsstrategie auf Landschaftsebene beprobt. Zehn Jahre nach Beginn der Totholzanreicherung spiegelten weder die Gamma-Diversität der xylobionten Organismen noch die Artenzusammensetzung der Käfer die früheren Bewirtschaftungstypen wider, und wiesen keine Unterschiede mehr zu den Naturwaldreservaten auf. Die Pilzartenzusammensetzung spiegelte jedoch noch immer den früheren Bewirtschaftungsgradienten wider. Die Ergebnisse zeigen, dass Totholzanreicherung auf Landschaftsebene positive Effekte auf xylobionte Artengemeinschaften haben kann. Somit stellt Totholzanreicherung eine Naturschutzmaßnahme dar, die zusätzlich zu permanenten Schutzgebieten, eine Grundlage schaffen kann, um holzabhängige Organismen in Europa zu schützen. In dieser Arbeit habe ich gezeigt, dass im Gegensatz zu dem, was bisher angenommen und zum Teil berichtet wurde, auch Wasserkäfer in stehenden Gewässern und nachtaktive Großschmetterlingen in Wäldern in den letzten drei bis vier Jahrzehnten an Artenreichtum, Abundanz und Biomasse abgenommen haben. In Übereinstimmung mit anderen Studien nahmen vor allem dunkel gefärbte Arten und Spezialisten stärker ab als hell gefärbte Arten und Generalisten. Die Gründe für diese Rückgänge konnten zum Teil auf natürliche Prozesse, Umweltverschmutzung und möglicherweise auf den Klimawandel zurückgeführt werden. Es sind jedoch weitere Studien, insbesondere experimentelle, erforderlich, um die Gründe für das „Insektensterben“ besser zu verstehen. Darüber hinaus sollten Zeitreihendaten mit Vorsicht interpretiert werden, insbesondere wenn die Anzahl der besammelten Jahre kleiner als zehn Jahre ist. Darüber hinaus sollten Validierungstechniken wie Links- und Rechts-Zensierung und Kreuzvalidierung eingesetzt werden, um die Robustheit der Analysen nachzuweisen. Der Mangel an Wissen, mit dem wir heute noch konfrontiert sind, sollte Wissenschaftler und Praktiker jedoch nicht davon abhalten, Naturschutzmaßnahmen anzuwenden. Um die Wirksamkeit solcher Maßnahmen nachzuweisen, ist eine langfristige Überprüfung von entscheidender Bedeutung. Solche Erfolgskontrollen sind für evidenzbasierte und damit angepasste Erhaltungsstrategien bedrohter Organismen unerlässlich. KW - climate change KW - insects KW - temporal development KW - nature conservation KW - entomology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235499 ER - TY - THES A1 - Solger, Franziska T1 - Central role of sphingolipids on the intracellular survival of \(Neisseria\) \(gonorrhoeae\) in epithelial cells T1 - Die zentrale Rolle von Sphingolipiden auf das intrazelluläre Überleben von \(Neisseria\) \(gonorrhoeae\) in Epithelzellen N2 - Neisseria gonorrhoeae are Gram-negative bacteria with diplococcal shape. As an obligate human pathogen, it is the causative agent of gonorrhoea, a sexually transmitted disease. Gonococci colonize a variety of mucosal tissues, mainly the urogenital tract in men and women. Occasionally N. gonorrhoeae invades the bloodstream, leading to disseminated gonococcal infection. These bacteria possess a repertoire of virulence factors, which expression patterns can be adapted to the environmental conditions of the host. Through the accumulation of antibiotic resistances and in absence of vaccines, some neisserial strains have the potential to spread globally and represent a major public health threat. Therefore, it is necessary to understand the exact molecular mechanisms underlying the successful infection and progression of gonococci within their host. This deeper understanding of neisserial infection and survival mechanisms is needed for the development of new therapeutic agents. In this work, the role of host-cell sphingolipids on the intracellular survival of N. gonorrhoeae was investigated. It was shown that different classes of sphingolipids strongly interact with invasive gonococci in epithelial cells. Therefore, novel and highly specific clickable sphingolipid analogues were applied to study these interactions with this pathogen. The formation of intra- and extracellular sphingosine vesicles, which were able to target gonococci, was observed. This direct interaction led to the uptake and incorporation of sphingosine into the neisserial membrane. Together with in vitro results, sphingosine was identified as a potential bactericidal reagent as part of the host cell defence. By using different classes of sphingolipids and their clickable analogues, essential structural features, which seem to trigger the bacterial uptake, were detected. Furthermore, effects of key enzymes of the sphingolipid signalling pathway were tested in a neutrophil infection model. In conclusion, the combination of click chemistry and infection biology made it possible to shed some light on the dynamic interplay between cellular sphingosine and N. gonorrhoeae. Thereby, a possible “catch-and-kill” mechanism could have been observed. N2 - Neisseria gonorrhoeae ist ein Gram-negatives Bakterium, welches als Diplokokke vorkommt. Als ein ausschließliches Humanpathogen sind Neisserien der Erreger für die sexuell übertragbare Infektionskrankheit Gonorrhö. Gonokokken besiedeln eine Vielzahl von Schleimhäuten, jedoch hauptsächlich den Urogenitaltrakt bei Männern und Frauen. Gelegentlich kann N. gonorrhoeae in die Blutbahn invadieren, was zu einer disseminierten Infektion führen kann. Diese Bakterien verfügen über ein Repertoire an Virulenzfaktoren, deren Expressionskombination den Umgebungsbedingungen des Wirts angepasst werden können. Durch die Anhäufung von Antibiotikaresistenzen und durch das Fehlen eines Impfstoffes, besteht die Gefahr, dass spezielle Neisserienstämme sich weltweit verbreiten und daher eine ernstzunehmende Bedrohung des Menschen sind. Daher ist es notwendig die zugrundeliegenden molekularen Mechanismen der erfolgreichen Infektion und Ausbreitung der Gonokokken im Wirt genauestens zu verstehen. Das detaillierte Wissen über die Neisserieninfektion und Überlebensmechanismen ist nötig für die Entwicklung neuer Therapieansätze. In dieser Arbeit wurde der Effekt von Sphingolipiden der Wirtszelle auf das intrazelluläre Überleben von N. gonorrhoeae untersucht. Es konnte gezeigt werden, dass unterschiedliche Klassen von Sphingolipiden stark mit invasiven Gonokokken in Epithelzellen interagieren. Um dies zu tun, wurden neue und hochspezifische clickbare Sphingolipidanaloge eingesetzt, um deren Interaktionen mit diesem Pathogen zu studieren. Die Formation von intra- als auch extrazellulären Sphingosinvesikeln, welche Gonokokken gezielt erreichten, konnte beobachtet werden. Diese direkte Interaktion führte zu einer Aufnahme und Einbau des Sphingosins in die Neisserienmembran. Zusammen mit in vitro Ergebnissen, konnte Sphingosin als potenzieller und antibakterieller Bestandteil des zellulären Abwehrsystems identifiziert werden. Weiterhin wurde durch die Verwendung unterschiedlicher Sphingolipidklassen und deren clickbaren Analoge wichtige Strukturen erkannt, die die bakterielle Aufnahme auslösen. Des Weiteren wurden die Auswirkungen von Schlüsselenzymen des Sphingolipidsignalwegs in einem Infektionsmodell mit Neutrophilen getestet. Abschließend ist zu sagen, dass die Kombination aus Click Chemie und Infektionsbiologie es ermöglicht hat, die dynamischen Wechselwirkungen zwischen zellulären Sphingosin und N. gonorrhoeae zu beleuchten. Dadurch konnte ein möglicher „catch-and-kill”-Mechanismus entdeckt werden. KW - Neisseria gonorrhoeae KW - Sphingosinkinase KW - Sphingosinanaloga KW - Click-Chemie KW - sphingosine KW - sphingolipids KW - Neisseria KW - intracellular KW - vesicles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-247534 ER - TY - JOUR A1 - Vogel, Sebastian A1 - Gossner, Martin M. A1 - Mergner, Ulrich A1 - Müller, Jörg A1 - Thorn, Simon T1 - Optimizing enrichment of deadwood for biodiversity by varying sun exposure and tree species: An experimental approach JF - Journal of Applied Ecology N2 - The enrichment of deadwood is essential for the conservation of saproxylic biodiversity in managed forests. However, existing strategies focus on a cost‐intensive increase of deadwood amount, while largely neglecting increasing deadwood diversity. Deadwood objects, that is logs and branches, from six tree species were experimentally sun exposed, canopy shaded and artificially shaded for 4 years, after which the alpha‐, beta‐ and gamma‐diversity of saproxylic beetles, wood‐inhabiting fungi and spiders were analysed. Analyses of beta‐diversity included the spatial distance between exposed deadwood objects. A random‐drawing procedure was used to identify the combination of tree species and sun exposure that yielded the highest gamma‐diversity at a minimum of exposed deadwood amount. In sun‐exposed plots, species numbers in logs were higher than in shaded plots for all taxa, while in branches we observed the opposite for saproxylic beetles. Tree species affected the species numbers only of saproxylic beetles and wood‐inhabiting fungi. The beta‐diversity of saproxylic beetles and wood‐inhabiting fungi among logs was influenced by sun exposure and tree species, but beta‐diversity of spiders by sun exposure only. For all saproxylic taxa recorded in logs, differences between communities increased with increasing spatial distance. A combination of canopy‐shaded Carpinus logs and sun‐exposed Populus logs resulted in the highest species numbers of all investigated saproxylic taxa among all possible combinations of tree species and sun‐exposure treatments. Synthesis and applications. We recommend incorporating the enrichment of different tree species and particularly the variation in sun exposure into existing strategies of deadwood enrichment. Based on the results of our study, we suggest to combine the logs of softwood broadleaf tree species (e.g. Carpinus, Populus), hardwood broadleaf tree species (e.g. Quercus) and coniferous tree species (e.g. Pinus) under different conditions of sun exposure and distribute them spatially in a landscape to maximize the beneficial effects on overall diversity. KW - broadleaf tree species KW - deadwood enrichment KW - forest conservation KW - forest management KW - saproxylic beetles KW - spiders KW - sun exposure KW - wood‐inhabiting fungi Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214614 VL - 57 IS - 10 SP - 2075 EP - 2085 ER - TY - JOUR A1 - Du, Baoguo A1 - Ma, Yuhua A1 - Yáñez‐Serrano, Ana Maria A1 - Arab, Leila A1 - Fasbender, Lukas A1 - Alfarraj, Saleh A1 - Albasher, Gadah A1 - Hedrich, Rainer A1 - White, Philip J. A1 - Werner, Christiane A1 - Rennenberg, Heinz T1 - Physiological responses of date palm (Phoenix dactylifera) seedlings to seawater and flooding JF - New Phytologist N2 - In their natural environment along coast lines, date palms are exposed to seawater inundation and, hence, combined stress by salinity and flooding. To elucidate the consequences of this combined stress on foliar gas exchange and metabolite abundances in leaves and roots, date palm seedlings were exposed to flooding with seawater and its major constituents under controlled conditions. Seawater flooding significantly reduced CO\(_{2}\) assimilation, transpiration and stomatal conductance, but did not affect isoprene emission. A similar effect was observed upon NaCl exposure. By contrast, flooding with distilled water or MgSO\(_{4}\) did not affect CO\(_{2}\)/H\(_{2}\)O gas exchange or stomatal conductance significantly, indicating that neither flooding itself, nor seawater sulfate, contributed greatly to stomatal closure. Seawater exposure increased Na and Cl contents in leaves and roots, but did not affect sulfate contents significantly. Metabolite analyses revealed reduced abundances of foliar compatible solutes, such as sugars and sugar alcohols, whereas nitrogen compounds accumulated in roots. Reduced transpiration upon seawater exposure may contribute to controlling the movement of toxic ions to leaves and, therefore, can be seen as a mechanism to cope with salinity. The present results indicate that date palm seedlings are tolerant towards seawater exposure to some extent, and highly tolerant to flooding. KW - compatible solutes and other metabolites KW - date palm KW - flooding KW - salinity KW - shoot–root interaction KW - stomatal conductance KW - sulfate Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228226 VL - 229 IS - 6 SP - 3318 EP - 3329 ER - TY - JOUR A1 - Seibold, Sebastian A1 - Hothorn, Torsten A1 - Gossner, Martin M. A1 - Simons, Nadja K. A1 - Blüthgen, Nico A1 - Müller, Jörg A1 - Ambarlı, Didem A1 - Ammer, Christian A1 - Bauhus, Jürgen A1 - Fischer, Markus A1 - Habel, Jan C. A1 - Penone, Caterina A1 - Schall, Peter A1 - Schulze, Ernst‐Detlef A1 - Weisser, Wolfgang W. T1 - Insights from regional and short‐term biodiversity monitoring datasets are valuable: a reply to Daskalova et al. 2021 JF - Insect Conservation and Diversity N2 - Reports of major losses in insect biodiversity have stimulated an increasing interest in temporal population changes. Existing datasets are often limited to a small number of study sites, few points in time, a narrow range of land‐use intensities and only some taxonomic groups, or they lack standardised sampling. While new monitoring programs have been initiated, they still cover rather short time periods. Daskalova et al. 2021 (Insect Conservation and Diversity, 14, 1‐18) argue that temporal trends of insect populations derived from short time series are biased towards extreme trends, while their own analysis of an assembly of shorter‐ and longer‐term time series does not support an overall insect decline. With respect to the results of Seibold et al. 2019 (Nature, 574, 671–674) based on a 10‐year multi‐site time series, they claim that the analysis suffers from not accounting for temporal pseudoreplication. Here, we explain why the criticism of missing statistical rigour in the analysis of Seibold et al. (2019) is not warranted. Models that include ‘year’ as random effect, as suggested by Daskalova et al. (2021), fail to detect non‐linear trends and assume that consecutive years are independent samples which is questionable for insect time‐series data. We agree with Daskalova et al. (2021) that the assembly and analysis of larger datasets is urgently needed, but it will take time until such datasets are available. Thus, short‐term datasets are highly valuable, should be extended and analysed continually to provide a more detailed understanding of insect population changes under the influence of global change, and to trigger immediate conservation actions. KW - Arthropod KW - biodiversity KW - insect decline KW - land use KW - time series Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228309 VL - 14 IS - 1 SP - 144 EP - 148 ER - TY - JOUR A1 - Peters, Birte A1 - Keller, Alexander A1 - Leonhardt, Sara Diana T1 - Diets maintained in a changing world: Does land-use intensification alter wild bee communities by selecting for flexible generalists? JF - Ecology and evolution N2 - Biodiversity loss, as often found in intensively managed agricultural landscapes, correlates with reduced ecosystem functioning, for example, pollination by insects, and with altered plant composition, diversity, and abundance. But how does this change in floral resource diversity and composition relate to occurrence and resource use patterns of trap-nesting solitary bees? To better understand the impact of land-use intensification on communities of trap-nesting solitary bees in managed grasslands, we investigated their pollen foraging, reproductive fitness, and the nutritional quality of larval food along a land-use intensity gradient in Germany. We found bee species diversity to decrease with increasing land-use intensity irrespective of region-specific community compositions and interaction networks. Land use also strongly affected the diversity and composition of pollen collected by bees. Lack of suitable pollen sources likely explains the absence of several bee species at sites of high land-use intensity. The only species present throughout, Osmia bicornis (red mason bee), foraged on largely different pollen sources across sites. In doing so, it maintained a relatively stable, albeit variable nutritional quality of larval diets (i.e., protein to lipid (P:L) ratio). The observed changes in bee–plant pollen interaction patterns indicate that only the flexible generalists, such as O. bicornis, may be able to compensate the strong alterations in floral resource landscapes and to obtain food of sufficient quality through readily shifting to alternative plant sources. In contrast, other, less flexible, bee species disappear. KW - bee decline KW - biodiversity exploratories KW - foraging KW - metabarcoding KW - pollen nutrients KW - solitary bees Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-312786 SN - 2045-7758 VL - 12 IS - 5 ER - TY - JOUR A1 - Britz, Sebastian A1 - Markert, Sebastian Matthias A1 - Witvliet, Daniel A1 - Steyer, Anna Maria A1 - Tröger, Sarah A1 - Mulcahy, Ben A1 - Kollmannsberger, Philip A1 - Schwab, Yannick A1 - Zhen, Mei A1 - Stigloher, Christian T1 - Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy JF - Frontiers in Neuroanatomy N2 - At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments. KW - FIB-SEM KW - 3D reconstruction KW - neuroanatomy KW - IL2 branching KW - amphids KW - Caenorhabditis elegans (C. elegans) KW - dauer Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249622 SN - 1662-5129 VL - 15 ER - TY - JOUR A1 - Pauli, Martin A1 - Paul, Mila M. A1 - Proppert, Sven A1 - Mrestani, Achmed A1 - Sharifi, Marzieh A1 - Repp, Felix A1 - Kürzinger, Lydia A1 - Kollmannsberger, Philip A1 - Sauer, Markus A1 - Heckmann, Manfred A1 - Sirén, Anna-Leena T1 - Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections JF - Communications Biology N2 - Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution. KW - mossy fiber synapses KW - CA3 pyrimidal cells KW - CA2+ channels KW - active zone KW - hippocampal KW - release KW - plasticity KW - proteins KW - platform KW - reveals Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259830 VL - 4 ER - TY - JOUR A1 - Widmann, Annekathrin A1 - Artinger, Marc A1 - Biesinger, Lukas A1 - Boepple, Kathrin A1 - Peters, Christina A1 - Schlechter, Jana A1 - Selcho, Mareike A1 - Thum, Andreas S. T1 - Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae JF - PLoS Genetics N2 - Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution. KW - genetic dissection KW - Drosophila KW - memory formation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166672 VL - 12 IS - 10 ER - TY - JOUR A1 - Ziegler, Alice A1 - Meyer, Hanna A1 - Otte, Insa A1 - Peters, Marcell K. A1 - Appelhans, Tim A1 - Behler, Christina A1 - Böhning-Gaese, Katrin A1 - Classen, Alice A1 - Detsch, Florian A1 - Deckert, Jürgen A1 - Eardley, Connal D. A1 - Ferger, Stefan W. A1 - Fischer, Markus A1 - Gebert, Friederike A1 - Haas, Michael A1 - Helbig-Bonitz, Maria A1 - Hemp, Andreas A1 - Hemp, Claudia A1 - Kakengi, Victor A1 - Mayr, Antonia V. A1 - Ngereza, Christine A1 - Reudenbach, Christoph A1 - Röder, Juliane A1 - Rutten, Gemma A1 - Schellenberger Costa, David A1 - Schleuning, Matthias A1 - Ssymank, Axel A1 - Steffan-Dewenter, Ingolf A1 - Tardanico, Joseph A1 - Tschapka, Marco A1 - Vollstädt, Maximilian G. R. A1 - Wöllauer, Stephan A1 - Zhang, Jie A1 - Brandl, Roland A1 - Nauss, Thomas T1 - Potential of airborne LiDAR derived vegetation structure for the prediction of animal species richness at Mount Kilimanjaro JF - Remote Sensing N2 - The monitoring of species and functional diversity is of increasing relevance for the development of strategies for the conservation and management of biodiversity. Therefore, reliable estimates of the performance of monitoring techniques across taxa become important. Using a unique dataset, this study investigates the potential of airborne LiDAR-derived variables characterizing vegetation structure as predictors for animal species richness at the southern slopes of Mount Kilimanjaro. To disentangle the structural LiDAR information from co-factors related to elevational vegetation zones, LiDAR-based models were compared to the predictive power of elevation models. 17 taxa and 4 feeding guilds were modeled and the standardized study design allowed for a comparison across the assemblages. Results show that most taxa (14) and feeding guilds (3) can be predicted best by elevation with normalized RMSE values but only for three of those taxa and two of those feeding guilds the difference to other models is significant. Generally, modeling performances between different models vary only slightly for each assemblage. For the remaining, structural information at most showed little additional contribution to the performance. In summary, LiDAR observations can be used for animal species prediction. However, the effort and cost of aerial surveys are not always in proportion with the prediction quality, especially when the species distribution follows zonal patterns, and elevation information yields similar results. KW - biodiversity KW - species richness KW - LiDAR KW - elevation KW - partial least square regression KW - arthropods KW - birds KW - bats KW - predictive modeling Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262251 SN - 2072-4292 VL - 14 IS - 3 ER - TY - JOUR A1 - Müller, Jörg A1 - Ulyshen, Mike A1 - Seibold, Sebastian A1 - Cadotte, Marc A1 - Chao, Anne A1 - Bässler, Claus A1 - Vogel, Sebastian A1 - Hagge, Jonas A1 - Weiß, Ingmar A1 - Baldrian, Petr A1 - Tláskal, Vojtěch A1 - Thorn, Simon T1 - Primary determinants of communities in deadwood vary among taxa but are regionally consistent JF - Oikos N2 - The evolutionary split between gymnosperms and angiosperms has far‐reaching implications for the current communities colonizing trees. The inherent characteristics of dead wood include its role as a spatially scattered habitat of plant tissue, transient in time. Thus, local assemblages in deadwood forming a food web in a necrobiome should be affected not only by dispersal ability but also by host tree identity, the decay stage and local abiotic conditions. However, experiments simultaneously manipulating these potential community drivers in deadwood are lacking. To disentangle the importance of spatial distance and microclimate, as well as host identity and decay stage as drivers of local assemblages, we conducted two consecutive experiments, a 2‐tree species and 6‐tree species experiment with 80 and 72 tree logs, respectively, located in canopy openings and under closed canopies of a montane and a lowland forest. We sampled saproxylic beetles, spiders, fungi and bacterial assemblages from logs. Variation partitioning for community metrics based on a unified framework of Hill numbers showed consistent results for both studies: host identity was most important for sporocarp‐detected fungal assemblages, decay stage and host tree for DNA‐detected fungal assemblages, microclimate and decay stage for beetles and spiders and decay stage for bacteria. Spatial distance was of minor importance for most taxa but showed the strongest effects for arthropods. The contrasting patterns among the taxa highlight the need for multi‐taxon analyses in identifying the importance of abiotic and biotic drivers of community composition. Moreover, the consistent finding of microclimate as the primary driver for saproxylic beetles compared to host identity shows, for the first time that existing evolutionary host adaptions can be outcompeted by local climate conditions in deadwood. KW - deadwood experiments KW - dispersal KW - forest management KW - habitat filter KW - wood-inhabiting fungi Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228201 VL - 129 IS - 10 SP - 1579 EP - 1588 ER - TY - JOUR A1 - Vollmuth, Nadine A1 - Schlicker, Lisa A1 - Guo, Yongxia A1 - Hovhannisyan, Pargev A1 - Janaki-Raman, Sudha A1 - Kurmasheva, Naziia A1 - Schmitz, Werner A1 - Schulze, Almut A1 - Stelzner, Kathrin A1 - Rajeeve, Karthika A1 - Rudel, Thomas T1 - c-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis JF - eLife N2 - Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3β axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism. KW - Chlamydia trachomatis Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301385 VL - 11 ER - TY - JOUR A1 - Hurd, Paul J. A1 - Grübel, Kornelia A1 - Wojciechowski, Marek A1 - Maleszka, Ryszard A1 - Rössler, Wolfgang T1 - Novel structure in the nuclei of honey bee brain neurons revealed by immunostaining JF - Scientific Reports N2 - In the course of a screen designed to produce antibodies (ABs) with affinity to proteins in the honey bee brain we found an interesting AB that detects a highly specific epitope predominantly in the nuclei of Kenyon cells (KCs). The observed staining pattern is unique, and its unfamiliarity indicates a novel previously unseen nuclear structure that does not colocalize with the cytoskeletal protein f-actin. A single rod-like assembly, 3.7-4.1 mu m long, is present in each nucleus of KCs in adult brains of worker bees and drones with the strongest immuno-labelling found in foraging bees. In brains of young queens, the labelling is more sporadic, and the rod-like structure appears to be shorter (similar to 2.1 mu m). No immunostaining is detectable in worker larvae. In pupal stage 5 during a peak of brain development only some occasional staining was identified. Although the cellular function of this unexpected structure has not been determined, the unusual distinctiveness of the revealed pattern suggests an unknown and potentially important protein assembly. One possibility is that this nuclear assembly is part of the KCs plasticity underlying the brain maturation in adult honey bees. Because no labelling with this AB is detectable in brains of the fly Drosophila melanogaster and the ant Camponotus floridanus, we tentatively named this antibody AmBNSab (Apis mellifera Brain Neurons Specific antibody). Here we report our results to make them accessible to a broader community and invite further research to unravel the biological role of this curious nuclear structure in the honey bee central brain. KW - mushroom body calyx KW - synaptic complexes KW - bodies KW - insect KW - plasticity KW - insights KW - genome KW - model KW - proteins KW - methylation KW - biological techniques KW - cell biology KW - developmental biology KW - molecular biology KW - neuroscience Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260059 VL - 11 ER - TY - JOUR A1 - Krüger, Timothy A1 - Maus, Katharina A1 - Kreß, Verena A1 - Meyer-Natus, Elisabeth A1 - Engstler, Markus T1 - Single-cell motile behaviour of Trypanosoma brucei in thin-layered fluid collectives JF - The European Physical Journal E N2 - We describe a system for the analysis of an important unicellular eukaryotic flagellate in a confining and crowded environment. The parasite Trypanosoma brucei is arguably one of the most versatile microswimmers known. It has unique properties as a single microswimmer and shows remarkable adaptations (not only in motility, but prominently so), to its environment during a complex developmental cycle involving two different hosts. Specific life cycle stages show fascinating collective behaviour, as millions of cells can be forced to move together in extreme confinement. Our goal is to examine such motile behaviour directly in the context of the relevant environments. Therefore, for the first time, we analyse the motility behaviour of trypanosomes directly in a widely used assay, which aims to evaluate the parasites behaviour in collectives, in response to as yet unknown parameters. In a step towards understanding whether, or what type of, swarming behaviour of trypanosomes exists, we customised the assay for quantitative tracking analysis of motile behaviour on the single-cell level. We show that the migration speed of cell groups does not directly depend on single-cell velocity and that the system remains to be simplified further, before hypotheses about collective motility can be advanced. KW - Trypanosoma brucei KW - motile behaviour KW - fluid collectives Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-273022 SN - 1292-895X VL - 44 IS - 3 ER - TY - JOUR A1 - Schäbler, Stefan A1 - Amatobi, Kelechi M. A1 - Horn, Melanie A1 - Rieger, Dirk A1 - Helfrich‑Förster, Charlotte A1 - Mueller, Martin J. A1 - Wegener, Christian A1 - Fekete, Agnes T1 - Loss of function in the Drosophila clock gene period results in altered intermediary lipid metabolism and increased susceptibility to starvation JF - Cellular and Molecular Life Sciences N2 - The fruit fly Drosophila is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between period\(^{01}\) (per\(^{01}\)) clock mutants and Canton-S wildtype (WT\(_{CS}\)) flies in an isogenic and non-isogenic background using LC–MS. In the non-isogenic background, metabo-lites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in per\(^{01}\) mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WTCS in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness. per\(^{01}\) mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in per\(^{01}\) did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in per\(^{01}\) mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants KW - circadian rhythms KW - metabolomics KW - mitochondrial activity KW - tryptophan KW - acylcarnitine KW - feeding Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232432 SN - 1420-682X VL - 77 ER - TY - JOUR A1 - Streinzer, Martin A1 - Chakravorty, Jharna A1 - Neumayer, Johann A1 - Megu, Karsing A1 - Narah, Jaya A1 - Schmitt, Thomas A1 - Bharti, Himender A1 - Spaethe, Johannes A1 - Brockmann, Axel T1 - Species composition and elevational distribution of bumble bees (Hymenoptera, Apidae, Bombus Latreille) in the East Himalaya, Arunachal Pradesh, India JF - ZooKeys N2 - The East Himalaya is one of the world’s most biodiverse ecosystems. However, very little is known about the abundance and distribution of many plant and animal taxa in this region. Bumble bees are a group of cold-adapted and high elevation insects that fulfil an important ecological and economical function as pollinators of wild and agricultural flowering plants and crops. The Himalayan mountain range provides ample suitable habitats for bumble bees. Systematic study of Himalayan bumble bees began a few decades ago and the main focus has centred on the western region, while the eastern part of the mountain range has received little attention and only a few species have been verified. During a three-year survey, more than 700 bumble bee specimens of 21 species were collected in Arunachal Pradesh, the largest of the north-eastern states of India. The material included a range of species that were previously known from a limited number of collected specimens, which highlights the unique character of the East Himalayan ecosystem. Our results are an important first step towards a future assessment of species distribution, threat, and conservation. Clear elevation patterns of species diversity were observed, which raise important questions about the functional adaptations that allow bumble bees to thrive in this particularly moist region in the East Himalaya. KW - Alpine habitats KW - Apidae KW - conservation KW - global change KW - insect collection KW - pollination Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201937 VL - 851 ER - TY - JOUR A1 - Stelzner, Kathrin A1 - Boyny, Aziza A1 - Hertlein, Tobias A1 - Sroka, Aneta A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kessie, David A1 - Mehling, Helene A1 - Potempa, Jan A1 - Ohlsen, Knut A1 - Fraunholz, Martin J. A1 - Rudel, Thomas T1 - Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A. KW - Staphylococcus aureus KW - Staphylococcal infection KW - host cells KW - HeLa cells KW - cytotoxicity KW - intracellular pathogens KW - apoptosis KW - epithelial cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263908 VL - 17 IS - 9 ER - TY - JOUR A1 - Trinkl, Moritz A1 - Kaluza, Benjamin F. A1 - Wallace, Helen A1 - Heard, Tim A. A1 - Keller, Alexander A1 - Leonhardt, Sara D. T1 - Floral Species Richness Correlates with Changes in the Nutritional Quality of Larval Diets in a Stingless Bee JF - Insects N2 - Bees need food of appropriate nutritional quality to maintain their metabolic functions. They largely obtain all required nutrients from floral resources, i.e., pollen and nectar. However, the diversity, composition and nutritional quality of floral resources varies with the surrounding environment and can be strongly altered in human-impacted habitats. We investigated whether differences in plant species richness as found in the surrounding environment correlated with variation in the floral diversity and nutritional quality of larval provisions (i.e., mixtures of pollen, nectar and salivary secretions) composed by the mass-provisioning stingless bee Tetragonula carbonaria (Apidae: Meliponini). We found that the floral diversity of larval provisions increased with increasing plant species richness. The sucrose and fat (total fatty acid) content and the proportion and concentration of the omega-6 fatty acid linoleic acid decreased, whereas the proportion of the omega-3 fatty acid linolenic acid increased with increasing plant species richness. Protein (total amino acid) content and amino acid composition did not change. The protein to fat (P:F) ratio, known to affect bee foraging, increased on average by more than 40% from plantations to forests and gardens, while the omega-6:3 ratio, known to negatively affect cognitive performance, decreased with increasing plant species richness. Our results suggest that plant species richness may support T. carbonaria colonies by providing not only a continuous resource supply (as shown in a previous study), but also floral resources of high nutritional quality. KW - floral resources KW - plant-insect interactions KW - nutrition KW - biodiversity KW - bee decline Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200605 SN - 2075-4450 VL - 11 IS - 2 ER - TY - JOUR A1 - Ditzel, Pia A1 - König, Sebastian A1 - Musembi, Peter A1 - Peters, Marcell K. T1 - Correlation between coral reef condition and the diversity and abundance of fishes and sea urchins on an East African coral reef JF - Oceans N2 - Coral reefs are one of the most diverse marine ecosystems, providing numerous ecosystem services. This present study investigated the relationship between coral reef condition and the diversity and abundance of fishes, on a heavily fished East African coral reef at Gazi Bay, Kenya. Underwater visual censuses were conducted on thirty 50 × 5 m belt transects to assess the abundance and diversity of fishes. In parallel, a 25-m length of each of the same transects was recorded with photo-quadrats to assess coral community structure and benthic characteristics. For statistical analyses, multi-model inference based on the Akaike Information Criterion was used to evaluate the support for potential predictor variables of coral reef and fish diversity. We found that coral genus richness was negatively correlated with the abundance of macroalgae, whereas coral cover was positively correlated with both the abundance of herbivorous invertebrates (sea urchins) and with fish family richness. Similarly, fish family richness appeared mainly correlated with coral cover and invertebrate abundance, although no correlates of fish abundance could be identified. Coral and fish diversity were very low, but it appears that, contrary to some locations on the same coast, sea urchin abundance was not high enough to be having a negative influence on coral and fish assemblages. Due to increasing threats to coral reefs, it is important to understand the relationship among the components of the coral reef ecosystem on overfished reefs such as that at Gazi Bay. KW - coral reef ecosystem KW - coral reef resilience KW - global warming KW - climate change KW - overfishing KW - indicator species Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284503 SN - 2673-1924 VL - 3 IS - 1 SP - 1 EP - 14 ER - TY - JOUR A1 - Danner, Nadja A1 - Keller, Alexander A1 - Härtel, Stephan A1 - Steffan-Dewenter, Ingolf T1 - Honey bee foraging ecology: Season but not landscape diversity shapes the amount and diversity of collected pollen JF - PLoS ONE N2 - The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes. KW - honey bees KW - pollen KW - season KW - foraging Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170424 VL - 12 IS - 8 ER - TY - JOUR A1 - Gröbner, Susanne N. A1 - Worst, Barbara C. A1 - Weischenfeldt, Joachim A1 - Buchhalter, Ivo A1 - Kleinheinz, Kortine A1 - Rudneva, Vasilisa A. A1 - Johann, Pascal D. A1 - Balasubramanian, Gnana Prakash A1 - Segura-Wang, Maia A1 - Brabetz, Sebastian A1 - Bender, Sebastian A1 - Hutter, Barbara A1 - Sturm, Dominik A1 - Pfaff, Elke A1 - Hübschmann, Daniel A1 - Zipprich, Gideon A1 - Heinold, Michael A1 - Eils, Jürgen A1 - Lawerenz, Christian A1 - Erkek, Serap A1 - Lambo, Sander A1 - Waszak, Sebastian A1 - Blattmann, Claudia A1 - Borkhardt, Arndt A1 - Kuhlen, Michaela A1 - Eggert, Angelika A1 - Fulda, Simone A1 - Gessler, Manfred A1 - Wegert, Jenny A1 - Kappler, Roland A1 - Baumhoer, Daniel A1 - Stefan, Burdach A1 - Kirschner-Schwabe, Renate A1 - Kontny, Udo A1 - Kulozik, Andreas E. A1 - Lohmann, Dietmar A1 - Hettmer, Simone A1 - Eckert, Cornelia A1 - Bielack, Stefan A1 - Nathrath, Michaela A1 - Niemeyer, Charlotte A1 - Richter, Günther H. A1 - Schulte, Johannes A1 - Siebert, Reiner A1 - Westermann, Frank A1 - Molenaar, Jan J. A1 - Vassal, Gilles A1 - Witt, Hendrik A1 - Burkhardt, Birgit A1 - Kratz, Christian P. A1 - Witt, Olaf A1 - van Tilburg, Cornelis M. A1 - Kramm, Christof M. A1 - Fleischhack, Gudrun A1 - Dirksen, Uta A1 - Rutkowski, Stefan A1 - Frühwald, Michael A1 - Hoff, Katja von A1 - Wolf, Stephan A1 - Klingebeil, Thomas A1 - Koscielniak, Ewa A1 - Landgraf, Pablo A1 - Koster, Jan A1 - Resnick, Adam C. A1 - Zhang, Jinghui A1 - Liu, Yanling A1 - Zhou, Xin A1 - Waanders, Angela J. A1 - Zwijnenburg, Danny A. A1 - Raman, Pichai A1 - Brors, Benedikt A1 - Weber, Ursula D. A1 - Northcott, Paul A. A1 - Pajtler, Kristian W. A1 - Kool, Marcel A1 - Piro, Rosario M. A1 - Korbel, Jan O. A1 - Schlesner, Matthias A1 - Eils, Roland A1 - Jones, David T. W. A1 - Lichter, Peter A1 - Chavez, Lukas A1 - Zapatka, Marc A1 - Pfister, Stefan M. T1 - The landscape of genomic alterations across childhood cancers JF - Nature N2 - Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7–8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials. KW - cancer genomics KW - oncogenesis KW - paediatric cancer KW - predictive markers KW - translational research Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229579 VL - 555 ER - TY - JOUR A1 - Letunic, Ivica A1 - Khedkar, Supriya A1 - Bork, Peer T1 - SMART: recent updates, new developments and status in 2020 JF - Nucleic Acids Research N2 - SMART (Simple Modular Architecture Research Tool) is a web resource (https://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 9 contains manually curatedmodels formore than 1300 protein domains, with a topical set of 68 new models added since our last update article (1). All the new models are for diverse recombinase families and subfamilies and as a set they provide a comprehensive overview of mobile element recombinases namely transposase, integrase, relaxase, resolvase, cas1 casposase and Xer like cellular recombinase. Further updates include the synchronization of the underlying protein databases with UniProt (2), Ensembl (3) and STRING (4), greatly increasing the total number of annotated domains and other protein features available in architecture analysis mode. Furthermore, SMART's vector-based protein display engine has been extended and updated to use the latest web technologies and the domain architecture analysis components have been optimized to handle the increased number of protein features available. KW - SMART KW - SMART version 9 KW - protein domains KW - protein domain architectures Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-363816 VL - 49 IS - D1 ER - TY - JOUR A1 - Kraus, Amelie J. A1 - Brink, Benedikt G. A1 - Siegel, T. Nicolai T1 - Efficient and specific oligo-based depletion of rRNA JF - Scientific Reports N2 - In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available. KW - parasite biology KW - RNA sequencing KW - transcriptomics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224829 VL - 9 ER - TY - JOUR A1 - Kim, Bo-Mi A1 - Amores, Angel A1 - Kang, Seunghyun A1 - Ahn, Do-Hwan A1 - Kim, Jin-Hyoung A1 - Kim, Il-Chan A1 - Lee, Jun Hyuck A1 - Lee, Sung Gu A1 - Lee, Hyoungseok A1 - Lee, Jungeun A1 - Kim, Han-Woo A1 - Desvignes, Thomas A1 - Batzel, Peter A1 - Sydes, Jason A1 - Titus, Tom A1 - Wilson, Catherine A. A1 - Catchen, Julian M. A1 - Warren, Wesley C. A1 - Schartl, Manfred A1 - Detrich, H. William III A1 - Postlethwait, John H. A1 - Park, Hyun T1 - Antarctic blackfin icefish genome reveals adaptations to extreme environments JF - Nature Ecology & Evolution N2 - Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments. KW - animal physiology KW - evolutionary genetics KW - genomics KW - ichthyology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325811 VL - 3 ER - TY - JOUR A1 - Heil, Hannah S. A1 - Schreiber, Benjamin A1 - Götz, Ralph A1 - Emmerling, Monika A1 - Dabauvalle, Marie-Christine A1 - Krohne, Georg A1 - Höfling, Sven A1 - Kamp, Martin A1 - Sauer, Markus A1 - Heinze, Katrin G. T1 - Sharpening emitter localization in front of a tuned mirror JF - Light: Science & Applications N2 - Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2,3,4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells. KW - imaging and sensing KW - super-resolution microscopy Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228080 VL - 7 ER - TY - THES A1 - Englmeier, Jana T1 - Consequences of climate change and land-use intensification for decomposer communities and decomposition processes T1 - Folgen von Klimawandel und intensiver Landnutzung für Zersetzergemeinschaften und Abbauprozesse N2 - The increase in intensively used areas and climate change are direct and indirect consequences of anthropogenic actions, caused by a growing population and increasing greenhouse gas emissions. The number of research studies, investigating the effects of land use and climate change on ecosystems, including flora, fauna, and ecosystem services, is steadily growing. This thesis contributes to this research area by investigating land-use and climate effects on decomposer communities (arthropods and microbes) and the ecosystem service ‘decomposition of dead material’. Chapter II deals with consequences of intensified land use and climate change for the ecosystem service ‘decomposition of dead organic material’ (necromass). Considering the severe decline in insects, we experimentally excluded insects from half of the study objects. The decomposition of both dung and carrion was robust to land-use changes. Dung decomposition, moreover, was unaffected by temperature and the presence/ absence of insects. Along the altitudinal gradient, however, highest dung decomposition was observed at medium elevation between 600 and 700 m above sea level (although insignificant). As a consequence, we assume that at this elevation there is an ideal precipitation:temperature ratio for decomposing organisms, such as earthworms or collembolans. Carrion decomposition was accelerated by increasing elevation and by the presence of insects, indicating that increasing variability in climate and an ongoing decline in insects could modify decomposition processes and consequently natural nutrient cycles. Moreover, we show that different types of dead organic material respond differently to environmental factors and should be treated separately in future studies. In Chapter III, we investigated land-use and climate effects on dung-visiting beetles and their resource specialization. Here, all beetles that are preferentially found on dung, carrion or other rotten material were included. Both α- and γ-diversity were strongly reduced in agricultural and urban areas. High precipitation reduced dung-visiting beetle abundance, whereas γ-diversity was lowest in the warmest regions. Resource specialization decreased with increasing temperatures. The results give evidence that land use as well as climate can alter dung-visiting beetle diversity and resource specialization and may hence influence the natural balance of beetle communities and their contribution to the ecosystem service ‘decomposition of dead material’. The following chapter, Chapter IV, contributes to the findings in Chapter II. Here, carrion decomposition is not only explained by land-use intensity and climate but also by diversity and community composition of two taxonomic groups found on carrion, beetles and bacteria. The results revealed a strong correlation between bacteria diversity and community composition with temperature. Carrion decomposition was to a great extent directed by bacterial community composition and precipitation. The role of beetles was neglectable in carrion decomposition. With this study, I show that microbes, despite their microscopic size, direct carrion decomposition and may not be neglected in future decomposition studies. In Chapter V a third necromass type is investigated, namely deadwood. The aim was to assess climate and land-use effects on deadwood-inhabiting fungi and bacteria. Main driver for microbial richness (measured as number of OTUs) was climate, including temperature and precipitation. Warmer climates promoted the diversity of bacteria, whereas fungi richness was unaffected by temperature. In turn, fungi richness was lower in urban landscapes compared to near-natural landscapes and bacteria richness was higher on meadows than on forest sites. Fungi were extremely specialized on their host tree, independent of land use and climate. Bacteria specialization, however, was strongly directed by land use and climate. These results underpin previous studies showing that fungi are highly specialized in contrast to bacteria and add new insights into the robustness of fungi specialization to climate and land use. I summarize that climate as well as intensive land use influence biodiversity. Temperature and precipitation, however, had positive and negative effects on decomposer diversity, while anthropogenic land use had mostly negative effects on the diversity of decomposers. N2 - Die Zunahme intensiv genutzter Landschaften und der Klimawandel sind direkte und indirekte Folgen menschlichen Handelns, verursacht durch eine wachsende Weltbevölkerung und zunehmende Mengen an Treibhausgasen. Die Zahl der wissenschaftlichen Studien, die sich mit den Veränderungen der Umwelt und den Konsequenzen für Ökosysteme, einschließlich Flora, Fauna und Ökosystemleistungen auseinandersetzen, steigt stetig. Mit dieser Thesis möchte ich meinen Beitrag zu diesem wichtigen und aktuellen Forschungsgebiet leisten. Dazu untersuche ich die Auswirkungen von Landnutzung und Klima auf die Ökosystemleistung „Zersetzung toten organischen Materials“ (Nekromasse) und die Auswirkungen auf die daran beteiligten Arthropoden- und Mikrobengemeinschaften. Kapitel II dieser Thesis setzt sich mit den Konsequenzen von intensiver Landnutzung und Klimawandel für die Ökosystemleistung „Zersetzung toten Materials“ auseinander. Unter Anbetracht des globalen Insektenrückgangs, wurde dieser Aspekt anhand eines Insektenausschluss-Experimentes zusätzlich simuliert. Es stellt sich heraus, dass sowohl der Abbau von Dung als auch von Aas sehr robust gegenüber landschaftlicher Nutzung war. Zudem blieb der Abbau von Dung unberührt von Temperaturänderungen und dem Ausschluss von Insekten. Entlang eines Höhengradienten wurde hingegen ein Trend zu einem unimodalen Muster mit maximaler Zersetzung bei ca. 600-700 m ü.M. beobachtet. Dieser Trend lässt vermuten, dass in dieser Höhe das Verhältnis von Niederschlag und Temperatur ideal für Dung zersetzende Gemeinschaften ist. Aas hingegen wurde in zunehmender Höhe und unter der Beteiligung von Insekten schneller zersetzt, was verdeutlich, dass Klimaänderungen und ein ansteigender Insektenrückgang starke Auswirkungen auf die Zersetzung von Aas und somit auf Nährstoffkreisläufe haben können. Hierbei wurde zudem ersichtlich, dass verschiedene Typen von Nekromasse unterschiedlich auf Umweltparameter reagieren und daher in künftigen Studien und Auswertungen separat betrachtet werden sollten. Kapitel III behandelt die Auswirkungen von Landnutzung und Klima auf die Biodiversität und Spezialisierung von Käfergemeinschaften an Dung. Hierbei wurden sämtliche Käfer berücksichtigt, welche vor allem an Dung, Aas oder sonstigem faulenden Material gefunden werden können. Sowohl α- als auch γ-Diversität von diesen Käfern wurde durch Agrarlandschaften und urbane Gebiete stark reduziert. Hohe Niederschlagsmengen wirkten sich negativ auf die Abundanz von Dungkäfern aus, wohingegen die γ-Diversität in warmen Regionen am niedrigsten war. Der Grad der Spezialisierung von Käfergemeinschaften auf verschiedene Dungressourcen nahm mit abnehmenden Temperaturen zu. Aus den Ergebnissen geht hervor, dass sowohl intensive Landnutzung als auch Klimaveränderungen Auswirkungen auf die Diversität und den Spezialisierungsgrad von Käfergemeinschaften an Dung haben können und somit das ökologische Gleichgewicht der