TY - JOUR A1 - Krauss, Jochen A1 - Vikuk, Veronika A1 - Young, Carolyn A. A1 - Krischke, Markus A1 - Mueller, Martin J. A1 - Baerenfaller, Katja T1 - Epichloë endophyte infection rates and alkaloid content in commercially available grass seed mixtures in Europe JF - Microorganisms N2 - Fungal endophytes of the genus Epichloë live symbiotically in cool season grass species and can produce alkaloids toxic to insects and vertebrates, yet reports of intoxication of grazing animals have been rare in Europe in contrast to overseas. However, due to the beneficial resistance traits observed in Epichloë infected grasses, the inclusion of Epichloë in seed mixtures might become increasingly advantageous. Despite the toxicity of fungal alkaloids, European seed mixtures are rarely tested for Epichloë infection and their infection status is unknown for consumers. In this study, we tested 24 commercially available seed mixtures for their infection rates with Epichloë endophytes and measured the concentrations of the alkaloids ergovaline, lolitrem B, paxilline, and peramine. We detected Epichloë infections in six seed mixtures, and four contained vertebrate and insect toxic alkaloids typical for Epichloë festucae var. lolii infecting Lolium perenne. As Epichloë infected seed mixtures can harm livestock, when infected grasses become dominant in the seeded grasslands, we recommend seed producers to test and communicate Epichloë infection status or avoiding Epichloë infected seed mixtures. KW - Epichloë spp. KW - grass endophytes KW - cool-season grass species KW - infection rates KW - alkaloids KW - toxicity KW - livestock KW - horses KW - Lolium perenne KW - perennial ryegrass Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203323 SN - 2076-2607 VL - 8 IS - 4 ER - TY - JOUR A1 - Thorn, Simon A1 - Chao, Anne A1 - Georgiev, Konstadin B. A1 - Müller, Jörg A1 - Bässler, Claus A1 - Campbell, John L. A1 - Jorge, Castro A1 - Chen, Yan-Han A1 - Choi, Chang-Yong A1 - Cobb, Tyler P. A1 - Donato, Daniel C. A1 - Durska, Ewa A1 - Macdonald, Ellen A1 - Feldhaar, Heike A1 - Fontaine, Jospeh B. A1 - Fornwalt, Paula J. A1 - Hernández Hernández, Raquel María A1 - Hutto, Richard L. A1 - Koivula, Matti A1 - Lee, Eun-Jae A1 - Lindenmayer, David A1 - Mikusinski, Grzegorz A1 - Obrist, Martin K. A1 - Perlík, Michal A1 - Rost, Josep A1 - Waldron, Kaysandra A1 - Wermelinger, Beat A1 - Weiß, Ingmar A1 - Zmihorski, Michal A1 - Leverkus, Alexandro B. T1 - Estimating retention benchmarks for salvage logging to protect biodiversity JF - Nature Communications N2 - Forests are increasingly affected by natural disturbances. Subsequent salvage logging, a widespread management practice conducted predominantly to recover economic capital, produces further disturbance and impacts biodiversity worldwide. Hence, naturally disturbed forests are among the most threatened habitats in the world, with consequences for their associated biodiversity. However, there are no evidence-based benchmarks for the proportion of area of naturally disturbed forests to be excluded from salvage logging to conserve biodiversity. We apply a mixed rarefaction/extrapolation approach to a global multi-taxa dataset from disturbed forests, including birds, plants, insects and fungi, to close this gap. We find that 757% (mean +/- SD) of a naturally disturbed area of a forest needs to be left unlogged to maintain 90% richness of its unique species, whereas retaining 50% of a naturally disturbed forest unlogged maintains 73 +/- 12% of its unique species richness. These values do not change with the time elapsed since disturbance but vary considerably among taxonomic groups. Salvage logging has become a common practice to gain economic returns from naturally disturbed forests, but it could have considerable negative effects on biodiversity. Here the authors use a recently developed statistical method to estimate that ca. 75% of the naturally disturbed forest should be left unlogged to maintain 90% of the species unique to the area. KW - natural disturbance KW - bird communities KW - forest KW - management KW - beetle KW - conservation KW - windthrow KW - diversity KW - impact KW - fire Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230512 VL - 11 ER - TY - JOUR A1 - Balkenhol, Johannes A1 - Kaltdorf, Kristin V. A1 - Mammadova-Bach, Elmina A1 - Braun, Attila A1 - Nieswandt, Bernhard A1 - Dittrich, Marcus A1 - Dandekar, Thomas T1 - Comparison of the central human and mouse platelet signaling cascade by systems biological analysis JF - BMC Genomics N2 - Background Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). Results The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12. Conclusions Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences. KW - interspecies comparison KW - transcriptome KW - proteome KW - platelet KW - network KW - signaling KW - mouse KW - human KW - interactome KW - cascade Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230377 VL - 21 ER - TY - JOUR A1 - Schlegel, Jan A1 - Sauer, Markus T1 - Hochaufgelöste Visualisierung einzelner Moleküle auf ganzen Zellen JF - BIOspektrum N2 - Biological systems are dynamic and three-dimensional but many techniques allow only static and two-dimensional observation of cells. We used three-dimensional (3D) lattice light-sheet single-molecule localization microscopy (dSTORM) to investigate the complex interactions and distribution of single molecules in the plasma membrane of whole cells. Different receptor densities of the adhesion receptor CD56 at different parts of the cell highlight the importance and need of three-dimensional observation and analysis techniques. KW - Visualisierung KW - Moleküle KW - Zellen Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232365 SN - 0947-0867 VL - 7 ER - TY - JOUR A1 - Fofanov, Mikhail V. A1 - Prokopov, Dmitry Yu. A1 - Kuhl, Heiner A1 - Schartl, Manfred A1 - Trifonov, Vladimir A. T1 - Evolution of microRNA biogenesis genes in the sterlet (Acipenser ruthenus) and other polyploid vertebrates JF - International Journal of Molecular Sciences N2 - MicroRNAs play a crucial role in eukaryotic gene regulation. For a long time, only little was known about microRNA-based gene regulatory mechanisms in polyploid animal genomes due to difficulties of polyploid genome assembly. However, in recent years, several polyploid genomes of fish, amphibian, and even invertebrate species have been sequenced and assembled. Here we investigated several key microRNA-associated genes in the recently sequenced sterlet (Acipenser ruthenus) genome, whose lineage has undergone a whole genome duplication around 180 MYA. We show that two paralogs of drosha, dgcr8, xpo1, and xpo5 as well as most ago genes have been retained after the acipenserid-specific whole genome duplication, while ago1 and ago3 genes have lost one paralog. While most diploid vertebrates possess only a single copy of dicer1, we strikingly found four paralogs of this gene in the sterlet genome, derived from a tandem segmental duplication that occurred prior to the last whole genome duplication. ago1,3,4 and exportins1,5 look to be prone to additional segment duplications producing up to four-five paralog copies in ray-finned fishes. We demonstrate for the first time exon microsatellite amplification in the acipenserid drosha2 gene, resulting in a highly variable protein product, which may indicate sub- or neofunctionalization. Paralogous copies of most microRNA metabolism genes exhibit different expression profiles in various tissues and remain functional despite the rediploidization process. Subfunctionalization of microRNA processing gene paralogs may be beneficial for different pathways of microRNA metabolism. Genetic variability of microRNA processing genes may represent a substrate for natural selection, and, by increasing genetic plasticity, could facilitate adaptations to changing environments. KW - sturgeon KW - whole genome duplication KW - microRNA KW - gene duplications Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285230 SN - 1422-0067 VL - 21 IS - 24 ER - TY - JOUR A1 - Naseem, Muhammad A1 - Osmanoğlu, Özge A1 - Kaltdorf, Martin A1 - Alblooshi, Afnan Ali M. A. A1 - Iqbal, Jibran A1 - Howari, Fares M. A1 - Srivastava, Mugdha A1 - Dandekar, Thomas T1 - Integrated framework of the immune-defense transcriptional signatures in the Arabidopsis shoot apical meristem JF - International Journal of Molecular Sciences N2 - The growing tips of plants grow sterile; therefore, disease-free plants can be generated from them. How plants safeguard growing apices from pathogen infection is still a mystery. The shoot apical meristem (SAM) is one of the three stem cells niches that give rise to the above ground plant organs. This is very well explored; however, how signaling networks orchestrate immune responses against pathogen infections in the SAM remains unclear. To reconstruct a transcriptional framework of the differentially expressed genes (DEGs) pertaining to various SAM cellular populations, we acquired large-scale transcriptome datasets from the public repository Gene Expression Omnibus (GEO). We identify here distinct sets of genes for various SAM cellular populations that are enriched in immune functions, such as immune defense, pathogen infection, biotic stress, and response to salicylic acid and jasmonic acid and their biosynthetic pathways in the SAM. We further linked those immune genes to their respective proteins and identify interactions among them by mapping a transcriptome-guided SAM-interactome. Furthermore, we compared stem-cells regulated transcriptome with innate immune responses in plants showing transcriptional separation among their DEGs in Arabidopsis. Besides unleashing a repertoire of immune-related genes in the SAM, our analysis provides a SAM-interactome that will help the community in designing functional experiments to study the specific defense dynamics of the SAM-cellular populations. Moreover, our study promotes the essence of large-scale omics data re-analysis, allowing a fresh look at the SAM-cellular transcriptome repurposing data-sets for new questions. KW - defense signaling KW - shoot apical meristem KW - CLV3p KW - meta-transcriptome KW - system inference KW - stem-cell-triggered immunity Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285730 SN - 1422-0067 VL - 21 IS - 16 ER - TY - JOUR A1 - Doll, Julia A1 - Kolb, Susanne A1 - Schnapp, Linda A1 - Rad, Aboulfazl A1 - Rüschendorf, Franz A1 - Khan, Imran A1 - Adli, Abolfazl A1 - Hasanzadeh, Atefeh A1 - Liedtke, Daniel A1 - Knaup, Sabine A1 - Hofrichter, Michaela AH A1 - Müller, Tobias A1 - Dittrich, Marcus A1 - Kong, Il-Keun A1 - Kim, Hyung-Goo A1 - Haaf, Thomas A1 - Vona, Barbara T1 - Novel loss-of-function variants in CDC14A are associated with recessive sensorineural hearing loss in Iranian and Pakistani patients JF - International Journal of Molecular Sciences N2 - CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss. KW - CDC14A KW - DFNB32 KW - autosomal recessive hearing loss KW - exome sequencing KW - splicing KW - frameshift KW - non-sense mediated mRNA decay Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285142 SN - 1422-0067 VL - 21 IS - 1 ER - TY - JOUR A1 - Stojanović, Stevan D. A1 - Fuchs, Maximilian A1 - Fiedler, Jan A1 - Xiao, Ke A1 - Meinecke, Anna A1 - Just, Annette A1 - Pich, Andreas A1 - Thum, Thomas A1 - Kunz, Meik T1 - Comprehensive bioinformatics identifies key microRNA players in ATG7-deficient lung fibroblasts JF - International Journal of Molecular Sciences N2 - Background: Deficient autophagy