TY - JOUR A1 - Rother, Lisa A1 - Kraft, Nadine A1 - Smith, Dylan B. A1 - El Jundi, Basil A1 - Gill, Richard J. A1 - Pfeiffer, Keram T1 - A micro-CT-based standard brain atlas of the bumblebee JF - Cell and Tissue Research N2 - In recent years, bumblebees have become a prominent insect model organism for a variety of biological disciplines, particularly to investigate learning behaviors as well as visual performance. Understanding these behaviors and their underlying neurobiological principles requires a clear understanding of brain anatomy. Furthermore, to be able to compare neuronal branching patterns across individuals, a common framework is required, which has led to the development of 3D standard brain atlases in most of the neurobiological insect model species. Yet, no bumblebee 3D standard brain atlas has been generated. Here we present a brain atlas for the buff-tailed bumblebee Bombus terrestris using micro-computed tomography (micro-CT) scans as a source for the raw data sets, rather than traditional confocal microscopy, to produce the first ever micro-CT-based insect brain atlas. We illustrate the advantages of the micro-CT technique, namely, identical native resolution in the three cardinal planes and 3D structure being better preserved. Our Bombus terrestris brain atlas consists of 30 neuropils reconstructed from ten individual worker bees, with micro-CT allowing us to segment neuropils completely intact, including the lamina, which is a tissue structure often damaged when dissecting for immunolabeling. Our brain atlas can serve as a platform to facilitate future neuroscience studies in bumblebees and illustrates the advantages of micro-CT for specific applications in insect neuroanatomy. KW - neuropils KW - Bombus terrestris KW - insect standard brain atlas KW - iterative shape averaging KW - reconstruction Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-267783 SN - 1432-0878 VL - 386 IS - 1 ER - TY - JOUR A1 - Leidinger, Ludwig A1 - Vedder, Daniel A1 - Cabral, Juliano Sarmento T1 - Temporal environmental variation may impose differential selection on both genomic and ecological traits JF - Oikos N2 - The response of populations and species to changing conditions determines how community composition will change functionally, including via trait shifts. Selection from standing variation has been suggested to be more efficient than acquiring new mutations. Yet, studies on community trait composition and trait selection largely focus on phenotypic variation in ecological traits, whereas the underlying genomic traits remain understudied. Using a genome‐explicit, niche‐ and individual‐based model, we address the potential interactions between genomic and ecological traits shaping communities under an environmental selective forcing, namely temporal positively autocorrelated environmental fluctuation. In this model, all ecological traits are explicitly coded by the genome. For our experiments, we initialized 90 replicate communities, each with ca 350 initial species, characterized by random genomic and ecological trait combinations, on a 2D spatially explicit landscape with two orthogonal gradients (temperature and resource use). We exposed each community to two contrasting scenarios: without (i.e. static environments) and with temporal variation. We then analyzed emerging compositions of both genomic and ecological traits at the community, population and genomic levels. Communities in variable environments were species poorer than in static environments, and populations more abundant, whereas genomes had lower genetic linkage, mean genetic variation and a non‐significant tendency towards higher numbers of genes. The surviving genomes (i.e. those selected by variable environments) coded for enhanced environmental tolerance and smaller biomass, which resulted in faster life cycles and thus also in increased potential for evolutionary rescue. Under temporal environmental variation, larger, less linked genomes retained more variation in mean dispersal ability at the population level than at genomic level, whereas the opposite trend emerged for biomass. Our results provide clues to how sexually‐reproducing diploid plant communities might react to variable environments and highlights the importance of genomic traits and their interaction with ecological traits for eco‐evolutionary responses to changing climates. KW - environmental variability KW - genomic traits KW - mechanistic model KW - rapid evolution KW - standing variation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238945 VL - 130 IS - 7 SP - 1100 EP - 1115 ER - TY - JOUR A1 - Sponsler, Douglas B. A1 - Bratman, Eve Z. T1 - Beekeeping in, of or for the city? A socioecological perspective on urban apiculture JF - People and Nature N2 - The term ‘urban beekeeping’ connotes a host of meanings—sociopolitical, commercial, ecological and personal—beyond the mere description of where bees and beekeepers happen to coincide. Yet, these meanings are seldom articulated explicitly or brought into critical engagement with the relevant fields of urban ecology and political ecology. Beginning with a brief account of the history of urban beekeeping in the United States, we draw upon urban ecological theory to construct a conceptual model of urban beekeeping that distinguishes beekeeping in, of and for the city. In our model, beekeeping in the city describes the mere importation of the traditionally rural practice of beekeeping into urban spaces for the private reasons of the individual beekeeper, whereas beekeeping of the city describes beekeeping that is consciously tailored to the urban context, often accompanied by (semi)professionalization of beekeepers and the formation of local expert communities (i.e. beekeeping associations). Beekeeping for the city describes a shift in mindset in which beekeeping is directed to civic ends beyond the boundaries of the beekeeping community per se. Using this framework, we identify and discuss specific socioecological assets and liabilities of urban beekeeping, and how these relate to beekeeping in, of and for the city. We then formulate actionable guidelines for maturing the practice of urban beekeeping into a beneficent and self‐critical form of urban ecological citizenship; these include fostering self‐regulation within the beekeeping community, harnessing beekeeping as a ‘gateway’ experience for a broader rapprochement between urban residents and nature, and recognizing the political‐ecological context of beekeeping with respect to matters of socioecological justice. KW - environmental justice KW - honey bee KW - multispecies studies KW - policy KW - pollinator KW - urban greening Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239949 VL - 3 IS - 3 SP - 550 EP - 559 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 - Wolf, Matthias T1 - How to teach about what is a species JF - Biology N2 - To ask students what a species is always has something rhetorical about it. Too quickly comes the rote answer, often learned by heart without ever thinking about it: “A species is a reproductive community of populations (reproductively isolated from others), which occupies a specific niche in nature” (Mayr 1982). However, do two people look alike because they are twins or are they twins because they look alike? “Two organisms do not belong to the same species because they mate and reproduce, but they only are able to do so because they belong to the same species” (Mahner and Bunge 1997). Unfortunately, most biology (pre-university) teachers have no opinion on whether species are real or conceptual, simply because they have never been taught the question themselves, but rather one answer they still pass on to their students today, learned by heart without ever thinking about it. Species are either real or conceptual and, in my opinion, it is this “or” that we should teach about. Only then can we discuss those fundamental questions such as who or what is selected, who or what evolves and, finally, what is biodiversity and phylogenetics all about? Individuals related to each other by the tree of life. KW - biospecies KW - species as individuals KW - species as natural kinds KW - species concept KW - species problem Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241052 SN - 2079-7737 VL - 10 IS - 6 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 - Loos, Jacqueline A1 - Krauss, Jochen A1 - Lyons, Ashley A1 - Föst, Stephanie A1 - Ohlendorf, Constanze A1 - Racky, Severin A1 - Röder, Marina A1 - Hudel, Lennart A1 - Herfert, Volker A1 - Tscharntke, Teja T1 - Local and landscape responses of biodiversity in calcareous grasslands JF - Biodiversity and Conservation N2 - Across Europe, calcareous grasslands become increasingly fragmented and their quality deteriorates through abandonment and land use intensification, both affecting biodiversity. Here, we investigated local and landscape effects on diversity patterns of several taxonomic groups in a landscape of highly fragmented calcareous grassland remnants. We surveyed 31 grassland fragments near Göttingen, Germany, in spring and summer 2017 for vascular plants, butterflies and birds, with sampling effort adapted to fragment area. Through regression modelling, we tested relationships between species richness and fragment size (from 314 to 51,395 m\(^2\)), successional stage, habitat connectivity and the per cent cover of arable land in the landscape at several radii. We detected 283 plant species, 53 butterfly species and 70 bird species. Of these, 59 plant species, 19 butterfly species and 9 bird species were grassland specialists. Larger fragments supported twice the species richness of plants than small ones, and hosted more species of butterflies, but not of birds. Larger grassland fragments contained more grassland specialist plants, but not butterfly or bird specialists. Increasing amounts of arable land in the landscape from 20 to 90% was related to the loss of a third of species of plants, and less so, of butterflies, but not of birds. Per cent cover of arable land negatively correlated to richness of grassland specialist plants and butterflies, but positively to grassland specialist birds. We found no effect by successional stages and habitat connectivity. Our multi-taxa approach highlights the need for conservation management at the local scale, complemented by measures at the landscape scale. KW - abandonment KW - birds KW - butterflies KW - land use intensification KW - nature conservation KW - vascular plants Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-308595 SN - 0960-3115 SN - 1572-9710 VL - 30 IS - 8-9 ER - TY - JOUR A1 - Riedmeier, Maria A1 - Decarolis, Boris A1 - Haubitz, Imme A1 - Müller, Sophie A1 - Uttinger, Konstantin A1 - Börner, Kevin A1 - Reibetanz, Joachim A1 - Wiegering, Armin A1 - Härtel, Christoph A1 - Schlegel, Paul-Gerhardt A1 - Fassnacht, Martin A1 - Wiegering, Verena T1 - Adrenocortical carcinoma in childhood: a systematic review JF - Cancers N2 - Adrenocortical tumors are rare in children. This systematic review summarizes the published evidence on pediatric adrenocortical carcinoma (ACC) to provide a basis for a better understanding of the disease, investigate new molecular biomarkers and therapeutic targets, and define which patients may benefit from a more aggressive therapeutic approach. We included 137 studies with 3680 ACC patients (~65% female) in our analysis. We found no randomized controlled trials, so this review mainly reflects retrospective data. Due to a specific mutation in the TP53 gene in ~80% of Brazilian patients, that cohort was analyzed separately from series from other countries. Hormone analysis was described in 2569 of the 2874 patients (89%). Most patients were diagnosed with localized disease, whereas 23% had metastasis at primary diagnosis. Only 72% of the patients achieved complete resection. In 334 children (23%), recurrent disease was reported: 81% — local recurrence, 19% (n = 65) — distant metastases at relapse. Patients < 4 years old had a different distribution of tumor stages and hormone activity and better overall survival (p < 0.001). Although therapeutic approaches are typically multimodal, no consensus is available on effective standard treatments for advanced ACC. Thus, knowledge regarding pediatric ACC is still scarce and international prospective studies are needed to implement standardized clinical stratifications and risk-adapted therapeutic strategies. KW - pediatric adrenocortical cancer KW - pediatric adrenocortical adenoma KW - pediatric adrenocortical tumor KW - prognostic factors KW - therapy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248507 SN - 2072-6694 VL - 13 IS - 21 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 - Broster Reix, Christine E. A1 - Florimond, Célia A1 - Cayrel, Anne A1 - Mailhé, Amélie A1 - Agnero-Rigot, Corentin A1 - Landrein, Nicolas A1 - Dacheux, Denis A1 - Havlicek, Katharina A1 - Bonhivers, Mélanie A1 - Morriswood, Brooke A1 - Robinson, Derrick R. T1 - Bhalin, an essential cytoskeleton-associated protein of Trypanosoma brucei linking TbBILBO1 of the flagellar pocket collar with the hook complex JF - Microorganisms N2 - Background: In most trypanosomes, endo and exocytosis only occur at a unique organelle called the flagellar pocket (FP) and the flagellum exits the cell via the FP. Investigations of essential cytoskeleton-associated structures located at this site have revealed a number of essential proteins. The protein TbBILBO1 is located at the neck of the FP in a structure called the flagellar pocket collar (FPC) and is essential for biogenesis of the FPC and parasite survival. TbMORN1 is a protein that is present on a closely linked structure called the hook complex (HC) and is located anterior to and overlapping the collar. TbMORN1 is essential in the bloodstream form of T. brucei. We now describe the location and function of BHALIN, an essential, new FPC-HC protein. Methodology/Principal Findings: Here, we show that a newly characterised protein, BHALIN (BILBO1 Hook Associated LINker protein), is localised to both the FPC and HC and has a TbBILBO1 binding domain, which was confirmed in vitro. Knockdown of BHALIN by RNAi in the bloodstream form parasites led to cell death, indicating an essential role in cell viability. Conclusions/Significance: Our results demonstrate the essential role of a newly characterised hook complex protein, BHALIN, that influences flagellar pocket organisation and function in bloodstream form T. brucei parasites. KW - trypanosoma KW - flagellar pocket KW - hook complex KW - endocytosis KW - cytoskeleton KW - protozoan KW - flagellar pocket collar Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250301 SN - 2076-2607 VL - 9 IS - 11 ER - TY - JOUR A1 - Uphus, Lars A1 - Lüpke, Marvin A1 - Yuan, Ye A1 - Benjamin, Caryl A1 - Englmeier, Jana A1 - Fricke, Ute A1 - Ganuza, Cristina A1 - Schwindl, Michael A1 - Uhler, Johannes A1 - Menzel, Annette T1 - Climate effects on vertical forest phenology of Fagus sylvatica L., sensed by Sentinel-2, time lapse camera, and visual ground observations JF - Remote Sensing N2 - Contemporary climate change leads to earlier spring phenological events in Europe. In forests, in which overstory strongly regulates the microclimate beneath, it is not clear if further change equally shifts the timing of leaf unfolding for the over- and understory of main deciduous forest species, such as Fagus sylvatica L. (European beech). Furthermore, it is not known yet how this vertical phenological (mis)match — the phenological difference between overstory and understory — affects the remotely sensed satellite signal. To investigate this, we disentangled the start of season (SOS) of overstory F.sylvatica foliage from understory F. sylvatica foliage in forests, within nine quadrants of 5.8 × 5.8 km, stratified over a temperature gradient of 2.5 °C in Bavaria, southeast Germany, in the spring seasons of 2019 and 2020 using time lapse cameras and visual ground observations. We explained SOS dates and vertical phenological (mis)match by canopy temperature and compared these to Sentinel-2 derived SOS in response to canopy temperature. We found that overstory SOS advanced with higher mean April canopy temperature (visual ground observations: −2.86 days per °C; cameras: −2.57 days per °C). However, understory SOS was not significantly affected by canopy temperature. This led to an increase of vertical phenological mismatch with increased canopy temperature (visual ground observations: +3.90 days per °C; cameras: +2.52 days per °C). These results matched Sentinel-2-derived SOS responses, as pixels of higher canopy height advanced more by increased canopy temperature than pixels of lower canopy height. The results may indicate that, with further climate change, spring phenology of F. sylvatica overstory will advance more than F. sylvatica understory, leading to increased vertical phenological mismatch in temperate deciduous forests. This may have major ecological effects, but also methodological consequences for the field of remote sensing, as what the signal senses highly depends on the pixel mean canopy height and the vertical (mis)match. KW - overstory KW - understory KW - Sentinel-2 KW - time lapse cameras KW - vertical mismatch KW - phenological escape KW - climate change KW - European beech Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248419 SN - 2072-4292 VL - 13 IS - 19 ER - TY - JOUR A1 - Berberich, Andreas A1 - Kurz, Andreas A1 - Reinhard, Sebastian A1 - Paul, Torsten Johann A1 - Burd, Paul Ray A1 - Sauer, Markus A1 - Kollmannsberger, Philip T1 - Fourier Ring Correlation and anisotropic kernel density estimation improve deep learning based SMLM reconstruction of microtubules JF - Frontiers in Bioinformatics N2 - Single-molecule super-resolution microscopy (SMLM) techniques like dSTORM can reveal biological structures down to the nanometer scale. The achievable resolution is not only defined by the localization precision of individual fluorescent molecules, but also by their density, which becomes a limiting factor e.g., in expansion microscopy. Artificial deep neural networks can learn to reconstruct dense super-resolved structures such as microtubules from a sparse, noisy set of data points. This approach requires a robust method to assess the quality of a predicted density image and to quantitatively compare it to a ground truth image. Such a quality measure needs to be differentiable to be applied as loss function in deep learning. We developed a new trainable quality measure based on Fourier Ring Correlation (FRC) and used it to train deep neural networks to map a small number of sampling points to an underlying density. Smooth ground truth images of microtubules were generated from localization coordinates using an anisotropic Gaussian kernel density estimator. We show that the FRC criterion ideally complements the existing state-of-the-art multiscale structural similarity index, since both are interpretable and there is no trade-off between them during optimization. The TensorFlow implementation of our FRC metric can easily be integrated into existing deep learning workflows. KW - dSTORM KW - deep learning–artificial neural network (DL-ANN) KW - single molecule localization microscopy KW - microtubule cytoskeleton KW - super-resolution Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261686 VL - 1 ER - TY - JOUR A1 - Alnusaire, Taghreed S. A1 - Sayed, Ahmed M. A1 - Elmaidomy, Abeer H. A1 - Al-Sanea, Mohammad M. A1 - Albogami, Sarah A1 - Albqmi, Mha A1 - Alowaiesh, Bassam F. A1 - Mostafa, Ehab M. A1 - Musa, Arafa A1 - Youssif, Khayrya A. A1 - Refaat, Hesham A1 - Othman, Eman M. A1 - Dandekar, Thomas A1 - Alaaeldin, Eman A1 - Ghoneim, Mohammed M. A1 - Abdelmohsen, Usama Ramadan T1 - An in vitro and in silico study of the enhanced antiproliferative and pro-oxidant potential of Olea europaea L. cv. Arbosana leaf extract via elastic nanovesicles (spanlastics) JF - Antioxidants N2 - The olive tree is a venerable Mediterranean plant and often used in traditional medicine. The main aim of the present study was to evaluate the effect of Olea europaea L. cv. Arbosana leaf extract (OLE) and its encapsulation within a spanlastic dosage form on the improvement of its pro-oxidant and antiproliferative activity against HepG-2, MCF-7, and Caco-2 human cancer cell lines. The LC-HRESIMS-assisted metabolomic profile of OLE putatively annotated 20 major metabolites and showed considerable in vitro antiproliferative activity against HepG-2, MCF-7, and Caco-2 cell lines with IC\(_{50}\) values of 9.2 ± 0.8, 7.1 ± 0.9, and 6.5 ± 0.7 µg/mL, respectively. The encapsulation of OLE within a (spanlastic) nanocarrier system, using a spraying method and Span 40 and Tween 80 (4:1 molar ratio), was successfully carried out (size 41 ± 2.4 nm, zeta potential 13.6 ± 2.5, and EE 61.43 ± 2.03%). OLE showed enhanced thermal stability, and an improved in vitro antiproliferative effect against HepG-2, MCF-7, and Caco-2 (IC\(_{50}\) 3.6 ± 0.2, 2.3 ± 0.1, and 1.8 ± 0.1 µg/mL, respectively) in comparison to the unprocessed extract. Both preparations were found to exhibit pro-oxidant potential inside the cancer cells, through the potential inhibitory activity of OLE against glutathione reductase and superoxide dismutase (IC\(_{50}\) 1.18 ± 0.12 and 2.33 ± 0.19 µg/mL, respectively). These inhibitory activities were proposed via a comprehensive in silico study to be linked to the presence of certain compounds in OLE. Consequently, we assume that formulating such a herbal extract within a suitable nanocarrier would be a promising improvement of its therapeutic potential. KW - olive KW - metabolomic profiling KW - antiproliferative KW - pro-oxidant KW - encapsulation KW - spanlastic KW - nanocarrier KW - docking KW - molecular dynamics simulation KW - Olea Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250064 SN - 2076-3921 VL - 10 IS - 12 ER - TY - RPRT A1 - Dandekar, Thomas T1 - A new cosmology of a crystallization process (decoherence) from the surrounding quantum soup provides heuristics to unify general relativity and quantum physics by solid state physics T1 - Eine neue Kosmologie eines Kristallisationsprozesses (Dekohärenz) vom umgebenden Quantenschaum bietet Heuristiken, um allgemeine Relativitätstheorie und Quantenphysik durch Festkörperphysik zu vereinen N2 - We explore a cosmology where the Big Bang singularity is replaced by a condensation event of interacting strings. We study the transition from an uncontrolled, chaotic soup (“before”) to a clearly interacting “real world”. Cosmological inflation scenarios do not fit current observations and are avoided. Instead, long-range interactions inside this crystallization event limit growth and crystal symmetries ensure the same laws of nature and basic symmetries over our domain. Tiny mis-arrangements present nuclei of superclusters and galaxies and crystal structure leads to the arrangement of dark (halo regions) and normal matter (galaxy nuclei) so convenient for galaxy formation. Crystals come and go, allowing an evolutionary cosmology where entropic forces from the quantum soup “outside” of the crystal try to dissolve it. These would correspond to dark energy and leads to a big rip scenario in 70 Gy. Preference of crystals with optimal growth and most condensation nuclei for the next generation of crystals may select for multiple self-organizing processes within the crystal, explaining “fine-tuning” of the local “laws of nature” (the symmetry relations formed within the crystal, its “unit cell”) to be particular favorable for self-organizing processes including life or even conscious observers in our universe. Independent of cosmology, a crystallization event may explain quantum-decoherence in general: The fact, that in our macroscopic everyday world we only see one reality. This contrasts strongly with the quantum world where you have coherence, a superposition of all quantum states. We suggest that a “real world” (so our everyday macroscopic world) happens only in our domain, i.e. inside a crystal. “Outside” of our domain and our observable universe there is the quantum soup of boiling quantum foam and superposition of all possibilities. In our crystallized world the vacuum no longer boils but is cooled down by the crystallization event and hence is 10**20 smaller, exactly as observed in our everyday world. As we live in a “solid” state, within a crystal, the different quanta which build our world have all their different states nicely separated. This theory postulates there are only n quanta and m states available for them (there is no Everett-like ever splitting multiverse after each decision). In the solid state we live in, there is decoherence, the states are nicely separated. The arrow of entropy for each edge of the crystal forms one fate, one worldline or clear development of a world, while the layers of the crystal are different system states. Some mathematical leads from loop quantum gravity point to required interactions and potentials. A complete mathematical treatment of this unified theory is far too demanding currently. Interaction potentials for strings or membranes of any dimension allow a solid state of quanta, so allowing decoherence in our observed world are challenging to calculate. However, if we introduce here the heuristic that any type of physical interaction of strings corresponds just to a type of calculation, there is already since 1898 the Hurwitz theorem showing that then only 1D, 2D, 4D and 8D (octonions) allow complex or hypercomplex number calculations. No other hypercomplex numbers and hence dimensions or symmetries are possible to allow calculations without yielding divisions by zero. However, the richest solution allowed by the Hurwitz theorem, octonions, is actually the observed symmetry of our universe, E8.   KW - Kosmologie KW - cosmology KW - Hurwitz-Theorem KW - Quantenschleifen-Gravitation KW - Verschränkung KW - Qubits KW - Hurwitz-Theorem KW - loop quantum gravity KW - entanglement KW - Qubits Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230769 ER - TY - JOUR A1 - Link, Fabian A1 - Borges, Alyssa R. A1 - Jones, Nicola G. A1 - Engstler, Markus T1 - To the Surface and Back: Exo- and Endocytic Pathways in Trypanosoma brucei JF - Frontiers in Cell and Developmental Biology N2 - Trypanosoma brucei is one of only a few unicellular pathogens that thrives extracellularly in the vertebrate host. Consequently, the cell surface plays a critical role in both immune recognition and immune evasion. The variant surface glycoprotein (VSG) coats the entire surface of the parasite and acts as a flexible shield to protect invariant proteins against immune recognition. Antigenic variation of the VSG coat is the major virulence mechanism of trypanosomes. In addition, incessant motility of the parasite contributes to its immune evasion, as the resulting fluid flow on the cell surface drags immunocomplexes toward the flagellar pocket, where they are internalized. The flagellar pocket is the sole site of endo- and exocytosis in this organism. After internalization, VSG is rapidly recycled back to the surface, whereas host antibodies are thought to be transported to the lysosome for degradation. For this essential step to work, effective machineries for both sorting and recycling of VSGs must have evolved in trypanosomes. Our understanding of the mechanisms behind VSG recycling and VSG secretion, is by far not complete. This review provides an overview of the trypanosome secretory and endosomal pathways. Longstanding questions are pinpointed that, with the advent of novel technologies, might be answered in the near future. KW - cell surface KW - African trypanosomes KW - endocytosis KW - exocytosis KW - membrane recycling KW - Rab KW - clathrin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244682 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Panzer, Sabine A1 - Zhang, Chong A1 - Konte, Tilen A1 - Bräuer, Celine A1 - Diemar, Anne A1 - Yogendran, Parathy A1 - Yu-Strzelczyk, Jing A1 - Nagel, Georg A1 - Gao, Shiqiang A1 - Terpitz, Ulrich T1 - Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates JF - Frontiers in Molecular Biosciences N2 - Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications. KW - black yeast KW - photoreceptor KW - microbial rhodopsins KW - optogenetics KW - proton channel KW - membrane trafficking KW - fungal rhodopsins KW - Aureobasidium Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249248 SN - 2296-889X VL - 8 ER - TY - JOUR A1 - Vogel, Sebastian A1 - Prinzing, Andreas A1 - Bußler, Heinz A1 - Müller, Jörg A1 - Schmidt, Stefan A1 - Thorn, Simon T1 - Abundance, not diversity, of host beetle communities determines abundance and diversity of parasitoids in deadwood JF - Ecology and Evolution N2 - Most parasites and parasitoids are adapted to overcome defense mechanisms of their specific hosts and hence colonize a narrow range of host species. Accordingly, an increase in host functional or phylogenetic dissimilarity is expected to increase the species diversity of parasitoids. However, the local diversity of parasitoids may be driven by the accessibility and detectability of hosts, both increasing with increasing host abundance. Yet, the relative importance of these two mechanisms remains unclear. We parallelly reared communities of saproxylic beetle as potential hosts and associated parasitoid Hymenoptera from experimentally felled trees. The dissimilarity of beetle communities was inferred from distances in seven functional traits and from their evolutionary ancestry. We tested the effect of host abundance, species richness, functional, and phylogenetic dissimilarities on the abundance, species richness, and Shannon diversity of parasitoids. Our results showed an increase of abundance, species richness, and Shannon diversity of parasitoids with increasing beetle abundance. Additionally, abundance of parasitoids increased with increasing species richness of beetles. However, functional and phylogenetic dissimilarity showed no effect on the diversity of parasitoids. Our results suggest that the local diversity of parasitoids, of ephemeral and hidden resources like saproxylic beetles, is highest when resources are abundant and thereby detectable and accessible. Hence, in some cases, resources do not need to be diverse to promote parasitoid diversity. KW - barcoding KW - deadwood KW - experiment KW - host–parasitoid interaction KW - natural enemy KW - specialization Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238892 VL - 11 IS - 11 SP - 6881 EP - 6888 ER - TY - JOUR A1 - Makbul, Cihan A1 - Kraft, Christian A1 - Grießmann, Matthias A1 - Rasmussen, Tim A1 - Katzenberger, Kilian A1 - Lappe, Melina A1 - Pfarr, Paul A1 - Stoffer, Cato A1 - Stöhr, Mara A1 - Wandinger, Anna-Maria A1 - Böttcher, Bettina T1 - Binding of a pocket factor to Hepatitis B virus capsids changes the rotamer conformation of Phenylalanine 97 JF - Viruses N2 - (1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses. KW - Hepatitis B Virus KW - pocket factor KW - Triton X 100 KW - envelopment KW - maturation signal KW - single strand blocking KW - electron cryo-microscopy KW - isothermal titration calorimetry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248565 SN - 1999-4915 VL - 13 IS - 11 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 - 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 - Andreska, Thomas T1 - Effects of dopamine on BDNF / TrkB mediated signaling and plasticity on cortico-striatal synapses T1 - Effekte von Dopamin auf BDNF / TrkB vermittelte Signalwege und Plastizität an cortico-striatalen Synapsen N2 - Progressive loss of voluntary movement control is the central symptom of Parkinson's disease (PD). Even today, we are not yet able to cure PD. This is mainly due to a lack of understanding the mechanisms of movement control, network activity and plasticity in motor circuits, in particular between the cerebral cortex and the striatum. Brain-derived neurotrophic factor (BDNF) has emerged as one of the most important factors for the development and survival of neurons, as well as for synaptic plasticity. It is thus an important target for the development of new therapeutic strategies against neurodegenerative diseases. Together with its receptor, the Tropomyosin receptor kinase B (TrkB), it is critically involved in development and function of the striatum. Nevertheless, little is known about the localization of BDNF within presynaptic terminals in the striatum, as well as the types of neurons that produce BDNF in the cerebral cortex. Furthermore, the influence of midbrain derived dopamine on the control of BDNF / TrkB interaction in striatal medium spiny neurons (MSNs) remains elusive so far. Dopamine, however, appears to play an important role, as its absence leads to drastic changes in striatal synaptic plasticity. This suggests that dopamine could regulate synaptic activity in the striatum via modulation of BDNF / TrkB function. To answer these questions, we have developed a sensitive and reliable protocol for the immunohistochemical detection of endogenous BDNF. We find that the majority of striatal BDNF is provided by glutamatergic, cortex derived afferents and not dopaminergic inputs from the midbrain. In fact, we found BDNF in cell bodies of neurons in layers II-III and V of the primary and secondary motor cortex as well as layer V of the somatosensory cortex. These are the brain areas that send dense projections to the dorsolateral striatum for control of voluntary movement. Furthermore, we could show that these projection neurons significantly downregulate the expression of BDNF during the juvenile development of mice between 3 and 12 weeks. In parallel, we found a modulatory effect of dopamine on the translocation of TrkB to the cell surface in postsynaptic striatal Medium Spiny Neurons (MSNs). In MSNs of the direct pathway (dMSNs), which express dopamine receptor 1 (DRD1), we observed the formation of TrkB aggregates in the 6-hydroxydopamine (6-OHDA) model of PD. This suggests that DRD1 activity controls TrkB surface expression in these neurons. In contrast, we found that DRD2 activation has opposite effects in MSNs of the indirect pathway (iMSNs). Activation of DRD2 promotes a rapid decrease in TrkB surface expression which was reversible and depended on cAMP. In parallel, stimulation of DRD2 led to induction of phospho-TrkB (pTrkB). This effect was significantly slower than the effect on TrkB surface expression and indicates that TrkB is transactivated by DRD2. Together, our data provide evidence that dopamine triggers dual modes of plasticity on striatal MSNs by acting on TrkB surface expression in DRD1 and DRD2 expressing MSNs. This surface expression of the receptor is crucial for the binding of BDNF, which is released from corticostriatal afferents. This leads to the induction of TrkB-mediated downstream signal transduction cascades and long-term potentiation (LTP). Therefore, the dopamine-mediated translocation of TrkB could be a mediator that modulates the balance between dopaminergic and glutamatergic signaling to allow synaptic plasticity in a spatiotemporal manner. This information and the fact that TrkB is segregated to persistent aggregates in PD could help to improve our understanding of voluntary movement control and to develop new therapeutic strategies beyond those focusing on dopaminergic supply. N2 - Der fortschreitende Verlust der willkürlichen Bewegungskontrolle ist ein zentrales Symptom der Parkinson-Krankheit (PD). Auch heute sind wir noch nicht in der Lage, PD zu heilen. Dafür verantwortlich ist hauptsächlich ein mangelndes Verständnis von Mechanismen der Bewegungskontrolle, Netzwerkaktivität und Plastizität in motorischen Schaltkreisen, insbesondere zwischen Hirnrinde und Striatum. Der neurotrophe Faktor BDNF ist einer der wichtigsten Faktoren für die Entwicklung und das Überleben von Neuronen sowie für synaptische Plastizität im zentralen Nervensystem. BDNF ist daher ein Target für die Entwicklung neuer therapeutischer Strategien gegen neurodegenerative Erkrankungen. Zusammen mit seinem Rezeptor, der Tropomyosin-Rezeptorkinase B (TrkB), ist BDNF maßgeblich an der Entwicklung und Funktion des Striatums beteiligt. Dennoch ist nur wenig bekannt, wo BDNF an Synapsen im Striatum lokalisiert ist, und wo BDNF in Neuronen der Hirnrinde synthetisiert wird. Außerdem ist der Einfluss von Dopamin aus dem Mittelhirn auf die Kontrolle der BDNF / TrkB-Interaktion in striatalen Medium-Spiny-Neuronen (MSNs) bisher unklar. Dopamin scheint jedoch eine wichtige Rolle zu spielen, da dessen Abwesenheit zu drastischen Veränderungen der striatalen Plastizität führt. Dopamin könnte synaptische Plastizität im Striatum über eine Modulation der BDNF / TrkB-Interaktion regulieren. Um diese Fragen beantworten zu können, haben wir ein sensitives und zuverlässiges Protokoll für den immunhistochemischen Nachweis von endogenem BDNF entwickelt. Wir fanden heraus, dass BDNF im Striatum vor allem in glutamatergen Synapsen von Projektion aus dem Kortex lokalisiert ist und nicht in Terminalen dopaminerger Neurone aus dem Mittelhirn. Tatsächlich fanden wir BDNF in den Zellkörpern von Neuronen in den Schichten II-III und V des primären und sekundären motorischen Kortex sowie Schicht V des somatosensorischen Kortex. Es sind jene Hirnareale, welche dichte Projektionen zum dorsolateralen Striatum senden und entscheidend an der Steuerung von willkürlichen Bewegungen beteiligt sind. Weiterhin konnten wir zeigen, dass eben jene Projektionsneurone die Bildung von BDNF während der juvenilen Entwicklung von Mäusen zwischen 3 und 12 Wochen signifikant herunter regulieren. In striatalen MSN fanden wir zudem einen modulatorischen Effekt von Dopamin auf die Translokation von TrkB zur Zelloberfläche. In MSNs des direkten Signalweges (dMSNs), welche Dopaminrezeptor 1 (DRD1) exprimieren, konnten wir die Bildung von TrkB-Aggregaten im 6-Hydroxydopamin (6-OHDA) - Rattenmodell der Parkinson Erkankung beobachten. Dies deutet darauf hin, dass die DRD1-Aktivität die TrkB-Oberflächenexpression in diesen Neuronen steuert. Im Gegensatz dazu fanden wir heraus, dass die DRD2-Aktivierung in MSNs des indirekten Signalweges (iMSNs) eine gegensätzliche Wirkung hat. Die Aktivierung von DRD2 führt zu einer schnellen Reduktion der TrkB-Oberflächenexpression, die reversibel und von cAMP abhängig ist. Außerdem führte die Stimulation von DRD2 zu einer Induktion von Phospho-TrkB (pTrkB). Dieser Effekt war deutlich langsamer als die Wirkung auf die TrkB-Oberflächenexpression und deutet auf eine Transaktivierung von TrkB über