Dungkäfergemeinschaften und ihren Ökosystemfunktionen beeinflussen können. Das darauffolgende Kapitel IV stellt eine Ergänzung zu Kapitel II dar. Hier wird die Zersetzung von Aas nicht nur anhand von Landnutzung und Klima erklärt, sondern auch anhand der α-Diversität und der Artenzusammensetzung von Käfern und Bakterien an Aas diskutiert. Es zeigte sich, dass Abundanz und Artenzusammensetzung der Bakteriengemeinschaft an Aas vor allem von der Temperatur abhingen. Außerdem wurde die Zersetzungsgeschwindigkeit maßgeblich von der Bakteriengemeinschaft und der Niederschlagsmenge bestimmt. Mit dieser Studie konnte ich zeigen, dass Bakterien trotz ihrer mikroskopischen Größe maßgeblich an der Zersetzung von Aas beteiligt sind und diese in Zersetzungsversuchen nicht vernachlässigt werden sollten. Das letzte Kapitel, Kapitel V, befasst sich mit den Konsequenzen von intensiver Landnutzung und Klimawandel auf mikrobielle Gemeinschaften in Totholz. Untersucht wurden hier sowohl Bakterien- als auch Pilzgemeinschaften. Haupttreiber der Artenvielfalt für beide Gruppen (gemessen als Anzahl an OTUs) war das Klima (Niederschlag und Temperatur). Ein wärmeres Klima kam der Vielfalt von Bakterien zugute, wohingegen die Pilzvielfalt nicht tangiert wurde. Außerdem reagierten Pilze negativ auf urbane Landnutzung, Bakterienvielfalt in Totholz war auf Wiesen jedoch höher als im Wald. Vor allem Pilze zeigten eine sehr starke Bindung zu ihrem Wirtsbaum, welche auch von äußeren Einflüssen wie Landnutzung und Klima nicht beeinflusst werden konnte. Die Spezialisierung von Bakterien hingegen wurde stark von Landnutzung und Klima beeinflusst. Diese Ergebnisse untermauern frühere Studien, die besagen, dass Pilze hoch spezialisiert sind und geben neue Erkenntnisse zur Robustheit der Spezialisierung gegenüber Landnutzungsintensität und Klima. Zusammenfassend kann ich sagen, dass sowohl Klima als auch Landnutzung Auswirkungen auf die Biodiversität haben. Während Temperatur und Niederschlag jedoch positive so wie negative Effekte hatten, wirkte sich anthropogene Landnutzung überwiegend negativ auf die Diversität von Zersetzergemeinschaften aus. KW - Mikroorganismus KW - decomposition KW - Klimaänderung KW - Zersetzungsprozess KW - microbes KW - dead organic material KW - Mikroben Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313994 ER - TY - JOUR A1 - Too, Chin Chin A1 - Keller, Alexander A1 - Sickel, Wiebke A1 - Lee, Sui Mae A1 - Yule, Catherine M. T1 - Microbial Community Structure in a Malaysian Tropical Peat Swamp Forest: The Influence of Tree Species and Depth JF - Frontiers in Microbiology N2 - Tropical peat swamp forests sequester globally significant stores of carbon in deep layers of waterlogged, anoxic, acidic and nutrient-depleted peat. The roles of microbes in supporting these forests through the formation of peat, carbon sequestration and nutrient cycling are virtually unknown. This study investigated physicochemical peat properties and microbial diversity between three dominant tree species: Shorea uliginosa (Dipterocarpaceae), Koompassia malaccensis (legumes associated with nitrogen-fixing bacteria), Eleiodoxa conferta (palm) and depths (surface, 45 and 90 cm) using microbial 16S rRNA gene amplicon sequencing. Water pH, oxygen, nitrogen, phosphorus, total phenolic contents and C/N ratio differed significantly between depths, but not tree species. Depth also strongly influenced microbial diversity and composition, while both depth and tree species exhibited significant impact on the archaeal communities. Microbial diversity was highest at the surface, where fresh leaf litter accumulates, and nutrient supply is guaranteed. Nitrogen was the core parameter correlating to microbial communities, but the interactive effects from various environmental variables displayed significant correlation to relative abundance of major microbial groups. Proteobacteria was the dominant phylum and the most abundant genus, Rhodoplanes, might be involved in nitrogen fixation. The most abundant methanogens and methanotrophs affiliated, respectively, to families Methanomassiliicoccaceae and Methylocystaceae. Our results demonstrated diverse microbial communities and provide valuable insights on microbial ecology in these extreme ecosystems. KW - tropical peat swamp forest KW - metabarcoding KW - microbial diversity and composition KW - tree species KW - depth KW - methanogens Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229000 VL - 9 ER - TY - THES A1 - Gabel, Martin Sebastian T1 - Behavioural resistance to \(Varroa\) \(destructor\) in the Western honeybee \(Apis\) \(mellifera\) - Mechanisms leading to decreased mite reproduction T1 - Resistenzverhalten der Westlichen Honigbiene \(Apis\) \(mellifera\) gegen \(Varroa\) \(destructor\) - Zu verringerter Milbenreproduktion führende Mechanismen N2 - The Western Honeybee (Apis mellifera) is among the most versatile species in the world. Its adaptability is rooted in thousands of the differently specialized individuals acting jointly together. Thus, bees that are able to handle a certain task or condition well can back up other individuals less capable to do so on the colony level. Vice versa, the latter individuals might perform better in other situations. This evolutionary recipe for success ensures the survival of colonies despite challenging habitat conditions. In this context, the ectoparasitic mite Varroa destructor reflects the most pronounced biotic challenge to honeybees worldwide. Without proper treatment, infested colonies rapidly dwindle and ultimately die. Nevertheless, resistance behaviours against this parasite have evolved in some populations through natural selection, enabling colonies to survive untreated. In this, different behaviours appear to be adapted to the respective habitat conditions and may complement each other. Yet, the why and how of this behavioural response to the mite remains largely unknown. My thesis focuses on the biological background of Varroa-resistance traits in honeybees and presents important findings for the comprehension of this complex host-parasite interaction. Based on this, I draw implications for both, applied bee breeding and scientific investigations in the field of Varroa-resistance. Specifically, I focus on two traits commonly found in resistant and, to a lower degree, also mite-susceptible colonies: decreased mite reproduction and the uncapping and subsequent recapping of sealed brood cells. Examining failures in the reproductive success of mites as a primary mechanism of Varroa-resistance, I was able to link them to specific bee behaviours and external factors. Since mite reproduction and the brood rearing of bees are inevitably connected, I first investigated the effects of brood interruption on the reproductive success of mites. Brood interruption decreased the reproductive success of mites both immediately and in the long term. By examining the causes of reproductive failure, I could show that this was mainly due to an increased share of infertile mites. Furthermore, I proved that interruption in brood rearing significantly increased the expression of recapping behaviour. These findings consequently showed a dynamic modulation of mite reproduction and recapping, as well as a direct effect of brood interruption on both traits. To further elucidate the plasticity in the expression of both traits, I studied mite reproduction, recapping behaviour and infestation levels over the course of three years. The resulting extensive dataset unveiled a significant seasonal variation in mite reproduction and recapping. In addition, I show that recapping decreases the reproductive success of mites by increasing delayed developing female offspring and cells lacking male offspring. By establishing a novel picture-based brood investigation method, I could furthermore show that both the removal of brood cells and recapping activity specifically target brood ages in which mite offspring would be expected. Recapping, however, did not cause infertility of mites. Considering the findings of my first study, this points towards complementary mechanisms. This underlines the importance of increased recapping behaviour and decreased mite reproduction as resistance traits, while at the same time emphasising the challenges of reliable data acquisition. To pave the way for a practical application of these findings in breeding, we then investigated the heritability (i.e., the share of genotypic variation on the observed phenotypic variation) of the accounted traits. By elaborating comparable test protocols and compiling data from over 4,000 colonies, we could, for the first time, demonstrate that recapping of infested cells and decreased reproductive success of mites are heritable (and thus selectable) traits in managed honeybee populations. My thesis proves the importance of recapping and decreased mite reproduction as resistance traits and therefore valuable goals for breeding efforts. In this regard, I shed light on the underlying mechanisms of both traits, and present clear evidence for their interaction and heritability. N2 - Die Westliche Honigbiene (Apis mellifera) zählt zu den anpassungsfähigsten Arten der Welt. Diese Anpassungsfähigkeit liegt in der Zusammenarbeit tausender unterschiedlich spezialisierter Individuen begründet. Auf Volksebene können Bienen, die mit einer bestimmten Aufgabe oder Situation gut umgehen können, andere Individuen, die dies weniger gut können, absichern. Andererseits können Letztere womöglich mit anderen Situationen besser umgehen. Dieses evolutionäre Erfolgskonzept sichert das Überleben der Völker selbst unter herausfordernden Habitatbedingungen. Die ektoparasitäre Milbe Varroa destructor stellt in diesem Zusammenhang weltweit die größte biotische Herausforderung dar. Ohne entsprechende Behandlung siechen die Völker rasch dahin und sterben schlussendlich. In einigen Populationen haben sich jedoch Resistenzmechanismen durch natürliche Selektion herausgebildet, die es den Völkern ermöglichen, ohne Behandlung zu überleben. Die verschiedenen Verhaltensweisen scheinen dabei an die jeweiligen Habitatbedingungen angepasst zu sein und sich gegenseitig zu ergänzen. Was diese Reaktion auf die Milben auslöst und wie sie funktioniert ist allerdings noch weitestgehend unbekannt. Meine Dissertation fokussiert den biologischen Hintergrund von Varroa-resistenzmechanismen bei Honigbienen und stellt dabei wichtige Erkenntnisse zum Verständnis dieser komplexen Parasit-Wirt-Beziehung vor. Darauf aufbauend leite ich Implikationen für die angewandte Bienenzucht und wissenschaftliche Untersuchungen auf dem Gebiet der Varroa-resistenz ab. Hierbei konzentriere ich mich insbesondere auf zwei Merkmale, die häufig in resistenten Völkern zu finden sind: die reduzierte Milbenreproduktion und das Entdeckeln und Wiederverdeckeln bereits verschlossener Brutzellen. Beide Merkmale treten in geringerem Umfang auch in milbenanfälligen Populationen auf und sind daher von besonderem Interesse für jedwede Zuchtbemühung mit dem Ziel der Varroa-resistenz. Durch die Untersuchung von Fehlern in der Reproduktion der Milben, konnte ich diesen Hauptmechanismus der Varroa-resistenz mit Verhaltensweisen der Bienen, sowie äußeren Faktoren in Verbindung setzen. Da die Milbenvermehrung untrennbar mit der Brutaufzucht der Bienen verbunden ist, habe ich zunächst die Einflüsse von Brutunterbrechungen auf den Vermehrungserfolg der Milben untersucht. Diese Untersuchung zeigte auf, dass Brutunterbrechungen den Vermehrungserfolg der Milben sowohl kurzfristig, als auch langfristig herabsetzen. Durch die Untersuchung der jeweils zugrundeliegenden Ursachen gescheiterter Milbenreproduktion konnte ich zeigen, dass dies vor Allem auf einen gesteigerten Anteil infertiler Milben zurückzuführen war. Des Weiteren konnte ich beweisen, dass die Unterbrechung der Brutaufzucht die Ausprägung des Wiederverdeckelns signifikant verstärkte. Folglich zeigten diese Ergebnisse eine dynamische Anpassung der Milbenreproduktion und des Wiederverdeckelns, sowie einen direkten Einfluss der Brutunterbrechungen auf beide Eigenschaften. Um die Plastizität der Ausprägung beider Merkmale genauer zu erklären, untersuchte ich daraufhin drei Jahre lang die Milbenvermehrung, das Verhalten des Wiederverdeckelns, sowie die Befallsentwicklung. Daraus resultierte ein umfangreicher Datensatz, der eine signifikante saisonale Variation der Milbenvermehrung und des Wiederverdeckelns belegte. Ich konnte außerdem eindeutig beweisen, dass das Wiederverdeckeln den Reproduktionserfolg der Milben herabsetzt, indem es die Anteile von verzögert heranwachsenden weiblichen Nachkommen und fehlenden Männchen steigert. Durch Anwendung einer neuartigen Bild-basierten Methode der Brutuntersuchung, konnte ich darüber hinaus zeigen, dass sich sowohl das Ausräumen, als auch das Wiederverdeckeln von Brutzellen auf Brutalter konzentriert, in denen Milbennachwuchs erwartet werden würde. Das Wiederverdeckeln trug jedoch nicht zur Infertilität der Milben bei, was zusammen mit den Ergebnissen meiner ersten Untersuchung auf komplementäre Mechanismen hinweist. Dies unterstreicht die Bedeutung des Wiederverdeckelns und der verminderten Milbenreproduktion als Resistenzmechanismen, hebt aber gleichzeitig auch die Herausforderungen einer verlässlichen Datenerhebung hervor. Um den Weg für die praktische Anwendung dieser Erkenntnisse in der Zuchtarbeit zu ebnen, untersuchten wir daraufhin die Erblichkeit (den Anteil der genotypischen Variation an der beobachteten phänotypischen Variation) der betrachteten Merkmale. Durch das Erarbeiten vergleichbarer Prüfprotokolle und Zusammenführen von Daten aus über 4000 Völkern, konnten wir erstmalig zeigen, dass das Wiederverdeckeln befallener Zellen und der verminderte Vermehrungserfolg der Milben erbliche und damit selektierbare Merkmale in bewirtschafteten Honigbienenpopulationen sind. Meine Dissertation beweist die Relevanz des Wiederverdeckelns und der verminderten Milbenreproduktion als Resistenzmerkmale und damit lohnende Ziele für Zuchtbemühungen. In diesem Zusammenhang beleuchtete ich verschiedene Mechanismen, die der Ausprägung beider Merkmale zugrunde liegen und lieferte eindeutige Beweise für deren Interaktion und Erblichkeit. KW - Varroa destructor KW - Resistenz KW - Biene KW - mite non-reproduction KW - recapping KW - Varroa resistance KW - biotechnical Varroa control KW - heritability KW - selection KW - honeybees KW - Varroa mites KW - Züchtung KW - Apis mellifera KW - Breeding Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-360536 ER - TY - JOUR A1 - Franchini, Paolo A1 - Jones, Julia C. A1 - Xiong, Peiwen A1 - Kneitz, Susanne A1 - Gompert, Zachariah A1 - Warren, Wesley C. A1 - Walter, Ronald B. A1 - Meyer, Axel A1 - Schartl, Manfred T1 - Long-term experimental hybridisation results in the evolution of a new sex chromosome in swordtail fish JF - Nature Communications N2 - The remarkable diversity of sex determination mechanisms known in fish may be fuelled by exceptionally high rates of sex chromosome turnovers or transitions. However, the evolutionary causes and genomic mechanisms underlying this variation and instability are yet to be understood. Here we report on an over 30-year evolutionary experiment in which we tested the genomic consequences of hybridisation and selection between two Xiphophorus fish species with different sex chromosome systems. We find that introgression and imposing selection for pigmentation phenotypes results in the retention of an unexpectedly large maternally derived genomic region. During the hybridisation process, the sex-determining region of the X chromosome from one parental species was translocated to an autosome in the hybrids leading to the evolution of a new sex chromosome. Our results highlight the complexity of factors contributing to patterns observed in hybrid genomes, and we experimentally demonstrate that hybridisation can catalyze rapid evolution of a new sex chromosome. KW - evolutionary genetics KW - experimental evolution KW - genome evolution Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228396 VL - 9 ER - TY - JOUR A1 - Hennrich, Marco L. A1 - Romanov, Natalie A1 - Horn, Patrick A1 - Jaeger, Samira A1 - Eckstein, Volker A1 - Steeples, Violetta A1 - Ye, Fei A1 - Ding, Ximing A1 - Poisa-Beiro, Laura A1 - Mang, Ching Lai A1 - Lang, Benjamin A1 - Boultwood, Jacqueline A1 - Luft, Thomas A1 - Zaugg, Judith B. A1 - Pellagatti, Andrea A1 - Bork, Peer A1 - Aloy, Patrick A1 - Gavin, Anne-Claude A1 - Ho, Anthony D. T1 - Cell-specific proteome analyses of human bone marrow reveal molecular features of age-dependent functional decline JF - Nature Communications N2 - Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models. KW - ageing KW - haematopoietic stem cells KW - mesenchymal stem cells Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319877 VL - 9 ER - TY - JOUR A1 - Hines, Rochelle M. A1 - Maric, Hans Michael A1 - Hines, Dustin J. A1 - Modgil, Amit A1 - Panzanelli, Patrizia A1 - Nakamura, Yasuko A1 - Nathanson, Anna J. A1 - Cross, Alan A1 - Deeb, Tarek A1 - Brandon, Nicholas J. A1 - Davies, Paul A1 - Fritschy, Jean-Marc A1 - Schindelin, Hermann A1 - Moss, Stephen J. T1 - Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin JF - Nature Communications N2 - Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development. KW - cellular neuroscience KW - ion channels in the nervous system KW - neurotransmitters KW - synaptic development Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320719 VL - 9 ER - TY - THES A1 - Hahn, Sarah T1 - Investigating non-canonical, 5' UTR-dependent translation of MYC and its impact on colorectal cancer development T1 - Untersuchung der nicht-kanonischen, 5' UTR-abhängigen Translation von MYC und ihres Einflusses auf die Entwicklung von Darmkrebs N2 - Colorectal cancer (CRC) is the second most common tumour disease in Germany, with the sequential accumulation of certain mutations playing a decisive role in the transition from adenoma to carcinoma. In particular, deregulation of the Wnt signalling pathway and the associated deregulated expression of the MYC oncoprotein play a crucial role. Targeting MYC thus represents an important therapeutic approach in the treatment of tumours. Since direct inhibition of MYC is challenging, various approaches have been pursued to date to target MYC indirectly. The MYC 5' UTR contains an internal ribosomal entry site (IRES), which has a particular role in the initiation of MYC translation, especially in multiple myeloma. As basis for this work, it was hypothesised on the basis of previous data that translation of MYC potentially occurs via its IRES in CRC as well. Based on this, two IRES inhibitors were tested for their potential to regulate MYC expression in CRC cells. In addition, alternative, 5’ UTR-dependent translation of MYC and interacting factors were investigated. EIF3D was identified as a MYC 5' UTR binding protein which has the potential to regulate MYC expression in CRC. The results of this work suggest that there is a link between eIF3D and MYC expression/translation, rendering eIF3D a potential therapeutic target for MYC-driven CRCs. N2 - Das kolorektale Karzinom (KRK) ist die zweithäufigste Tumorerkrankung in Deutschland, wobei die sequenzielle Akkumulation bestimmter Mutationen eine entscheidende Rolle beim Übergang vom Adenom zum Karzinom spielt. Insbesondere die Deregulation des Wnt-Signalweges und die damit verbundene deregulierte Expression des MYC-Onkoproteins spielen eine entscheidende Rolle. MYC ist ein zentraler Vermittler von Zellfunktionen und reguliert als Transkriptionsfaktor die Expression fast aller Gene sowie verschiedener RNA-Spezies. Selbst kleine Veränderungen der zellulären MYC-Konzentration können das Proliferationsverhalten beeinflussen und die Entstehung und das Fortschreiten von Tumoren fördern. Die gezielte Beeinflussung von MYC stellt daher einen wichtigen therapeutischen Ansatz für die Behandlung von Tumoren dar. Da eine direkte Hemmung von MYC aufgrund seiner Struktur herausfordernd ist, wurden bisher verschiedene Ansätze verfolgt, um MYC indirekt zu beeinflussen, etwa über seinen Interaktionspartner MAX oder auf Ebene der Stabilität, Transkription oder Translation. In unserer eigenen Forschungsgruppe lag der Schwerpunkt in den letzten Jahren speziell auf der Translation von MYC im KRK. Es konnte gezeigt werden, dass die Hemmung der kanonischen cap-abhängigen Translation nicht wie erwartet zu einer Verringerung der zellulären MYC-Level führt, was auf einen alternativen Mechanismus der MYC-Translation hindeutet, der unabhängig vom eIF4F-Komplex abläuft. Die 5'-UTR von MYC enthält eine interne ribosomale Eintrittsstelle (IRES), die eine besondere Rolle bei der Initiierung der MYC-Translation spielt, insbesondere im Multiplen Myelom. Als Grundlage für diese Arbeit wurde daher die Hypothese aufgestellt, dass die Translation von MYC im KRK möglicherweise ebenfalls über die IRES erfolgt. Auf dieser Grundlage wurden zunächst zwei publizierte IRES-Inhibitoren auf ihr Potenzial zur Regulierung der MYC-Expression in KRK-Zellen getestet. J007-IRES hatte keine Auswirkungen auf die MYC-Proteinmenge, und Cymarin scheint weitaus globalere Auswirkungen zu haben, die nicht ausschließlich auf die Verringerung der MYC-Proteinmenge zurückzuführen sind. Daher wurde weiter untersucht, inwieweit die alternative Translation von MYC generell von der 5'-UTR und damit interagierenden Faktoren abhängig ist. EIF3D wurde als MYC-5'-UTR-Bindungsprotein identifiziert, dessen Knockdown zu reduzierten MYC-Leveln, einem Proliferationsdefizit sowie einer Verringerung der globalen Proteinsynthese in KRK-Zellen führte. Darüber hinaus führte die Depletion von EIF3D zu ähnlichen Veränderungen im zellulären Genexpressionsmuster wie die Depletion von MYC, wobei viele tumorassoziierte Signalwege betroffen waren. Mittels eCLIP-seq wurde die Bindung von eIF3D an die MYC mRNA nachgewiesen, der genaue Mechanismus einer möglicherweise durch eIF3D vermittelten Translation von MYC muss jedoch weiter untersucht werden. Die Ergebnisse dieser Arbeit deuten darauf hin, dass eine Verbindung zwischen eIF3D und der MYC-Expression/Translation besteht, wodurch eIF3D zu einem potenziellen therapeutischen Ziel für MYC-getriebene KRKs wird. KW - Myc KW - Translation KW - Colorectal cancer KW - 5' UTR Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-364202 ER - TY - THES A1 - Hartmann, Oliver T1 - Development of somatic modified mouse models of Non-Small cell lung cancer T1 - Entwicklung von somatisch veränderten Mausmodellen für nichtkleinzelligen Lungenkrebs N2 - In 2020, cancer was the leading cause of death worldwide, accounting for nearly 10 million deaths. Lung cancer was the most common cancer, with 2.21 million cases per year in both sexes. This non-homogeneous disease is further subdivided into small cell lung cancer (SCLC, 15%) and non-small cell lung cancer (NSCLC, 85%). By 2023, the American Cancer Society estimates that NSCLC will account for 13% of all new cancer cases and 21% of all estimated cancer deaths. In recent years, the treatment of patients with NSCLC has improved with the development of new therapeutic interventions and the advent of targeted and personalised therapies. However, these advances have only marginally improved the five-year survival rate, which remains alarmingly low for patients with NSCLC. This observation highlights the importance of having more appropriate experimental and preclinical models to recapitulate, identify and test novel susceptibilities in NSCLC. In recent years, the Trp53fl/fl KRaslsl-G12D/wt mouse model developed by Tuveson, Jacks and Berns has been the main in vivo model used to study NSCLC. This model mimics ADC and SCC to a certain extent. However, it is limited in its ability to reflect the genetic complexity of NSCLC. In this work, we use CRISPR/Cas9 genome editing with targeted mutagenesis and gene deletions to recapitulate the conditional model. By comparing the Trp53fl/fl KRaslsl- G12D/wt with the CRISPR-mediated Trp53mut KRasG12D, we demonstrated that both showed no differences in histopathological features, morphology, and marker expression. Furthermore, next-generation sequencing revealed a very high similarity in their transcriptional profile. Adeno-associated virus-mediated tumour induction and the modular design of the viral vector allow us to introduce additional mutations in a timely manner. CRISPR-mediated mutation of commonly mutated tumour suppressors in NSCLC reliably recapitulated the phenotypes described in patients in the animal model. Lastly, the dual viral approach could induce the formation of lung tumours not only in constitutive Cas9 