has been recently implicated as a driver of pulmonary fibrosis, yet bioinformatics approaches to study this cellular process are lacking. Autophagy-related 5 and 7 (ATG5/ATG7) are critical elements of macro-autophagy. However, an alternative ATG5/ATG7-independent macro-autophagy pathway was recently discovered, its regulation being unknown. Using a bioinformatics proteome profiling analysis of ATG7-deficient human fibroblasts, we aimed to identify key microRNA (miR) regulators in autophagy. Method: We have generated ATG7-knockout MRC-5 fibroblasts and performed mass spectrometry to generate a large-scale proteomics dataset. We further quantified the interactions between various proteins combining bioinformatics molecular network reconstruction and functional enrichment analysis. The predicted key regulatory miRs were validated via quantitative polymerase chain reaction. Results: The functional enrichment analysis of the 26 deregulated proteins showed decreased cellular trafficking, increased mitophagy and senescence as the major overarching processes in ATG7-deficient lung fibroblasts. The 26 proteins reconstitute a protein interactome of 46 nodes and miR-regulated interactome of 834 nodes. The miR network shows three functional cluster modules around miR-16-5p, miR-17-5p and let-7a-5p related to multiple deregulated proteins. Confirming these results in a biological setting, serially passaged wild-type and autophagy-deficient fibroblasts displayed senescence-dependent expression profiles of miR-16-5p and miR-17-5p. Conclusions: We have developed a bioinformatics proteome profiling approach that successfully identifies biologically relevant miR regulators from a proteomics dataset of the ATG-7-deficient milieu in lung fibroblasts, and thus may be used to elucidate key molecular players in complex fibrotic pathological processes. The approach is not limited to a specific cell-type and disease, thus highlighting its high relevance in proteome and non-coding RNA research. KW - bioinformatics KW - miR KW - proteomics KW - functional network analysis KW - senescence KW - lung fibrosis KW - autophagy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285181 SN - 1422-0067 VL - 21 IS - 11 ER - TY - JOUR A1 - Hesselbach, Hannah A1 - Seeger, Johannes A1 - Schilcher, Felix A1 - Ankenbrand, Markus A1 - Scheiner, Ricarda T1 - Chronic exposure to the pesticide flupyradifurone can lead to premature onset of foraging in honeybees Apis mellifera JF - Journal of Applied Ecology N2 - 1.Honeybees Apis mellifera and other pollinating insects suffer from pesticides in agricultural landscapes. Flupyradifurone is the active ingredient of a novel pesticide by the name of ‘Sivanto’, introduced by Bayer AG (Crop Science Division, Monheim am Rhein, Germany). It is recommended against sucking insects and marketed as ‘harmless’ to honeybees. Flupyradifurone binds to nicotinergic acetylcholine receptors like neonicotinoids, but it has a different mode of action. So far, little is known on how sublethal flupyradifurone doses affect honeybees. 2. We chronically applied a sublethal and field‐realistic concentration of flupyradifurone to test for long‐term effects on flight behaviour using radio‐frequency identification. We examined haematoxylin/eosin‐stained brains of flupyradifurone‐treated bees to investigate possible changes in brain morphology and brain damage. 3. A field‐realistic flupyradifurone dose of approximately 1.0 μg/bee/day significantly increased mortality. Pesticide‐treated bees initiated foraging earlier than control bees. No morphological damage in the brain was observed. 4. Synthesis and applications. The early onset of foraging induced by a chronical application of flupyradifurone could be disadvantageous for honeybee colonies, reducing the period of in‐hive tasks and life expectancy of individuals. Radio‐frequency identification technology is a valuable tool for studying pesticide effects on lifetime foraging behaviour of insects. KW - radiofrequency identification KW - flight behaviour KW - flupyradifurone KW - foraging KW - histology KW - honeybee KW - insecticide KW - mortality Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212769 VL - 57 IS - 3 ER - TY - JOUR A1 - Sajko, Sara A1 - Grishkovskaya, Irina A1 - Kostan, Julius A1 - Graewert, Melissa A1 - Setiawan, Kim A1 - Trübestein, Linda A1 - Niedermüller, Korbinian A1 - Gehin, Charlotte A1 - Sponga, Antonio A1 - Puchinger, Martin A1 - Gavin, Anne-Claude A1 - Leonard, Thomas A. A1 - Svergun, Dimitri I. A1 - Smith, Terry K. A1 - Morriswood, Brooke A1 - Djinovic-Carugo, Kristina T1 - Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats JF - PLoS One N2 - MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation. KW - recognition nexus domain KW - trypanosoma brucei KW - blood stream KW - phosphatidylserine transport KW - biological macromolecules KW - membrane occupation KW - solution scattering KW - molecular cloning KW - flagellar pocket KW - endocytosis Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231261 VL - 15 IS - 23 ER - TY - JOUR A1 - Krauss, Jochen A1 - Vikuk, Veronika A1 - Young, Carolyn A. A1 - Krischke, Markus A1 - Mueller, Martin J. A1 - Baerenfaller, Katja T1 - Correction: Krauss, J., et al. Epichloë endophyte infection rates and alkaloid content in commercially available grass seed mixtures in Europe. Microorganisms 2020, 8, 498 JF - Microorganisms N2 - No abstract available. KW - Epichloë KW - endophyte Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216254 SN - 2076-2607 VL - 8 IS - 10 ER - TY - JOUR A1 - Baur, Florentin A1 - Nietzer, Sarah L. A1 - Kunz, Meik A1 - Saal, Fabian A1 - Jeromin, Julian A1 - Matschos, Stephanie A1 - Linnebacher, Michael A1 - Walles, Heike A1 - Dandekar, Thomas A1 - Dandekar, Gudrun T1 - Connecting cancer pathways to tumor engines: a stratification tool for colorectal cancer combining human in vitro tissue models with boolean in silico models JF - Cancers N2 - To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification. KW - in silico simulation KW - 3D tissue models KW - colorectal cancer KW - BRAF mutation KW - targeted therapy KW - stratification Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193798 SN - 2072-6694 VL - 12 IS - 1 ER - TY - JOUR A1 - Beer, Katharina A1 - Helfrich-Förster, Charlotte T1 - Model and Non-model Insects in Chronobiology JF - Frontiers in Behavioral Neuroscience N2 - The fruit fly Drosophila melanogaster is an established model organism in chronobiology, because genetic manipulation and breeding in the laboratory are easy. The circadian clock neuroanatomy in D. melanogaster is one of the best-known clock networks in insects and basic circadian behavior has been characterized in detail in this insect. Another model in chronobiology is the honey bee Apis mellifera, of which diurnal foraging behavior has been described already in the early twentieth century. A. mellifera hallmarks the research on the interplay between the clock and sociality and complex behaviors like sun compass navigation and time-place-learning. Nevertheless, there are aspects of clock structure and function, like for example the role of the clock in photoperiodism and diapause, which can be only insufficiently investigated in these two models. Unlike high-latitude flies such as Chymomyza costata or D. ezoana, cosmopolitan D. melanogaster flies do not display a photoperiodic diapause. Similarly, A. mellifera bees do not go into “real” diapause, but most solitary bee species exhibit an obligatory diapause. Furthermore, sociality evolved in different Hymenoptera independently, wherefore it might be misleading to study the social clock only in one social insect. Consequently, additional research on non-model insects is required to understand the circadian clock in Diptera and Hymenoptera. In this review, we introduce the two chronobiology model insects D. melanogaster and A. mellifera, compare them with other insects and show their advantages and limitations as general models for insect circadian clocks. KW - circadian clock KW - complex behavior KW - diapause KW - sociality KW - Drosophila melanogaster KW - Apis mellifera Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-218721 SN - 1662-5153 VL - 14 ER - TY - JOUR A1 - Bae, Soyeon A1 - Heidrich, Lea A1 - Levick, Shaun R. A1 - Gossner, Martin M. A1 - Seibold, Sebastian A1 - Weisser, Wolfgang W. A1 - Magdon, Paul A1 - Serebryanyk, Alla A1 - Bässler, Claus A1 - Schäfer, Deborah A1 - Schulze, Ernst-Detlef A1 - Doerfler, Inken A1 - Müller, Jörg A1 - Jung, Kirsten A1 - Heurich, Marco A1 - Fischer, Markus A1 - Roth, Nicolas A1 - Schall, Peter A1 - Boch, Steffen A1 - Wöllauer, Stephan A1 - Renner, Swen C. A1 - Müller, Jörg T1 - Dispersal ability, trophic position and body size mediate species turnover processes: Insights from a multi-taxa and multi-scale approach JF - Diversity and Distribution N2 - Aim: Despite increasing interest in β-diversity, that is the spatial and temporal turnover of species, the mechanisms underlying species turnover at different spatial scales are not fully understood, although they likely differ among different functional groups. We investigated the relative importance of dispersal limitations and the environmental filtering caused by vegetation for local, multi-taxa forest communities differing in their dispersal ability, trophic position and body size. Location: Temperate forests in five regions across Germany. Methods: In the inter-region analysis, the independent and shared effects of the regional spatial structure (regional species pool), landscape spatial structure (dispersal limitation) and environmental factors on species turnover were quantified with a 1-ha grain across 11 functional groups in up to 495 plots by variation partitioning. In the intra-region analysis, the relative importance of three environmental factors related to vegetation (herb and tree layer composition and forest physiognomy) and spatial structure for species turnover was determined. Results: In the inter-region analysis, over half of the explained variation in community composition (23% of the total explained 35%) was explained by the shared effects of several factors, indicative of spatially structured environmental filtering. Among the independent effects, environmental factors were the strongest on average over 11 groups, but the importance of landscape spatial structure increased for less dispersive functional groups. In the intra-region analysis, the independent effect of plant species composition had a stronger influence on species turnover than forest physiognomy, but the relative importance of the latter increased with increasing trophic position and body size. Main conclusions: Our study revealed that the mechanisms structuring assemblage composition are associated with the traits of functional groups. Hence, conservation frameworks targeting biodiversity of multiple groups should cover both environmental and biogeographical gradients. Within regions, forest management can enhance β-diversity particularly by diversifying tree species composition and forest physiognomy. KW - body size KW - dispersal ability KW - environmental filtering KW - forest physiognomy KW - neutral processes KW - plant composition KW - regional species pool KW - species turnover KW - trophic position KW - β-diversity Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236117 VL - 27 IS - 3 ER - TY - JOUR A1 - Jessen, Christina A1 - Kreß, Julia K. C. A1 - Baluapuri, Apoorva A1 - Hufnagel, Anita A1 - Schmitz, Werner A1 - Kneitz, Susanne A1 - Roth, Sabine