DRD2 hin. Insgesamt scheint Dopamin entgegengesetzte Plastizitätsmodi in striatalen MSNs auszulösen, indem es auf die TrkB-Oberflächenexpression in DRD1- und DRD2-exprimierenden MSNs einwirkt. Diese Oberflächenexpression des Rezeptors ist entscheidend für die Bindung von BDNF, welches aus kortiko-striatalen Afferenzen freigesetzt wird. Dies führt zur Induktion von TrkB-vermittelten-Signaltransduktionskaskaden und Langzeitpotenzierung (LTP). Daher könnte die dopamin-vermittelte Translokalisation von TrkB das Gleichgewicht zwischen dopaminergen und glutamatergen Signalen modulieren, um die synaptische Plastizität in einer räumlich-zeitlich abgestimmten Weise zu ermöglichen. Diese Information und die Tatsache, dass TrkB bei PD stabile Aggregate bildet, könnte dazu beitragen, unser Verständnis der willkürlichen Bewegungskontrolle zu verbessern und neue therapeutische Strategien zu entwickeln, die über jene hinausgehen, welche sich auf die dopaminerge Versorgung konzentrieren. KW - Brain-derived neurotrophic factor KW - Parkinson Krankheit KW - Plastizität KW - Motorisches Lernen KW - Basalganglien KW - Brain-derived neurotrophic factor KW - TrkB KW - Basal Ganglia KW - Motor learning KW - Parkinson's disease KW - Synaptic plasticity KW - Striatum KW - Medium spiny neurons KW - Cortico-striatal projection neurons Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-174317 ER - TY - JOUR A1 - Pütz, Stephanie M. A1 - Kram, Jette A1 - Rauh, Elisa A1 - Kaiser, Sophie A1 - Toews, Romy A1 - Lueningschroer-Wang, Yi A1 - Rieger, Dirk A1 - Raabe, Thomas T1 - Loss of p21-activated kinase Mbt/PAK4 causes Parkinson-like symptoms in Drosophila JF - Disease Models & Mechanisms N2 - Parkinson's disease (PD) provokes bradykinesia, resting tremor, rigidity and postural instability, and also non-motor symptoms such as depression, anxiety, sleep and cognitive impairments. Similar phenotypes can be induced in Drosophila melanogaster through modification of PD-relevant genes or the administration of PD inducing toxins. Recent studies correlated deregulation of human p21-activated kinase 4 (PAK4) with PD, leaving open the question of a causative relationship of mutations in this gene for manifestation of PD symptoms. To determine whether flies lacking the PAK4 homolog Mushroom bodies tiny (Mbt) show PD-like phenotypes, we tested for a variety of PD criteria. Here, we demonstrate that mbt mutant flies show PD-like phenotypes including age-dependent movement deficits, reduced life expectancy and fragmented sleep. They also react to a stressful situation with higher immobility, indicating an influence of Mbt on emotional behavior. Loss of Mbt function has a negative effect on the number of dopaminergic protocerebral anterior medial (PAM) neurons, most likely caused by a proliferation defect of neural progenitors. The age-dependent movement deficits are not accompanied by a corresponding further loss of PAM neurons. Previous studies highlighted the importance of a small PAM subgroup for age-dependent PD motor impairments. We show that impaired motor skills are caused by a lack of Mbt in this PAM subgroup. In addition, a broader re-expression of Mbt in PAM neurons improves life expectancy. Conversely, selective Mbt knockout in the same cells shortens lifespan. We conclude that mutations in Mbt/PAK4 can play a causative role in the development of PD phenotypes. KW - Sleep fragmentation KW - Life expectancy KW - Emotional behavior KW - Dopaminergic PAM cluster neurons KW - Drosophila KW - Parkinson's disease KW - Mbt KW - PAK4 KW - Negative geotaxis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259222 VL - 14 IS - 6 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 - 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 - 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 - Castañeda Londono, Paula Andrea A1 - Banholzer, Nicole A1 - Bannermann, Bridget A1 - Kramer, Susanne T1 - Is mRNA decapping activity of ApaH like phosphatases (ALPH’s) the reason for the loss of cytoplasmic ALPH’s in all eukaryotes but Kinetoplastida? JF - BMC Ecology and Evolution N2 - Background: ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and are present in eukaryotes of all eukaryotic super-groups; still, only two proteins have been functionally characterised. One is ALPH1 from the Kinetoplastid Trypanosoma brucei that we recently found to be the mRNA decapping enzyme of the parasite. mRNA decapping by ALPHs is unprecedented in eukaryotes, which usually use nudix hydrolases, but the bacterial ancestor protein ApaH was recently found to decap non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or more widespread among eukaryotes. Results: We screened 824 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to refine phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of ALPHs. We found that most eukaryotes have either no ALPH (500/824) or very short ALPHs, consisting almost exclusively of the catalytic domain. These ALPHs had mostly predicted non-cytoplasmic localisations, often supported by the presence of transmembrane helices and signal peptides and in two cases (one in this study) by experimental data. The only exceptions were ALPH1 homologues from Kinetoplastida, that all have unique C-terminal and mostly unique N-terminal extension, and at least the T. brucei enzyme localises to the cytoplasm. Surprisingly, despite of these non-cytoplasmic localisations, ALPHs from all eukaryotic super-groups had in vitro mRNA decapping activity. Conclusions: ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme since, or use it exclusively outside the cytoplasm in organelles in a version consisting of the catalytic domain only. While our data provide no evidence for the presence of further mRNA decapping enzymes among eukaryotic ALPHs, the broad substrate range of ALPHs that includes mRNA caps provides an explanation for the selection against the presence of a cytoplasmic ALPH protein as a mean to protect mRNAs from unregulated degradation. Kinetoplastida succeeded to exploit ALPH as their mRNA decapping enzyme, likely using the Kinetoplastida-unique N- and C-terminal extensions for regulation. KW - ApaH like phosphatase KW - ApaH KW - ALPH KW - Trypanosoma brucei KW - mRNA decapping KW - m7G cap KW - mRNA cap KW - ALPH1 KW - Kinetoplastida Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261180 VL - 21 ER - TY - THES A1 - Mayr, Antonia Veronika T1 - Following Bees and Wasps up Mt. Kilimanjaro: From Diversity and Traits to hidden Interactions of Species T1 - Auf den Spuren von Bienen und Wespen auf den Kilimandscharo: Eine Studie über die Diversität, Merkmale und verborgenen Wechselwirkungen zwischen Arten N2 - Chapter 1 – General Introduction One of the greatest challenges of ecological research is to predict the response of ecosystems to global change; that is to changes in climate and land use. A complex question in this context is how changing environmental conditions affect ecosystem processes at different levels of communities. To shed light on this issue, I investigate drivers of biodiversity on the level of species richness, functional traits and species interactions in cavity-nesting Hymenoptera. For this purpose, I take advantage of the steep elevational gradient of Mt. Kilimanjaro that shows strong environmental changes on a relatively small spatial scale and thus, provides a good environmental scenario for investigating drivers of diversity. In this thesis, I focus on 1) drivers of species richness at different trophic levels (Chapter 2); 2) seasonal patterns in nest-building activity, life-history traits and ecological rates in three different functional groups and at different elevations (Chapter 3) and 3) changes in cuticular hydrocarbons, pollen composition and microbiomes in Lasioglossum bees caused by climatic variables (Chapter 4). Chapter 2 – Climate and food resources shape species richness and trophic interactions of cavity-nesting Hymenoptera Drivers of species richness have been subject to research for centuries. Temperature, resource availability and top-down regulation as well as the impact of land use are considered to be important factors in determining insect diversity. Yet, the relative importance of each of these factors is unknown. Using trap nests along the elevational gradient of Mt. Kilimanjaro, we tried to disentangle drivers of species richness at different trophic levels. Temperature was the major driver of species richness across trophic levels, with increasing importance of food resources at higher trophic levels in natural antagonists. Parasitism rate was both related to temperature and trophic level, indicating that the relative importance of bottom-up and top-down forces might shift with climate change. Chapter 3 – Seasonal variation in the ecology of tropical cavity-nesting Hymenoptera Natural populations fluctuate with the availability of resources, presence of natural enemies and climatic variations. But tropical mountain seasonality is not yet well investigated. We investigated seasonal patterns in nest-building activity, functional traits and ecological rates in three different insect groups at lower and higher elevations separately. Insects were caught with trap nests which were checked monthly during a 17 months period that included three dry and three rainy seasons. Insects were grouped according to their functional guilds. All groups showed strong seasonality in nest-building activity which was higher and more synchronised among groups at lower elevations. Seasonality in nest building activity of caterpillar-hunting and spider-hunting wasps was linked to climate seasonality while in bees it was strongly linked to the availability of flowers, as well as for the survival rate and sex ratio of bees. Finding adaptations to environmental seasonality might imply that further changes in climatic seasonality by climate change could have an influence on life-history traits of tropical mountain species. Chapter 4 – Cryptic species and hidden ecological interactions of halictine bees along an elevational Gradient Strong environmental gradients such as those occurring along mountain slopes are challenging for species. In this context, hidden adaptations or interactions have rarely been considered. We used bees of the genus Lasioglossum as model organisms because Lasioglossum is the only bee genus occurring with a distribution across the entire elevational gradient at Mt. Kilimanjaro. We asked if and how (a) cuticular hydrocarbons (CHC), which act as a desiccation barrier, change in composition and chain length along with changes in temperature and humidity (b), Lasioglossum bees change their pollen diet with changing resource availability, (c) gut microbiota change with pollen diet and climatic conditions, and surface microbiota change with CHC and climatic conditions, respectively, and if changes are rather influenced by turnover in Lasioglossum species along the elevational gradient. We found physiological adaptations with climate in CHC as well as changes in communities with regard to pollen diet and microbiota, which also correlated with each other. These results suggest that complex interactions and feedbacks among abiotic and biotic conditions determine the species composition in a community. Chapter 5 – General Discussion Abiotic and biotic factors drove species diversity, traits and interactions and they worked differently depending on the functional group that has been studied, and whether spatial or temporal units were considered. It is therefore likely, that in the light of global change, different species, traits and interactions will be affected differently. Furthermore, increasing land use intensity could have additional or interacting effects with climate change on biodiversity, even though the potential land-use effects at Mt. Kilimanjaro are still low and not impairing cavity-nesting Hymenoptera so far. Further studies should address species networks which might reveal more sensitive changes. For that purpose, trap nests provide a good model system to investigate effects of global change on multiple trophic levels and may also reveal direct effects of climate change on entire life-history traits when established under different microclimatic conditions. The non-uniform effects of abiotic and biotic conditions on multiple aspects of biodiversity revealed with this study also highlight that evaluating different aspects of biodiversity can give a more comprehensive picture than single observations. N2 - Kapitel 1 – Allgemeine Einführung Eine der größten Herausforderungen der ökologischen Forschung ist es, die Reaktion der Ökosysteme auf den globalen Wandel, d.h. auf Veränderungen von Klima und Landnutzung, vorherzusagen. Eine komplexe Frage in diesem Zusammenhang ist, wie sich verändernde Umweltbedingungen auf die Ökosystemprozesse auf verschiedenen Ebenen von Gemeinschaften auswirken. Um dieses Thema näher zu beleuchten, untersuche ich die Triebkräfte der Biodiversität auf der Ebene des Artenreichtums, der funktionellen Eigenschaften und der Wechselwirkungen zwischen Arten bei Hautflüglern, die in Hohlräumen nisten. Zu diesem Zweck nutze ich den steilen Höhengradienten des Kilimandscharo, der starke Umweltveränderungen auf relativ kleinem Raum mit sich bringt und somit ein gutes System für die Untersuchung von Triebkräften der biologischen Vielfalt bietet. In dieser Arbeit konzentriere ich mich auf 1) Triebkräfte des Artenreichtums auf verschiedenen trophischen Ebenen (Kapitel 2); 2) saisonale Muster in der Nestbauaktivität, lebensgeschichtliche Merkmale und ökologische Raten in drei verschiedenen funktionellen Gruppen und in verschiedenen Höhenlagen (Kapitel 3) und 3) Veränderungen in kutikulären Kohlenwasserstoffen, Pollenzusammensetzung und Mikrobiomen bei Lasioglossum Bienen, die durch klimatische Faktoren verursacht werden (Kapitel 4). Kapitel 2 – Klima und Nahrungsressourcen prägen den Artenreichtum und die trophischen Wechselwirkungen von hohlraumnistenden Hautflüglern Die Triebkräfte des Artenreichtums werden seit Jahrhunderten erforscht. Temperatur, Ressourcenverfügbarkeit und Top-Down-Regulierung sowie die Auswirkungen der Landnutzung werden als wichtige Faktoren für die Bestimmung der Insektenvielfalt angesehen. Die relative Bedeutung jedes dieser Faktoren ist jedoch unbekannt. Mit Hilfe von Nisthilfen entlang des Höhengradienten des Kilimandscharo versuchten wir, die Triebkräfte des Artenreichtums auf verschiedenen trophischen Ebenen zu enträtseln. Die Temperatur war der Hauptfaktor für den Artenreichtum auf allen trophischen Ebenen, wobei die Bedeutung der Nahrungsressourcen auf den höheren trophischen Ebenen der natürlichen Antagonisten zunahm. Die Parasitierungsrate wurde sowohl durch die Temperatur als auch durch die trophische Ebene bestimmt, was darauf hindeutet, dass sich die relative Bedeutung der Bottom-up- und Top-down-Kräfte mit dem Klimawandel verschieben könnte. Kapitel 3 – Saisonale Schwankungen in der Ökologie von tropischen hohlraumnistenden Hautflüglern Natürliche Populationen schwanken mit der Verfügbarkeit von Ressourcen, dem Vorhandensein natürlicher Feinde und klimatischen Schwankungen. Die Saisonalität ist jedoch auf tropischen Bergen noch nicht gut untersucht. Wir untersuchten saisonale Muster in der Nestbauaktivität, funktionale Merkmale und ökologische Raten bei drei verschiedenen Insektengruppen in niedrigeren und höheren Höhenlagen. Insekten wurden mit Nisthilfen gefangen, die während eines Zeitraums von 17 Monaten, der drei Trocken- und drei Regenzeiten umfasste, monatlich überprüft wurden. Die Insekten wurden nach ihren funktionalen Gilden eingeteilt. Alle Gruppen zeigten eine starke Saisonalität im Nestbau, die in niedrigeren Lagen höher war und dort zwischen den Gruppen stärker synchronisiert war. Die Saisonalität im Nestbau von Raupen- und Spinnen- jagenden Wespen war mit saisonalen Klimaschwankungen verbunden, während sie bei Bienen stark von der Verfügbarkeit von Blüten abhing, genauso wie die Überlebensrate und das Geschlechterverhältnis der Bienen von der Blütenmenge abhing. Die Anpassung an die Saisonalität der Umwelt könnte bedeuten, dass weitere Veränderungen der saisonalen Klimaschwankungen durch den Klimawandel einen Einfluss auf die lebensgeschichtlichen Merkmale tropischer Bergarten haben könnten. Kapitel 4 – Kryptische Arten und versteckte ökologische Wechselwirkungen bei Schmalbienen entlang eines Höhengradienten Starke Umweltgradienten, wie sie an Berghängen auftreten, stellen für Arten eine Herausforderung dar. Versteckte Anpassungen oder Interaktionen wurden in diesem Zusammenhang selten berücksichtigt. Als Modellorganismen haben wir Bienen der Gattung Lasioglossum verwendet, da Lasioglossum die einzige Bienengattung ist, die über den gesamten Höhengradienten am Kilimandscharo weit verbreitet ist. Wir fragten, ob und wie (a) kutikuläre Kohlenwasserstoffe (CHC), die als Barriere gegen Austrocknung wirken, sich in ihrer Zusammensetzung und Kettenlänge entlang von Temperatur- und Feuchtigkeitsänderungen verändern; (b) Lasioglossum Bienen ihre Pollennahrung mit wechselnder Ressourcenverfügbarkeit ändern; (c) Änderungen von Darm-Mikrobiota mit Pollennahrung und Klimabedingungen und Änderungen von Oberflächen-Mikrobiota mit CHC und Klimabedingungen zusammen hängen, und ob die Veränderungen eher durch den Wechsel von Lasioglossum Arten entlang des Höhengradienten beeinflusst werden. Wir fanden physiologische Anpassungen an das Klima in CHC, sowie Veränderungen in der Zusammensetzung von Pollennahrung und Mikrobiota, die auch miteinander korrelierten. Diese Ergebnisse deuten darauf hin, dass komplexe Wechselwirkungen und Rückkopplungen zwischen abiotischen und biotischen Bedingungen die Artenzusammensetzung in einer Gemeinschaft bestimmen. Kapitel 5 – Allgemeine Diskussion Abiotische und biotische Faktoren förderten die Artenvielfalt, Eigenschaften und Wechselwirkungen von Arten und sie wirkten unterschiedlich, je nachdem, welche funktionelle Gruppe untersucht wurde und ob räumliche oder zeitliche Einheiten berücksichtigt worden sind. Es ist daher wahrscheinlich, dass im Lichte des globalen Wandels verschiedene Arten, Merkmale und Wechselwirkungen unterschiedlich betroffen sein werden. Darüber hinaus könnte eine zunehmende Landnutzungsintensität zusätzliche Auswirkungen oder Wechselwirkungen mit dem Klimawandel auf die Biodiversität haben, auch wenn die potenziellen Landnutzungseffekte am Kilimandscharo noch gering sind und bis jetzt die hohlraumnistenden Hautflüglern nicht beeinträchtigen. Weitere Studien sollten sich mit Nahrungsnetzwerken befassen, die empfindlichere Veränderungen aufzeigen könnten. Nisthilfen bieten dafür ein gutes Modellsystem, um die Auswirkungen des globalen Wandels auf mehreren trophischen Ebenen zu untersuchen, und können auch direkte Auswirkungen des Klimawandels auf ganze lebensgeschichtliche Merkmale aufzeigen, wenn sie unter verschiedenen mikroklimatischen Bedingungen etabliert werden. Die nicht einheitlichen Auswirkungen abiotischer und biotischer Bedingungen auf mehrere Aspekte der Biodiversität, die in dieser Studie gezeigt wurden, zeigen auch, dass die Untersuchung verschiedener Aspekte der Biodiversität ein umfassenderes Bild vermitteln kann als Einzelbetrachtungen. KW - land use KW - Landnutzung KW - climate change KW - bees KW - wasps KW - biodiversity KW - Klimawandel KW - Bienen KW - Wespen KW - Biodiversität Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-182922 ER - TY - JOUR A1 - Mrestani, Achmed A1 - Pauli, Martin A1 - Kollmannsberger, Philip A1 - Repp, Felix A1 - Kittel, Robert J. A1 - Eilers, Jens A1 - Doose, Sören A1 - Sauer, Markus A1 - Sirén, Anna-Leena A1 - Heckmann, Manfred A1 - Paul, Mila M. T1 - Active zone compaction correlates with presynaptic homeostatic potentiation JF - Cell Reports N2 - Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier. KW - active zone KW - Bruchpilot KW - RIM-binding protein KW - compaction KW - homeostasis KW - presynaptic plasticity KW - super-resolution microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265497 VL - 37 IS - 1 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 - Lehenberger, Maximilian A1 - Benkert, Markus A1 - Biedermann, Peter H. W. T1 - Ethanol-Enriched Substrate Facilitates Ambrosia Beetle Fungi, but Inhibits Their Pathogens and Fungal Symbionts of Bark Beetles JF - Frontiers in Microbiology N2 - Bark beetles (sensu lato) colonize woody tissues like phloem or xylem and are associated with a broad range of micro-organisms. Specific fungi in the ascomycete orders Hypocreales, Microascales and Ophistomatales as well as the basidiomycete Russulales have been found to be of high importance for successful tree colonization and reproduction in many species. While fungal mutualisms are facultative for most phloem-colonizing bark beetles (sensu stricto), xylem-colonizing ambrosia beetles are long known to obligatorily depend on mutualistic fungi for nutrition of adults and larvae. Recently, a defensive role of fungal mutualists for their ambrosia beetle hosts was revealed: Few tested mutualists outcompeted other beetle-antagonistic fungi by their ability to produce, detoxify and metabolize ethanol, which is naturally occurring in stressed and/or dying trees that many ambrosia beetle species preferentially colonize. Here, we aim to test (i) how widespread beneficial effects of ethanol are among the independently evolved lineages of ambrosia beetle fungal mutualists and (ii) whether it is also present in common fungal symbionts of two bark beetle species (Ips typographus, Dendroctonus ponderosae) and some general fungal antagonists of bark and ambrosia beetle species. The majority of mutualistic ambrosia beetle fungi tested benefited (or at least were not harmed) by the presence of ethanol in terms of growth parameters (e.g., biomass), whereas fungal antagonists were inhibited. This confirms the competitive advantage of nutritional mutualists in the beetle’s preferred, ethanol-containing host material. Even though most bark beetle fungi are found in the same phylogenetic lineages and ancestral to the ambrosia beetle (sensu stricto) fungi, most of them were highly negatively affected by ethanol and only a nutritional mutualist of Dendroctonus ponderosae benefited, however. This suggests that ethanol tolerance is a derived trait in nutritional fungal mutualists, particularly in ambrosia beetles that show cooperative farming of their fungi. KW - ambrosia fungi KW - bark and ambrosia beetles KW - symbiont selection KW - ethanol KW - detoxification KW - Ips typographus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222222 SN - 1664-302X VL - 11 ER - TY - JOUR A1 - Schuster, Sarah A1 - Lisack, Jaime A1 - Subota, Ines A1 - Zimmermann, Henriette A1 - Reuter, Christian A1 - Mueller, Tobias A1 - Morriswood, Brooke A1 - Engstler, Markus T1 - Unexpected plasiticty in the life cycle of Trypanosoma brucei JF - eLife N2 - African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host’s circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia. KW - trypanosoma KW - sleeping sickness KW - tsetse fly KW - transmission KW - life cycle KW - development Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261744 VL - 10 ER - TY - THES A1 - Zachary, Marie T1 - Functional characterization of small non-coding RNAs of \(Neisseria\) \(gonorrhoeae\) T1 - Funktionelle Charakterisierung kleiner nicht-kodierender RNAs in \(Neisseria\) \(gonorrhoeae\) N2 - During infection, bacteria need to adapt to a changing environment and have to endure various stress conditions. Small non-coding RNAs are considered as important regulators of bacterial gene expression and so allow quick adaptations by altering expression of specific target genes. Regulation of gene expression in the human-restricted pathogen Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhoea, is only poorly understood. The present study aims a better understanding of gene regulation in N. gonorrhoeae by studying small non-coding RNAs. The discovery of antisense RNAs for all opa genes led to the hypothesis of asRNA-mediated degradation of out-of-frame opa transcripts. Analysis of asRNA expression revealed a very low abundance of the transcripts and inclusion of another phase-variable gene in the study indicates that the asRNAs are not involved in degradation of out-of-frame transcripts. This doctoral thesis focuses on the analysis of trans-acting sRNAs. The sibling sRNAs NgncR_162 and NgncR_163 were discovered as post-transcriptional regulators altering expression of genes involved in metabolic processes, amino acid uptake and transcriptional regulation. A more detailed analysis by in silico and transcriptomic approaches showed that the sRNAs regulate a broad variety of genes coding for proteins of central metabolism, amino acid biosynthesis and degradation and several transport processes. Expression levels of the sibling sRNAs depend on the growth phase of the bacteria and on the growth medium. This indicates that NgncR_162 and NgncR_163 are involved in the adaptation of the gonococcal metabolism to specific growth conditions. This work further initiates characterisation of the sRNA NgncR_237. An in silico analysis showed details on sequence conservation and a possible secondary structure. A combination of in silico target prediction and differential RNA sequencing resulted in the identification of several target genes involved in type IV pilus biogenesis and DNA recombination. However, it was not successful to find induction conditions for sRNA expression. Interestingly, a possible sibling sRNA could be identified that shares the target interaction sequence with NgncR_237 and could therefore target the same mRNAs. In conclusion, this thesis provides further insights in gene regulation by non-coding RNAs in N. gonorrhoeae by analysing two pairs of sibling sRNAs modulating bacterial metabolism or possibly type IV pilus biogenesis. N2 - Bakterien müssen sich während des Infektionsprozesses an eine sich veränderte Umgebung anpassen und sind dabei zahlreichen Stressfaktoren ausgesetzt. Kleine, nicht-kodierende RNAs gelten als wichtige Regulatoren der bakteriellen Genexpression und ermöglichen daher eine schnelle Anpassung durch eine Veränderung der Expression spezifischer Ziel-Gene. Die Regulation der Genexpression des Humanpathogens Neisseria gonorrhoeae, Auslöser der Geschlechtskrankheit Gonorrhö, ist bis jetzt kaum verstanden. Die vorliegende Studie soll durch die Analyse kleiner, nicht-kodierender RNAs zum besseren Verständnis der Genregulation in Gonokokken beitragen. Durch die Entdeckung von antisense-RNAs für alle opa Gene wurde die Hypothese entwickelt, dass diese für den Abbau von opa Transkripten außerhalb des Leserahmens verantwortlich sind. Eine Analyse der asRNA Expression zeigte jedoch, dass diese sehr wenig exprimiert werden und auch die Untersuchung eines anderen phasenvariablen Gens weist darauf hin, dass die asRNAs keine Bedeutung für den Abbau von Transkripten außerhalb des Leserahmens haben. Der Schwerpunkt der Doktorarbeit liegt auf der Untersuchung trans-codierter sRNAs. Die Zwillings-sRNAs NgncR_162 und NgncR_163 agieren als post-transkriptionelle Regulatoren, die die Expression von Genen verändern, die bei Stoffwechselprozessen, Aminosäureaufnahme und transkriptioneller Regulation eine Rolle spielen. Eine detailliertere Analyse durch in silico- und Transkriptom-Studien zeigte, dass die sRNAs ein großes Spektrum an Genen regulieren, die für Proteine des Zentralstoffwechsels, der Aminosäurebiosynthese und des –abbaus, sowie zahlreicher Transportprozesse kodieren. Die Expressionslevel der Zwillings-sRNAs hängen von der Wachstumsphase der Bakterien und dem Wachstumsmedium ab. Das weist darauf hin, dass NgncR_162 und NgncR_163 eine Rolle bei der Adaptation des Stoffwechsels von Gonokokken zu bestimmten Wachstumsbedingungen spielen. In dieser Arbeit wird zudem die Charakterisierung der sRNA NgncR_237 initiiert. Im Rahmen von in silico Analysen wurde die Sequenzkonservierung und mögliche Sekundärstruktur untersucht. Eine Kombination aus in silico Zielgen-Vorhersage und differentieller RNA Sequenzierung führte zur Identifizierung zahlreicher Zielgene, die in der Biogenese von Typ IV Pili und DNA Rekombination eine Rolle spielen. Allerdings konnten keine Induktionsbedingungen für die sRNA Expression gefunden werden. Interessanterweise konnte eine mögliche Zwillings-sRNA identifiziert werden, die dieselbe Targetinteraktionsdomäne wie NgncR_237 hat und somit dieselben Zielgene regulieren könnte. Zusammenfassend ermöglicht diese Arbeit neue Einblicke in die Genregulation durch nicht-kodierende RNAs in Gonokokken, indem zwei Paare Zwillings-sRNAs analysiert wurden, die den bakteriellen Stoffwechsel anpassen oder möglicherweise eine Rolle in der Typ IV Pilus Biogenese spielen. KW - Neisseria gonorrhoeae KW - Non-coding RNA KW - Genregulation KW - regulation of gene expression Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245826 ER - TY - THES A1 - Schubert, Jonathan T1 - Bildgebende Zweifarben-Einzelmolekül-PET-Fluoreszenzspektroskopie am molekularen Chaperon Hsp90 T1 - Two-color single-molecule PET fluorescence imaging spectroscopy on the molecular chaperone Hsp90 N2 - Im Forschungsfeld der Proteindynamik häufen sich in den letzten Jahren Untersuchungen an einzelnen Molekülen. Damit können molekulare Ereignisse, die in konventioneller Spektroskopie durch stochastische Prozesse unentdeckt bleiben, durch direkte Beobachtung identifiziert und analysiert werden, was zu tieferem mechanistischem Verständnis des untersuchten Systems beitragen kann. Die Implikation des molekularen Chaperons Hsp90 in die korrekte Faltung und Aktivierung einer Vielzahl davon abhängiger Klientenproteine machen es zu einem zentralen Knotenpunkt der zellulären Proteinhomöostase, allerdings ist der Mechanismus seiner breiten Klientenerkennung und -prozessierung bisher nur lückenhaft untersucht. Mit der Erkenntnis, dass Hsp90 ATP abhängig große, ratenlimitierende Umstrukturierungen erfährt, wurden Reportersysteme entwickelt, die auf dem Förster-Resonanzenergietransfer mit einer räumlichen Auflösung von ca. 2-10 nm basieren. Diese dokumentieren einen Klammerschluss des Chaperons und prognostizieren einen intermediatbbasierten Konformations-Zyklus. Details über den Mechanismus der Umstrukturierungen wurden mit der Entwicklung von Reportersystemen ermittelt, die auf dem photoinduzierten Elektronentransfer zwischen der Aminosäure Tryptophan und einem organischen Farbstoff basieren. Die Technik beruht auf kontaktinduzierter Fluoreszenzlöschung und damit verbundenen digitalen Intensitätsübergängen, dabei ermöglicht die räumliche Sensitivität von < 1 nm die Beobachtung von lokalen Umstrukturierungen. In Hsp90 wurden damit mittels konventioneller Spektroskopie drei kritische lokale Umlagerungen untersucht und daraus ein Modell mit heterogenen apo-Konformationen sowie ein kooperativer Konformationszyklus abgeleitet, der dem intermediatbasierten Modell gegenübersteht. Im Rahmen dieser Dissertation wurde anhand des Hsp90-Chaperons eine Methode entwickelt, die eine bildgebende PET Fluoreszenzspektroskopie von mehreren Umstrukturierungen gleichzeitig an einzelnen Molekülen erlaubt. Ein umfangreiches Farbstoffscreening führte zur Identifizierung eines Farbstoffpaars, das die PET-basierte simultane Aufzeichnung zweier Konformations-Koordinaten ermöglicht. Über verschiedene Modifikationen des Chaperons konnten einzelmolekültaugliche Oberflächen hergestellt werden, auf denen zweifach markierte Hsp90-Proteine immobilisiert sind. Fluoreszenzintensitätszeitspuren einzelner Chaperone und entsprechende Kontrollkonstrukte bestätigen qualitativ den Erfolg der Methode, für die quantitative Analyse wurde eine Routine in der Programmiersprache Python entwickelt, mit welcher kinetische Informationen ermittelt werden konnten. Diese legen eine enge wechselseitige Abhängigkeit der drei lokalen Elemente nahe, wobei der Großteil der Konformationsübergänge zweier simultan aufgezeichneter Umstrukturierungen Synchronität innerhalb von zwei Sekunden zeigt. Im Vergleich zur Hydrolyse von einem ATP in mehreren Minuten deutet das auf eine enge Kopplung hin. Weiter konnte eine Beschleunigung der Dynamiken durch aromatische Modifikation des N-Terminus von Hsp90 beobachtet werden, zudem erlaubt der Einzelmolekülansatz die Verwendung des nativen Nukleotids ATP, wodurch auch die lokalen Öffnungsdynamiken zugänglich werden. Die zur Bestimmung der Zeitkonstanten durchgeführte Analyse unterstützt die Ansicht heterogener apo-Zustände und einer einheitlich geschlossenen Konformation. Die bildgebende Zweifarben-Einzelmolekül-PET-Spektroskopie konnte insgesamt zu einem Komplement der Einzelmolekül-FRET-Spektroskopie entwickelt werden, um damit lokale Konformationsdynamiken zu untersuchen. Der bildgebende Ansatz erlaubt eine einfache Implementierung in einen experimentellen Einzelmolekül-FRET Aufbau bei gleichzeitiger Erweiterung der beobachteten Koordinaten und wird so zu einem breit anwendbaren Werkzeug multidimensionaler Dynamikuntersuchungen einzelner Proteine. N2 - Over the past years, the number of investigations of single molecules has risen in the field of protein dynamics studies. Direct observation of molecular events that are obscured by stochastic processes in bulk measurements can provide a deeper mechanistic understanding of the systems under study. The molecular chaperone Hsp90, as being involved in the correct folding and activation of client proteins, thereby acting at late-stage folding, is a central node of cellular protein homeostasis. The mechanistic understanding of its broad client recognition and processing capability still remains elusive. The discovery of large conformational changes that drive the chaperone through a rate limiting conformational cycle as a reaction of ATP binding led to the development of reporter systems that probe the global rearrangement. As the reporters are based on Förster resonance energy transfer, they are active on a spatial scale of 2-10 nm and report on the molecular clamp closure. The predicted conformational cycle implicates several intermediate states. Details of the underlying rearrangements were obtained by the development of reporter systems based on photoinduced electron transfer between the amino acid tryptophan and an organic dye. As the technique relies on contact-induced quenching of fluorescence, which is accompanied by digital intensity transitions, the resulting spatial resolution of < 1 nm enables probing of local conformational rearrangements. In bulk experiments, three critical local dynamics were probed in Hsp90, leading to the assumption of heterogeneous apo conformations and an associated cooperative cycle which faces the intermediate-based model. Within the scope of this doctoral thesis, two color single-molecule PET fluorescence imaging spectroscopy was developed using the Hsp90 chaperone to study multiple conformational rearrangements simultaneously on individual proteins. Extensive dye screening identified a dye pair suitable for the PET-based investigation of two different conformational coordinates simultaneously. Modifications on the chaperone protein enabled the immobilization of double-labeled Hsp90 molecules on glass surfaces that are suited for single-molecule studies. Fluorescence intensity time traces of single chaperones and related control constructs validated qualitatively the success of the method. For quantitative analysis, a routine was developed in the programming language Python to obtain kinetic information. Derived kinetics pointed to a close interdependence between the three local elements. Furthermore, the majority of state transitions of rearrangements studied at the same time occurred simultaneously within a two-second window, thereby suggesting synchronicity. Compared to the hydrolysis of one ATP molecule taking minutes, this suggests a tight coupling of motions. Further, an aromatic modification of the Hsp90 N-Terminus resulted in accelerated local dynamics. Besides investigating the dynamics accompanying clamp closure, clamp opening kinetics also became accessible through the use of native nucleotide ATP. The analysis performed as part of the determination of time constants supports the view of a heterogeneous apo and a uniformly closed conformation. Two-color single-molecule PET fluorescence imaging spectroscopy was developed into a technique complement to single-molecule FRET spectroscopy that enables the probing of local conformational dynamics in immobilized proteins. The imaging approach allows for easy implementation in a single-molecule FRET setup while expanding the observed coordinates, making the PET-based technique a widely applicable tool for multidimensional dynamics studies of single proteins. KW - Fluoreszenzspektroskopie KW - Einzelmolekülspektroskopie KW - Hitzeschock-Proteine KW - Proteinsynthese KW - Photoinduzierter Elektronentransfer KW - photoinduced electron transfer KW - Hsp90 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244938 ER - TY - THES A1 - Rajab, Suhaila T1 - Untersuchung von Sub-Millisekunden Dynamiken und allosterischer Kommunikation in Ligandenbindedomänen ionotroper Glutamatrezeptoren T1 - Investigation of sub-millisecond dynamics and allosteric communication in ionotropic glutamate receptor ligand binding domains N2 - Ionotrope Glutamatrezeptoren (iGluRs) sind ligandengesteuerte Ionenkanäle und vermitteln den Großteil der exzitatorischen Signalweiterleitung im gesamten zentralen Nervensystem. Darüber hinaus spielen iGluRs eine entscheidende Rolle bei der neuronalen Entwicklung und Funktion, einschließlich Lernprozessen und Gedächtnisbildung. Da eine Fehlfunktion dieser Rezeptoren mit zahlreichen neurodegenerativen Erkrankungen verbunden ist, stellen iGluRs zudem wichtige Zielproteine für die pharmakologische Wirkstoffentwicklung dar. Im Allgemeinen wird zwischen drei Untergruppen ionotroper Glutamatrezeptoren unterschieden, welche aufgrund ihrer Selektivität für einen bestimmten Liganden benannt sind: AMPA-, Kainate-, und NMDA-Rezeptoren. Die iGluRs jeder dieser Untergruppen bestehen in der Regel aus vier Untereinheiten, welche wiederum aus vier semiautonomen Domänen aufgebaut sind: (i) die aminoterminale Domäne (ATD), (ii) die Ligandenbindedomäne (LBD), (iii) die Transmembrandomäne (TMD) und (iv) die carboxyterminale Domäne (CTD). Die Ligandenbindedomäne, welche wiederum aus zwei Lobes (D1 und D2) besteht und in ihrer Struktur einer Muschelschale ähnelt, vollzieht bei Bindung eines Neurotransmitters eine Konformationsänderung, wobei sie sich um den gebundenen Agonisten herumschließt. Diese Konformationsänderung der