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 - JOUR A1 - Kuhlemann, Alexander A1 - Beliu, Gerti A1 - Janzen, Dieter A1 - Petrini, Enrica Maria A1 - Taban, Danush A1 - Helmerich, Dominic A. A1 - Doose, Sören A1 - Bruno, Martina A1 - Barberis, Andrea A1 - Villmann, Carmen A1 - Sauer, Markus A1 - Werner, Christian T1 - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy JF - Frontiers in Synaptic Neuroscience N2 - Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft. KW - super-resolution microscopy (SRM) KW - click-chemistry KW - dSTORM KW - GABA-A receptor KW - genetic code expansion Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035 SN - 1663-3563 VL - 13 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 - Krones, David A1 - Rühling, Marcel A1 - Becker, Katrin Anne A1 - Kunz, Tobias C. A1 - Sehl, Carolin A1 - Paprotka, Kerstin A1 - Gulbins, Erich A1 - Fraunholz, Martin T1 - Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line JF - Frontiers in Microbiology N2 - Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins. KW - acid sphingomyelinase KW - staphylococcal alpha-toxin KW - sphingomyelinase release KW - lysosomal recruitment KW - Staphylococcus aureus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244843 SN - 1664-302X VL - 12 ER - TY - THES A1 - Krimmer, Elena T1 - Agri-environment schemes and ecosystem services: The influence of different sown flower field characteristics on pollination, natural pest control and crop yield T1 - Agrarumweltmaßnahmen und Ökosystemdienstleistungen: der Einfluss unterschiedlicher Blühflächen Merkmale auf Bestäubung, natürliche Schädlingskontrolle und Erträge N2 - Insects are responsible for the major part of the ecosystem services pollination and natural pest control. If insects decline, these ecosystem services can not longer be reliably delivered. Agricultural intensification and the subsequent loss and fragmentation of habitats has among others been identified to cause insect decline. Ecological intensification aims to promote alternative and sustainable management practices in agricultural farming, for example to decrease the use of external inputs such as pesticides. Agri-environment schemes make amends for farmers if they integrate ecologically beneficial measures into their farming regime and can therefore promote ecological intensification. There is a wide variety of agri-environment schemes, but the implementation of sown flower fields on crop fields is often included. Flower fields offer foraging resources as well as nesting sites for many different insect species and should be able to support insect populations as well as to increase ecosystem services to adjacent fields. However, the potential of flower fields to exhibit these effects is depending on many factors. Among others, the age and size of the flower field can influence if and how different insects profit from the measure. Additionally, the complexity of the surrounding landscape and therefore the existing biodiversity is influencing the potential of flower fields to increase ecosystem services locally. The goal of this study is to disentangle to which degree these factors influence the ecosystem services pollination and natural pest control and if these factors interact with each other. Furthermore, it will be examined if and how flower fields and ecosystem services influence crop yield. Additional factors examined in this study are distance decay and pesticide use. The abundance of beneficial insects can decrease strongly with increasing distance to suitable habitats. Pesticide use in turn could abrogate positive effects of flower fields on beneficial insects. To examine these different aspects and to be able to make recommendations for flower field implementation, field experiments were conducted on differently composed sown flower fields and adjacent oilseed rape fields. Flower fields differed in their age and continuity as well as in their size. Additionally, flower and oilseed rape fields were chosen in landscapes with different amounts of semi-natural habitat. Oilseed rape fields adjacent to calcareous grasslands and conventional crop fields served as controls. Pollinator observations and pollen beetle and parasitism surveys were conducted in the oilseed rape fields. Additionally, different yield parameters of the oilseed rape plants were recorded. Observations were conducted and samples taken in increasing distance to the flower fields to examine distance decay functions. Spray windows were established to inspect the influence of pesticides on ecosystem services and crop yields. Linear mixed models were used for statistical analysis. The results show, that newly established flower fields with high amounts of flower cover are very attractive for pollinators. If the flower fields reached a certain size (> 1.5ha), the pollinators tended to stay in these fields and did not distribute into the surroundings. High amounts of semi-natural habitat in the surrounding landscape increased the value of small flower fields as starting points for pollinators and their subsequent spillover into crop fields. Additionally, high amounts of semi-natural habitat decreased the decay of pollinators with increasing distance to the flower fields. Based on these results, it can be recommended to establish many small flower fields in landscapes with high amounts of semi-natural habitat and large flower fields in landscapes with low amounts of semi-natural habitat. However, it is mentionable that flower fields are no substitute for perennial semi-natural habitats. These still must be actively conserved to increase pollination to crop fields. Furthermore, the lowest amount of pollen beetle infestation was found on oilseed rape fields adjacent to continuous flower fields aged older than 6 years. Flower fields and calcareous grasslands in general increased pollen beetle parasitism in adjacent oilseed rape fields compared to conventional crop fields. The threshold for effective natural pest control could only be reached in the pesticide free areas in the oilseed rape fields adjacent to continuous flower fields and calcareous grasslands. Parasitism and superparasitism declined with increasing distance to the adjacent fields in pesticide treated areas of the oilseed rape fields. However, they remained on a similar level in spray windows without pesticides. Large flower fields increased parasitism and superparasitism more than small flower fields. Flower fields generally have the potential to increase pollen beetle parasitism rates, but pesticides can abrogate these positive effects of flower fields on natural pest control. Last but not least, effects of flower fields and ecosystem services on oilseed rape yield were examined. No positive effects of pollination on oilseed rape yield could be found. Old and continuous flower fields increased natural pest control in oilseed rape fields, which in turn increased seed set and total seed weight of oilseed rape plants. The pesticide treatment had negative effects on natural pest control, but positive effects on crop yield. Pollination and natural pest control decreased with increasing distance to the field edge, but fruit set slightly increased. The quality of the field in terms of soil and climatic conditions did not influence the yield parameters examined in this study. Yield formation in oilseed rape plants is a complex process with many factors involved, and it is difficult to disentangle indirect effects of flower fields on yield. However, perennial flower fields can promote ecological intensification by increasing crop yield via natural pest control. This study contributes to a better understanding of the effects of differently composed flower fields on pollination, natural pest control and oilseed rape yield. N2 - Insekten sind für einen Großteil der Ökosystemdienstleistungen Bestäubung und natürliche Schädlingskontrolle zuständig. Schwinden die Insekten, so können diese Dienstleistungen nicht mehr zuverlässig gewährleistet werden. Als Ursachen für den Rückgang an Insekten wurde unter anderem die Intensivierung der Landwirtschaft und damit einhergehend der Verlust und die Fragmentierung von Lebensraum identifiziert. Ökologische Intensivierung hat das Ziel, alternative und nachhaltige Bewirtschaftungsmethoden in der Landwirtschaft zu fördern und beispielsweise den Einsatz von Spritzmitteln zu verringern. Agrarumweltmaßnahmen entschädigen Landwirte, wenn sie ökologisch wertvolle Maßnahmen in ihren Betrieb integrieren und können dadurch ökologische Intensivierung unterstützen. Die Bandbreite an Agrarumweltmaßnahmen ist groß, beinhaltet aber häufig das Anlegen von Blühflächen auf Ackerflächen. Blühflächen liefern Nahrungsressourcen und Lebensraum für eine Vielzahl von Insekten und sollten daher in der Lage sein Insektenpopulationen zu unterstützen und Ökosystemdienstleistungen auf angrenzenden Feldern zu verstärken. Jedoch ist das ökologische Potential von Blühflächen von einer Vielzahl von Faktoren abhängig. Unter anderem können das Alter und die Größe der Blühfläche entscheidend beeinflussen, inwiefern unterschiedliche Insektengruppen profitieren. Zusätzlich hat die Landschaftskomplexität der direkten Umgebung, und damit die potentiell vorhandene Biodiversität, großen Einfluss auf die Fähigkeit von Blühflächen Ökosystemdienstleistungen lokal zu erhöhen. In dieser Studie geht es darum zu entschlüsseln, wie sich diese verschiedenen Faktoren sich auf die beiden Ökosystemdienstleistungen Bestäubung und natürliche Schädlingskontrolle auswirken und ob sie sich gegenseitig beeinflussen. Zusätzlich soll untersucht werden, inwiefern Blühflächen und Ökosystemdienstleistungen Erträge beeinflussen können. Weitere in dieser Studie untersuchte Einflussfaktoren sind die Distanz zur Blühfläche und der Einsatz von Pestiziden. Die Abundanz von Nützlingen kann mit der Distanz zu geeigneten Habitaten stark abnehmen. Der Einsatz von Spritzmitteln wiederum könnte die positiven Einflüsse der Blühflächen auf Nützlinge aufheben. Um diese verschiedenen Aspekte zu untersuchen und letztendlich Empfehlung für die Etablierung von Blühflächen geben zu können, wurden Feldversuche auf Blühflächen mit unterschiedlicher Beschaffenheit und auf angrenzenden Rapsflächen durchgeführt. Die Blühflächen unterschieden sich hierbei in ihrem Alter und ihrer Kontinuität. Zusätzlich wurden Blühflächen mit unterschiedlicher Größe getestet. Außerdem wurden die Blühflächen und ihre benachbarten Rapsfelder so ausgewählt, dass sie sich in Landschaften mit unterschiedlichem Anteil an halbnatürlichen Habitaten befinden. Rapsflächen neben Kalkmagerrasen und Äckern mit konventionellen Feldfrüchten dienten als Kontrollflächen. Auf den Rapsflächen wurden Bestäuberbeobachtungen sowie Aufnahmen von Rapsglanzkäferbefall und deren Parasitierung durchgeführt. Zusätzlich wurden verschiedene Ertragsparameter von Raps aufgenommen. Die Untersuchungen fanden jeweils in unterschiedlichen Distanzen zur Blühfläche innerhalb des Rapsfeldes statt, um Distanz-Abnahme Funktionen zu untersuchen. Spritzfenster wurden etabliert, um den Einfluss von Pestiziden auf Ökosystemdienstleistungen und Erträge zu untersuchen. Für die statistische Auswertung wurden lineare gemischte Modelle verwendet. Die Ergebnisse haben zum einen gezeigt, dass frisch angelegte Blühflächen mit hoher Blütendeckung sehr attraktiv für Bestäuber sind. Jedoch blieben die Bestäuber in den Blühflächen, wenn diese eine gewisse Größe hatten (> 1.5ha) und verteilten sich nicht auf die umgebenden Flächen. Ein hoher Anteil an halbnatürlichen Habitaten in der umgebenden Landschaft erhöhte den Wert von kleinen Blühflächen als Ausgangspunkt für Bestäuber und ihren anschließenden Übergang auf Ackerflächen. Hohe Mengen an halbnatürlichen Habitaten verringerten außerdem den Rückgang der Bestäuber mit steigender Entfernung zur Blühfläche. Auf Grundlage dieser Ergenisse wäre es zu empfehlen, kleine Blühflächen in Landschaften mit viel halbnatürlichem Habitat und große Blühflächen in Landschaften mit wenig halbnatürlichem Habitat anzulegen. Außerdem ist anzumerken, dass Blühflächen keinen adequaten Ersatz für dauerhafte halbantürliche Habitate darstellen. Diese müssen weiterhin aktiv geschützt und erhalten werden, um Bestäubung auf Ackerflächen zu fördern. Des Weiteren wurde auf Rapsflächen neben kontinuierlichen Blühflächen mit einem Alter über 6 Jahre der niedrigste Befall mit Rapsglanzkäferlarven festgestellt. Blühflächen und Kalkmagerrasen erhöhten die Parasitierung von Rapsglanzkäfern in benachbarten Rapsflächen im Vergleich zu Rapsflächen die neben Ackerflächen liegen. Der Schwellenwert für eine effektive natürliche Schädlingskontrolle wurde nur in den pestizidfreien Bereichen in Rapsflächen neben kontinuierlichen Blühflächen und Kalkmagerasen erreicht. In mit Pestiziden behandelten Bereichen nahmen Parasitismus und Superparasitismus mit zunehmender Entfernung zum benachbarten Feld ab. In den Spritzfenstern ohne Pestizide blieben sie jedoch auf dem gleichen Niveau. Große Blühflächen erhöhten Parasitismus und Superparasitismus mehr als kleine. Insgesamt können Blühflächen die Parasitierungsraten von Rapsglanzkäfern auf Rapsflächen erhöhen, jedoch können Pestizide diese positiven Effekte aufheben. Zuletzt wurden die Effekte von Blühflächen und Ökosystemdienstleistungen auf den Rapsertrag untersucht. Hier stellte sich heraus, dass Bestäubung keine positiven Effekte auf den Rapsertrag hatte. Alte und kontinuierliche Blühflächen erhöhten die natürliche Schädlingskontrolle in den Rapsfeldern, welche wiederrum den Samenansatz und das absolute Samengewicht erhöhten. Die Behandlung mit Pestiziden hatte negative Asuwirkungen auf natürliche Schädlingskontrolle, aber positive Auswirkungen auf den Ertrag. Bestäubung und natürliche Schädlingskontrolle nahmen mit der zunehmenden Entfernung zum Feldrand ab, aber der Fruchtansatz nahm leicht zu. Die Feldqualität hatte keine Auswirkungen auf die im Modell untersuchten Rapsertrag Messwerte. Ertragsbildung bei Rapspflanzen ist ein komplexer Vorgang an dem viele Faktoren beteiligt sind. Mehrjährige Blühflächen können ökologische Intensivierung fördern indem sie den Ertrag durch natürliche Schädlingskontrolle erhöhen. Diese Studie leistet einen wertvollen Beitrag zum besseren Verständnis der Auswirkungen von unterschiedlich beschaffenen Blühflächen auf Bestäubung, natürliche Schädlingskontrolle und Rapsertrag. KW - Ökologie KW - Agrarumweltmaßnahmen KW - Ökosystemdienstleistung Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-206577 ER - TY - JOUR A1 - Kriegel, Peter A1 - Matevski, Dragan A1 - Schuldt, Andreas T1 - Monoculture and mixture-planting of non-native Douglas fir alters species composition, but promotes the diversity of ground beetles in a temperate forest system JF - Biodiversity and Conservation N2 - Planting non-native tree species, like Douglas fir in temperate European forest systems, is encouraged to mitigate effects of climate change. However, Douglas fir monocultures often revealed negative effects on forest biota, while effects of mixtures with native tree species on forest ecosystems are less well understood. We investigated effects of three tree species (Douglas fir, Norway spruce, native European beech), on ground beetles in temperate forests of Germany. Beetles were sampled in monocultures of each tree species and broadleaf-conifer mixtures with pitfall traps, and environmental variables were assessed around each trap. We used linear mixed models in a two-step procedure to disentangle effects of environment and tree species identity on ground beetle abundance, species richness, functional diversity and species assemblage structure. Contradictory to our expectations, ground beetle abundance and functional diversity was highest in pure Douglas fir stands, while tree mixtures showed intermediate values between pure coniferous and pure beech stands. The main drivers of these patterns were only partially dependent on tree species identity, which highlights the importance of structural features in forest stands. However, our study revealed distinct shifts in assemblage structure between pure beech and pure Douglas fir stands, which were only partially eased through mixture planting. Our findings suggest that effects of planting non-native trees on associated biodiversity can be actively modified by promoting beneficial forest structures. Nevertheless, integrating non-native tree species, even in mixtures with