expressing animals, but also in wildtype animals. Thus, the implementation of CRISPR genome editing can rapidly advance the repertoire of in vivo models for NSCLC research. Furthermore, it can reduce the necessity of extensive breeding. N2 - Krebs war mit fast 10 Millionen Todesfällen weltweit die häufigste Todesursache in 2020. Mit 2,21 Millionen Fällen pro Jahr in beiden Geschlechtern kombiniert war Lungenkrebs die häufigste Unterart. Auszeichnend für dieses Krankheit ist die hohe Komplexität und Heterogenität. Daher wird diese weiter in kleinzelligen Lungenkrebs (SCLC, 15 %) und nicht-kleinzelligen Lungenkrebs (NSCLC, 85 %) unterteilt. Die American Cancer Society schätzt, dass bis 2023 13 % aller neuen Krebsfälle und 21 % aller geschätzten Krebstodesfälle auf das nicht-kleinzellige Lungenkarzinom entfallen werden. In den letzten Jahren hat sich die Behandlung von Patienten mit nicht-kleinzelligem Lungenkarzinom durch die Entwicklung neuer therapeutischer Maßnahmen und das Anwenden personalisierter Therapien verbessert. Allerdings haben diese Fortschritte die Fünfjahresüberlebensrate nur geringfügig verbessert, die für Patienten mit NSCLC nach wie vor alarmierend niedrig ist. Diese macht deutlich, wie wichtig es ist, über geeignetere experimentelle und präklinische Modelle zu verfügen, um neue Therapieansätze beim NSCLC zu rekapitulieren, zu identifizieren und zu testen. In der letzten Dekade war das von Tuveson, Jacks und Berns entwickelte Trp53fl/fl KRaslsl-G12D/wt-Mausmodell das wichtigste In-vivo-Modell zur Untersuchung von NSCLC. Dieses kann grundlegend das Krankheitsbild von NSCLC wiederspiegeln. Es ist jedoch nur begrenzt in der Lage, die genetische Komplexität von NSCLC im vollen Umfang zu refelktieren. In dieser Arbeit verwenden wir CRISPR/Cas9 Genome Editing mit gezielter Mutagenese und Gendeletionen, um das konditionale Modell zu rekapitulieren. Durch den Vergleich des Trp53fl/fl KRaslsl-G12D/wt mit dem CRISPR-vermittelten Trp53mut KRasG12D konnten wir zeigen, dass beide keine Unterschiede in Bezug auf histopathologische Merkmale, Morphologie und Markerexpression aufweisen. Darüber hinaus ergab die Analyse mittels Next Generation Sequencing 8Hochdruchsatz.Sequenzierung) eine sehr große Ähnlichkeit in ihrem Transkriptionsprofil. Die Adeno-assoziierte Virus-vermittelte Tumorinduktion und der modulare Aufbau des viralen Vektors ermöglichen es uns, zusätzliche Mutationen zeitnah einzuführen. Die CRISPR-vermittelte Mutation von häufig mutierten Tumorsuppressoren bei NSCLC rekapitulierte zuverlässig die bei Patienten beschriebenen Phänotypen im Tiermodell. Schließlich konnte der duale virale Ansatz die Bildung von Lungentumoren nicht nur in konstitutiv Cas9 exprimierenden Tieren, sondern auch in Wildtyp-Tieren induzieren. Somit kann die Anwendung von CRISPR-Genome Editing das Repertoire an In-vivo- Modellen für die NSCLC-Forschung rasch erweitern. Darüber hinaus kann es die Notwendigkeit umfangreicher Züchtungen verringern. KW - CRISPR/Cas-Methode KW - in vivo KW - Lung Cancer KW - CRISPR/Cas9 KW - in vivo genome editing KW - Immunohistochemistry KW - Nicht-kleinzelliges Bronchialkarzinom KW - NSCLC KW - Mouse Model KW - CRISPR Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-363401 ER - TY - JOUR A1 - Link, Jana A1 - Paouneskou, Dimitra A1 - Velkova, Maria A1 - Daryabeigi, Anahita A1 - Laos, Triin A1 - Labella, Sara A1 - Barroso, Consuelo A1 - Pacheco Piñol, Sarai A1 - Montoya, Alex A1 - Kramer, Holger A1 - Woglar, Alexander A1 - Baudrimont, Antoine A1 - Markert, Sebastian Mathias A1 - Stigloher, Christian A1 - Martinez-Perez, Enrique A1 - Dammermann, Alexander A1 - Alsheimer, Manfred A1 - Zetka, Monique A1 - Jantsch, Verena T1 - Transient and Partial Nuclear Lamina Disruption Promotes Chromosome Movement in Early Meiotic Prophase JF - Developmental Cell N2 - Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis. KW - meiosis KW - C. elegans KW - chromosome movement KW - chromosome pairing KW - nuclear envelope KW - lamin Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236901 VL - 45 ER - TY - JOUR A1 - Kraus, Michael A1 - Grimm, Clemens A1 - Seibel, Jürgen T1 - Reversibility of a Point Mutation Induced Domain Shift: Expanding the Conformational Space of a Sucrose Phosphorylase JF - Scientific Reports N2 - Despite their popularity as enzyme engineering targets structural information about Sucrose Phosphorylases remains scarce. We recently clarified that the Q345F variant of Bifidobacterium adolescentis Sucrose Phosphorylase is able to accept large polyphenolic substrates like resveratrol via a domain shift. Here we present a crystal structure of this variant in a conformation suitable for the accommodation of the donor substrate sucrose in excellent agreement with the wild type structure. Remarkably, this conformation does not feature the previously observed domain shift which is therefore reversible and part of a dynamic process rather than a static phenomenon. This crystallographic snapshot completes our understanding of the catalytic cycle of this useful variant and will allow for a more rational design of further generations of Sucrose Phosphorylase variants. KW - biocatalysis KW - X-ray crystallography Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224845 VL - 8 ER - TY - JOUR A1 - Müller, Laura S. M. A1 - Cosentino, Raúl O. A1 - Förstner, Konrad U. A1 - Guizetti, Julien A1 - Wedel, Carolin A1 - Kaplan, Noam A1 - Janzen, Christian J. A1 - Arampatzi, Panagiota A1 - Vogel, Jörg A1 - Steinbiss, Sascha A1 - Otto, Thomas D. A1 - Saliba, Antoine-Emmanuel A1 - Sebra, Robert P. A1 - Siegel, T. Nicolai T1 - Genome organization and DNA accessibility control antigenic variation in trypanosomes JF - Nature N2 - Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation. KW - histone variants KW - genome architecture KW - single molecule real time (SMRT) KW - brucei genome KW - distance-dependent decay Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224265 VL - 563 ER - TY - THES A1 - Aroko, Erick Onyango T1 - Trans-regulation of \(Trypanosoma\) \(brucei\) variant surface glycoprotein (VSG) mRNA and structural analysis of a \(Trypanosoma\) \(vivax\) VSG using X-ray crystallography T1 - Trans-regulierung der mRNA des variablen Oberflächenglykoprotein (VSG) von \(Trypanosoma\) \(brucei\) und strukturelle Analyse eines \(Trypanosoma\) \(vivax\) VSG mittels Kristallstrukturanalyse N2 - African trypanosomes are unicellular parasites that cause nagana and sleeping sickness in livestock and man, respectively. The major pathogens for the animal disease include Trypanosoma vivax, T. congolense, and T. brucei brucei, whereas T. b. gambiense and T. b. rhodesiense are responsible for human infections. Given that the bloodstream form (BSF) of African trypanosomes is exclusively extracellular, its cell surface forms a critical boundary with the host environment. The cell surface of the BSF African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). This surface protein acts as an impenetrable shield that protects the cells from host immune factors and is also involved in antibody clearance and antigenic variation, which collectively ensure that the parasite stays ahead of the host immune system. Gene expression in T. brucei is markedly different from other eukaryotes: most genes are transcribed as long polycistronic units, processed by trans-splicing a 39-nucleotide mini exon at the 5′ and polyadenylation at the 3′ ends of individual genes to generate the mature mRNA. Therefore, gene expression in T. brucei is regulated post-transcriptionally, mainly by the action of RNA binding proteins (RBPs) and conserved elements in the 3′ untranslated regions (UTR) of transcripts. The expression of VSGs is highly regulated, and only a single VSG gene is expressed at a time from one of the ~15 subtelomeric domains termed bloodstream expression sites (BES). When cells are engineered to simultaneously express two VSGs, the total VSG mRNA do not exceed the wild type amounts. This suggests that a robust VSG mRNA balancing mechanism exists in T. brucei. The present study uses inducible and constitutive expression of ectopic VSG genes to show that the endogenous VSG mRNA is regulated only if the second VSG is properly targeted to the ER. Additionally, the endogenous VSG mRNA response is triggered when high amounts of the GFP reporter with a VSG 3′UTR is targeted to the ER. Further evidence that non-VSG ER import signals can efficiently target VSGs to the ER is presented. This study suggests that a robust trans-regulation of the VSG mRNA is elicited at the ER through a feedback loop to keep the VSG transcripts in check and avoid overshooting the secretory pathway capacity. Further, it was shown that induction of expression of the T. vivax VSG ILDat1.2 in T. brucei causes a dual cell cycle arrest, with concomitant upregulation of the protein associated with differentiation (PAD1) expression. It could be shown that T. vivax VSG ILDat1.2 can only be sufficiently expressed in T. brucei after replacing its native GPI signal peptide with that of a T. brucei VSG. Taken together, these data indicate that inefficient VSG GPI anchoring and expression of low levels of the VSG protein can trigger differentiation from slender BSF to stumpy forms. However, a second T. vivax VSG, ILDat2.1, is not expressed in T. brucei even after similar modifications to its GPI signals. An X-ray crystallography approach was utilized to solve the N-terminal domain (NTD) structure of VSG ILDat1.2. This is first structure of a non-T. brucei VSG, and the first of a surface protein of T. vivax to be solved. VSG ILDat1.2 NTD maintains the three-helical bundle scaffold conserved in T. brucei surface proteins. However, it is likely that there are variations in the architecture of the membrane proximal region of the ILDat1.2 NTD and its CTD from T. brucei VSGs. The tractable T. brucei system is presented as a model that can be used to study surface proteins of related trypanosome species, thus creating avenues for further characterization of trypanosome surface coats. N2 - Afrikanische Trypanosomen sind einzellige Parasiten, die Nagana in Nutzvieh und die Schlafkrankheit im Menschen verursachen. Zu den Hauptverursachern der Tierkrankheit gehören Trypanosoma vivax, T. congolense und T. brucei brucei, während T. b. gambiense und T. b. rhodesiense für Infektionen im Menschen verantwortlich sind. Da die Blutstromform (BSF) der afrikanischen Trypanosomen rein extrazellulär vorkommt, bildet die Zelloberfläche eine kritische Grenzregion mit der Wirtsumgebung. Die Zelloberoberfläche der BSF afrikanischer Trypanosomen ist mit einem dichten Mantel an immunogenen variablen Oberflächenglykoproteinen (variant surface glycoprotein, VSG) umgeben. Dieses Oberflächenprotein dient als Barriere zum Schutz gegen Faktoren des Wirtsimmunsystems und spielt ebenfalls eine Rolle in Antikörper-Clearance und antigener Variation, welche gemeinsam dafür sorgen, dass der Parasit dem Wirtsimmunsystem stets einen Schritt voraus bleibt. Die Genexpression von T. brucei weist dezidierte Unterschiede im Vergleich zu anderen Eukaryoten auf: Die meisten Gene werden als lange polyzystronische Einheiten transkribiert, die durch trans-Splicing eines Miniexons aus 39 Nukleotiden am 5′ und Polyadenylierung am 3′ Ende der individuellen Gene prozessiert wird. Daher wird die Genexpression in T. brucei posttranskriptionell reguliert, zumeist durch RNA Bindeproteine (RBPs) und konservierte Elemente in der 3′ untranslatierten Region (UTR). Die Expression der VSGs ist stark reguliert, so wird zu einer gegebenen Zeit stets nur ein VSG Gen aus einer von ~15 Subtelomerregionen, die Blutstrom Expressionsorte (bloodstream expression sites, BES) genannt werden, exprimiert. Zellen, die gentechnisch manipuliert wurden um zwei VSGs zu exprimieren, produzieren die gleiche Menge an VSG mRNA wie Wildtyp Zellen. Dies deutet auf die Existenz eines robusten Mechanismus zur Regulierung der Gesamt-VSG mRNA Menge in T. brucei hin. Diese Arbeit verwendet induzierbare sowie konstitutive Expression eines ektopischen VSG Gens um zu zeigen, dass die endogene VSG mRNA nur reguliert wird, wenn das zweite VSG zum ER gelangt. Außerdem wird die endogene VSG mRNA Antwort auch ausgelöst, wenn hohe Mengen eines GFP Reporters, der eine VSG 3′UTR enthält, zum ER geleitet wird. Weiterhin, wird gezeigt, dass ER Importsignale anderer Proteine VSGs effizient zum ER dirigieren können. Das Ergebnis dieser Studie deutet darauf hin, dass eine Rückkopplungsschleife am ER eine robuste trans-Regulation der VSG mRNA auslöst, die die VSG Transkripte limitiert und somit eine Überlastung des sekretorischen Wegs verhindert. Weiterhin konnte gezeigt werden, dass es nach Induktion der Expression des T. vivax VSGs ILDat1.2 in T. brucei zu einem doppelten Zellzyklusarrest mit gleichzeitiger Hochregulation der Expression des protein associated with differentation (PAD1) kam und dass dieses T. vivax VSG nur nach Austausch des GPI Signalpeptids durch das eines T. brucei VSGs effizient exprimiert werden konnte. Zusammengenommen suggerieren diese Daten, dass eine ineffiziente GPI-Verankerung und wenig abundante Expression des VSGs die Differenzierung der sogenannten slender BSF zur sogenannten stumpy Form einleiten kann. Ein zweites T. vivax VSG, ILDat2.1, konnte hingegen auch nach Austausch des GPI Signals nicht in T. brucei exprimiert werden. Mit Hilfe der Röntgenstrukturanalyse wurde die Struktur der N-terminalen Domäne (NTD) des ILDat1.2 VSGs gelöst. Es handelt sich hierbei um die erste Proteinstruktur eines VSGs, welches nicht aus T. brucei stammt und die erste Struktur eines Oberflächenproteins von T. vivax. Das in T. brucei Oberflächenproteinen konservierte drei-Helix Grundgerüst ist auch in der NTD des ILDat1.2 VSGs enthalten. Die Architektur der Membranproximalen Gegend der IlDat1.2 NTD und CTD unterscheiden sich aber vermutlich von der der T. brucei VSGs. Das leicht handhabbare T. brucei System bietet somit ein geeignetes Modell um die Oberflächenproteine anderer afrikanischer Trypanosomen Spezies zu untersuchen und eröffnet neue Wege zur Charakterisierung ihrer Oberflächenmäntel. KW - Trypanosoma vivax KW - Trypanosoma brucei KW - Variant surface glycoprotein KW - messenger RNA KW - Regulation of expression KW - messenger RNA regulation KW - VSG structure Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241773 ER - TY - THES A1 - Heimberger, Kevin T1 - Regulation pathways of c-MYC under glutamine-starving conditions in colon carcinoma cells T1 - Regulierungsmechanismen von c-MYC in Darmkrebszellen unter Glutaminmangelbedingungen N2 - Colon carcinomas (CRC) are statistically among the most fatal cancer types and hence one of the top reasons for premature mortality in the developed world. CRC cells are characterized by high proliferation rates caused by deregulation of gene transcription of proto-oncogenes and general chromosomal instability. On macroscopic level, CRC cells show a strongly altered nutrient and energy metabolism. This work presents research to understand general links between the metabolism and transcription alteration. Mainly focussing on glutamine dependency, shown in colon carcinoma cells and expression pathways of the pro-proliferation protein c-MYC. Previous studies showed that a depletion of glutamine in the cultivation medium of colon carcinoma cell lines caused a proliferation arrest and a strong decrease of overall c-MYC levels. Re-addition of glutamine quickly replenished c-MYC levels through an unknown mechanism. Several proteins altering this regulation mechanism were identified and proposed as possible starting point for further in detail studies to unveil the precise biochemical pathway controlling c-MYC translation repression and reactivation in a rapid manner. On a transcriptional level the formation of RNA:DNA hybrids, so called R-loops, was observed under glutamine depleted conditions. The introduction and overexpression of RNaseH1, a R-loop degrading enzyme, in combination with an ectopically expressed c-MYC variant, independent of cellular regulation mechanisms by deleting the regulatory 3’-UTR of the c-MYC gene, lead to a high rate of apoptotic cells in culture. Expression of a functionally inactive variant of RNaseH1 abolished this effect. This indicates a regulatory function of R-loops formed during glutamine starvation in the presence of c-MYC protein in a cell. Degradation of R-loops and high c-MYC levels in this stress condition had no imminent effect on the cell cycle progression is CRC cells but disturbed the nucleotide metabolism. Nucleotide triphosphates were strongly reduced in comparison to starving cells without R-loop degradation and proliferating cells. This study proposes a model of a terminal cycle of transcription termination, unregulated initiation and elongation of transcription leading to a depletion of energy resources of cells. This could finally lead to high apoptosis of the cells. Sequencing experiments to determine a coinciding of termination sites and R-loop formation sides failed so far but show a starting point for further studies in this essential survival mechanism involving R-loop formation and c-MYC downregulation. N2 - Darmkrebs gehört statistisch zu den Krebsarten mit den höchsten Sterblichkeitsraten und zählen somit zu den häufigsten Todesursachen der entwickelten Länder. Darmkrebszellen zeichnen sich durch chromosomale Instabilität und hohe Proliferationsraten aus, die durch eine Deregulierung der Expression verschiedener Proto-Onkogene zustande kommen. Generell besitzen diese Krebszellen einen stark veränderten Nährstoff- und Energiestoffwechsel im Vergleich zu gesunden somatischen Zellen. Diese Arbeit strebt ein besseres Verständnis der Verbindung zwischen dem Metabolismus und der Gen-Expression an. Das Hauptaugenmerk liegt hierbei auf dem Mechanismus der Expression des proliferationsfördernden Proteins c-MYC und der Abhängigkeit von Glutamin, die Darmkrebszellen charakterisiert. Frühere Studien haben gezeigt, dass der Entzug von Glutamin aus dem Kulturmedium von Darmkrebszelllinien eine Arretierung des Zellzyklus bewirkt sowie die Konzentration des Proteins c-MYC reduziert. Erneute Zugabe von Glutamin zum Medium stellt die MYC-Konzentration schnell wieder her. Die Hintergründe dieses Mechanismus sind bislang aber kaum verstanden. Einige Proteine wurden hier als potenzielle Kandidaten identifiziert, die einen Einfluss auf den biochemischen Prozess haben könnten, der die schnelle Wiederaufnahme der c-MYC Translation gewährleistet. Auf Translationsebene wurden RNA:DNA-Hybriden, sogenannte R-loops, gefunden, die sich unter anderem unter Glutamin-Mangelbedingungen im Genom bilden können. Ein gezielter Abbau dieser R-loops mithilfe des Enzyms RNaseH1, in Kombination mit der ektopischen Expression einer c-MYC-Variante, die unempfindlich gegenüber der zelleigenen Regulationsmechanismen ist, führte zu einer erhöhten Anzahl an apoptotischen Zellen in Kultur. Exprimiert man eine funktionell inaktive Variante der RNaseH1, statt der funktionellen, so kann dieser Apoptose-fördernde Prozess nicht beobachtet werden. Dies bestärkt die Hypothese, dass die R-loops, die sich während eines Glutamin-Mangels und hoher c-MYC-Konzentration bilden, eine regulatorische Funktion innehaben. Als Ursache für die Apoptose konnte ein Effekt der veränderten Expression auf das Fortschreiten des Zellzyklus ausgeschlossen werden. Jedoch zeigte sich eine Veränderung im Nukleotid-Metabolismus. Betroffene Zellen zeigten deutlich reduzierte Nuklotidtriphosphat-Konzentrationen im Vergleich zu Zellen unter Glutaminmangelbedingungen ohne R-loop-Abbau. In dieser Arbeit wurde ein Modell entwickelt, das einen sich selbst negativ verstärkenden Zyklus vorschlägt, der die Zellen zur Apoptose führt. Transkriptionstermination und eine unkontrollierte Initiation der Transkription im Wechsel führt zu einem Verbrauch der lebensnotwendigen Energieressourcen der Zellen. Sequenzierungsexperimente zur Lokalisierung der R-loops und Terminationsstellen sind bislang fehlgeschlagen, bieten jedoch Ansätze für künftige Forschung. KW - Myc KW - cMYC regulation KW - Colorectal Cancer KW - Glutamine KW - Translation regulation Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-363316 ER - TY - JOUR A1 - Selkrig, Joel A1 - Mohammad, Farhan A1 - Ng, Soon Hwee A1 - Chua, Jia Yi A1 - Tumkaya, Tayfun A1 - Ho, Joses A1 - Chiang, Yin Ning A1 - Rieger, Dirk A1 - Pettersson, Sven A1 - Helfrich-Förster, Charlotte A1 - Yew, Joanne Y. A1 - Claridge-Chang, Adam T1 - The Drosophila microbiome has a limited influence on sleep, activity, and courtship behaviors JF - Scientific Reports N2 - In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this dependency is present in other animal species. We proposed the hypothesis that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal’s survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved. KW - behavioural ecology KW - sleep Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235891 VL - 8 ER - TY - JOUR A1 - Nerreter, Thomas A1 - Letschert, Sebastian A1 - Götz, Ralph A1 - Doose, Sören A1 - Danhof, Sophia A1 - Einsele, Hermann A1 - Sauer, Markus A1 - Hudecek, Michael T1 - Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T JF - Nature Communications N2 - Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens. KW - cancer imaging KW - cancer immunotherapy KW - imaging Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232258 VL - 10 ER - TY - JOUR A1 - Steuer Costa, Wagner A1 - Van der Auwera, Petrus A1 - Glock, Caspar A1 - Liewald, Jana F. A1 - Bach, Maximilian A1 - Schüler, Christina A1 - Wabnig, Sebastian A1 - Oranth, Alexandra A1 - Masurat, Florentin A1 - Bringmann, Henrik A1 - Schoofs, Liliane A1 - Stelzer, Ernst H. K. A1 - Fischer, Sabine C. A1 - Gottschalk, Alexander T1 - A GABAergic and peptidergic sleep neuron as a locomotion stop neuron with compartmentalized Ca2+ dynamics JF - Nature Communications N2 - Animals must slow or halt locomotion to integrate sensory inputs or to change direction. In Caenorhabditis elegans, the GABAergic and peptidergic neuron RIS mediates developmentally timed quiescence. Here, we show RIS functions additionally as a locomotion stop neuron. RIS optogenetic stimulation caused acute and persistent inhibition of locomotion and pharyngeal pumping, phenotypes requiring FLP-11 neuropeptides and GABA. RIS photoactivation allows the animal to maintain its body posture by sustaining muscle tone, yet inactivating motor neuron oscillatory activity. During locomotion, RIS axonal Ca2+ signals revealed functional compartmentalization: Activity in the nerve ring process correlated with locomotion stop, while activity in a branch correlated with induced reversals. GABA was required to induce, and FLP-11 neuropeptides were required to sustain locomotion stop. RIS attenuates neuronal activity and inhibits movement, possibly enabling sensory integration and decision making, and exemplifies dual use of one cell across development in a compact nervous system. KW - cellular neuroscience KW - neural circuits Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223273 VL - 10 ER - TY - JOUR A1 - Carradec, Quentin A1 - Pelletier, Eric A1 - Da Silva, Corinne A1 - Alberti, Adriana A1 - Seeleuthner, Yoann A1 - Blanc-Mathieu, Romain A1 - Lima-Mendez, Gipsi A1 - Rocha, Fabio A1 - Tirichine, Leila A1 - Labadie, Karine A1 - Kirilovsky, Amos A1 - Bertrand, Alexis A1 - Engelen, Stefan A1 - Madoui, Mohammed-Amin A1 - Méheust, Raphaël A1 - Poulain, Julie A1 - Romac, Sarah A1 - Richter, Daniel J. A1 - Yoshikawa, Genki A1 - Dimier, Céline A1 - Kandels-Lewis, Stefanie A1 - Picheral, Marc A1 - Searson, Sarah A1 - Jaillon, Olivier A1 - Aury, Jean-Marc A1 - Karsenti, Eric A1 - Sullivan, Matthew B. A1 - Sunagawa, Shinichi A1 - Bork, Peer A1 - Not, Fabrice A1 - Hingamp, Pascal A1 - Raes, Jeroen A1 - Guidi, Lionel A1 - Ogata, Hiroyuki A1 - de Vargas, Colomban A1 - Iudicone, Daniele A1 - Bowler, Chris A1 - Wincker, Patrick T1 - A global ocean atlas of eukaryotic gene JF - Nature Communications N2 - While our knowledge about the roles of microbes and viruses in the ocean has increased tremendously due to recent advances in genomics and metagenomics, research on marine microbial eukaryotes and zooplankton has benefited much less from these new technologies because of their larger genomes, their enormous diversity, and largely unexplored physiologies. Here, we use a metatranscriptomics approach to capture expressed genes in open ocean Tara Oceans stations across four organismal size fractions. The individual sequence reads cluster into 116 million unigenes representing the largest reference collection of eukaryotic transcripts from