A1 - Marquardt, André A1 - Appenzeller, Silke A1 - Ade, Casten P. A1 - Glutsch, Valerie A1 - Wobser, Marion A1 - Friedmann-Angeli, José Pedro A1 - Mosteo, Laura A1 - Goding, Colin R. A1 - Schilling, Bastian A1 - Geissinger, Eva A1 - Wolf, Elmar A1 - Meierjohann, Svenja T1 - The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression JF - Oncogene N2 - The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H\(_2\)O\(_2\) or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma. KW - NRF2 KW - melanoma malignancy KW - COX2 expression Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235064 SN - 0950-9232 VL - 39 ER - TY - JOUR A1 - Isaacs, Darren A1 - Mikasi, Sello Given A1 - Obasa, Adetayo Emmanuel A1 - Ikomey, George Mondinde A1 - Shityakov, Sergey A1 - Cloete, Ruben A1 - Jacobs, Graeme Brendon T1 - Structural comparison of diverse HIV-1 subtypes using molecular modelling and docking analyses of integrase inhibitors JF - Viruses N2 - The process of viral integration into the host genome is an essential step of the HIV-1 life cycle. The viral integrase (IN) enzyme catalyzes integration. IN is an ideal therapeutic enzyme targeted by several drugs; raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and bictegravir (BIC) having been approved by the USA Food and Drug Administration (FDA). Due to high HIV-1 diversity, it is not well understood how specific naturally occurring polymorphisms (NOPs) in IN may affect the structure/function and binding affinity of integrase strand transfer inhibitors (INSTIs). We applied computational methods of molecular modelling and docking to analyze the effect of NOPs on the full-length IN structure and INSTI binding. We identified 13 NOPs within the Cameroonian-derived CRF02_AG IN sequences and further identified 17 NOPs within HIV-1C South African sequences. The NOPs in the IN structures did not show any differences in INSTI binding affinity. However, linear regression analysis revealed a positive correlation between the Ki and EC50 values for DTG and BIC as strong inhibitors of HIV-1 IN subtypes. All INSTIs are clinically effective against diverse HIV-1 strains from INSTI treatment-naïve populations. This study supports the use of second-generation INSTIs such as DTG and BIC as part of first-line combination antiretroviral therapy (cART) regimens, due to a stronger genetic barrier to the emergence of drug resistance. KW - integrase KW - naturally occurring polymorphisms KW - HIV-1 KW - molecular modelling KW - molecular docking KW - diversity Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211170 SN - 1999-4915 VL - 12 IS - 9 ER - TY - THES A1 - Lakovic, Milica T1 - Evolution of animal dispersal: Putting timing in perspective T1 - Evolution von Ausbreitungsstrategien: Die Fitnesskonsequenzen des Zeitpunkts von Emigration N2 - Dispersal is a life-history trait affecting dynamics and persistence of populations; it evolves under various known selective pressures. Theoretical studies on dispersal typically assume 'natal dispersal', where individuals emigrate right after birth. But emigration may also occur during a later moment within a reproductive season ('breeding dispersal'). For example, some female butterflies first deposit eggs in their natal patch before migrating to other site(s) to continue egg-laying there. How breeding compared to natal dispersal influences the evolution of dispersal has not been explored. To close this gap we used an individual-based simulation approach to analyze (i) the evolution of timing of breeding dispersal in annual organisms, (ii) its influence on dispersal (compared to natal dispersal). Furthermore, we tested (iii) its performance in direct evolutionary contest with individuals following a natal dispersal strategy. Our results show that evolution should typically result in lower dispersal under breeding dispersal, especially when costs of dispersal are low and population size is small. By distributing offspring evenly across two patches, breeding dispersal allows reducing direct sibling competition in the next generation whereas natal dispersal can only reduce trans-generational kin competition by producing highly dispersive offspring in each generation. The added benefit of breeding dispersal is most prominent in patches with small population sizes. Finally, the evolutionary contests show that a breeding dispersal strategy would universally out-compete natal dispersal. N2 - Emigration und die daraus resultierende Ausbreitung („dispersal“) ist ein wichtiges Ereignis im Lebenszyklus von Insekten, mit grundlegenden öko-evolutionären Folgen. Fortschreitender globaler Wandel hinterlässt viele Arten in stark fragmentierten Habitaten; der Verbreitungsstrategie kommt deshalb eine Schlüsselrolle im Fortbestehen von Populationen zu. Insekten sind besonders anfällig gegenüber Habitatzerstörungen, da viele von ihnen Spezialisten sind und daher z.B. stark von Präsenz bestimmter Wirtsarten und deren Verteilung abhängen. Zum Schutz dieser Arten ist es folglich entscheidend die Ursachen und Folgen verschiedener Ausbreitungsstrategien zu verstehen. Zudem können Arten mit unterschiedlichen Lebenszyklen spezifische Ausbreitungsstrategien aufweisen. Natale Emigration („natal dispersal“) ist definiert als das Verlassen des Ortes der Geburt, um an einem neuen Ort zu reproduzieren, während „breeding dispersal“ Ausbreitung zwischen zwei aufeinanderfolgenden Paarungen bedeutet. Natal dispersal kann während des Larval- und Adultstadiums stattfinden, breeding dispersal nur während des Adultstadiums. Weiterhin ist der Zeitpunkt der Verpaarung, entweder vor oder nach Ausbreitung, besonders wichtig für Weibchen, die nicht nur die eigenen Gene transportieren, sondern eventuell auch die eines verpaarten Männchens. Es ist eindeutig, dass sich Genfluss und ökoevolutionäre Dynamik zwischen diesen Ausbreitungsstrategien unterscheiden. Schließlich erhielt nformationsverarbeitung durch Insekten und dessen Rolle in emigrationsbezogenen Entscheidungen in jüngster Zeit viel Aufmerksamkeit. Dennoch wurde der Zeitraum der Informationsbeschaffung (z.B. während des Larven- oder Adultstadiums) und folglich die Verfügbarkeit von Information zum Zeitpunkt der Emigration von Theoretikern und Empirikern größtenteils nicht beachtet. Meine Doktorarbeit liefert theoretische Einsichten in den optimalen Zeitpunkt der Emigration, des Zeitpunktes der Paarung (in Relation zu Emigration) und die Rolle von Informationsbeschaffung in Insekten- Metapopulationen. Mit Individuen basierten Modellen analysierte ich zuerst die Evolution des Emigrationszeitpunktes in Metapopulationen, gefolgt von der Evolution des (optimalen) Emigrations- und Paarungszeitpunktes in Metapopulationen von Insekten. Abschließend untersuchte ich, wie sich die Investition von Zeit in das Sammeln von Informationen auf den Zeitpunkt und die Häufigkeit von Emigration auswirkt. Ergebnisse meiner Thesis zeigen, dass die Vermeidung von Konkurrenz innerhalb der Art eine entscheidende Rolle in der Evolution des Zeitpunktes der Emigration einnimmt; weiterhin konnte ich zeigen, dass Insekten Informationen über die Populationsdichte nutzen können, um daran angepasst Entscheidungen bezüglich ihrer Emigration zu treffen; in heterogener Umwelt bestimmt die Toleranz gegenüber der Habitate die Evolution der Ausbreitungsstrategie und des Paarungszeitpunktes, was folglich die lokal Anpassung innerhalb ganzer Landschaften bestimmt. Meine Thesis bietet neue Einsichten in die Evolution von Ausbreitung, insbesondere auf den richtigen Zeitpunkt und die Reihenfolge von Emigration, Verpaarung und dem Sammeln von Informationen. Dieser Aspekt des Timings wurde bisher von theoretischen und empirischen Ökologen größtenteils ignoriert. Um die Populationsdynamik und die Ausbreitung einer Art verstehen zu können, ist es essentiell den Lebenszyklus und die Zeitpunkte der wichtigsten Lebensereignisse (Verbreitung, Reproduktion) zu kennen. Dies ist zwingend nötig, wenn eine erfolgreiche Umsetzung von Naturschutzmaßnahmen (z.B. Wiedereinführung von Arten) oder biologischer Schädlingsbekämpfung (z.B. Einführung von Prädatoren zur Bekämpfung von Schädlingen) angestrebt wird. KW - dispersal timing KW - metapopulation KW - individual-based simulation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-154522 ER - TY - THES A1 - Kaltdorf, Martin Ernst T1 - Analyse von regulatorischen Netzwerken bei Zelldifferenzierung und in der Infektionsbiologie T1 - Analysis of Regulatory Networks during Cell Differentiation and in Infection Biology N2 - Das zentrale Paradigma der Systembiologie zielt auf ein möglichst umfassendes Ver-ständnis der komplexen Zusammenhänge biologischer Systeme. Die in dieser Arbeit angewandten Methoden folgen diesem Grundsatz. Am Beispiel von drei auf Basis von Datenbanken und aktueller Literatur rekonstruier-ten Netzwerkmodellen konnte in der hier vorliegenden Arbeit die Gültigkeit analyti-scher und prädiktiver Algorithmen nachgewiesen werden, die in Form der Analy-sesoftware Jimena angewandt wurden. Die daraus resultierenden Ergebnisse sowohl für die Berechnung von stabilen Systemzuständen, der dynamischen Simulation, als auch der Identifikation zentraler Kontrollknoten konnten experimentell validiert wer-den. Die Ergebnisse wurden in einem iterativen Prozess verwendet werden um das entsprechende Netzwerkmodell zu optimieren. Beim Vergleich des Verhaltens des semiquantitativ ausgewerteten regulatorischen Netzwerks zur Kontrolle der Differenzierung humaner mesenchymaler Stammzellen in Chondrozyten (Knorpelbildung), Osteoblasten (Knochenbildung) und Adipozyten (Fett-zellbildung) konnten 12 wichtige Faktoren (darunter: RUNX2, OSX/SP7, SOX9, TP53) mit Hilfe der Berechnung der Bedeutung (Kontrollzentralität der Netzwerkknoten identifi-ziert werden). Der Abgleich des simulierten Verhaltens dieses Netzwerkes ergab eine Übereinstimmung mit experimentellen Daten von 47,2%, bei einem widersprüchlichen Verhalten von ca. 25%, dass unter anderem durch die temporäre Natur experimentel-ler Messungen im Vergleich zu den terminalen Bedingungen des Berechnung der stabilen Systemzustände erklärt werden kann. Bei der Analyse des Netzwerkmodells der menschlichen Immunantwort auf eine Infek-tion durch A. fumigatus konnten vier Hauptregulatoren identifiziert werden (A. fumi-gatus, Blutplättchen, hier Platelets genannt, und TNF), die im Zusammenspiel mit wei-teren Faktoren mit hohen Zentralitätswerten (CCL5, IL1, IL6, Dectin-1, TLR2 und TLR4) fähig sind das gesamte Netzwerkverhalten zu beeinflussen. Es konnte gezeigt werden, dass sich das Aktivitätsverhalten von IL6 in Reaktion auf A. fumigatus und die regulato-rische Wirkung von Blutplättchen mit den entsprechenden experimentellen Resultaten deckt. Die Simulation, sowie die Berechnung der stabilen Systemzustände der Immunantwort von A. thaliana auf eine Infektion durch Pseudomonas syringae konnte zeigen, dass die in silico Ergebnisse mit den experimentellen Ergebnissen übereinstimmen. Zusätzlich konnten mit Hilfe der Analyse der Zentralitätswerte des Netzwerkmodells fünf Master-regulatoren identifiziert werden: TGA Transkriptionsfaktor, Jasmonsäure, Ent-Kaurenoate-Oxidase, Ent-kaurene-Synthase und Aspartat-Semialdehyd-Dehydrogenase. Während die ersteren beiden bereits lange als wichtige Regulatoren für die Gib-berellin-Synthese bekannt sind, ist die immunregulatorische Funktion von Aspartat-Semialdehyd-Dehydrogenase bisher weitgehend unbekannt. N2 - The central paradigm of systems biology aims at a comprehensive understanding in complex relationships of biological systems. The methods