native trees, will invariably alter assemblage structures of associated biota, which can compromise conservation efforts targeted at typical species composition. KW - mixed-species forestry KW - exotic species KW - Pseudotsuga menziesii KW - functional diversity KW - insects KW - microhabitats Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-269017 SN - 1572-9710 VL - 30 IS - 5 ER - TY - JOUR A1 - Kriegel, Peter A1 - Fritze, Michael‐Andreas A1 - Thorn, Simon T1 - Surface temperature and shrub cover drive ground beetle (Coleoptera: Carabidae) assemblages in short‐rotation coppices JF - Agricultural and Forest Entomology N2 - Increasing demand for biomass has led to an on‐going intensification of fuel wood plantations with possible negative effects on open land biodiversity. Hence, ecologists increasingly call for measures that reduce those negative effects on associated biodiversity. However, our knowledge about the efficiency of such measures remains scarce. We investigated the effects of gap implementation in short rotation coppices (SRCs) on carabid diversity and assemblage composition over 3 years, with pitfall traps in gaps, edges and interiors. In parallel, we quantified soil surface temperature, shrub‐ and herb cover. Edges had the highest number of species and abundances per trap, whereas rarefied species richness was significantly lower in short rotation coppice interiors than in other habitat types. Carabid community composition differed significantly between habitat types. The main environmental drivers were temperature for number of species and abundance and shrub cover for rarefied species richness. We found significantly higher rarefied species richness in gaps compared with interiors. Hence, we argue that gap implementation benefits overall diversity in short rotation coppices. Furthermore, the differences in species community composition between habitat types through increased species turnover support carabid diversity in short rotation coppices. These positive effects were largely attributed to microclimate conditions. However, to maintain positive effects, continuous management of herb layer might be necessary. KW - Carabidae KW - fuel wood KW - short‐rotation coppice KW - shrub‐cover KW - temperature Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239873 VL - 23 IS - 4 SP - 400 EP - 410 ER - TY - JOUR A1 - Kramer, Susanne A1 - Meyer-Natus, Elisabeth A1 - Stigloher, Christian A1 - Thoma, Hanna A1 - Schnaufer, Achim A1 - Engstler, Markus T1 - Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples JF - Nucleic Acids Research N2 - Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans. KW - mRNA Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230647 VL - 49 IS - 3 ER - TY - JOUR A1 - Kouhestani, Dina A1 - Geis, Maria A1 - Alsouri, Saed A1 - Bumm, Thomas G. P. A1 - Einsele, Hermann A1 - Sauer, Markus A1 - Stuhler, Gernot T1 - Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager JF - Cellular & Molecular Immunology N2 - No abstract available. KW - immunotherapy KW - tumour immunology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241189 VL - 18 ER - TY - JOUR A1 - Kessie, David K. A1 - Lodes, Nina A1 - Oberwinkler, Heike A1 - Goldman, William E. A1 - Walles, Thorsten A1 - Steinke, Maria A1 - Gross, Roy T1 - Activity of Tracheal Cytotoxin of Bordetella pertussis in a Human Tracheobronchial 3D Tissue Model JF - Frontiers in Cellular and Infection Microbiology N2 - Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model. KW - tracheal cytotoxin KW - airway epithelia KW - tissue model KW - ciliostasis KW - tight junction KW - Bordetella pertussis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222736 SN - 2235-2988 VL - 10 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 - Kastner, Carolin A1 - Hendricks, Anne A1 - Deinlein, Hanna A1 - Hankir, Mohammed A1 - Germer, Christoph-Thomas A1 - Schmidt, Stefanie A1 - Wiegering, Armin T1 - Organoid Models for Cancer Research — From Bed to Bench Side and Back JF - Cancers N2 - Simple Summary Despite significant strides in multimodal therapy, cancers still rank within the first three causes of death especially in industrial nations. A lack of individualized approaches and accurate preclinical models are amongst the major barriers that limit the development of novel therapeutic options and drugs. Recently, the 3D culture system of organoids was developed which stably retains the genetic and phenotypic characteristics of the original tissue, healthy as well as diseased. In this review, we summarize current data and evidence on the relevance and reliability of such organoid culture systems in cancer research, focusing on their role in drug investigations (in a personalized manner). Abstract Organoids are a new 3D ex vivo culture system that have been applied in various fields of biomedical research. First isolated from the murine small intestine, they have since been established from a wide range of organs and tissues, both in healthy and diseased states. Organoids genetically, functionally and phenotypically retain the characteristics of their tissue of origin even after multiple passages, making them a valuable tool in studying various physiologic and pathophysiologic processes. The finding that organoids can also be established from tumor tissue or can be engineered to recapitulate tumor tissue has dramatically increased their use in cancer research. In this review, we discuss the potential of organoids to close the gap between preclinical in vitro and in vivo models as well as clinical trials in cancer research focusing on drug investigation and development. KW - cancer KW - tumor disease KW - organoid KW - patient-derived organoid (PDOs) KW - patient-derived tumor organoid (PDTO) Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246307 SN - 2072-6694 VL - 13 IS - 19 ER - TY - JOUR A1 - Karimi, Sohail M. A1 - Freund, Matthias A1 - Wager, Brittney M. A1 - Knoblauch, Michael A1 - Fromm, Jörg A1 - M. Mueller, Heike A1 - Ache, Peter A1 - Krischke, Markus A1 - Mueller, Martin J. A1 - Müller, Tobias A1 - Dittrich, Marcus A1 - Geilfus, Christoph-Martin A1 - Alfaran, Ahmed H. A1 - Hedrich, Rainer A1 - Deeken, Rosalia T1 - Under salt stress guard cells rewire ion transport and abscisic acid signaling JF - New Phytologist N2 - Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity. KW - soil KW - stomata KW - abscisic acid (ABA) KW - glycophyte Arabidopsis KW - guard cell KW - halophyte Thellungiella/Eutrema KW - ion transport KW - salt stress Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259635 VL - 231 IS - 3 ER - TY - THES A1 - Jessen, Christina T1 - NRF2 links antioxidant and immune-relevant features in melanoma T1 - NRF2 verknüpft antioxidative und immunrelevante Eigenschaften im Melanom N2 - The transcription factor NRF2 is considered as the master regulator of cytoprotective and ROS-detoxifying gene expression. Due to their vulnerability to accumulating reactive oxygen species, melanomas are dependent on an efficient oxidative stress response, but to what extent melanomas rely on NRF2 is only scarcely investigated so far. In tumor entities harboring activating mutations of NRF2, such as lung adenocarcinoma, NRF2 activation is closely connected to therapy resistance. In melanoma, activating mutations are rare and triggers and effectors of NRF2 are less well characterized. This work revealed that NRF2 is activated by oncogenic signaling, cytokines and pro-oxidant triggers, released cell-autonomously or by the tumor microenvironment. Moreover, silencing of NRF2 significantly reduced melanoma cell proliferation and repressed well-known NRF2 target genes, indicating basal transcriptional activity of NRF2 in melanoma. Transcriptomic analysis showed a large set of deregulated gene sets, besides the well-known antioxidant effectors. NRF2 suppressed the activity of MITF, a marker for the melanocyte lineage, and induced expression of epidermal growth factor receptor (EGFR), thereby stabilizing the dedifferentiated melanoma phenotype and limiting pigmentation markers and melanoma-associated antigens. In general, the dedifferentiated melanoma phenotype is associated with a reduced tumor immunogenicity. Furthermore, stress-inducible cyclooxygenase 2 (COX2) expression, a crucial immune-modulating gene, was regulated by NRF2 in an ATF4-dependent manner. Only in presence of both transcription factors was COX2 robustly induced by H2O2 or TNFα. COX2 catalyzes the first step of the prostaglandin E2 (PGE2) synthesis, which was described to be associated with tumor immune evasion and reduction of the innate immune response. In accordance with these potentially immune-suppressive features, immunocompetent mice injected with NRF2 knockout melanoma cells had a strikingly longer tumor-free survival compared to NRF2-proficient cells. In line with the in vitro data, NRF2-deficient tumors showed suppression of COX2 and induction of MITF. Furthermore, transcriptomic analyses of available tumors revealed a strong induction of genes belonging to the innate immune response, such as RSAD2 and IFIH1. The expression of these genes strongly correlated with immune evasion parameters in human melanoma datasets and NRF2 activation or PGE2 supplementation limited the innate immune response in vitro. In summary, the stress dependent NRF2 activation stabilizes the dedifferentiated melanoma phenotype and facilitates the synthesis of PGE2. As a result, NRF2 reduces gene expression of the innate immune response and promotes the generation of an immune-cold tumor microenvironment. Therefore, NRF2 not only elevated the ROS resilience, but also strongly contributed to tumor growth, maintenance, and immune control in cutaneous melanoma. N2 - Der Transkriptionsfaktor NRF2 gilt als Masterregulator der antioxidativen Zellantwort. Im Melanom ist die Rolle von NRF2 bisher nur wenig untersucht worden, obwohl das Melanom anfällig für oxidativen Stress ist und somit eine besondere Abhängigkeit von antioxidativen Prozessen besteht. In Tumorentitäten mit NRF2 aktivierende Mutationen, wie z.B. dem Lungenadenokarzinom, ist die NRF2 Aktivierung mit einer Therapieresistenz verbunden. Allerdings sind diese aktivierenden Mutationen im Melanom selten und Mechanismen, die zu einer NRF2 Aktivierung führen, sind kaum bekannt. Die vorliegende Arbeit zeigt, dass NRF2 hier vor allem durch onkogene Signalwege, Zytokine und pro-oxidative Trigger aktiviert wird. Zudem verringerte die NRF2 Inhibierung die Zellproliferation und reduzierte die Expression von bekannten NRF2 Zielgenen. Dies weist darauf hin, dass die basale Transkriptionsaktivität von NRF2 im Melanom wichtig ist. Neben den bekannten Zielgenen waren außerdem eine Vielzahl von ROS-unabhängigen Gen-Sets dereguliert. Zum einen reduzierte NRF2 die Aktivität von MITF, dem melanozytären Lineage Marker und induzierte die Expression des epidermalen Wachstumsfaktorrezeptors, EGFR. Dadurch stabilisiert NRF2 den undifferenzierten Melanom-Phänotyp, welcher allgemein mit einer verminderten Expression von Pigmentierungsmarkern und Melanom-assoziierten Antigenen verbunden ist. Zum anderen regulierte NRF2 die Expression von Cyclooxygenase 2 (COX2), in Abhängigkeit von dem Transkriptionsfaktor ATF4. COX2 wurde basal und nach H2O2 oder TNFα Stimulation nur in Anwesenheit beider Transkriptionsfaktoren exprimiert. COX2 katalysiert die Prostaglandin E2 (PGE2) Synthese und es wurde beschrieben, dass hohe Konzentrationen an PGE2, sowohl die Immunevasion von Tumoren erleichtert als auch die angeborenen Immunantwort reduziert. In Übereinstimmung mit diesen potenziell immun-evasiven Eigenschaften zeigten immunkompetente Mäuse, denen NRF2-knockout Melanomzellen injiziert wurden, im Vergleich zu Kontrollzellen, ein deutlich längeres tumorfreies Überleben. Die NRF2-abhängige COX2 Erhöhung und MITF Hemmung, wurde zudem in den Maustumoren bestätigt. Darüber hinaus zeigten die NRF2-defizienten Tumore eine starke Induktion von Genen des angeborenen Immunsystems, wie z.B. RSAD2 und IFIH1. Die Expression dieser Gene korrelierte mit Immunevasionsparametern in Datensätzen des humanen Melanoms und eine NRF2 Aktivierung oder PGE2 Zugabe unterdrückte die Genexpression der angeborene Immunantwort bereits in vitro. Somit stabilisiert stress-induziertes NRF2 den undifferenzierten Melanom-Phänotyp und fördert die immunmodulierende PGE2 Produktion. Infolgedessen reduziert NRF2 die angeborene Immunantwort und unterstützt die Entstehung einer immun-suppressiven Tumormikroumgebung, welche das Tumorwachstum erleichtert. Die endogene NRF2 Aktivierung im Melanom fördert somit nicht nur die ROS-Resilienz, sondern auch die Tumoraufrechterhaltung und das Tumorwachstum im immunkompetenten Organismus. KW - Melanom KW - Oxidativer Stress KW - Genexpression KW - Krebsforschung KW - NRF2 KW - Antioxidantien KW - melanoma dedifferentiation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233495 ER - TY - JOUR A1 - Jansch, Charline A1 - Ziegler, Georg C. A1 - Forero, Andrea A1 - Gredy, Sina A1 - Wäldchen, Sina A1 - Vitale, Maria Rosaria A1 - Svirin, Evgeniy A1 - Zöller, Johanna E. M. A1 - Waider, Jonas A1 - Günther, Katharina A1 - Edenhofer, Frank A1 - Sauer, Markus A1 - Wischmeyer, Erhard A1 - Lesch, Klaus-Peter T1 - Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly JF - Journal of Neural Transmission N2 - Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders. KW - neuropsychiatric disorders KW - human induced pluripotent stem cell (hiPSC) KW - serotonin-specific neurons KW - median and dorsal raphe KW - synapse formation KW - Cadherin-13 (CDH13) Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-268519 SN - 1435-1463 VL - 128 IS - 2 ER - TY - JOUR A1 - Höhne, Christin A1 - Prokopov, Dmitry A1 - Kuhl, Heiner A1 - Du, Kang A1 - Klopp, Christophe A1 - Wuertz, Sven A1 - Trifonov, Vladimir A1 - Stöck, Matthias T1 - The immune system of sturgeons and paddlefish (Acipenseriformes): a review with new data from a chromosome‐scale sturgeon genome JF - Reviews in Aquaculture N2 - Sturgeon immunity is relevant for basic evolutionary and applied research, including caviar‐ and meat‐producing aquaculture, protection of wild sturgeons and their re‐introduction through conservation aquaculture. Starting from a comprehensive overview of immune organs, we discuss pathways of innate and adaptive immune systems in a vertebrate phylogenetic and genomic context. The thymus as a key organ of adaptive immunity in sturgeons requires future molecular studies. Likewise, data on immune functions of sturgeon‐specific pericardial and meningeal tissues are largely missing. Integrating immunological and endocrine functions, the sturgeon head kidney resembles that of teleosts. Recently identified pattern recognition receptors in sturgeon require research on downstream regulation. We review first acipenseriform data on Toll‐like receptors (TLRs), type I transmembrane glycoproteins expressed in membranes and endosomes, initiating inflammation and host defence by molecular pattern‐induced activation. Retinoic acid‐inducible gene‐I‐like (RIG‐like) receptors of sturgeons present RNA and key sensors of virus infections in most cell types. Sturgeons and teleosts share major components of the adaptive immune system, including B cells, immunoglobulins, major histocompatibility complex and the adaptive cellular response by T cells. The ontogeny of the sturgeon innate and onset of adaptive immune genes in different organs remain understudied. In a genomics perspective, our new data on 100 key immune genes exemplify a multitude of evolutionary trajectories after the sturgeon‐specific genome duplication, where some single‐copy genes contrast with many duplications, allowing tissue specialization, sub‐functionalization or both. Our preliminary conclusion should be tested by future evolutionary bioinformatics, involving all >1000 immunity genes. This knowledge update about the acipenseriform immune system identifies several important research gaps and presents a basis for future applications. KW - evolution KW - genomics KW - immune genes KW - immune organs KW - immune system KW - sturgeon Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239865 VL - 13 IS - 3 SP - 1709 EP - 1729 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 - Hojsgaard, Diego A1 - Schartl, Manfred T1 - Skipping sex: A nonrecombinant genomic assemblage of complementary reproductive modules JF - BioEssays N2 - The unusual occurrence and developmental