any single biome. The catalog is used to unveil functions expressed by eukaryotic marine plankton, and to assess their functional biogeography. Almost half of the sequences have no similarity with known proteins, and a great number belong to new gene families with a restricted distribution in the ocean. Overall, the resource provides the foundations for exploring the roles of marine eukaryotes in ocean ecology and biogeochemistry. KW - genomics KW - marine biology KW - microbial ecology KW - water microbiology Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222250 VL - 9 ER - TY - JOUR A1 - Brunk, Michael A1 - Sputh, Sebastian A1 - Doose, Sören A1 - van de Linde, Sebastian A1 - Terpitz, Ulrich T1 - HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi JF - Scientific Reports N2 - The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains. KW - bioinformatics KW - cell growth KW - fungal biology KW - microscopy Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221691 VL - 8 ER - TY - JOUR A1 - Dammert, Marcel A. A1 - Brägelmann, Johannes A1 - Olsen, Rachelle R. A1 - Böhm, Stefanie A1 - Monhasery, Niloufar A1 - Whitney, Christopher P. A1 - Chalishazar, Milind D. A1 - Tumbrink, Hannah L. A1 - Guthrie, Matthew R. A1 - Klein, Sebastian A1 - Ireland, Abbie S. A1 - Ryan, Jeremy A1 - Schmitt, Anna A1 - Marx, Annika A1 - Ozretić, Luka A1 - Castiglione, Roberta A1 - Lorenz, Carina A1 - Jachimowicz, Ron D. A1 - Wolf, Elmar A1 - Thomas, Roman K. A1 - Poirier, John T. A1 - Büttner, Reinhard A1 - Sen, Triparna A1 - Byers, Lauren A. A1 - Reinhardt, H. Christian A1 - Letai, Anthony A1 - Oliver, Trudy G. A1 - Sos, Martin L. T1 - MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer JF - Nature Communications N2 - MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients. KW - genetic engineering KW - oncogenes KW - small-cell lung cancer KW - targeted therapies Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223569 VL - 10 ER - TY - JOUR A1 - Krah, Franz-Sebastian A1 - Büntgen, Ulf A1 - Schaefer, Hanno A1 - Müller, Jörg A1 - Andrew, Carrie A1 - Boddy, Lynne A1 - Diez, Jeffrey A1 - Egli, Simon A1 - Freckleton, Robert A1 - Gange, Alan C. A1 - Halvorsen, Rune A1 - Heegaard, Einar A1 - Heideroth, Antje A1 - Heibl, Christoph A1 - Heilmann-Clausen, Jacob A1 - Høiland, Klaus A1 - Kar, Ritwika A1 - Kauserud, Håvard A1 - Kirk, Paul M. A1 - Kuyper, Thomas W. A1 - Krisai-Greilhuber, Irmgard A1 - Norden, Jenni A1 - Papastefanou, Phillip A1 - Senn-Irlet, Beatrice A1 - Bässler, Claus T1 - European mushroom assemblages are darker in cold climates JF - Nature Communications N2 - Thermal melanism theory states that dark-colored ectotherm organisms are at an advantage at low temperature due to increased warming. This theory is generally supported for ectotherm animals, however, the function of colors in the fungal kingdom is largely unknown. Here, we test whether the color lightness of mushroom assemblages is related to climate using a dataset of 3.2 million observations of 3,054 species across Europe. Consistent with the thermal melanism theory, mushroom assemblages are significantly darker in areas with cold climates. We further show differences in color phenotype between fungal lifestyles and a lifestyle differentiated response to seasonality. These results indicate a more complex ecological role of mushroom colors and suggest functions beyond thermal adaption. Because fungi play a crucial role in terrestrial carbon and nutrient cycles, understanding the links between the thermal environment, functional coloration and species’ geographical distributions will be critical in predicting ecosystem responses to global warming. KW - evolutionary ecology KW - fungal ecology KW - fungal evolution KW - macroecology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224815 VL - 10 ER - TY - JOUR A1 - Milanese, Alessio A1 - Mende, Daniel R A1 - Paoli, Lucas A1 - Salazar, Guillem A1 - Ruscheweyh, Hans-Joachim A1 - Cuenca, Miguelangel A1 - Hingamp, Pascal A1 - Alves, Renato A1 - Costea, Paul I A1 - Coelho, Luis Pedro A1 - Schmidt, Thomas S. B. A1 - Almeida, Alexandre A1 - Mitchell, Alex L A1 - Finn, Robert D. A1 - Huerta-Cepas, Jaime A1 - Bork, Peer A1 - Zeller, Georg A1 - Sunagawa, Shinichi T1 - Microbial abundance, activity and population genomic profiling with mOTUs2 JF - Nature Communications N2 - Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites). KW - microbiome KW - software Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224089 VL - 10 ER - TY - THES A1 - Schwebs, Marie T1 - Structure and dynamics of the plasma membrane: a single-molecule study in \(Trypanosoma\) \(brucei\) T1 - Die Struktur und Dynamik der Plasmamembran: eine Einzelmolekülstudie in \(Trypanosoma\) \(brucei\) N2 - The unicellular, flagellated parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and nagana in livestock. In the last decades, it has become an established eukaryotic model organism in the field of biology, as well as in the interdisciplinary field of biophysics. For instance, the dense variant surface glycoprotein (VSG) coat offers the possibility to study the dynamics of GPI-anchored proteins in the plasma membrane of living cells. The fluidity of the VSG coat is not only an interesting object of study for its own sake, but is critically important for the survival of the parasite in the mammalian host. In order to maintain the integrity of the coat, the entire VSG coat is recycled within a few minutes. This is surprisingly fast for a purely diffusive process with the flagellar pocket (FP) as the sole site for endo- and exocytosis. Previous studies characterising VSG dynamics using FRAP reported diffusion coefficients that were not sufficient to to enable fast turnover based on passive VSG randomisation on the trypanosome surface. In this thesis, live-cell single-molecule fluorescence microscopy (SMFM) was employed to elucidate whether VSG diffusion coefficients were priorly underestimated or whether directed forces could be involved to bias VSGs towards the entrance of the FP. Embedding the highly motile trypanosomes in thermo-stable hydrogels facilitated the investigation of VSG dynamics on living trypanosomes at the mammalian host's temperature of 37°C. To allow for a spatial correlation of the VSG dynamics to the FP entrance, a cell line was employed harbouring a fluorescently labelled structure as a reference. Sequential two-colour SMFM was then established to allow for recording and registration of the dynamic and static single-molecule information. In order to characterise VSG dynamics, an algorithm to obtain reliable information from short trajectories was adapted (shortTrAn). It allowed for the quantification of the local dynamics in two distinct scenarios: diffusion and directed motion. The adaptation of the algorithm to the VSG data sets required the introduction of an additional projection filter. The algorithm was further extended to take into account the localisation errors inherent to single-particle tracking. The results of the quantification of diffusion and directed motion were presented in maps of the trypanosome surface, including an outline generated from a super-resolved static structure as a reference. Information on diffusion was displayed in one map, an ellipse plot. The colour code represented the local diffusion coefficient, while the shape of the ellipses provided an indication of the diffusion behaviour (aniso- or isotropic diffusion). The eccentricity of the ellipses was used to quantify deviations from isotropic diffusion. Information on directed motion was shown in three maps: A velocity map, representing the amplitude of the local velocities in a colour code. A quiver plot, illustrating the orientation of directed motion, and a third map which indicated the relative standard error of the local velocities colour-coded. Finally, a guideline based on random walk simulations was used to identify which of the two motion scenarios dominated locally. Application of the guideline to the VSG dynamics analysed by shortTrAn yielded supermaps that showed the locally dominant motion mode colour-coded. I found that VSG dynamics are dominated by diffusion, but several times faster than previously determined. The diffusion behaviour was additionally characterised by spatial heterogeneity. Moreover, isolated regions exhibiting the characteristics of round and elongated traps were observed on the cell surface. Additionally, VSG dynamics were studied with respect to the entrance of the FP. VSG dynamics in this region displayed similar characteristics compared to the remainder of the cell surface and forces biasing VSGs into the FP were not found. Furthermore, I investigated a potential interference of the attachment of the cytoskeleton to the plasma membrane with the dynamics of VSGs which are anchored to the outer leaflet of the membrane. Preliminary experiments were conducted on osmotically swollen trypanosomes and trypanosomes depleted for a microtubule-associated protein anchoring the subpellicular microtubule cytoskeleton to the plasma membrane. The measurements revealed a trend that detachment of the cytoskeleton could be associated with a reduction in the VSG diffusion coefficient and a loss of elongated traps. The latter could be an indication that these isolated regions were caused by underlying structures associated with the cytoskeleton. The measurements on cells with an intact cytoskeleton were complemented by random walk simulations of VSG dynamics with the newly determined diffusion coefficient on long time scales not accessible in experiments. Simulations showed that passive VSG randomisation is fast enough to allow for a turnover of the full VSG coat within a few minutes. According to an estimate based on the known rate of endocytosis and the newly determined VSG diffusion coefficient, the majority of exocytosed VSGs could escape from the FP to the cell surface without being immediately re-endocytosed. N2 - Der einzellige, begeißelte Parasit Trypanosoma brucei ist der Erreger der humanen Afrikanischen Schlafkrankheit und Nagana bei Nutztieren. In den vergangenen Jahrzehnten hat er sich sowohl in der Biologie als auch im interdisziplinären Bereich der Biophysik als eukaryotischer Modellorganismus etabliert. So bietet der dichte variant surface glycoprotein (VSG) Mantel beispielsweise die Möglichkeit, die Dynamik von GPI-verankerten Proteinen in der Plasmamembran von lebenden Zellen zu untersuchen. Die Fluidität des VSG-Mantels ist nicht nur um ihrer selbst Willen ein interessantes Studienobjekt, sondern auch von entscheidender Bedeutung für das Überleben des Parasiten im Säugetierwirt. Damit die Integrität des Mantels erhalten bleibt, wird der gesamte VSG Mantel kontinuierlich innerhalb weniger Minuten ausgetauscht. Dies ist erstaunlich schnell für einen rein diffusiven Prozess, bei welchem die Geißeltasche (GT) der einzige Ort für Endo- und Exozytose ist. Bisherige Studien zur Charakterisierung der VSG Dynamik mit FRAP ermittelten Diffusionskoeffizienten, welche nicht ausreichten, um einen schnellen Austausch durch eine passive Randomisierung der VSG auf der Trypanosomenoberfläche zu ermöglichen. In dieser Arbeit wurde die Einzelmolekül-Fluoreszenzmikroskopie (EMFM) an lebenden Zellen eingesetzt, um herauszufinden, ob die VSG Diffusionskoeffizienten zuvor unterschätzt wurden oder ob gerichtete Kräfte beteiligt sein könnten, um VSGs zum Eingang der GT zu leiten. Die Einbettung der hochmotilen Trypanosomen in thermostabilen Hydrogelen erlaubte die Analyse der VSG Dynamik auf lebenden Trypanosomen bei einer Temperatur des Säugetierwirts von 37°C. Um eine räumliche Korrelation der VSG Dynamik mit dem Eingang zur GT zu ermöglichen, wurde eine Zelllinie verwendet, die eine fluoreszenzmarkierte Struktur als Referenz besaß. Anschließend wurde die sequenzielle EMFM in zwei Farben etabliert, um sowohl die Aufzeichnung als auch die Registrierung der dynamischen und statischen Einzelmolekülinformationen zu gewährleisten. Um die VSG Dynamik zu charakterisieren, wurde ein Algorithmus zur Gewinnung von zuverlässigen Informationen aus kurzen Trajektorien adaptiert (shortTrAn). Dieser ließ die Quantifizierung der lokalen Dynamik anhand zweier unterschiedlicher Szenarien zu: Diffusion und gerichtete Bewegung. Die Anpassung des Algorithmus an die VSG Datensätze erforderte die Einführung eines zusätzlichen Projektionsfilters. Darüber hinaus wurde der Algorithmus erweitert, um die Lokalisierungsfehler zu berücksichtigen, die bei der Verfolgung von Einzelpartikeln unvermeidbar auftreten. Anschließend wurden die Ergebnisse der Quantifizierung von Diffusion und gerichteter Bewegung in Karten präsentiert, die die Trypanosomenoberfläche abbildeten, einschließlich eines Umrisses, der als Referenz aus einer hochaufgelösten statischen Struktur generiert wurde. Die Informationen zur Diffusion wurden in einer Karte, einem Ellipsenplot, dargestellt. Dabei repräsentierte eine Farbkodierung die lokalen Diffusionskoeffizienten, während die Form der Ellipsen einen Hinweis auf das Diffusionsverhalten (aniso- oder isotrope Diffusion) gab. Die Exzentrizität der Ellipsen wurde hierbei genutzt, um die Abweichung von isotroper Diffusion zu quantifizieren. Die Informationen zur gerichteten Bewegung wurden in drei Karten wiedergegeben: Eine Karte für die Geschwindigkeit zeigte die Amplitude der lokalen Geschwindigkeiten farbkodiert. Ein Köcherplot veranschaulichte die Richtung der Geschwindigkeit und eine dritte Karte zeigte den relativen Standardfehler der lokalen Geschwindigkeiten farblich kodiert an. Abschließend wurde ein auf Random-Walk-Simulationen basierender Leitfaden herangezogen, um zu entscheiden, welches der beiden Szenarien lokal dominierte. Die Anwendung des Leitfadens auf die mit shortTrAn analysierte VSG Dynamik ergab Übersichtskarten, in denen der lokal dominierende Bewegungsmodus farblich kodiert war. Ich konnte zeigen, dass die VSG Dynamik von der Diffusion dominiert wird. Jedoch war diese um ein Vielfaches schneller als bisher angenommen. Das Diffusionsverhalten war zudem durch eine räumliche Heterogenität charakterisiert. Des Weiteren wurden auf der Zelloberfläche isolierte Regionen beobachtet, die die Eigenschaften von runden und länglichen Fallen aufwiesen. Zusätzlich wurde die VSG Dynamik in Bezug auf den Eingang der GT untersucht. Die VSG Dynamik in dieser Region wies ähnliche Kennwerte auf wie die restliche Zelloberfläche, und es konnten keine Kräfte festgestellt werden, welche die VSGs in die GT dirigieren. Des Weiteren habe ich den potenziellen Einfluss der Verankerung des Zytoskeletts an der Plasmamembran auf die Dynamik der VSGs untersucht, die in der äußeren Membranschicht verankert sind. Hierzu wurden vorläufige Experimente auf osmotisch geschwollenen Trypanosomen und Trypanosomen durchgeführt, denen ein Mikrotubuli assoziiertes Protein fehlte, welches das subpellikuläre Mikrotubuli-Zytoskelett an der Plasmamembran verankert. Bei den Messungen wurde ein Trend festgestellt, wonach die Ablösung des Zytoskeletts mit einer Verringerung des VSG Diffusionskoeffizienten und dem Verlust der länglichen Fallen korrelieren könnte. Letzteres könnte ein Hinweis darauf sein, dass diese isolierten Regionen durch darunter liegende, mit dem Zytoskelett verbundene Strukturen verursacht wurden. Die Messungen auf Zellen mit intaktem Zytoskelett wurden durch Random-Walk-Simulationen von VSG Trajektorien mit dem neu ermittelten Diffusionskoeffizienten auf langen, experimentell nicht zugänglichen Zeitskalen ergänzt. Die Simulationen zeigten, dass die passive Randomisierung der VSGs schnell genug ist, um einen Austausch des gesamten VSG Mantels innerhalb weniger Minuten zu ermöglichen. Einer Schätzung zufolge, die auf der bekannten Endozytoserate und dem neu ermittelten VSG Diffusionskoeffizienten basierte, könnte der Großteil der exozytierten VSGs aus der GT zur Zelloberfläche gelangen, ohne unmittelbar wieder endozytiert zu werden. KW - Trypanosoma brucei KW - Einzelmolekülmikroskopie KW - Membranproteine KW - Diffusionskoeffizient KW - Single-molecule fluorescence microscopy KW - Single-molecule tracking KW - Variant surface glycoprotein KW - GPI-anchored protein KW - Diffusion coefficient KW - Zellskelett KW - Zytoskelett Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275699 ER - TY - JOUR A1 - Prusty, Bhupesh K. A1 - Gulve, Nitish A1 - Govind, Sheila A1 - Krueger, Gerhard R. F. A1 - Feichtinger, Julia A1 - Larcombe, Lee A1 - Aspinall, Richard A1 - Ablashi, Dharam V. A1 - Toro, Carla T. T1 - Active HHV-6 Infection of Cerebellar Purkinje Cells in Mood Disorders JF - Frontiers in Microbiology N2 - Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD. KW - HHV-6 KW - bipolar disorder KW - schizophrenia KW - major depressive disorder KW - Purkinje cells Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369222 VL - 9 ER - TY - THES A1 - Amini, Emad T1 - How central and peripheral clocks and the neuroendocrine system interact to time eclosion behavior in \(Drosophila\) \(melanogaster\) T1 - Wie zentrale und periphere Uhren und das neuroendokrine System zusammenwirken, um das Schlupfverhalten von \(Drosophila\) \(melanogaster\) zeitlich festzulegen N2 - To grow larger, insects must shed their old rigid exoskeleton and replace it with a new one. This process is called molting and the motor behavior that sheds the old cuticle is called ecdysis. Holometabolic insects have pupal stages in between their larval and adult forms, during which they perform metamorphosis. The pupal stage ends with eclosion, i.e., the emergence of the adult from the pupal shell. Insects typically eclose at a specific time during the day, likely when abiotic conditions are at their optimum. A newly eclosed insect is fragile and needs time to harden its exoskeleton. Hence, eclosion is regulated by sophisticated developmental and circadian timing mechanisms. In Drosophila melanogaster, eclosion is limited to a daily time window in the morning, regarded as the “eclosion gate”. In a population of laboratory flies entrained by light/dark cycles, most of the flies eclose around lights on. This rhythmic eclosion pattern is controlled by the circadian clock and persists even under constant conditions. Developmental timing is under the control of complex hormonal signaling, including the steroid ecdysone, insulin-like peptides, and prothoracicotropic hormone (PTTH). The interactions of the central circadian clock in the brain and a peripheral clock in the prothoracic gland (PG) that produces ecdysone are important for the circadian timing of eclosion. These two clocks are connected by a bilateral pair of peptidergic PTTH neurons (PTTHn) that project to the PG. Before each molt, the ecdysone level rises and then falls shortly before ecdysis. The falling ecdysone level must fall below a certain threshold value for the eclosion gate to open. The activity of PTTHn is inhibited by short neuropeptide F (sNPF) from the small ventrolateral neurons (sLNvs) and inhibition is thought to lead to a decrease in ecdysone production. The general aim of this thesis is to further the understanding of how the circadian clock and neuroendocrinal pathways are coordinated to drive eclosion rhythmicity and to identify when these endocrinal signaling pathways are active. In Chapter I, a series of conditional PTTHn silencing-based behavioral assays, combined with neuronal activity imaging techniques such as non-invasive ARG-Luc show that PTTH signaling is active and required shortly before eclosion and may serve to phase-adjust the activity of the PG at the end of pupal development. Trans-synaptic anatomical stainings identified the sLNvs, dorsal neurons 1 (DN1), dorsal neurons 2 (DN2), and lateral posterior neurons (LPNs) clock neurons as directly upstream of the PTTHn. Eclosion motor behavior is initiated by Ecdysis triggering hormone (ETH) which activates a pair of ventromedial (Vm) neurons to release eclosion hormone (EH) which positively feeds back to the source of ETH, the endocrine Inka cells. In Chapter II trans-synaptic tracing showed that most clock neurons provide input to the Vm and non-canonical EH neurons. Hence, clock can potentially influence the ETH/EH feedback loop. The activity profile of the Inka cells and Vm neurons before eclosion is described. Vm and Inka cells are active around seven hours before eclosion. Interestingly, all EH neurons appear to be exclusively peptidergic. In Chapter III, using chemoconnectomics, PTTHns were found to express receptors for sNPF, allatostatin A (AstA), allatostatin C (AstC), and myosuppressin (Ms), while EH neurons expressed only Ms and AstA receptors. Eclosion assays of flies with impaired AstA, AstC, or Ms signaling do not show arrhythmicity under constant conditions. However, optogenetic activation of the AstA neurons strongly suppresses eclosion. Chapter IV focuses on peripheral ventral’ Tracheal dendrite (v’Td) and class IV dendritic arborization (C4da) neurons. The C4da neurons mediate larval light avoidance through endocrine PTTH signaling. The v’Td neurons mainly receive O2/CO2 input from the trachea and are upstream of Vm neurons but are not required for eclosion rhythmicity. Conditional ablation of the C4da neurons or torso (receptor of PTTH) knock-out in the C4da neurons impaired eclosion rhythmicity. Six to seven hours before eclosion, PTTHn, C4da, and Vm neurons are active based on ARG-Luc imaging. Thus, C4da neurons may indirectly connect the PTTHn to the Vm neurons. In summary, this thesis advances our knowledge of the temporal activity and role of PTTH signaling during pupal development and rhythmic eclosion. It further provides a comprehensive characterization of the synaptic and peptidergic inputs from clock neurons to PTTHn and EH neurons. AstA, AstC, and Ms are identified as potential modulators of eclosion circuits and suggest an indirect effect of PTTH signaling on EH signaling via the peripheral sensory C4da neurons. N2 - Um zu wachsen, müssen Insekten ihr altes, starres Exoskelett abwerfen und durch ein neues ersetzen. Dieser Vorgang wird als Häutung bezeichnet, und das motorische Verhalten, bei dem die alte Kutikula abgestoßen wird, heißt Ekdysis. Holometabole Insekten haben zwischen ihrer Larven- und Erwachsenenform ein Puppenstadium, in welchem sie eine Metamorphose durchlaufen. Das Puppenstadium endet mit dem Schlüpfen des erwachsenen Tieres aus der Puppenhülle. Die Insekten schlüpfen in der Regel zu einem bestimmten Zeitpunkt am Tag, wenn die abiotischen Bedingungen optimal sind, da das frisch geschlüpfte Insekt zerbrechlich ist und Zeit braucht, um sein Exoskelett auszuhärten. Daher wird der Schlupf durch ausgeklügelte Mechanismen der Entwicklung und der inneren Uhr gesteuert. Bei Drosophila melanogaster ist der Sclupf auf ein tägliches Zeitfenster am Morgen beschränkt, das als "Schlupffenster" bezeichnet wird. In einer Population von Laborfliegen, die durch Licht/Dunkel-Zyklen gesteuert wird, schlüpfen die meisten Fliegen in etwa um das Einschalten der Beleuchtung. Dieses rhythmische Schlupfmuster wird von der inneren Uhr gesteuert und bleibt auch unter konstanten Bedingungen bestehen. Das Timing der Entwicklung wird von komplexen hormonellen Signalen gesteuert, darunter das Steroid Ecdyson, insulinähnliche Peptide und das prothorakotrope Hormon (PTTH). Die Wechselwirkungen zwischen der zentralen zirkadianen Uhr im Gehirn und einer peripheren Uhr in der Prothorakaldrüse (PG), die Ecdyson produziert, sind wichtig für die zirkadiane Zeitsteuerung des Schlupfs. Diese beiden Uhren sind durch ein bilaterales Paar peptiderger PTTH-Neuronen (PTTHn) verbunden, die in die PG projizieren. Vor jeder Häutung steigt der Ecdysonspiegel an und fällt dann kurz vor danach wieder ab. Der fallende Ecdysonspiegel muss einen bestimmten Schwellenwert unterschreiten, damit sich das Schlupffenster öffnen kann. Die Aktivität der PTTHn wird durch das kurze Neuropeptid F (sNPF) aus den kleinen ventrolateralen Neuronen (sLNvs) gehemmt, und es wird angenommen, dass die Hemmung zu einer Abnahme der Ecdysonproduktion führt. Das allgemeine Ziel dieser Thesis besteht darin, die Koordination zwischen der zirkadianen Uhr und den neuroendokrinen Signalwegen zur Steuerung der Eklosionsrhythmik weiter zu charakterisieren und zu ermitteln, wann diese endokrinen Signalwege aktiv sind. In Kapitel I zeigen eine Reihe von Verhaltenstests, die auf der konditionalen Ausschaltung von PTTHn basieren, in Kombination mit Techniken zur Darstellung neuronaler Aktivität, wie z. B. nicht-invasives ARG-Luc imaging, dass PTTH-Signale kurz vor dem Schlupf aktiv und erforderlich sind und zur Phasenanpassung der Aktivität der PG am Ende der Puppenentwicklung dienen