LBD wird auf die Transmembrandomäne, welche den membranüberspannenden Ionenkanal ausbildet, übertragen, was in einer Umlagerung der Transmembranhelices und infolgedessen der Öffnung des Ionenkanals resultiert. Die Konformationsänderung der LBD ist demnach die treibende Kraft, welche dem Öffnen und Schließen des Ionenkanals zugrunde liegt. Aus diesem Grund stellt die isolierte Ligandenbindedomäne, welche als lösliches Protein hergestellt werden kann, ein etabliertes Modellsystem zur Untersuchung der strukturellen und funktionellen Zusammenhänge innerhalb des Funktionsmechanismus ionotroper Glutamatrezeptoren dar. Im Rahmen dieser Arbeit wurden die Konformationsdynamiken der in Escherichia coli-Bakterien exprimierten isolierten Ligandenbindedomänen der drei homologen Untergruppen – AMPA-, Kainate- und NMDA-Rezeptoren – sowohl als Monomer als auch als Dimer untersucht. Hierbei wurden im ungebundenen Apo-Zustand der Proteine signifikante Kinetiken im Bereich von Nanosekunden bis Mikrosekunden festgestellt, welche bei Bindung eines Agonisten sowie bei Dimerisierung erheblichen Veränderungen zeigen. Darüber hinaus wurde allosterische Kommunikation zwischen den LBDs der NMDA-Untergruppe untersucht, wobei in der Tat ein deutlicher allosterischer Effekt in Bezug auf die Konformationsdynamiken der Proteine gemessen werden konnte. Weiterhin wurde ein PET-FCS-basiertes Verfahren zur Messung der Dissoziationskonstante der Bindung eines Liganden an die LBD eines AMPA-Rezeptors entwickelt. Zuletzt wurde außerdem ermittelt, ob ein Unterschied zwischen vollen und partiellen Agonisten hinsichtlich ihres Einflusses auf die Konformationsdynamiken einer AMPA-Rezeptor LBD besteht, was nachgewiesenermaßen nicht der Fall ist. Alle Messungen wurden auf Einzelmolekülebene auf Zeitskalen von Nanosekunden bis Millisekunden basierend auf Fluoreszenzfluktuationen unter Verwendung des photoinduzierten Elektronentransfers (PET) in Kombination mit Korrelationsspektroskopie (PET-FCS) durchgeführt. Zu diesem Zweck wurden PET-basierte Fluoreszenzsonden entwickelt, um Konformationsänderungen auf einer räumlichen Skala von einem Nanometer zu detektieren. Durch die Experimente innerhalb dieser Arbeit konnte gezeigt werden, dass die PET-FCS-Methode eine vielversprechende Ergänzung zu allen bisher bestehenden Methoden zur Untersuchung der Konformationsdynamiken der Ligandenbindedomäne ionotroper Glutamatrezeptoren darstellt und daher eine aussichtsreiche Möglichkeit zur Erweiterung des zukünftigen Verständnisses der Funktionsweise von iGluRs bietet. N2 - Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate most of the excitatory signal transmission throughout the central nervous system. In addition, iGluRs play a crucial role in neural development and function, including learning and memory. Since receptor malfunction contributes to a variety of neurological diseases, iGluRs are key targets for drug development in pharmacology. Furthermore, ionotropic glutamate receptors are divided into three major subgroups, all of which are named due to their selectivity for a certain ligand: AMPA, Kainate and NMDA. Members of each subgroup usually consist of four subunits, which in turn comprise four semi-autonomous domains: (i) the amino terminal domain (ATD), (ii) the ligand binding domain (LBD), (iii) the transmembrane domain (TMD), and (iv) the carboxy terminal domain (CTD). Upon binding a neurotransmitter the ligand binding domain, which adopts a clamshell-like structure consisting of two domains (D1 and D2), undergoes a conformational change by closing around the ligand and trapping it within the binding cleft. The conformational change of the LBD is then transferred to the transmembrane domain which forms the membrane-spanning ion channel, which results in rearrangement of the transmembrane helices and consequently in opening of the ion channel. Accordingly, the conformational change of the LBD is the driving force underlying opening and closing of the ion channel. The isolated ligand binding domain can be produced as soluble protein and represents a well-established model system for exploring structural and functional relationships within the functional mechanism of ionotropic glutamate receptors. As part of this thesis, ligand binding domains of all three homologues – AMPAR, KainateR and NMDAR – have been expressed in Escherichia coli bacterial cells and conformational dynamics of the proteins both as monomer and as dimer have been investigated. In the unbound apo state of the proteins, significant kinetics have been observed in the nanosecond to microsecond time range which undergo considerable changes upon agonist binding or dimerization. In addition, allosteric communication between LBDs of the NMDA subgroup has been investigated, whereby a distinct allosteric effect regarding the conformational dynamics of the protein could actually be measured. Furthermore, a PET-FCS-based tool for measuring the dissociation constant of a ligand for an AMPA receptor LBD has been developed. Finally, it has been investigated whether full and partial agonists have different effects on the conformational dynamics of an AMPA receptor LBD, which has been found clearly not to be the case. All measurements have been performed at the single-molecule level on time scales from nanoseconds to milliseconds based on fluorescence fluctuations using photoinduced electron transfer (PET) fluorescence quenching in combination with correlation spectroscopy (PET-FCS). To this end, PET-based fluorescence probes have been engineered to monitor conformational changes on the one-nanometer scale. The experiments that have been carried out within this thesis introduce PET-FCS as a promising tool to complement all previously existing methods for studying conformational dynamics of ionotropic glutamate receptor ligand binding domains and hence offer a promising opportunity to expand future understanding of how iGluRs work. KW - Fluoreszenzkorrelationsspektroskopie KW - Glutamatrezeptor KW - Einzelmolekülspektroskopie KW - Proteinsynthese KW - Photoinduzierter Elektronentransfer KW - photoinduced electron transfer KW - Ionotrope Glutamatrezeptoren KW - ionotropic glutamate receptors KW - Ligandenbindedomäne KW - ligand binding domain Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244946 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 - THES A1 - López Arboleda, William Andrés T1 - Global Genetic Heterogeneity in Adaptive Traits T1 - Globale genetische Heterogenität in adaptiven Merkmalen N2 - Genome Wide Association Studies (GWAS) have revolutionized the way on how genotype-phenotype relations are assessed. In the 20 years long history of GWAS, multiple challenges from a biological, computational, and statistical point of view have been faced. The implementation of this technique using the model plant species Arabidopsis thaliana, has enabled the detection of many association for multiple traits. Despite a lot of studies implementing GWAS have discovered new candidate genes for multiple traits, different samples are used across studies. In many cases, either globally diverse samples or samples composed of accessions from a geographically restricted area are used. With the aim of comparing GWAS outcomes between populations from different geographic areas, this thesis describes the performance of GWAS in different European samples of A. thaliana. Here, association mapping results for flowering time were compared. Chapter 2 describes the analyses of random resampling from this original sample. The aim was to establish reduced subsamples to later carry out GWAS and compare the outcomes between these subsamples. In Chapter 3, the European sample was split into eight equally-sized local samples representing different geographic regions. Next, GWAS was carried out and an attempt was made to clarify the differences in GWAS outcomes. Chapter 4 contains the results of a collaboration with Prof. Dr. Wolfgang Dröge- Laser, in which my mainly task was the analysis of RNAseq data from A. thaliana plants infected by pathogenic fungi. Finally, Appendix A presents a very short description of my participation in the GHP Project on Access to Care for Cardiometabolic Diseases (HPACC) at the university of Heidelberg. N2 - Die genomweiten Assoziationsstudien (GWAS) haben die Art und Weise revolutionierten, wie genotypische-phänotypische Zusammenhänge untersucht werden. In der 20-jährigen Geschichte dieser Analysen, gab es zahlreiche biologische, mathematische und statistische Herausforderungen. Die Anwendung dieser Methodik in der Modellpflanze Arabidopsis thaliana ermöglichte die Erkennung neuer Zusammenhänge für zahlreicher Merkmale. Obwohl viele Studien, die GWAS implementieren, neue Kandidatengene für verschiedene Merkmale entdeckt haben, werden in den verschiedenen Analysen oft unterschiedliche Populationen verwendet. Es werden entweder global unterschiedliche Accessionen oder alternative welche aus einem geografisch begrenzten Gebiet als Population für die Anaylsen verwendet. Mit dem Ziel, GWAS-Ergebnisse zwischen Populationen aus verschiedenen geografischen Gebieten zu vergleichen, beschreibt diese Arbeit die Eigenschaften der Analyse in verschiedenen europäischen Populationen von A. thaliana. Verglichen wurden die Ergebnisse der Assoziationskartierung für die Blütezeit. Kapitel 2 beschreibt die Analysen von zufälligen Populationen im Vergleich zur gesamten europäischen Population. Ziel war es, reduzierte Stichproben zu erstellen, um später GWAS durchzuführen und die Ergebnisse zwischen diesen Stichproben zu vergleichen. In Kapitel 3 wurde die europäische Population in acht gleich große lokale Subpopulationen aufgeteilt. Diese repräsentieren verschiedene geografische Regionen. Als nächstes wurde GWAS durchgeführt und die Unterschiede in den jeweilgen GWAS-Ergebnissen beschrieben. Kapitel 4 behinhaltet die Ergebnisse aus einer Zusammenarbeit mit Prof. Dr. Wolfgang Dröge-Laser: Hier war meine Hauptaufgabe die Analyse von RNAs Sequenzierungsdaten von mit pathogenen Pilzen befallenen A. thaliana-Pflanzen. Schließlich enthält Anhang A eine zusammenfassende Beschreibung meiner Mitarbeit am GHP-Projekt zum Zugang zur Versorgung bei kardiometabolischen Erkrankungen (HPACC) an der Universität Heidelberg KW - Genotype-phenotype relationship KW - GWAS KW - adaptive traits KW - local adaptation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242468 ER - TY - JOUR A1 - Kühnemundt, Johanna A1 - Leifeld, Heidi A1 - Scherg, Florian A1 - Schmitt, Matthias A1 - Nelke, Lena C. A1 - Schmitt, Tina A1 - Bauer, Florentin A1 - Göttlich, Claudia A1 - Fuchs, Maximilian A1 - Kunz, Meik A1 - Peindl, Matthias A1 - Brähler, Caroline A1 - Kronenthaler, Corinna A1 - Wischhusen, Jörg A1 - Prelog, Martina A1 - Walles, Heike A1 - Dandekar, Thomas A1 - Dandekar, Gudrun A1 - Nietzer, Sarah L. T1 - Modular micro-physiological human tumor/tissue models based on decellularized tissue for improved preclinical testing JF - ALTEX N2 - High attrition-rates entailed by drug testing in 2D cell culture and animal models stress the need for improved modeling of human tumor tissues. In previous studies our 3D models on a decellularized tissue matrix have shown better predictivity and higher chemoresistance. A single porcine intestine yields material for 150 3D models of breast, lung, colorectal cancer (CRC) or leukemia. The uniquely preserved structure of the basement membrane enables physiological anchorage of endothelial cells and epithelial-derived carcinoma cells. The matrix provides different niches for cell growth: on top as monolayer, in crypts as aggregates and within deeper layers. Dynamic culture in bioreactors enhances cell growth. Comparing gene expression between 2D and 3D cultures, we observed changes related to proliferation, apoptosis and stemness. For drug target predictions, we utilize tumor-specific sequencing data in our in silico model finding an additive effect of metformin and gefitinib treatment for lung cancer in silico, validated in vitro. To analyze mode-of-action, immune therapies such as trispecific T-cell engagers in leukemia, as well as toxicity on non-cancer cells, the model can be modularly enriched with human endothelial cells (hECs), immune cells and fibroblasts. Upon addition of hECs, transmigration of immune cells through the endothelial barrier can be investigated. In an allogenic CRC model we observe a lower basic apoptosis rate after applying PBMCs in 3D compared to 2D, which offers new options to mirror antigen-specific immunotherapies in vitro. In conclusion, we present modular human 3D tumor models with tissue-like features for preclinical testing to reduce animal experiments. KW - modular tumor tissue models KW - invasiveness KW - bioreactor culture KW - combinatorial drug predictions KW - immunotherapies Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231465 VL - 38 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 - 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 - 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 - Peters, Simon A1 - Kaiser, Lena A1 - Fink, Julian A1 - Schumacher, Fabian A1 - Perschin, Veronika A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Stigloher, Christian A1 - Kleuser, Burkhard A1 - Seibel, Juergen A1 - Schubert-Unkmeir, Alexandra T1 - Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria JF - Scientific Reports N2 - Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity. KW - antimicrobials KW - biological techniques KW - imaging KW - microbiology KW - microbiology techniques KW - microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259147 VL - 11 IS - 1 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 - Scherer, Marc A1 - Fleishman, Sarel J. A1 - Jones, Patrik R. A1 - Dandekar, Thomas A1 - Bencurova, Elena T1 - Computational Enzyme Engineering Pipelines for Optimized Production of Renewable Chemicals JF - Frontiers in Bioengineering and Biotechnology N2 - To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO2 or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways. KW - computational KW - enzyme KW - engineering KW - design KW - biomanufacturing KW - biofuel KW - microbes KW - metabolism Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-240598 SN - 2296-4185 VL - 9 ER - TY - JOUR A1 - Makbul, Cihan A1 - Khayenko, Vladimir A1 - Maric, Hans Michael A1 - Böttcher, Bettina T1 - Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype JF - Microorganisms N2 - Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes. KW - hepatitis B core protein KW - hepatitis B virus KW - peptide inhibitor of envelopment KW - isothermal titration calorimetry KW - electron cryo microscopy KW - low-secretion phenotype mutants KW - peptide microarray Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236720 SN - 2076-2607 VL - 9 IS - 5 ER - TY - JOUR A1 - Osmanoglu, Özge A1 - Khaled AlSeiari, Mariam A1 - AlKhoori, Hasa Abduljaleel A1 - Shams, Shabana A1 - Bencurova, Elena A1 - Dandekar, Thomas A1 - Naseem, Muhammad T1 - Topological Analysis of the Carbon-Concentrating CETCH Cycle and a Photorespiratory Bypass Reveals Boosted CO\(_2\)-Sequestration by Plants JF - Frontiers in Bioengineering and Biotechnology N2 - Synthetically designed alternative photorespiratory pathways increase the biomass of tobacco and rice plants. Likewise, some in planta–tested synthetic carbon-concentrating cycles (CCCs) hold promise to increase plant biomass while diminishing atmospheric carbon dioxide burden. Taking these individual contributions into account, we hypothesize that the integration of bypasses and CCCs will further increase plant productivity. To test this in silico, we reconstructed a metabolic model by integrating photorespiration and photosynthesis with the synthetically designed alternative pathway 3 (AP3) enzymes and transporters. We calculated fluxes of the native plant system and those of AP3 combined with the inhibition of the glycolate/glycerate transporter by using the YANAsquare package. The activity values corresponding to each enzyme in photosynthesis, photorespiration, and for synthetically designed alternative pathways were estimated. Next, we modeled the effect of the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle (CETCH), which is a set of natural and synthetically designed enzymes that fix CO₂ manifold more than the native Calvin–Benson–Bassham (CBB) cycle. We compared estimated fluxes across various pathways in the native model and under an introduced CETCH cycle. Moreover, we combined CETCH and AP3-w/plgg1RNAi, and calculated the fluxes. We anticipate higher carbon dioxide–harvesting potential in plants with an AP3 bypass and CETCH–AP3 combination. We discuss the in vivo implementation of these strategies for the improvement of C3 plants and in natural high carbon harvesters. KW - CO2-sequestration KW - photorespiration KW - elementary modes KW - synthetic pathways KW - carboxylation KW - metabolic modeling KW - CETCH cycle Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249260 SN - 2296-4185 VL - 9 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 - Meir, Michael A1 - Kannapin, Felix A1 - Diefenbacher, Markus A1 - Ghoreishi, Yalda A1 - Kollmann, Catherine A1 - Flemming, Sven A1 - Germer, Christoph-Thomas A1 - Waschke, Jens A1 - Leven, Patrick A1 - Schneider, Reiner A1 - Wehner, Sven A1 - Burkard, Natalie A1 - Schlegel, Nicolas T1 - Intestinal epithelial barrier maturation by enteric glial cells is GDNF-dependent JF - International Journal of Molecular Sciences N2 - Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAP\(^{cre}\) x Ai14\(^{floxed}\) mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor. KW - enteric glial cells KW - neurotrophic factors KW - intestinal epithelial barrier KW - GDNF5 KW - RET6 KW - inflammatory bowel disease KW - enteric nervous system KW - gut barrier KW - intercellular junctions Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-258913 SN - 1422-0067 VL - 22 IS - 4 ER - TY - THES A1 - Lapuente, Juan M. T1 - The Chimpanzees of the Comoé National Park, Ivory Coast. Status, distribution, ecology and behavior T1 - Die Schimpansen im Comoé Nationalpark, Elfenbeinküste. Status, Verbreitung, Ökologie und Verhalten N2 - Although wild chimpanzees (Pan troglodytes) have been studied intensely for more than 50 years, there are still many aspects of their ecology and behavior that are not well understood. Every time that a new population of chimpanzees has been studied, new behaviors and unknown aspects of their ecology have been discovered. All this accumulated knowledge is helping us to piece together a model of how could last human and chimpanzee common ancestors have lived and behaved between seven and five million years ago. Comoé chimpanzees had never been studied in depth, until we started our research in October 2014, only a few censuses had been realized. The last surveys prior our work, stated that the population was so decimated that was probably functionally extinct. When we started this research, we had to begin with a new intensive survey, using new methods, to ascertain the real status and distribution of the chimpanzees living in Comoé National Park (CNP). During the last five years, we have realized a deep study aiming to know more about their ecology and behavior. We combined transects and reconnaissance marches (recces) with the use of camera traps, for the first time in CNP, obtaining a wealth of data that is not fully comprised in this dissertation. With this research, we determined that there is a sustainable continuous population of Western chimpanzees (Pan troglodytes verus) in CNP and the adjacent area of Mont Tingui, to the West, with a minimum of 127 weaned chimpanzees living in our main 900 km2 study area, SW of CNP. We found that this population is formed by a minimum of eight different chimpanzee communities, of which we studied seven, four of them more in detail. These chimpanzees spent much more time in the forest than in the savanna habitats. We also found that Comoé chimpanzees consumed at least 58 different food items in their dit, which they obtained both from forest and savanna habitats. Another finding was that insectivory had an important role in their diet, with at least four species of ants, three of termites and some beetle larvae. These chimpanzees also hunted at least three species of monkeys and maybe rodents and duikers and occasionally consumed the big land snails of genus Achatina. We found that, during the fruit scarcity period in the late rainy season, they intensely consumed the cambium of Ceiba pentandra, as fallback food, much more than the bark or cambium of any other tree species. Another interesting finding was that all the chimpanzees in the studied area realized this particular bark-peeling behavior and had been repeatedly peeling the trees of this species for years. This did not increase tree mortality and the damage caused to the trees was healed in two years, not reducing the growth, thus being a sustainable use of the trees. We found that Comoé chimpanzees produced and used a great variety of tools, mainly from wooden materials, but also from stone and herbaceous vegetation. Their tool repertory included stick tools to dip for Dorylus burmeisteri ants, to fish for Camponotus and Crematogaster ants, to dip for honey, mainly from Meliponini stingless bees, but sometimes from honey bees (Apis mellifera). It also included the use of stick tools to fish termites of Macrotermes subhyalinus and Odontotermes majus (TFTs), to dip for water from tree holes and investigatory probes for multiple purposes. Additionally, these chimpanzees used leaf-sponges to drink from tree holes and to collect clayish water from salt-licks. They also used stones to hit the buttresses of trees during displays, the so called accumulative stone throwing behavior and probably used stones as hammers, to crack open hard-shelled Strichnos spinosa and Afraegle paniculata fruits and Achatina snails. The chimpanzees also used objects that are not generally accepted as animal tools, for being attached to the substrate, with different purposes: they drummed buttresses of trees with hands and/or feet to produce sound during male displays and they pounded open hard-shelled fruits, Achatina snails and Cubitermes termite mounds on stone or root anvils. We finally measured the stick tools and found significant differences between them suggesting that they were specialized tools made specifically for every purpose. We studied more in detail the differences between apparently similar tools, the honey dipping tools and the water dipping tools, often with brushes made at their tips to collect the fluids. These last tools were exclusive from Comoé and have not been described at any other site. We found that total length, diameter and brush length were significantly different, suggesting that they were specialized tools. We concluded that Comoé chimpanzees had a particular culture, different from those of other populations of Western chimpanzees across Africa. Efficient protection, further research and permanent presence of research teams are required to avoid that this unique population and its culture disappears by the poaching pressure and maybe by the collateral effects of climate change. N2 - Obwohl wild lebende Schimpansen (Pan troglodytes) seit mehr als 50 Jahren intensiv untersucht werden, gibt es noch zahlreiche Aspekte ihrer Ökologie und ihres Verhaltens, die nicht gut verstanden werden. Jedes Mal, wenn eine neue Population von Schimpansen studiert wurde, wurden neue Verhaltensweisen und unbekannte Aspekte ihrer Ökologie entdeckt. All dieses gesammelte Wissen hilft uns, ein Modell zu erstellen, wie lange die gemeinsamen Vorfahren von Menschen und Schimpansen vor sieben bis fünf Millionen Jahren gelebt und sich verhalten haben könnten. Als wir im Oktober 2014 mit unserer Forschung begannen waren die Comoé-Schimpansen, bis auf einige Populations-Zensus von Schimpansen in der Elfenbeinküste, noch nie eingehend untersucht worden. Die letzte Zählung bevor unsere Arbeit began ergab, dass die Schimansenpopulation so stark dezimiert war, dass sie als funktionell ausgestorben erarchtet werden konnte. Zum Beginn unserer Forschung, führten wir zuerst mit neusten Methoden einen neuen detailierten Zensus durch, um den tatsächlichen Status und die Verteilung der im Comoé-Nationalpark (CNP) lebenden Schimpansen zu ermitteln. In den folgenden fünf Jahren haben wir zudem eine umfassende Studie durchgeführt, um mehr über ihre Ökologie und ihr Verhalten zu erfahren. Wir haben im CNP erstmals systematische Transekte und Datenerhebungen mittels Kamerafallen kombiniert, um eine Fülle von Erkenntnissen zu erhalten, die in dieser Dissertation nicht vollständig enthalten sind. Wir stellten fest, dass es in CNP und dem westlich angrenzenden Gebiet des Mont Tingui nach wie vor eine nachhaltige und kontinuierliche Population westlicher Schimpansen (Pan troglodytes verus) existiert, wobei mindestens 131 adulte (entwöhnte) Schimpansen in unserem 900 km² großen Hauptuntersuchungsgebiet südwestlich des CNP leben. Diese Population besteht aus mindestens acht verschiedenen Schimpansengruppen, von denen wir sieben untersuchten, vier davon genauer. Wir konnten zeigen dass diese Schimpansen deutlich mehr Zeit im Wald als in den angrenzenden Savannenhabitaten verbringen. Wir stellten fest, dass Comoé- Schimpansen mindestens 58 verschiedene Futtermittel aus Wald- als auch aus Savannenhabitaten nutzen. Zudem spielt der Konsum von Insekten, bestelhend aus mindestens vier Ameisen-, drei Termiten- und verschiedenene Käferlarven eine wichtige Rolle in ihrem Ernährungsreportoire. Die Comoé-Schimpansen jagen zudem mindestens drei Affena sowie möglicherweise Nagetiere und Duiker, und fraßen gelegentlich die großen Schnecken der Gattung Achatina. Wir fanden heraus, dass sie den typischen Mangel an reifen Fuechten in der \späten Regenzeit durch den intensiven Konsum der Rinde (Kambium) von Ceiba pentandra kompensieren. Alle Schimpansen im untersuchten Gebiet zeigten dieses besondere Verhalten, bei dem sie die Rinde von Ceiba Bäumen schälen. Wir konnte zeigen, dass die Schimpansen diese Bäume seit Jahren wiederholt geschält hatten, was offenbar den Bäumen keinen nachhaltigen Schaden zugefügte. Innerhalb von zwei Jahren ware die Schäden geheilt and das Wachstum nicht verringert, was schlussfolgern lässt dass die Nutzung der Baumrinde nachhaltig ist. Wir fanden heraus, dass Comoé-Schimpansen eine Vielzahl von Werkzeugen aus Vegetation aber auch Steinen herstellten und verwendeten. Das Werkzeugrepertoire umfasste Stöckchen zur Gewinning von on Ameisen der Art Dorylus burmeisteri, sowie Ameisen der Gattungen Camponotus und Crematogaster, aber auch von Bienenhonig produziert von der stachellosen Gattung Melipoa sowie von Apis mellifera. Die Schimpansen nutzen ausserdem Pflanzenwerkzeuge zum Termitenfischen von Macrotermes subhyalinus und Odontotermes majus, um an das Wasser in Baumvertiefungen zu gelangen, sowie für diverse andere Untersuchungszwecke. Zusätzlich verwenden die Comoé-Schimpansen Blattschwämme, um aus Baumlöchern zu trinken und lehmiges Wasser von den Salzlecken zu sammeln. Im Rahmen ihres Imponierverhaltens schleuden sie Steine an die Brettwurzeln spezieller grosser Bäume, ein neu entdecktes Verhalten das als akkumulatives Steinerfen bezeichnet wird. Es ist wahrscheinlich dass sie Steine auch als Hammerwerkzeuge nutzen, um hartschalige Früchte wie Strichnos spinosa und Afraegle paniculata sowie grosse Landschnecken aufzubrechen. Die Schimpansen verwenden Gegenstände auch in anderen Zusammenhängen, die nicht unbedingt als Werkzeuggebrauch definiert werden können: Sie trommlen im Rahmen vom männlichen Imponierverhalten laut mit Händen und Füßen auf die Brettwurzeln von Bäumen, und zerschmettern harte Früchte, Schneckenhäuser und Cubitermes-Termitenhügel auf Ambossen aus Gestein oder Wurzeln. Wir haben signifikante Unterschiede beim Vermessen der Stabwerkzeuge festgestellt, was darauf hindeutet, dass es sich um Spezialwerkzeuge handelt, die speziell für verschiedene Zwecke hergestellt werden. Wir haben insbesondere die Unterschiede zwischen scheinbar ähnlichen Pinselwerkzeugen für den Konsum von Flüssigkeiten (H zu verhindern, sowie die möglichen Nebeneffekte des Klimawandels zu dokumentieren.onig, Wasser) genauer untersucht. Diese Pinselwerkzeuge der Comoé-Schimpansen sind offenbar einzigartig und bislang nicht in der Literatur beschrieben. Gesamtlänge, Durchmesser und Bürstenlänge weichen je nach Verwendungszweck der Pinsel erheblich voneinander ab, was darauf hindeutet, dass es sich um Spezialwerkzeuge handelt. Wir schlussfolgern, dass die Kultur der Comoé-Schimpansen einzigartig innerhalb der der westlichen Schimpansen ist. Um diese einzigartige Population von Schimpansen effektiv zu schützen benötigt es weitere Forschung sowie die ständige Präsenz von Forschungsteams, um Wilderei KW - Chimpanzee KW - ecology KW - distribution KW - behavior KW - Comoé National Park KW - tool-use KW - primate KW - tool KW - diet KW - habitat KW - Parc National de la Comoé KW - Schimpanse KW - Verhalten Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223180 ER - TY - THES A1 - Eisenhuth, Nicole Juliana T1 - Novel and conserved roles of the histone methyltransferase DOT1B in trypanosomatid parasites T1 - Neue und konservierte Rollen der Histonmethyltransferase DOT1B in Parasiten der Ordnung Trypanosomatida N2 - The family of trypanosomatid parasites, including the human pathogens Trypanosoma brucei and Leishmania, has evolved sophisticated strategies to survive in harmful host environments. While Leishmania generate a safe niche inside the host’s macrophages, Trypanosoma brucei lives extracellularly in the mammalian bloodstream, where it is constantly exposed to the attack of the immune system. Trypanosoma brucei ensures its survival by periodically changing its protective surface coat in a process known as antigenic variation. The surface coat is composed of one species of ‘variant surface glycoprotein’ (VSG). Even though the genome possesses a large repertoire of different VSG isoforms, only one is ever expressed at a time from one out of the 15 specialized subtelomeric ‘expression sites’ (ES). Switching the coat can be accomplished either by a recombination-based exchange of the actively-expressed VSG with a silent VSG, or by a transcriptional switch to a previously silent ES. The conserved histone methyltransferase DOT1B methylates histone H3 on lysine 76 and is involved in ES regulation in T. brucei. DOT1B ensures accurate transcriptional silencing of the inactive ES VSGs and influences the kinetics of a transcriptional switch. The molecular machinery that enables DOT1B to execute these regulatory functions at the ES is still elusive, however. To learn more about DOT1B-mediated regulatory processes, I wanted to identify DOT1B-associated proteins. Using two complementary approaches, specifically affinity purification and proximity-dependent biotin identification (BioID), I identified several novel DOT1B-interacting candidates. To validate these data, I carried out reciprocal co-immunoprecipitations with the most promising candidates. An interaction of DOT1B with the Ribonuclease H2 protein complex, which has never been described before in any other organism, was confirmed. Trypanosomal Ribonuclease H2 maintains genome integrity by resolving RNA-DNA hybrids, structures that if not properly processed might initiate antigenic variation. I then investigated DOT1B’s contribution to this novel route to antigenic variation. Remarkably, DOT1B depletion caused an increased RNA-DNA hybrid abundance, accumulation of DNA damage, and increased VSG switching. Deregulation of VSGs from throughout the silent repertoire was observed, indicating that recombination-based switching events occurred. Encouragingly, the pattern of deregulated VSGs was similar to that seen in Ribonuclease H2-depleted cells. Together these data support the hypothesis that both proteins act together in modulating RNA-DNA hybrids to contribute to the tightly-regulated process of antigenic variation. The transmission of trypanosomatid parasites to mammalian hosts is facilitated by insect vectors. Parasites need to adapt to the extremely different environments encountered during transmission. To ensure their survival, they differentiate into various specialized forms adapted to each tissue microenvironment. Besides antigenic variation, DOT1B additionally affects the developmental differentiation from the mammalian-infective to the insect stage of Trypanosoma brucei. However, substantially less is known about the influence of chromatin-associated proteins such as DOT1B on survival and adaptation strategies of related Leishmania parasites. To elucidate whether DOT1B’s functions are conserved in Leishmania, phenotypes after gene deletion were analyzed. As in Trypanosoma brucei, generation of a gene deletion mutant demonstrated that DOT1B is not essential for the cell viability in vitro. DOT1B deletion was accompanied with a loss of histone H3 lysine 73 trimethylation (the lysine homologous to trypanosomal H3K76), indicating that Leishmania DOT1B is also solely responsible for catalyzing this post-translational modification. As in T. brucei, dimethylation could only be observed during mitosis/cytokinesis, while trimethylation was detectable throughout the cell cycle in wild-type cells. In contrast to the trypanosome DOT1B, LmxDOT1B was not essential for differentiation in vitro. However, preliminary data indicate that the enzyme is required for effective macrophage infection. In conclusion, this study demonstrated that the identification of protein networks and the characterization of protein functions of orthologous proteins from related parasites are effective tools to improve our understanding of the parasite survival strategies. Such insights are a necessary step on the road to developing better treatments for the devastating diseases they cause. N2 - Vertreter der Familie der Trypanosomatidae einschließlich der humanpathogenen Trypanosoma brucei und Leishmania Arten entwickelten eine Reihe von ausgeklügelten Strategien, um in ihren Wirten zu überleben. Während sich Leishmanien eine sichere Nische in den Makrophagen ihrer Wirte aufbauen, lebt Trypanosoma brucei ausschließlich extrazellulär im Blutkreislauf der Säugetiere. Dort ist der Parasit ständig dem Angriff des Immunsystems ausgesetzt. Um sein Überleben zu sichern, wechselt er regelmäßig seine variablen Oberflächenproteine (VSG), eine Strategie, die auch als antigene Variation bekannt ist. Obwohl das Genom des Parasiten über ein enormes Repertoire an VSG Genen verfügt, wird immer nur eine einzige Art von einer von 15 spezialisierten telomerproximalen Expressionsstellen (ES) transkribiert. Um die VSG-Zelloberfläche zu wechseln, können Trypanosomen das VSG Gen der aktiven ES gegen ein inaktives VSG aus dem gigantischen Repertoire mittels Rekombination eintauschen. Eine weitere Möglichkeit ist der Transkriptionswechsel zu einer zuvor stillen ES. Die konservierte Histonmethyltransferase DOT1B katalysiert die Methylierung von Histon H3 am Lysin 76 und ist an der ES-Regulation beteiligt. DOT1B gewährleistet den transkriptionell inaktiven Status der ES und beeinflusst die Kinetik eines transkriptionellen ES Wechsels. Die molekularen Komponenten, die DOT1B diese regulatorischen Funktionen an der ES ermöglichen, sind jedoch noch unbekannt. Um mehr über die von DOT1B vermittelten Mechanismen zu erfahren, ist es notwendig, DOT1B-assoziierte Proteine zu identifizieren. Durch die Anwendung von komplementären biochemischen Proteinaufreinigungsmethoden gelang es mir, mehrere potentielle Proteininteraktionen zu DOT1B zu entdecken. Um die Daten zu validieren, führte ich weitere Proteinaufreinigungen mit den vielversprechendsten Kandidaten durch. Eine Interaktion zwischen DOT1B und der Ribonuklease H2 konnte bestätigt werden - eine Interaktion, die noch nie zuvor in anderen Organismen beschrieben wurde. In Trypanosomen gewährleistet Ribonuklease H2 die Genomintegrität, indem das Enzym RNA-DNA-Hybride auflöst. Diese Strukturen können zudem, wenn sie nicht richtig prozessiert werden, antigene Variation initiieren. In dieser Studie wurde daher außerdem DOT1B’s Beitrag zu diesem Weg der Initiation der antigenen Variation analysiert. In der Tat konnte gezeigt werden, dass DOT1B RNA-DNA-Hybride moduliert und die Genomintegrität sowie VSG-Wechselrate beeinflusst. Die Tatsache, dass in DOT1B-Mutanten VSG Isoformen von den unterschiedlichsten Genomregionen exprimiert wurden, deutet darauf hin, dass rekombinations-basierte Ereignisse dem VSG-Wechsel zu Grunde lagen. Da in den DOT1B-Mutanten ähnliche VSG exprimiert wurden wie in Ribonuklease H2-Mutanten, kann vermutet werden, dass beide Proteine bei der Modulation der RNA-DNA-Hybride zusammenwirken, um antigene Variation zu regulieren. Trypanosomen und Leishmanien werden mittels Insektenvektoren auf den nächsten Säugerwirt übertragen. Sie müssen daher nicht nur im Säugerwirt überleben, sondern sich auch an die extrem unterschiedliche Umgebung im Vektor anpassen. Dafür differenzieren sich die Parasiten in speziell angepasste Zellstadien. Zusätzlich zu der antigenen Variation beeinflusst DOT1B die Entwicklungsdifferenzierung in Trypanosoma brucei. In Leishmanien hingegen ist über den Einfluss von chromatin-assoziierten Proteinen wie DOT1B auf die Überlebens- und Anpassungsstrategien wesentlich weniger bekannt. Um herauszufinden, ob die Funktionen von DOT1B in Leishmanien konserviert sind, wurden Phänotypen nach Gendeletion analysiert. Wie auch in Trypanosoma brucei konnte gezeigt werden, dass DOT1B für das Überleben der Parasiten nicht essentiell ist. Die Deletion von DOT1B ging mit einem Verlust der Trimethylierung von Histon H3 am Lysin 73 (dem zum trypanosomalen H3K76 homologen Lysin) einher, was darauf hinweist, dass DOT1B auch in Leishmanien allein für die Katalyse dieser posttranslationalen Modifikation verantwortlich ist. Wie in Trypanosoma brucei konnte eine Dimethylierung nur in der Mitose/Zytokinese beobachtet werden, wobei die Trimethylierung während des gesamten Zellzyklus in Wildtyp-Zellen nachweisbar war. Im Gegensatz zum trypanosomalen DOT1B war LmxDOT1B für die Differenzierung in vitro entbehrlich. Vorläufige Daten zeigen jedoch, dass das Enzym für eine wirksame Makrophageninfektion wesentlich ist. Zusammenfassend zeigte diese Studie, dass die Identifizierung von Proteinnetzwerken und die Charakterisierung von Funktionen orthologer Proteine aus verwandten Parasiten wirksame Werkzeuge sind, um unser Verständnis der Überlebensstrategien der Parasiten zu verbessern. Solche Erkenntnisse