diversity of asexual eukaryotes remain a puzzle. De novo formation of a functioning asexual genome requires a unique assembly of sets of genes or gene states to disrupt cellular mechanisms of meiosis and gametogenesis, and to affect discrete components of sexuality and produce clonal or hemiclonal offspring. We highlight two usually overlooked but essential conditions to understand the molecular nature of clonal organisms, that is, a nonrecombinant genomic assemblage retaining modifiers of the sexual program, and a complementation between altered reproductive components. These subtle conditions are the basis for physiologically viable and genetically balanced transitions between generations. Genomic and developmental evidence from asexual animals and plants indicates the lack of complementation of molecular changes in the sexual reproductive program is likely the main cause of asexuals' rarity, and can provide an explanatory frame for the developmental diversity and lability of developmental patterns in some asexuals as well as for the discordant time to extinction estimations. KW - amphimixis KW - apomixis KW - automixis KW - gynogenesis KW - hybridogenesis KW - parthenogenesis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225818 VL - 43 IS - 1 ER - TY - JOUR A1 - Heydarian, Motaharehsadat A1 - Schweinlin, Matthias A1 - Schwarz, Thomas A1 - Rawal, Ravisha A1 - Walles, Heike A1 - Metzger, Marco A1 - Rudel, Thomas A1 - Kozjak-Pavlovic, Vera T1 - Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity JF - Journal of Tissue Engineering N2 - Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment. KW - Triple co-culture KW - biomimetic 3D tissue model KW - Neisseria gonorrhoeae KW - perfusion-based bioreactor system KW - neutrophil transmigration Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259032 VL - 12 ER - TY - JOUR A1 - Hepbasli, Denis A1 - Gredy, Sina A1 - Ullrich, Melanie A1 - Reigl, Amelie A1 - Abeßer, Marco A1 - Raabe, Thomas A1 - Schuh, Kai T1 - Genotype- and Age-Dependent Differences in Ultrasound Vocalizations of SPRED2 Mutant Mice Revealed by Machine Deep Learning JF - Brain Sciences N2 - Vocalization is an important part of social communication, not only for humans but also for mice. Here, we show in a mouse model that functional deficiency of Sprouty-related EVH1 domain-containing 2 (SPRED2), a protein ubiquitously expressed in the brain, causes differences in social ultrasound vocalizations (USVs), using an uncomplicated and reliable experimental setting of a short meeting of two individuals. SPRED2 mutant mice show an OCD-like behaviour, accompanied by an increased release of stress hormones from the hypothalamic–pituitary–adrenal axis, both factors probably influencing USV usage. To determine genotype-related differences in USV usage, we analyzed call rate, subtype profile, and acoustic parameters (i.e., duration, bandwidth, and mean peak frequency) in young and old SPRED2-KO mice. We recorded USVs of interacting male and female mice, and analyzed the calls with the deep-learning DeepSqueak software, which was trained to recognize and categorize the emitted USVs. Our findings provide the first classification of SPRED2-KO vs. wild-type mouse USVs using neural networks and reveal significant differences in their development and use of calls. Our results show, first, that simple experimental settings in combination with deep learning are successful at identifying genotype-dependent USV usage and, second, that SPRED2 deficiency negatively affects the vocalization usage and social communication of mice. KW - SPRED KW - SPRED2 KW - mice KW - neural networks KW - ultrasound vocalizations KW - DeepSqueak Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248525 SN - 2076-3425 VL - 11 IS - 10 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 - INPR A1 - Hennig, Thomas A1 - Prusty, Archana B. A1 - Kaufer, Benedikt A1 - Whisnant, Adam W. A1 - Lodha, Manivel A1 - Enders, Antje A1 - Thomas, Julius A1 - Kasimir, Francesca A1 - Grothey, Arnhild A1 - Herb, Stefanie A1 - Jürges, Christopher A1 - Meister, Gunter A1 - Erhard, Florian A1 - Dölken, Lars A1 - Prusty, Bhupesh K. T1 - Selective inhibition of microRNA processing by a herpesvirus-encoded microRNA triggers virus reactivation from latency N2 - Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a novel cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the latent-lytic switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture, which impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 was sufficient to trigger virus reactivation from latency thereby identifying it as a readily drugable master regulator of the herpesvirus latent-lytic switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders like myalgic encephalitis/chronic fatigue syndrome (ME/CFS) and Long-COVID. KW - Herpesvirus KW - HHV-6 KW - miRNA processing KW - miR-30 KW - mitochondria KW - fusion and fission KW - type I interferon KW - latency KW - virus reactivation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-267858 UR - https://doi.org/10.21203/rs.3.rs-820696/v1 ET - submitted version ER - TY - JOUR A1 - Hempelmann, Alexander A1 - Hartleb, Laura A1 - van Straaten, Monique A1 - Hashemi, Hamidreza A1 - Zeelen, Johan P. A1 - Bongers, Kevin A1 - Papavasiliou, F. Nina A1 - Engstler, Markus A1 - Stebbins, C. Erec A1 - Jones, Nicola G. T1 - Nanobody-mediated macromolecular crowding induces membrane fission and remodeling in the African trypanosome JF - Cell Reports N2 - The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host’s antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells. KW - African trypanosome KW - host-pathogen interaction KW - variant surface glycoproteins KW - immune epitope mapping KW - structural biology KW - nanovesicle formation KW - nanotube formation KW - protein crowding KW - membrane fission Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270285 VL - 37 IS - 5 ER - TY - JOUR A1 - Helmprobst, Frederik A1 - Kneitz, Susanne A1 - Klotz, Barbara A1 - Naville, Magali A1 - Dechaud, Corentin A1 - Volff, Jean-Nicolas A1 - Schartl, Manfred T1 - Differential expression of transposable elements in the medaka melanoma model JF - PLoS One N2 - Malignant melanoma incidence is rising worldwide. Its treatment in an advanced state is difficult, and the prognosis of this severe disease is still very poor. One major source of these difficulties is the high rate of metastasis and increased genomic instability leading to a high mutation rate and the development of resistance against therapeutic approaches. Here we investigate as one source of genomic instability the contribution of activation of transposable elements (TEs) within the tumor. We used the well-established medaka melanoma model and RNA-sequencing to investigate the differential expression of TEs in wildtype and transgenic fish carrying melanoma. We constructed a medaka-specific TE sequence library and identified TE sequences that were specifically upregulated in tumors. Validation by qRT- PCR confirmed a specific upregulation of a LINE and an LTR element in malignant melanomas of transgenic fish. KW - melanoma KW - genomics KW - transposable elements KW - cancer genomics KW - malignant tumors KW - gene prediction KW - human genomics KW - retrotransposons Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260615 VL - 16 IS - 10 ER - TY - JOUR A1 - Helfrich-Förster, C. A1 - Monecke, S. A1 - Spiousas, I. A1 - Hovestadt, T. A1 - Mitesser, O. A1 - Wehr, T. A. T1 - Women temporarily synchronize their menstrual cycles with the luminance and gravimetric cycles of the Moon JF - Science Advances N2 - Many species synchronize reproductive behavior with a particular phase of the lunar cycle to increase reproductive success. In humans, a lunar influence on reproductive behavior remains controversial, although the human menstrual cycle has a period close to that of the lunar cycle. Here, we analyzed long-term menstrual recordings of individual women with distinct methods for biological rhythm analysis. We show that women’s menstrual cycles with a period longer than 27 days were intermittently synchronous with the Moon’s luminance and/or gravimetric cycles. With age and upon exposure to artificial nocturnal light, menstrual cycles shortened and lost this synchrony. We hypothesize that in ancient times, human reproductive behavior was synchronous with the Moon but that our modern lifestyles have changed reproductive physiology and behavior. KW - moon KW - menstrual cycles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231479 VL - 7 IS - 5 ER - TY - JOUR A1 - Heidrich, Lea A1 - Pinkert, Stefan A1 - Brandl, Roland A1 - Bässler, Claus A1 - Hacker, Hermann A1 - Roth, Nicolas A1 - Busse, Annika A1 - Müller, Jörg A1 - Friess, Nicolas T1 - Noctuid and geometrid moth assemblages show divergent elevational gradients in body size and color lightness JF - Ecography N2 - Previous macroecological studies have suggested that larger and darker insects are favored in cold environments and that the importance of body size and color for the absorption of solar radiation is not limited to diurnal insects. However, whether these effects hold true for local communities and are consistent across taxonomic groups and sampling years remains unexplored. This study examined the variations in body size and color lightness of the two major families of nocturnal moths, Geometridae and Noctuidae, along an elevational gradient of 700 m in Southern Germany. An assemblage-based analysis was performed using community-weighted means and a fourth-corner analysis to test for variations in color and body size among communities as a function of elevation. This was followed by a species-level analysis to test whether species occurrence and abundance along an elevation gradient were related to these traits, after controlling for host plant availability. In both 2007 and 2016, noctuid moth assemblages became larger and darker with increasing elevation, whereas geometrids showed an opposite trend in terms of color lightness and no clear trend in body size. In single species models, the abundance of geometrids, but not of noctuids, was driven by habitat availability. In turn, the abundance of dark-colored noctuids, but not geometrids increased with elevation. While body size and color lightness affect insect physiology and the ability to cope with harsh conditions, divergent trait–environment relationships between both families underline that findings of coarse-scale studies are not necessarily transferable to finer scales. Local abundance and occurrence of noctuids are shaped by morphological traits, whereas that of geometrids are rather shaped by local habitat availability, which can modify their trait–environment-relationship. We discuss potential explanations such as taxon-specific flight characteristics and the effect of microclimatic conditions. KW - insects KW - color lightness KW - body size KW - elevation KW - habitat availability KW - flight characteristics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256694 VL - 44 IS - 8 ER - TY - THES A1 - Heidrich, Lea T1 - The effect of environmental heterogeneity on communities T1 - Der Einfluss von Heterogenität in Umweltbedingungen auf Artgemeinschaften N2 - How diversity of life is generated, maintained, and distributed across space and time is the central question of community ecology. Communities are shaped by three assembly processes: (I) dispersal, (II) environ-mental, and (III) interaction filtering. Heterogeneity in environmental conditions can alter these filtering processes, as it increases the available niche space, spatially partitions the resources, but also reduces the effective area available for individual species. Ultimately, heterogeneity thus shapes diversity. However, it is still unclear under which conditions heterogeneity has positive effects on diversity and under which condi-tions it has negative or no effects at all. In my thesis, I investigate how environmental heterogeneity affects the assembly and diversity of diverse species groups and whether these effects are mediated by species traits. In Chapter II, I first examine how much functional traits might inform about environmental filtering pro-cesses. Specifically, I examine to which extent body size and colour lightness, both of which are thought to reflect the species thermal preference, shape the distribution and abundance of two moth families along elevation. The results show, that assemblages of noctuid moths are more strongly driven by abiotic filters (elevation) and thus form distinct patterns in colour lightness and body size, while geometrid moths are driven by biotic filters (habitat availability), and show no decline in body size nor colour lightness along elevation. Thus, one and the same functional trait can have quite different effects on community assembly even between closely related taxonomic groups. In Chapter III, I elucidate how traits shift the relative importance of dispersal and environmental filtering in determining beta diversity between forests. Environmental filtering via forest heterogeneity had on aver-age higher independent effects than dispersal filtering within and among regions, suggesting that forest heterogeneity determines species turnover even at country-wide extents. However, the relative importance of dispersal filtering increased with decreasing dispersal ability of the species group. From the aspects of forest heterogeneity covered, variations in herb or tree species composition had overall stronger influence on the turnover of species than forest physiognomy. Again, this ratio was influenced by species traits, namely trophic position, and body size, which highlights the importance of ecological properties of a taxo-nomic group in community assembly. In Chapter IV, I assess whether such ecological properties ultimately determine the level of heterogeneity which maximizes species richness. Here, I considered several facets of heterogeneity in forests. Though the single facets of heterogeneity affected diverse species groups both in positive and negative ways, we could not identify any generalizable mechanism based on dispersal nor the trophic position of the species group which would dissolve these complex relationships. In Chapter V, I examine the effect of environmental heterogeneity of the diversity of traits itself to evalu-ate, whether the effects of environmental heterogeneity on species richness are truly based on increases in the number of niches. The results revealed that positive effects of heterogeneity on species richness are not necessarily based on an increased number of niches alone, but proposedly also on a spatially partition of resources or sheltering effects. While ecological diversity increased overall, there were also negative trends which indicate filtering effects via heterogeneity. In Chapter VI, I present novel methods in measuring plot-wise heterogeneity of forests across continental scales via Satellites. The study compares the performance of Sentinel-1 and LiDar-derived measurements in depicting forest structures and heterogeneity and to their predictive power in modelling diversity. Senti-nel-1 could match the performance of Lidar and shows high potential to assess free yet detailed infor-mation about forest structures in temporal resolutions for modelling the diversity of species. Overall, my thesis supports the notion that heterogeneity in environmental conditions is an important driv-er of beta-diversity, species richness, and ecological diversity. However, I could not identify any general-izable mechanism which direction and form this effect will have. N2 - Eine zentrale Frage in der Ökologie ist es, wie die Diversität von Artgemeinschaften generiert, aufrecht-erhalten, und über Zeit und Raum verteilt wird. Die Zusammensetzung von Artgemeinschaften wird durch drei Prozesse bestimmt, die einzelne Arten herausfiltern: (I) Ausbreitung, sowie (II) Umweltbedin-gungen und (III) Interaktionen mit anderen Arten. Heterogenität in Umweltbedingungen verändert das Zusammenspiel dieser Filterprozesse, da es die Anzahl verfügbarer Nischen erhöht und Ressourcen räum-lich aufteilt, aber auch den für die jeweilige Art verfügbaren Raum reduziert, was schlussendlich die Diver-sität der Artgemeinschaft beeinflusst. Es ist jedoch immer noch unklar, wann Heterogenität die Diversität positiv und wann negativ oder sogar überhaupt nicht