used in this work support this aim. By the example of three network models reconstructed on the basis of databases and current literature, the validity of analytical and predictive algorithms could be demon-strated in this work. As simulation software the framework Jimena was applied. The results for the calculation of stable system states, the dynamic simulation as well as the identification of central control nodes could be validated experimentally. The re-sults were used in an iterative process to further optimize the corresponding network model. Comparing the behavior of the semi-quantitatively evaluated regulatory network to control the differentiation of human mesenchymal stem cells into chondrocytes (carti-lage formation), osteoblasts (bone formation) and adipocytes (fatty cell formation), 12 important factors (including: RUNX2, OSX/SP7, SOX9, TP53) could be identified by the calculation of the control centralities of the network nodes. The comparison of the simulated behavior of these nodes showed an agreement with experimental data of 47.2%. We found a contradictory behavior of approximately 25%. Differing results can be explained due to the temporary nature of experimental measurements compared to the terminal conditions of the calculation the stable system states. Analyzing the network model of the human immune response to A. fumigatus infec-tion, four major regulators could be identified (A. fumigatus, platelets, and TNF), which interact with other factors with high control centrality values (CCL5, IL1, IL6, Dectin1). TLR2 and TLR4) are capable of affecting the overall network behavior. It could be shown that the activity behavior of IL6 in response to the modular activity of the plate-lets as well as A. fumigatus coincides with the corresponding experimental results. The simulation, as well as the calculation of the stable system states of the immune response of A. thaliana to an infection by Pseudomonas syringae, showed that in silico results are in agreement with the experimental results. By analyzing the control cen-trality values of the network model, five main regulators could be: TGA transcription factor, jasmonic acid, ent-kaurene-Oxidase, ent-kaurene synthase and aspartate semi-aldehyde. While the former two have long been recognized as important regulators of gibberel-lin synthesis, the immunoregulatory function of aspartate semialdehyde dehydrogen-ase has been largely unknown. KW - Netzwerksimulation KW - Immunbiologie KW - Zelldifferenzierung KW - Systembiologie KW - Signaltransduktion KW - Mesenchymale Stammzelldifferenzierung KW - Netzwerkrekonstruktion KW - network simulation KW - network reconstruction KW - immunology KW - mesenchymal stem cell differentiation KW - signal transduction Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-198526 ER - TY - JOUR A1 - Floren, Andreas A1 - von Rintelen, Thomas A1 - Herbert, Paul D. N. A1 - de Araujo, Bruno Cancian A1 - Schmidt, Stefan A1 - Balke, Michael A1 - Narakusumo, Raden Pramesa A1 - Peggie, Djunijanti A1 - Ubaidillah, Rosichon A1 - von Rintelen, Kristina A1 - Müller, Tobias T1 - Integrative ecological and molecular analysis indicate high diversity and strict elevational separation of canopy beetles in tropical mountain forests JF - Scientific Reports N2 - Tropical mountain forests contribute disproportionately to terrestrial biodiversity but little is known about insect diversity in the canopy and how it is distributed between tree species. We sampled tree-specific arthropod communities from 28 trees by canopy fogging and analysed beetle communities which were first morphotyped and then identified by their DNA barcodes. Our results show that communities from forests at 1100 and 1700 m a.s.l. are almost completely distinct. Diversity was much lower in the upper forest while community structure changed from many rare, less abundant species to communities with a pronounced dominance structure. We also found significantly higher beta-diversity between trees at the lower than higher elevation forest where community similarity was high. Comparisons on tree species found at both elevations reinforced these results. There was little species overlap between sites indicating limited elevational ranges. Furthermore, we exploited the advantage of DNA barcodes to patterns of haplotype diversity in some of the commoner species. Our results support the advantage of fogging and DNA barcodes for community studies and underline the need for comprehensive research aimed at the preservation of these last remaining pristine forests. KW - beta-diversity KW - community data KW - gradients KW - insects KW - hypthesis KW - evolution KW - passes KW - ants Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230565 VL - 10 ER - TY - JOUR A1 - Götz, Ralph A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Solger, Franziska A1 - Schlegel, Jan A1 - Seibel, Jürgen A1 - Kozjak-Pavlovic, Vera A1 - Rudel, Thomas A1 - Sauer, Markus T1 - Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy JF - Nature Communications N2 - Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 +/- 7.7nm. Imaging of lipid bilayers using light microscopy is challenging. Here the authors label cells using a short chain click-compatible ceramide to visualize mammalian and bacterial membranes with expansion microscopy. KW - nanoscale imaging KW - bacterial infection KW - sphingolipid expansion microscopy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231248 VL - 11 ER - TY - JOUR A1 - Diers, J. A1 - Wagner, J. A1 - Baum, P. A1 - Lichthardt, S. A1 - Kastner, C. A1 - Matthes, N. A1 - Matthes, H. A1 - Germer, C.‐T. A1 - Löb, S. A1 - Wiegering, A. T1 - Nationwide in‐hospital mortality rate following rectal resection for rectal cancer according to annual hospital volume in Germany JF - BJS Open N2 - Background The impact of hospital volume after rectal cancer surgery is seldom investigated. This study aimed to analyse the impact of annual rectal cancer surgery cases per hospital on postoperative mortality and failure to rescue. Methods All patients diagnosed with rectal cancer and who had a rectal resection procedure code from 2012 to 2015 were identified from nationwide administrative hospital data. Hospitals were grouped into five quintiles according to caseload. The absolute number of patients, postoperative deaths and failure to rescue (defined as in‐hospital mortality after a documented postoperative complication) for severe postoperative complications were determined. Results Some 64 349 patients were identified. The overall in‐house mortality rate was 3·9 per cent. The crude in‐hospital mortality rate ranged from 5·3 per cent in very low‐volume hospitals to 2·6 per cent in very high‐volume centres, with a distinct trend between volume categories (P < 0·001). In multivariable logistic regression analysis using hospital volume as random effect, very high‐volume hospitals (53 interventions/year) had a risk‐adjusted odds ratio of 0·58 (95 per cent c.i. 0·47 to 0·73), compared with the baseline in‐house mortality rate in very low‐volume hospitals (6 interventions per year) (P < 0·001). The overall postoperative complication rate was comparable between different volume quintiles, but failure to rescue decreased significantly with increasing caseload (15·6 per cent after pulmonary embolism in the highest volume quintile versus 38 per cent in the lowest quintile; P = 0·010). Conclusion Patients who had rectal cancer surgery in high‐volume hospitals showed better outcomes and reduced failure to rescue rates for severe complications than those treated in low‐volume hospitals. KW - rectal resection KW - rectal cancer KW - mortality rate Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212878 VL - 4 IS - 2 SP - 310 EP - 319 ER - TY - JOUR A1 - Polidori, Carlo A1 - Ballesteros, Yolanda A1 - Wurdack, Mareike A1 - Asís, Josep Daniel A1 - Tormos, José A1 - Baños-Picón, Laura A1 - Schmitt, Thomas T1 - Low host specialization in the cuckoo wasp, Parnopes grandior, weakens chemical mimicry but does not lead to local adaption JF - Insects N2 - Insect brood parasites have evolved a variety of strategies to avoid being detected by their hosts. Few previous studies on cuckoo wasps (Hymenoptera: Chrysididae), which are natural enemies of solitary wasps and bees, have shown that chemical mimicry, i.e., the biosynthesis of cuticular hydrocarbons (CHC) that match the host profile, evolved in several species. However, mimicry was not detected in all investigated host-parasite pairs. The effect of host range as a second factor that may play a role in evolution of mimicry has been neglected, since all previous studies were carried out on host specialists and at nesting sites where only one host species occurred. Here we studied the cuckoo wasp Parnopes grandior, which attacks many digger wasp species of the genus Bembix (Hymenoptera: Crabronidae). Given its weak host specialization, P. grandior may either locally adapt by increasing mimicry precision to only one of the sympatric hosts or it may evolve chemical insignificance by reducing the CHC profile complexity and/or CHCs amounts. At a study site harbouring three host species, we found evidence for a weak but appreciable chemical deception strategy in P. grandior. Indeed, the CHC profile of P. grandior was more similar to all sympatric Bembix species than to a non-host wasp species belonging to the same tribe as Bembix. Furthermore, P. grandior CHC profile was equally distant to all the hosts' CHC profiles, thus not pointing towards local adaptation of the CHC profile to one of the hosts' profile. We conducted behavioural assays suggesting that such weak mimicry is sufficient to reduce host aggression, even in absence of an insignificance strategy, which was not detected. Hence, we finally concluded that host range may indeed play a role in shaping the level of chemical mimicry in cuckoo wasps. KW - Chrysididae KW - Bembix KW - chemical mimicry KW - cuticular hydrocarbons Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200651 SN - 2075-4450 VL - 11 IS - 2 ER - TY - JOUR A1 - Rabl, Dominik A1 - Alonso-Rodríguez, Aura M. A1 - Brehm, Gunnar A1 - Fiedler, Konrad T1 - Trait variation in moths mirrors small-scaled ecological gradients in a tropical forest landscape JF - Insects N2 - Along environmental gradients, communities are expected to be filtered from the regional species pool by physical constraints, resource availability, and biotic interactions. This should be reflected in species trait composition. Using data on species-rich moth assemblages sampled by light traps in a lowland rainforest landscape in Costa Rica, we show that moths in two unrelated clades (Erebidae-Arctiinae; Geometridae) are much smaller-sized in oil palm plantations than in nearby old-growth forest, with intermediate values at disturbed forest sites. In old-growth forest, Arctiinae predominantly show aposematic coloration as a means of anti-predator defense, whereas this trait is much reduced in the prevalence in plantations. Similarly, participation in Müllerian mimicry rings with Hymenoptera and Lycidae beetles, respectively, is rare in plantations. Across three topographic types of old-growth forests, community-weighted means of moth traits showed little variation, but in creek forest, both types of mimicry were surprisingly rare. Our results emphasize that despite their mobility, moth assemblages are strongly shaped by local environmental conditions through the interplay of bottom–up and top–down processes. Assemblages in oil palm plantations are highly degraded not only in their biodiversity, but also in terms of trait expression. KW - Costa Rica KW - body size KW - mimicry rings KW - aposematism KW - oil palm plantations KW - lowland rainforest Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213016 SN - 2075-4450 VL - 11 IS - 9 ER - TY - JOUR A1 - Scheiner, Ricarda A1 - Strauß, Sina A1 - Thamm, Markus A1 - Farré-Armengol, Gerard A1 - Junker, Robert R. T1 - The bacterium Pantoea ananatis modifies behavioral responses to sugar solutions in honeybees JF - Insects N2 - 1. Honeybees, which are among the most important pollinators globally, do not only collect pollen and nectar during foraging but may also disperse diverse microbes. Some of these can be deleterious to agricultural crops and forest trees, such as the bacterium Pantoea ananatis, an emerging pathogen in some systems. P. ananatis infections can lead to leaf blotches, die-back, bulb rot, and fruit rot. 2. We isolated P. ananatis bacteria from flowers with the aim of determining whether honeybees can sense these bacteria and if the bacteria affect behavioral responses of the bees to sugar solutions. 