könnten. Trans-synaptische anatomische Färbungen identifizierten die sLNvs, die dorsalen Neuronen 1 (DN1), die dorsalen Neuronen 2 (DN2) und die lateralen posterioren Neuronen (LPNs) als Uhrneuronen, die dem PTTHn direkt vorgeschaltet sind. Das motorische Schlupfverhalten wird durch das Ecdysis-auslösende Hormon (ETH) ausgelöst, das ein Paar ventromedialer (Vm) Neuronen zur Freisetzung des Eklosionshormons (EH) anregt, welches positiv an die Quelle des ETH, die endokrinen Inka-Zellen, zurückkoppelt. In Kapitel II zeigte die trans-synaptische Nachverfolgung, dass die meisten Uhrneuronen Input für die Vm- und nicht-kanonischen EH-Neuronen liefern, sodass die Uhr möglicherweise die ETH/EH-Rückkopplungsschleife beeinflussen kann. Das Aktivitätsprofil der Inka-Zellen und Vm-Neuronen vor dem Schlupf wird beschrieben. Vm- und Inka-Zellen sind etwa sieben Stunden vor dem Schlupf aktiv. Interessanterweise scheinen alle EH-Neuronen ausschließlich peptiderg zu sein. In Kapitel III wurde mit Hilfe von Chemoconnectomics festgestellt, dass PTTH-Neuronen Rezeptoren für sNPF, Allatostatin A (AstA), Allatostatin C (AstC) und Myosuppressin (Ms) exprimieren, während EH nur Ms- und AstA-Rezeptoren exprimieren. Eklosionsversuche mit Fliegen, deren AstA-, AstC- oder Ms-Signalübertragung beeinträchtigt ist, zeigen unter konstanten Bedingungen keine Arrhythmie. Eine optogenetische Aktivierung der AstA-Neuronen führt jedoch zu einer starken Unterdrückung des Schlupfs. Kapitel IV konzentriert sich auf die peripheren ventralen Trachealdendritischen Neurone (v'Td) und dendritische Verzweigungsneurone der Klasse IV (C4da). Die C4da-Neuronen vermitteln die Lichtvermeidung der Larven durch endokrine PTTH-Signale. Die v'Td-Neuronen erhalten hauptsächlich O2/CO2-Input aus den Tracheen und sind den Vm-Neuronen vorgeschaltet, werden aber für die Schlupfrhythmik nicht benötigt. Die bedingte Ablation der C4da-Neuronen und das Knock-out von torso (Rezeptor für PTTH) in den C4da-Neuronen beeinträchtigten die Schlupfrhythmik. Sechs bis sieben Stunden vor dem Schlupf sind die PTTHn-, C4da- und Vm-Neuronen aktiv. Somit könnten C4da-Neuronen indirekt die PTTHn mit den Vm-Neuronen verbinden. Zusammenfassend lässt sich sagen, dass diese Arbeit unser Wissen über das zeitliche Aktivitätsmuster und der Rolle des PTTH signalling während der Puppenentwicklung und dem rhythmisches Schlupf erweitert. Sie liefert auch eine umfassende Charakterisierung der synaptischen und peptidergen Eingänge von Uhrneuronen zu PTTHn- und EH-Neuronen. AstA, AstC und Ms wurden als potenzielle Modulatoren der neuronalen Schlupfschaltkreise identifiziert und deuten auf einen indirekten Effekt der PTTH-Signalgebung auf das EH signalling über die peripheren sensorischen C4da-Neuronen hin. KW - Prothoracicotropic hormone KW - Prothoracic gland KW - Eclosion KW - Eclosion hormone KW - C4da KW - v’Td KW - Neuropeptide KW - Neuroendokrines System KW - Taufliege Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-361309 ER - TY - THES A1 - Dehmer, Markus T1 - A novel USP11-TCEAL1-mediated mechanism protects transcriptional elongation by RNA Polymerase II T1 - Ein neuer USP11-TCEAL1 vermittelter Mechanismus schützt die transkriptionelle Elongation der RNA Polymerase II N2 - Deregulated expression of MYC oncoproteins is a driving event in many human cancers. Therefore, understanding and targeting MYC protein-driven mechanisms in tumor biology remain a major challenge. Oncogenic transcription in MYCN-amplified neuroblastoma leads to the formation of the MYCN-BRCA1-USP11 complex that terminates transcription by evicting stalling RNAPII from chromatin. This reduces cellular stress and allows reinitiation of new rounds of transcription. Basically, tumors with amplified MYC genes have a high demand on well orchestration of transcriptional processes-dependent and independent from MYC proteins functions in gene regulation. To date, the cooperation between promoter-proximal termination and transcriptional elongation in cancer cells remains still incomplete in its understanding. In this study the putative role of the dubiquitinase Ubiquitin Specific Protease 11 (USP11) in transcription regulation was further investigated. First, several USP11 interaction partners involved in transcriptional regulation in neuroblastoma cancer cells were identified. In particular, the transcription elongation factor A like 1 (TCEAL1) protein, which assists USP11 to engage protein-protein interactions in a MYCN-dependent manner, was characterized. The data clearly show that TCEAL1 acts as a pro-transcriptional factor for RNA polymerase II (RNAPII)-medi- ated transcription. In detail, TCEAL1 controls the transcription factor S-II (TFIIS), a factor that assists RNAPII to escape from paused sites. The findings claim that TCEAL1 outcompetes the transcription elongation factor TFIIS in a non-catalytic manner on chromatin of highly expressed genes. This is reasoned by the need regulating TFIIS function in transcription. TCEAL1 equili- brates excessive backtracking and premature termination of transcription caused by TFIIS. Collectively, the work shed light on the stoichiometric control of TFIIS demand in transcriptional regulation via the USP11-TCEAL1-USP7 complex. This complex protects RNAPII from TFIIS-mediated termination helping to regulate productive transcription of highly active genes in neuroblastoma. N2 - Die deregulierte Expression von MYC Onkoproteinen ist ein zentrales Event in vielen huma-nen Krebsarten. Aus diesem Grund sind das Verständnis und die gezielte Bekämpfung MYC-getriebener Mechanismen in der Tumorbiologie nach wie vor eine große Herausforderung. In MYCN-amplifizierten Neuroblastomen führt eine übermäßig hohe Transkriptionsrate zur stress-bedingten Rekrutierung des MYCN-BRCA1-USP11-Komplexes. Dieser Komplex be-endet vorzeitig die Transkription, indem er RNAPII Moleküle vom Chromatin wirft. Durch diesen Mechanismus wird zellulärer Stress reduziert und ermöglicht dadurch einen erneuten Start der Transkription. Grundsätzlich stellen Tumoren mit einer Amplifikation von einem der MYC Proteine hohe Anforderungen an eine feine Abstimmung der einzelnen Schritte in der Transkription. Dies ist sowohl abhängig als auch unabhängig von den bereits beschriebe-nen Funktionen der MYC-Proteine in der Genregulation. Bis heute ist das Zusammenspiel zwischen promoter-proximaler Termination und transkriptioneller Elongation noch nicht vollständig aufgeklärt. In dieser Studie wurde eine potenzielle Rolle von USP11 in der Regulation der Transkription weitergehend untersucht. Zunächst wurden mehrere Interaktionspartner von USP11, die an der Regulation der Transkription in Neuroblastom Krebszellen beteiligt sind, identifiziert. Es wurde insbesondere das Transcription Elongation Factor A Like 1 (TCEAL1) Protein charak-terisiert. Dieses Protein unterstützt USP11 dabei, Protein-Protein-Interaktionen MYCN-vermittelt einzugehen. Die Daten zeigen, dass TCEAL1 als pro-transkriptioneller Faktor für die RNA-Polymerase II (RNAPII) -vermittelte Transkription fungiert. Genauer, TCEAL1 kontrolliert den Transkriptionsfaktor S-II (TFIIS), einen Faktor, der der RNAPII dabei hilft, die Transkription nach einem kurzen Pausieren („pausing“) fortzusetzen. Die Ergebnisse zei-gen, dass TCEAL1 den Elongationsfaktor TFIIS auf nicht-katalytische Weise von dem Chromatin von hochexprimierten Genen verdrängt. Dies ist darin begründet, dass die Funkti-on von TFIIS bei der Transkription reguliert werden muss. TCEAL1 gleicht übermäßiges Zurückwandern der RNAPII und die vorzeitige Beendigung der Transkription, das durch TFIIS vermittelt wird, aus. Diese Arbeit gibt Aufschluss über die stöchiometrische Kontrolle des TFIIS-Bedarfs bei der Transkriptionsregulation durch den USP11-TCEAL1-USP7-Komplex. Dieser Komplex schützt die RNAPII vor der TFIIS-vermittelter Termination der Transkription und trägt zur Regulierung einer produktiven Transkription hochaktiver Gene im Neuroblastom bei. KW - Transkription KW - N-Myc KW - Transcription Regulation KW - Pause Release KW - Ubiquitin Specific Protease 11 KW - transcription elongation factor A (SII)-like 1 (TCEAL1) KW - RNA Polymerase II (RNAPII) KW - Transcriptional Stress Response Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-360544 ER - TY - THES A1 - Adhikari, Bikash T1 - Targeted degradation of Myc-interacting oncoproteins T1 - Gezielte Degradation von mit Myc interagierenden Onkoproteinen N2 - The hallmark oncoprotein Myc is a major driver of tumorigenesis in various human cancer entities. However, Myc’s structural features make it challenging to develop small molecules against it. A promising strategy to indirectly inhibit the function of Myc is by targeting its interactors. Many Myc-interacting proteins have reported scaffolding functions which are difficult to target using conventional occupancy- driven inhibitors. Thus, in this thesis, the proteolysis targeting chimera (PROTAC) approach was used to target two oncoproteins interacting with Myc which promote the oncogenicity of Myc, Aurora-A and WDR5. PROTACs are bifunctional small molecules that bind to the target protein with one ligand and recruit a cellular E3- ligase with the other ligand to induce target degradation via the ubiquitin- proteasome system. So far, the most widely used E3-ligases for PROTAC development are Cereblon (CRBN) and von Hippel–Lindau tumor suppressor (VHL). Furthermore, there are cases of incompatibility between some E3-ligases and proteins to bring about degradation. Hence there is a need to explore new E3- ligases and a demand for a tool to predict degradative E3-ligases for the target protein in the PROTAC field. In the first part, a highly specific mitotic kinase Aurora-A degrader, JB170, was developed. This compound utilized Aurora-A inhibitor alisertib as the target ligand and thalidomide as the E3-ligase CRBN harness. The specificity of JB170 and the ternary complex formation was supported by the interactions between Aurora-A and CRBN. The PROTAC-mediated degradation of Aurora-A induced a distinct S- phase defect rather than mitotic arrest, shown by its catalytic inhibition. The finding demonstrates that Aurora-A has a non-catalytic role in the S-phase. Furthermore, the degradation of Aurora-A led to apoptosis in various cancer cell lines. In the second part, two different series of WDR5 PROTACs based on two protein- protein inhibitors of WDR5 were evaluated. The most efficient degraders from both series recruited VHL as a E3-ligase and showed partial degradation of WDR5. In addition, the degradation efficiency of the PROTACs was significantly affected by the linker nature and length, highlighting the importance of linker length and composition in PROTAC design. The degraders showed modest proliferation defects at best in cancer cell lines. However, overexpression of VHL increased the degradation efficiency and the antiproliferative effect of the PROTACs. In the last part, a rapamycin-based assay was developed to predict the degradative E3-ligase for a target. The assay was validated using the WDR5/VHL and Aurora- A/CRBN pairs. The result that WDR5 is degraded by VHL but not CRBN and Aurora-A is degraded by CRBN, matches observations made with PROTACs. This technique will be used in the future to find effective tissue-specific and essential E3-ligases for targeted degradation of oncoproteins using PROTACs. Collectively, the work presented here provides a strategy to improve PROTAC development and a starting point for developing Aurora-A and WDR5 PROTACs for cancer therapy. N2 - Das Onkoprotein Myc ist ein wichtiger Faktor bei der Tumorentstehung in verschiedenen menschlichen Krebsarten. Die strukturellen Merkmale von Myc machen es jedoch schwierig, kleine Moleküle gegen dieses Protein zu entwickeln. Eine vielversprechende Strategie zur indirekten Hemmung der Funktion von Myc besteht darin, auf seine Interaktoren abzuzielen. Viele Proteine, die mit Myc interagieren, haben Gerüstfunktionen, die mit herkömmlichen Inhibitoren nur schwer zu hemmen sind. Daher wurde in dieser Arbeit der PROTAC-Ansatz (Proteolysis Targeting Chimera) verwendet, um zwei Onkoproteine, die mit Myc interagieren und die Onkogenität von Myc fördern, ins Visier zu nehmen: Aurora-A und WDR5. PROTACs sind bifunktionale kleine Moleküle, die mit einem Liganden an das Zielprotein binden und mit dem anderen Liganden eine zelluläre E3-Ligase rekrutieren, um den Abbau des Zielproteins über das Ubiquitin-Proteasom-System einzuleiten. Die bisher am häufigsten verwendeten E3-Ligasen für die Entwicklung von PROTACs sind Cereblon (CRBN) und der von Hippel-Lindau-Tumorsuppressor (VHL). Außerdem gibt es Fälle von Inkompatibilität zwischen einigen E3-Ligasen und Proteinen, die abgebaut werden sollen. Daher besteht die Notwendigkeit, neue E3-Ligasen zu erforschen und Werkzeuge zur Vorhersage abbauender E3-Ligasen für das Zielprotein zu entwickeln. Im ersten Teil wurde ein hochspezifischer Degrader der mitotischen Kinase Aurora-A, JB170, entwickelt. Bei dieser Verbindung wurde der Aurora-A-Inhibitor Alisertib als Zielligand und Thalidomid als Binder für die E3-Ligase CRBN verwendet. Die Spezifität von JB170 und die ternäre Komplexbildung wurden durch die Wechselwirkungen zwischen Aurora-A und CRBN unterstützt. Der durch PROTAC vermittelte Abbau von Aurora-A führte zu einem deutlichen Defekt in der S-Phase und nicht zu einem mitotischen Stillstand, wie es für dessen katalytische Hemmung beobachtet wurde. Dies zeigt, dass Aurora-A eine nicht-katalytische Funktion in der S-Phase hat. Außerdem führte der Abbau von Aurora-A in verschiedenen Krebszelllinien zur Apoptose. Im zweiten Teil wurden zwei verschiedene Serien von WDR5 PROTACs auf der Grundlage von zwei Protein-Protein-Inhibitoren von WDR5 untersucht. Die effizientesten Degrader aus beiden Serien rekrutierten VHL als E3-Ligase und zeigten einen teilweisen Abbau von WDR5. Darüber hinaus wurde die Abbaueffizienz der PROTACs erheblich von der Art und Länge des Linkers beeinflusst, was die Bedeutung der Linkerlänge und -zusammensetzung bei der Entwicklung von PROTACs unterstreicht. Die Abbauprodukte zeigten bestenfalls bescheidene Proliferationsdefekte in Krebszelllinien. Eine Überexpression von VHL erhöhte jedoch die Abbaueffizienz und den antiproliferativen Effekt der PROTACs. Im letzten Teil wurde ein auf Rapamycin basierender Assay entwickelt, um die abbauende E3-Ligase für ein Target vorherzusagen. Der Assay wurde anhand der Paare WDR5/VHL und Aurora-A/CRBN validiert. Das Ergebnis, dass WDR5 von VHL, aber nicht von CRBN abgebaut wird und Aurora-A von CRBN abgebaut wird, stimmt mit den Beobachtungen überein, die mit PROTACs gemacht wurden. Diese Technik wird in Zukunft eingesetzt werden, um wirksame gewebespezifische und essentielle E3-Ligasen für den gezielten Abbau von Onkoproteinen mit Hilfe von PROTACs zu finden. Insgesamt bieten die hier vorgestellten Arbeiten eine Strategie zur Verbesserung der PROTAC-Entwicklung und einen Ausgangspunkt für die Entwicklung von Aurora-A- und WDR5-PROTACs für die Krebstherapie. KW - Degradation KW - PROTACs KW - Oncoprotein KW - Cancer KW - Onkoprotein Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-317326 ER - TY - JOUR A1 - Snaebjornsson, Marteinn T A1 - Schulze, Almut T1 - Non-canonical functions of enzymes facilitate cross-talk between cell metabolic and regulatory pathways JF - Experimental & Molecular Medicine N2 - The metabolic rewiring that occurs during cell transformation is a hallmark of cancer. It is diverse in different cancers as it reflects different combinations of oncogenic drivers, tumor suppressors, and the microenvironment. Metabolic rewiring is essential to cancer as it enables uncontrolled proliferation and adaptation to the fluctuating availability of nutrients and oxygen caused by poor access to the vasculature due to tumor growth and a foreign microenvironment encountered during metastasis. Increasing evidence now indicates that the metabolic state in cancer cells also plays a causal role in tumor growth and metastasis, for example through the action of oncometabolites, which modulate cell signaling and epigenetic pathways to promote malignancy. In addition to altering the metabolic state in cancer cells, some multifunctional enzymes possess non-metabolic functions that also contribute to cell transformation. Some multifunctional enzymes that are highly expressed in cancer, such as pyruvate kinase M2 (PKM2), have non-canonical functions that are co-opted by oncogenic signaling to drive proliferation and inhibit apoptosis. Other multifunctional enzymes that are frequently downregulated in cancer, such as fructose-bisphosphatase 1 (FBP1), are tumor suppressors, directly opposing mitogenic signaling via their non-canonical functions. In some cases, the enzymatic and non-canonical roles of these enzymes are functionally linked, making the modulation of non-metabolic cellular processes dependent on the metabolic state of the cell. KW - cancer metabolism Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238763 VL - 50 ER - TY - JOUR A1 - Sommerfeld, Andreas A1 - Senf, Cornelius A1 - Buma, Brian A1 - D'Amato, Anthony W. A1 - Després, Tiphaine A1 - Díaz-Hormazábal, Ignacio A1 - Fraver, Shawn A1 - Frelich, Lee E. A1 - Gutiérrez, Álvaro G. A1 - Hart, Sarah J. A1 - Harvey, Brian J. A1 - He, Hong S. A1 - Hlásny, Tomáš A1 - Holz, Andrés A1 - Kitzberger, Thomas A1 - Kulakowski, Dominik A1 - Lindenmayer, David A1 - Mori, Akira S. A1 - Müller, Jörg A1 - Paritsis, Juan A1 - Perry, George L. W. A1 - Stephens, Scott L. A1 - Svoboda, Miroslav A1 - Turner, Monica G. A1 - Veblen, Thomas T. A1 - Seidl, Rupert T1 - Patterns and drivers of recent disturbances across the temperate forest biome JF - Nature Communications N2 - Increasing evidence indicates that forest disturbances are changing in response to global change, yet local variability in disturbance remains high. We quantified this considerable variability and analyzed whether recent disturbance episodes around the globe were consistently driven by climate, and if human influence modulates patterns of forest disturbance. We combined remote sensing data on recent (2001–2014) disturbances with in-depth local information for 50 protected landscapes and their surroundings across the temperate biome. Disturbance patterns are highly variable, and shaped by variation in disturbance agents and traits of prevailing tree species. However, high disturbance activity is consistently linked to warmer and drier than average conditions across the globe. Disturbances in protected areas are smaller and more complex in shape compared to their surroundings affected by human land use. This signal disappears in areas with high recent natural disturbance activity, underlining the potential of climate-mediated disturbance to transform forest landscapes. KW - forest ecology KW - forestry Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239157 VL - 9 ER - TY - JOUR A1 - Vujanić, Gordan M. A1 - Gessler, Manfred A1 - Ooms, Ariadne H. A. G. A1 - Collini, Paola A1 - Coulomb-l'Hermine, Aurore A1 - D'Hooghe, Ellen A1 - de Krijger, Ronald R. A1 - Perotti, Daniela A1 - Pritchard-Jones, Kathy A1 - Vokuhl, Christian A1 - van den Heuvel-Eibrink, Marry M. A1 - Graf, Norbert T1 - The UMBRELLA SIOP–RTSG 2016 Wilms tumour pathology and molecular biology protocol JF - Nature Reviews Urology N2 - On the basis of the results of previous national and international trials and studies, the Renal Tumour Study Group of the International Society of Paediatric Oncology (SIOP–RTSG) has developed a new study protocol for paediatric renal tumours: the UMBRELLA SIOP–RTSG 2016 protocol (the UMBRELLA protocol). Currently, the overall outcomes of patients with Wilms tumour are excellent, but subgroups with poor prognosis and increased relapse rates still exist. The identification of these subgroups is of utmost importance to improve treatment stratification, which might lead to reduction of the direct and late effects of chemotherapy. The UMBRELLA protocol aims to validate new prognostic factors, such as blastemal tumour volume and molecular markers, to further improve outcome. To achieve this aim, large, international, high-quality databases are needed, which dictate optimization and international harmonization of specimen handling and comprehensive sampling of biological material, refine definitions and improve logistics for expert review. To promote broad implementation of the UMBRELLA protocol, the updated SIOP–RTSG pathology and molecular biology protocol for Wilms tumours has been outlined, which is a consensus from the SIOP–RTSG pathology panel. KW - molecular biology KW - paediatric cancer KW - pathology KW - renal cancer Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233265 VL - 15 ER - TY - JOUR A1 - Woodcock, B. A. A1 - Garratt, M. P. D. A1 - Powney, G. D. A1 - Shaw, R. F. A1 - Osborne, J. L. A1 - Soroka, J. A1 - Lindström, S. A. M. A1 - Stanley, D. A1 - Ouvrard, P. A1 - Edwards, M. E. A1 - Jauker, F. A1 - McCracken, M. E. A1 - Zou, Y. A1 - Potts, S. G. A1 - Rundlöf, M. A1 - Noriega, J. A. A1 - Greenop, A. A1 - Smith, H. G. A1 - Bommarco, R. A1 - van der Werf, W. A1 - Stout, J. C. A1 - Steffan-Dewenter, I. A1 - Morandin, L. A1 - Bullock, J. M. A1 - Pywell, R. F. T1 - Meta-analysis reveals that pollinator functional diversity and abundance enhance crop pollination and yield JF - Nature Communications N2 - How insects promote crop pollination remains poorly understood in terms of the contribution of functional trait differences between species. We used meta-analyses to test for correlations between community abundance, species richness and functional trait metrics with oilseed rape yield, a globally important crop. While overall abundance is consistently important in predicting yield, functional divergence between species traits also showed a positive correlation. This result supports the complementarity hypothesis that pollination function is maintained by non-overlapping trait distributions. In artificially constructed communities (mesocosms), species richness is positively correlated with yield, although this effect is not seen under field conditions. As traits of the dominant species do not predict yield above that attributed to the effect of abundance alone, we find no evidence in support of the mass ratio hypothesis. Management practices increasing not just pollinator abundance, but also functional divergence, could benefit oilseed rape agriculture. KW - agroecology KW - agriculture KW - ecosystem services KW - environmental sciences Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233787 VL - 10 ER - TY - JOUR A1 - Wegert, Jenny A1 - Vokuhl, Christian A1 - Collord, Grace A1 - Del Castillo Velasco-Herrera, Martin A1 - Farndon, Sarah J. A1 - Guzzo, Charlotte A1 - Jorgensen, Mette A1 - Anderson, John A1 - Slater, Olga A1 - Duncan, Catriona A1 - Bausenwein, Sabrina A1 - Streitenberger, Heike A1 - Ziegler, Barbara A1 - Furtwängler, Rhoikos A1 - Graf, Norbert A1 - Stratton, Michael R. A1 - Campbell, Peter J. A1 - Jones, David TW A1 - Koelsche, Christian A1 - Pfister, Stefan M. A1 - Mifsud, William A1 - Sebire, Neil A1 - Sparber-Sauer, Monika A1 - Koscielniak, Ewa A1 - Rosenwald, Andreas A1 - Gessler, Manfred A1 - Behjati, Sam T1 - Recurrent intragenic rearrangements of EGFR and BRAF in soft tissue tumors of infants JF - Nature Communications N2 - Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors. KW - cancer KW - genetics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233446 VL - 9 ER - TY - JOUR A1 - Annunziata, Ida A1 - van de Vlekkert, Diantha A1 - Wolf, Elmar A1 - Finkelstein, David A1 - Neale, Geoffrey A1 - Machado, Eda A1 - Mosca, Rosario A1 - Campos, Yvan A1 - Tillman, Heather A1 - Roussel, Martine F. A1 - Weesner, Jason Andrew A1 - Fremuth, Leigh Ellen A1 - Qiu, Xiaohui A1 - Han, Min-Joon A1 - Grosveld, Gerard C. A1 - d'Azzo, Alessandra T1 - MYC competes with MiT/TFE in regulating lysosomal biogenesis and autophagy through an epigenetic rheostat JF - Nature Communications N2 - Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically. KW - autophagy KW - cancer KW - cancer metabolism KW - cell biology KW - mechanisms of disease Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221189 VL - 10 ER - TY - JOUR A1 - Lu, Yuan A1 - Boswell, Wiliam A1 - Boswell, Mikki A1 - Klotz, Barbara A1 - Kneitz, Susanne A1 - Regneri, Janine A1 - Savage, Markita A1 - Mendoza, Cristina A1 - Postlethwait, John A1 - Warren, Wesley C. A1 - Schartl, Manfred A1 - Walter, Ronald B. T1 - Application of the Transcriptional Disease Signature (TDSs) to Screen Melanoma-Effective Compounds in a Small Fish Model JF - Scientific Reports N2 - Cell culture and protein target-based compound