beeinflusst. In dieser Dissertation werde ich der Fra-ge nachgehen, wie Heterogenität die Artzusammensetzung und Diversität verschiedenster Artengruppen beeinflusst und ob deren Reaktion auf Heterogenität durch Artmerkmale beeinflusst wird. In Kapitel II untersuche ich zunächst inwieweit funktionale Merkmale den Einfluss von Umweltbedingun-gen auf Arten widerspiegeln. Dazu untersuchte ich den Einfluss von Körpergröße und Helligkeit auf die Verbreitung und Abundanz zweier Nachtfalterfamilien entlang eines Höhengradienten. Es zeigte sich, dass Noctuidae stärker von abiotischen Filterprozessen, d.h. Höhe, betroffen waren und klare Zu- bzw. Ab-nahmen in Körpergröße und Helligkeit entlang der Höhe aufwiesen, während Geometridae eher von bioti-schen Filterprozessen, d.h. der Verfügbarkeit ihres Habitats, beeinflusst wurden und keine Merkmalsmus-ter entlang der Höhe aufwiesen. Entsprechend kann ein- und dasselbe Merkmal selbst innerhalb nah-verwandter Artgruppen unterschiedliche Effekte auf die Zusammensetzung von Arten haben. In Kapitel III erläutere ich, wie funktionelle Merkmale die relative Wichtigkeit von Ausbreitungs- und Umweltfiltern für beta-Diversität verschieben können. Sowohl innerhalb als auch zwischen den untersuch-ten Regionen beeinflusste Heterogenität in Wäldern die beta-Diversität stärker als die räumliche Distanz. Letztere wurde allerdings immer bedeutender, je schlechter die Ausbreitungsfähigkeit der jeweiligen Arten-gruppe war. Wenn die Heterogenität in Wäldern nach floristischen und strukturellen Aspekten aufgeteilt wird, so hatte erstere alles in allem einen stärkeren Einfluss auf Unterschiede zwischen Artgemeinschaften. Bei Artengruppen höheren trophischen Levels und größeren Körperbaus hatten die strukturellen Aspekte jedoch einen stärkeren Einfluss. Diese Ergebnisse verdeutlichen, dass die Artzusammensetzung von be-stimmte Merkmale beeinflusst werden kann. In Kapitel IV untersuche ich ob solche Merkmale das Level an Heterogenität festlegen, an welchen Arten-reichtum am höchsten ist. Dazu betrachtete ich mehrere Aspekte von Heterogenität in Wäldern. Obwohl Heterogenität in diesen Aspekten sowohl positive als auch negative Einfluss auf den Artenreichtum der verschiedensten Artengruppen hatte, konnten wir diese nicht anhand der Ausbreitungsfähigkeit oder des trophischen Levels der Artengruppen ableiten. In Kapitel V untersuche ich schließlich den Effekt von Heterogenität auf die Vielfalt von funktionalen Merkmalen. Dieser Ansatz soll helfen zu evaluieren, ob eventuelle Anstiege in der Artenzahl mit Hetero-genität einem Zuwachs in der Anzahl der ökologischen Nischen zurückzuführen sind. Die Ergebnisse legen nahe, dass ein Anstieg von Artenreichtum nicht dadurch beeinflusst wird, sondern auch durch ande-re Mechanismen wie die räumliche Aufteilung von Ressourcen oder durch die Schaffung von Zufluchts-räumen. Obwohl Heterogenität die ökologische Diversität überwiegend positiv beeinflusste, gab es auch einige negative Reaktionen die darauf hindeuten, dass Heterogenität auch bestimmte Merkmale aus einer Artgemeinschaft herausfiltern kann. In Kapitel VI präsentiere ich neue, Satelliten-gestützte Methoden in der Erfassung von Waldstrukturen. In dieser Studie werden die Eignung von LiDar (Lasergestützte Waldvermessungen aus der Luft) und Senti-nel-1 (Satellitenscan durch Radiowellen) verglichen, Waldstrukturen und deren Heterogenität zu messen sowie verschiedene Diversitäts-indices zu modellieren. Hierbei schnitt Sentinel-1 ähnlich gut ab wie LiDar. Somit zeigt Sentinel-1 großes Potential zukünftige Biodiversitätsaufnahmen zu unterstützen, auch aufgrund der kostenfreie Verfügbarkeit von Daten, deren globalen Abdeckung und hohen zeitlichen Auflösung. Insgesamt unterstützen die Ergebnisse meiner Arbeit die große Bedeutung von Heterogenität, insbesonde-re von Waldstrukturen, für beta-Diversität, Artenreichtum und funktionaler Diversität. Allerdings konnte keine generelle Regel identifiziert werden, nach der sich vorhersagen lassen würde welche genaue Richtung dieser Effekt haben wird. KW - Heterogenität KW - Wald KW - Artenvielfalt KW - Waldstruktur Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221781 ER - TY - THES A1 - Heiby, Julia T1 - Insight into molecular mechanisms of folding and self-association of spider silk protein domains T1 - Einblicke in molekulare Mechanismen der Faltung und Selbstassoziation von Spinnenseidenproteindomänen N2 - Spider silk is a biomaterial of extraordinary toughness paired with elasticity. The assembly of silk proteins, so-called spidroins (from “spider” and “fibroin”), generates the silk threads we typically see in our garden or the corners of our houses. Although spider webs from different species vary considerably in geometry and size, many sections of spidroin sequences are conserved. Highly conserved regions, found in all spidroins, relate to the terminal domains of the protein, i.e., the N-terminal (NTD) and C-terminal domains (CTD). Both have an essential function in the silk fibre association and polymerisation. The NTD is a 14 kDa five-helix bundle, which self-associates via a pH-driven mechanism. This process is critical for starting the polymerisation of the fibre. However, detailed insights into how conserved this mechanism is in different species and the quantitative thermodynamic comparison between homologous NTDs was missing. For this reason, four homologous NTDs of the major ampullate gland (MaSp) from spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus were investigated. I analysed and quantified equilibrium thermodynamics, kinetics of folding, and self-association. Methods involved dynamic light scattering (MALS), stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. The results showed conserved, cooperative two-state folding on a sub-millisecond time scale. All homologous NTDs showed a similarly fast association in the order of 10^9 M^−1 s^−1, while the resulting equilibrium dissociation constants were in the low nanomolar range. Electrostatic forces were found to be of great importance for protein association. Monomeric protein stability increased with salt concentration while enhancing its folding speed. However, due to Debye-Hückel effects, we found intermolecular electrostatics to be shielded, which reduced the NTDs association capacity significantly at high ionic strength. Altogether, the energetics and kinetics of the NTD dimerisation was conserved for all analysed homologs. Comparable to the NTD, the spider silks CTD is also a α-helix bundle, which covalently links two spidroins. The orientation of the domains predetermines the future fibre geometry. Here again, the detailed quantitative characterisation of the folding and dimerisation was missing. Therefore, the CTD from the E. australis was analysed in-depth. The protein folded via a three-state mechanism and was placed in the family of knotted proteins. By analysing the amino acid composition of the NTD of the MaSp1 of the Euprosthenops australis, we found an unusually high content of methionine residues (Met). To elucidate why this protein exhibits so many Met residues, I mutated all core Mets simultaneously to leucine (Leu). Results revealed a dramatically stabilised NTD, which now folded 50 times faster. After solving the tertiary structure of the mutant by NMR (nuclear magnetic resonance) spectroscopy, the structure of the monomeric mutant was found to be identical with the wild-type protein. However, when probing the dimerisation of the NTD, I could show that the association capacity was substantially impaired for the mutant. Our findings lead to the conclusion that Met provides the NTD with enhanced conformational dynamics and thus mobilises the protein, which results in tightly associated dimers. In additional experiments, I first re-introduced new Met residues into the Met-depleted protein at sequence positions containing native Leu. Hence, the mutated NTD protein was provided with the same number of Leu, which were previously removed by mutation. However, the protein did not regain wild-type characteristics. The functionality was not restored, but its stability was decreased as expected. To probe our hypothesis gained from the MaSp NTD, I transferred the experiment to another protein, namely the Hsp90 chaperone. Therefore, I incorporated methionine residues in the protein, which resulted in a slight improvement of its function. Finally, trial experiments were performed aiming at the synthesis of shortened spidroin constructs containing less repetitive middle-segments than the wild-type protein. The objective was to study the findings of the terminal domains in the context of an intact spidroin. The synthesis of these engineered spidroins was challenging. Nevertheless, preliminary results encourage the assumption that the characteristics observed in the isolated domains hold true in the context of a full-length spidroin. N2 - Spinnenseide ist ein Biomaterial mit außergewöhnlicher Widerstandsfähigkeit welche gepaart ist mit Elastizität. Das Zusammenfügen von Seidenproteinen aus so ge-nannten Spidroinen (ein Kunstwort aus „Spinne“ und „Fibroin“) erzeugt die Seiden-fäden, die wir typischerweise in unseren Gärten oder in den Ecken unserer Häuser finden. Obwohl Spinnennetze von verschiedenen Spinnenarten in Geometrie und Größe stark variieren, sind große Teile der Spidroin-Sequenzen konserviert. Stark konservierte Bereiche, die in allen Spidroinen vorkommen, sind die endständigen Bereiche des Proteins, die N-terminale (NTD) und C-terminale Domäne (CTD) ge-nannt werden. Beide haben wichtige Funktionen in der Assoziation der Proteine im Spinnkanal und deren Polymerisation zur Ausbildung des Seidenfadens. Die NTD ist ein kleines 14 kDa Protein, bestehend aus einem Bündel aus fünf Helices, dessen Selbstorganisation pH-abhängig ist. Dieser Prozess leitet die Poly-merisation der Faser ein. Allerdings fehlten bis heute Informationen darüber, ob dieser Mechanismus bei homologen Domänen aus verschiedenen Spinnenarten konser¬viert ist, da kaum quantitative biophysikalische Daten vorhanden sind. Aus diesem Grund wurden vier homologe NTDs der Spinnenarten Euprosthenops australis, Nephila clavipes, Latrodectus hesperus und Latrodectus geometricus vergleichend untersucht und deren Gleichgewichts-Thermodynamik, die Kinetik der Faltung und die Selbstassoziation quantitativ analysiert. Dazu wurden Methoden wie dynamische Mehrwinkel-Lichtstreuung (MALS), Stopped-Flow Fluoreszenz-spektroskopie und Zirkulardichroismus in Kombination mit thermischen und chemischen Denaturierungs¬experimenten angewandt. Die Ergebnisse lieferten die Erkenntnis einer kooperativen Zwei-Zustands-Faltung, die auf einer Zeitskala von weniger als einer Millisekunde stattfand. Alle homologen NTDs zeigten eine schnelle Assoziationsratenkonstante in der Größenordnung von 10^9 M^-1 s^-1, während die Gleichgewichts-Dissoziationskonstante für alle Homologe im nied¬rigen nano-molaren Bereich lag. Die Proteinassoziation wurde durch elektrostatische Kräfte gesteuert, wobei hohe Salzkonzentrationen die Stabilität des monomeren Proteins und dessen Faltungsgeschwindigkeit erhöhten. Die Assoziation zweier Domänen wurde jedoch durch Abschirmung intermolekularer elektrostatischer Kräfte, dem Debye-Hückel-Gesetz zufolge, reduziert. Die Energetik und Kinetik der NTD-Dimerisierung aller untersuchten Homologen erwies sich konserviert. Ebenso wie die NTD, ist auch die CTD der Spinnenseide ein α-helikales Bündel, welche jedoch zwei Spidroine kovalent miteinander verbindet. Die Orientierung der verknüpften Domäne bestimmt bereits die zukünftige Faserstruktur. Ähnlich wie bei der NTD, waren Faltung und Dimerisierung der CTD bisher nicht im Detail be-schrieben. Durch eine detaillierte Analyse der CTD der E. australis konnte gezeigt werden, dass das Protein sich in einem dreistufigen Mechanismus faltet und außerdem der Familie der geknoteten Proteine angehört. Bei genauerer Betrachtung der Aminosäurezusammensetzung der E. australis NTD konnte ein ungewöhnlich hoher Anteil der Aminosäure Methionin (Met) festge¬stellt werden. Um diesen überraschenden Sachverhalt zu verstehen, habe ich alle im Kern liegenden Met zu Leucin (Leu) mutiert. Die Ergebnisse zeigten eine extrem stabilisierte NTD, welche sich nun 50-fach schneller faltete. Die Protein¬struktur der Mutante wurde in Lösung mittels NMR Spektroskopie ermittelt. Das Ergebnis lieferte deckungsgleiche Strukturen von Mutante und Wildtyp im monomeren Zustand. Allerdings zeigten NTD Dimerisierungs-Versuche, dass die Assoziations-fähigkeit der Mutante erheblich beeinträchtigt war. Untersuchungen der nativen Dynamik mittels NMR und Fluoreszenzkorrelationsspektroskopie zeigten, dass Met diese entscheidend verstärkt, was zu einem eng assoziierten Dimer führte. Im Versuch die Dynamik wieder künstlich herzustellen, habe ich neue Met in die Mutante eingeführt, auf Sequenzpositionen welche natürlicherweise Leu aufweisen. Somit wurde die ursprüngliche Anzahl an Met in der NTD wiederher¬gestellt, jedoch an anderen Positionen. Obwohl das Protein wie erwartet an Stabilität verlor, konnte dessen Funktionalität nicht wiederhergestellt werden. Um unsere Erkenntnisse auf andere Proteine zu übertragen, wurden Met Reste künstlich in ein Hsp90 Protein eingeführt. Es konnte eine leicht verbesserte Funktionalität des Proteins beobachtet werden. Schließlich wurde versucht, die für die CTD und NTD gewonnen Erkenntnisse auf intakte, jedoch verkürzte Spidroine zu übertragen. Dazu wurden Spidroine mit weniger repetitiven Mittelsegmenten mittels rekombinanten Methoden hergestellt. Die Synthese dieser Spidroine erwies sich als Herausforderung. Allerdings zeigten die vorläufigen Ergebnisse, dass eine Verallgemeinerung der Erkenntnisse der isolierten Domänen auf das Volllängen-Spidroin möglich ist. KW - Spinnenseide KW - Fluoreszenzspektroskopie KW - Terminale Domaine KW - Spider Silk KW - Fluorescence spectroscopy KW - Terminal domains Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193455 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 - Hartke, Juliane A1 - Waldvogel, Ann‐Marie A1 - Sprenger, Philipp P. A1 - Schmitt, Thomas A1 - Menzel, Florian A1 - Pfenninger, Markus A1 - Feldmeyer, Barbara T1 - Little parallelism in genomic signatures of local adaptation in two sympatric, cryptic sister species JF - Journal of Evolutionary Biology N2 - Species living in sympatry and sharing a similar niche often express parallel phenotypes as a response to similar selection pressures. The degree of parallelism within underlying genomic levels is often unexplored, but can give insight into the mechanisms of natural selection and adaptation. Here, we use multi‐dimensional genomic associations to assess the basis of local and climate adaptation in two sympatric, cryptic Crematogaster levior ant species along a climate gradient. Additionally, we investigate the genomic basis of chemical communication in both species. Communication in insects is mainly mediated by cuticular hydrocarbons (CHCs), which also protect against water loss and, hence, are subject to changes via environmental acclimation or adaptation. The combination of environmental and chemical association analyses based on genome‐wide Pool‐Seq data allowed us to identify single nucleotide polymorphisms (SNPs) associated with climate and with chemical differences. Within species, CHC changes as a response to climate seem to be driven by phenotypic plasticity, since there is no overlap between climate‐ and CHC‐associated SNPs. The only exception is the odorant receptor OR22c, which may be a candidate for population‐specific CHC recognition in one of the species. Within both species, climate is significantly correlated with CHC differences, as well as to allele frequency differences. However, associated candidate SNPs, genes and functions are largely species‐specific and we find evidence for minimal parallel evolution only on the level of genomic regions (J = 0.04). This highlights that even closely related species may follow divergent evolutionary trajectories when expressing similar adaptive phenotypes. KW - BayPass KW - environmental association analysis KW - Formicidae KW - mutualism KW - parallel evolution KW - population divergence Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228355 VL - 34 IS - 6 SP - 937 EP - 952 ER - TY - JOUR A1 - Hagge, Jonas A1 - Müller, Jörg A1 - Birkemoe, Tone A1 - Buse, Jörn A1 - Christensen, Rune Haubo Bojesen A1 - Gossner, Martin