sind ein notwendiger Schritt auf dem Weg zu effektiveren Behandlungsmethoden für die verheerenden Krankheiten, die diese Parasiten verursachen. KW - Trypanosoma brucei KW - Leishmania KW - Chromatin KW - Histon-Methyltransferase KW - DNA repair KW - developmental differentiation KW - DOT1 KW - Ribonuclease H2 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219936 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 - THES A1 - Zwettler, Fabian Ulrich T1 - Expansionsmikroskopie kombiniert mit hochauflösender Fluoreszenzmikroskopie T1 - Expansion Microscopy combined with Super-Resolution Fluorescence Microscopy N2 - Fluorescence microscopy is a form of light microscopy that has developed during the 20th century and is nowadays a standard tool in Molecular and Cell biology for studying the structure and function of biological molecules. High-resolution fluorescence microscopy techniques, such as dSTORM (direct Stochastic Optical Reconstruction Microscopy) allow the visualization of cellular structures at the nanometre scale (10−9 m). This has already made it possible to decipher the composition and function of various biopolymers, such as proteins, lipids and nucleic acids, up to the three-dimensional (3D) structure of entire organelles. In practice, however, it has been shown that these imaging methods and their further developments still face great challenges in order to achieve an effective resolution below ∼ 10 nm. This is mainly due to the nature of labelling biomolecules. For the detection of molecular structures, immunostaining is often performed as a standard method. Antibodies to which fluorescent molecules are coupled, recognize and bind specifcally and with high affnity to the molecular section of the target structure, also called epitope or antigen. The fluorescent molecules serve as reporter molecules which are imaged with the use of a fluorescence microscope. However, the size of these labels with a length of about 10-15 nm in the case of immunoglobulin G (IgG) antibodies, cause a detection of the fluorescent molecules shifted to the real position of the studied antigen. In dense regions where epitopes are located close to each other, steric hindrance between antibodies can also occur and leads to an insuffcient label density. Together with the shifted detection of fluorescent molecules, these factors can limit the achievable resolution of a microscopy technique. Expansion microscopy (ExM) is a recently developed technique that achieves a resolution improvement by physical expansion of an investigated object. Therefore, biological samples such as cultured cells, tissue sections, whole organs or isolated organelles are chemically anchored into a swellable polymer. By absorbing water, this so-called superabsorber increases its own volume and pulls the covalently bound biomolecules isotropically apart. Routinely, this method achieves a magnifcation of the sample by about four times its volume. But protocol variants have already been developed that result in higher expansion factors of up to 50-fold. Since the ExM technique includes in the frst instance only the sample treatment for anchoring and magnifcation of the sample, it can be combined with various standard methods of fluorescence microscopy. In theory, the resolution of the used imaging technique improves linearly with the expansion factor of the ExM treated sample. However, an insuffcient label density and the size of the antibodies can here again impair the effective achievable resolution. The combination of ExM with high-resolution fluorescence microscopy methods represents a promising strategy to increase the resolution of light microscopy. In this thesis, I will present several ExM variants I developed which show the combination of ExM with confocal microscopy, SIM (Structured Illumination Microscopy), STED (STimulated Emission Depletion) and dSTORM. I optimized existing ExM protocols and developed different expansion strategies, which allow the combination with the respective imaging technique. Thereby, I gained new structural insights of isolated centrioles from the green algae Chlamydomonas reinhardtii by combining ExM with STED and confocal microscopy. In another project, I combined 3D-SIM imaging with ExM and investigated the molecular structure of the so-called synaptonemal complex. This structure is formed during meiosis in eukaryotic cells and contributes to the exchange of genetic material between homologous chromosomes. Especially in combination with dSTORM, the ExM method showed its high potential to overcome the limitations of modern fluorescence microscopy techniques. In this project, I expanded microtubules in mammalian cells, a polymer of the cytoskeleton as well as isolated centrioles from C. reinhardtii. By labelling after expansion of the samples, I was able to signifcantly reduce the linkage error of the label and achieve an improved label density. In future, these advantages together with the single molecule sensitivity and high resolution obtained by the dSTORM method could pave the way for achieving molecular resolution in fluorescence microscopy N2 - Die Fluoreszenzmikroskopie ist eine Form der Lichtmikroskopie, die sich im Laufe des 20. Jahrhunderts entwickelt hat und heutzutage standardmäßig in der Molekular-und Zellbiologie zur Erforschung von Aufbau und Funktion biologischer Moleküle eingesetzt wird. Hochauflösende Verfahren der Fluoreszenzmikroskopie, wie die dSTORM (direct Stochastic Optical Reconstruction Microscopy) Technik, ermöglichen die Visualisierung zellulärer Strukturen im Nanometer-Größenbereich (10−9 m). Dadurch konnte bereits die Zusammensetzung und Funktion unterschiedlicher Biopolymere, wie die von Proteinen, Lipiden und Nukleinsäuren, bis hin zum dreidimensionalen (3D) Aufbau ganzer Organellen entschlüsselt werden. In der Praxis zeigt sich jedoch, dass diese Bildgebungsverfahren und ihre Weiterentwicklungen immer noch vor großen Herausforderungen stehen, bevor eine effektive Auflösung von unter ∼10 nm erreicht werden kann. Die größte Hürde stellt die Art und Weise der Markierung von Biomolekülen dar. Bei dieser wird zum Nachweis molekularer Strukturen häufig die sogenannte Immunfärbung als Standardmethode eingesetzt. Antikörper, welche mit Fluoreszenzmolekülen gekoppelt werden, erkennen und binden hierbei spezifisch und mit hoher Affinität den Molekülabschnitt der Zielstruktur, auch Epitop oder Antigen genannt. Die Fluoreszenzmoleküle dienen als Reportermoleküle, welche mit Hilfe eines Fluoreszenzmikroskops abgebildet werden. Die Größe der Antikörper, mit einer Länge von etwa 10-15 nm im Falle von Immunglobulin G (IgG) Antikörpern, bewirkt jedoch eine Detektion der fluoreszierenden Moleküle verschoben zur eigentlichen Lage des untersuchten Antigens. In Regionen mit räumlich dicht nebeneinander liegenden Epitopen kann es zusätzlich zur sterischen Hinderung zwischen den Antikörpern kommen. Dies führt zu einer unzureichenden Markierungsdichte und stellt - zusammen mit der verschobenen Detektion der Fluoreszenzmoleküle - eine Limitierung der zu erreichenden Auflösung dar. Die Expansionsmikroskopie (ExM) ist ein neu entwickeltes Verfahren, welches eine Auflösungsverbesserung durch die physikalische Expansion eines untersuchten Objekts erreicht. Hierbei werden biologische Proben, wie beispielsweise kultivierte Zellen, Gewebeschnitte, ganze Organe oder isolierte Organellen, chemisch in ein quellbares Polymer verankert. Durch Absorption von Wasser vergrößert dieser sogenannte Superabsorber sein eigenes Volumen und zieht während der räumlichen Expansion die kovalent gebundenen Biomoleküle isotrop auseinander. Standardmäßig wird durch dieses Verfahren eine Vergrößerung der Proben um etwa das vierfache Volumen erreicht, wobei bereits Protokollvarianten entwickelt wurden, die eine bis zu 50-fache Expansion erzielt haben. Da die ExM-Technik zunächst nur die Probenbehandlung zur Verankerung und Vergrößerung der Probe selbst beinhaltet, kann sie mit unterschiedlichen Standardmethoden der Fluoreszenzmikroskopie kombiniert werden. Dadurch verbessert sich die Auflösung des verwendeten Bildgebungsverfahrens theoretisch linear um den Faktor der Volumenvergrößerung der ExM behandelten Probe. Eine unzureichende Markierungsdichte und die Größe der verwendeten Antikörper können auch hier die effektiv erreichbare Auflösung beeinträchtigen. Die Kombination der ExM mit hochauflösenden Verfahren der Fluoreszenzmikroskopie stellt eine vielversprechende Strategie zur Erhöhung der bisher erreichbaren Auflösung in der Lichtmikroskopie dar. In dieser Arbeit werde ich mehrere von mir entwickelte ExM Varianten vorstellen, welche die Kombination von ExM mit konfokaler Mikroskopie, SIM (Structured Illumination Microscopy), STED (STimulated Emission Depletion) und dSTORM zeigen. Um die Verbindung mit dem jeweiligen Bildgebungsverfahren zu ermöglichen, optimierte ich bestehende ExM-Protokolle und entwickelte unterschiedliche Expansionsstrategien. Dadurch konnte ich neue strukturelle Erkenntnisse von isolierten Zentriolen aus der Grünalge Chlamydomonas reinhardtii durch die Verbindung von ExM mit STED und konfokaler Mikroskopie gewinnen. In einem weiteren Projekt kombinierte ich 3D-SIM mit ExM und untersuchte den molekularen Aufbau des sogenannten synaptonemalen Komplexes. Diese Struktur bildet sich in eukaryotischen Zellen während der Reifeteilung (Meiose) aus und trägt zum Austausch des genetischen Materials zwischen homologen Chromosomen bei. Vor allem in Verbindung mit dSTORM zeigte sich das hohe Potential der ExM-Methode, die bisherigen Limitierungen moderner Techniken der Fluoreszenzmikroskopie zu überwinden. In diesem Projekt expandierte ich Mikrotubuli in Säugetierzellen, ein Polymer des Zytoskeletts, sowie isolierte Zentriolen aus C. reinhardtii. Dadurch, dass die Markierung erst nach dem Expandieren der Proben erfolgte, gelang es, den Abstandsfehler der Markierung deutlich zu verringern und eine verbesserte Markierungsdichte zu erreichen. Diese Vorteile könnten in Verbindung mit der Einzelmolekülsensititvität und hohen Auflösung der dSTORM Methode Wegbereiter zur Erreichung einer molekularen Auflösung sein KW - Fluoreszenzmikroskopie KW - Expansionsmikroskopie KW - Einzelmolekül-Lokalisationsmikroskopie KW - Zentriolen KW - Synaptonemaler Komplex Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212362 ER - TY - THES A1 - Kehrberger, Sandra T1 - Effects of climate warming on the timing of flowering and emergence in a tritrophic relationship: plants - bees - parasitoids T1 - Auswirkungen der Klimaerwärmung auf die zeitliche Regulierung der Blüte und des Schlupfes in einer tritrophischen Beziehung: Pflanzen - Bienen - Parasitoide N2 - The right timing of phenological events is crucial for species fitness. Species should be highly synchronized with mutualists, but desynchronized with antagonists. With climate warming phenological events advance in many species. However, often species do not respond uniformly to warming temperatures. Species-specific responses to climate warming can lead to asynchrony or even temporal mismatch of interacting species. A temporal mismatch between mutualists, which benefit from each other, can have negative consequences for both interaction partners. For host-parasitoid interactions temporal asynchrony can benefit the host species, if it can temporally escape its parasitoid, with negative consequences for the parasitoid species, but benefit the parasitoid species if it increases synchrony with its host, which can negatively affect the host species. Knowledge about the drivers of phenology and the species-specific responses to these drivers are important to predict future effects of climate change on trophic interactions. In this dissertation I investigated how different drivers act on early flowering phenology and how climate warming affects the tritrophic relationship of two spring bees (Osmia cornuta & Osmia bicornis), an early spring plant (Pulsatilla vulgaris), which is one of the major food plants of the spring bees, and three main parasitoids of the spring bees (Cacoxenus indagator, Anthrax anthrax, Monodontomerus). In Chapter II I present a study in which I investigated how different drivers and their change over the season affect the reproductive success of an early spring plant. For that I recorded on eight calcareous grasslands around Würzburg, Germany the intra-seasonal changes in pollinator availability, number of co-flowering plants and weather conditions and studied how they affect flower visitation rates, floral longevity and seed set of the early spring plant P. vulgaris. I show that bee abundances and the number of hours, which allowed pollinator foraging, were low at the beginning of the season, but increased over time. However, flower visitation rates and estimated total number of bee visits were higher on early flowers of P. vulgaris than later flowers. Flower visitation rates were also positively related to seed set. Over time and with increasing competition for pollinators by increasing numbers of co-flowering plants flower visitation rates decreased. My data shows that a major driver for early flowering dates seems to be low interspecific competition for pollinators, but not low pollinator abundances and unfavourable weather conditions. Chapter III presents a study in which I investigated the effects of temperature on solitary bee emergence and on the flowering of their food plant and of co-flowering plants in the field. Therefore I placed bee cocoons of two spring bees (O. cornuta & O. bicornis) on eleven calcareous grasslands which differed in mean site temperature. On seven of these grasslands the early spring plant P. vulgaris occurred. I show that warmer temperatures advanced mean emergence in O. cornuta males. However, O. bicornis males and females of both species did not shift their emergence. Compared to the bees P. vulgaris advanced its flowering phenology more strongly with warmer temperatures. Co-flowering plants did not shift flowering onset. I suggest that with climate warming the first flowers of P. vulgaris face an increased risk of pollinator limitation whereas for bees a shift in floral resources may occur. In Chapter IV I present a study in which I investigated the effects of climate warming on host-parasitoid relationships. I studied how temperature and photoperiod affect emergence phenology in two spring bees (O. cornuta & O. bicornis) and three of their main parasitoids (C. indagator, A. anthrax, Monodontomerus). In a climate chamber experiment with a crossed design I exposed cocoons within nest cavities and cocoons outside of nest cavities to two different temperature regimes (long-term mean of Würzburg, Germany and long-term mean of Würzburg + 4 °C) and three photoperiods (Würzburg vs. Snåsa, Norway vs. constant darkness) and recorded the time of bee and parasitoid emergence. I show that warmer temperatures advanced emergence in all studied species, but bees advanced less strongly than parasitoids. Consequently, the time period between female bee emergence and parasitoid emergence decreased in the warm temperature treatment compared to the cold one. Photoperiod influenced the time of emergence only in cocoons outside of nest cavities (except O. bicornis male emergence). The data also shows that the effect of photoperiod compared to the effect of temperature on emergence phenology was much weaker. I suggest that with climate warming the synchrony of emergence phenologies of bees and their parasitoids will amplify. Therefore, parasitism rates in solitary bees might increase which can negatively affect reproductive success and population size. In this dissertation I show that for early flowering spring plants low interspecific competition for pollinators with co-flowering plants is a major driver of flowering phenology, whereas other drivers, like low pollinator abundances and unfavourable weather conditions are only of minor importance. With climate warming the strength of different drivers, which act on the timing of phenological events, can change, like temperature. I show that warmer temperatures advance early spring plant flowering more strongly than bee emergence and flowering phenology of later co-flowering plants. Furthermore, I show that warmer temperatures advance parasitoid emergence more strongly than bee emergence. Whereas temperature changes can lead to non-uniform temporal shifts, I demonstrate that geographic range shifts and with that altered photoperiods will not change emergence phenology in bees and their parasitoids. In the tritrophic system I investigated in this dissertation climate warming may negatively affect the reproductive success of the early spring plant and the spring bees but not of the parasitoids, which may even benefit from warming temperatures. N2 - Der richtige Zeitpunkt von phänologischen Ereignissen ist maßgeblich für das Überleben und die Fortpflanzung einer Art. Arten sollten eine möglichst hohe Synchronisation mit Mutualisten aufweisen, aber eine möglichst geringe mit Antagonisten. Die Klimaerwärmung führt dazu, dass sich bei vielen Arten phänologische Ereignisse verfrühen. Allerdings reagieren Arten unterschiedlich auf wärmere Temperaturen. Artspezifische Reaktionen auf die Klimaerwärmung können zu Asynchronität oder sogar zu zeitlicher Diskrepanz bei interagierenden Arten führen. Eine zeitliche Diskrepanz zwischen Mutualisten, die voneinander profitieren, kann sich negativ auf beide Interaktionspartner auswirken. Bei Wirt-Parasitoid Beziehungen kann der Wirt von einer zeitlichen Diskrepanz profitieren, wenn er seinem Parasitoid zeitlich entfliehen kann, was wiederum negative Folgen für den Parasitoid haben kann. Jedoch kann der Parasitoid profitieren, wenn er die Synchronisation mit seinem Wirt erhöhen kann, was wiederum den Wirt negativ beeinflussen kann. Das Wissen über die Treiber von phänologischen Ereignissen und die artspezifischen Reaktionen auf diese Treiber sind von Bedeutung um die Auswirkungen des Klimawandels auf trophische Beziehungen vorherzusagen. In meiner Doktorarbeit habe ich untersucht, wie verschiedene Treiber mit einer frühen Blüte zusammenhängen und wie der Klimawandel die tritrophische Beziehung von zwei Frühlingsbienen (Osmia cornuta & Osmia bicornis), einer Frühlingspflanzenart (Pulsatilla vulgaris), die eine der wichtigen Futterpflanzen der Bienen ist, und der drei Hauptparasitoiden der Frühlingsbienen (Cacoxenus indagator, Anthrax anthrax, Monodontomerus) beeinflusst. In Kapitel II präsentiere ich eine Studie, in der ich den Einfluss verschiedener Treiber und ihre saisonale Veränderung auf den Fortpflanzungserfolg einer Frühlingspflanzenart untersucht habe. Dazu habe ich auf acht Kalkmagerrasen bei Würzburg (Deutschland) die innersaisonalen Veränderungen der Bestäuberverfügbarkeit, der Anzahl an gleichzeitig blühenden Pflanzenarten und die Wetterbedingungen aufgezeichnet. Des Weiteren habe ich erforscht wie diese Faktoren die Blütenbesuchsrate, die Blütenlanglebigkeit und den Samenansatz der Frühlingspflanze P. vulgaris beeinflussen. Ich konnte zeigen, dass die Anzahl an Bienen und die Anzahl an Stunden, die ein Furagieren von Bestäubern ermöglicht hätten, am Anfang der Saison niedrig waren und mit der Zeit zunahmen. Jedoch war die Blütenbesuchsrate und die geschätzte Anzahl an Bienenbesuchen höher bei frühen als bei späten P. vulgaris Blüten. Die Blütenbesuchsrate wirkte sich auch positiv auf den Samenansatz aus. Die Blütenbesuchsrate nahm mit der Zeit und mit zunehmender Konkurrenz um Bestäuber durch eine zunehmende Anzahl an gleichzeitig blühenden Pflanzenarten ab. Meine Daten zeigen, dass ein Hauptreiber von frühen Blühzeitpunkten die geringe zwischenartliche Konkurrenz um Bestäuber ist, aber nicht die niedrige Bestäuberanzahl und ungünstige Wetterbedingungen. Kapitel III präsentiert eine Studie, in welcher ich die Auswirkungen der Temperatur auf den Schlupf von Solitärbienen und die Blüte ihrer Futterpflanzen und gleichzeitig blühenden Pflanzen im Freiland untersucht habe. Dafür habe ich Bienenkokons von zwei Frühlingsbienen (O. cornuta & O. bicornis) auf elf Kalkmagerrasen, die sich in der mittleren Flächentemperatur unterschieden, platziert. Auf sieben dieser Kalkmagerrasen kam die Frühlingspflanzenart P. vulgaris vor. Ich konnte zeigen, dass wärmere Temperaturen den mittleren Schlupf von O. cornuta Männchen verfrühen. Die Männchen von O. bicornis und die Weibchen beider Arten haben ihren Schlupfzeitpunkt jedoch nicht verschoben. Im Vergleich zu den Bienen verfrühte P. vulgaris seine Blühphänologie bei warmen Temperaturen stärker. Die gleichzeitig blühenden Pflanzenarten verschoben ihren Blühbeginn nicht. Die Daten zeigen, dass wärmere Temperaturen den Bienenschlupf weniger stark verfrühen als die Blüte ihrer Futterpflanze. Das lässt darauf schließen, dass mit dem Klimawandel die ersten Blüten von P. vulgaris ein erhöhtes Risiko haben nicht bestäubt zu werden, während die Bienen möglicherweise auf andere Blühressourcen ausweichen müssen. Kapitel IV beschreibt eine Studie, in welcher ich die Auswirkungen der Klimaerwärmung auf Wirt-Parasitoid Beziehungen untersucht habe. Dabei habe ich die Auswirkungen von Temperatur und Photoperiode auf die Schlupfphänologie zweier Frühlingsbienen (O. cornuta & O. bicornis) und drei ihrer Hauptparasitoide (C. indagator, A. anthrax, Monodontomerus) erforscht. In einem Klimakammerexperiment mit gekreuztem Design habe ich Kokons in Nesthöhlen und Kokons außerhalb von Nesthöhlen, zwei verschiedenen Temperaturregimen (Langzeitmittel von Würzburg, Deutschland und Langzeitmittel von Würzburg + 4 °C) und drei Photoperioden (Würzburg, Deutschland contra Snåsa, Norwegen contra Dauerdunkel) ausgesetzt und die Zeitpunkte des Bienen- und Parasitoidenschlupfes aufgezeichnet. Ich konnte zeigen, dass warme Temperaturen in allen untersuchten Arten den Schlupfzeitpunkt verfrühten, jedoch bei den Bienen weniger stark als bei den Parasitoiden. Eine Folge daraus ist, dass sich die Zeitspanne zwischen dem Schlupf der Bienenweibchen und dem Schlupf der Parasitoide im warmen Temperaturregime im Vergleich zum kalten verkürzte. Die Photoperiode hatte auf den Zeitpunkt des Schlupfes nur in Kokons außerhalb von Nisthöhlen einen Effekt (außer beim Schlupf von O. bicornis Männchen). Die Daten zeigen auch, dass der Effekt der Photoperiode auf die Schlupfphänologie im Vergleich zu dem Effekt der Temperatur viel schwächer war. Daraus schließe ich, dass sich im Zuge der Klimaerwärmung die Synchronisation der Schlupfphänologien von Bienen und ihren Parasitoiden verstärken wird. Eine Folge davon könnten erhöhte Parasitierungsraten bei Solitärbienen sein, welche den Reproduktionserfolg und die Populationsgröße negativ beeinflussen können. In dieser Doktorarbeit habe ich gezeigt, dass einer der Haupttreiber einer frühen Blüte bei Frühlingspflanzen geringe zwischenartliche Konkurrenz um Bestäuber mit später gleichzeitig blühenden Pflanzenarten ist, während andere Treiber, wie geringe Bestäuberabundanzen und ungünstige Wetterbedingungen nur von geringer Bedeutung sind. Im Zuge des Klimawandels könnte sich die Stärke verschiedener Treiber, die den Zeitpunkt von phänologischen Ereignissen beeinflussen, verändern. Ich konnte außerdem zeigen, dass wärmere Temperaturen die Blüte von frühen Frühlingspflanzen stärker verfrühen, als den Schlupf von Bienen und die Blüte von später gleichzeitig blühenden Pflanzenarten. Des Weiteren zeigte ich, dass wärmere Temperaturen den Schlupf von Parasitoiden stärker verfrühen als den Schlupf der Bienen. Ich konnte zeigen, dass während Temperaturveränderungen zu verschieden starken zeitlichen Verschiebungen führen können, Verschiebungen von geografischen Verbreitungsgebieten und damit geänderten Photoperioden die Schlupfphänologie von Bienen und ihren Parasitoiden wahrscheinlich nicht ändern werden. In dem tritrophischen System, das ich in dieser Doktorarbeit untersucht habe, könnte die Erwärmung des Klimas den Fortpflanzungserfolg der frühen Frühlingspflanze und der Frühlingsbienen negativ beeinflussen, aber wahrscheinlich nicht die der Parasitoide, die vielleicht sogar davon profitieren können. KW - Biene KW - Klimaänderung KW - Interaktion KW - Parasitoid KW - Pflanzen KW - Klimaerwärmung KW - Mutualismus KW - Antagonismus KW - Synchronisation KW - Phänologie Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213932 ER - TY - THES A1 - Maistrenko, Oleksandr T1 - Pangenome analysis of bacteria and its application in metagenomics T1 - Bakterielle Pan-Genome und ihre Anwendungen in der Metagenomik N2 - The biosphere harbors a large quantity and diversity of microbial organisms that can thrive in all environments. Estimates of the total number of microbial species reach up to 1012, of which less than 15,000 have been characterized to date. It has been challenging to delineate phenotypically, evolutionary and ecologically meaningful lineages such as for example, species, subspecies and strains. Even within recognized species, gene content can vary considerably between sublineages (for example strains), a problem that can be addressed by analyzing pangenomes, defined as the non-redundant set of genes within a phylogenetic clade, as evolutionary units. Species considered to be ecologically and evolutionary coherent units, however to date it is still not fully understood what are primary habitats and ecological niches of many prokaryotic species and how environmental preferences drive their genomic diversity. Majority of comparative genomics studies focused on a single prokaryotic species in context of clinical relevance and ecology. With accumulation of sequencing data due to genomics and metagenomics, it is now possible to investigate trends across many species, which will facilitate understanding of pangenome evolution, species and subspecies delineation. The major aims of this thesis were 1) to annotate habitat preferences of prokaryotic species and strains; 2) investigate to what extent these environmental preferences drive genomic diversity of prokaryotes and to what extent phylogenetic constraints limit this diversification; 3) explore natural nucleotide identity thresholds to delineate species in bacteria in metagenomics gene catalogs; 4) explore species delineation for applications in subspecies and strain delineation in metagenomics. The first part of the thesis describes methods to infer environmental preferences of microbial species. This data is a prerequisite for the analyses performed in the second part of the thesis which explores how the structure of bacterial pangenomes is predetermined by past evolutionary history and how is it linked to environmental preferences of the species. The main finding in this subchapter that habitat preferences explained up to 49% of the variance for pangenome structure, compared to 18% by phylogenetic inertia. In general, this trend indicates that phylogenetic inertia does not limit evolution of pangenome size and diversity, but that convergent evolution may overcome phylogenetic constraints. In this project we show that core genome size is associated with higher environmental ubiquity of species. It is likely this is due to the fact that species need to have more versatile genomes and most necessary genes need to be present in majority of genomes of that species to be highly prevalent. Taken together these findings may be useful for future predictive analyses of ecological niches in newly discovered species. The third part of the thesis explores data-driven, operational species boundaries. I show that homologous genes from the same species from different genomes tend to share at least 95% of nucleotide identity, while different species within the same genus have lower nucleotide identity. This is in line with other studies showing that genome-wide natural species boundary might be in range of 90-95% of nucleotide identity. Finally, the fourth part of the thesis discusses how challenges in species delineation are relevant for the identification of meaningful within-species groups, followed by a discussion on how advancements in species delineation can be applied for classification of within-species genomic diversity in the age of metagenomics. N2 - Die Biosphäre beherbergt eine große Zahl verschiedener Mikroorganismen, die fast alle bekannten Lebensräume besiedeln können. Die Gesamtzahl mikrobieller Spezies liegt Schätzungen zu Folge bei bis zu 1012, von denen jedoch bis heute erst 15.000 beschrieben worden sind. Die Beschreibung von phänotypisch, evolutionsbiologisch und ökologisch kohärenten Spezies, Sub-Spezies oder Stämmen stellt Forscher vor konzeptionelle Herausforderungen. Selbst innerhalb anerkannter Spezies kann die Kombination einzelner Gene oft stark variieren. Diese Beobachtung ist die Grundlage der Analyse von Pan-Genomen. also der Konstellation originärer Gene innerhalb einer Abstammunsglinie, als evolutionsbiologische Einheiten. Spezies entsprechen prinzipiell ökologisch und evolutionär kohärenten Einheiten, jedoch sind die primären Habitate und ökologischen Nischen vieler prokaryotischer Spezies bis heute nur unzureichend beschrieben, insbesondere mit Blick auf den Einfluss ökologischer Präferenzen auf die Evolution von Genomen. Die Mehrheit vergleichender genomischer Studien untersucht einzelne prokaryotische Spezies mit Bezug auf deren klinische oder ökologische Relevanz. Aufgrund der wachsenden Verfügbarkeit genomischer Daten ist es nun jedoch möglich, vergleichende Studien über Speziesgrenzen hinweg durchzuführen, um allgemeine Prinzipien der Evolution von Pan-Genomen, Spezies und Sub-Spezies zu untersuchen. Die wesentlichen Ziele der vorliegenden Arbeit waren 1) die Annotation von Habitatpräferenzen prokaryotischer Spezies und Stämme; 2) die Quantifizierung des Einflusses von Umwelt und Evolutionsgeschichte (Phylogenie) auf die genomische Diversität von Prokaryoten; 3) die Bestimmung natürlicher Schwellenwerte der Genomsequenzähnlichkeit zwischen Spezies, auch anhand von Genkatalogen; 4) die Untersuchung der Abgrenzung zwischen Spezies, Sub-Spezies und Stämmen mithilfe metagenomischer Daten. Im ersten Teil der Arbeit werden Methoden zur Bestimmung ökologischer Präferenzen mikrobieller Spezies beschrieben. Die so gewonnenen Daten dienen in der Folge als Grundlage für die Quantifizierung von Umwelt- und evolutionsgeschichtlichen Einflüssen auf die Struktur und Evolution bakterieller Pan-Genome im zweiten Teil der Arbeit. Ein zentrales Ergebnis dieser Untersuchung war, dass bis zu 49% der strukturellen Varianz in Pan-Genomen durch Habitatpräferenzen erklärt werden kann, im Gegensatz zu lediglich 18% durch phylogenetische Trägheitseffekte. Dies zeigt, dass die Größe und Diversität von Pan-Genomen nicht phylogenetisch limitiert ist, insbesondere in Fällen von konvergenter Evolution. Große Kern-Genome sind ferner mit einer weiten ökologischen Verbreitung von Spezies assoziiert; eine mögliche Erklärung ist, dass weit verbreitete Spezies vielseitigere Genome mit mehr notwendigen Genen besitzen, die ein Überleben in vielfältigen Umgebungen ermöglichen. Die vorgelegte Arbeit kann weiterhin einen Beitrag zur Vorhersage ökologischer Profile neu beschriebener Spezies leisten. Im dritten Teil der Arbeit werden datenbezogene, operationelle Definition von Spezies-Grenzen untersucht. Es konnte gezeigt werden, dass Gene verschiedener Genome innerhalb derselben Spezies normalerweise mindestens 95% Ähnlichkeit der Nukleotidsequenz aufweisen, während die Ähnlichkeit zwischen Spezies desselben Genus geringer ausfällt. Dieser Wert liegt im Rahmen früherer Schätzungen. Der vierte Teil der Arbeit beschreibt abschließend die Herausforderungen bei der Bestimmung von evolutionären Linien innerhalb von Spezies und diskutiert anschließend, wie konzeptionelle Entwicklungen in dieser Frage für die Klassifizierung und Quantifizierung von Diversität anhand metagenomischer Daten genutzt werden kann. KW - Pangenom KW - phylogenetische Trägheit KW - Lebensraum KW - Stammvielfalt KW - mikrobielle Ökologie und Evolution KW - pangenome KW - phylogenetic inertia KW - habitat KW - strain diversity KW - microbial ecology and evolution KW - metagenomics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214996 ER - TY - JOUR A1 - Schneider-Schaulies, Sibylle A1 - Schumacher, Fabian A1 - Wigger, Dominik A1 - Schöl, Marie A1 - Waghmare, Trushnal A1 - Schlegel, Jan A1 - Seibel, Jürgen A1 - Kleuser, Burkhard T1 - Sphingolipids: effectors and Achilles heals in viral infections? JF - Cells N2 - As viruses are obligatory intracellular parasites, any step during their life cycle strictly depends on successful interaction with their particular host cells. In particular, their interaction with cellular membranes is of crucial importance for most steps in the viral replication cycle. Such interactions are initiated by uptake of viral particles and subsequent trafficking to intracellular compartments to access their replication compartments which provide a spatially confined environment concentrating viral and cellular components, and subsequently, employ cellular membranes for assembly and exit of viral progeny. The ability of viruses to actively modulate lipid composition such as sphingolipids (SLs) is essential for successful completion of the viral life cycle. In addition to their structural and biophysical properties of cellular membranes, some sphingolipid (SL) species are bioactive and as such, take part in cellular signaling processes involved in regulating viral replication. It is especially due to the progress made in tools to study accumulation and dynamics of SLs, which visualize their compartmentalization and identify interaction partners at a cellular level, as well as the availability of genetic knockout systems, that the role of particular SL species in the viral replication process can be analyzed and, most importantly, be explored as targets for therapeutic intervention. KW - glycosphingolipids KW - ceramides KW - sphingosine 1-phosphate KW - sphingomyelinase KW - HIV KW - SARS-CoV-2 KW - measles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245151 SN - 2073-4409 VL - 10 IS - 9 ER - TY - JOUR A1 - Leverkus, Alexandro B. A1 - Thorn, Simon A1 - Gustafsson, Lena A1 - Noss, Reed A1 - Müller, Jörg A1 - Pausas, Juli G. A1 - Lindenmayer, David B. T1 - Environmental policies to cope with novel disturbance regimes–steps to address a world scientists’ warning to humanity JF - Environmental Research Letters N2 - No abstract available. KW - global change KW - novel disturbance KW - regime shift KW - forest management KW - risk management Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254180 SN - 1748-9326 VL - 16 IS - 2 ER - TY - JOUR A1 - Colizzi, Francesca Sara A1 - Beer, Katharina A1 - Cuti, Paolo A1 - Deppisch, Peter A1 - Martínez Torres, David A1 - Yoshii, Taishi A1 - Helfrich-Förster, Charlotte T1 - Antibodies Against the Clock Proteins Period and Cryptochrome Reveal the Neuronal Organization of the Circadian Clock in the Pea Aphid JF - Frontiers in Physiology N2 - Circadian clocks prepare the organism to cyclic environmental changes in light, temperature, or food availability. Here, we characterized the master clock in the brain of a strongly photoperiodic insect, the aphid Acyrthosiphon pisum, immunohistochemically with antibodies against A. pisum Period (PER), Drosophila melanogaster Cryptochrome (CRY1), and crab Pigment-Dispersing Hormone (PDH). The latter antibody detects all so far known PDHs and PDFs (Pigment-Dispersing Factors), which play a dominant role in the circadian system of many arthropods. We found that, under long days, PER and CRY are expressed in a rhythmic manner in three regions of the brain: the dorsal and lateral protocerebrum and the lamina. No staining was detected with anti-PDH, suggesting that aphids lack PDF. All the CRY1-positive cells co-expressed PER and showed daily PER/CRY1 oscillations of high amplitude, while the PER oscillations of the CRY1-negative PER neurons were of considerable lower amplitude. The CRY1 oscillations were highly synchronous in all neurons, suggesting that aphid CRY1, similarly to Drosophila CRY1, is light sensitive and its oscillations are synchronized by light-dark cycles. Nevertheless, in contrast to Drosophila CRY1, aphid CRY1 was not degraded by light, but steadily increased during the day and decreased during the night. PER was always located in the nuclei of the clock neurons, while CRY was predominantly cytoplasmic and revealed the projections of the PER/CRY1-positive neurons. We traced the PER/CRY1-positive neurons through the aphid protocerebrum discovering striking similarities with the circadian clock of D. melanogaster: The CRY1 fibers innervate the dorsal and lateral protocerebrum and putatively connect the different PER-positive neurons with each other. They also run toward the pars intercerebralis, which controls hormone release via the neurohemal organ, the corpora cardiaca. In contrast to Drosophila, the CRY1-positive fibers additionally travel directly toward the corpora cardiaca and the close-by endocrine gland, corpora allata. This suggests a direct link between the circadian clock and the photoperiodic control of hormone release that can be studied in the future. KW - aphids KW - circadian clock KW - cryptochrome KW - period KW - hemiptera KW - insects KW - photoperiodism Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242909 SN - 1664-042X VL - 12 ER - TY - JOUR A1 - Geisinger, Adriana A1 - Rodríguez-Casuriaga, Rosana A1 - Benavente, Ricardo T1 - Transcriptomics of Meiosis in the Male Mouse JF - Frontiers in Cell and Developmental Biology N2 - Molecular studies of meiosis in mammals have been long relegated due to some intrinsic obstacles, namely the impossibility to reproduce the process in vitro, and the difficulty to obtain highly pure isolated cells of the different meiotic stages. In the recent years, some technical advances, from the improvement of flow cytometry sorting protocols to single-cell RNAseq, are enabling to profile the transcriptome and its fluctuations along the meiotic process. In this mini-review we will outline the diverse methodological approaches that have been employed, and some of the main findings that have started to arise from these studies. As for practical reasons most studies have been carried out in males, and mostly using mouse as a model, our focus will be on murine male meiosis, although also including specific comments about humans. Particularly, we will center on the controversy about gene expression during early meiotic prophase; the widespread existing gap between transcription and translation in meiotic cells; the expression patterns and potential roles of meiotic long non-coding RNAs; and