3. Honeybees decreased their responsiveness to different sugar solutions when these contained high concentrations of P. ananatis but were not deterred by solutions from which bacteria had been removed. This suggests that their reduced responsiveness was due to the taste of bacteria and not to the depletion of sugar in the solution or bacteria metabolites. Intriguingly, the bees appeared not to taste ecologically relevant low concentrations of bacteria. 4. Synthesis and applications. Our data suggest that honeybees may introduce P.ananatis bacteria into nectar in field-realistic densities during foraging trips and may thus affect nectar quality and plant fitness. KW - plant bacteria KW - bacterial spread KW - sucrose responsiveness KW - Apis mellifera Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216247 SN - 2075-4450 VL - 11 IS - 10 ER - TY - JOUR A1 - Venjakob, Christine A1 - Leonhardt, Sara A1 - Klein, Alexandra-Maria T1 - Inter-individual nectar chemistry changes of field scabious, Knautia arvensis JF - Insects N2 - Nectar is crucial to maintain plant-pollinator mutualism. Nectar quality (nutritional composition) can vary strongly between individuals of the same plant species. The factors driving such inter-individual variation have however not been investigated closer. We investigated nectar quality of field scabious, Knautia arvensis in different grassland plant communities varying in species composition and richness to assess whether nectar quality can be affected by the surrounding plant community. We analyzed (with high performance liquid chromatography) the content of carbohydrates, overall amino acids, and essential amino acids. Amino acid and carbohydrate concentrations and proportions varied among plant individuals and with the surrounding plant community but were not related to the surrounding plant species richness. Total and individual carbohydrate concentrations were lowest, while proportions of the essential amino acids, valine, isoleucine, leucine (all phagostimulatory), and lysine were highest in plant species communities of the highest diversity. Our results show that K. arvensis nectar chemistry varies with the composition of the surrounding plant community, which may alter the taste and nutritional value and thus affect the plant’s visitor spectrum and visitation rate. However, the strong inter-individual variation in nectar quality requires additional studies (e.g., in semi-field studies) to disentangle different biotic and abiotic factors contributing to inter-individual nectar chemistry in a plant-community context. KW - amino acids KW - carbohydrates KW - flower-visiting insects KW - insect nutrition KW - Jena Experiment Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200866 SN - 2075-4450 VL - 11 IS - 2 ER - TY - JOUR A1 - Jahed, Razieh Rafiei A1 - Kavousi, Mohammad Reza A1 - Farashiani, Mohammad Ebrahim A1 - Sagheb-Talebi, Khosro A1 - Babanezhad, Manoochehr A1 - Courbaud, Benoit A1 - Wirtz, Roland A1 - Müller, Jörg A1 - Larrieu, Laurent T1 - A comparison of the formation rates and composition of tree-related microhabitats in beech-dominated primeval Carpathian and Hyrcanian forests JF - Forests N2 - Primeval forests in the temperate zone exist only as a few remnants, but theses serve as important reference areas for conservation. As key habitats, tree-related microhabitats (TreMs) are of intense interest to forest ecologists, but little is known about their natural composition and dynamics in different tree species. Beech forms a major part of the temperate forests that extend from Europe, home to European beech Fagus sylvatica L. (Fs), eastward to Iran, where Oriental beech Fagus orientalis Lipsky (Fo) is the dominant species. In this study, we compared TreMs in primeval forests of both species, using data from Fo growing in 25 inventory plots throughout the Hyrcanian forest belt in Iran and from Fs growing in a 9 ha permanent plot in the Uholka Forest of Ukraine. TreMs based on 47 types and 11 subgroups were recorded. Beech trees in the Hyrcanian forest had a higher mean diameter at breast height (dbh) than beech trees in Uholka and contained twice as many TreMs per hectare. Although the mean richness of TreMs per TreM bearing tree was similar in the two species, on the basis of the comparison single trees in two groups (n = 405 vs. 2251), the composition of the TreMs clearly differed, as the proportions of rot holes, root-buttress concavities, and crown deadwood were higher in the Hyrcanian Forest, and those of bark losses, exposed heartwood, and burrs and cankers higher in Uholka Forest. Estimates of TreMs dynamics based on dbh and using Weibull models showed a significantly faster cumulative increase of TreMs in Fo, in which saturation occurred already in trees with a dbh of 70–80 cm. By contrast, the increase in TreMs in Fs was continuous. In both species, the probability density was highest at a dbh of about 30 cm, but was twice as high in Fo. Because of limitations of our study design, the reason behind observed differences of TreM formation and composition between regions remains unclear, as it could be either result of the tree species or the environment, or their interaction. However, the observed differences were more likely the result of differences in the environment than in the two tree species. Nevertheless, our findings demonstrate that the Hyrcanian Forest, recently designated as a natural heritage site in Iran, is unique, not only as a tertiary relict or due to its endemic trees, herbs and arthropods, but also because of its TreMs, which form a distinct and rich habitat for associated taxa, including endemic saproxylic species. KW - TreMs KW - Fagus orientalis KW - Fagus sylvatica KW - primeval forest Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200849 SN - 1999-4907 VL - 11 IS - 2 ER - TY - JOUR A1 - Oelschlaegel, Diana A1 - Weiss Sadan, Tommy A1 - Salpeter, Seth A1 - Krug, Sebastian A1 - Blum, Galia A1 - Schmitz, Werner A1 - Schulze, Almut A1 - Michl, Patrick T1 - Cathepsin inhibition modulates metabolism and polarization of tumor-associated macrophages JF - Cancers N2 - Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH\(_2\), led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE\(_2\), which correlated with the upregulation of numerous NF\(_k\)B-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu. KW - cathepsin KW - activity-based probes KW - tumor-associated macrophage KW - autophagy KW - lysosome KW - lipid metabolism KW - inflammation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213040 SN - 2072-6694 VL - 12 IS - 9 ER - TY - JOUR A1 - Leonhardt, Sara D. A1 - Lihoreau, Mathieu A1 - Spaethe, Johannes T1 - Mechanisms of nutritional resource exploitation by insects JF - Insects N2 - Insects have evolved an extraordinary range of nutritional adaptations to exploit other animals, plants, bacteria, fungi and soils as resources in terrestrial and aquatic environments. This special issue provides some new insights into the mechanisms underlying these adaptations. Contributions comprise lab and field studies investigating the chemical, physiological, cognitive and behavioral mechanisms that enable resource exploitation and nutrient intake regulation in insects. The collection of papers highlights the need for more studies on the comparative sensory ecology, underlying nutritional quality assessment, cue perception and decision making to fully understand how insects adjust resource selection and exploitation in response to environmental heterogeneity and variability. KW - nutritional adaptations Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211161 SN - 2075-4450 VL - 11 IS - 9 ER - TY - JOUR A1 - Schmidt, Stefanie A1 - Denk, Sarah A1 - Wiegering, Armin T1 - Targeting protein synthesis in colorectal cancer JF - Cancers N2 - Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy. KW - colorectal cancer KW - protein synthesis KW - translation initiation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-206014 SN - 2072-6694 VL - 12 IS - 5 ER - TY - JOUR A1 - Kann, Simone A1 - Kunz, Meik A1 - Hansen, Jessica A1 - Sievertsen, Jürgen A1 - Crespo, Jose J. A1 - Loperena, Aristides A1 - Arriens, Sandra A1 - Dandekar, Thomas T1 - Chagas disease: detection of Trypanosoma cruzi by a new, high-specific real time PCR JF - Journal of Clinical Medicine N2 - Background: Chagas disease (CD) is a major burden in Latin America, expanding also to non-endemic countries. A gold standard to detect the CD causing pathogen Trypanosoma cruzi is currently not available. Existing real time polymerase chain reactions (RT-PCRs) lack sensitivity and/or specificity. We present a new, highly specific RT-PCR for the diagnosis and monitoring of CD. Material and Methods: We analyzed 352 serum samples from Indigenous people living in high endemic CD areas of Colombia using three leading RT-PCRs (k-DNA-, TCZ-, 18S rRNA-PCR), the newly developed one (NDO-PCR), a Rapid Test/enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence. Eighty-seven PCR-products were verified by sequence analysis after plasmid vector preparation. Results: The NDO-PCR showed the highest sensitivity (92.3%), specificity (100%), and accuracy (94.3%) for T. cruzi detection in the 87 sequenced samples. Sensitivities and specificities of the kDNA-PCR were 89.2%/22.7%, 20.5%/100% for TCZ-PCR, and 1.5%/100% for the 18S rRNA-PCR. The kDNA-PCR revealed a 77.3% false positive rate, mostly due to cross-reactions with T. rangeli (NDO-PCR 0%). TCZ- and 18S rRNA-PCR showed a false negative rate of 79.5% and 98.5% (NDO-PCR 7.7%), respectively. Conclusions: The NDO-PCR demonstrated the highest specificity, sensitivity, and accuracy compared to leading PCRs. Together with serologic tests, it can be considered as a reliable tool for CD detection and can improve CD management significantly. KW - Chagas disease KW - Chagas diagnosis KW - Chagas monitoring KW - Chagas real time PCR KW - Trypanosoma cruzi Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-205746 SN - 2077-0383 VL - 9 IS - 5 ER - TY - JOUR A1 - Grund-Mueller, Nils A1 - Ruedenauer, Fabian A. A1 - Spaethe, Johannes A1 - Leonhardt, Sara D. T1 - Adding amino acids to a sucrose diet is not sufficient to support longevity of adult bumble bees JF - Insects N2 - Dietary macro-nutrients (i.e., carbohydrates, protein, and fat) are important for bee larval development and, thus, colony health and fitness. To which extent different