M. A1 - Gruppe, Axel A1 - Heibl, Christoph A1 - Jarzabek‐Müller, Andrea A1 - Seibold, Sebastian A1 - Siitonen, Juha A1 - Soutinho, João Gonçalo A1 - Sverdrup‐Thygeson, Anne A1 - Thorn, Simon A1 - Drag, Lukas T1 - What does a threatened saproxylic beetle look like? Modelling extinction risk using a new morphological trait database JF - Journal of Animal Ecology N2 - The extinction of species is a non‐random process, and understanding why some species are more likely to go extinct than others is critical for conservation efforts. Functional trait‐based approaches offer a promising tool to achieve this goal. In forests, deadwood‐dependent (saproxylic) beetles comprise a major part of threatened species, but analyses of their extinction risk have been hindered by the availability of suitable morphological traits. To better understand the mechanisms underlying extinction in insects, we investigated the relationships between morphological features and the extinction risk of saproxylic beetles. Specifically, we hypothesised that species darker in colour, with a larger and rounder body, a lower mobility, lower sensory perception and more robust mandibles are at higher risk. We first developed a protocol for morphological trait measurements and present a database of 37 traits for 1,157 European saproxylic beetle species. Based on 13 selected, independent traits characterising aspects of colour, body shape, locomotion, sensory perception and foraging, we used a proportional‐odds multiple linear mixed‐effects model to model the German Red List categories of 744 species as an ordinal index of extinction risk. Six out of 13 traits correlated significantly with extinction risk. Larger species as well as species with a broad and round body had a higher extinction risk than small, slim and flattened species. Species with short wings had a higher extinction risk than those with long wings. On the contrary, extinction risk increased with decreasing wing load and with higher mandibular aspect ratio (shorter and more robust mandibles). Our study provides new insights into how morphological traits, beyond the widely used body size, determine the extinction risk of saproxylic beetles. Moreover, our approach shows that the morphological characteristics of beetles can be comprehensively represented by a selection of 13 traits. We recommend them as a starting point for functional analyses in the rapidly growing field of ecological and conservation studies of deadwood. KW - deadwood KW - extinction risk KW - forest biodiversity KW - forestry KW - functional traits KW - morphometry KW - red lists KW - saproxylic beetles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244717 VL - 90 IS - 8 SP - 1934 EP - 1947 ER - TY - JOUR A1 - Habenstein, Jens A1 - Thamm, Markus A1 - Rössler, Wolfgang T1 - Neuropeptides as potential modulators of behavioral transitions in the ant Cataglyphis nodus JF - Journal of Comparative Neurology N2 - Age‐related behavioral plasticity is a major prerequisite for the ecological success of insect societies. Although ecological aspects of behavioral flexibility have been targeted in many studies, the underlying intrinsic mechanisms controlling the diverse changes in behavior along the individual life history of social insects are not completely understood. Recently, the neuropeptides allatostatin‐A, corazonin, and tachykinin have been associated with the regulation of behavioral transitions in social insects. Here, we investigated changes in brain localization and expression of these neuropeptides following major behavioral transitions in Cataglyphis nodus ants. Our immunohistochemical analyses in the brain revealed that the overall branching pattern of neurons immunoreactive (ir) for the three neuropeptides is largely independent of the behavioral stages. Numerous allatostatin‐A‐ and tachykinin‐ir neurons innervate primary sensory neuropils and high‐order integration centers of the brain. In contrast, the number of corazonergic neurons is restricted to only four neurons per brain hemisphere with cell bodies located in the pars lateralis and axons extending to the medial protocerebrum and the retrocerebral complex. Most interestingly, the cell‐body volumes of these neurons are significantly increased in foragers compared to freshly eclosed ants and interior workers. Quantification of mRNA expression levels revealed a stage‐related change in the expression of allatostatin‐A and corazonin mRNA in the brain. Given the presence of the neuropeptides in major control centers of the brain and the neurohemal organs, these mRNA‐changes strongly suggest an important modulatory role of both neuropeptides in the behavioral maturation of Cataglyphis ants. KW - allatostatin‐A KW - corazonin KW - division of labor KW - neuropeptides KW - social insects KW - tachykinin KW - Cataglyphis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244751 VL - 529 IS - 12 SP - 3155 EP - 3170 ER - TY - JOUR A1 - Habenstein, Jens A1 - Schmitt, Franziska A1 - Liessem, Sander A1 - Ly, Alice A1 - Trede, Dennis A1 - Wegener, Christian A1 - Predel, Reinhard A1 - Rössler, Wolfgang A1 - Neupert, Susanne T1 - Transcriptomic, peptidomic, and mass spectrometry imaging analysis of the brain in the ant Cataglyphis nodus JF - Journal of Neurochemistry N2 - Behavioral flexibility is an important cornerstone for the ecological success of animals. Social Cataglyphis nodus ants with their age‐related polyethism characterized by age‐related behavioral phenotypes represent a prime example for behavioral flexibility. We propose neuropeptides as powerful candidates for the flexible modulation of age‐related behavioral transitions in individual ants. As the neuropeptidome of C. nodus was unknown, we collected a comprehensive peptidomic data set obtained by transcriptome analysis of the ants’ central nervous system combined with brain extract analysis by Q‐Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different regions of the brain by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. In total, we identified 71 peptides with likely bioactive function, encoded on 49 neuropeptide‐, neuropeptide‐like, and protein hormone prepropeptide genes, including a novel neuropeptide‐like gene (fliktin). We next characterized the spatial distribution of a subset of peptides encoded on 16 precursor proteins with high resolution by MALDI MS imaging (MALDI MSI) on 14 µm brain sections. The accuracy of our MSI data were confirmed by matching the immunostaining patterns for tachykinins with MSI ion images from consecutive brain sections. Our data provide a solid framework for future research into spatially resolved qualitative and quantitative peptidomic changes associated with stage‐specific behavioral transitions and the functional role of neuropeptides in Cataglyphis ants. KW - brain KW - MALDI imaging KW - neuropeptides KW - neuropeptidomics KW - social insect KW - transcriptomics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239917 VL - 158 IS - 2 SP - 391 EP - 412 ER - TY - THES A1 - Habenstein, Jens T1 - Neuropeptides in the brain of \(Cataglyphis\) \(nodus\) ants and their role as potential modulators of behavior T1 - Neuropeptide im Gehirn von \(Cataglyphis\) \(nodus\) Ameisen und ihre Rolle als potenzielle Modulatoren von Verhalten N2 - An adequate task allocation among colony members is of particular importance in large insect societies. Some species exhibit distinct polymorphic worker classes which are responsible for a specific range of tasks. However, much more often the behavior of the workers is related to the age of the individual. Ants of the genus Cataglyphis (Foerster 1850) undergo a marked age-related polyethism with three distinct behavioral stages. Newly emerged ants (callows) remain more or less motionless in the nest for the first day. The ants subsequently fulfill different tasks inside the darkness of the nest for up to four weeks (interior workers) before they finally leave the nest to collect food for the colony (foragers). This thesis focuses on the neuronal substrate underlying the temporal polyethism in Cataglyphis nodus ants by addressing following major objectives: (1) Investigating the structures and neuronal circuitries of the Cataglyphis brain to understand potential effects of neuromodulators in specific brain neuropils. (2) Identification and localization of neuropeptides in the Cataglyphis brain. (3) Examining the expression of suitable neuropeptide candidates during behavioral maturation of Cataglyphis workers. The brain provides the fundament for the control of the behavioral output of an insect. Although the importance of the central nervous system is known beyond doubt, the functional significance of large areas of the insect brain are not completely understood. In Cataglyphis ants, previous studies focused almost exclusively on major neuropils while large proportions of the central protocerebrum have been often disregarded due to the lack of clear boundaries. Therefore, I reconstructed a three-dimensional Cataglyphis brain employing confocal laser scanning microscopy. To visualize synapsin-rich neuropils and fiber tracts, a combination of fluorescently labeled antibodies, phalloidin (a cyclic peptide binding to filamentous actin) and anterograde tracers was used. Based on the unified nomenclature for insect brains, I defined traceable criteria for the demarcation of individual neuropils. The resulting three-dimensional brain atlas provides information about 33 distinct synapse-rich neuropils and 30 fiber tracts, including a comprehensive description of the olfactory and visual tracts in the Cataglyphis brain. This three-dimensional brain atlas further allows to assign present neuromodulators to individual brain neuropils. Neuropeptides represent the largest group of neuromodulators in the central nervous system of insects. They regulate important physiological and behavioral processes and have therefore recently been associated with the regulation of the temporal polyethism in social insects. To date, the knowledge of neuropeptides in Cataglyphis ants has been mainly derived from neuropeptidomic data of Camponotus floridanus ants and only a few neuropeptides have been characterized in Cataglyphis. Therefore, I performed a comprehensive transcriptome analysis in Cataglyphis nodus ants and identified peptides by using Q-Exactive Orbitrap mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. This resulted in the characterization of 71 peptides encoded on 49 prepropeptide genes, including a novel neuropeptide-like gene (fliktin). In addition, high-resolution MALDI-TOF MS imaging (MALDI-MSI) was applied for the first time in an ant brain to localize peptides on thin brain cryosections. Employing MALDI-MSI, I was able to visualize the spatial distribution of 35 peptides encoded on 16 genes. To investigate the role of neuropeptides during behavioral maturation, I selected suitable neuropeptide candidates and analyzed their spatial distributions and expression levels following major behavioral transitions. Based on recent studies, I suggested the neuropeptides allatostatin-A (Ast-A), corazonin (Crz) and tachykinin (TK) as potential regulators of the temporal polyethism. The peptidergic neurons were visualized in the brain of C. nodus ants using immunohistochemistry. Independent of the behavioral stages, numerous Ast-A- and TK-immunoreactive (-ir) neurons innervate important high-order integration centers and sensory input regions with cell bodies dispersed all across the cell body rind. In contrast, only four corazonergic neurons per hemisphere were found in the Cataglyphis brain. Their somata are localized in the pars lateralis with axons projecting to the medial protocerebrum and the retrocerebral complex. Number and branching patterns of the Crz-ir neurons were similar across behavioral stages, however, the volume of the cell bodies was significantly larger in foragers than in the preceding behavioral stages. In addition, quantitative PCR analyses displayed increased Crz and Ast-A mRNA levels in foragers, suggesting a concomitant increase of the peptide levels. The task-specific expression of Crz and Ast-A along with the presence in important sensory input regions, high-order integration center, and the neurohormonal organs indicate a sustaining role of the neuropeptides during behavioral maturation of Cataglyphis workers. The present thesis contains a comprehensive reference work for the brain anatomy and the neuropeptidome of Cataglyphis ants. I further demonstrated that neuropeptides are suitable modulators for the temporal polyethism of Cataglyphis workers. The complete dataset provides a solid framework for future neuroethological studies in Cataglyphis ants as well as for comparative studies on insects. This may help to improve our understanding of the functionality of individual brain neuropils and the role of neuropeptides, particularly during behavioral maturation in social insects. N2 - Eine adäquate Aufgabenverteilung unter den Koloniemitgliedern ist in großen Insektengesellschaften von besonderer Bedeutung. Einige Arten weisen polymorphe Arbeiterklassen auf, die jeweils für einen bestimmten Aufgabenbereich zuständig sind. Viel häufiger jedoch steht das Verhalten der Arbeiterinnen im Zusammenhang mit dem Alter der Individuen. Ameisen der Gattung Cataglyphis (Foerster 1850) weisen einen ausgeprägten alterskorrelierten Polyethismus auf, der sich durch drei unterschiedliche Verhaltensstadien kennzeichnet. Neu geschlüpfte Ameisen (Callows) verharren den ersten Tag mehr oder weniger bewegungslos im Nest. Anschließend erfüllen die Ameisen in der Dunkelheit des Nestes bis zu vier Wochen lang verschiedene Aufgaben (Interior), bevor sie schließlich das Nest verlassen, um Nahrung für die Kolonie zu sammeln (Forager). Diese Arbeit konzentriert sich auf die neuronalen Grundlagen, die dem alterskorrelierten Polyethismus bei Cataglyphis nodus Ameisen zugrunde liegt, indem folgende Hauptziele verfolgt werden: (1) Untersuchung der Strukturen und der neuronalen Schaltkreise des Cataglyphis-Gehirns, um mögliche Effekte von Neuromodulatoren in spezifischen Hirnneuropilen besser zu verstehen. (2) Identifizierung und Lokalisierung von Neuropeptiden im Gehirn von Cataglyphis Ameisen. (3) Untersuchung der Expression geeigneter Neuropeptid-Kandidaten im Zuge der Verhaltensreifung von Cataglyphis Arbeitern. Das Gehirn bildet die Grundlage für die Steuerung des Verhaltens von Insekten. Obwohl die tragende Rolle des zentralen Nervensystems für das Verhalten zweifelsfrei bekannt ist, sind die funktionellen Aufgaben großer Bereiche des Insektengehirns nicht vollständig erforscht. Bei Cataglyphis Ameisen konzentrierten sich vorangegangene Studien fast ausschließlich auf die Hauptneuropile, während große Teile des zentralen Protocerebrums mangels klarer Abgrenzungen weitgehend unberücksichtigt geblieben sind. Daher habe ich ein dreidimensionales Cataglyphis-Gehirn mit Hilfe der konfokalen Laser-Scanning-Mikroskopie rekonstruiert. Um die synapsinreichen Neuropile und Nerventrakte zu visualisieren, wurde eine Kombination aus fluoreszenzgekoppelten Antikörpern, Phalloidin (ein zyklisches Peptid, das an filamentöses Aktin bindet) und anterograden Tracern verwendet. Basierend auf der einheitlichen Nomenklatur für Insektengehirne definierte ich nachvollziehbare Kriterien für die Abgrenzung der einzelnen Neuropile. Die resultierende dreidimensionale neuronale Karte liefert Informationen über 33 verschiedene synapsinreiche Neuropile und 30 Nerventrakte, einschließlich einer umfassenden Beschreibung der olfaktorischen und visuellen Trakte im Cataglyphis-Gehirn. Dieser dreidimensionale Hirnatlas erlaubt es darüber hinaus, die vorhandenen Neuromodulatoren einzelnen Neuropilen des Gehirns zuzuordnen. Neuropeptide stellen die umfangreichste Gruppe an Neuromodulatoren im zentralen Nervensystem von Insekten dar. Sie regulieren wichtige physiologische Prozesse und Verhaltensweisen und wurden deshalb in jüngerer Vergangenheit mit der Regulation des alterskorrelierenden Polyethismus bei sozialen Insekten in Verbindung gebracht. Bislang wurde das Wissen über Neuropeptide bei Cataglyphis Ameisen hauptsächlich aus neuropeptidomischen Daten von Camponotus floridanus Ameisen abgeleitet und nur wenige Neuropeptide wurden bei Cataglyphis charakterisiert. Daher führte ich eine umfassende Transkriptomanalyse bei Cataglyphis nodus Ameisen durch und identifizierte