the visualization of meiotic sex chromosome inactivation from the RNAseq perspective. KW - meiosis KW - transcriptomics KW - RNAseq KW - meiotic prophase KW - spermatogenesis KW - lncRNAs KW - MSCI KW - spermatogenic cell sorting Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231032 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Maier, Carina R. A1 - Fischer, Thomas A1 - Prieto-Garcia, Cristian A1 - Baluapuri, Apoorva A1 - Schwarz, Jessica A1 - Schmitz, Werner A1 - Garrido-Rodriguez, Martin A1 - Pahor, Nikolett A1 - Davies, Clare C. A1 - Bassermann, Florian A1 - Orian, Amir A1 - Wolf, Elmar A1 - Schulze, Almut A1 - Calzado, Marco A. A1 - Rosenfeldt, Mathias T. A1 - Diefenbacher, Markus E. T1 - Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease JF - Frontiers in Cell and Developmental Biology N2 - Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research. KW - non-small cell lung cancer KW - CRISPR-Cas9 KW - mouse model KW - lung cancer KW - MYC KW - JUN Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230949 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Yu, Yidong A1 - Wolf, Ann-Katrin A1 - Thusek, Sina A1 - Heinekamp, Thorsten A1 - Bromley, Michael A1 - Krappmann, Sven A1 - Terpitz, Ulrich A1 - Voigt, Kerstin A1 - Brakhage, Axel A. A1 - Beilhack, Andreas T1 - Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms JF - Journal of Fungi N2 - Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates. KW - fungal infection model KW - calcofluor white staining KW - Aspergillus KW - Lichtheimia KW - silkworm Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228855 SN - 2309-608X VL - 7 IS - 2 ER - TY - JOUR A1 - Hensgen, Ronja A1 - England, Laura A1 - Homberg, Uwe A1 - Pfeiffer, Keram T1 - Neuroarchitecture of the central complex in the brain of the honeybee: Neuronal cell types JF - Journal of Comparative Neurology N2 - The central complex (CX) in the insect brain is a higher order integration center that controls a number of behaviors, most prominently goal directed locomotion. The CX comprises the protocerebral bridge (PB), the upper division of the central body (CBU), the lower division of the central body (CBL), and the paired noduli (NO). Although spatial orientation has been extensively studied in honeybees at the behavioral level, most electrophysiological and anatomical analyses have been carried out in other insect species, leaving the morphology and physiology of neurons that constitute the CX in the honeybee mostly enigmatic. The goal of this study was to morphologically identify neuronal cell types of the CX in the honeybee Apis mellifera. By performing iontophoretic dye injections into the CX, we traced 16 subtypes of neuron that connect a subdivision of the CX with other regions in the bee's central brain, and eight subtypes that mainly interconnect different subdivisions of the CX. They establish extensive connections between the CX and the lateral complex, the superior protocerebrum and the posterior protocerebrum. Characterized neuron classes and subtypes are morphologically similar to those described in other insects, suggesting considerable conservation in the neural network relevant for orientation. KW - RRID: AB_2337244 KW - RRID: AB_2315425 KW - central complex KW - insect brain KW - neuroanatomy KW - sky compass KW - Apis mellifera Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215566 VL - 529 ER - TY - JOUR A1 - Kunz, Tobias C. A1 - Rühling, Marcel A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kozjak-Pavlovic, Vera A1 - Rudel, Thomas A1 - Fraunholz, Martin T1 - The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy JF - Frontiers in Cellular and Infection Microbiology N2 - Expansion Microscopy (ExM) is a novel tool improving the resolution of fluorescence microscopy by linking the sample into a hydrogel that gets physically expanded in water. Previously, we have used ExM to visualize the intracellular Gram-negative pathogens Chlamydia trachomatis, Simkania negevensis, and Neisseria gonorrhoeae. Gram-positive bacteria have a rigid and thick cell wall that impedes classic expansion strategies. Here we developed an approach, which included a series of enzymatic treatments resulting in isotropic 4× expansion of the Gram-positive pathogen Staphylococcus aureus. We further demonstrate the suitability of the technique for imaging of planktonic bacteria as well as endocytosed, intracellular bacteria at a spatial resolution of approximately 60 nm with conventional confocal laser scanning microscopy. KW - high-resolution imaging KW - endosomes KW - autophagosomes KW - host-pathogen interaction KW - expansion microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232292 SN - 2235-2988 VL - 11 ER - TY - THES A1 - Roth, Nicolas Mériadec Max André T1 - Temporal development of communities with a focus on insects, in time series of one to four decades T1 - Entwicklung von Artengemeinschaften in der Zeit mit einem Fokus auf Insekten in Zeitreihen von einer bis vier Dekaden N2 - Changes and development are fundamental principles in biocenoses and can affect a multitude of ecological processes. In insect communities phenological and density changes, changes in species richness and community composition, as well as interactions between those changes, are the most important macro processes. However, climate change and other factors like habitat degradation and loss alter these processes leading to shifts and general biodiversity declines. Even though knowledge about insect decline in central Europe increased during the last decades, there are significant knowledge gaps about the development of insect communities in certain habitats and taxa. For example, insect communities in small lentic as well as in forested habitats are under-sampled and reported to be less endangered than communities in other habitats. Furthermore, the changes within habitats and taxa are additionally influenced by certain traits, like host or feeding specialization. To disentangle these influences and to increase the knowledge about the general long-term development of insect communities, comprehensive long-term monitoring studies are needed. In addition, long-term effects of conservation strategies should also be evaluated on large time scales in order to be able to decide on a scientific base which strategies are effective in promoting possibly declining taxa. Hence, this thesis also tackles the effects of an integrative conservation strategy on wood dependent beetle and fungi, beside the development of water beetle and macro moth communities over multiple decades. In Chapter 2 I present a study on the development of water beetle communities (Dytiscidae, Haliplidae, Noteridae) in 33 water bodies in Southern Germany from 1991 to 2018. Time-standardized capture per waterbody was used during three periods: between 1991 and 1995, 2007 and 2008, and 2017 and 2018. Results showed annual declines in both species number (ca. -1%) and abundance (ca. -2%). In addition, community composition shifted over time in part due to changing pH values. Hence, the recorded changes during the 28-year study period partly reflect natural succession processes. However, since also moor-related beetle species decreased significantly, it is likely that water beetles in southern Germany are also threatened by non-successional factors, including desiccation, increased nitrogen input and/or mineralization, as well as the loss of specific habitats. The results suggest, that in small to midsize lentic waterbodies, current development should aim for constant creation of new water bodies and protection of moor waterbodies in order to protect water beetle communities on a landscape scale. In Chapter 3 I present an analysis of the development of nocturnal macro moth species richness, abundance and biomass over four decades in forests of southern Germany. Two local scale data sets featuring a coppiced oak forest as well as an oak high forest were analysed separately from a regional data set representing all forest types in the temperate zone of Central Europe. At the regional scale species richness, abundance and biomass showed annual declines of ca. 1 %, 1.3 % and 1.4 %, respectively. These declines were more pronounced in plant host specialists and in dark coloured species. In contrast, species richness increased by ca. 1.5 % annually in the coppiced forest, while no significant trends were found in the high forest. In contrast to past assumptions, insect decline apparently affects also hyper diverse insect groups in forests. Since host specialists and dark coloured species were affected more heavily by the decline than other groups, habitat loss and climate change seem to be potential drivers of the observed trends. However, the positive development of species richness in the coppiced oak forest indicates that maintaining complex and diverse forest ecosystems through active management might compensate for negative trends in biodiversity. Chapter 4 features a study specifically aiming to investigate the long-term effect of deadwood enrichment as an integrative conservation strategy on saproxylic beetles and fungi in a central European beech forest at a landscape scale. A before–after control–impact design, was used to compare assemblages and gamma diversities of saproxylic organisms (beetles and fungi) in strictly protected old-growth forest areas (reserves) and previously moderately and intensively managed forest areas. Forests were sampled one year before and a decade after starting a landscape-wide strategy of dead-wood enrichment. Ten years after the start of the dead-wood enrichment, neither gamma diversities of saproxylic organisms nor species composition of beetles did reflect the previous management types anymore. However, fungal species composition still mirrored the previous management gradient. The results demonstrated that intentional enrichment of dead wood at the landscape scale can effectively restore communities of saproxylic organisms and may thus be a suitable strategy in addition to permanent strict reserves in order to protect wood dependent organisms in Europe. In this thesis I showed, that in contrast to what was assumed and partly reported so far, also water beetles in lentic water bodies and macro moths in forests decreased in species richness, abundance and biomass during the last three to four decades. In line with earlier studies, especially dark coloured species and specialists decreased more than light-coloured species and generalists. The reasons for these declines could partly be attributed to natural processes and pollution and possibly to climate change. However, further studies, especially experimental ones, will be needed to achieve a better understanding of the reasons for insect decline. Furthermore, analyses of time series data should be interpreted cautiously especially if the number of sampling years is smaller than ten years. In addition, validation techniques such as left- and right- censoring and cross validation should be used in order to proof the robustness of the analyses. However, the lack of knowledge, we are still facing today, should not prevent scientists and practitioners from applying conservation measures. In order to prove the effectiveness of such measures, long-term monitoring is crucial. Such control of success is essential for evidence based and thus adapted conservation strategies of threatened organisms. N2 - Veränderungen und Entwicklung sind grundlegende Prinzipien in Biozönosen und können eine Vielzahl von ökologischen Prozessen beeinflussen. In Insektengemeinschaften stellen Veränderungen in der Phänologie und Dichte, Veränderungen des Artenreichtums und der Artenzusammensetzung sowie die Wechselwirkungen zwischen diesen, die wichtigsten Makroprozesse dar. Klimawandel und andere Faktoren wie der Verlust von Lebensräumen oder deren Qualitätsverschlechterung beeinflussen diese Prozesse jedoch und führen zu Veränderungen und allgemeinen Rückgängen der Biodiversität. Auch wenn die Erkenntnisse zum „Insektensterben“ in Mitteleuropa in den letzten Jahrzehnten zugenommen haben, gibt es erhebliche Wissenslücken über die Entwicklung von Insektengemeinschaften in bestimmten Lebensräumen und Taxa. Beispielsweise ist die Entwicklung von Insektengemeinschaften in kleinen, stehenden Gewässern und in Wäldern wenig erforscht. Darüber hinaus werden die Veränderungen innerhalb von Habitaten und Taxa zusätzlich durch bestimmte Merkmale, wie Wirts- oder Nahrungsspezialisierung, beeinflusst. Um diese verschiedenen Einflüsse auseinanderhalten zu können und das Wissen über die allgemeine Langzeitentwicklung von Insektengemeinschaften zu vergrößern, sind umfassende Langzeitstudien erforderlich. Darüber hinaus sollten auch die langfristigen Auswirkungen von Naturschutzstrategien über lange Zeiträume evaluiert werden, um auf wissenschaftlicher Grundlage entscheiden zu können, welche Strategien zur Förderung bedrohter Taxa wirksam sind. Daher befasst sich diese Arbeit neben der Entwicklung von Wasserkäfer- und Großschmetterlingsgemeinschaften über mehrere Jahrzehnte auch mit den Auswirkungen einer integrativen Naturschutzmaßnahme auf xylobionte Käfer und Pilze. In Kapitel 2 stelle ich eine Studie über die Entwicklung von Wasserkäfergemeinschaften (Dytiscidae, Haliplidae, Noteridae) in 33 Gewässern Süddeutschlands von 1991 bis 2018 vor. Die zeitstandardisierte Erfassung pro Wasserkörper erfolgte in drei Zeiträumen: zwischen 1991 und 1995, 2007 und 2008 sowie 2017 und 2018. Die Ergebnisse zeigten einen jährlichen Rückgang sowohl der Artenzahl (ca. -1%) als auch der Abundanz (ca. -2%). Darüber hinaus verschob sich die Artenzusammensetzung im Laufe der Zeit zum Teil aufgrund sich ändernder pH-Werte. Daraus lässt sich schlussfolgern, dass die erfassten Veränderungen während des 28- jährigen Untersuchungszeitraums teilweise natürliche Sukzessionsprozesse widerspiegeln. Da aber auch an moorige Gewässer gebundene Käferarten deutlich abgenommen haben, ist es wahrscheinlich, dass die Wasserkäfer Süddeutschlands auch durch Faktoren wie Austrocknung, erhöhten Stickstoffeintrag und/oder Mineralisierung sowie durch den Verlust spezifischer Lebensräume bedroht sind. Aufgrund dieser Entwicklungen ist es empfehlenswert, auf Landschaftsebene auf die ständige Schaffung neuer Gewässer und den besonderen Schutz von Moorgewässern zu setzen, um Wasserkäfergemeinschaften erfolgreich schützen zu können. In Kapitel 3 präsentiere ich eine Analyse der Diversitäts-, Abundanz- und Biomassenentwicklung von nachtaktiven Großschmetterlingen über vier Jahrzehnte in Wäldern Süddeutschlands. Neben einem bayernweiten Datensatz, der alle typischen Waldtypen der gemäßigten Zone Mitteleuropas beinhaltet, wurden zwei lokale, besonders regelmäßig besammelte Gebiete getrennt analysiert. In diesen Gebieten werden die Eichenwälder als Hoch- bzw. als Mittelwald bewirtschaftet. Bayernweit wiesen Artenreichtum, Abundanz und Biomasse jährliche Rückgänge von ca. 1 %, 1,3 % bzw. 1,4 % auf. Diese Rückgänge waren bei Wirtspflanzenspezialisten und bei dunkel gefärbten Arten besonders stark ausgeprägt. Im Gegensatz dazu nahm der Artenreichtum im Mittelwald jährlich um ca. 1,5 % zu, während im Hochwald keine signifikanten Trends festgestellt werden konnten. Im Gegensatz zu früheren Annahmen betrifft der Insektenrückgang offenbar auch hyperdiverse Insektengruppen im Wald. Da Wirtspflanzenspezialisten und dunkel gefärbte Arten vom Rückgang stärker betroffen waren als andere, scheinen Lebensraumverlust und Klimawandel potentielle Treiber der beobachteten Trends zu sein. Die positive Entwicklung des Artenreichtums im Mittelwald zeigt jedoch, dass der Erhalt komplexer und vielfältiger Waldökosysteme durch aktives Management, negative Biodiversitätstrends zum Teil kompensieren könnte. Kapitel 4 enthält eine Studie, die die Langzeitwirkung von Totholzanreicherung als integrative Naturschutzmaßnahme auf xylobionte Käfer und Pilze in einem mitteleuropäischen Buchenwald auf der Landschaftsebene untersucht. Dabei wurde die Gamma-Diversität und die Artenzusammensetzung dieser beiden Gruppen anhand einer Vorher-Nachher Untersuchung mit Kontrollflächen (Naturwaldreservate) untersucht. Die bewirtschafteten Flächen wurden weiterhin in zuvor mäßig und intensiv bewirtschaftete Flächen eingeteilt. Die Wälder wurden ein Jahr vor und ein Jahrzehnt nach Beginn einer Totholzanreicherungsstrategie auf Landschaftsebene beprobt. Zehn Jahre nach Beginn der Totholzanreicherung spiegelten weder die Gamma-Diversität der xylobionten Organismen noch die Artenzusammensetzung der Käfer die früheren Bewirtschaftungstypen wider, und wiesen keine Unterschiede mehr zu den Naturwaldreservaten auf. Die Pilzartenzusammensetzung spiegelte jedoch noch immer den früheren Bewirtschaftungsgradienten wider. Die Ergebnisse zeigen, dass Totholzanreicherung auf Landschaftsebene positive Effekte auf xylobionte Artengemeinschaften haben kann. Somit stellt Totholzanreicherung eine Naturschutzmaßnahme dar, die zusätzlich zu permanenten Schutzgebieten, eine Grundlage schaffen kann, um holzabhängige Organismen in Europa zu schützen. In dieser Arbeit habe ich gezeigt, dass im Gegensatz zu dem, was bisher angenommen und zum Teil berichtet wurde, auch Wasserkäfer in stehenden Gewässern und nachtaktive Großschmetterlingen in Wäldern in den letzten drei bis vier Jahrzehnten an Artenreichtum, Abundanz und Biomasse abgenommen haben. In Übereinstimmung mit anderen Studien nahmen vor allem dunkel gefärbte Arten und Spezialisten stärker ab als hell gefärbte Arten und Generalisten. Die Gründe für diese Rückgänge konnten zum Teil auf natürliche Prozesse, Umweltverschmutzung und möglicherweise auf den Klimawandel zurückgeführt werden. Es sind jedoch weitere Studien, insbesondere experimentelle, erforderlich, um die Gründe für das „Insektensterben“ besser zu verstehen. Darüber hinaus sollten Zeitreihendaten mit Vorsicht interpretiert werden, insbesondere wenn die Anzahl der besammelten Jahre kleiner als zehn Jahre ist. Darüber hinaus sollten Validierungstechniken wie Links- und Rechts-Zensierung und Kreuzvalidierung eingesetzt werden, um die Robustheit der Analysen nachzuweisen. Der Mangel an Wissen, mit dem wir heute noch konfrontiert sind, sollte Wissenschaftler und Praktiker jedoch nicht davon abhalten, Naturschutzmaßnahmen anzuwenden. Um die Wirksamkeit solcher Maßnahmen nachzuweisen, ist eine langfristige Überprüfung von entscheidender Bedeutung. Solche Erfolgskontrollen sind für evidenzbasierte und damit angepasste Erhaltungsstrategien bedrohter Organismen unerlässlich. KW - climate change KW - insects KW - temporal development KW - nature conservation KW - entomology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235499 ER - TY - THES A1 - Solger, Franziska T1 - Central role of sphingolipids on the intracellular survival of \(Neisseria\) \(gonorrhoeae\) in epithelial cells T1 - Die zentrale Rolle von Sphingolipiden auf das intrazelluläre Überleben von \(Neisseria\) \(gonorrhoeae\) in Epithelzellen N2 - Neisseria gonorrhoeae are Gram-negative bacteria with diplococcal shape. As an obligate human pathogen, it is the causative agent of gonorrhoea, a sexually transmitted disease. Gonococci colonize a variety of mucosal tissues, mainly the urogenital tract in men and women. Occasionally N. gonorrhoeae invades the bloodstream, leading to disseminated gonococcal infection. These bacteria possess a repertoire of virulence factors, which expression patterns can be adapted to the environmental conditions of the host. Through the accumulation of antibiotic resistances and in absence of vaccines, some neisserial strains have the potential to spread globally and represent a major public health threat. Therefore, it is necessary to understand the exact molecular mechanisms underlying the successful infection and progression of gonococci within their host. This deeper understanding of neisserial infection and survival mechanisms is needed for the development of new therapeutic agents. In this work, the role of host-cell sphingolipids on the intracellular survival of N. gonorrhoeae was investigated. It was shown that different classes of sphingolipids strongly interact with invasive gonococci in epithelial cells. Therefore, novel and highly specific clickable sphingolipid analogues were applied to study these interactions with this pathogen. The formation of intra- and extracellular sphingosine vesicles, which were able to target gonococci, was observed. This direct interaction led to the uptake and incorporation of sphingosine into the neisserial membrane. Together with in vitro results, sphingosine was identified as a potential bactericidal reagent as part of the host cell defence. By using different classes of sphingolipids and their clickable analogues, essential structural features, which seem to trigger the bacterial uptake, were detected. Furthermore, effects of key enzymes of the sphingolipid signalling pathway were tested in a neutrophil infection model. In conclusion, the combination of click chemistry and infection biology made it possible to shed some light on the dynamic interplay between cellular sphingosine and N. gonorrhoeae. Thereby, a possible “catch-and-kill” mechanism could have been observed. N2 - Neisseria gonorrhoeae ist ein Gram-negatives Bakterium, welches als Diplokokke vorkommt. Als ein ausschließliches Humanpathogen sind Neisserien der Erreger für die sexuell übertragbare Infektionskrankheit Gonorrhö. Gonokokken besiedeln eine Vielzahl von Schleimhäuten, jedoch hauptsächlich den Urogenitaltrakt bei Männern und Frauen. Gelegentlich kann N. gonorrhoeae in die Blutbahn invadieren, was zu einer disseminierten Infektion führen kann. Diese Bakterien verfügen über ein Repertoire an Virulenzfaktoren, deren Expressionskombination den Umgebungsbedingungen des Wirts angepasst werden können. Durch die Anhäufung von Antibiotikaresistenzen und durch das Fehlen eines Impfstoffes, besteht die Gefahr, dass spezielle Neisserienstämme sich weltweit verbreiten und daher eine ernstzunehmende Bedrohung des Menschen sind. Daher ist es notwendig die zugrundeliegenden molekularen Mechanismen der erfolgreichen Infektion und Ausbreitung der Gonokokken im Wirt genauestens zu verstehen. Das detaillierte Wissen über die Neisserieninfektion und Überlebensmechanismen ist nötig für die Entwicklung neuer Therapieansätze. In dieser Arbeit wurde der Effekt von Sphingolipiden der Wirtszelle auf das intrazelluläre Überleben von N. gonorrhoeae untersucht. Es konnte gezeigt werden, dass unterschiedliche Klassen von Sphingolipiden stark mit invasiven Gonokokken in Epithelzellen interagieren. Um dies zu tun, wurden neue und hochspezifische clickbare Sphingolipidanaloge eingesetzt, um deren Interaktionen mit diesem Pathogen zu studieren. Die Formation von intra- als auch extrazellulären Sphingosinvesikeln, welche Gonokokken gezielt erreichten, konnte beobachtet werden. Diese direkte Interaktion führte zu einer Aufnahme und Einbau des Sphingosins in die Neisserienmembran. Zusammen mit in vitro Ergebnissen, konnte Sphingosin als potenzieller und antibakterieller Bestandteil des zellulären Abwehrsystems identifiziert werden. Weiterhin wurde durch die Verwendung unterschiedlicher Sphingolipidklassen und deren clickbaren Analoge wichtige Strukturen erkannt, die die bakterielle Aufnahme auslösen. Des Weiteren wurden die Auswirkungen von Schlüsselenzymen des Sphingolipidsignalwegs in einem Infektionsmodell mit Neutrophilen getestet. Abschließend ist zu sagen, dass die Kombination aus Click Chemie und Infektionsbiologie es ermöglicht hat, die dynamischen Wechselwirkungen zwischen zellulären Sphingosin und N. gonorrhoeae zu beleuchten. Dadurch konnte ein möglicher „catch-and-kill”-Mechanismus entdeckt werden. KW - Neisseria gonorrhoeae KW - Sphingosinkinase KW - Sphingosinanaloga KW - Click-Chemie KW - sphingosine KW - sphingolipids KW - Neisseria KW - intracellular KW - vesicles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-247534 ER - TY - JOUR A1 - Du, Baoguo A1 - Ma, Yuhua A1 - Yáñez‐Serrano, Ana Maria A1 - Arab, Leila A1 - Fasbender, Lukas A1 - Alfarraj, Saleh A1 - Albasher, Gadah A1 - Hedrich, Rainer A1 - White, Philip J. A1 - Werner, Christiane A1 - Rennenberg, Heinz T1 - Physiological responses of date palm (Phoenix dactylifera) seedlings to seawater and flooding JF - New Phytologist N2 - In their natural environment along coast lines, date palms are exposed to seawater inundation and, hence, combined stress by salinity and flooding. To elucidate the consequences of this combined stress on foliar gas exchange and metabolite abundances in leaves and roots, date palm seedlings were exposed to flooding with seawater and its major constituents under controlled conditions. Seawater flooding significantly reduced CO\(_{2}\) assimilation, transpiration and stomatal conductance, but did not affect isoprene emission. A similar effect was observed upon NaCl exposure. By contrast, flooding with distilled water or MgSO\(_{4}\) did not affect CO\(_{2}\)/H\(_{2}\)O gas exchange or stomatal conductance significantly, indicating that neither flooding itself, nor seawater sulfate, contributed greatly to stomatal closure. Seawater exposure increased Na and Cl contents in leaves and roots, but did not affect sulfate contents significantly. Metabolite analyses revealed reduced abundances of foliar compatible solutes, such as sugars and sugar alcohols, whereas nitrogen compounds accumulated in roots. Reduced transpiration upon seawater exposure may contribute to controlling the movement of toxic ions to leaves and, therefore, can be seen as a mechanism to cope with salinity. The present results indicate that date palm seedlings are tolerant towards seawater exposure to some extent, and highly tolerant to flooding. KW - compatible solutes and other metabolites KW - date palm KW - flooding KW - salinity KW - shoot–root interaction KW - stomatal conductance KW - sulfate Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228226 VL - 229 IS - 6 SP - 3318 EP - 3329 ER - TY - JOUR A1 - Seibold, Sebastian A1 - Hothorn, Torsten A1 - Gossner, Martin M. A1 - Simons, Nadja K. A1 - Blüthgen, Nico A1 - Müller, Jörg A1 - Ambarlı, Didem A1 - Ammer, Christian A1 - Bauhus, Jürgen A1 - Fischer, Markus A1 - Habel, Jan C. A1 - Penone, Caterina A1 - Schall, Peter A1 - Schulze, Ernst‐Detlef A1 - Weisser, Wolfgang W. T1 - Insights from regional and short‐term biodiversity monitoring datasets are valuable: a reply to Daskalova et al. 2021 JF - Insect Conservation and Diversity N2 - Reports of major losses in insect biodiversity have stimulated an increasing interest in temporal population changes. Existing datasets are often limited to a small number of study sites, few points in time, a narrow range of land‐use intensities and only some taxonomic groups, or they lack standardised sampling. While new monitoring programs have been initiated, they still cover rather short time periods. Daskalova et al. 2021 (Insect Conservation and Diversity, 14, 1‐18) argue that temporal trends of insect populations derived from short time series are biased towards extreme trends, while their own analysis of an assembly of shorter‐ and longer‐term time series does not support an overall insect decline. With respect to the results of Seibold et al. 2019 (Nature, 574, 671–674) based on a 10‐year multi‐site time series, they claim that the analysis suffers from not accounting for temporal pseudoreplication. Here, we explain why the criticism of missing statistical rigour in the analysis of Seibold et al. (2019) is not warranted. Models that include ‘year’ as random effect, as suggested by Daskalova et al. (2021), fail to detect non‐linear trends and assume that consecutive years are independent samples which is questionable for insect time‐series data. We agree with Daskalova et al. (2021) that the assembly and analysis of larger datasets is urgently needed, but it will take time until such datasets are available. Thus, short‐term datasets are highly valuable, should be extended and analysed continually to provide a more detailed understanding of insect population changes under the influence of global change, and to trigger immediate conservation actions. KW - Arthropod KW - biodiversity KW - insect decline KW - land use KW - time series Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228309 VL - 14 IS - 1 SP - 144 EP - 148 ER - TY - JOUR A1 - Britz, Sebastian A1 - Markert, Sebastian Matthias A1 - Witvliet, Daniel A1 - Steyer, Anna Maria A1 - Tröger, Sarah A1 - Mulcahy, Ben A1 - Kollmannsberger, Philip A1 - Schwab, Yannick A1 - Zhen, Mei A1 - Stigloher, Christian T1 - Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy JF - Frontiers in Neuroanatomy N2 - At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments. KW - FIB-SEM KW - 3D reconstruction KW - neuroanatomy KW - IL2 branching KW - amphids KW - Caenorhabditis elegans (C. elegans) KW - dauer Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249622 SN - 1662-5129 VL - 15 ER - TY - JOUR A1 - Pauli, Martin A1 - Paul, Mila M. A1 - Proppert, Sven A1 - Mrestani, Achmed A1 - Sharifi, Marzieh A1 - Repp, Felix A1 - Kürzinger, Lydia A1 - Kollmannsberger, Philip A1 - Sauer, Markus A1 - Heckmann, Manfred A1 - Sirén, Anna-Leena T1 - Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections JF - Communications Biology N2 - Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution. KW - mossy fiber synapses KW - CA3 pyrimidal cells KW - CA2+ channels KW - active zone KW - hippocampal KW - release KW - plasticity KW - proteins KW - platform KW - reveals Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259830 VL - 4 ER - TY - JOUR A1 - Hurd, Paul J. A1 - Grübel, Kornelia A1 - Wojciechowski, Marek A1 - Maleszka, Ryszard A1 - Rössler, Wolfgang T1 - Novel structure in the nuclei of honey bee brain neurons revealed by immunostaining JF - Scientific Reports N2 - In the course of a screen designed to produce antibodies (ABs) with affinity to proteins in the honey bee brain we found an interesting AB that detects a highly specific epitope predominantly in the nuclei of Kenyon cells (KCs). The observed staining pattern is unique, and its unfamiliarity indicates a novel previously unseen nuclear structure that does not colocalize with the cytoskeletal protein f-actin. A single rod-like assembly, 3.7-4.1 mu m long, is present in each nucleus of KCs in adult brains of worker bees and drones with the strongest immuno-labelling found in foraging bees. In brains of young queens, the labelling is more sporadic, and the rod-like structure appears to be shorter (similar to 2.1 mu m). No immunostaining is detectable in worker larvae. In pupal stage 5 during a peak of brain development only some occasional staining was identified. Although the cellular function of this unexpected structure has not been determined, the unusual distinctiveness of the revealed pattern suggests an unknown and potentially important protein assembly. One possibility is that this nuclear assembly is part of the KCs plasticity underlying the brain maturation in adult honey bees. Because no labelling with this AB is detectable in brains of the fly Drosophila melanogaster and the ant Camponotus floridanus, we tentatively named this antibody AmBNSab (Apis mellifera Brain Neurons Specific antibody). Here we report our results to make them accessible to a broader community and invite further research to unravel the biological role of this curious nuclear structure in the honey bee central brain. KW - mushroom body calyx KW - synaptic complexes KW - bodies KW - insect KW - plasticity KW - insights KW - genome KW - model KW - proteins KW - methylation KW - biological techniques KW - cell biology KW - developmental biology KW - molecular biology KW - neuroscience Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260059 VL - 11 ER - TY - JOUR A1 - Krüger, Timothy A1 - Maus, Katharina A1 - Kreß, Verena A1 - Meyer-Natus, Elisabeth A1 - Engstler, Markus T1 - Single-cell motile behaviour of Trypanosoma brucei in thin-layered fluid collectives JF - The European Physical Journal E N2 - We describe a system for the analysis of an important unicellular eukaryotic flagellate in a confining and crowded environment. The parasite Trypanosoma brucei is arguably one of the most versatile microswimmers known. It has unique properties as a single microswimmer and shows remarkable adaptations (not only in motility, but prominently so), to its environment during a complex developmental cycle involving two different hosts. Specific life cycle stages show fascinating collective behaviour, as millions of cells can be forced to move together in extreme confinement. Our goal is to examine such motile behaviour directly in the context of the relevant environments. Therefore, for the first time, we analyse the motility behaviour of trypanosomes directly in a widely used assay, which aims to evaluate the parasites behaviour in collectives, in response to as yet unknown parameters. In a step towards understanding whether, or what type of, swarming behaviour of trypanosomes exists, we customised the assay for quantitative tracking analysis of motile behaviour on the single-cell level. We show that the migration speed of cell groups does not directly depend on single-cell velocity and that the system remains to be simplified further, before hypotheses about collective motility can be advanced. KW - Trypanosoma brucei KW - motile behaviour KW - fluid collectives Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-273022 SN - 1292-895X VL - 44 IS - 3 ER - TY - JOUR A1 - Stelzner, Kathrin A1 - Boyny, Aziza A1 - Hertlein, Tobias A1 - Sroka, Aneta A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kessie, David A1 - Mehling, Helene A1 - Potempa, Jan A1 - Ohlsen, Knut A1 - Fraunholz, Martin J. A1 - Rudel, Thomas T1 - Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A. KW - Staphylococcus aureus KW - Staphylococcal infection KW - host cells KW - HeLa cells KW - cytotoxicity KW - intracellular pathogens KW - apoptosis KW - epithelial cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263908 VL - 17 IS - 9 ER - TY - JOUR A1 - Letunic, Ivica A1 - Khedkar, Supriya A1 - Bork, Peer T1 - SMART: recent updates, new developments and status in 2020 JF - Nucleic Acids Research N2 - SMART (Simple Modular Architecture Research Tool) is a web resource (https://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 9 contains manually curatedmodels formore than 1300 protein domains, with a topical set of 68 new models added since our last update article (1). All the new models are for diverse recombinase families and subfamilies and as a set they provide a comprehensive overview of mobile element recombinases namely transposase, integrase, relaxase, resolvase, cas1 casposase and Xer like cellular recombinase. Further updates include the synchronization of the underlying protein databases with UniProt (2), Ensembl (3) and STRING (4), greatly increasing the total number of annotated domains and other protein features available in architecture analysis mode. Furthermore, SMART's vector-based protein display engine has been extended and updated to use the latest web technologies and the domain architecture analysis components have been optimized to handle the increased number of protein features available. KW - SMART KW - SMART version 9 KW - protein domains KW - protein domain architectures Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-363816 VL - 49 IS - D1 ER - TY - THES A1 - Kühl, Julia T1 - FAAP100, der FA/BRCA-Signalweg für genomische Stabilität und das DNA-Reparatur-Netzwerk T1 - FAAP100, the FA/BRCA pathway for genomic stability and the DNA repair network N2 - Die Fanconi-Anämie (FA) ist eine seltene, heterogene Erbkrankheit. Sie weist ein sehr variables klinisches Erscheinungsbild auf, das sich aus angeborenen Fehlbildungen, hämatologischen Funktionsstörungen, einem erhöhten Risiko für Tumorentwicklung und endokrinen Pathologien zusammensetzt. Die Erkrankung zählt zu den genomischen Instabilitätssyndromen, welche durch eine fehlerhafte DNA-Schadensreparatur gekennzeichnet sind. Bei der FA zeigt sich dies vor allem in einer charakteristischen Hypersensitivität gegenüber DNA-quervernetzenden Substanzen (z. B. Mitomycin C, Cisplatin). Der zelluläre FA-Phänotyp zeichnet sich durch eine erhöhte Chromosomenbrüchigkeit und einen Zellzyklusarrest in der G2-Phase aus. Diese Charakteristika sind bereits spontan vorhanden und werden durch Induktion mit DNA-quervernetzenden Substanzen verstärkt. Der Gendefekt ist dabei in einem der 22 bekannten FA-Gene (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U, -V, -W) oder in noch unbekannten FA-Genen zu finden. Die FA-Gendefekte werden mit Ausnahme von FANCR (dominant-negative de novo Mutationen) und FANCB (X-chromosomal) autosomal rezessiv vererbt. Die FA-Genprodukte bilden zusammen mit weiteren Proteinen den FA/BRCA-Signalweg. Das Schlüsselereignis dieses Signalwegs stellt die Monoubiquitinierung von FANCD2 und FANCI (ID2-Komplex) dar. Ausgehend davon lässt sich zwischen upstream- und downstream-gelegenen FA-Proteinen unterscheiden. Letztere sind direkt an der DNA-Schadensreparatur beteiligt. Zu den upstream-gelegenen Proteinen zählt der FA-Kernkomplex, der sich aus bekannten FA-Proteinen und aus FA-assoziierten-Proteinen (FAAPs) zusammensetzt und für die Monoubiquitinierung des ID2-Komplexes verantwortlich ist. Für FAAPs wurden bisher keine pathogenen humanen Mutationen beschrieben. Zu diesen Proteinen gehört auch FAAP100, das mit FANCB und FANCL innerhalb des FA-Kernkomplexes den Subkomplex LBP100 bildet. Durch die vorliegende Arbeit wurde eine nähere Charakterisierung dieses Proteins erreicht. In einer Amnion-Zelllinie konnte eine homozygote Missense-Mutation identifiziert werden. Der Fetus zeigte einen typischen FA-Phänotyp und auch seine Zellen wiesen charakteristische FA-Merkmale auf. Der zelluläre Phänotyp ließ sich durch FAAP100WT komplementieren, sodass die Pathogenität der Mutation bewiesen war. Unterstützend dazu wurden mithilfe des CRISPR/Cas9-Systems weitere FAAP100-defiziente Zelllinien generiert. Diese zeigten ebenfalls einen typischen FA-Phänotyp, welcher sich durch FAAP100WT komplementieren ließ. Die in vitro-Modelle dienten als Grundlage dafür, die Funktion des FA-Kernkomplexes im Allgemeinen und die des Subkomplexes LBP100 im Besonderen besser zu verstehen. Dabei kann nur durch intaktes FAAP100 das LBP100-Modul gebildet und dieses an die DNA-Schadensstelle transportiert werden. Dort leistet FAAP100 einen essentiellen Beitrag für den FANCD2-Monoubiquitinierungsprozess und somit für die Aktivierung der FA-abhängigen DNA-Schadensreparatur. Um die