screening strategies, though broadly utilized in selecting candidate compounds, often fail to eliminate candidate compounds with non-target effects and/or safety concerns until late in the drug developmental process. Phenotype screening using intact research animals is attractive because it can help identify small molecule candidate compounds that have a high probability of proceeding to clinical use. Most FDA approved, first-in-class small molecules were identified from phenotypic screening. However, phenotypic screening using rodent models is labor intensive, low-throughput, and very expensive. As a novel alternative for small molecule screening, we have been developing gene expression disease profiles, termed the Transcriptional Disease Signature (TDS), as readout of small molecule screens for therapeutic molecules. In this concept, compounds that can reverse, or otherwise affect known disease-associated gene expression patterns in whole animals may be rapidly identified for more detailed downstream direct testing of their efficacy and mode of action. To establish proof of concept for this screening strategy, we employed a transgenic strain of a small aquarium fish, medaka (Oryzias latipes), that overexpresses the malignant melanoma driver gene xmrk, a mutant egfr gene, that is driven by a pigment cell-specific mitf promoter. In this model, melanoma develops with 100% penetrance. Using the transgenic medaka malignant melanoma model, we established a screening system that employs the NanoString nCounter platform to quantify gene expression within custom sets of TDS gene targets that we had previously shown to exhibit differential transcription among xmrk-transgenic and wild-type medaka. Compound-modulated gene expression was identified using an internet-accessible custom-built data processing pipeline. The effect of a given drug on the entire TDS profile was estimated by comparing compound-modulated genes in the TDS using an activation Z-score and Kolmogorov-Smirnov statistics. TDS gene probes were designed that target common signaling pathways that include proliferation, development, toxicity, immune function, metabolism and detoxification. These pathways may be utilized to evaluate candidate compounds for potential favorable, or unfavorable, effects on melanoma-associated gene expression. Here we present the logistics of using medaka to screen compounds, as well as, the development of a user-friendly NanoString data analysis pipeline to support feasibility of this novel TDS drug-screening strategy. KW - bioinformatics KW - phenotypic screening Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-237322 VL - 9 ER - TY - JOUR A1 - Mercier, Rebecca A1 - Wolmarans, Annemarie A1 - Schubert, Jonathan A1 - Neuweiler, Hannes A1 - Johnson, Jill L. A1 - LaPointe, Paul T1 - The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation JF - Nature Communications N2 - Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis. KW - biophysics KW - cell growth KW - chaperones KW - enzymes Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224007 VL - 10 ER - TY - JOUR A1 - Lübcke, Paul M. A1 - Ebbers, Meinolf N. B. A1 - Volzke, Johann A1 - Bull, Jana A1 - Kneitz, Susanne A1 - Engelmann, Robby A1 - Lang, Hermann A1 - Kreikemeyer, Bernd A1 - Müller-Hilke, Brigitte T1 - Periodontal treatment prevents arthritis in mice and methotrexate ameliorates periodontal bone loss JF - Scientific Reports N2 - Recent studies indicate a causal relationship between the periodontal pathogen P. gingivalis and rheumatoid arthritis involving the production of autoantibodies against citrullinated peptides. We therefore postulated that therapeutic eradication P. gingivalis may ameliorate rheumatoid arthritis development and here turned to a mouse model in order to challenge our hypothesis. F1 (DBA/1 x B10.Q) mice were orally inoculated with P. gingivalis before collagen-induced arthritis was provoked. Chlorhexidine or metronidazole were orally administered either before or during the induction phase of arthritis and their effects on arthritis progression and alveolar bone loss were compared to intraperitoneally injected methotrexate. Arthritis incidence and severity were macroscopically scored and alveolar bone loss was evaluated via microcomputed tomography. Serum antibody titres against P. gingivalis were quantified by ELISA and microbial dysbiosis following oral inoculation was monitored in stool samples via microbiome analyses. Both, oral chlorhexidine and metronidazole reduced the incidence and ameliorated the severity of collagen-induced arthritis comparable to methotrexate. Likewise, all three therapies attenuated alveolar bone loss. Relative abundance of Porphyromonadaceae was increased after oral inoculation with P. gingivalis and decreased after treatment. This is the first study to describe beneficial effects of non-surgical periodontal treatment on collagen-induced arthritis in mice and suggests that mouthwash with chlorhexidine or metronidazole may also be beneficial for patients with rheumatoid arthritis and a coexisting periodontitis. Methotrexate ameliorated periodontitis in mice, further raising the possibility that methotrexate may also positively impact on the tooth supporting tissues of patients with rheumatoid arthritis. KW - rheumatic diseases KW - rheumatology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-237355 VL - 9 ER - TY - THES A1 - Gaballa, Abdallah Hatem Hassan Hosny Ahmed T1 - PAF1c drives MYC-mediated immune evasion in pancreatic ductal adenocarcinoma T1 - PAF1c treibt die MYC-vermittelte Immunevasion im duktalen Adenokarzinom der Bauchspeicheldrüse an N2 - The expression of the MYC proto-oncogene is elevated in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC). Previous findings in PDAC have shown that this increased MYC expression mediates immune evasion and promotes S-phase progression. How these functions are mediated and whether a downstream factor of MYC mediates these functions has remained elusive. Recent studies identifying the MYC interactome revealed a complex network of interaction partners, highlighting the need to identify the oncogenic pathway of MYC in an unbiased manner. In this work, we have shown that MYC ensures genomic stability during S-phase and prevents transcription-replication conflicts. Depletion of MYC and inhibition of ATR kinase showed a synergistic effect to induce DNA damage. A targeted siRNA screen targeting downstream factors of MYC revealed that PAF1c is required for DNA repair and S-phase progression. Recruitment of PAF1c to RNAPII was shown to be MYC dependent. PAF1c was shown to be largely dispensable for cell proliferation and regulation of MYC target genes. Depletion of CTR9, a subunit of PAF1c, caused strong tumor regression in a pancreatic ductal adenocarcinoma model, with long-term survival in a subset of mice. This effect was not due to induction of DNA damage, but to restoration of tumor immune surveillance. Depletion of PAF1c resulted in the release of RNAPII with transcription elongation factors, including SPT6, from the bodies of long genes, promoting full-length transcription of short genes. This resulted in the downregulation of long DNA repair genes and the concomitant upregulation of short genes, including MHC class I genes. These data demonstrate that a balance between long and short gene transcription is essential for tumor progression and that interference with PAF1c levels shifts this balance toward a tumor-suppressive transcriptional program. It also directly links MYC-mediated S-phase progression to immune evasion. Unlike MYC, PAF1c has a stable, known folded structure; therefore, the development of a small molecule targeting PAF1c may disrupt the immune evasive function of MYC while sparing its physiological functions in cellular growth. N2 - Die Expression des MYC-Proto-Onkogens ist bei einem großen Teil der Patienten mit duktalem Adenokarzinom der Bauchspeicheldrüse (PDAC) erhöht. Bisherige Erkenntnisse in der Erforschung des ankreaskarzinoms zeigen, dass die erhöhte MYCExpression die Umgehung des Immunsystems bewirkt und die Progression der S-Phase fördert. Wie diese Funktionen vermittelt werden und ob ein nachgeschalteter Faktor von MYC für diese Funktion verantwortlich ist, blieb jedoch bisher ungeklärt. Jüngste Studien zur Identifizierung des MYC-Interaktoms haben ein sehr komplexes Netzwerk an Interaktionspartnern von MYC aufgedeckt, was die Notwendigkeit unterstreicht, die onkogenen Eigenschaften von MYC und seinen Interaktionspartnern unvoreingenommen und genau zu untersuchen. In dieser Arbeit konnte gezeigt werden, dass MYC die genomische Stabilität während der S-Phase herstellt und Konflikte zwischen Transkription und Replikation verhindert. Die Depletion von MYC und die Hemmung der ATR-Kinase zeigten bei der Induktion von DNA Schäden eine synergistische Wirkung. Ein siRNA-Screen, der Gene beinhaltete, die MYC nachgeschaltet sind, ergab, dass PAF1c für die DNA-Reparatur und die S-PhasenProgression erforderlich ist. Es zeigte sich außerdem, dass die Rekrutierung von PAF1c an RNAPII von MYC abhängig ist. Für die Zellproliferation und die Regulierung von MYCZielgenen ist PAF1c jedoch weitgehend entbehrlich. Es konnte gezeigt werden, dass die Depletion von CTR9, einer Untereinheit von PAF1c, in einem murinen Modell des duktalen Adenokarzinoms der Bauchspeicheldrüse zu einer starken Tumorregression mit langfristigem Überleben einiger Mäuse führte. Diese Wirkung war nicht auf die Induktion von DNA-Schäden zurückzuführen, sondern auf die Wiederherstellung der Immunüberwachung des Tumors. Die Deletion von PAF1c führte zu einer Umverteilung von RNAPII und Trankriptionselongationsfaktoren wie SPT6, von langen Genen hin zu kurzen Genen. Dadurch wurden lange Gene wie zum Beispiel DNA Reparaturgene nicht vollständig transkribiert, kurze Gene wie MHC-Klasse-I-Gene hingegen schon. Diese Daten zeigen, dass ein Gleichgewicht zwischen der Transkription langer und kurzer Gene für die Tumorprogression wichtig ist und dass eine Verminderung der PAF1c-Konzentration dieses Gleichgewicht in Richtung eines tumorsuppressiven Transkriptionsprogramms verschiebt. Außerdem besteht ein direkter Zusammenhang zwischen der MYCvermittelten S-Phasen-Progression und der Umgehung des Immunsystems. Im Gegensatz zu MYC verfügt PAF1c über eine stabile und gut bekannte gefaltete Struktur. Daher könnte die Entwicklung eines kleinen Moleküls, das PAF1c hemmt, die Funktion von MYC zur Umgehung des Immunsystems stören und gleichzeitig seine physiologischen Funktionen für das Zellwachstum nicht beeinträchtigen. KW - Myc KW - Transkription KW - PAF1c KW - Transcription elongation KW - Immune evasion KW - Immunevasion Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-360459 ER - TY - JOUR A1 - Chen, Wei-Hua A1 - Lu, Guanting A1 - Chen, Xiao A1 - Zhao, Xing-Ming A1 - Bork, Peer T1 - OGEE v2: an update of the online gene essentiality database with special focus on differentially essential genes in human cancer cell lines JF - Nucleic Acids Research N2 - OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased number of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info. KW - human cancer cell lines KW - gene essentiality database KW - OGEE v2 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181334 VL - 45 IS - D1 ER - TY - JOUR A1 - Seitz, Nicola A1 - vanEngelsdorp, Dennis A1 - Leonhardt, Sara D. T1 - Conserving bees in destroyed landscapes: The potentials of reclaimed sand mines JF - Global Ecology and Conservation N2 - Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more ‘sand loving’ bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats. KW - bee conservation KW - bee decline KW - habitat restoration KW - land use KW - wild bees KW - ground nesters Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235877 VL - 19 ER - TY - JOUR A1 - Hofrichter, Michaela A. H. A1 - Doll, Julia A1 - Habibi, Haleh A1 - Enayati, Samaneh A1 - Mehrjardi, Mohammad Yahya Vahidi A1 - Müller, Tobias A1 - Dittrich, Marcus A1 - Haaf, Thomas A1 - Vona, Barbara T1 - Exome-wide copy number variation analysis identifies a COL9A1 in frame deletion that is associated with hearing loss JF - European Journal of Medical Genetics N2 - Pathogenic variants in COL9A1 are primarily associated with autosomal recessive Stickler syndrome. Patients with COL9A1-associated Stickler syndrome (STL) present hearing loss (HL), ophthalmic manifestations and skeletal abnormalities. However, the clinical spectrum of patients with COL9A1 variants can also include multiple epiphyseal dysplasia, as well as non-syndromic HL that was observed in one previously reported proband. Exome sequencing was performed on the genomic DNA of an Iranian patient and his affected brother who both report non-syndromic HL. A 44.6 kb homozygous in-frame deletion spanning exons 6 to 33 of COL9A1 was detected via exome-based copy number variation analysis. The deleted exons were confirmed by PCR in the patient and his affected brother, who both have non-syndromic HL. Segregation analysis via qPCR confirmed the parents as heterozygous deletion carriers. Breakpoint analysis mapped the homozygous deletion spanning introns 5 to 33 (g.70,948,188_70,997,277del, NM_001851.4(COL9A1):c.697–3754_2112+769del, p.(Phe233_Ser704del), with an additional 67 bp of inserted intronic sequence that may have originated due to a fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) mechanism. This mechanism has not been previously implicated in HL or STL. This is also the first reported copy number variation in COL9A1 that was identified through an exome data set in an Iranian family with apparent non-syndromic HL. The present study emphasizes the importance of exome-wide copy number variation analysis in molecular diagnosis and provides supporting evidence to associate COL9A1 with autosomal recessive non-syndromic HL. KW - COL9A1 KW - copy number variation KW - FoSTeS/MMBIR mechanism KW - non-syndromic hearing loss KW - Stickler syndrome Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-322008 VL - 62 ER - TY - JOUR A1 - van de Peppel, L. J. J. A1 - Aanen, D. K. A1 - Biedermann, P. H. W. T1 - Low intraspecific genetic diversity indicates asexuality and vertical transmission in the fungal cultivars of ambrosia beetles JF - Fungal Ecology N2 - Ambrosia beetles farm ascomycetous fungi in tunnels within wood. These ambrosia fungi are regarded asexual, although population genetic proof is missing. Here we explored the intraspecific genetic diversity of Ambrosiella grosmanniae and Ambrosiella hartigii (Ascomycota: Microascales), the mutualists of the beetles Xylosandrus germanus and Anisandrus dispar. By sequencing five markers (ITS, LSU, TEF1α, RPB2, β-tubulin) from several fungal strains, we show that X. germanus cultivates the same two clones of A. grosmanniae in the USA and in Europe, whereas A. dispar is associated with a single A. hartigii clone across Europe. This low genetic diversity is consistent with predominantly asexual vertical transmission of Ambrosiella cultivars between beetle generations. This clonal agriculture is a remarkable case of convergence with fungus-farming ants, given that both groups have a completely different ecology and evolutionary history. KW - clonal fungiculture KW - ambrosia fungus KW - Ambrosiella KW - vertical transmission KW - symbiosis KW - Xylosandrus KW - Anisandrus KW - asexuality KW - genetic diversity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232161 VL - 32 ER - TY - JOUR A1 - Baluapuri, Apoorva A1 - Hofstetter, Julia A1 - Dudvarski Stankovic, Nevenka A1 - Endres, Theresa A1 - Bhandare, Pranjali A1 - Vos, Seychelle Monique A1 - Adhikari, Bikash A1 - Schwarz, Jessica Denise A1 - Narain, Ashwin A1 - Vogt, Markus A1 - Wang, Shuang-Yan A1 - Düster, Robert A1 - Jung, Lisa Anna A1 - Vanselow, Jens Thorsten A1 - Wiegering, Armin A1 - Geyer, Matthias A1 - Maric, Hans Michael A1 - Gallant, Peter A1 - Walz, Susanne A1 - Schlosser, Andreas A1 - Cramer, Patrick A1 - Eilers, Martin A1 - Wolf, Elmar T1 - MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation JF - Molecular Cell N2 - The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth. KW - MYC KW - SPT5 KW - SUPT5H KW - SPT6 KW - RNA polymerase II KW - transcription KW - elongation rate KW - processivity KW - directionality KW - tumorigenesis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221438 VL - 74 ER - TY - THES A1 - Kuklovsky [former Finke], Valerie T1 - Are some bees smarter than others? An examination of consistent individual differences in the cognitive abilities of honey bees T1 - Sind manche Bienen schlauer als andere? Eine Untersuchung von konsistenten individuellen Unterschieden in den kognitiven Fähigkeiten von Honigbienen N2 - Cognition refers to the ability to of animals to acquire, process, store and use vital information from the environment. Cognitive processes are necessary to predict the future and reduce the uncertainty of the ever-changing environment. Classically, research on animal cognition focuses on decisive cognitive tests to determine the capacity of a species by the testing the ability of a few individuals. This approach views variability between these tested key individuals as unwanted noise and is thus often neglected. However, inter-individual variability provides important insights to behavioral plasticity, cognitive specialization and brain modularity. Honey bees Apis mellifera are a robust and traditional model for the study of learning, memory and cognition due to their impressive capabilities and rich behavioral repertoire. In this thesis I have applied a novel view on the learning abilities of honey bees by looking explicitly at individual differences in a variety of learning tasks. Are some individual bees consistently smarter than some of her sisters? If so, will a smart individual always perform good independent of the time, the context and the cognitive requirements or do bees show distinct isolated ‘cognitive modules’? My thesis presents the first comprehensive investigation of consistent individual differences in the cognitive abilities of honey bees. To speak of an individual as behaving consistently, a crucial step is to test the individual multiple times to examine the repeatability of a behavior. I show that free-flying bees remain consistent in a visual discrimination task for three consecutive days. Successively, I explored individual consistency in cognitive proficiency across tasks involving different sensory modalities, contexts and cognitive requirements. I found that free-flying bees show a cognitive specialization between visual and olfactory learning but remained consistent across a simple discrimination task and a complex concept learning task. I wished to further explore individual consistency with respect to tasks of different cognitive complexity, a question that has never been tackled before in an insect. I thus performed a series of four experiments using either visual or olfactory stimuli and a different training context (free-flying and restrained) and tested bees in a discrimination task, reversal learning and negative patterning. Intriguingly, across all these experiments I evidenced the same results: The bees’ performances were consistent across the discrimination task and reversal learning and negative patterning respectively. No association was evidenced between reversal learning and negative patterning. After establishing the existence of consistent individual differences in the cognitive proficiency of honey bees I wished to determine factors which could underlie these differences. Since genetic components are known to underlie inter-individual variability in learning abilities, I studied the effects of genetics on consistency in cognitive proficiency by contrasting bees originating from either from a hive with a single patriline (low genetic diversity) or with multiple patrilines (high genetic diversity). These two groups of bees showed differences in the patterns of individually correlated performances, indicating a genetic component accounts for consistent cognitive individuality. Another major factor underlying variability in learning performances is the individual responsiveness to sucrose solution and to visual stimuli, as evidenced by many studies on restrained bees showing a positive correlation between responsiveness to task relevant stimuli and learning performances. I thus tested whether these relationships between sucrose/visual responsiveness and learning performances are applicable for free-flying bees. Free-flying bees were again subjected to reversal learning and negative patterning and subsequently tested in the laboratory for their responsiveness to sucrose and to light. There was no evidence of a positive relationship between sucrose/visual responsiveness and neither performances of free-flying bees in an elemental discrimination, reversal learning and negative patterning. These findings indicate that relationships established between responsiveness to task relevant stimuli and learning proficiency established in the laboratory with restrained bees might not hold true for a completely different behavioral context i.e. for free-flying bees in their natural environment. These results show that the honey bee is an excellent insect model to study consistency in cognitive proficiency and to identify the underlying factors. I mainly discuss the results with respect to the question of brain modularity in insects and the adaptive significance of individuality in cognitive abilities for honey bee colonies. I also provide a proposition of research questions which tie in this theme of consistent cognitive proficiency and could provide fruitful areas for future research. N2 - Unter Kognition versteht man die Fähigkeit von Tieren, essenzielle Informationen aus der Umwelt zu erfassen, zu verarbeiten, zu speichern und zu nutzen. Kognitive Prozesse sind notwendig, um die Zukunft vorherzusagen und die Unvorhersehbarkeit der sich ständig verändernden Umwelt zu verringern. Die Forschung der Kognition von Tieren konzentriert sich klassischerweise auf entscheidende kognitive Tests, um die Fähigkeit einer Spezies anhand der Leistungen einiger weniger Individuen zu bestimmen. Bei diesem Ansatz wird die Variabilität zwischen Individuen als unerwünschtes Rauschen betrachtet und daher vernachlässigt. Die interindividuelle Variabilität liefert jedoch wichtige Erkenntnisse über die Plastizität des Verhaltens, die kognitive Spezialisierung und die Modularität des Gehirns. Die Honigbiene Apis mellifera ist aufgrund ihrer eindrucksvollen Fähigkeiten und ihres reichen Verhaltensrepertoires ein robuster und traditioneller Modellorganismus für die Untersuchung von Lernen, Gedächtnis und Kognition. In dieser Arbeit habe ich das Lernverhalten von Honigbienen in einem neuen Blickwinkel betrachtet, indem ich explizit die individuellen Unterschiede bei diversen Lernaufgaben untersucht habe. Zeigen manche Bienen durchweg eine erhöhte Lernleistung im Vergleich zu ihren Schwestern? Wenn ja, erbringt ein Individuum unabhängig von der Zeit, dem Kontext und den kognitiven Anforderungen der Lernaufgaben immer gute Leistungen, oder zeigen Bienen ausgeprägte unabhängige "kognitive Module"? Die vorliegende Doktorarbeit stellt die erste umfassende Untersuchung konsistenter individueller Unterschiede in den kognitiven Fähigkeiten von Honigbienen dar. Um von einem konsistenten Verhalten sprechen zu können, ist es entscheidend das Individuum mehrfach zu testen, um die Wiederholbarkeit eines Verhaltens zu untersuchen. Ich konnte zeigen, dass frei fliegende Bienen bei einer visuellen Unterscheidungsaufgabe an drei aufeinanderfolgenden Tagen eine konsistente Lernleistung zeigen. Im Anschluss untersuchte ich die individuelle Konsistenz der kognitiven Fähigkeiten bei Lernaufgaben mit unterschiedlichen sensorischen Modalitäten, Kontexten und kognitiven Anforderungen. Frei fliegende Bienen zeigten eine kognitive Spezialisierung zwischen visuellem und olfaktorischem Lernen, während sie bei einer einfachen Unterscheidungsaufgabe und einer komplexen Konzeptlernaufgabe konsistent im Lernverhalten blieben. Anschließend wollte ich die individuelle Konsistenz im Lernverhalten bei Aufgaben unterschiedlicher kognitiver Komplexität weiter erforschen, eine Frage, die bisher noch nie bei einem Insekt behandelt wurde. Ich führte dazu eine Reihe von vier Experimenten durch, bei denen entweder visuelle oder olfaktorische Stimuli und ein unterschiedlicher Trainingskontext (frei fliegend oder eingespannt) verwendet wurden. Die Bienen wurden in einer Unterscheidungsaufgabe, einer Umlernaufgabe und in Negative Patterning getestet. Erstaunlicherweise