Peptide mit Hilfe der Q-Exactive Orbitrap Massenspektrometrie (MS) und der Matrix-assistierte Laser Desorption-Ionisierung Time-of-Flight (MALDI-TOF) MS. Hierdurch konnten insgesamt 71 Peptide charakterisiert werden, die auf 49 Präpropeptid-Genen kodiert sind, einschließlich eines neuartigen Neuropeptid-ähnlichen Gens (Fliktin). Darüber hinaus wurde das hochauflösende MALDI-TOF MS-Imaging (MALDI-MSI) zum ersten Mal in einem Ameisenhirn angewandt, um Peptide auf dünnen Hirnkryoschnitten zu lokalisieren. Mittels MALDI-MSI konnte ich die räumliche Verteilung von 35 Peptiden sichtbar machen, die auf 16 Genen kodiert sind. Um die Rolle der Neuropeptide während der Verhaltensreifung zu untersuchen, wählte ich geeignete Neuropeptid-Kandidaten aus und analysierte deren räumliche Verteilung und Expressionsniveaus im Zuge wichtiger Verhaltensübergänge. Basierend auf aktuellen Studien schlug ich die Neuropeptide Allatostatin-A (Ast-A), Corazonin (Crz) und Tachykinin (TK) als mögliche Regulatoren des alterskorrelierenden Polyethismus vor. Die peptidergen Neurone wurden im Gehirn von C. nodus Ameisen mittels Immunhistochemie sichtbar gemacht. Unabhängig von den Verhaltensstadien innervieren die zahlreichen Ast-A- und TK-immunreaktiven (-ir) Neuronen wichtige Integrationszentren höherer Ordnung sowie sensorische Eingangsregionen, während ihre Zellkörper über die gesamte Zellkörperschicht verteilt sind. Im Gegensatz dazu wurden im Cataglyphis-Gehirn nur vier corazonerge Neuronen pro Hemisphäre gefunden. Ihre Somata sind in der Pars lateralis lokalisiert, deren Axone in das mediale Protocerebrum und den retrozerebralen Komplex projizieren. Anzahl und Verzweigungsmuster der Crz-ir Neuronen waren in allen Verhaltensstadien ähnlich, jedoch war das Volumen der Zellkörper bei Foragern signifikant größer als in den vorangegangenen Verhaltensstadien. Darüber hinaus zeigten quantitative PCR Analysen erhöhte Crz- und Ast-A mRNA-Level in Foragern, was auf einen gleichzeitigen Anstieg der Peptidspiegel schließen lässt. Die aufgabenspezifische Expression von Crz und Ast-A sowie deren Präsenz in wichtigen sensorischen Eingangsbereichen, Integrationszentren höherer Ordnung und den neurohormonellen Organen weisen auf eine tragende Rolle der Neuropeptide während der Verhaltensreifung von Cataglyphis Arbeiterinnen hin. Die vorliegende Arbeit beinhaltet ein umfassendes Nachschlagewerk für die Hirnanatomie und das Neuropeptidom von Cataglyphis Ameisen. Zudem konnte ich demonstrieren, dass Neuropeptide geeignete Modulatoren für den alterskorrelierenden Polyethismus von Cataglyphis Arbeitern sind. Der komplette Datensatz bietet eine solide Grundlage für zukünftige, neuroethologische Studien an Cataglyphis Ameisen sowie vergleichenden Studien in Insekten. Hierdurch kann unser Verständnis über die Funktionalität einzelner Hirnneuropile und die Rolle von Neuropeptiden, insbesondere während der Verhaltensreifung sozialer Insekten, in Zukunft verbessert werden. KW - Cataglyphis KW - Neuropeptide KW - Neuroethologie KW - Insektenstaaten KW - polyethism KW - neuropeptides KW - neuroethology KW - neuromodulation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249618 ER - TY - JOUR A1 - Haack, Stephanie A1 - Baiker, Sarah A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Sparwasser, Tim A1 - Langenhorst, Daniela A1 - Beyersdorf, Niklas T1 - Superagonistic CD28 stimulation induces IFN‐γ release from mouse T helper 1 cells in vitro and in vivo JF - European Journal of Immunology N2 - Like human Th1 cells, mouse Th1 cells also secrete IFN‐γ upon stimulation with a superagonistic anti‐CD28 monoclonal antibody (CD28‐SA). Crosslinking of the CD28‐SA via FcR and CD40‐CD40L interactions greatly increased IFN‐γ release. Our data stress the utility of the mouse as a model organism for immune responses in humans. KW - CD28 KW - Th1 cells KW - cytokine release KW - interferon γ KW - Superagonistic antibody Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239028 VL - 51 IS - 3 SP - 738 EP - 741 ER - TY - JOUR A1 - Gupta, Shishir K. A1 - Srivastava, Mugdha A1 - Osmanoglu, Özge A1 - Xu, Zhuofei A1 - Brakhage, Axel A. A1 - Dandekar, Thomas T1 - Aspergillus fumigatus versus genus Aspergillus: conservation, adaptive evolution and specific virulence genes JF - Microorganisms N2 - Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes. KW - molecular evolution KW - phylogenetic analysis KW - adaptation KW - recombination KW - positive selection KW - human pathogenic fungi KW - genus Aspergillus KW - Aspergillus fumigatus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246318 SN - 2076-2607 VL - 9 IS - 10 ER - TY - JOUR A1 - Gupta, Shishir K. A1 - Srivastava, Mugdha A1 - Minocha, Rashmi A1 - Akash, Aman A1 - Dangwal, Seema A1 - Dandekar, Thomas T1 - Alveolar regeneration in COVID-19 patients: a network perspective JF - International Journal of Molecular Sciences N2 - A viral infection involves entry and replication of viral nucleic acid in a host organism, subsequently leading to biochemical and structural alterations in the host cell. In the case of SARS-CoV-2 viral infection, over-activation of the host immune system may lead to lung damage. Albeit the regeneration and fibrotic repair processes being the two protective host responses, prolonged injury may lead to excessive fibrosis, a pathological state that can result in lung collapse. In this review, we discuss regeneration and fibrosis processes in response to SARS-CoV-2 and provide our viewpoint on the triggering of alveolar regeneration in coronavirus disease 2019 (COVID-19) patients. KW - COVID-19 KW - SARS-CoV-2 KW - alveolar regeneration KW - alveolar fibrosis KW - signaling pathway KW - network biology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284307 SN - 1422-0067 VL - 22 IS - 20 ER - TY - THES A1 - Gründl, Marco T1 - Biochemical characterization of the MMB-Hippo crosstalk and its physiological relevance for heart development T1 - Biochemische Charakterisierung des MMB-Hippo Signalweges und dessen physiologische Rolle in der Herzentwicklung N2 - The Myb-MuvB (MMB) complex plays an essential role in the time-dependent transcriptional activation of mitotic genes. Recently, our laboratory identified a novel crosstalk between the MMB-complex and YAP, the transcriptional coactivator of the Hippo pathway, to coregulate a subset of mitotic genes (Pattschull et al., 2019). Several genetic studies have shown that the Hippo-YAP pathway is essential to drive cardiomyocyte proliferation during cardiac development (von Gise et al., 2012; Heallen et al., 2011; Xin et al., 2011). However, the exact mechanisms of how YAP activates proliferation of cardiomyocytes is not known. This doctoral thesis addresses the physiological role of the MMB-Hippo crosstalk within the heart and characterizes the YAP-B-MYB interaction with the overall aim to identify a potent inhibitor of YAP. The results reported in this thesis indicate that complete loss of the MMB scaffold protein LIN9 in heart progenitor cells results in thinning of ventricular walls, reduced cardiomyocyte proliferation and early embryonic lethality. Moreover, genetic experiments using mice deficient in SAV1, a core component of the Hippo pathway, and LIN9-deficient mice revealed that the correct function of the MMB complex is critical for proliferation of cardiomyocytes due to Hippo-deficiency. Whole genome transcriptome profiling as well as genome wide binding studies identified a subset of Hippo-regulated cell cycle genes as direct targets of MMB. By proximity ligation assay (PLA), YAP and B-MYB were discovered to interact in embryonal cardiomyocytes. Biochemical approaches, such as co-immunoprecipitation assays, GST-pulldown assays, and µSPOT-based peptide arrays were employed to characterize the YAP-B-MYB interaction. Here, a PY motif within the N-terminus of B-MYB was found to directly interact with the YAP WW-domains. Consequently, the YAP WW-domains were important for the ability of YAP to drive proliferation in cardiomyocytes and to activate MMB target genes in differentiated C2C12 cells. The biochemical information obtained from the interaction studies was utilized to develop a novel competitive inhibitor of YAP called MY-COMP (Myb-YAP competition). In MY-COMP, the protein fragment of B-MYB containing the YAP binding domain is fused to a nuclear localization signal. Co-immunoprecipitation studies as well as PLA revealed that the YAP-B-MYB interaction is robustly blocked by expression of MY-COMP. Adenoviral overexpression of MY-COMP in embryonal cardiomyocytes suppressed entry into mitosis and blocked the pro-proliferative function of YAP. Strikingly, characterization of the cellular phenotype showed that ectopic expression of MY-COMP led to growth defects, nuclear abnormalities and polyploidization in HeLa cells. Taken together, the results of this thesis reveal the mechanism of the crosstalk between the Hippo signaling pathway and the MMB complex in the heart and form the basis for interference with the oncogenic activity of the Hippo coactivator YAP. N2 - Der Myb-MuvB Komplex spielt eine essenzielle Rolle in der transkriptionellen Aktivierung von Zellzyklusgenen. Unser Labor hat kürzlich einen bis dahin unbekannten Mechanismus zwischen dem MMB-Komplex und Hippo-YAP Signalweg, der zur Aktivierung von Mitosegenen beiträgt, identifiziert. Der Hippo-YAP Signalweg ist beteiligt an der Gewebehomöostase und am Wachstum von Organen. So reguliert der Hippo-YAP Signalweg zum Beispiel während der Herzentwicklung die Proliferation von Herzmuskelzellen. Der exakte Mechanismus wie YAP die Zellteilung von Kardiomyozyten aktiviert, ist jedoch bisher nicht bekannt. In der vorliegenden Doktorarbeit wird das Zusammenspiel zwischen dem Hippo-Signalweg und dem MMB-Komplex im Herzen untersucht. Außerdem wird die Interaktion zwischen YAP und B-MYB biochemisch charakterisiert, um einen Inhibitor zu entwickeln, der die Aktivität von YAP vermindert. Die Ergebnisse dieser Doktorarbeit zeigen, dass der Verlust der zentralen Untereinheit des MMB-Komplexes, LIN9, in Vorläuferzellen der Kardiomyozyten zu einer Reduktion der Herzwand sowie zu einer niedrigeren Proliferationsrate von Herzmuskelzellen und einer erhöhten Embryonalsterblichkeit führt. Außerdem wurde in genetischen Experimenten mit Hippo- und LIN9-defizienten Mäusen gezeigt, dass der MMB-Komplex wichtig für die Aktivierung der Proliferation in Hippo-defizienten Kardiomyozyten ist. Eine globale Analyse der Transkription und Chromatinbindung von YAP und LIN9 im Herzen zeigte, dass eine Untergruppe von Zellzyklusgenen, die nach Inaktivierung des Hippo-Signalwegs vermehrt exprimiert werden, gleichzeitig den MMB-Komplex am Promoter gebunden haben. Durch Interaktionsstudien konnte gezeigt werden, dass YAP und B-MYB in embryonalen Kardiomyozyten miteinander interagieren. Die Bindung der beiden Transkriptionsfaktoren wurde durch Co-Immunpräzipitation, GST-Pulldown-Analysen und Peptid-Arrays biochemisch untersucht. Dabei wurde gezeigt, dass ein PY-Motiv im N-terminus von B-MYB direkt an die WW-Domänen von YAP bindet. Im Umkehrschluss wurde festgestellt, dass die WW-Domänen von YAP essenziell sind, um sowohl die Proliferation in Herzmuskelzellen als auch die Expression von Mitosegenen in differenzierten C2C12 Zellen zu aktivieren. Letztendlich wurden die Ergebnisse der Interaktionsstudie genutzt, um einen neuartigen kompetitiven Inhibitor von YAP zu entwickeln. Für MY-COMP (Myb-YAP Competition) wurde der Proteinabschnitt von B-MYB, der die YAP Bindedomäne enthält, mit einer Kernlokalisierungssequenz fusioniert. Bindestudien zeigten, dass MY-COMP die Interaktion zwischen YAP und B-MYB effektiv blockiert. Eine durch Adenoviren vermittelte Überexpression von MY-COMP in embryonalen Herzmuskelzellen resultierte in einer verminderten Anzahl von mitotischen Zellen. Somit wird durch Expression von MY-COMP, die proliferative Fähigkeit von YAP vermindert. Interessanterweise wurden in HeLa Zellen, die mit MY-COMP behandelt wurden, vermehrt Abnormalitäten der Zellkerne, polyploide Zellen sowie ein Wachstumsdefizit beobachtet. Zusammengefasst verdeutlichen die Ergebnisse dieser Doktorarbeit die Bedeutung des Zusammenspiels zwischen dem MMB-Komplex und dem Hippo-YAP-Signalweg für die Herzentwicklung und bilden die Grundlage, für die effektive Inhibierung der onkogenen Eigenschaften des Hippo-Coaktivators YAP. KW - Zellzyklus KW - Heart development KW - Hippo pathway KW - Myb-MuvB complex KW - Cardiomyocyte Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213328 ER - TY - THES A1 - Groma, Michaela T1 - Identification of a novel LysR-type transcriptional regulator in \(Staphylococcus\) \(aureus\) T1 - Identifizierung eines neuen Transkriptionsregulators vom LysR-Typ in \(Staphylococcus\) \(aureus\) N2 - Staphylococcus aureus is a facultative pathogen which causes a variety of infections. The treatment of staphylococcal infections is complicated because the bacteria is resistant to multiple common antibiotics. S. aureus is also known to express a variety of virulence factors which modulate the host’s immune response in order to colonize and invade certain host cells, leading to the host cell’s death. Among the virulence factors is a LysR-type transcriptional regulator (lttr) which is required for efficient colonization of secondary organs. In a recent report, which used transposon screening on S. aureus-infected mice, it was found that the amount of a novel lttr852 mutant bacteria recovered from the kidneys was significantly lower compared to the wildtype strains. This doctoral thesis therefore focused on phenotypical and molecular characterization of lttr852. An assessment of the S. aureus biofilm formation and the hemolysis revealed that lttr852 was not involved in the regulation of these virulence processes. RNA-sequencing for potential target genes of lttr852 identified differentially expressed genes that are involved in branched chain amino-acid biosynthesis, methionine sulfoxide reductase and copper transport, as well as a reduced transcription of genes encoding urease and of components of pyrimidine nucleotides. Promoter fusion with GFP reporters as as well as OmniLog were used to identify conditions under which the lttr852 was active. The promoter studies showed that glucose and high temperatures diminish the lttr852 promoter activity in a time-dependent manner, while micro-aerobic conditions enhanced the promoter activity. Copper was found to be a limiting factor. In addition, the impact on promoter activity of the lttr852 was tested in the presence of various regulators, but no central link to the genes involved in virulence was identified. The present work, thus, showed that lttr852, a new member of the class of LysR-type transcriptional regulators in S. aureus, has an important role in the rapid adaptation of S. aureus to the changing microenvironment of the host. N2 - Staphylococcus aureus ist ein fakultativer Erreger, der eine Vielzahl von Infektionen verursacht. Die Behandlung von Staphylokokken-Infektionen ist aufgrund des Auftretens einer Resistenz gegen mehrere gängige Antibiotika kompliziert. Es ist bekannt, dass S. aureus eine Vielzahl von Virulenzfaktoren exprimiert, um die Immunantwort des Wirts zu umgehen, und so in bestimmte Wirtszellen einzudringen und diese zu kolonisieren, was zum Tod von Wirtszellen führen kann. Unter den Virulenzfaktoren befindet sich ein Transkriptions-regulator vom LysR-Typ (lttr), der für eine effiziente Besiedlung von Sekundärorganen erforderlich ist. In einem kürzlich durchgeführten Transposon-Screen, bei dem Mäuse mit S. aureus infiziert wurden, wurde ein neuartiger lttr, der lttr852 identifiziert, bei dem aus den Nieren gewonnenen Bakterien signifikant dezimiert waren. Diese Doktorarbeit befasste sich mit der phänotypischen und molekularen Charakterisierung von lttr852. Die Auswertung der Biofilmbildung und der Hämolyse von S. aureus ergab, dass lttr852 nicht an der Regulation dieser Virulenzprozesse beteiligt war. Die RNA-Sequenzierung für potenzielle Zielgene von lttr852 identifizierte sowohl eine erhöhte Expression von Genen, die in der Aminosäuren-Biosynthese, Methionin-Sulfoxid-Reduktase und dem Kupfertransport involviert sind, als auch eine verringerte Transkription von codierenden Genen der Urease und Komponenten der Pyrimidin Nukleotide. Die Promotorfusion mit dem GFP-Reporter sowie das OmniLog System wurden verwendet, um Bedingungen zu identifizieren unter denen das