Funktion von FAAP100 auch in vivo zu untersuchen, wurde ein Faap100-/--Mausmodell generiert, das einen mit anderen FA-Mausmodellen vergleichbaren, relativ schweren FA-Phänotyp aufwies. Aufgrund der Ergebnisse lässt sich FAAP100 als neues FA-Gen klassifizieren. Zudem wurde die Rolle des Subkomplexes LBP100 innerhalb des FA-Kernkomplexes weiter aufgeklärt. Beides trägt zu einem besseren Verständnis des FA/BRCA-Signalweges bei. Ein weiterer Teil der vorliegenden Arbeit beschäftigt sich mit der Charakterisierung von FAAP100138, einer bisher nicht validierten Isoform von FAAP100. Durch dieses Protein konnte der zelluläre FA-Phänotyp von FAAP100-defizienten Zelllinien nicht komplementiert werden, jedoch wurden Hinweise auf einen dominant-negativen Effekt von FAAP100138 auf den FA/BRCA-Signalweg gefunden. Dies könnte zu der Erklärung beitragen, warum und wie der Signalweg, beispielsweise in bestimmtem Gewebearten, herunterreguliert wird. Zudem wäre eine Verwendung in der Krebstherapie denkbar. N2 - Fanconi Anemia (FA) is a rare heterogeneous hereditary disease. It shows a highly variable clinical presentation including congenital malformations, bone marrow failure and increased risk for cancer and endocrine pathologies. The disease is classified as one of the genomic instability disorders that are characterized by failure of DNA damage repair processes. FA shows a typical hypersensitivity toward DNA crosslinking agents (e.g. Mitomycin C, cisplatin). There is an increased rate of chromosomal breakage and cell cycle arrest in the G2 phase. These characteristics are present spontaneously and after incubation with DNA crosslinking agents. The genetic defect can be found in one of the 22 reported FA genes (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U, -V, -W) or yet unknown FA genes. FA gene defects are inherited in an autosomal recessive way with the exceptions of FANCR (dominant negative de novo mutations) and FANCB (X-linked). Together with other proteins, the FA gene products establish the FA/BRCA pathway. The key event of this pathway is the monoubiquitination of FANCD2 and FANCI (ID2 complex). From this point it is possible to differentiate between upstream and downstream FA proteins. The latter are directly involved in FA-dependent DNA repair processes. The upstream positioned FA proteins form the FA core complex that includes FA and FA-associated proteins (FAAPs). The FA core complex is responsible for the monoubiquitination of FANCD2 and FANCI. To date no pathogenic human mutations of the FAAPs have been described. Among these proteins is FAAP100 which together with FANCB and FANCL forms the subcomplex LBP100 within the FA core complex. In the present thesis a closer characterization of this protein has been achieved. In an amniotic cell line a homozygous missense mutation could be identified. The affected fetus displayed a typical FA phenotype and the cells also showed characteristics of FA. The cellular phenotype was complemented by FAAP100WT, thus proving the pathogenicity of the mutation. Supporting this result, additional FAAP100-deficient cell lines have been generated using the CRISPR/Cas9 system. These also exhibited a typical FA cellular phenotype which could be complemented by FAAP100WT. In vitro models served as a basis for better understanding the function of the FA core complex in general and of the LBP100 subcomplex in particular. Only in the presence of an intact FAAP100 the LBP100 module can be formed and transported to sites of DNA interstrand crosslinks. There, FAAP100 significantly contributes to the FANCD2 monoubiquitination process and thus to the activation of FA-dependent DNA damage repair. In order to also examine the function of FAAP100 in vivo, an Faap100-/- mouse model has been generated which shows a relatively severe FA phenotype comparable to other FA mouse models. Because of these results FAAP100 can be categorized as a new FA gene. Moreover, the role of the LBP100 subcomplex within the FA core complex was further elucidated and a better understanding of the FA/BRCA pathway was achieved. Another part of this thesis deals with the characterization of FAAP100138, a hitherto not validated isoform of FAAP100. The cellular FA phenotype of FAAP100-deficient cell lines could not be complemented by this isoform. However, there are clues pointing to a dominant negative effect of FAAP100138 on the FA/BRCA pathway. This finding could serve as a potential explanation of how and why the FA signaling pathway is downregulated in certain tissues. A therapeutic application for cancer of FAAP100138 appears possible. KW - Fanconi-Anämie KW - DNA-Reparatur KW - FAAP100 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171669 ER - TY - THES A1 - Kortmann, Mareike T1 - Biodiversity and recreation – Optimizing tourism and forest management in forests affected by bark beetles T1 - Biodiversität und Erholungsfunktionen – Optimierung von Tourismus- und Waldmanagement in Borkenkäferwäldern N2 - Forests are multi-functional system, which have to fulfil different objectives at the same time. The main functions include the production of wood, storage of carbon, the promotion of biological diversity and the provision of recreational space. Yet, global forests are affected by large and intense natural disturbances, like bark beetle infestations. While natural disturbances threaten wood production and are perceived as ‘catastrophe’ diminishing recreational value, biodiversity can benefit from the disturbance-induced changes in forest structures. This trade-off poses a dilemma to managers of bark beetle affected stands, particularly in protected areas designated to both nature conservation and recreation. Forest landscapes need a sustainable management concept aligning these different objectives. In order to support this goal with scientific knowledge, the aim of this work is to analyse ecological and social effects along a gradient of different disturbance severities. In this context, I studied the effects of a disturbance severity gradient on the diversity of different taxonomic groups including vascular plants, mosses, lichens, fungi, arthropods and birds in five national parks in Central Europe. To analyse the recreational value of the landscape I conducted visitor surveys in the same study areas in which the biodiversity surveys were performed. To analyse possible psychological or demographic effects on preferences for certain disturbance intensities, an additional online survey was carried out. N2 - Wälder müssen unterschiedliche Zielsetzungen zur gleichen Zeit erfüllen. Zu den wichtigsten Zielsetzungen zählen Produktion von Holz, Speicherung von CO2, die Förderung der biologischen Vielfalt und die Bereitstellung von Erholungsgebieten. Wälder sind jedoch global von intensiven natürlichen Störungen wie Borkenkäferbefall betroffen. Während natürliche Störungen die Holzproduktion bedrohen und von der Bevölkerung als „Katastrophe“ wahrgenommen werden, die den Erholungswert verringert, kann die biologische Vielfalt von den störungsbedingten Veränderungen der Waldstrukturen profitieren. Dieser Kompromiss stellt die Manager der von Borkenkäfern betroffenen Bestände vor ein Dilemma, insbesondere in Schutzgebieten, die sowohl dem Naturschutz als auch der Erholung gewidmet sind, und fordert ein nachhaltiges Bewirtschaftungskonzept, das diese unterschiedlichen Ziele in Einklang bringt. Um diese Vorhaben durch wissenschaftliche Erkenntnisse zu unterstützen, ist das Ziel dieser Arbeit, ökologische und soziale Effekte entlang eines Gradienten verschiedener Störungsintensitätsgrade zu analysieren. In diesem Zusammenhang wurden die Auswirkungen verschiedener Störungsintensitäten auf die Biodiversität verschiedener taxonomischer Gruppen, einschließlich Gefäßpflanzen, Moosen, Flechten, Pilzen, Arthropoden und Vögeln untersucht. Außerdem Befragungen von Nationalpark Besuchern durchgeführt, um den Erholungswert der Landschaft zu analysieren. Um mögliche psychologische oder demografische Auswirkungen auf Präferenzen für bestimmte Störungsintensitäten zu analysieren, wurde zudem eine Online-Umfrage durchgeführt. KW - Borkenkäfer KW - Nationalpark KW - Biodiversität KW - natural disturbance KW - nature conservation KW - national park KW - biodiversity KW - recreation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-240317 ER - TY - THES A1 - Kuhlemann, Alexander T1 - Bioorthogonal labeling of neuronal proteins using super-resolution fluorescence microscopy T1 - Bioorthogonale Markierung von neuronalen Proteinen mittels hochauflösender Fluoreszenzmikroskopie N2 - The synaptic cleft is of central importance for synaptic transmission, neuronal plasticity and memory and thus well studied in neurobiology. To target proteins of interest with high specificity and strong signal to noise conventional immunohistochemistry relies on the use of fluorescently labeled antibodies. However, investigations on synaptic receptors remain challenging due to the defined size of the synaptic cleft of ~20 nm between opposing pre- and postsynaptic membranes. At this limited space, antibodies bear unwanted side effects such as crosslinking, accessibility issues and a considerable linkage error between fluorophore and target of ~10 nm. With recent single molecule localization microscopy (SMLM) methods enabling localization precisions of a few nanometers, the demand for labeling approaches with minimal linkage error and reliable recognition of the target molecules rises. Within the scope of this work, different labeling techniques for super-resolution fluorescence microscopy were utilized allowing site-specific labeling of a single amino acid in synaptic proteins like kainate receptors (KARs), transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory proteins (TARPs), γ-aminobutyric acid type A receptors (GABA-ARs) and neuroligin 2 (NL2). The method exploits the incorporation of unnatural amino acids (uAAs) in the protein of interest using genetic code expansion (GCE) via amber suppression technology and subsequent labeling with tetrazine functionalized fluorophores. Implementing this technique, hard-to-target proteins such as KARs, TARPs and GABA-ARs could be labeled successfully, which could only be imaged insufficiently with conventional labeling approaches. Furthermore, functional studies involving electrophysiological characterization, as well as FRAP and FRET experiments validated that incorporation of uAAs maintains the native character of the targeted proteins. Next, the method was transferred into primary hippocampal neurons and in combination with super-resolution microscopy it was possible to resolve the nanoscale organization of γ2 and γ8 TARPs. Cluster analysis of dSTORM localization data verified synaptic accumulation of γ2, while γ8 was homogenously distributed along the neuron. Additionally, GCE and bioorthogonal labeling allowed visualization of clickable GABA-A receptors located at postsynaptic compartments in dissociated hippocampal neurons. Moreover, saturation experiments and FRET imaging of clickable multimeric receptors revealed successful binding of multiple tetrazine functionalized fluorophores to uAA-modified dimeric GABA-AR α2 subunits in close proximity (~5 nm). Further utilization of tetrazine-dyes via super-resolution microscopy methods such as dSTORM and click-ExM will provide insights to subunit arrangement in receptors in the future. This work investigated the nanoscale organization of synaptic proteins with minimal linkage error enabling new insights into receptor assembly, trafficking and recycling, as well as protein-protein interactions at synapses. Ultimately, bioorthogonal labeling can help to understand pathologies such as the limbic encephalitis associated with GABA-AR autoantibodies and is already in application for cancer therapies. N2 - Der synaptische Spalt ist von zentraler Bedeutung für die synaptische Reizweiterleitung, neuronale Plastizität und Gedächtnis und dadurch neurobiologisch sehr gut charakterisiert. Um Zielproteine mit hoher Spezifität und einem guten Signal-zu-Rauschen Verhältnis zu adressieren, wird konventionell auf Immunhistochemie mittels Fluoreszenzfarbstoff-markierter Antikörper zurückgegriffen. Untersuchungen synaptischer Rezeptoren bleiben dabei jedoch aufgrund der limitierten Zugänglichkeit des synaptischen Spalts mit einem Abstand von ~20 nm zwischen gegenüberliegenden pre- und postsynaptischen Membranen herausfordernd. Speziell in einem räumlich begrenzten Umfeld können bei der Verwendung von Antikörpern unerwünschte Artefakte auftreten, die durch Kreuzverlinkung, eine verminderte Zugänglichkeit und einen erheblichen Markierungsabstand zwischen Fluorophor und Probe von ~10 nm entstehen. Aktuelle Verfahren der Einzelmolekül-Lokalisations-Mikroskopie (SMLM), die eine Lokalisationsgenauigkeit von wenigen Nanometern ermöglichen, erhöhen die Nachfrage an Markierungsstrategien mit minimalem Markierungsabstand und zuverlässiger Erkennung der Zielstruktur. Im Rahmen dieser Arbeit wurden daher verschiedene Markierungsmethoden für die hochauflösende Fluoreszenz-Mikroskopie erprobt. Dies ermöglichte die ortsspezifische Markierung einer einzigen Aminosäure in synaptischen Proteinen wie Kainat-Rezeptoren (KARs), Transmembran-α-Amino-3-hydroxy-5-methyl-4-isoxazol-Propionsäure-Rezeptor regulierenden Proteinen (TARPs), γ-Aminobuttersäure-Typ-A-Rezeptoren (GABA-ARs) oder Neuroligin 2 (NL2). Die angewandte Methodik nutzt den Einbau von unnatürlichen Aminosäuren (uAAs) in das Zielprotein mittels Erweiterung des genetischen Codes (GCE) durch Unterdrückung des Amber-Stop-Codons. Durch Anwendung dieser Strategie gelang es, schwer adressierbare Proteine wie KARs, TARPs und GABA-ARs, welche zuvor mittels konventioneller Markierungsversuche nur unzureichend abgebildet werden konnten, erfolgreich zu markieren. Funktionelle Studien wie elektrophysiologische Charakterisierungen, aber auch FRAP und FRET Experimente zeigten, dass dabei der native Zustand der Zielproteine auch nach dem Einbau von uAAs erhalten bleibt. Schließlich wurde die Methode in primäre hippocampale Neuronen überführt und in Kombination mit hochauflösender Mikroskopie konnte die Organisation von γ2 und γ8 TARPs im Nanobereich aufgelöst werden. Eine Cluster-Analyse von dSTORM Lokalisationsdaten bestätigte die Anreicherung von γ2 in Synapsen, während γ8 homogen entlang des Neurons verteilt vorliegt. Die Erweiterung des genetischen Codes in Kombination mit bioorthogonaler Markierung erlaubte zusätzlich die Visualisierung von clickbaren GABA-A Rezeptoren in Postsynapsen von dissoziierten hippocampalen Neuronen. Außerdem zeigten Saturierungs-Experimente und FRET-Bildgebung die erfolgreiche Bindung von mehreren Tetrazin-gekoppelten Fluorophoren an uAA-modifizierten, dimerischen GABA-AR α2-Untereinheiten in geringem Abstand (~5 nm). Auf der Basis dieser Resultate werden zukünftig hochauflösende mikroskopische Verfahren, wie dSTORM und click-ExM, in Kombination mit Tetrazin-Farbstoffen die Visualisierung von multimerischen Rezeptoren ermöglichen. Im Rahmen dieser Arbeit konnte die Organisation von synaptischen Proteinen mit minimalem Markierungsabstand im Nanobereich untersucht werden und dadurch neue Einsichten in Rezeptor-Zusammenbau, -Bewegungen und -Wiederverwertung, aber auch Protein-Protein Interaktionen in Synapsen gewonnen werden. Die Weiterentwicklung bioorthogonaler Markierungsstrategien kann in Zukunft dazu beitragen Krankheiten, wie die Limbische Enzephalitis, welche mit GABA-AR Autoantikörpern in Verbindung steht, besser zu verstehen und findet zudem bereits heute Anwendung in Krebstherapien. KW - microscopy KW - bioorthogonal labeling KW - super-resolution fluorescence microscopy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-243731 ER - TY - THES A1 - Bötzl, Fabian Alexander T1 - The influence of crop management and adjacent agri-environmental scheme type on natural pest control in differently structured landscapes T1 - Der Effekt von Feldkultur und angrenzenden Agrarumweltmaßnahmen auf natürliche Schädlingskontrolle in unterschiedlich strukturierten Landschaften N2 - Summary Chapters I & II: General Introduction & General Methods Agriculture is confronted with a rampant loss of biodiversity potentially eroding ecosystem service potentials and adding up to other stressors like climate change or the consequences of land-use change and intensive management. To counter this ‘biodiversity crisis’, agri-environment schemes (AES) have been introduced as part of ecological intensification efforts. These AES combine special management regimes with the establishment of tailored habitats to create refuges for biodiversity in agricultural landscapes and thus ensure biodiversity mediated ecosystem services such as pest control. However, little is known about how well different AES habitats fulfil this purpose and whether they benefit ecosystem services in adjacent crop fields. Here I investigated how effective different AES habitats are for restoring biodiversity in different agricultural landscapes (Chapter V) and whether they benefit natural pest control in adjacent oilseed rape (Chapter VI) and winter cereal fields (Chapter VII). I recorded biodiversity and pest control potentials using a variety of different methods (Chapters II, V, VI & VII). Moreover, I validated the methodology I used to assess predator assemblages and predation rates (Chapters III & IV). Chapter III: How to record ground dwelling predators? Testing methodology is critical as it ensures scientific standards and trustworthy results. Pitfall traps are widely used to record ground dwelling predators, but little is known about how different trap types affect catches. I compared different types of pitfall traps that had been used in previous studies in respect to resulting carabid beetle assemblages. While barrier traps collected more species and deliver more complete species inventories, conventional simple pitfall traps provide reliable results with comparatively little handling effort. Placing several simple pitfall traps in the field can compensate the difference while still saving handling effort.   Chapter IV: How to record predation rates? A plethora of methods has been proposed and used for recording predation rates, but these have rarely been validated before use. I assessed whether a novel approach to record predation, the use of sentinel prey cards with glued on aphids, delivers realistic results. I compared different sampling efforts and showed that obtained predation rates were similar and could be linked to predator (carabid beetle) densities and body-sizes (a proxy often used for food intake rates). Thus, the method delivers reliable and meaningful predation rates. Chapter V: Do AES habitats benefit multi-taxa biodiversity? The main goal of AES is the conservation of biodiversity in agricultural landscapes. I investigated how effectively AES habitats with different temporal continuity fulfil this goal in differently structured landscapes. The different AES habitats investigated had variable effects on local biodiversity. Temporal continuity of AES habitats was the most important predictor with older, more temporally continuous habitats harbouring higher overall biodiversity and different species assemblages in most taxonomic groups than younger AES habitats. Results however varied among taxonomic groups and natural enemies were equally supported by younger habitats. Semi-natural habitats in the surrounding landscape and AES habitat size were of minor importance for local biodiversity and had limited effects. This stresses that newly established AES habitats alone cannot restore farmland biodiversity. Both AES habitats as well as more continuous semi-natural habitats synergistically increase overall biodiversity in agricultural landscapes. Chapter VI: The effects of AES habitats on predators in adjacent oilseed rape fields Apart from biodiversity conservation, ensuring ecosystem service delivery in agricultural landscapes is a crucial goal of AES. I therefore investigated the effects of adjacent AES habitats on ground dwelling predator assemblages in oilseed rape fields. I found clear distance decay effects from the field edges into the field centres on both richness and densities of ground dwelling predators. Direct effects of adjacent AES habitats on assemblages in oilseed rape fields however were limited and only visible in functional traits of carabid beetle assemblages. Adjacent AES habitats doubled the proportion of predatory carabid beetles indicating a beneficial role for pest control. My results show that pest control potentials are largest close to the field edges and beneficial effects are comparably short ranged. Chapter VII: The effects of AES habitats on pest control in adjacent cereal fields Whether distance functions and potential effects of AES habitats are universal across crops is unknown. Therefore, I assessed distance functions of predators, pests, predation rates and yields after crop rotation in winter cereals using the same study design as in the previous year. Resulting distance functions were not uniform and differed from those found in oilseed rape in the previous year, indicating that the interactions between certain adjacent habitats vary with habitat and crop types. Distance functions of cereal-leaf beetles (important cereal pests) and parasitoid wasps were moreover modulated by semi-natural habitat proportion in the surrounding landscapes. Field edges buffered assemblage changes in carabid beetle assemblages over crop rotation confirming their important function as refuges for natural enemies. My results emphasize the beneficial role of field edges for pest control potentials. These findings back the calls for smaller field sizes and more diverse, more heterogeneously structured agricultural landscapes. Chapter VIII: General Discussion Countering biodiversity loss and ensuring ecosystem service provision in agricultural landscapes is intricate and requires strategic planning and restructuring of these landscapes. I showed that agricultural landscapes could benefit maximally from (i) a mixture of AES habitats and semi-natural habitats to support high levels of overall biodiversity and from (ii) smaller continuously managed agricultural areas (i.e. smaller field sizes or the insertion of AES elements within large fields) to maximize natural pest control potentials in crop fields. I propose a mosaic of younger AES habitats and semi-natural habitats to support ecosystem service providers and increase edge density for ecosystem service spillover into adjacent crops. The optimal extent and density of this network as well as the location in which AES and semi-natural habitats interact most beneficially with adjacent crops need further investigation. My results provide a further step towards more sustainable agricultural landscapes that simultaneously allow biodiversity to persist and maintain agricultural production under the framework of ecological intensification. N2 - Zusammenfassung Kapitel I & II: Allgemeine Einleitung & Allgemeine Methodik Die Landwirtschaft sieht sich einem gravierenden Verlust an biologischer Vielfalt gegenüber, der möglicherweise Ökosystemdienstleistungen erodiert und zusätzlich zu anderen Stressoren wirkt, wie etwa dem globalen Klimawandel oder den Folgen veränderter Landnutzung und intensiven Managements. Um dieser ‚Biodiversitätskrise‘ entgegen zu wirken wurden im Rahmen der ökologischen Intensivierung Agrarumweltmaßnahmen (AES) eingeführt. Diese AES verbinden spezielle Managementregime mit der Schaffung designter Habitate, die als Refugien für Biodiversität in Agrarlandschaften deinen und dadurch Ökosystemdienstleistungen, die auf Biodiversität beruhen, wie natürliche Schädlingskontrolle, sicherstellen sollen. Wie gut verschiedene AES jedoch diese Ziele erfüllen und ob Ökosystemdienstleistungen in angrenzenden Feldern tatsächlich davon profitieren, ist weitgehend unbekannt. In meiner Doktorarbeit untersuche ich wie effektiv verschiedene AES Habitate darin sind, Biodiversität in unterschiedlichen Agrarlandschaften wieder her zu stellen (Kapitel V) und ob diese natürliche Schädlingskontrolle in angrenzenden Rapsfeldern (Kapitel VI) und Wintergetreidefeldern (Kapitel VII) von diesen Habitaten profitiert. Biodiversität und Potentiale natürlicher Schädlingskontrolle wurden mit diversen unterschiedlichen Methoden erfasst (Kapitel II, V, VI & VII). Zusätzlich habe ich Methoden, die ich zur Erfassung von Räubergesellschaften und Prädationsraten verwendet habe, validiert (Kapitel III & IV). Kapitel III: Wie erfasst man bodenaktive Räuber? Das Testen von Methoden ist essenziell, da es wissenschaftliche Standards und vertrauenswürdige Ergebnisse sicherstellt. Bodenfallen werden häufig verwendet, um bodenaktive Prädatoren zu erfassen, aber wie verschiedene Bodenfallentypen das Fangergebnis beeinflussen ist weitgehend unbekannt. Ich habe verschiedene, in früheren Studien verwendete, Bodenfallentypen hinsichtlich der resultierenden Laufkäfergesellschaften verglichen. Während Fallen mit Leitschienen die meisten Arten fingen und dadurch die vollständigsten Artenlisten ergaben, lieferten einfache Bodenfallen verlässliche Ergebnisse bei vergleichsweise geringem Aufwand. Das Platzieren einiger einfacher Bodenfallen kann bei immer noch geringerem Aufwand die Unterschiede kompensieren. Kapitel IV: Wie erfasst man Prädationsraten? Eine Fülle verschiedener Methoden zur Erfassung von Prädationsraten wurde vorgeschlagen und verwendet, jedoch wurden diese meist nicht validiert, bevor sie verwendet wurden. Ich habe getestet ob eine neuartige Methode zur Erfassung von Prädationsraten, die Verwendung von Prädationskarten mit aufgeklebten Blattläusen, realistische Resultate liefert. Dazu wurden verschiedene Karten mit unterschiedlichem Aufwand getestet. Die resultierenden Prädationsraten waren vergleichbar und durch Räuber- (Laufkäfer-) Dichten sowie deren mittlere Körpergröße (ein oft genutzter Indikator für Nahrungsaufnahmeraten) erklärt werden konnten. Daher liefert diese Methode verlässliche und sinnvolle Prädationsraten. Kapitel V: Profitiert multi-Taxa Biodiversität von AES? Das Hauptziel von AES ist der Erhalt der Biodiversität in Agrarlandschaften. Ich habe untersucht, wie effektiv AES Habitate mit verschiedener zeitlicher Kontinuität dieses Ziel in unterschiedlich strukturierten Landschaften erfüllen. Die verschiedenen AES Habitate hatten variierende Effekte auf die lokale Biodiversität. Zeitliche Kontinuität der AES Habitate war der wichtigste Einfluss da ältere, kontinuierlichere Habitate eine höhere Gesamtbiodiversität und in den meisten taxonomischen Gruppen andere Artengemeinschaften beherbergten als jüngere AES Habitate. Die Ergebnisse variierten jedoch zwischen den taxonomischen Gruppen und natürliche Feinde von Agrarschädlingen wurden auch durch jüngere AES Habitate gleichwertig unterstützt. Halbnatürliche Habitate in der Landschaft sowie die Größe des AES Habitats waren von geringerer Bedeutung für die lokale Biodiversität und hatten lediglich begrenzte Effekte. Diese Ergebnisse betonen, dass neu angelegte AES Habitate allein die Biodiversität in der Agrarlandschaft nicht wiederherstellen können. AES Habitate wirken synergistisch zusammen mit kontinuierlicheren halbnatürlichen Habitaten und sichern mit diesen ein Maximum an biologischer Vielfalt in Agrarlandschaften Kapitel VI: Die Effekte von AES Habitaten auf Räuber in angrenzenden Rapsfeldern Neben dem Erhalt der Artenvielfalt ist das Sicherstellen von Ökosystemdienstleistungen in Agrarlandschaften ein essenzielles Ziel von AES. Ich untersuchte daher die Effekte angrenzender AES Habitate auf bodenaktive Prädatoren in Rapsfeldern. Für die Artenvielfalt als auch für die Dichten von bodenaktiven Prädatoren zeigten sich klare Distanzfunktionen von den Feldrändern abnehmend zur Feldmitte. Direkte Effekte angrenzender AES auf die Räubergesellschaften in Rapsfeldern waren hingegen limitiert und nur auf der Ebene der funktionellen Merkmale von Laufkäfergesellschaften festzustellen. Angrenzende AES Habitate verdoppelten den Anteil räuberischer Laufkäfer in den Gesellschaften, was auf einen positiven Effekt auf natürliche Schädlingsbekämpfung schließen lässt. Meine Ergebnisse deuten darauf hin, dass Potentiale natürlicher Schädlingsbekämpfung nahe den Feldrändern am größten sind und nicht relativ weit ins Feld hinein reichen. Kapitel VII: Die Effekte von AES Habitaten auf natürliche Schädlingskontrolle in angrenzenden Getreidefeldern Es ist allerdings noch gänzlich unbekannt, ob Distanzfunktionen und potenzielle Effekte angrenzender AES Habitate universell auf andere Feldfrüchte übertragbar sind. Ich habe daher im gleichen Studiendesign wie im vorangegangenen Jahr Distanzfunktionen von natürlichen Feinden, Schädlingen, Prädationsraten und Erträgen nach dem Fruchtwechsel in Wintergetreide erfasst. Die gefundenen Distanzfunktionen waren verschieden und unterschieden sich von den im Vorjahr im Raps erfassten Distanzfunktionen, was darauf schließen lässt, dass die Interaktion zwischen verschiedenen Feldfrüchten und Nachbarhabitaten variieren. Distanzfunktionen von Getreidehähnchen (wichtigen Getreideschädlingen) und parasitoiden Wespen waren zusätzlich durch den Anteil halbnatürlicher Habitate in der Landschaft moduliert. Feldränder pufferten die Veränderungen in Laufkäfergesellschaften über den Fruchtwechsel ab, was deren wichtige Funktion als Refugialhabitate für natürliche Schädlingsbekämpfer verdeutlicht. Meine Ergebnisse betonen die Rolle von Feldrändern für die natürliche Schädlingsbekämpfung. Die Ergebnisse stärken die Forderung nach kleineren Feldgrößen und diverseren, heterogener strukturierten Agrarlandschaften. Kapitel VIII: Allgemeine Diskussion Der Kampf gegen den Verlust der biologischen Vielfalt und das Sicherstellen von Ökosystemdienstleistungen in Agrarlandschaften ist komplex und erfordert ein strategisches Planen und eine Transformation dieser Landschaften. Ich habe gezeigt, dass Agrarlandschaften von (i) einer Mischung aus AES Habitaten und halbnatürlichen Habitaten, die zusammen ein große Artenvielfalt unterstützen, und von (ii) einer geringeren kontinuierlich bewirtschafteten Agrarfläche (d.h. kleineren Feldgrößen oder dem Einfügen von AES Habitaten in bestehende große Felder) um natürliche Schädlingskontrolle zu maximieren, profitieren würden. Ich schlage vor ein Mosaik aus jüngeren AES Habitaten und halbnatürlichen Habitaten zu schaffen, um Ökosystemdienstleister zu unterstützen und das Netzwerk an Feldrändern zu vergrößern wodurch Ökosystemdienstleistungen in angrenzenden Feldkulturen maximiert werden könnten. Um die optimale Ausdehnung und Dichte dieses Netzwerks wie auch die optimale Platzierung, in der AES und halbnatürliche Habitate die größtmöglichen Effekte auf angrenzende Feldkulturen haben, zu klären, bedarf es weiterer Forschung. Meine Ergebnisse liefern einen weiteren Schritt hin zu nachhaltigeren Agrarlandschaften die im Rahmen der ökologischen Intensivierung gleichzeitig sowohl ein Fortbestehen der Biodiversität als auch landwirtschaftliche Produktion erlauben. KW - Ökologie KW - Ecology KW - Natural pest control KW - Biodiversity conservation KW - Ecosystem services KW - Carabid beetles Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241400 ER - TY - THES A1 - Vellmer, Tim T1 - New insights into the histone variant H2A.Z incorporation pathway in \(Trypanosoma\) \(brucei\) T1 - Neue Erkenntnisse zum Einbau der Histonvariante H2A.Z in \(Trypanosoma\) \(brucei\) N2 - The histone variant H2A.Z is a key player in transcription regulation in eukaryotes. Histone acetylations by the NuA4/TIP60 complex are required to enable proper incorporation of the histone variant and to promote the recruitment of other complexes and proteins required for transcription initiation. The second key player in H2A.Z-mediated transcription is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant. By the time this project started little was known about H2A.Z in the unicellular parasite Trypanosoma brucei. Like in other eukaryotes H2A.Z was exclusively found in the transcription start sites of the polycistronic transcription units where it keeps the chromatin in an open conformation to enable RNA-polymerase II-mediated transcription. Previous studies showed the variant colocalizing with an acetylation of lysine on histone H4 and a methylation of lysine 4 on histone H3. Data indicated that HAT2 is linked to H2A.Z since it is required for acetylation of lyinse 10 on histone H4. A SWR1-like complex and a complex homologous to the NuA4/TIP60 could not be identified yet. This study aimed at identifying a SWR1-like remodelling complex in T. brucei and at identifying a protein complex orthologous to NuA4/TIP60 as well as at answering the question whether HAT2 is part of this complex or not. To this end, I performed multiple mass spectrometry-coupled co-Immunoprecipitation assays with potential subunits of a SWR1 complex, HAT2 and a putative homolog of a NuA4/TIP60 subunit. In the course of these experiments, I was able to identify the TbSWR1 complex. Subsequent cell fractionation and chromatin immunoprecipitation-coupled sequencing analysis experiments confirmed, that this complex is responsible for the incorporation of the histone variant H2A.Z in T. brucei. In addition to this chromatin remodelling complex, I was also able to identify two histone acetyltransferase complexes assembled around HAT1 and HAT2. In the course of my study data were published by the research group of Nicolai Siegel that identified the histone acetyltransferase HAT2 as being responsible for histone H4 acetylation, in preparation to promote H2A.Z incorporation. The data also indicated that HAT1 is responsible for acetylation of H2A.Z. According to the literature, this acetylation is required for proper transcription initiation. Experimental data generated in this study indicated, that H2A.Z and therefore TbSWR1 is involved in the DNA double strand break response of T. brucei. The identification of the specific complex composition of all three complexes provided some hints about how they could interact with each other in the course of transcription regulation and the DNA double strand break response. A proximity labelling approach performed with one of the subunits of the TbSWR1 complex identified multiple transcription factors, PTM writers and proteins potentially involved in chromatin maintenance. Overall, this work will provide some interesting insights about the composition of the complexes involved in H2A.Z incorporation in T. brucei. Furthermore, it is providing valuable information to set up experiments that could shed some light on RNA-polymerase II-mediated transcription and chromatin remodelling in T. brucei in particular and Kinetoplastids in general. N2 - Die Histonvariante H2A.Z ist ein Schlüsselelement bei der Transkriptionsregulation in Eukaryoten. Histonacetylierungen die vom NuA4/Tip60 Komplex prozessiert werden, sind für den korrekten Einbau der Variante unerlässlich. Darüber hinaus erlauben diese posttranslationellen Modifikationen die Rekrutierung weiterer Proteine und Komplexe die für die Transkription notwendig sind. Ein weiteres Schlüsselelement der mittels H2A.Z regulierten Transkription ist der Komplex zur Umstrukturierung des Chromatins SWR1, welcher das kanonische Histon H2A gegen seine Variante austauscht. Zu Beginn dieses Projektes war der Wissenstand bezüglich der Histonvariante H2A.Z in dem einzelligen Parasiten Trypanosoma brucei limitiert. Wie in anderen eukaryotischen Organismen wurde die Variante ausschließlich an den Startpunkten der polyzistronischen Transkriptionseinheiten gefunden, an denen es für die Öffnung des Chromatins verantwortlich ist und so die Transkription mittels RNAPolymerase II ermöglicht. Vorangegangene Studien konnten zeigen, dass die Variante mit einer Acetylierung des Lysins 10 im Histon H4 und einer Methylierung des Lysins 4 im Histon H3 co-lokalisiert. Einige Daten lieferten den Hinwies, dass die Histon-Acetyltransferase HAT2 mit H2A.Z in Zusammenhang steht, da diese die Acetylierung des Lysins 10 im Hinston H4 prozessiert. Komplexe die in ihrer Funktion dem SWR1 oder dem NuA4/TIP60 Komplex entsprechen, konnten bisher noch nicht gefunden werden. Die vorliegende Arbeit zielt darauf ab Komplexe zu identifizieren, die in ihrer Funktion dem SWR1 sowie dem NuA4/TIP60 Komplex entsprechen. Zudem soll die Frage geklärt werden ob HAT2 Teil eines möglichen NuA4/TIP60 Komplexes ist. In diesem Zusammenhang habe ich mehrere Massenspektrometrie gekoppelte Co-Immunopräzipitationen mit potenziellen Untereinheiten eines SWR1 Komplexes sowie HAT2 und einem Protein welches otholog zu einer NuA4/TIP60 Untereinheit ist, durchgeführt. Im Verlauf dieser Experimente konnte der SWR1 Komplex in T. brucei (TbSWR1) identifiziert werden. Anschließende Zellfraktionierungen sowie Chromatin Immunopräzipitationen gekoppelte Sequenzanalysen konnten bestätigen, dass der identifizierte Komplex für den Einbau der Histonvariante H2A.Z zuständig ist. Darüber hinaus konnten neben diesem Komplex noch zwei weitere Komplexe identifiziert werden, die jeweils die Histonacetyltransferasen HAT1 und HAT2 als Kernkomponenten enthalten. Im Verlauf meiner Arbeit wurden von der Arbeitsgruppe von Nicolai Siegel Daten publiziert die zeigten, dass die Histonacetyltransferase HAT2, in Vorbereitung auf den Einbau von H2A.Z, für die Acetylierung des Histons H4 verantwortlich ist. Im Gegenzug ist HAT1 für die Acetylierung von H2A.Z notwendig, welche wiederum für die korrekte Initiation der Transkription benötigt wird. Damit entspricht die Funktion der Acetylierung von H2A.Z in T. brucei der in der Literatur beschriebenen Funktion. Experimentelle Daten die im Verlauf dieser Arbeit generiert wurden, lieferten einen Hinweis darauf, dass H2A.Z auch an der Reparatur von DNS Doppelstrangbrüchen beteiligt ist. Die Aufschlüsselung der spezifischen Zusammensetzung aller drei Komplexe gab einige Hinweise darauf, wie sie sowohl während der Transkriptionsregulation als auch der Reparatur von DNS Doppelstrangbrüchen miteinander interagieren. Im Zuge einer molekularen Umgebungskartierung, die mit einer der Untereinheiten des TbSWR1 Komplexes durchgeführt wurde, konnten mehrere Transkriptionsfaktoren und Enzyme zur Histonmodifizierung identifiziert werden. Dabei wurden auch einige Proteine identifiziert, welche möglicherweise mit der Umformung des Chromatins in Zusammenhang stehen. Abschließend ist festzuhalten, dass diese Arbeit einige äußerst interessante Einsichten über die Zusammensetzung der Komplexe, die am H2A.Z Einbau in T. brucei beteiligt sind, liefern konnte. Darüber hinaus stellt sie