diets (varying in macro-nutrient composition) affect adult bees and whether they can thrive on nectar as the sole amino acid source has, however, been little investigated. We investigated how diets varying in protein concentration and overall nutrient composition affected consumption, longevity, and breeding behavior of the buff-tailed bumble bee, Bombus terrestris (Hymenoptera: Apidae). Queenless micro-colonies were fed either natural nutrient sources (pollen), nearly pure protein (i.e., the milk protein casein), or sucrose solutions with low and with high essential amino acid content in concentrations as can be found in nectar. We observed micro-colonies for 110 days. We found that longevity was highest for pure pollen and lowest for pure sucrose solution and sucrose solution supplemented with amino acids in concentrations as found in the nectar of several plant species. Adding higher concentrations of amino acids to sucrose solution did only slightly increase longevity compared to sucrose alone. Consequently, sucrose solution with the applied concentrations and proportions of amino acids or other protein sources (e.g., casein) alone did not meet the nutritional needs of healthy adult bumble bees. In fact, longevity was highest and reproduction only successful in micro-colonies fed pollen. These results indicate that, in addition to carbohydrates and protein, adult bumble bees, like larvae, need further nutrients (e.g., lipids and micro-nutrients) for their well-being. An appropriate nutritional composition seemed to be best provided by floral pollen, suggesting that pollen is an essential dietary component not only for larvae but also for adult bees. KW - nutrition KW - nutrients KW - foraging KW - pollen KW - resources KW - adult bees Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203866 SN - 2075-4450 VL - 11 IS - 4 ER - TY - JOUR A1 - Grubbs, Kirk J. A1 - Surup, Frank A1 - Biedermann, Peter H. W. A1 - McDonald, Bradon R. A1 - Klassen, Jonathan L. A1 - Carlson, Caitlin M. A1 - Clardy, Jon A1 - Currie, Cameron R. T1 - Cycloheximide-Producing Streptomyces Associated With Xyleborinus saxesenii and Xyleborus affinis Fungus-Farming Ambrosia Beetles JF - Frontiers in Microbiology N2 - Symbiotic microbes help a myriad of insects acquire nutrients. Recent work suggests that insects also frequently associate with actinobacterial symbionts that produce molecules to help defend against parasites and predators. Here we explore a potential association between Actinobacteria and two species of fungus-farming ambrosia beetles, Xyleborinus saxesenii and Xyleborus affinis. We isolated and identified actinobacterial and fungal symbionts from laboratory reared nests, and characterized small molecules produced by the putative actinobacterial symbionts. One 16S rRNA phylotype of Streptomyces (XylebKG-1) was abundantly and consistently isolated from the galleries and adults of X. saxesenii and X. affinis nests. In addition to Raffaelea sulphurea, the symbiont that X. saxesenii cultivates, we also repeatedly isolated a strain of Nectria sp. that is an antagonist of this mutualism. Inhibition bioassays between Streptomyces griseus XylebKG-1 and the fungal symbionts from X. saxesenii revealed strong inhibitory activity of the actinobacterium toward the fungal antagonist Nectria sp. but not the fungal mutualist R. sulphurea. Bioassay guided HPLC fractionation of S. griseus XylebKG-1 culture extracts, followed by NMR and mass spectrometry, identified cycloheximide as the compound responsible for the observed growth inhibition. A biosynthetic gene cluster putatively encoding cycloheximide was also identified in S. griseus XylebKG-1. The consistent isolation of a single 16S phylotype of Streptomyces from two species of ambrosia beetles, and our finding that a representative isolate of this phylotype produces cycloheximide, which inhibits a parasite of the system but not the cultivated fungus, suggests that these actinobacteria may play defensive roles within these systems. KW - symbiosis KW - mutualism KW - insect fungal interactions KW - antimicrobial KW - Insect symbiois Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212449 VL - 11 ER - TY - JOUR A1 - Fathy, Moustafa A1 - Okabe, Motonori A1 - Othman, Eman M. A1 - Saad Eldien, Heba M. A1 - Yoshida, Toshiko T1 - Preconditioning of adipose-derived mesenchymal stem-like cells with eugenol potentiates their migration and proliferation in vitro and therapeutic abilities in rat hepatic fibrosis JF - Molecules N2 - Mesenchymal stem cells (MSCs) have considerable therapeutic abilities in various disorders, including hepatic fibrosis. They may be affected with different culture conditions. This study investigated, on molecular basics, the effect of pretreatment with eugenol on the characteristics of adipose tissue-derived MSCs (ASCs) in vitro and the implication of eugenol preconditioning on the in vivo therapeutic abilities of ASCs against CCl\(_4\)-induced hepatic fibrosis in rats. The effect of eugenol on ASCs was assessed using viability, scratch migration and sphere formation assays. Expressions of genes and proteins were estimated by immunofluorescence or qRT-PCR. For the in vivo investigations, rats were divided into four groups: the normal control group, fibrotic (CCl\(_4\)) group, CCl\(_4\)+ASCs group and CCl\(_4\) + eugenol-preconditioned ASCs (CCl\(_4\)+E-ASCs) group. Eugenol affected the viability of ASCs in a concentration- and time-dependent manner. Eugenol improved their self-renewal, proliferation and migration abilities and significantly increased their expression of c-Met, reduced expression 1 (Rex1), octamer-binding transcription factor 4 (Oct4) and nanog genes. Furthermore, E-ASCs showed more of a homing ability than ASCs and improved the serum levels of ALT, AST, albumin, total bilirubin and hyaluronic acid more efficient than ASCs in treating CCl\(_4\)-induced hepatic fibrosis, which was confirmed with histopathology. More interestingly, compared to the CCl\(_4\)+ASCs group, CCl\(_4\)+E-ASCs group showed a lower expression of inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), cluster of differentiation 163 (CD163) and tumor necrosis factor-α (TNF-α) genes and higher expression of matrix metalloproteinase (MMP)-9 and MMP-13 genes. This study, for the first time, revealed that eugenol significantly improved the self-renewal, migration and proliferation characteristics of ASCs, in vitro. In addition, we demonstrated that eugenol-preconditioning significantly enhanced the therapeutic abilities of the injected ASCs against CCl\(_4\)-induced hepatic fibrosis. KW - adipose tissue-derived MSCs KW - eugenol KW - migration KW - self-renewal KW - hepatic fibrosis KW - CCl\(_4\) Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-203662 SN - 1420-3049 VL - 25 IS - 9 ER - TY - JOUR A1 - Biltueva, Larisa S. A1 - Prokopov, Dmitry Yu. A1 - Romanenko, Svetlana A. A1 - Interesova, Elena A. A1 - Schartl, Manfred A1 - Trifonov, Vladimir A. T1 - Chromosome distribution of highly conserved tandemly arranged repetitive DNAs in the Siberian sturgeon (Acipenser baerii) JF - Genes N2 - Polyploid genomes present a challenge for cytogenetic and genomic studies, due to the high number of similar size chromosomes and the simultaneous presence of hardly distinguishable paralogous elements. The karyotype of the Siberian sturgeon (Acipenser baerii) contains around 250 chromosomes and is remarkable for the presence of paralogs from two rounds of whole-genome duplications (WGD). In this study, we applied the sterlet-derived acipenserid satDNA-based whole chromosome-specific probes to analyze the Siberian sturgeon karyotype. We demonstrate that the last genome duplication event in the Siberian sturgeon was accompanied by the simultaneous expansion of several repetitive DNA families. Some of the repetitive probes serve as good cytogenetic markers distinguishing paralogous chromosomes and detecting ancestral syntenic regions, which underwent fusions and fissions. The tendency of minisatellite specificity for chromosome size groups previously observed in the sterlet genome is also visible in the Siberian sturgeon. We provide an initial physical chromosome map of the Siberian sturgeon genome supported by molecular markers. The application of these data will facilitate genomic studies in other recent polyploid sturgeon species. KW - Acipenser baerii KW - sturgeon karyotype KW - whole-genome duplication KW - paralogs KW - polyploidy KW - acipenserid minisatellite KW - satellite DNA KW - tandem repeats Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219371 SN - 2073-4425 VL - 11 IS - 11 ER - TY - THES A1 - Thelen, David T1 - Erstellung eines genregulatorischen Netzwerkes zur Simulation der Entstehung von Zahnhartsubstanz T1 - Construction of a gene regulatory network to simulate the formation of dental hard tissue N2 - In this dissertation, the author describes the creation of a basic bioinformatic model of human enamel maturation. Supported by the interactions found in the KEGG Pathway database, we were able to establish a gene regulatory network (GRN) that focuses primarily on the signal transduction pathways apoptosis, cell cycle, hedgehog signaling pathway, MAP kinase pathway, mTOR signaling pathway, Notch signaling pathway, TGF-β signaling pathway and Wnt signaling pathway. We extended this through further verified interactions and implicated the tooth-specific genes AMELX, AMELY, AMBN, ENAM and DSPP. In the subsequent simulation of the network by the simulation tool Jimena, six stable states could be identified. These are examined in more detail and juxtaposed with results of a GEO dataset. The long-term goal is to draw conclusions about the odontogenesis of humans through consistent optimization of the bioinformatics network. N2 - In dieser Dissertation beschreibt der Autor die Erstellung eines grundlegenden bioinformatischen Modelles der menschlichen Zahnschmelzreifung. Mithilfe der KEGG Pathway-Datenbank wurde ein genregulatorisches Netzwerk (GRN) erstellt, welches maßgeblich auf den Signaltransduktionswegen Apoptose, Zellzyklus, Hedgehog-Signalweg, MAP-Kinase-Weg, mTOR-Signalweg Notch-Signalweg Signalweg, TGF-β-Signalweg und Wnt-Signalweg basiert. Im Weiteren wurde dieses Netzwerk durch zahlreiche verifizierte Wechselwirkungen erweitert und die zahnspezifischen Gene AMELX, AMELY, AMBN, ENAM und DSPP implementiert. In der anschließenden Simulation des Netzwerks mit dem Simulations-Tool Jimena konnten sechs stabile Zustände identifiziert werden. Diese wurden genauer untersucht und den Erkenntnissen eines GEO-Datensatzes gegenübergestellt. Langfristiges Ziel ist es, durch konsequente Optimierung des bioinformatischen Netzwerks Rückschlüsse auf die Odontogenese des Menschen zu ziehen. KW - Universität Würzburg. Lehrstuhl für Bioinformatik KW - Boolesches Netz KW - Zahnentwicklung KW - Amelogenese KW - Genregulation KW - genregulatorisches Netzwerk Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204068 ER - TY - JOUR A1 - Koch, Rebecca-Diana A1 - Hörner, Eva-Maria A1 - Münch, Nadine A1 - Maier, Elke A1 - Kozjak-Pavlovic, Vera T1 - Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis JF - Frontiers in Cellular and Infection Microbiology N2 - Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion. KW - exit KW - release KW - cell death KW - caspases Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215158 SN - 2235-2988 VL - 10 ER - TY - JOUR A1 - Hardulak, Laura A. A1 - Morinière, Jérôme A1 - Hausmann, Axel A1 - Hendrich, Lars A1 - Schmidt, Stefan A1 - Doczkal, Dieter A1 - Müller, Jörg A1 - Hebert, Paul D. N. A1 - Haszprunar, Gerhard T1 - DNA metabarcoding for biodiversity monitoring in a national park: Screening for invasive and pest species JF - Molecular Ecology Resources N2 - DNA metabarcoding was utilized for a large‐scale, multiyear assessment of biodiversity in Malaise trap collections from the Bavarian Forest National Park (Germany, Bavaria). Principal component analysis of read count‐based biodiversities revealed clustering in concordance with whether collection sites were located inside or outside of the National Park. Jaccard distance matrices of the presences of barcode index numbers (BINs) at collection sites in the two survey years (2016 and 2018) were significantly correlated. Overall similar patterns in the presence of total arthropod BINs, as well as BINs belonging to four major arthropod orders across the study area, were observed in both survey years, and are also comparable with results of a previous study based on DNA barcoding of Sanger‐sequenced specimens. A custom reference sequence library was assembled from publicly available data to screen for pest or invasive arthropods among the specimens or from the preservative ethanol. A single 98.6% match to the invasive bark beetle Ips duplicatus was detected in an ethanol sample. This species has not previously been detected in the National Park. KW - biodiversity KW - DNA barcoding KW - invasive species KW - metabarcoding KW - monitoring KW - pest species Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217812 VL - 20 IS - 6 SP - 1542 EP - 1557 ER - TY - THES A1 - Beliu, Gerti T1 - Bioorthogonale Tetrazin-Farbstoffe für die Lebendzell-Markierung und hochaufgelöste Fluoreszenzmikroskopie T1 - Bioorthogonal tetrazine-dyes for live-cell labeling and super-resolution fluorescence microscopy N2 - Der genetische Code beschreibt die Ver- und Entschlüsselung der Erb-information für das universelle Prinzip der Proteinbiosynthese aus einzelnen Aminosäuren. Durch Erweiterung des genetischen Codes lassen sich unna-türliche Aminosäuren (uAA) mit einzigartigen biophysikalischen Eigenschaf-ten ortsspezifisch in Proteine einführen und ermöglichen die spezifische Ma-nipulation von Proteinen. Die Click-Reaktion zwischen der unnatürlichen Aminosäure TCO*-Lysin und Tetrazin besitzt eine außergewöhnliche Reaktionskinetik (≥800 M-1s-1) und ermöglicht eine spezifische und bioorthogonale Markierung von Bio- ¬molekülen unter physiologischen Bedingungen. Im Fokus dieser Arbeit stand zunächst die Markierung von Membran- ¬rezeptoren durch Click-Chemie in lebenden Zellen sowie die Untersuchung der Wechselwirkung 22 bekannter und neuartiger Tetrazin-Farbstoff- Konjugate. Darüber hinaus wurde die Anwendbarkeit von bioorthogonalen Click-Reaktionen für die hochauflösende Fluoreszenzmikroskopie untersucht. Durch Erweiterung des genetischen Codes in Proteine aus der Klasse der ionotropen Glutamatrezeptoren (iGluR), TNF-Rezeptoren oder Mikrotubu-li-assoziierten Proteinen (MAP) wurde ortspezifisch die unnatürliche Amino-säure TCO*-Lysin eingeführt und dadurch die Fluoreszenzmarkierung durch Tetrazin-Farbstoffe ermöglicht. Die direkte chemische Kopplung von TCO an Liganden wie Phalloidin und Docetaxel, welche spezifisch das Aktin-Zytoskelett bzw. Mikrotubuli-Filamente binden können, ermöglichte zudem die Click-Färbungen von fixierten und lebenden Zellen ohne genetische Ver-änderungen der Zielproteine. Des Weiteren wurden die spektroskopischen Eigenschaften von 22 Tetrazin-Farbstoffen, verteilt über den gesamten sichtbaren Wellenlängenbereich, untersucht. Ein charakteristisches Kennzeichen der Click-Reaktion mit Tet-razin-Farbstoffen ist dabei ihre Fluorogenität. Das Tetrazin fungiert nicht nur als reaktive Gruppe während der Click-Reaktion mit Alkenen, sondern führt in vielen Tetrazin-Farbstoff-Konjugaten zur Fluoreszenzlöschung. Während bei grün-absorbierenden Farbstoffe vor allem FRET-basierte Löschprozesse dominieren, konnte photoinduzierter Elektronentransfer (PET) vom angeregten Farbstoff zum Tetrazin als Hauptlöschmechanismus bei rot-absorbierenden Oxazin- und Rhodamin-Derivaten identifiziert werden. Die effiziente und spezifische Markierung aller untersuchten Tetrazin- Farbstoffe ermöglichte die Visualisierung von Aktin-Filamenten, Mikrotubuli und Membranrezeptoren sowohl durch konventionelle Fluoreszenzmikrosko-pie als auch durch hochauflösende Verfahren, wie z.B. dSTORM, auf Ein-zelmolekülebene. Die unterschiedliche Zellpermeabilität von Tetrazin-Farbstoffen kann dabei vorteilhaft für die spezifische intra- und extrazelluläre Markierung von Proteinen in fixierten und lebenden Zellen genutzt werden. N2 - The genetic code describes the encoding and decoding of genetic infor-mation for the universal principle of protein biosynthesis from individual amino acids. By expanding the genetic code, unnatural amino acids (uAA) with unique biophysical properties can be introduced site-specifically into pro-teins and enable the selective manipulation of proteins. The click reaction of the unnatural amino acid TCO*-lysine and tetrazine has an extraordinary reaction kinetic (≥800 M-1s-1) enabling the specific and bioorthogonal labeling of biomolecules under physiological conditions. The main focus of this work was the labeling of membrane receptors by click chemistry in living cells and the investigation of the interaction of 22 known and novel tetrazine dye conjugates. In addition, the applicability of bioorthogonal click reactions for high-resolution fluorescence microscopy was investigated. For this purpose, the unnatural amino acid TCO*-lysine was introduced site-specifically via genetic code expansion into proteins from the class of iono-tropic glutamate receptors (iGluR), TNF receptors or microtubule- associated proteins (MAP), thereby enabling fluorescence labeling with tetrazine dyes. The direct chemical coupling of TCO to ligands such as phalloidin and docetaxel, which can specifically bind the actin cytoskeleton or microtubule filaments, allowed click staining of fixed and living cells without genetic modifications of the target proteins. Furthermore, the spectroscopic properties of 22 tetrazine dyes spanning the entire visible wavelength range were investigated. A hallmark of the click reaction using tetrazine dyes is their fluorogenicity. Thus, the tetrazine not only functions as a reactive group during the click reaction with alkenes, but also leads to fluorescence quenching in many tetrazine-dye conjugates. While FRET-based quenching processes dominate in green-absorbing dyes, photoinduced electron transfer (PET) from excited dye to tetrazine has been identified as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. The efficient and specific labeling of all investigated tetrazine dyes facilitates the visualization of actin filaments, microtubules and membrane receptors by conventional fluorescence microscopy as well as by super-resolution microscopy techniques, e.g. dSTORM, also at single molecule level. The different cell permeability of tetrazine dyes can be used advantageously for the specific intra- and extracellular labeling of proteins in fixed and living cells. KW - Hochaufgelöste Fluoreszenzmikroskopie KW - Tetrazin Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189628 ER - TY - JOUR A1 - Biedermann, Peter H. W. T1 - Cooperative Breeding in the Ambrosia Beetle Xyleborus affinis and Management of Its Fungal Symbionts JF - Frontiers in Ecology and Evolution N2 - Fungus-farming is known from attine ants, macrotermites, and ambrosia beetles (Scolytinae, Platypodinae). Farming ant and termite societies are superorganismal and grow fungal cultivars in monocultures. Social organization of ambrosia beetle groups and their farming systems are poorly studied, because of their enigmatic life within tunnel systems inside of wood. Ambrosia beetle-fungus symbioses evolved many times independently in both the beetles and their fungal cultivars. Observations suggest that there is evolutionary convergence between these lineages, but also a high variation in the degree of sociality and the modes of fungiculture. Using a laboratory observation technique, I here tried to give insights into the social system and fungus symbiosis of the sugar-cane borer, Xyleborus affinis Eichhoff (Scolytinae: Curculionidae), a currently poorly studied ambrosia beetle. The study revealed a cooperatively breeding system characterized by delayed dispersal of adult daughters, alloparental brood care by larvae and adults, and about half of the totipotent adult daughters laying eggs within the natal nest. Most interesting, there was a tendency of egg-laying females to engage more commonly in mutually beneficial behaviors than non-egg-layers. Fungus gardens covering gallery walls composed of five different filamentous fungi. A Raffaelea isolate was predominant and together with an unidentified fungus likely served as the main food for adults and larvae. Three isolates, a Mucor, a Fusarium and a Phaeoacremonium isolate were most abundant in the oldest gallery part close to the entrance; Mucor, Fusarium and the Raffaelea isolate in diseased individuals. Additionally, there was correlative evidence for some fungal isoaltes influencing beetle feeding and hygienic behaviors. Overall, X. affinis is now the second ambrosia beetle that can be classified as a cooperative breeder with division of labor among and between adults and larvae. KW - cooperative breeding KW - bark beetle KW - insect agriculture KW - symbiosis KW - fungus community KW - social behavior KW - fungus-farming KW - mutualism Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215662 SN - 2296-701X VL - 8 ER - TY - JOUR A1 - Solger, Franziska A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Paprotka, Kerstin A1 - Pfister, Pauline A1 - Hagen, Franziska A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Seibel, Jürgen A1 - Rudel, Thomas T1 - A Role of Sphingosine in the Intracellular Survival of Neisseria gonorrhoeae JF - Frontiers in Cellular and Infection Microbiology N2 - Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells. KW - sphingosine KW - sphingolipids KW - sphingosine kinases KW - invasion KW - survival KW - click chemistry Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204111 SN - 2235-2988 VL - 10 ER - TY - THES A1 - Stelzner, Kathrin T1 - Identification of factors involved in Staphylococcus aureus- induced host cell death T1 - Identifizierung von Faktoren, die am Staphylococcus aureus-induzierten Wirtszelltod beteiligt sind N2 - Staphylococcus aureus is a Gram-positive commensal bacterium, that asymptomatically colonizes human skin and mucosal surfaces. Upon opportune conditions, such as immunodeficiency or breached barriers of the host, it can cause a plethora of infections ranging from local, superficial infections to life-threatening diseases. Despite being regarded as an extracellular pathogen, S. aureus can invade and survive within non-phagocytic and phagocytic cells. Eventually, the pathogen escapes from the host cell resulting in killing of the host cell, which is associated with tissue destruction and spread of infection. However, the exact molecular mechanisms underlying S. aureus-induced host cell death remain to be elucidated. In the present work, a genome-wide haploid genetic screen was performed to identify host cell genes crucial for S. aureus intracellular cytotoxicity. A mutant library of the haploid cell line HAP1 was infected with the pathogen and cells surviving the infection were selected. Twelve genes were