lttr852 aktiv ist. Die Promotorstudien ergaben, dass die Anwesenheit von Glucose und eine erhöhte Temperatur die Promotoraktivität von lttr852 zeitabhängig herabsetzt, wobei die Aktivität durch mikroaerobe Bedingungen begünstigt wird. Kupfer wurde als limitierender Faktor identifiziert. Außerdem wurde der Einfluss diverser Regulatoren auf die transkriptionelle Regulation von lttr852 kontrolliert, jedoch keine zentrale Rolle in der Regulation von Virulenzgenen zugewiesen. Damit konnte innerhalb der vorliegenden Arbeit gezeigt werden, dass lttr852, ein neues Mitglied der Klasse der LysR-Typ Transkriptionsregulatoren in S. aureus, eine wichtige Rolle in der schnellen Adaption von S. aureus an die wechselnde Mikroumgebungen des Wirts hat. KW - Staphylococcus aureus KW - transcriptional regulation KW - metabolic adaptation KW - secondary site infection KW - LysR-type Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246757 ER - TY - JOUR A1 - Grob, Robin A1 - Tritscher, Clara A1 - Grübel, Kornelia A1 - Stigloher, Christian A1 - Groh, Claudia A1 - Fleischmann, Pauline N. A1 - Rössler, Wolfgang T1 - Johnston's organ and its central projections in Cataglyphis desert ants JF - Journal of Comparative Neurology N2 - The Johnston's organ (JO) in the insect antenna is a multisensory organ involved in several navigational tasks including wind‐compass orientation, flight control, graviception, and, possibly, magnetoreception. Here we investigate the three dimensional anatomy of the JO and its neuronal projections into the brain of the desert ant Cataglyphis, a marvelous long‐distance navigator. The JO of C. nodus workers consists of 40 scolopidia comprising three sensory neurons each. The numbers of scolopidia slightly vary between different sexes (female/male) and castes (worker/queen). Individual scolopidia attach to the intersegmental membrane between pedicel and flagellum of the antenna and line up in a ring‐like organization. Three JO nerves project along the two antennal nerve branches into the brain. Anterograde double staining of the antennal afferents revealed that JO receptor neurons project to several distinct neuropils in the central brain. The T5 tract projects into the antennal mechanosensory and motor center (AMMC), while the T6 tract bypasses the AMMC via the saddle and forms collaterals terminating in the posterior slope (PS) (T6I), the ventral complex (T6II), and the ventrolateral protocerebrum (T6III). Double labeling of JO and ocellar afferents revealed that input from the JO and visual information from the ocelli converge in tight apposition in the PS. The general JO anatomy and its central projection patterns resemble situations in honeybees and Drosophila. The multisensory nature of the JO together with its projections to multisensory neuropils in the ant brain likely serves synchronization and calibration of different sensory modalities during the ontogeny of navigation in Cataglyphis. KW - ant brain KW - chordotonal organ KW - graviception KW - magnetic compass KW - multisensory integration KW - navigation KW - wind compass Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225679 VL - 529 IS - 8 SP - 2138 EP - 2155 ER - TY - JOUR A1 - Grob, Robin A1 - Heinig, Niklas A1 - Grübel, Kornelia A1 - Rössler, Wolfgang A1 - Fleischmann, Pauline N. T1 - Sex-specific and caste-specific brain adaptations related to spatial orientation in Cataglyphis ants JF - Journal of Comparative Neurology N2 - Cataglyphis desert ants are charismatic central place foragers. After long-ranging foraging trips, individual workers navigate back to their nest relying mostly on visual cues. The reproductive caste faces other orientation challenges, i.e. mate finding and colony foundation. Here we compare brain structures involved in spatial orientation of Cataglyphis nodus males, gynes, and foragers by quantifying relative neuropil volumes associated with two visual pathways, and numbers and volumes of antennal lobe (AL) olfactory glomeruli. Furthermore, we determined absolute numbers of synaptic complexes in visual and olfactory regions of the mushroom bodies (MB) and a major relay station of the sky-compass pathway to the central complex (CX). Both female castes possess enlarged brain centers for sensory integration, learning, and memory, reflected in voluminous MBs containing about twice the numbers of synaptic complexes compared with males. Overall, male brains are smaller compared with both female castes, but the relative volumes of the optic lobes and CX are enlarged indicating the importance of visual guidance during innate behaviors. Male ALs contain greatly enlarged glomeruli, presumably involved in sex-pheromone detection. Adaptations at both the neuropil and synaptic levels clearly reflect differences in sex-specific and caste-specific demands for sensory processing and behavioral plasticity underlying spatial orientation. KW - antennal lobe KW - synaptic plasticity KW - polymorphism KW - optic lobes KW - mushroom bodies KW - learning and memory KW - central complex Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257299 VL - 529 IS - 18 ER - TY - THES A1 - Grebinyk, Anna T1 - Synergistic Chemo- and Photodynamic Treatment of Cancer Cells with C\(_{60}\) Fullerene Nanocomplexes T1 - Synergistische chemo- und photodynamische Behandlung von Krebszellen mit C\(_{60}\)-Fulleren-Nanokomplexen N2 - Recent progress in nanotechnology has attracted interest to a biomedical application of the carbon nanoparticle C60 fullerene (C60) due to its unique structure and versatile biological activity. In the current study the dual functionality of C60 as a photosensitizer and a drug nanocarrier was exploited to improve the efficiency of chemotherapeutic drugs towards human leukemic cells. Pristine C60 demonstrated time-dependent accumulation with predominant mitochondrial localization in leukemic cells. C60’s effects on leukemic cells irradiated with high power single chip LEDs of different wavelengths were assessed to find out the most effective photoexcitation conditions. A C60-based noncovalent nanosized system as a carrier for an optimized drug delivery to the cells was evaluated in accordance to its physicochemical properties and toxic effects. Finally, nanomolar amounts of C60-drug nanocomplexes in 1:1 and 2:1 molar ratios were explored to improve the efficiency of cell treatment, complementing it with photodynamic approach. A proposed treatment strategy was developed for C60 nanocomplexes with the common chemotherapeutic drug Doxorubicin, whose intracellular accumulation and localization, cytotoxicity and mechanism of action were investigated. The developed strategy was revealed to be transferable to an alternative potent anticancer drug – the herbal alkaloid Berberine. Hereafter, a strong synergy of treatments arising from the combination of C60-mediated drug delivery and C60 photoexcitation was revealed. Presented data indicate that a combination of chemo- and photodynamic treatments with C60-drug nanoformulations could provide a promising synergetic approach for cancer treatment. N2 - Kürzliche Fortschritte in der Nanotechnologie haben Interesse an einer biomedizinischen Anwendung des Kohlenstoffnanopartikels C60 Fulleren (C60) aufgrund seiner einzigartigen Struktur und breiten biologischen Aktivität geweckt. In der aktuellen Studie wurde die doppelte Funktionalität von C60 als Photosensibilisator und als Wirkstoff-Nanoträger genutzt, um die Wirkung von Chemotherapeutika auf menschliche Leukämiezellen zu verbessern. C60 alleine zeigte in den Zellen eine zeitabhängige Akkumulation mit vorherrschender mitochondrialer Lokalisation. Die Wirkung von C60 auf Leukämiezellen, die mit unterschiedlicher Wellenlänge bestrahlt wurden, wurde bewertet, um die effektivsten Photoanregungsbedingungen zu finden. Die physikochemischen Eigenschaften und toxischen Wirkungen von C60 auf die Leukämiezellen wurden nach nicht kovalenter Bindung von Arzneistoffen bewertet. Schließlich wurden nanomolare Mengen von C60-Wirkstoff-Nanokomplexen in Molverhältnissen von 1:1 und 2:1 untersucht, um die Effizienz der Behandlung von Zellen zu verbessern und sie durch photodynamischen Ansatz zu ergänzen. Mit dem gängigen Chemotherapeutikum Doxorubicin wurde eine Behandlungsstrategie entwickelt und dessen intrazelluläre Akkumulation und Lokalisation, Zytotoxizität und Wirkmechanismus untersucht wurden. Es wurde gezeigt, dass die entwickelte Strategie auch auf ein alternatives Krebsmedikament übertragbar ist – das pflanzliche Alkaloid Berberin. Die erhaltenen Daten deuten darauf hin, dass eine Kombination von chemo- und photodynamischen Behandlungen mit C60-Nanokomplexen einen vielversprechenden synergetischen Ansatz für die Krebsbehandlung bieten könnte. KW - cancer KW - drug delivery KW - photodynamic therapy KW - fullerene Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222075 ER - TY - JOUR A1 - Grassinger, Julia Maria A1 - Floren, Andreas A1 - Müller, Tobias A1 - Cerezo-Echevarria, Argiñe A1 - Beitzinger, Christoph A1 - Conrad, David A1 - Törner, Katrin A1 - Staudacher, Marlies A1 - Aupperle-Lellbach, Heike T1 - Digital lesions in dogs: a statistical breed analysis of 2912 cases JF - Veterinary Sciences N2 - Breed predispositions to canine digital neoplasms are well known. However, there is currently no statistical analysis identifying the least affected breeds. To this end, 2912 canine amputated digits submitted from 2014–2019 to the Laboklin GmbH & Co. KG for routine diagnostics were statistically analyzed. The study population consisted of 155 different breeds (most common: 634 Mongrels, 411 Schnauzers, 197 Labrador Retrievers, 93 Golden Retrievers). Non-neoplastic processes were present in 1246 (43%), tumor-like lesions in 138 (5%), and neoplasms in 1528 cases (52%). Benign tumors (n = 335) were characterized by 217 subungual keratoacanthomas, 36 histiocytomas, 35 plasmacytomas, 16 papillomas, 12 melanocytomas, 9 sebaceous gland tumors, 6 lipomas, and 4 bone tumors. Malignant neoplasms (n = 1193) included 758 squamous cell carcinomas (SCC), 196 malignant melanomas (MM), 76 soft tissue sarcomas, 52 mast cell tumors, 37 non-specified sarcomas, 29 anaplastic neoplasms, 24 carcinomas, 20 bone tumors, and 1 histiocytic sarcoma. Predisposed breeds for SCC included the Schnauzer (log OR = 2.61), Briard (log OR = 1.78), Rottweiler (log OR = 1.54), Poodle (log OR = 1.40), and Dachshund (log OR = 1.30). Jack Russell Terriers (log OR = −2.95) were significantly less affected by SCC than Mongrels. Acral MM were significantly more frequent in Rottweilers (log OR = 1.88) and Labrador Retrievers (log OR = 1.09). In contrast, Dachshunds (log OR = −2.17), Jack Russell Terriers (log OR = −1.88), and Rhodesian Ridgebacks (log OR = −1.88) were rarely affected. This contrasted with the well-known predisposition of Dachshunds and Rhodesian Ridgebacks to oral and cutaneous melanocytic neoplasms. Further studies are needed to explain the underlying reasons for breed predisposition or “resistance” to the development of specific acral tumors and/or other sites. KW - canine KW - subungual KW - toe KW - tumor KW - inflammation KW - breed predisposition Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242690 SN - 2306-7381 VL - 8 IS - 7 ER - TY - THES A1 - Goos, Carina T1 - Nuclear periphery granules of trypanosomes - A characterization of composition and function T1 - Nuclear periphery granules in Trypanosomen - Eine Charakterisierung in Komposition und Funktion N2 - The nuclear envelope serves as important mRNA surveillance system. In yeast and humans, several control mechanisms act in parallel to prevent nuclear export of unprocessed mRNAs. However, trypanosomes lack homologues to most of the proteins involved. In addition, gene expression in trypanosomes relies almost completely on post-transcriptional regulation as they transcribe mRNAs as long polycistrons, which are subsequently processed into individual mRNA molecules by trans-splicing. As trans-splicing is not error-free, unspliced mRNAs may be recognized and prevented from reaching the cytoplasm by a yet unknown mechanism. When trans-splicing is inhibited in trypanosomes, the formation of a novel RNA granule type at the cytoplasmic periphery of the nucleus, so called nuclear periphery granules (NPGs) was previously observed. To identify potential regulators of nuclear export control, changes in protein localization which occur when trans-splicing is inhibited, were globally analyzed during this work. For this, trypanosome nuclei were purified under conditions maintaining NPG attachment to the nucleus, in the absence and presence of trans-splicing. Mass spectrometry analyses identified 128 proteins which are specifically enriched in nuclear preparations of cells inhibited for trans-splicing. Amongst them are proteins, which change their localization to the nucleus or to the nuclear pores as well as many proteins that move into NPGs. Some of these proteins are promising candidates for nuclear export control proteins, as the changes in localization (to the nucleus or nuclear pores) were specific to the accumulation of unspliced mRNAs. The NPG proteome almost exclusively contains proteins involved in mRNA metabolism, mostly unique to trypanosomes, notably major translation initiation factors were absent. These data indicate that NPGs are RNP complexes which have started or completed nuclear export, but not yet entered translation. As a byproduct of these proteomic studies, a high-quality dataset of the yet unknown T. brucei nuclear proteome is provided, closing an important gap in knowledge to study trypanosome biology, in particular nuclear related processes. NPGs were characterized in more detail by microscopy. The granules are cytoplasmic and present in at least two different trypanosome life cycle stages. There are at least two distinct granule subsets, with differences in protein composition. A closer analysis of NPGs by electron microscopy revealed that the granules are electron dense structures, which are connected to nuclear pores by string-like structures. In order to approach the function of NPGs, on the one hand, the hypothesis that NPGs might be related to perinuclear germ granules of adult gonads of C. elegans was tested: we found no relation between the two granule types. On the other hand, initial single molecule mRNA FISH experiments performed in trypanosomes showed no accumulation of unspliced transcripts in NPGs, arguing against an involvement of the granules in mRNA quality control. N2 - Die Kernhülle um den Zellkern dient als wichtiges mRNA-Überwachungssystem in Eukaryoten. Bei Hefen und Menschen wirken dabei beispielsweise mehrere Kontrollmechanismen parallel, um den Export von unverarbeiteten mRNAs aus dem Kern heraus zu verhindern. Trypanosomen fehlen jedoch Homologe zu den Meisten hierbei beteiligten Proteinen. Außerdem basiert Genexpression in Trypanosomen fast ausschließlich auf posttranskriptioneller Kontrolle, da die Parasiten mRNAs als lange Polycistrons transkribieren, die anschließend durch Transspleißen zu einzelnen mRNA-Molekülen verarbeitet werden. Da der Prozess des Transspleißen nicht fehlerfrei zu sein scheint, gibt es möglicherweise einen noch unbekannten Mechanismus, der nicht gespleißte mRNAs erkennt und diese daran hindert, das Zytoplasma zu erreichen. Unter Bedingungen, in denen Transspleißen in Trypanosomen blockiert ist, konnte eine neue Art von RNA-Granula an der zytoplasmatischen Peripherie des Zellkerns beobachtet werden, sogenannte Nuclear Periphery Granules (NPGs). Um potentielle Regulatoren einer Kontrolle des mRNA-Exports zu identifizieren, wurde während dieser Arbeit umfassend analysiert, inwieweit sich die Lokalisation von Proteinen ändert, wenn Transspleißen gehemmt wird. Zu diesem Zweck wurden Zellkerne von Trypanosomen unter Bedingungen aufgereinigt, bei denen die Bindung der NPGs an den Zellkern in Abwesenheit und Gegenwart von Transspleißen erhalten blieb. Mit Hilfe von massen-spektrometrischen Analysen konnten 128 Proteine identifiziert werden, die spezifisch in den Kernpräparaten von Zellen angereichert sind, in denen Transspleißen blockiert ist. Darunter befinden sich Proteine, die ihre Lokalisation in den Kern hinein oder zu den Kernporen hin verändern, sowie viele Proteine, die sich in NPGs bewegen. Einige dieser Proteine stellen vielversprechende Kandidaten für eine potenzielle Rolle in der Kernexportkontrolle dar, da die Veränderungen in der Lokalisation (zum Kern oder zu den Kernporen) spezifisch für die Akkumulation von nicht gespleißten mRNAs waren. Das hier erarbeitete NPG-Proteom enthält fast ausschließlich Proteine, die am mRNA-Metabolismus beteiligt sind und nur in Trypanosomen vorkommen. Insbesondere fehlen im NPG-Proteom wichtige Translationsinitiationsfaktoren. Die Daten zeigen, dass NPGs RNP-Komplexe sind, die den Export aus dem Zellkern bereits begonnen oder abgeschlossen haben, aber noch nicht mit dem Translationsprozess begonnen haben. Als Nebenprodukt dieser proteomischen Analyse, kann ein qualitativ hochwertiger Datensatz des noch unbekannten Kernproteoms von T. brucei bereitgestellt werden. Damit wird eine wichtige Wissenslücke bei der Forschung zur Trypanosomenbiologie, insbesondere zu nuklearen Prozessen geschlossen. Im Rahmen dieser Arbeit wurden außerdem NPGs anhand mikroskopischer Untersuchungen detaillierter charakterisiert. Die Granula sind zytoplasmatisch und liegen in mindestens zwei verschiedenen Lebenszyklusstadien von Trypanosomen vor. Es gibt mindestens zwei Untergruppen der Granula mit Unterschieden in der Proteinzusammensetzung. Eine genauere elektronenmikroskopische Analyse ergab, dass es sich bei NPGs um elektronendichte Strukturen handelt, die durch fadenartige Strukturen mit den Kernporen verbunden sind. Um mehr über die potenzielle Funktion von NPGs herauszufinden, wurde zum einen die Hypothese untersucht, dass NPGs mit perinuklearen Keimgranula adulter Gonaden in C. elegans verwandt sind: Es konnte keine Beziehung zwischen den beiden Granulatypen gefunden werden. Zum anderen zeigten erste Experimente mittels Einzelmolekül-mRNA-FISH in Trypanosomen keine Akkumulation von ungespleißten Transkripten in NPGs, was gegen eine Beteiligung an mRNA-Qualitätskontrollmechanismen spricht. KW - Trypanosoma brucei KW - Kinetoplastida KW - Rnsstoffwechsel KW - Nuclear periphery granules KW - RNA granules KW - Nuclear export control KW - Trypanosomes Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234368 ER - TY - THES A1 - Georgiev, Kostadin T1 - Sustainable management of naturally disturbed forests T1 - Nachhaltiges Management von natürlichen Störungen in Wäldern N2 - Owing to climate change, natural forest disturbances and consecutive salvage logging are drastically increasing worldwide, consequently increasing the importance of understanding how these disturbances would affect biodiversity conservation and provision of ecosystem services. In chapter II, I used long-term water monitoring data and mid-term data on α-diversity of twelve species groups to quantify the effects of natural disturbances (windthrow and bark beetle) and salvage logging on concentrations of nitrate and dissolved organic carbon (DOC) in streamwater and α-diversity. I found that natural disturbances led to a temporal increase of nitrate concentrations in streamwater, but these concentrations remained within the health limits recommended by the World Health Organization for drinking water. Salvage logging did not exert any additional impact on nitrate and DOC concentrations, and hence did not affect streamwater quality. Thus, neither natural forest disturbances in watersheds nor associated salvage logging have a harmful effect on the quality of the streamwater used for drinking water. Natural disturbances increased the α-diversity in eight out of twelve species groups. Salvage logging additionally increased the α-diversity of five species groups related to open habitats, but decreased the biodiversity of three deadwood-dependent species groups. In chapter III, I investigated whether salvage logging following natural disturbances (wildfire and windthrow) altered the natural successional trajectories of bird communities. I compiled data on breeding bird assemblages from nine study areas in North America, Europe and Asia, over a period of 17 years and tested whether bird community dissimilarities changed over time for taxonomic, functional and phylogenetic diversity when rare, common and dominant species were weighted differently. I found that salvage logging led to significantly larger dissimilarities than expected by chance and that these dissimilarities persisted over time for rare, common and dominant species, evolutionary lineages, and for rare functional groups. Dissimilarities were highest for rare, followed by common and dominant species. In chapter IV, I investigated how β-diversity of 13 taxonomic groups would differ in intact, undisturbed forests, disturbed, unlogged forests and salvage-logged forests 11 years after a windthrow and salvage logging. The study suggests that both windthrow and salvage logging drive changes in between-treatment β-diversity, whereas windthrow alone seems to drive changes in within-treatment β-diversity. Over a decade after the windthrow at the studied site, the effect of subsequent salvage logging on within-treatment β-diversity was no longer detectable but the effect on between-treatment β-diversity persisted, with more prominent changes in saproxylic groups and rare species than in non-saproxylic groups or common and dominant species. Based on these results, I suggest that salvage logging needs to be carefully weighed against its long-lasting impact on communities of rare species. Also, setting aside patches of naturally disturbed areas is a valuable management alternative as these patches would enable post-disturbance succession of bird communities in unmanaged patches and would promote the conservation of deadwood-dependent species, without posing health risks to drinking water sources. N2 - In Folge des Klimawandels treten in Wäldern vermehrt natürliche Störungen auf, wodurch wiederum die Zahl an nachfolgenden Sanitärhieben (Räumungen) drastisch gestiegen ist. Wie sich natürliche Störungen und Sanitärhiebe auf die biologische Vielfalt und die Bereitstellung von Ökosystemleistungen auswirken können, ist bisher jedoch nur unzureichend bekannt. In Kapitel II nutzte ich langfristige Wassermonitoringdaten und mittelfristige Biodiversitätsdaten über zwölf Artengruppen, um die Effekte von natürlichen Störungen (Windwurf und Borkenkäfer) und Sanitärhieben auf die Konzentrationen von Nitraten und gelöster organischer Kohlenstoffe (GOK) in Bächen und Artenzahl zu quantifizieren. Die Ergebnisse zeigen, heraus, dass natürliche Störungen zu einer temporären Erhöhung der Nitratwerte führen, welche dennoch laut Angaben der Weltgesundheitsorganisation immer noch als unbedenklich eingestuft werden können. Die Sanitärhiebe hatten keinen zusätzlichen Einfluss auf die Nitrat- und GOK-Konzentrationen und daher keinen Einfluss auf die Wasserqualität. Daraus lässt sich schließen, dass sich weder natürliche Waldstörungen in Wassereinzugsgebieten noch die damit verbundenen Sanitärhiebe auf die Trinkwasserqualität aus auswirken. Natürliche Störungen erhöhten die Artenzahlen in acht von zwölf Artengruppen. Zusätzlich erhöhten die Sanitärhiebe die Artenzahlen von fünf Artengruppen, welche auf offene Lebensräume angewiesen sind, verringerte jedoch die Artenzahlen von drei xylobionte Artengruppen. In Kapitel III habe ich untersucht, ob Sanitärhiebe nach natürlichen Waldstörungen zu sukzessiven Veränderungen der Vogelgemeinschaften führen. Hierzu habe ich die taxonomische, funktionelle und phylogenetische Diversität von Brutvogelgemeinschaften aus neun Untersuchungsregionen in Nordamerika, Europa und Asien über die Zeit von 17 Jahren verglichen und analysiert, ob sich das jeweilige Diversitätsmaß verändert, wenn seltene, häufige und dominante Arten unterschiedlich gewichtet werden. Ich konnte zeigen, dass Sanitärhiebe zu signifikant größeren Unterschieden geführt haben als zufällig zu erwarten gewesen sind und dass diese Unterschiede über die Zeit sowohl für seltene, häufige und dominante Arten, als auch für evolutionäre Linien, und funktionelle Gruppen fortdauern. Diese Unterschiede waren am größten für seltene, gefolgt von häufigen und dominanten Arten. In Kapitel IV untersuchte ich wie sich die β-Diversität von 13 taxonomischen Gruppen zwischen ungestörten Wäldern, gestörten und ungeräumten Wäldern sowie gestörten und geräumten Wäldern 11 Jahre nach Windwurf und anschließender Räumung unterscheidet. Die Ergebnisse deuten darauf hin, dass sowohl Windwurf als auch Räumung Änderungen in der β-Diversität bewirken. Windwurf allein jedoch scheint diese Änderungen in der β-Diversität innerhalb der Behandlung bewirken zu können. Über ein Jahrzehnt nach dem Windwurf war der Effekt des Sanitärhiebes auf die β-Diversität innerhalb der Behandlung nicht mehr nachweisbar. Der Effekt auf die β-Diversität zwischen den Behandlungen blieb jedoch bestehen, wobei sich die xylobionten Gruppen und seltenen Arten stärker veränderten als die nicht-xylobionten Gruppen oder häufigen und dominanten Arten. Basierend auf diesen Ergebnissen schlage ich vor, dass der Einsatz von Sanitärhieben sorgfältig gegen ihre langfristigen Auswirkungen auf Gemeinschaften seltener Arten abgewogen werden muss. Zusätzlich, besteht mit dem Belassen von natürlich gestörten Waldgebieten eine wertvolle Managementalternative, da diese Flächen eine natürliche Entwicklung von Vogelgemeinschaften ermöglichen und xylobionte Arten fördern, ohne dass die Trinkwasserqualität negativ beeinträchtigt wird. KW - species richness KW - water quality KW - beta diversity KW - Hill numbers KW - post-disturbance logging KW - biodiversity response KW - ecosystem services Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242854 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 - Garitano-Trojaola, Andoni A1 - Sancho, Ana A1 - Götz, Ralph A1 - Eiring, Patrick A1 - Walz, Susanne A1 - Jetani, Hardikkumar A1 - Gil-Pulido, Jesus A1 - Da Via, Matteo Claudio A1 - Teufel, Eva A1 - Rhodes, Nadine A1 - Haertle, Larissa A1 - Arellano-Viera, Estibaliz A1 - Tibes, Raoul A1 - Rosenwald, Andreas A1 - Rasche, Leo A1 - Hudecek, Michael A1 - Sauer, Markus A1 - Groll, Jürgen A1 - Einsele, Hermann A1 - Kraus, Sabrina A1 - Kortüm, Martin K. T1 - Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia JF - Communications Biology N2 - The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD+AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD+AML. Garitano-Trojaola et al. used a combination of human acute myeloid leukemia (AML) cell lines and primary samples to show that RAC1-dependent actin cytoskeleton remodeling through BCL2 family plays a key role in resistance to the FLT3 inhibitor, Midostaurin in AML. They showed that by targeting RAC1 and BCL2, Midostaurin resistance was diminished, which potentially paves the way for an innovate treatment approach for FLT3 mutant AML. KW - actin KW - acute myeloid leukaemia Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260709 VL - 4 IS - 1 ER - TY - JOUR A1 - Fuss, Carmina Teresa A1 - Other, Katharina A1 - Heinze, Britta A1 - Landwehr, Laura-Sophie A1 - Wiegering, Armin A1 - Kalogirou, Charis A1 - Hahner, Stefanie A1 - Fassnacht, Martin T1 - Expression of the chemokine receptor CCR7 in the normal adrenal gland and adrenal tumors and its correlation with clinical outcome in adrenocortical carcinoma JF - Cancers N2 - Background: The chemokine receptor CCR7 is crucial for an intact immune function, but its expression is also associated with clinical outcome in several malignancies. No data exist on the expression of CCR7 in adrenocortical tumors. Methods: CCR7 expression was investigated by qRT-PCR and immunohistochemistry in 4 normal adrenal glands, 59 adrenocortical adenomas, and 181 adrenocortical carcinoma (ACC) samples. Results: CCR7 is highly expressed in the outer adrenocortical zones and medulla. Aldosterone-producing adenomas showed lower CCR7 protein levels (H-score 1.3 ± 1.0) compared to non-functioning (2.4 ± 0.5) and cortisol-producing adenomas (2.3 ± 0.6), whereas protein expression was variable in ACC (1.8 ± 0.8). In ACC, CCR7 protein expression was significantly higher in lymph node metastases (2.5 ± 0.5) compared to primary tumors (1.8±0.8) or distant metastases (2.0 ± 0.4; p < 0.01). mRNA levels of CCR7 were not significantly different between ACCs, normal adrenals, and adrenocortical adenomas. In contrast to other tumor entities, neither CCR7 protein nor mRNA expression significantly impacted patients' survival. Conclusion: We show that CCR7 is expressed on mRNA and protein level across normal adrenals, benign adrenocortical tumors, as well as ACCs. Given that CCR7 did not influence survival in ACC, it is probably not involved in tumor progression, but it could play a role in adrenocortical homeostasis. KW - CCR7 KW - chemokine receptor KW - adrenocortical carcinoma KW - adrenal tumors Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250112 SN - 2072-6694 VL - 13 IS - 22 ER - TY - JOUR A1 - Ferreira, Eliana Aparecida A1 - Boff, Samuel A1 - Verza, Sandra S. A1 - Mussury, Rosilda Mara T1 - Bioecological and behavioral interaction between pollinating bees and the pioneer shrub Ludwigia nervosa in degraded area suggests an exotic bee as its major pollinator JF - Biology N2 - The flowers of plants of the genus Ludwigia are an important source of food for several species of bees. In the current study, we conducted an experiment with the aim to describe the reproductive biology and phenology of L. nervosa; to identify the species of visiting bees; analyze the foraging behavior of bees; and to investigate whether the reproductive success of the species is related to the foraging activity of bees. We found that the flowers received visits from several native bee species (n = 7), in addition of the exotic honey bees which came to be the dominant species. During visits the majority of the bees foraged in both resources, pollen and nectar. The significantly higher production of fruits in open pollinated pollination experiment compared to artificial cross pollination, suggests honey bees as effective pollinator of this plant species in the study site. Pollen deposition occurs efficiently, given the absence of pollen limitation. Despite massive visitation of honey bees, Ludwigianervosa is attractive to native bees, and therefore it may help to sustain population of both native and exotic pollinators in fragmented humid areas. KW - cross pollination KW - disturbed humid area KW - germination speed KW - honey bees and native bees KW - pollen limitation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228757 SN - 2079-7737 VL - 10 IS - 2 ER - TY - JOUR A1 - Eisenreich, Wolfgang A1 - Rudel, Thomas A1 - Heesemann, Jürgen A1 - Goebel, Werner T1 - Persistence of Intracellular Bacterial Pathogens—With a Focus on the Metabolic Perspective JF - Frontiers in Cellular and Infection Microbiology N2 - Persistence has evolved as a potent survival strategy to overcome adverse environmental conditions. This capability is common to almost all bacteria, including all human bacterial pathogens and likely connected to chronic infections caused by some of these pathogens. Although the majority of a bacterial cell population will be killed by the particular stressors, like antibiotics, oxygen and nitrogen radicals, nutrient starvation and others, a varying subpopulation (termed persisters) will withstand the stress situation and will be able to revive once the stress is removed. Several factors and pathways have been identified in the past that apparently favor the formation of persistence, such as various toxin/antitoxin modules or stringent response together with the alarmone (p)ppGpp. However, persistence can occur stochastically in few cells even of stress-free bacterial populations. Growth of these cells could then be induced by the stress conditions. In this review, we focus on the persister formation of human intracellular bacterial pathogens, some of which belong to the most successful persister producers but lack some or even all of the assumed persistence-triggering factors and pathways. We propose a mechanism for the persister formation of these bacterial pathogens which is based on their specific intracellular bipartite metabolism. We postulate that this mode of metabolism ultimately leads, under certain starvation conditions, to the stalling of DNA replication initiation which may be causative for the persister state. KW - persistence KW - mechanisms of persister formation KW - intracellular bacterial pathogens KW - stress conditions KW - ATP-DnaA complex KW - DNA replication initiation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222348 SN - 2235-2988 VL - 10 ER -