wurden bei diesen Experimenten die gleichen Ergebnisse festgestellt: Die Lernleitung der Bienen in der Unterscheidungsaufgabe zeigte eine positive Korrelation mit der Lernleistung im Umlernen und Negative Patterning. Zwischen dem Umkehrlernen und Negative Patterning konnte jedoch kein Zusammenhang festgestellt werden. Nachdem ich festgestellt hatte, dass es konsistente individuelle Unterschiede in den kognitiven Fähigkeiten von Bienen gibt, wollte ich die Faktoren ermitteln, die diesen Unterschieden zugrunde liegen könnten. Es war bereits bekannt, dass genetische Komponenten der interindividuellen Variabilität im Lernverhalten zugrunde liegen. Deshalb untersuchte ich den Einfluss von genetischer Vielfalt auf die Beständigkeit von kognitiven Fähigkeiten, indem ich Bienen gegenüberstellte, die entweder aus einem Bienenstock mit einer einzigen Patriline (geringe genetische Vielfalt) oder mit mehreren Patrilinen (hohe genetische Vielfalt) stammten. Diese beiden Gruppen von Bienen wiesen Unterschiede in den Mustern der individuellen korrelierten Lernleistungen auf, was darauf hindeutet, dass eine genetische Komponente für kognitive Individualität verantwortlich ist. Ein weiterer wichtiger Faktor, welcher der Variabilität im Lernverhalten zugrunde liegt, ist die individuelle Reaktionsfähigkeit auf Saccharose Lösungen und auf visuelle Stimuli. Dies wurde durch viele Studien an eingespannten Bienen gezeigt, die eine positive Korrelation zwischen der Reaktionsfähigkeit auf aufgabenrelevante Reize und den Lernfähigkeiten feststellten. Ich habe daher untersucht, ob diese Beziehungen zwischen der Reaktionsfähigkeit auf Saccharose und visuellen Stimuli und den Lernleistungen auch für frei fliegende Bienen zutreffen. Die individuellen Lernleistungen im Umlernen und Negative patterning von frei fliegenden Bienen wurden erneut ermittelt und anschließend wurde im Labor die Reaktionsfähigkeit auf Saccharose und Licht getestet. Es gab keine Hinweise auf eine positive Korrelation zwischen der Reaktionsfähigkeit auf Saccharose und Licht und den Lernleistungen von frei fliegenden Bienen. Diese Ergebnisse deuten darauf hin, dass Beziehungen zwischen der Reaktionsfähigkeit auf aufgabenrelevante Stimuli und der Lernleistung, die im Labor mit eingespannten Bienen festgestellt wurden, möglicherweise nicht für einen anderen Verhaltenskontext gelten, d. h. für frei fliegende Bienen in ihrer natürlichen Umgebung. Diese Ergebnisse zeigen, dass die Honigbiene ein hervorragendes Insektenmodell ist, um die Konsistenz kognitiver Fähigkeiten zu untersuchen und die zugrunde liegenden Faktoren zu ermitteln. Ich diskutiere die Ergebnisse vor allem im Hinblick auf die Frage der Modularität des Gehirns bei Insekten und die adaptive Bedeutung von individuellen konsistenten kognitiven Fähigkeiten für Honigbienenvölker. Ich schlage auch Forschungsfragen vor, die mit individuellen konsistenten kognitiven Fähigkeiten zusammenhängen und wertvolle Bereiche für künftige Forschungen darstellen könnten. KW - Lernen KW - Biene KW - Kognition KW - Individual differences KW - Cognitive consistency KW - Cognitive profile KW - Learning KW - Honeybee KW - Cognition Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323012 ER - TY - JOUR A1 - Becher, Isabelle A1 - Andrés-Pons, Amparo A1 - Romanov, Natalie A1 - Stein, Frank A1 - Schramm, Maike A1 - Baudin, Florence A1 - Helm, Dominic A1 - Kurzawa, Nils A1 - Mateus, André A1 - Mackmull, Marie-Therese A1 - Typas, Athanasios A1 - Müller, Christoph W. A1 - Bork, Peer A1 - Beck, Martin A1 - Savitski, Mikhail M. T1 - Pervasive Protein Thermal Stability Variation during the Cell Cycle JF - Cell N2 - Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions. KW - thermal proteome profiling KW - cell cycle KW - proteomics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221565 VL - 173 ER - TY - JOUR A1 - Mooij, Wolf M A1 - van Wijk, Dianneke A1 - Beusen, Arthur HW A1 - Brederveld, Robert J A1 - Chang, Manqi A1 - Cobben, Marleen MP A1 - DeAngelis, Don L A1 - Downing, Andrea S A1 - Green, Pamela A1 - Gsell, Alena S A1 - Huttunen, Inese A1 - Janse, Jan H A1 - Janssen, Annette BG A1 - Hengeveld, Geerten M A1 - Kong, Xiangzhen A1 - Kramer, Lilith A1 - Kuiper, Jan J A1 - Langan, Simon J A1 - Nolet, Bart A A1 - Nuijten, Rascha JM A1 - Strokal, Maryna A1 - Troost, Tineke A A1 - van Dam, Anne A A1 - Teurlincx, Sven T1 - Modeling water quality in the Anthropocene: directions for the next-generation aquatic ecosystem models JF - Current Opinion in Environmental Sustainability N2 - “Everything changes and nothing stands still” (Heraclitus). Here we review three major improvements to freshwater aquatic ecosystem models — and ecological models in general — as water quality scenario analysis tools towards a sustainable future. To tackle the rapid and deeply connected dynamics characteristic of the Anthropocene, we argue for the inclusion of eco-evolutionary, novel ecosystem and social-ecological dynamics. These dynamics arise from adaptive responses in organisms and ecosystems to global environmental change and act at different integration levels and different time scales. We provide reasons and means to incorporate each improvement into aquatic ecosystem models. Throughout this study we refer to Lake Victoria as a microcosm of the evolving novel social-ecological systems of the Anthropocene. The Lake Victoria case clearly shows how interlinked eco-evolutionary, novel ecosystem and social-ecological dynamics are, and demonstrates the need for transdisciplinary research approaches towards global sustainability. Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224173 VL - 36 ER - TY - JOUR A1 - Flunkert, Julia A1 - Maierhofer, Anna A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Horvath, Steve A1 - Nanda, Indrajit A1 - Haaf, Thomas T1 - Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts JF - Experimental Cell Research N2 - To study delayed genetic and epigenetic radiation effects, which may trigger radiation-induced carcinogenesis, we have established single-cell clones from irradiated and non-irradiated primary human fibroblasts. Stable clones were endowed with the same karyotype in all analyzed metaphases after 20 population doublings (PDs), whereas unstable clones displayed mosaics of normal and abnormal karyotypes. To account for variation in radiation sensitivity, all experiments were performed with two different fibroblast strains. After a single X-ray dose of 2 Gy more than half of the irradiated clones exhibited radiation-induced genome instability (RIGI). Irradiated clones displayed an increased rate of loss of chromosome Y (LOY) and copy number variations (CNVs), compared to controls. CNV breakpoints clustered in specific chromosome regions, in particular 3p14.2 and 7q11.21, coinciding with common fragile sites. CNVs affecting the FHIT gene in FRA3B were observed in independent unstable clones and may drive RIGI. Bisulfite pyrosequencing of control clones and the respective primary culture revealed global hypomethylation of ALU, LINE-1, and alpha-satellite repeats as well as rDNA hypermethylation during in vitro ageing. Irradiated clones showed further reduced ALU and alpha-satellite methylation and increased rDNA methylation, compared to controls. Methylation arrays identified several hundred differentially methylated genes and several enriched pathways associated with in vitro ageing. Methylation changes in 259 genes and the MAP kinase signaling pathway were associated with delayed radiation effects (after 20 PDs). Collectively, our results suggest that both genetic (LOY and CNVs) and epigenetic changes occur in the progeny of exposed cells that were not damaged directly by irradiation, likely contributing to radiation-induced carcinogenesis. We did not observe epigenetic differences between stable and unstable irradiated clones. The fact that the DNA methylation (DNAm) age of clones derived from the same primary culture varied greatly suggests that DNAm age of a single cell (represented by a clone) can be quite different from the DNAm age of a tissue. We propose that DNAm age reflects the emergent property of a large number of individual cells whose respective DNAm ages can be highly variable. KW - copy number variation (CNV) KW - delayed radiation effects KW - DNA methylation (DNAm) age KW - global DNA methylation KW - loss of chromosome Y (LOY); KW - methylation array analysis KW - radiation-induced genome instability (RIGI) Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228177 VL - 370 ER - TY - JOUR A1 - Schubert, Frank K. A1 - Hagedorn, Nicolas A1 - Yoshii, Taishi A1 - Helfrich-Förster, Charlotte A1 - Rieger, Dirk T1 - Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system JF - Journal of Comparative Neurology N2 - Drosophila melanogaster is a long-standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. In our recent work, we focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor-labeling technique Flybow we were able to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s-LNv) and one ITP positive dorsolateral neuron (LNd). These two clock neurons share the same spatial and functional properties. We found both neurons innervating the same brain areas with similar pre- and postsynaptic sites in the brain. Here the anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF-neurons (l-LNvs). We could show that the four hardly distinguishable l-LNvs consist of two subgroups with different innervation patterns. While three of the neurons reflect the well-known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla-surface. We named this neuron “extra” l-LNv (l-LNvx). We suggest the anatomical findings reflect different functional properties of the two l-LNv subgroups. KW - circadian clock neurons KW - Drosophila melanogaster KW - flybow KW - morphology KW - RRID: AB_760350 KW - RRID: AB_2315460 KW - RRID: AB_2314242 KW - RRID: AB_2315311 KW - RRID: AB_2314041 KW - RRID: AB_300798 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234477 VL - 526 ER - TY - THES A1 - Korte, Pamela T1 - Die funktionelle Bedeutung des Lipidstoffwechsels für die Stomataöffnung bei Hitzestress in \(Arabidopsis\) \(thaliana\) T1 - The functional significance of lipid metabolism for stomatal opening during heat stress in \(Arabidopsis\) \(thaliana\) N2 - Pflanzen sind verschiedenen Umweltbedingungen ausgesetzt, die zu suboptimalen Wachstumsbedingungen führen können. Dies gilt für eine Vielzahl von biotischen und abiotischen Faktoren. In der hier vorgelegten Arbeit wird der Effekt von erhöhten Temperaturen und Hitze genauer analysiert. Hitze ist einer der wichtigsten abiotischen Stressfaktoren, der das Pflanzenwachstum und die Reproduktion beeinflusst. Viele wichtige Kulturpflanzen zeigen immense Ertragseinbußen, die durch Hitze hervorgerufen werden. Durch den fortschreitenden Klimawandel werden jedoch Hitzeperioden immer häufiger und somit die Folgen für die Nahrungsproduktion immer gravierender. Zur Züchtung von Pflanzen die hitzetolerant sind und weniger hohe Ertragseinbußen unter diesem Stress aufweisen, ist es essenziell die grundlegenden molekularen Mechanismen der Hitzetoleranz zu verstehen. Es müssen die verschiedenen physiologischen und biochemischen Prozesse identifiziert werden, die es Pflanzen ermöglichen, sich anzupassen. Es ist bekannt, dass die Anpassungsmechanismen von Pflanzen komplex sind und sowohl Veränderungen auf zellulärer wie auch auf organismischer Ebene beinhalten. Ziel dieser Arbeit war es, weitere Erkenntnisse zu gewinnen, wie diese Anpassung vonstattengeht und welche molekularen Prozesse an ihr beteiligt sind. Ein Hauptaugenmerk lag dabei auf dem Einfluss des Lipidmetabolismus und den daran beteiligten Enzymen. Es konnte bereits gezeigt werden, dass die Akkumulation von Triacylglycerolen bei hohen Temperaturen die basale Thermotoleranz bei Arabidopsis thaliana erhöht. Wie jedoch der genaue Mechanismus dieser durch Triacylglycerole vermittelten Thermotoleranz funktioniert, war bis dato nicht bekannt. Ich konnte zeigen, dass die angesammelten Triacylglycerole genutzt werden können, um die Stomata während des Hitzestress zu öffnen. Dies führt zu einer erhöhten Transpiration und somit einer Kühlung der Blätter. Der Abbau von Triacylglycerolen und Stärke am Morgen ist notwendig, um die Stomata zu öffnen. Zusätzlich dient der Abbau der Aufrechterhaltung des Citratzyklus und somit der Energieversorgung. In weiteren Experimenten konnte ich durch Fütterung mit stabil markierter Laurinsäure zeigen, dass die Triacylglycerole auch dem Aufbau neuer Aminosäuren unter Stressbedingungen dienen. Die hier vorgestellten Arbeiten bieten die Grundlage, um den Mechanismus der Thermotoleranz besser zu verstehen. Das Verständnis der in dieser Arbeit beschriebenen molekularen Signalwege und Enzyme kann langfristig dazu beitragen hitzeresistentere Nutzpflanzen zu züchten. N2 - Plants are exposed to various environmental conditions that can lead to suboptimal growth conditions. This applies to a variety of biotic and abiotic factors. In the work presented here, the effect of elevated temperatures and heat is analyzed in more detail. Heat is one of the most important abiotic stress factors affecting plant growth and reproduction. Many important crops show immense yield losses caused by heat. However, as climate change progresses, periods of heat are becoming more frequent and the consequences for food production are becoming increasingly serious. Understanding the basic molecular mechanisms of heat tolerance is essential to breed plants that are heat tolerant and show less yield loss under this stress. The various physiological and biochemical processes that enable plants to adapt need to be identified. It is known that the adaptation mechanisms of plants are complex and involve changes at both the cellular and organismal level. The aim of this work was to gain further insights into how this adaptation takes place and which molecular processes are involved. The main focus was on the influence of lipid metabolism and the enzymes involved. It has already been shown that the accumulation of triacylglycerols at high temperatures increases basal thermotolerance in Arabidopsis thaliana. However, the exact mechanism of this triacylglycerol mediated thermotolerance was not known until now. I was able to show that the accumulated triacylglycerols can be used to open the stomata during heat stress. This leads to increased transpiration and thus cooling of the leaves. The degradation of triacylglycerols and starch in the morning is necessary to open the stomata. In addition, the degradation serves to maintain the citrate cycle and thus the energy supply. In further experiments, I was able to show by feeding stably labeled lauric acid that the triacylglycerols also serve to build up new amino acids under stress conditions. The work presented here provides the basis for a better understanding of the mechanism of thermotolerance. Understanding the molecular signaling pathways and enzymes described in this work could - in the long term - contribute to breeding of more heat-resistant crops. KW - Hitzestress KW - Ackerschmalwand KW - Arabidopsis thaliana KW - Triacylglycerol Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-370461 ER - TY - JOUR A1 - Göttlich, Claudia A1 - Kunz, Meik A1 - Zapp, Cornelia A1 - Nietzer, Sarah L. A1 - Walles, Heike A1 - Dandekar, Thomas A1 - Dandekar, Gudrun T1 - A combined tissue-engineered/in silico signature tool patient stratification in lung cancer JF - Molecular Oncology N2 - Patient-tailored therapy based on tumor drivers is promising for lung cancer treatment. For this, we combined in vitro tissue models with in silico analyses. Using individual cell lines with specific mutations, we demonstrate a generic and rapid stratification pipeline for targeted tumor therapy. We improve in vitro models of tissue conditions by a biological matrix-based three-dimensional (3D) tissue culture that allows in vitro drug testing: It correctly shows a strong drug response upon gefitinib (Gef) treatment in a cell line harboring an EGFR-activating mutation (HCC827), but no clear drug response upon treatment with the HSP90 inhibitor 17AAG in two cell lines with KRAS mutations (H441, A549). In contrast, 2D testing implies wrongly KRAS as a biomarker for HSP90 inhibitor treatment, although this fails in clinical studies. Signaling analysis by phospho-arrays showed similar effects of EGFR inhibition by Gef in HCC827 cells, under both 2D and 3D conditions. Western blot analysis confirmed that for 3D conditions, HSP90 inhibitor treatment implies different p53 regulation and decreased MET inhibition in HCC827 and H441 cells. Using in vitro data (western, phospho-kinase array, proliferation, and apoptosis), we generated cell line-specific in silico topologies and condition-specific (2D, 3D) simulations of signaling correctly mirroring in vitro treatment responses. Networks predict drug targets considering key interactions and individual cell line mutations using the Human Protein Reference Database and the COSMIC database. A signature of potential biomarkers and matching drugs improve stratification and treatment in KRAS-mutated tumors. In silico screening and dynamic simulation of drug actions resulted in individual therapeutic suggestions, that is, targeting HIF1A in H441 and LKB1 in A549 cells. In conclusion, our in vitro tumor tissue model combined with an in silico tool improves drug effect prediction and patient stratification. Our tool is used in our comprehensive cancer center and is made now publicly available for targeted therapy decisions. KW - 3D lung tumor model KW - Boolean signaling network KW - chemoresistance KW - HSP90 inhibitor KW - insilico drug screening too KW - KRAS mutation signature Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233137 VL - 12 ER - TY - JOUR A1 - Grebinyk, Anna A1 - Grebinyk, Sergii A1 - Prylutska, Svitlana A1 - Ritter, Uwe A1 - Matyshevska, Olga A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - C60 fullerene accumulation in human leukemic cells and perspectives of LED-mediated photodynamic therapy JF - Free Radical Biology and Medicine N2 - Recent progress in nanobiotechnology has attracted interest to a biomedical application of the carbon nanostructure C60 fullerene since it possesses a unique structure and versatile biological activity. C60 fullerene potential application in the frame of cancer photodynamic therapy (PDT) relies on rapid development of new light sources as well as on better understanding of the fullerene interaction with cells. The aim of this study was to analyze C60 fullerene effects on human leukemic cells (CCRF-CEM) in combination with high power single chip light-emitting diodes (LEDs) light irradiation of different wavelengths: ultraviolet (UV, 365 nm), violet (405 nm), green (515 nm) and red (632 nm). The time-dependent accumulation of fullerene C60 in CCRF-CEM cells up to 250 ng/106 cells at 24 h with predominant localization within mitochondria was demonstrated with immunocytochemical staining and liquid chromatography mass spectrometry. In a cell viability assay we studied photoexcitation of the accumulated C60 nanostructures with ultraviolet or violet LEDs and could prove that significant phototoxic effects did arise. A less pronounced C60 fullerene phototoxic effect was observed after irradiation with green, and no effect was detected with red light. A C60 fullerene photoactivation with violet light induced substantial ROS generation and apoptotic cell death, confirmed by caspase3/7 activation and plasma membrane phosphatidylserine externalization. Our work proved C60 fullerene ability to induce apoptosis of leukemic cells after photoexcitation with high power single chip 405 nm LED as a light source. This underlined the potential for application of C60 nanostructure as a photosensitizer for anticancer therapy. KW - C-60 fullerene KW - photodanamic therapy KW - LEDs KW - leukemic cells KW - immunocytochemistry KW - HPLC-ESI-MS KW - apoptosis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228245 VL - 124 ER - TY - JOUR A1 - Minden, Vanessa A1 - Schnetger, Bernhard A1 - Pufal, Gesine A1 - Leonhardt, Sara D. T1 - Antibiotic-induced effects on scaling relationships and on plant element contents in herbs and grasses JF - Ecology and Evolution N2 - Plant performance is correlated with element concentrations in plant tissue, which may be impacted by adverse chemical soil conditions. Antibiotics of veterinary origin can adversely affect plant performance. They are released to agricultural fields via grazing animals or manure, taken up by plants and may be stored, transformed or sequestered by plant metabolic processes. We studied the potential effects of three antibiotics (penicillin, sulfadiazine, and tetracycline) on plant element contents (macro- and microelements). Plant species included two herb species (Brassica napus and Capsella bursa-pastoris) and two grass species (Triticum aestivum and Apera spica-venti), representing two crop species and two noncrop species commonly found in field margins, respectively. Antibiotic concentrations were chosen as to reflect in vivo situations, that is, relatively low concentrations similar to those detected in soils. In a greenhouse experiment, plants were raised in soil spiked with antibiotics. After harvest, macro- and microelements in plant leaves, stems, and roots were determined (mg/g). Results indicate that antibiotics can affect element contents in plants. Penicillin exerted the greatest effect both on element contents and on scaling relationships of elements between plant organs. Roots responded strongest to antibiotics compared to stems and leaves. We conclude that antibiotics in the soil, even in low concentrations, lead to low-element homeostasis, altering the scaling relationships between roots and other plant organs, which may affect metabolic processes and ultimately the performance of a plant. KW - antibiotics KW - homeostasis KW - scaling relationships KW - standardized major axis regression KW - tissue nutrient contents Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224094 VL - 8 ER - TY - JOUR A1 - Stein, Katharina A1 - Stenchly, Kathrin A1 - Coulibaly, Drissa A1 - Pauly, Alain A1 - Dimobe, Kangbeni A1 - Steffan-Dewenter, Ingolf A1 - Konaté, Souleymane A1 - Goetze, Dethardt A1 - Porembski, Stefan A1 - Linsenmair, K. Eduard T1 - Impact of human disturbance on bee pollinator communities in savanna and agricultural sites in Burkina Faso, West Africa JF - Ecology and Evolution N2 - All over the world, pollinators are threatened by land-use change involving degradation of seminatural habitats or conversion into agricultural land. Such disturbance often leads to lowered pollinator abundance and/or diversity, which might reduce crop yield in adjacent agricultural areas. For West Africa, changes in bee communities across disturbance gradients from savanna to agricultural land are mainly unknown. In this study, we monitored for the impact of human disturbance on bee communities in savanna and crop fields. We chose three savanna areas of varying disturbance intensity (low, medium, and high) in the South Sudanian zone of Burkina Faso, based on land-use/land cover data via Landsat images, and selected nearby cotton and sesame fields. During 21 months covering two rainy and two dry seasons in 2014 and 2015, we captured bees using pan traps. Spatial and temporal patterns of bee species abundance, richness, evenness and community structure were assessed. In total, 35,469 bee specimens were caught on 12 savanna sites and 22 fields, comprising 97 species of 32 genera. Bee abundance was highest at intermediate disturbance in the rainy season. Species richness and evenness did not differ significantly. Bee communities at medium and highly disturbed savanna sites comprised only subsets of those at low disturbed sites. An across-habitat spillover of bees (mostly abundant social bee species) from savanna into crop fields was observed during the rainy season when crops are mass-flowering, whereas most savanna plants are not in bloom. Despite disturbance intensification, our findings suggest that wild bee communities can persist in anthropogenic landscapes and that some species even benefitted disproportionally. West African areas of crop production such as for cotton and sesame may serve as important food resources for bee species in times when resources in the savanna are scarce and receive at the same time considerable pollination service. KW - bee communities KW - cotton KW - sesame KW - species spillover KW - sub-Saharan Africa Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239999 VL - 8 ER -