einige wertvolle Informationen zur Verfügung. Diese könnten zur gezielten Planung von Experimenten genutzt werden, um mehr über RNA-Polymerase II vermittelte Transkription und Chromatin Umstrukturierung in T. brucei im speziellen und in Kinetoplastiden im Allgemeinen zu erfahren. KW - Chromatinremodelling KW - Histone KW - Transkription KW - Chromatinremodeling KW - Histones KW - Variants KW - Complexes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257960 ER - TY - THES A1 - Lasway, Julius Vincent T1 - Impact of human land use on bee diversity and plant-pollinator interactions in Tanzania savannah ecosystems T1 - Auswirkungen der Landnutzung durch den Menschen auf die Bienendiversität und die Wechselwirkungen zwischen Pflanze und Bestäuber in den Savannenökosystemen Tansanias N2 - One of the pronounced global challenges facing ecologists is how to feed the current growing human population while sustaining biodiversity and ecosystem services. To shed light on this, I investigated the impact of human land use on bee diversity and plant-pollinator interactions in Tanzania Savannah ecosystems. The thesis comprises the following chapters: Chapter I: General Introduction This chapter provides the background information including the study objectives and hypotheses. It highlights the ecological importance of bees and the main threats facing bee pollinators with a focus on two land-use practices namely livestock grazing and agriculture. It also highlights the diversity and global distribution of bees. It further introduces the tropical savannah ecosystem, its climate, and vegetation characteristics and explains spectacular megafauna species of the system that form centers of wildlife tourism and inadequacy knowledge on pollinators diversity of the system. Finally, this chapter describes the study methodology including, the description of the study area, study design, and data collection. Chapter II: Positive effects of low livestock grazing intensity on East African bee assemblages mediated by increases in floral resources The impact of livestock grazing intensity on bee assemblage has been subjected to research over decades. Moreover, most of these studies have been conducted in temperate Europe and America leaving the huge tropical savannah of East Africa less studied. Using sweep netting and pan traps, a total of 183 species (from 2,691 individuals) representing 55 genera and five families were collected from 24 study sites representing three levels of livestock grazing intensity in savannah ecosystem of northern Tanzania. Results have shown that moderate livestock grazing slightly increased bee species richness. However, high livestock grazing intensity led to a strong decline. Besides, results revealed a unimodal distribution pattern of bee species richness and mean annual temperature. It was also found that the effect of livestock grazing and environmental temperature on bee species richness was mediated by a positive effect of moderate grazing on floral resource richness. The study, therefore, reveals that bee communities of the African savannah zone may benefit from low levels of livestock grazing as this favors the growth of flowering plant species. A high level of livestock grazing intensity will cause significant species losses, an effect that may increase with climatic warming. Chapter III: Agricultural intensification with seasonal fallow land promotes high bee diversity in Afrotropical drylands This study investigated the impact of local agriculture intensification on bee diversity in the Afro tropical drylands of northern Tanzania. Using sweep netting and pan traps, a total of 219 species (from 3,428 individuals) representing 58 genera and six families were collected from 24 study sites (distributed from 702 to 1708 m. asl) representing three levels of agriculture intensity spanning an extensive gradient of mean annual temperature. Results showed that bee species richness increased with agricultural intensity and with increasing temperature. However, the effects of agriculture intensity and temperature on bee species richness were mediated by the positive effects of agriculture and temperature on floral resource richness used by bee pollinators. Moreover, results showed that variation of bee body sizes increases with agricultural intensification, “that effect”, however, diminished in environments with higher temperatures. This study reveals that bee assemblages in Afrotropical drylands benefit from agriculture intensification in the way it is currently practiced. Further intensification, including year-round irrigated crop monocultures and extensive use of agrochemicals, is likely to exert a negative impact on bee diversity and pollination services, as reported in temperate regions. Moreover, several bee species were restricted to natural savannah habitats. Therefore, to conserve bee communities in Afro tropical drylands and guarantee pollination services, a mixture of savannah and agriculture, with long periods of fallow land should be maintained. Chapter IV: Impact of land use intensification and local features on plants and pollinators in Sub-Saharan smallholder farms For the first time in the region, this study explores the impact of land-use intensification on plants and pollinators in Sub-Saharan smallholder farms. The study complemented field surveys of bees with a modern DNA metabarcoding approach to characterize the foraged plants and thus built networks describing plant-pollinator interactions at the individual insect level. This information was coupled with quantitative traits of landscape composition and floral availability surrounding each farm. The study found that pollinator richness decreased with increasing impervious and agricultural cover in the landscape, whereas the flower density at each farm correlated with pollinator richness. The intensification of agricultural land use and urbanization correlated with a higher foraging niche overlap among pollinators due to the convergence of individuals' flower-visiting strategies. Furthermore, within farms, the higher availability of floral resources drove lower niche overlap among individuals, greater abundance of flower visitors shaped higher generalization at the networks level (H2I), possibly due to increased competition. These mechanistic understandings leading to individuals’ foraging niche overlap and generalism at the network level, could imply stability of interactions and the pollination ecosystem service. The integrative survey proved that plant-pollinator systems are largely affected by land use intensification and by local factors in smallholder farms of Sub-Saharan Africa. Thus, policies promoting nature-based solutions, among which the introduction of more pollinator-friendly practices by smallholder farmers, could be effective in mitigating the intensification of both urban and rural landscapes in this region, as well as in similar Sub-Saharan contexts. Chapter V: A synopsis of the Bee occurrence data of northern Tanzania This study represents a synopsis of the bee occurrence data of northern Tanzania obtained from a survey in the Kilimanjaro, Arusha, and Manyara regions. Bees were sampled using two standardized methods, sweep netting and colored pan traps. The study summed up 953 species occurrences of 45 species belonging to 20 genera and four families (Halictidae, Apidae, Megachilidae, and andrenidae) A. This study serves as the baseline information in understanding the diversity and distribution of bees in the northern parts of the country. Understanding the richness and distribution of bees is a critical step in devising robust conservation and monitoring strategies for their populations since limited taxonomic information of the existing and unidentified bee species makes their conservation haphazard. Chapter VI: General discussion In general, findings obtained in these studies suggest that livestock grazing and agriculture intensification affects bee assemblages and floral resources used by bee pollinators. Results have shown that moderate livestock grazing intensity may be important in preserving bee diversity. However, high level of livestock grazing intensity may result in a strong decline in bee species richness and abundance. Moreover, findings indicate that agriculture intensification with seasonal fallow lands supports high floral resource richness promoting high bee diversity in Afrotropical drylands. Nonetheless, natural savannahs were found to contain unique bee species. Therefore, agriculture intensification with seasonal fallow should go in hand with conserving remnant savannah in the landscapes to increase bee diversity and ensure pollination services. Likewise, findings suggest that increasing urbanization and agriculture cover at the landscape level reduce plant and pollinator biodiversity with negative impacts on their complex interactions with plants. Conversely, local scale availability of floral resources has shown the positive effects in buffering pollinators decline and mitigating all detrimental effects induced by land-use intensification. Moreover, findings suggest that the impact of human land use (livestock grazing and agriculture) do not act in isolation but synergistically interacts with climatic factors such as mean annual temperature, MAT. The impact of MAT on bee species richness in grazing gradient showed to be more detrimental than in agriculture habitats. This could probably be explained by the remaining vegetation cover following anthropogenic disturbance. Meaning that the remaining vegetation cover in the agricultural gradient probably absorbs the solar radiations hence reducing detrimental effect of mean annual temperature on bee species richness. This one is not the case in grazing gradient since the impact of livestock grazing is severe, leaving the bare land with no vegetation cover. Finally, our findings conclude that understanding the interplay of multiple anthropogenic activities and their interaction with MAT as a consequence of ongoing climate change is necessary for mitigating their potential consequences on bee assemblages and the provision of ecosystem services. Morever, future increases in livestock grazing and agriculture intensification (including year-round crop irrigated monocultures and excessive use of agrochemicals) may lead to undesirable consequences such as species loss and impair provision of pollination services. N2 - Eine der größten globalen Herausforderungen für Ökologen ist die Beantwortung der Frage, wie die wachsende menschliche Bevölkerung ernährt und gleichzeitig die biologische Vielfalt und die Ökosystemleistungen erhalten werden können. Um dies zu beleuchten, habe ich die Auswirkungen der menschlichen Landnutzung auf die Bienenvielfalt und die Wechselwirkungen zwischen Pflanzen und Bestäubern in den Ökosystemen der Tansania-Savanne untersucht. Die Arbeit umfasst die folgenden Kapitel: Kapitel I: Allgemeine Einführung Dieses Kapitel enthält die Hintergrundinformationen, einschließlich der Studienziele und Hypothesen. Es hebt die ökologische Bedeutung von Bienen und die Hauptbedrohungen für Bienenbestäuber hervor, wobei der Schwerpunkt auf zwei Landnutzungspraktiken liegt, nämlich Viehbeweidung und Landwirtschaft. Außerdem werden die Vielfalt und die globale Verbreitung der Bienen herausgearbeitet. Des Weiteren werden das Ökosystem der tropischen Savanne, sein Klima und seine Vegetationscharakteristika vorgestellt und die spektakulären Megafauna-Arten des Systems erläutert, die Zentren des Wildtiertourismus bilden, sowie die unzureichenden Kenntnisse über die Vielfalt der Bestäuber in diesem System. Schließlich wird in diesem Kapitel die Methodik der Studie beschrieben, einschließlich der Beschreibung des Untersuchungsgebiets, des Studiendesigns und der Datenerhebung. Kapitel II: Positive Auswirkungen einer geringen Beweidungsintensität auf ostafrikanische Bienengemeinschaften, vermittelt durch eine Zunahme der floralen Ressourcen Die Auswirkungen der Weideintensität auf die Bienenbestände sind seit Jahrzehnten Gegenstand von empirischen Untersuchungen. Die meisten dieser Studien wurden jedoch in den gemäßigten Breiten Europas und Amerikas durchgeführt, während die riesigen tropischen Savannen Ostafrikas weniger untersucht wurden. Mit Hilfe von Wurfnetzen und Schwenkfallen wurden insgesamt 183 Arten (von 2.691 Individuen) aus 55 Gattungen und fünf Familien an 24 Untersuchungsstandorten, die drei Stufen der Viehweideintensität im Savannen-Ökosystem im Norden Tansanias repräsentieren, gesammelt. Die Ergebnisse zeigen, dass eine mäßige Beweidung mit Weidevieh den Artenreichtum der Bienen leicht erhöht. Eine hohe Beweidungsintensität führte jedoch zu einem starken Rückgang. Außerdem zeigten die Ergebnisse ein unimodales Verteilungsmuster des Bienenartenreichtums und der mittleren Jahrestemperatur. Es wurde auch festgestellt, dass die Auswirkungen von Viehbeweidung und Umwelttemperatur auf den Bienenartenreichtum durch eine positive Auswirkung von mäßiger Beweidung auf den Reichtum an floralen Ressourcen vermittelt wurden. Die Studie zeigt daher, dass Bienengemeinschaften in der afrikanischen Savanne von einer geringen Beweidung durch Vieh profitieren können, da dies das Wachstum blühender Pflanzenarten fördert. Eine hohe Beweidungsintensität führt zu erheblichen Artenverlusten, die sich infolge der Klimaerwärmung noch verstärken können. Kapitel III: Intensivierung der Landwirtschaft mit saisonalem Brachland fördert hohe Bienenvielfalt in afrotropischen Trockengebieten In dieser Studie wurden die Auswirkungen der Intensivierung der lokalen Landwirtschaft auf die Bienenvielfalt in den afrotropischen Trockengebieten im Norden Tansanias untersucht. An 24 Untersuchungsstandorten (zwischen 702 und 1.708 m ü.N.N.), die drei Intensitätsstufen der Landwirtschaft repräsentieren und einen weiten Gradienten der Jahresmitteltemperatur abdecken, wurden 219 Arten (von 3.428 Individuen) gesammelt, die 58 Gattungen und sechs Familien repräsentieren. Die Ergebnisse zeigten, dass der Artenreichtum der Bienen mit der Intensität der Landwirtschaft und mit steigender Temperatur zunahm. Die Auswirkungen der Intensität der Landwirtschaft und der Temperatur auf den Artenreichtum der Bienen wurden jedoch durch die positiven Auswirkungen der Landwirtschaft und der Temperatur auf den Reichtum der von den Bienenbestäubern genutzten Blütenressourcen vermittelt. Außerdem zeigten die Ergebnisse, dass die Variation der Körpergröße der Bienen mit der Intensivierung der Landwirtschaft zunimmt, diese jedoch in Umgebungen mit höheren Temperaturen abnimmt. Diese Studie zeigt, dass die Bienengemeinschaften in afrotropischen Trockengebieten von der Intensivierung der Landwirtschaft, wie sie derzeit praktiziert wird, profitieren. Eine weitere Intensivierung, einschließlich ganzjährig bewässerter Monokulturen und intensiver Einsatz von Agrochemikalien, wird sich wahrscheinlich negativ auf die Bienenvielfalt und die Bestäubungsleistung auswirken, wie dies auch in den gemäßigten Regionen beobachtet wurde. Außerdem war das Vorkommen einiger Bienenarten auf natürliche Savannenlebensräume beschränkt. Um die Bienengemeinschaften in afrotropischen Trockengebieten zu erhalten und die Bestäubungsleistungen zu gewährleisten, sollte daher eine Mischung aus Savanne und Landwirtschaft mit Langzeitig-Brachflächen beibehalten werden. Kapitel IV: Auswirkungen der Intensivierung der Landnutzung und lokaler Gegebenheiten auf Pflanzen und Bestäuber in kleinbäuerlichen Betrieben südlich der Sahara In dieser Studie werden zum ersten Mal in der Region die Auswirkungen der Intensivierung der Landnutzung auf Pflanzen und Bestäuber in kleinbäuerlichen Betrieben südlich der Sahara untersucht. Hierbei wurden Felduntersuchungen von Bienen um einen modernen DNA-Metabarcoding-Ansatz ergänzt, um die beflogenen Pflanzen zu charakterisieren und so Netzwerke aufzudecken, die die Interaktionen zwischen Pflanzen und Bestäubern auf der Ebene einzelner Insekten beschreiben. Diese Informationen wurden mit quantitativen Merkmalen der Landschaftszusammensetzung und der Blütenverfügbarkeit in der Umgebung der einzelnen landwirtschaftlichen Betriebe verknüpft. Die Studie ergab, dass der Reichtum an Bestäubern mit zunehmendem Landschaftsanteil an undurchlässiger und landwirtschaftlicher Fläche abnahm, während die Blütendichte mit dem Reichtum an Bestäubern korrelierte. Die Intensivierung der landwirtschaftlichen Nutzung und die Urbanisierung korrelierten mit einer stärkeren Überlappung der Nischen für die Nahrungssuche von Bestäubern, was auf die Konvergenz der Strategien der Individuen bei der Suche nach Blüten zurückzuführen ist. Darüber hinaus führte innerhalb der landwirtschaftlichen Betriebe die höhere Verfügbarkeit von Blütenressourcen zu einer geringeren Nischenüberschneidung zwischen den Individuen, während eine größere Anzahl von Blütenbesuchern zu einer stärkeren Generalisierung auf der Ebene der Netzwerke führte (H2I), was möglicherweise auf einen erhöhten Wettbewerb zurückzuführen ist. Diese mechanistischen Erkenntnisse, die zur Überlappung der Nischen der Individuen bei der Nahrungssuche und zum Generalismus auf der Netzwerkebene führen, könnten die Stabilität der Interaktionen und der Ökosystemdienstleistung Bestäubung implizieren. Die integrative Untersuchung hat gezeigt, dass die Bestäubersysteme in den kleinbäuerlichen Betrieben Afrikas südlich der Sahara weitgehend von der Intensivierung der Landnutzung und von lokalen Faktoren beeinflusst werden. Daher könnten politische Maßnahmen zur Förderung naturbasierter Lösungen, zu denen auch die Einführung bestäuberfreundlicher Praktiken durch Kleinbauern gehört, die Intensivierung sowohl städtischer als auch ländlicher Landschaften in dieser Region wie auch in ähnlichen Kontexten südlich der Sahara wirksam abmildern. Kapitel V: Ein Überblick über die Daten zum Bienenvorkommen im Norden Tansanias Diese Studie gibt einen Überblick über die Daten zum Bienenvorkommen im Norden Tansanias, die im Rahmen einer Erhebung in den Regionen Kilimanjaro, Arusha und Manyara gewonnen wurden. Die Bienen wurden mit zwei standardisierten Methoden erfasst: mit Keschern und Farbschalen. Im Rahmen der Studie wurden 953 Individuen aus 45 Arten aus 20 Gattungen und vier Familien (Halictidae, Apidae, Megachilidae und Andrenidae) nachgewiesen. Diese Studie dient als Grundlage für das Verständnis der Vielfalt und Verbreitung von Bienen in den nördlichen Teilen des Landes. Das Verständnis des Reichtums und der Verbreitung von Bienen ist ein entscheidender Schritt bei der Entwicklung robuster Erhaltungs- und Überwachungsstrategien für deren Populationen, da die begrenzten taxonomischen Informationen über die vorhandenen und nicht identifizierten Bienenarten deren Erhaltung ungewiss erscheinen lassen. Kapitel VI: Allgemeine Diskussion Im Allgemeinen deuten die Ergebnisse dieser Studien darauf hin, dass die Beweidung mit Vieh und die Intensivierung der Landwirtschaft Auswirkungen auf die Bienenbestände und die von Bienenbestäubern genutzten Blütenressourcen haben. Die Ergebnisse haben gezeigt, dass eine mäßige Beweidungsintensität für die Erhaltung der Bienenvielfalt von Bedeutung sein kann. Eine hohe Beweidungsintensität kann jedoch zu einem starken Rückgang des Artenreichtums und der Abundanz von Bienen führen. Außerdem deuten die Ergebnisse darauf hin, dass die Intensivierung der Landwirtschaft mit saisonalem Brachland einen hohen Reichtum an floralen Ressourcen aufweist, der eine hohe Bienenvielfalt in afrotropischen Trockengebieten fördert. Nichtsdestotrotz zeigte sich, dass natürliche Savannen eine einzigartige Artenzusammensetzung aufweisen. Daher sollte die Intensivierung der Landwirtschaft mit saisonalem Brachland mit der Erhaltung von Savannenresten in den Landschaften einhergehen, um die Bienenvielfalt zu erhöhen und die Bestäubungsleistung sicherzustellen. Ebenso deuten die Ergebnisse darauf hin, dass die zunehmende Urbanisierung und landwirtschaftliche Nutzung auf Landschaftsebene die biologische Vielfalt von Pflanzen und Bestäubern verringert, was sich negativ auf ihre komplexen Interaktionen mit Pflanzen auswirkt. Umgekehrt hat sich die Verfügbarkeit von Blütenressourcen auf lokaler Ebene als positiv erwiesen, da sie den Rückgang der Bestäuber abpuffert und alle durch die Intensivierung der Flächennutzung verursachten negativen Auswirkungen abmildert. Darüber hinaus deuten die Ergebnisse darauf hin, dass die Auswirkungen der menschlichen Landnutzung (Viehbeweidung und Landwirtschaft) nicht isoliert wirken, sondern synergetisch mit Klimafaktoren wie der mittleren Jahrestemperatur (MAT) zusammenwirken. Die Auswirkung von MAT auf den Artenreichtum der Bienen in Weidegebieten erwies sich als nachteiliger als in landwirtschaftlich genutzten Lebensräumen. Dies könnte wahrscheinlich durch die verbleibende Vegetationsdeckung nach einer anthropogenen Störung erklärt werden. Das bedeutet, dass die verbleibende Vegetationsdeckung im landwirtschaftlichen Gradienten wahrscheinlich die Sonneneinstrahlung absorbiert und damit die nachteiligen Auswirkungen der mittleren Jahrestemperatur auf den Artenreichtum der Bienen verringert. Dies ist im Weidegradienten nicht der Fall, da die Auswirkungen der Beweidung durch das Weidevieh schwerwiegend sind und kahles Land ohne nennenswerte Vegetationsbedeckung zurücklassen. Unsere Ergebnisse lassen den Schluss zu, dass ein Verständnis des Zusammenspiels verschiedener anthropogener Aktivitäten und ihrer Interaktion mit MAT als Folge des fortschreitenden Klimawandels notwendig ist, um die potenziellen Folgen für die Bienenbestände und die Bereitstellung von Ökosystemleistungen zu mildern. Darüber hinaus können die künftige Zunahme der Viehbeweidung und die Intensivierung der Landwirtschaft (einschließlich ganzjährig bewässerter Monokulturen und übermäßiger Einsatz von Agrochemikalien) zu unerwünschten Folgen wie dem Verlust von Arten und Bestäubungsleistungen führen. KW - Human land use KW - Plant-pollinator interactions KW - Tanzania KW - Savannah ecosystems KW - Livestock grazing KW - Agriculture intensification KW - Bee assemblages KW - Bee abundance KW - Bee species richness Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257726 ER - TY - JOUR A1 - Bahena, Paulina A1 - Daftarian, Narsis A1 - Maroofian, Reza A1 - Linares, Paola A1 - Villalobos, Daniel A1 - Mirrahimi, Mehraban A1 - Rad, Aboulfazl A1 - Doll, Julia A1 - Hofrichter, Michaela A. H. A1 - Koparir, Asuman A1 - Röder, Tabea A1 - Han, Seungbin A1 - Sabbaghi, Hamideh A1 - Ahmadieh, Hamid A1 - Behboudi, Hassan A1 - Villanueva-Mendoza, Cristina A1 - Cortés-Gonzalez, Vianney A1 - Zamora-Ortiz, Rocio A1 - Kohl, Susanne A1 - Kuehlewein, Laura A1 - Darvish, Hossein A1 - Alehabib, Elham A1 - La Arenas-Sordo, Maria de Luz A1 - Suri, Fatemeh A1 - Vona, Barbara A1 - Haaf, Thomas T1 - Unraveling the genetic complexities of combined retinal dystrophy and hearing impairment JF - Human Genetics N2 - Usher syndrome, the most prevalent cause of combined hereditary vision and hearing impairment, is clinically and genetically heterogeneous. Moreover, several conditions with phenotypes overlapping Usher syndrome have been described. This makes the molecular diagnosis of hereditary deaf-blindness challenging. Here, we performed exome sequencing and analysis on 7 Mexican and 52 Iranian probands with combined retinal degeneration and hearing impairment (without intellectual disability). Clinical assessment involved ophthalmological examination and hearing loss questionnaire. Usher syndrome, most frequently due to biallelic variants in MYO7A (USH1B in 16 probands), USH2A (17 probands), and ADGRV1 (USH2C in 7 probands), was diagnosed in 44 of 59 (75%) unrelated probands. Almost half of the identified variants were novel. Nine of 59 (15%) probands displayed other genetic entities with dual sensory impairment, including Alström syndrome (3 patients), cone-rod dystrophy and hearing loss 1 (2 probands), and Heimler syndrome (1 patient). Unexpected findings included one proband each with Scheie syndrome, coenzyme Q10 deficiency, and pseudoxanthoma elasticum. In four probands, including three Usher cases, dual sensory impairment was either modified/aggravated or caused by variants in distinct genes associated with retinal degeneration and/or hearing loss. The overall diagnostic yield of whole exome analysis in our deaf-blind cohort was 92%. Two (3%) probands were partially solved and only 3 (5%) remained without any molecular diagnosis. In many cases, the molecular diagnosis is important to guide genetic counseling, to support prognostic outcomes and decisions with currently available and evolving treatment modalities. KW - Usher syndrome KW - hearing impairment KW - combined retinal dystrophy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-267750 SN - 1432-1203 VL - 141 IS - 3-4 ER - TY - THES A1 - Niehörster, Thomas T1 - Spektral aufgelöste Fluoreszenzlebensdauer-Mikroskopie mit vielen Farben T1 - Spectrally resolved fluorescence lifetime imaging microscopy with many colours N2 - Die Fluoreszenzmikroskopie ist eine vielseitig einsetzbare Untersuchungsmethode für biologische Proben, bei der Biomoleküle selektiv mit Fluoreszenzfarbstoffen markiert werden, um sie dann mit sehr gutem Kontrast abzubilden. Dies ist auch mit mehreren verschiedenartigen Zielmolekülen gleichzeitig möglich, wobei üblicherweise verschiedene Farbstoffe eingesetzt werden, die über ihre Spektren unterschieden werden können. Um die Anzahl gleichzeitig verwendbarer Färbungen zu maximieren, wird in dieser Arbeit zusätzlich zur spektralen Information auch das zeitliche Abklingverhalten der Fluoreszenzfarbstoffe mittels spektral aufgelöster Fluoreszenzlebensdauer-Mikroskopie (spectrally resolved fluorescence lifetime imaging microscopy, sFLIM) vermessen. Dazu wird die Probe in einem Konfokalmikroskop von drei abwechselnd gepulsten Lasern mit Wellenlängen von 485 nm, 532nm und 640nm angeregt. Die Detektion des Fluoreszenzlichtes erfolgt mit einer hohen spektralen Auflösung von 32 Kanälen und gleichzeitig mit sehr hoher zeitlicher Auflösung von einigen Picosekunden. Damit wird zu jedem detektierten Fluoreszenzphoton der Anregungslaser, der spektrale Kanal und die Ankunftszeit registriert. Diese detaillierte multidimensionale Information wird von einem Pattern-Matching-Algorithmus ausgewertet, der das Fluoreszenzsignal mit zuvor erstellten Referenzpattern der einzelnen Farbstoffe vergleicht. Der Algorithmus bestimmt so für jedes Pixel die Beiträge der einzelnen Farbstoffe. Mit dieser Technik konnten pro Anregungslaser fünf verschiedene Färbungen gleichzeitig dargestellt werden, also theoretisch insgesamt 15 Färbungen. In der Praxis konnten mit allen drei Lasern zusammen insgesamt neun Färbungen abgebildet werden, wobei die Anzahl der Farben vor allem durch die anspruchsvolle Probenvorbereitung limitiert war. In anderen Versuchen konnte die sehr hohe Sensitivität des sFLIM-Systems genutzt werden, um verschiedene Zielmoleküle voneinander zu unterscheiden, obwohl sie alle mit demselben Farbstoff markiert waren. Dies war möglich, weil sich die Fluoreszenzeigenschaften eines Farbstoffmoleküls geringfügig in Abhängigkeit von seiner Umgebung ändern. Weiterhin konnte die sFLIM-Technik mit der hochauflösenden STED-Mikroskopie (STED: stimulated emission depletion) kombiniert werden, um so hochaufgelöste zweifarbige Bilder zu erzeugen, wobei nur ein einziger gemeinsamer STED-Laser benötigt wurde. Die gleichzeitige Erfassung von mehreren photophysikalischen Messgrößen sowie deren Auswertung durch den Pattern-Matching-Algorithmus ermöglichten somit die Entwicklung von neuen Methoden der Fluoreszenzmikroskopie für Mehrfachfärbungen. N2 - Fluorescence microscopy is an important and near-universal technique to examine biological samples. Typically, biomolecules are selectively labelled with fluorophores and then imaged with high contrast. This can be done for several target molecules simultaneously, using different fluorophores that are usually distinguished by their spectra. This thesis describes a method to maximize the number of simultaneous stainings. Not only the spectral information but also the temporal information of the fluorescence decay is exploited by means of spectrally resolved fluorescence lifetime imaging microscopy (sFLIM). Using a confocal laser scanning microscope, the sample is excited by three alternatingly pulsed lasers at 485 nm, 532 nm, and 640 nm. Fluorescence light is detected on 32 spectrally separated detection channels with high time resolution of a few picoseconds. Thus, in this setup, we record the excitation laser, the spectral channel, and the time of arrival for each fluorescence photon. This detailed multi-dimensional information is then processed by a pattern-matching algorithm that compares the fluorescence signal with reference patterns of the used fluorophores to determine the contribution of each fluorophore in each pixel. Using this technique we imaged five different stainings per excitation laser, implying that 15 simultaneous stainings should theoretically be achievable. Current constraints in the sample preparation procedure limited the number of simultaneous stainings to nine. In additional experiments, we exploited the sensitivity of the sFLIM system to image several different target molecules simultaneously with the same fluorophore, taking advantage of slight changes in the fluorescence behaviour of the fluorophore due to environmental changes. We also combined sFLIM with stimulated emission depletion (STED) to perform super-resolution multi-target imaging with two stainings that operated with one common STED laser. Thus, the simultaneous exploitation of several photophysical parameters, in combination with algorythmic evaluation, allowed us to devise novel modes of multi-target imaging in fluorescence microscopy. KW - Fluoreszenzmikroskopie KW - Fluoreszenzlebensdauer-Mikroskopie KW - Konfokale Mikroskopie KW - STED-Mikroskopie KW - Fluoreszenz KW - Mustervergleich KW - Pattern Matching KW - sFLIM KW - TCSPC KW - Mikroskopie KW - Microscopy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-296573 ER - TY - JOUR A1 - Lichter, Katharina A1 - Paul, Mila Marie A1 - Pauli, Martin A1 - Schoch, Susanne A1 - Kollmannsberger, Philip A1 - Stigloher, Christian A1 - Heckmann, Manfred A1 - Sirén, Anna-Leena T1 - Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse JF - Cell Reports N2 - Rab3A-interacting molecule (RIM) is crucial for fast Ca\(^{2+}\)-triggered synaptic vesicle (SV) release in presynaptic active zones (AZs). We investigated hippocampal giant mossy fiber bouton (MFB) AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1α\(^{−/−}\) and wild-type mice. In RIM1α\(^{−/−}\), AZs are larger with increased synaptic cleft widths and a 3-fold reduced number of tightly docked SVs (0–2 nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron-dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1α\(^{−/−}\) is more heterogeneous. Thus, RIM1α, besides its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs. KW - active zone KW - acute brain slices KW - CA3 KW - electron tomography KW - high-pressure freezing KW - hippocampal mossy fiber bouton KW - RIM1α KW - SV pool KW - synaptic ultrastructure KW - presynaptic Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300913 VL - 40 IS - 12 ER - TY - JOUR A1 - Jeanclos, Elisabeth A1 - Schlötzer, Jan A1 - Hadamek, Kerstin A1 - Yuan-Chen, Natalia A1 - Alwahsh, Mohammad A1 - Hollmann, Robert A1 - Fratz, Stefanie A1 - Yesilyurt-Gerhards, Dilan A1 - Frankenbach, Tina A1 - Engelmann, Daria A1 - Keller, Angelika A1 - Kaestner, Alexandra A1 - Schmitz, Werner A1 - Neuenschwander, Martin A1 - Hergenröder, Roland A1 - Sotriffer, Christoph A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Gohla, Antje T1 - Glycolytic flux control by drugging phosphoglycolate phosphatase JF - Nature Communications N2 - Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates. KW - phosphoglycolate phosphatase KW - glycolytic flux control KW - intrinsic metabolism Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300928 VL - 13 IS - 1 ER - TY - JOUR A1 - Kaya-Zeeb, Sinan A1 - Engelmayer, Lorenz A1 - Straßburger, Mara A1 - Bayer, Jasmin A1 - Bähre, Heike A1 - Seifert, Roland A1 - Scherf-Clavel, Oliver A1 - Thamm, Markus T1 - Octopamine drives honeybee thermogenesis JF - eLife N2 - In times of environmental change species have two options to survive: they either relocate to a new habitat or they adapt to the altered environment. Adaptation requires physiological plasticity and provides a selection benefit. In this regard, the Western honeybee (Apis mellifera) protrudes with its thermoregulatory capabilities, which enables a nearly worldwide distribution. Especially in the cold, shivering thermogenesis enables foraging as well as proper brood development and thus survival. In this study, we present octopamine signaling as a neurochemical prerequisite for honeybee thermogenesis: we were able to induce hypothermia by depleting octopamine in the flight muscles. Additionally, we could restore the ability to increase body temperature by administering octopamine. Thus, we conclude that octopamine signaling in the flight muscles is necessary for thermogenesis. Moreover, we show that these effects are mediated by β octopamine receptors. The significance of our results is highlighted by the fact the respective receptor genes underlie enormous selective pressure due to adaptation to cold climates. Finally, octopamine signaling in the service of thermogenesis might be a key strategy to survive in a changing environment. KW - honeybee KW - octopamine KW - thermogenesis Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301327 VL - 11 ER - TY - JOUR A1 - Reinhard, Nils A1 - Bertolini, Enrico A1 - Saito, Aika A1 - Sekiguchi, Manabu A1 - Yoshii, Taishi A1 - Rieger, Dirk A1 - Helfrich‐Förster, Charlotte T1 - The lateral posterior clock neurons of Drosophila melanogaster express three neuropeptides and have multiple connections within the circadian clock network and beyond JF - Journal of Comparative Neurology N2 - Drosophila’s lateral posterior neurons (LPNs) belong to a small group of circadian clock neurons that is so far not characterized in detail. Thanks to a new highly specific split‐Gal4 line, here we describe LPNs’ morphology in fine detail, their synaptic connections, daily bimodal expression of neuropeptides, and propose a putative role of this cluster in controlling daily activity and sleep patterns. We found that the three LPNs are heterogeneous. Two of the neurons with similar morphology arborize in the superior medial and lateral protocerebrum and most likely promote sleep. One unique, possibly wakefulness‐promoting, neuron with wider arborizations extends from the superior lateral protocerebrum toward the anterior optic tubercle. Both LPN types exhibit manifold connections with the other circadian clock neurons, especially with those that control the flies’ morning and evening activity (M‐ and E‐neurons, respectively). In addition, they form synaptic connections with neurons of the mushroom bodies, the fan‐shaped body, and with many additional still unidentified neurons. We found that both LPN types rhythmically express three neuropeptides, Allostatin A, Allostatin C, and Diuretic Hormone 31 with maxima in the morning and the evening. The three LPN neuropeptides may, furthermore, signal to the insect hormonal center in the pars intercerebralis and contribute to rhythmic modulation of metabolism, feeding, and reproduction. We discuss our findings in the light of anatomical details gained by the recently published hemibrain of a single female fly on the electron microscopic level and of previous functional studies concerning the LPN. KW - activity KW - circadian clock neurons KW - insect brain KW - neuropeptides KW - sleep KW - trans‐Tango Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-276456 VL - 530 IS - 9 SP - 1507 EP - 1529 ER - TY - JOUR A1 - Lasway, Julius V. A1 - Peters, Marcell K. A1 - Njovu, Henry K. A1 - Eardley, Connal A1 - Pauly, Alain A1 - Steffan‐Dewenter, Ingolf T1 - Agricultural intensification with seasonal fallow land promotes high bee diversity in Afrotropical drylands JF - Journal of Applied Ecology N2 - The exponential increase in the human population in tandem with increased food demand has caused agriculture to be the global‐dominant form of land use. Afrotropical drylands are currently facing the loss of natural savannah habitats and agricultural intensification with largely unknown consequences for bees. Here we investigate the effects of agricultural intensification on bee assemblages in the Afrotropical drylands of northern Tanzania. We disentangled the direct effects of agricultural intensification and temperature on bee richness from indirect effects mediated by changes in floral resources. We collected data from 24 study sites representing three levels of management intensity (natural savannah, moderate intensive and highly intensive agriculture) spanning an extensive gradient of mean annual temperature (MAT) in northern Tanzania. We used ordinary linear models and path analysis to test the effects of agricultural intensity and MAT on bee species richness, bee species composition and body‐size variation of bee communities. We found that bee species richness increased with agricultural intensity and with increasing temperature. The effects of agricultural intensity and temperature on bee species richness were mediated by the positive effects of agriculture and temperature on the richness of floral resources used by bees. During the off‐growing season, agricultural land was characterized by an extensive period of fallow land holding a very high density of flowering plants with unique bee species composition. The increase in bee diversity in agricultural habitats paralleled an increasing variation of bee body sizes with agricultural intensification that, however, diminished in environments with higher temperatures. Synthesis and applications. Our study reveals that bee assemblages in Afrotropical drylands benefit from agricultural intensification in the way it is currently practiced. However, further land‐use intensification, including year‐round irrigated crop monocultures and excessive use of agrochemicals, is likely to exert a negative impact on bee diversity and pollination services, as reported in temperate regions. Moreover, several bee species were restricted to natural savannah habitats. To conserve bee communities and guarantee pollination services in the region, a mixture of savannah and agriculture, with long periods of fallow land should be maintained. KW - bee abundance KW - bee body size KW - bee species richness KW - forage resources KW - inter‐tegular distance KW - mean annual temperature KW - northern Tanzania KW - species community composition Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311877 VL - 59 IS - 12 SP - 3014 EP - 3026 ER - TY - JOUR A1 - Sponsler, Douglas B. A1 - Requier, Fabrice A1 - Kallnik, Katharina A1 - Classen, Alice A1 - Maihoff, Fabienne A1 - Sieger, Johanna A1 - Steffan‐Dewenter, Ingolf T1 - Contrasting patterns of richness, abundance, and turnover in mountain bumble bees and their floral hosts JF - Ecology N2 - Environmental gradients generate and maintain biodiversity on Earth. Mountain slopes are among the most pronounced terrestrial environmental gradients, and the elevational structure of species and their interactions can provide unique insight into the processes that govern community assembly and function in mountain ecosystems. We recorded bumble bee–flower interactions over 3 years along a 1400‐m elevational gradient in the German Alps. Using nonlinear modeling techniques, we analyzed elevational patterns at the levels of abundance, species richness, species β‐diversity, and interaction β‐diversity. Though floral richness exhibited a midelevation peak, bumble bee richness increased with elevation before leveling off at the highest sites, demonstrating the exceptional adaptation of these bees to cold temperatures and short growing seasons. In terms of abundance, though, bumble bees exhibited divergent species‐level responses to elevation, with a clear separation between species preferring low versus high elevations. Overall interaction β‐diversity was mainly caused by strong turnover in the floral community, which exhibited a well‐defined threshold of β‐diversity rate at the tree line ecotone. Interaction β‐diversity increased sharply at the upper extreme of the elevation gradient (1800–2000 m), an interval over which we also saw steep decline in floral richness and abundance. Turnover of bumble bees along the elevation gradient was modest, with the highest rate of β‐diversity occurring over the interval from low‐ to mid‐elevation sites. The contrast between the relative robustness bumble bee communities and sensitivity of plant communities to the elevational gradient in our study suggests that the strongest effects of climate change on mountain bumble bees may be indirect effects mediated by the responses of their floral hosts, though bumble bee species that specialize in high‐elevation habitats may also experience significant direct effects of warming. KW - alpine plants KW - climate KW - elevation gradient KW - mountain ecology KW - pollination network Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-287199 VL - 103 IS - 7 ER - TY - JOUR A1 - Gebert, Friederike A1 - Steffan‐Dewenter, Ingolf A1 - Kronbach, Patrick A1 - Peters, Marcell K. T1 - The role of diversity, body size and climate in dung removal: A correlative and experimental approach JF - Journal of Animal Ecology N2 - The mechanisms by which climatic changes influence ecosystem functions, that is, by a direct climatic control of ecosystem processes or by modifying richness and trait compositions of species communities, remain unresolved. This study is a contribution to this discourse by elucidating the linkages between climate, land use, biodiversity, body size and ecosystem functions. We disentangled direct climatic from biodiversity‐mediated effects by using dung removal by dung beetles as a model system and by combining correlative field data and exclosure experiments along an extensive elevational gradient on Mt. Kilimanjaro, Tanzania. Dung removal declined with increasing elevation, being associated with a strong reduction in the richness and body size traits of dung beetle communities. Climate influenced dung removal rates by modifying biodiversity rather than by direct effects. The biodiversity–ecosystem effect was driven by a change in the mean body size of dung beetles. Dung removal rates were strongly reduced when large dung beetles were experimentally excluded. This study underscores that climate influences ecosystem functions mainly by modifying biodiversity and underpins the important role of body size for dung removal. KW - altitudinal gradients KW - biodiversity–ecosystem functioning relationship KW - body size KW - diversity gradients KW - ecosystem services KW - land use KW - Scarabaeidae KW - temperature Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-293907 VL - 91 IS - 11 SP - 2181 EP - 2191 ER - TY - JOUR A1 - Kortmann, Mareike A1 - Roth, Nicolas A1 - Buse, Jörn A1 - Hilszczański, Jacek A1 - Jaworski, Tomasz A1 - Morinière, Jérôme A1 - Seidl, Rupert A1 - Thorn, Simon A1 - Müller, Jörg C. T1 - Arthropod dark taxa provide new insights into diversity responses to bark beetle infestations JF - Ecological Applications N2 - Natural disturbances are increasing around the globe, also impacting protected areas. Although previous studies have indicated that natural disturbances result in mainly positive effects on biodiversity, these analyses mostly focused on a few well established taxonomic groups, and thus uncertainty remains regarding the comprehensive impact of natural disturbances on biodiversity. Using Malaise traps and meta‐barcoding, we studied a broad range of arthropod taxa, including dark and cryptic taxa, along a gradient of bark beetle disturbance severities in five European national parks. We identified order‐level community thresholds of disturbance severity and classified barcode index numbers (BINs; a cluster system for DNA sequences, where each cluster corresponds to a species) as negative or positive disturbance indicators. Negative indicator BINs decreased above thresholds of low to medium disturbance severity (20%–30% of trees killed), whereas positive indicator BINs benefited from high disturbance severity (76%–98%). BINs allocated to a species name contained nearly as many positive as negative disturbance indicators, but dark and cryptic taxa, particularly Diptera and Hymenoptera in our data, contained higher numbers of negative disturbance indicator BINs. Analyses of changes in the richness of BINs showed variable responses of arthropods to disturbance severity at lower taxonomic levels, whereas no significant signal was detected at the order level due to the compensatory responses of the underlying taxa. We conclude that the analyses of dark taxa can offer new insights into biodiversity responses to disturbances. Our results suggest considerable potential for forest management to foster arthropod diversity, for example by maintaining both closed‐canopy forests (>70% cover) and open forests (<30% cover) on the landscape. KW - arthropods KW - biodiversity KW - conservation KW - metabarcoding KW - national park KW - natural disturbance KW - threshold indicator taxa analysis Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-276392 VL - 32 IS - 2 ER - TY - JOUR A1 - Uhler, Johannes A1 - Haase, Peter A1 - Hoffmann, Lara A1 - Hothorn, Torsten A1 - Schmidl, Jürgen A1 - Stoll, Stefan A1 - Welti, Ellen A. R. A1 - Buse, Jörn A1 - Müller, Jörg T1 - A comparison of different Malaise trap types JF - Insect Conservation and Diversity N2 - Recent reports on insect decline have highlighted the need for long‐term data on insect communities towards identifying their trends and drivers. With the launch of many new insect monitoring schemes to investigate insect communities over large spatial and temporal scales, Malaise traps have become one of the most important tools due to the broad spectrum of species collected and reduced capture bias through passive sampling of insects day and night. However, Malaise traps can vary in size, shape, and colour, and it is unknown how these differences affect biomass, species richness, and composition of trap catch, making it difficult to compare results between studies. We compared five Malaise trap types (three variations of the Townes and two variations of the Bartak Malaise trap) to determine their effects on biomass and species richness as identified by metabarcoding. Insect biomass varied by 20%–55%, not strictly following trap size but varying with trap type. Total species richness was 20%–38% higher in the three Townes trap models compared to the Bartak traps. Bartak traps captured lower richness of highly mobile taxa but increased richness of ground‐dwelling taxa. The white roofed Townes trap captured a higher richness of pollinators. We find that biomass, total richness, and taxa group specific richness are all sensitive to Malaise trap type. Trap type should be carefully considered and aligned to match monitoring and research questions. Additionally, our estimates of trap type effects can be used to adjust results to facilitate comparisons across studies. KW - Bartak KW - biodiversity KW - insect communities KW - insect monitoring KW - Malaise trap KW - Townes KW - trap selectivity Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-293694 VL - 15 IS - 6 SP - 666 EP - 672 ER - TY - JOUR A1 - Kohl, Patrick L. A1 - Steffan‐Dewenter, Ingolf T1 - Nectar robbing rather than pollinator availability constrains reproduction of a bee‐flowered plant at high elevations JF - Ecosphere N2 - Abiotic factors are generally assumed to determine whether species can exist at the extreme ends of environmental gradients, for example, at high elevations, whereas the role of biotic interactions is less clear. On temperate mountains, insect‐pollinated plant species with bilaterally symmetrical flowers exhibit a parallel elevational decline in species richness and abundance with bees. This suggests that the lack of mutualistic interaction partners sets the elevational range limits of plants via a reduction in reproductive success. We used the bee‐pollinated mountain plant Clinopodium alpinum (Lamiaceae), which blooms along a continuous 1000‐m elevational gradient and has bilaterally symmetrical flowers, as a model to test the predicted parallel elevational decline in flower visitation and seed production. Although the community of flower visitors changed with elevation, the flower visitation rate by the most frequent visitors, bumble bees (33.8% of legitimate visits), and the overall rate of flower visitation by potential pollinators did not vary significantly with elevation. However, we discovered that nectar robbing by bumble bees and nectar theft by ants, two interactions with potentially negative effects on flowers, sharply increased with elevation. Seed set depended on pollinators across elevations and followed a weak hump‐shaped pattern, peaking at mid‐elevations and decreasing by about 20% toward both elevational range edges. Considering the mid‐ and high elevations, elevational variation in seed production could not be explained by legitimate bee visitation rates but was inversely correlated with the frequency of nectar robbing. Our observations challenge the hypothesis that a decrease in the availability of pollinators limits seed production of bee‐flowered plants at high elevations but suggest that an increase in negative interactions (nectar robbing and larceny) constrains reproductive success. KW - altitudinal gradients KW - bee pollination KW - chalcidoid wasps KW - climatic gradients KW - elevational diversity patterns KW - floral larceny KW - fly pollination KW - mountain ecosystems KW - plant–pollinator interactions KW - range limits KW - zygomorphy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-287141 VL - 13 IS - 6 ER - TY - JOUR A1 - Jones, Jeffrey J. A1 - Huang, Shouguang A1 - Hedrich, Rainer A1 - Geilfus, Christoph‐Martin A1 - Roelfsema, M. Rob G. T1 - The green light gap: a window of opportunity for optogenetic control of stomatal movement JF - New Phytologist N2 - Green plants are equipped with photoreceptors that are capable of sensing radiation in the ultraviolet‐to‐blue and the red‐to‐far‐red parts of the light spectrum. However, plant cells are not particularly sensitive to green light (GL), and light which lies within this part of the spectrum does not efficiently trigger the opening of stomatal pores. Here, we discuss the current knowledge of stomatal responses to light, which are either provoked via photosynthetically active radiation or by specific blue light (BL) signaling pathways. The limited impact of GL on stomatal movements provides a unique option to use this light quality to control optogenetic tools. Recently, several of these tools have been optimized for use in plant biological research, either to control gene expression, or to provoke ion fluxes. Initial studies with the BL‐activated potassium channel BLINK1 showed that this tool can speed up stomatal movements. Moreover, the GL‐sensitive anion channel GtACR1 can induce stomatal closure, even at conditions that provoke stomatal opening in wild‐type plants. Given that crop plants in controlled‐environment agriculture and horticulture are often cultivated with artificial light sources (i.e. a combination of blue and red light from light‐emitting diodes), GL signals can be used as a remote‐control signal that controls stomatal transpiration and water consumption. KW - anion channel KW - channelrhodopsin KW - Chl KW - guard cell KW - ion channel KW - light‐gated KW - membrane potential KW - phototropin Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-293724 VL - 236 IS - 4 SP - 1237 EP - 1244 ER - TY - JOUR A1 - Wech, Tobias A1 - Ankenbrand, Markus Johannes A1 - Bley, Thorsten Alexander A1 - Heidenreich, Julius Frederik T1 - A data-driven semantic segmentation model for direct cardiac functional analysis based on undersampled radial MR cine series JF - Magnetic Resonance in Medicine N2 - Purpose Image acquisition and subsequent manual analysis of cardiac cine MRI is time-consuming. The purpose of this study was to train and evaluate a 3D artificial neural network for semantic segmentation of radially undersampled cardiac MRI to accelerate both scan time and postprocessing. Methods A database of Cartesian short-axis MR images of the heart (148,500 images, 484 examinations) was assembled from an openly accessible database and radial undersampling was simulated. A 3D U-Net architecture was pretrained for segmentation of undersampled spatiotemporal cine MRI. Transfer learning was then performed using samples from a second database, comprising 108 non-Cartesian radial cine series of the midventricular myocardium to optimize the performance for authentic data. The performance was evaluated for different levels of undersampling by the Dice similarity coefficient (DSC) with respect to reference labels, as well as by deriving ventricular volumes and myocardial masses. Results Without transfer learning, the pretrained model performed moderately on true radial data [maximum number of projections tested, P = 196; DSC = 0.87 (left ventricle), DSC = 0.76 (myocardium), and DSC =0.64 (right ventricle)]. After transfer learning with authentic data, the predictions achieved human level even for high undersampling rates (P = 33, DSC = 0.95, 0.87, and 0.93) without significant difference compared with segmentations derived from fully sampled data. Conclusion A 3D U-Net architecture can be used for semantic segmentation of radially undersampled cine acquisitions, achieving a performance comparable with human experts in fully sampled data. This approach can jointly accelerate time-consuming cine image acquisition and cumbersome manual image analysis. KW - undersampling KW - cardiovascular magnetic resonance (CMR) KW - deep learning KW - radial KW - semantic segmentation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257616 VL - 87 IS - 2 ER - TY - JOUR A1 - Fleischmann, Pauline N. A1 - Grob, Robin A1 - Rössler, Wolfgang T1 - Magnetosensation during re-learning walks in desert ants (Cataglyphis nodus) JF - Journal of Comparative Physiology A N2 - At the beginning of their foraging careers, Cataglyphis desert ants calibrate their compass systems and learn the visual panorama surrounding the nest entrance. For that, they perform well-structured initial learning walks. During rotational body movements (pirouettes), naïve ants (novices) gaze back to the nest entrance to memorize their way back to the nest. To align their gaze directions, they rely on the geomagnetic field as a compass cue. In contrast, experienced ants (foragers) use celestial compass cues for path integration during food search. If the panorama at the nest entrance is changed, foragers perform re-learning walks prior to heading out on new foraging excursions. Here, we show that initial learning walks and re-learning walks are structurally different. During re-learning walks, foragers circle around the nest entrance before leaving the nest area to search for food. During pirouettes, they do not gaze back to the nest entrance. In addition, foragers do not use the magnetic field as a compass cue to align their gaze directions during re-learning walk pirouettes. Nevertheless, magnetic alterations during re-learning walks under manipulated panoramic conditions induce changes in nest-directed views indicating that foragers are still magnetosensitive in a cue conflict situation. KW - path integration KW - landmark panorama KW - learning and memory KW - magnetic compass KW - navigation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-266556 SN - 1432-1351 VL - 208 IS - 1 ER - TY - JOUR A1 - Schilcher, Felix A1 - Hilsmann, Lioba A1 - Ankenbrand, Markus J. A1 - Krischke, Markus A1 - Mueller, Martin J. A1 - Steffan-Dewenter, Ingolf A1 - Scheiner, Ricarda T1 - Honeybees are buffered against undernourishment during larval stages JF - Frontiers in Insect Science N2 - The negative impact of juvenile undernourishment on adult behavior has been well reported for vertebrates, but relatively little is known about invertebrates. In honeybees, nutrition has long been known to affect task performance and timing of behavioral transitions. Whether and how a dietary restriction during larval development affects the task performance of adult honeybees is largely unknown. We raised honeybees in-vitro, varying the amount of a standardized diet (150 µl, 160 µl, 180 µl in total). Emerging adults were marked and inserted into established colonies. Behavioral performance of nurse bees and foragers was investigated and physiological factors known to be involved in the regulation of social organization were quantified. Surprisingly, adult honeybees raised under different feeding regimes did not differ in any of the behaviors observed. No differences were observed in physiological parameters apart from weight. Honeybees were lighter when undernourished (150 µl), while they were heavier under the overfed treatment (180 µl) compared to the control group raised under a normal diet (160 µl). These data suggest that dietary restrictions during larval development do not affect task performance or physiology in this social insect despite producing clear effects on adult weight. We speculate that possible effects of larval undernourishment might be compensated during the early period of adult life. KW - nutrition KW - juvenile hormone KW - nurse bees KW - foragers KW - triglycerides KW - undernourishment KW - task allocation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304646 SN - 2673-8600 VL - 2 ER - TY - JOUR A1 - Pietro-Garcia, Christian A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Fischer, Thomas A1 - Maier, Carina R. A1 - Rosenfeldt, Mathias A1 - Schülein-Völk, Christina A1 - Klann, Kevin A1 - Kalb, Reinhard A1 - Dikic, Ivan A1 - Münch, Christian A1 - Diefenbacher, Markus E. T1 - Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway JF - Cell Death and Differentiation N2 - Squamous cell carcinomas (SCC) frequently have an exceptionally high mutational burden. As consequence, they rapidly develop resistance to platinum-based chemotherapy and overall survival is limited. Novel therapeutic strategies are therefore urgently required. SCC express ∆Np63, which regulates the Fanconi Anemia (FA) DNA-damage response in cancer cells, thereby contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 is recruited to sites of DNA damage in cisplatin-treated cells. ATR phosphorylates USP28 and increases its enzymatic activity. This phosphorylation event is required to positively regulate the DNA damage repair in SCC by stabilizing ∆Np63. Knock-down or inhibition of USP28 by a specific inhibitor weakens the ability of SCC to cope with DNA damage during platin-based chemotherapy. Hence, our study presents a novel mechanism by which ∆Np63 expressing SCC can be targeted to overcome chemotherapy resistance. Limited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ∆Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-∆Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment. KW - USP28 KW - Cisplatin KW - squamous tumors Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-273014 SN - 1476-5403 VL - 29 IS - 3 ER - TY - THES A1 - Vogel, Cassandra Ezra T1 - The effects of land-use and agroecological practices on biodiversity and ecosystem services in tropical smallholder farms T1 - Die Effekte von Landnutzung und Agroökologie auf Biodiversität und Ökosystemdienstleistungen in der tropischen Subsistenzlandwirtschaft N2 - Biodiversity is in rapid decline worldwide. These declines are more pronounced in areas that are currently biodiversity rich, but economically poor – essentially describing many tropical regions in the Global South where landscapes are dominated by smallholder agriculture. Agriculture is an important driver of biodiversity decline, through habitat destruction and unsustainable practices. Ironically, agriculture itself is dependent on a range of ecosystem services, such as pollination and pest control, provided by biodiversity. Biodiversity on fields and the delivery of ecosystem services to crops is often closely tied to the composition of the surrounding landscape – complex landscapes with a higher proportion of (semi-)natural habitats tend to support a high abundances and biodiversity of pollinators and natural enemies that are beneficial to crop production. However, past landscape scale studies have focused primarily on industrialized agricultural landscapes in the Global North, and context dependent differences between regions and agricultural systems are understudied. Smallholder agriculture supports 2 billion people worldwide and contributes to over half the world’s food supply. Yet smallholders, particularly in sub-Saharan Africa, are underrepresented in research investigating the consequences of landscape change and agricultural practices. Where research in smallholder agriculture is conducted, the focus is often on commodity crops, such as cacao, and less on crops that are directly consumed by smallholder households, though the loss of services to these crops could potentially impact the most vulnerable farmers the hardest. Agroecology – a holistic and nature-based approach to agriculture, provides an alternative to unsustainable input-intensive agriculture. Agroecology has been found to benefit smallholders through improved agronomical and food-security outcomes. Co-benefits of agroecological practices with biodiversity and ecosystem services are assumed, but not often empirically tested. In addition, the local and landscape effects on biodiversity and ecosystem services are more commonly studied in isolation, but their potentially interactive effects are so far little explored. Our study region in northern Malawi exemplifies many challenges experienced by smallholder farmers throughout sub-Saharan Africa and more generally in the Global South. Malawi is located in a global biodiversity hotspot, but biodiversity is threatened by rapid habitat loss and a push for input-intensive agriculture by government and other stakeholders. In contrast, agroecology has been effectively promoted and implemented in the study region. We investigated how land-use differences and the agroecological practices affects biodiversity and ecosystem services of multiple taxa in a maize-bean intercropping system (Chapter 2), and pollination of pumpkin (Chapter 3) and pigeon pea (Chapter 4). Additionally, the effects of local and landscape scale shrub- to farmland habitat conversion was investigated on butterfly communities, as well as the potential for agroecology to mitigate these effects (Chapter 5). N2 - Die globale Biodiversität nimmt rapide ab. Dieser Biodiversitätsverlust ist in Regionen die reich an Biodiversität aber wirtschaftlich arm sind besonders stark ausgeprägt, insbesondere in vielen tropischen Regionen, die durch Subsistenzlandwirtschaft geprägt sind. Durch die Zerstörung natürlicher Lebensräume und nicht nachhaltige Land Nutzung ist Landwirtschaft eine der Hauptursachen dieses Biodiversitätsrückgangs. Dabei ist gerade landwirtschaftliche Produktion abhängig von Biodiversität, da Biodiversität Ökosystemdienstleistungen wie Bestäubung und natürliche Schädlingskontrolle bereitstellt. Biodiversität und Ökosystemdienstleistungen auf Feldern werden stark durch die umliegende Landschaft beeinflusst - komplexe Landschaften mit einen großen Anteil (halb-)natürlicher haben in der Regel höhere Abudanzen und eine größere Biodiversität von Bestäubern und natürlichen Feinden die vorteilhaft für die landwirtschaftliche Produktion sind. Forschung auf Landschaftebene hat bisher jedoch vorrangig auf die industrialisierte Landwirtschaft in z.B. Europa oder die USA fokussiert und kontextabhängige Unterschiede zwischen Regionen und landwirtschaftlichen Systemen sind nicht ausreichend studiert..Weltweit sind etwa 2 Milliarden Menschen von Subsistenzlandwirtschaft abhängig. Jedoch sind diese Kleinbauern, in der Forschung über die Konsequenzen von Landnutzung und landwirtschaftlichen Managements auf Biodiversität und Ökosystemdienstleistungen unterrepräsentiert, insbesondere Kleinbauern aus Subsahara-Afrika. Die wenigen verfügbaren Studien legend den Fokus oft auf wirtschaftlich wichtige Kulturpflanzen, wie etwa Kakao, und selten auf Kulturpflanzen, die für Ernährungssicherheit der Kleinbauern wichtig sind, obwohl der Verlust der Ökosystemdienstleistungen diese möglicherweise am härtesten trifft. Agroökologie ist eine nachhaltigere Form des landwirtschaftlichen Managements als die konventionelle Landwirtschaft, und will den Einsatz von Agrochemie zu reduzieren und eine holistische Landwirtschaft fördern. Agroökologie steigert die Ernährungssicherheit von Kleinbauern, insbesondere wenn die Bauern viele verschiedene agroökologische Verfahren nutzen. Vorteile der Agroökologie für Biodiversität und Ökosystemdienstleistungen werden oft vermutet, wurden bislang jedoch selten empirisch getestet. Zusätzlich wurden Effekte auf Biodiversität und Ökosystemdienstleistungen vorrangig getrennt zwischen der lokalen und der Landschaftsebene betrachtet, was das Erkennen potentieller Interaktionen erschwert. Unsere Studienregion in Nord Malawi spiegelt die viele Herausforderungen der afrikanischen Zusammenfassung Subsistenzlandwirtschaft wider. Malawi liegt in einem Biodiversitäts-Hotspot, jedoch ist diese Biodiversität durch einen schnellen Rückgang natürlicher Lebensräume und durch die Intensivierung der Landwirtschaft stark gefährdet. Dem gegenüber stehen erfolgreicher Ausbau und Umsetzung von Agroökologie in der Region. Das gab mir die Möglichkeit, die Effekte von Landnutzung und Agroökologie auf Biodiversität und Ökosystemdienstleistungen in Malawi zu untersuchen. Dafür habe ich in Mais und Bohnen in Einzel- und Mischkultur 7 taxonomische Gruppen die verschiedene Ökosystemdienstleistungen erbringen erfasst (Kapitel 2) sowie Bestäuber und Bestäubung auf Kürbis (Kapitel 3) und Straucherbsen studiert (Kapitel 4). Zusätzlich habe ich an Schmetterlingen die Effekte von Lebensraumverlust auf der lokalen und auf Landschaftsebene studiert, und untersucht, ob Agroökologie potenziell negative Effekte mindern kann (Kapitel 5). KW - biodiversity KW - ecosystem services KW - landscape ecology KW - smallholder agriculture KW - pollination KW - pest control KW - agroecology KW - tropical ecology Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290661 ER - TY - JOUR A1 - Maihoff, Fabienne A1 - Bohlke, Kyte A1 - Brockmann, Axel A1 - Schmitt, Thomas T1 - Increased complexity of worker CHC profiles in Apis dorsata correlates with nesting ecology JF - PLoS ONE N2 - Cuticular hydrocarbons (CHC) are known to serve as discrimination cues and will trigger defence behaviour in a plethora of eusocial insects. However, little is known how about nestmate recognition ability selects for CHC diversification. In this study we investigate differences in CHC composition of four major honey bee species with respect to the differences in their nesting behavior. In contrast to A. mellifera, A. cerana and A. florea, the giant honey bee A. dorsata prefers to build their nests in aggregations with very small spatial distances between nests, which increases the probability of intrusions. Thus, A. dorsata exhibits a particularly challenging nesting behavior which we hypothesize should be accompanied with an improved nestmate recognition system. Comparative analyses of the worker CHC profiles indicate that A. dorsata workers exhibit a unique and more complex CHC profile than the other three honey bee species. This increased complexity is likely based on a developmental process that retains the capability to synthesize methyl-branched hydrocarbons as adults. Furthermore, two sets of behavioral experiments provide evidence that A. dorsata shows an improved nestmate discrimination ability compared to the phylogenetically ancestral A. florea, which is also open-nesting but does not form nest aggregations. The results of our study suggest that ecological traits like nesting in aggregation might be able to drive CHC profile diversification even in closely related insect species. KW - Apis dorsata KW - cuticular hydrocarbons KW - nesting Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301353 VL - 17 IS - 7 ER - TY - JOUR A1 - Reinhard, Sebastian A1 - Helmerich, Dominic A. A1 - Boras, Dominik A1 - Sauer, Markus A1 - Kollmannsberger, Philip T1 - ReCSAI: recursive compressed sensing artificial intelligence for confocal lifetime localization microscopy JF - BMC Bioinformatics N2 - Background Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method. Results Here we introduce ReCSAI, a compressed sensing neural network to reconstruct localizations for confocal dSTORM, together with a simulation tool to generate training data. We implemented and compared different artificial network architectures, aiming to combine the advantages of compressed sensing and deep learning. We found that a U-Net with a recursive structure inspired by iterative compressed sensing showed the best results on realistic simulated datasets with noise, as well as on real experimentally measured confocal lifetime scanning data. Adding a trainable wavelet denoising layer as prior step further improved the reconstruction quality. Conclusions Our deep learning approach can reach a similar reconstruction accuracy for confocal dSTORM as frame binning with traditional fitting without requiring the acquisition of multiple frames. In addition, our work offers generic insights on the reconstruction of sparse measurements from noisy experimental data by combining compressed sensing and deep learning. We provide the trained networks, the code for network training and inference as well as the simulation tool as python code and Jupyter notebooks for easy reproducibility. KW - compressed sensing KW - AI KW - SMLM KW - FLIMbee KW - dSTORM Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299768 VL - 23 IS - 1 ER - TY - JOUR A1 - Widder, Anna A1 - Kelm, Matthias A1 - Reibetanz, Joachim A1 - Wiegering, Armin A1 - Matthes, Niels A1 - Germer, Christoph-Thomas A1 - Seyfried, Florian A1 - Flemming, Sven T1 - Robotic-assisted versus laparoscopic left hemicolectomy — postoperative inflammation status, short-term outcome and cost effectiveness JF - International Journal of Environmental Research and Public Health N2 - Robotic-assisted colon surgery may contain advantages over the laparoscopic approach, but clear evidence is sparse. This study aimed to analyze postoperative inflammation status, short-term outcome and cost-effectiveness of robotic-assisted versus laparoscopic left hemicolectomy. All consecutive patients who received minimal-invasive left hemicolectomy at the Department of Surgery I at the University Hospital of Wuerzburg in 2021 were prospectively included. Importantly, no patient selection for either procedure was carried out. The robotic-assisted versus laparoscopic approaches were compared head to head for postoperative short-term outcomes as well as cost-effectiveness. A total of 61 patients were included, with 26 patients having received a robotic-assisted approach. Baseline characteristics did not differ among the groups. Patients receiving a robotic-assisted approach had a significantly decreased length of hospital stay as well as lower rates of complications in comparison to patients who received laparoscopic surgery (n = 35). In addition, C-reactive protein as a marker of systemic stress response was significantly reduced postoperatively in patients who were operated on in a robotic-assisted manner. Consequently, robotic-assisted surgery could be performed in a cost-effective manner. Thus, robotic-assisted left hemicolectomy represents a safe and cost-effective procedure and might improve patient outcomes in comparison to laparoscopic surgery. KW - robotic surgery KW - colon resection KW - postoperative inflammation KW - cost-effectiveness KW - left hemicolectomy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286203 SN - 1660-4601 VL - 19 IS - 17 ER - TY - THES A1 - Wagner, Martin T1 - Zyto- und Gentoxizität von Zinkoxid-Nanopartikeln in humanen mesenchymalen Stammzellen nach repetitiver Exposition und im Langzeitversuch T1 - Time-Dependent Toxic and Genotoxic Effects of Zinc Oxide Nanoparticles after Long-Term and Repetitive Exposure to Human Mesenchymal Stem Cells N2 - Zinkoxid-Nanopartikel (ZnO-NP) finden in vielen Produkten des täglichen Verbrauchs Verwendung. Daten über die toxikologischen Eigenschaften von ZnO-NP werden kontrovers diskutiert. Die menschliche Haut ist in Bezug auf die ZnO-NP Exposition das wichtigste Kontakt-Organ. Intakte Haut stellt eine suffiziente Barriere gegenüber NP dar. Bei defekter Haut ist ein Kontakt zu den proliferierenden Stammzellen möglich, sodass diese als wichtiges toxikologische Ziel für NP darstellen. Das Ziel dieser Dissertation war die Bewertung der genotoxischen und zytotoxischen Effekte an humanen mesenchymalen Stammzellen (hMSC) durch niedrig dosierte ZnO-NP nach 24 stündiger Exposition, repetitiven Expositionen und im Langzeitversuch bis zu 6 Wochen. Zytotoxische Wirkungen von ZnO-NP wurden mit 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid-Test (MTT) gemessen. Darüber hinaus wurde die Genotoxizität durch den Comet-Assay bewertet. Zur Langzeitbeobachtung bis zu 6 Wochen wurde die Transmissionselektronenmikroskopie (TEM) verwendet. Zytotoxizität nach 24-stündiger ZnO-NP-Exposition war ab einer Konzentration von 50 µg/ml nachweisbar. Genotoxizität konnten bereits bei Konzentrationen von 1 und 10 µg/ml ZnO-NP beschrieben werden. Wiederholte Exposition verstärkte die Zyto-, aber nicht die Genotoxizität. Eine intrazelluläre NP-Akkumulation mit Penetration der Zellorganelle wurde bei einer Exposition bis zu 6 Wochen beobachtet. Die Ergebnisse deuten auf zytotoxische und genotoxisches Effekte von ZnO-NP hin. Bereits geringe Dosen von ZnO-NP können bei wiederholter Exposition toxische Wirkungen hervorrufen sowie eine langfristige Zellakkumulation. Diese Daten sollten bei der Verwendung von ZnO-NP an geschädigter Haut berücksichtigt werden. N2 - Zinc oxide nanoparticles (ZnO-NP) are widely used in many products of daily consumption. Data on the toxicological properties of the ZnO-NP used are discussed controversially. Human skin is the most important organ in terms of ZnO-NP exposure. Intact skin has been shown to provide an adequate barrier against NPs, while defective skin allows NP contact with proliferating cells. Among proliferating cells, stem cells are the main toxicological target for NPs. Therefore, the aim of this dissertation was to evaluate the genotoxic and cytotoxic effects of human mesenchymal stem cells (hMSC) by low-dose ZnO-NP after 24 hours of exposure, repetitive exposures and in long-term experiments up to 6 weeks. Cytotoxic effects of ZnO-NP were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT). In addition, genotoxicity was assessed by the comet assay. Transmission electron microscopy (TEM) was used for long-term observation after 6 exposure periods. The results of the study show that ZnO-NP has a cytotoxic effect starting at high concentrations of 50 µg/mL and could demonstrate genotoxic effects in hMSC exposed to 1 and 10 µg/ml ZnO-NP. Repeated exposure enhanced cytotoxicity but not genotoxicity. Intracellular NP accumulation with penetration of the cell organelles was observed at exposure up to 6 weeks. The results indicate the cytotoxic and genotoxic potential of ZnO-NP. Even small doses of ZnO-NP can cause toxic effects with repeated exposure and long-term cell accumulation. These data should be considered when using ZnO-NP on damaged skin. KW - nanoparticle KW - zinc oxid KW - stem cells KW - nanotoxicology KW - human skin KW - Nanopartikel KW - humane mesenchymale Stammzellen KW - Genotoxizität KW - Zytotoxizität KW - Repetitive Exposition KW - Elektronenmikroskopie Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275726 ER - TY - JOUR A1 - Dannhäuser, Sven A1 - Mrestani, Achmed A1 - Gundelach, Florian A1 - Pauli, Martin A1 - Komma, Fabian A1 - Kollmannsberger, Philip A1 - Sauer, Markus A1 - Heckmann, Manfred A1 - Paul, Mila M. T1 - Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation JF - Frontiers in Cellular Neuroscience N2 - Introduction Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. Methods We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Results and discussion Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13\(^{GFSTF}\) 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13\(^{GFSTF}\), Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13\(^{GFSTF}\) subclusters that move toward the AZ center during PHP with unaltered Unc-13\(^{GFSTF}\) protein levels. KW - active zone KW - Unc-13 KW - MiMIC KW - presynaptic homeostasis KW - nanoarchitecture KW - localization microscopy KW - STORM KW - HDBSCAN Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299440 SN - 1662-5102 VL - 16 ER -