identified, which were significantly enriched when compared to an infection with a non-cytotoxic S. aureus strain. Additionally, characteristics of regulated cell death pathways and the role of Ca2+ signaling in S. aureus-infected cells were investigated. Live cell imaging of Ca2+ reporter cell lines was used to analyze single cells. S. aureus-induced host cell death exhibited morphological features of apoptosis and activation of caspases was detected. Cellular H2O2 levels were elevated during S. aureus intracellular infection. Further, intracellular S. aureus provoked cytosolic Ca2+ overload in epithelial cells. This resulted from Ca2+ release from endoplasmic reticulum and Ca2+ influx via the plasma membrane and led to mitochondrial Ca2+ overload. The final step of S. aureus-induced cell death was plasma membrane permeabilization, a typical feature of necrotic cell death. In order to identify bacterial virulence factors implicated in S. aureus-induced host cell killing, the cytotoxicity of selected mutants was investigated. Intracellular S. aureus employs the bacterial cysteine protease staphopain A to activate an apoptosis-like cell death characterized by cell contraction and membrane bleb formation. Phagosomal escape represents a prerequisite staphopain A-induced cell death, whereas bacterial intracellular replication is dispensable. Moreover, staphopain A contributed to efficient colonization of the lung in a murine pneumonia model. In conclusion, this work identified at least two independent cell death pathways activated by intracellular S. aureus. While initially staphopain A mediates S. aureus-induced host cell killing, cytosolic Ca2+-overload follows later and leads to the final demise of the host cell. N2 - Staphylococcus aureus ist ein Gram-positives, kommensales Bakterium, welches menschliche Haut- und Schleimhautoberflächen asymptomatisch kolonisiert. Unter günstigen Bedingungen, wie z. B. Immunschwäche oder verletzten Barrieren des Wirtes, kann es eine Vielzahl von Infektionen verursachen, die von lokalen, oberflächlichen Infektionen bis hin zu lebensbedrohlichen Krankheiten reichen. Obwohl S. aureus als extrazellulärer Erreger angesehen wird, kann das Bakterium von nicht-phagozytischen und phagozytischen Zellen aufgenommen werden und dort überleben. Schließlich bricht das Pathogen aus der Wirtszelle aus und die damit einhergehende Tötung der Wirtszelle wird mit Gewebezerstörung und Ausbreitung der Infektion in Verbindung gebracht. Die genauen molekularen Mechanismen, die dem S. aureus induzierten Wirtszelltod zugrunde liegen, müssen jedoch noch geklärt werden. In dieser Arbeit wurde ein genomweiter haploid genetischer Screen durchgeführt, um Wirtszellgene zu identifizieren, die für die intrazelluläre Zytotoxizität von S. aureus entscheidend sind. Eine Mutantenbibliothek der haploiden Zelllinie HAP1 wurde mit dem Erreger infiziert und die Zellen, die die Infektion überlebten, wurden selektiert. Dabei wurden zwölf Gene identifiziert, die signifikant angereichert waren gegenüber einer Infektion mit einem nicht-zytotoxischen S. aureus Stamm. Des Weiteren wurden Eigenschaften regulierter Zelltod-Signalwege und die Rolle der Ca2+-Signalübertragung in S. aureus infizierten Zellen untersucht. Lebendzellbildgebung von Ca2+-Reporterzelllinien wurde zur Analyse von einzelnen Zellen eingesetzt. Der S. aureus induzierte Wirtszelltod wies morphologische Merkmale von Apoptose auf und die Aktivierung von Caspasen wurde nachgewiesen. Der zelluläre H2O2-Spiegel wurde durch die intrazelluläre Infektion mit S. aureus erhöht. Zusätzlich rief der intrazelluläre S. aureus eine zytosolische Ca2+-Überbelastung in Epithelzellen hervor. Dies resultierte aus der Ca2+-Freisetzung vom endoplasmatischen Retikulum und dem Einstrom von Ca2+ über die Plasmamembran und führte zu einer mitochondrialen Ca2+-Überbelastung. Der finale Schritt des durch S. aureus induzierten Zelltods war die Permeabilisierung der Plasmamembran, ein typisches Merkmal des nekrotischen Zelltods. Um bakterielle Virulenzfaktoren zu identifizieren, die am S. aureus-induzierten Wirtszelltod beteiligt sind, wurde die Zytotoxizität von ausgewählten Mutanten untersucht. Der intrazelluläre S. aureus nutzt die bakterielle Cysteinprotease Staphopain A, um einen Apoptose-artigen Zelltod zu aktivieren, der durch Zellkontraktion und Blasenbildung der Membran gekennzeichnet ist. Der phagosomale Ausbruch stellt eine Voraussetzung für den Staphopain A-induzierten Zelltod da, während die intrazelluläre Replikation der Bakterien nicht notwendig ist. Darüber hinaus trug Staphopain A zu einer effizienten Kolonisation der Lunge in einem murinen Pneumonie-Modell bei. Zusammenfassend lässt sich sagen, dass diese Arbeit mindestens zwei unabhängige Zelltod-Signalwege identifiziert hat, die durch den intrazellulären S. aureus aktiviert werden. Während zunächst Staphopain A den Tod der Wirtszelle einleitet, folgt später die zytosolische Ca2+-Überlastung und führt zum endgültigen Untergang der Wirtszelle. KW - Staphylococcus aureus KW - Zelltod KW - Wirtszelle KW - cell death KW - host cell Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188991 N1 - Zusatzmaterial (Videos) befinden sich auch auf einer CD in der gedruckten Ausgabe ER - TY - THES A1 - Hartlieb, Heiko T1 - Functional analysis of Mushroom body miniature’s RGG-box and its role in neuroblast proliferation in Drosophila melanogaster T1 - Funktionelle Analyse der RGG-Box von Mushroom body miniature und deren Rolle in der Neuroblastenproliferation in Drosophila melanogaster N2 - Development of the central nervous system in Drosophila melanogaster relies on neural stem cells called neuroblasts. Neuroblasts divide asymmetrically to give rise to a new neuroblast as well as a small daughter cell which eventually generates neurons or glia cells. Between each division, neuroblasts have to re-grow to be able to divide again. In previous studies, it was shown that neuroblast proliferation, cell size and the number of progeny cells is negatively affected in larvae carrying a P-element induced disruption of the gene mushroom body miniature (mbm). This mbm null mutation called mbmSH1819 is homozygously lethal during pupation. It was furthermore shown that the nucleolar protein Mbm plays a role in the processing of ribosomal RNA (rRNA) as well as the translocation of ribosomal protein S6 (RpS6) in neuroblasts and that it is a transcriptional target of Myc. Therefore, it was suggested that Mbm might regulate neuroblast proliferation through a role in ribosome biogenesis. In the present study, it was attempted to further elucidate these proposed roles of Mbm and to identify the protein domains that are important for those functions. Mbm contains an arginine/glycine rich region in which a di-RG as well as a di-RGG motif could be found. Together, these two motifs were defined as Mbm’s RGG-box. RGG-boxes can be found in many proteins of different families and they can either promote or inhibit protein-RNA as well as protein-protein interactions. Therefore, Mbm’s RGG-box is a likely candidate for a domain involved in rRNA binding and RpS6 translocation. It could be shown by deletion of the RGG-box, that MbmdRGG is unable to fully rescue survivability and neuroblast cell size defects of the null mutation mbmSH1819. Furthermore, Mbm does indeed rely on its RGG-box for the binding of rRNA in vitro and in mbmdRGG as well as mbmSH1819 mutants RpS6 is partially delocalized. Mbm itself also seems to depend on the RGG-box for correct localization since MbmdRGG is partially delocalized to the nucleus. Interestingly, protein synthesis rates are increased in mbmdRGG mutants, possibly induced by an increase in TOR expression. Therefore, Mbm might possess a promoting function in TOR signaling in certain conditions, which is regulated by its RGG-box. Moreover, RGG-boxes often rely on methylation by protein arginine methyltransferases (in Drosophila: Darts – Drosophila arginine methyltransferases) to fulfill their functions. Mbm might be symmetrically dimethylated within its RGG-box, but the results are very equivocal. In any case, Dart1 and Dart5 do not seem to be capable of Mbm methylation. Additionally, Mbm contains two C2HC type zinc-finger motifs, which could be involved in rRNA binding. In an earlier study, it was shown that the mutation of the zinc-fingers, mbmZnF, does not lead to changes in neuroblast cell size, but that MbmZnF is delocalized to the cytoplasm. In the present study, mbmZnF mutants were included in most experiments. The results, however, are puzzling since mbmZnF mutant larvae exhibit an even lower viability than the mbm null mutants and MbmZnF shows stronger binding to rRNA than wild-type Mbm. This suggests an unspecific interaction of MbmZnF with either another protein, DNA or RNA, possibly leading to a dominant negative effect by disturbing other interaction partners. Therefore, it is difficult to draw conclusions about the zinc-fingers’ functions. In summary, this study provides further evidence that Mbm is involved in neuroblast proliferation as well as the regulation of ribosome biogenesis and that Mbm relies on its RGG-box to fulfill its functions. N2 - Die Entwicklung des zentralen Nervensystems von Drosophila melanogaster beruht auf neuronalen Stammzellen genannt Neuroblasten. Neuroblasten teilen sich asymmetrisch und bringen dabei sowohl einen neuen Neuroblasten als auch eine kleinere Tochterzelle hervor, die wiederum letztlich Neuronen oder Gliazellen generiert. Zwischen jeder Zellteilung müssen die Neuroblasten wieder auf ihre ursprüngliche Größe wachsen, sodass sie zur erneuten Teilung in der Lage sind. In vorhergehenden Studien konnte gezeigt werden, dass sowohl die Proliferation der Neuroblasten, deren Zellgröße als auch die Anzahl ihrer Tocherzellen reduziert ist in Larven, die eine P-Element-induzierte Unterbrechung des Gens mushroom body miniature (mbm) tragen. Diese mbm-Nullmutation, genannt mbmSH1819, ist homozygot letal während des Puppenstadiums. Es konnte außerdem gezeigt werden, dass das nucleoläre Protein Mbm eine Rolle in der Prozessierung ribosomaler RNA (rRNA), sowie der Translokation des ribosomalen Proteins S6 (RpS6) in Neuroblasten erfüllt und dass seine Transkription durch Myc reguliert wird. Daher wurde geschlussfolgert, dass Mbm die Proliferation von Neuroblasten durch eine Funktion in der Ribosomenbiogenese regulieren könnte. In der vorliegenden Studie wurde das Ziel verfolgt, weitere Hinweise auf diese möglichen Funktionen von Mbm zu finden und die Proteindomänen zu identifizieren, die dafür benötigt werden. Mbm beinhaltet einen Arginin/Glycin-reichen Abschnitt, der ein di-RG sowie ein di-RGG Motiv enthält. Diese beiden Motive wurden zusammen zu Mbms RGG-Box definiert. RGG-Boxen finden sich in vielen Proteinen verschiedener Familien und sie können sich sowohl verstärkend als auch inhibierend auf Protein-RNA- sowie Protein-Protein-Interaktionen auswirken. Somit stellt Mbms RGG-Box einen vielversprechenden Kandidaten dar für eine Proteindomäne, die in die rRNA-Bindung sowie die Translokation von RpS6 involviert ist. Es konnte gezeigt werden, dass Mbm mit deletierter RGG-Box (MbmdRGG) nicht in der Lage ist, die Überlebensfähigkeit und die Neuroblastengröße der Nullmutation mbmSH1819 vollständig zu retten. Des Weiteren benötigt Mbm die RGG-Box, um rRNA in vitro zu binden und in mbmdRGG sowie mbmSH1819 Mutanten konnte eine partielle Delokalisation von RpS6 beobachtet werden. Die korrekte Lokalisation von Mbm selbst scheint auch von der RGG-Box abzuhängen, da MbmdRGG teilweise in den Nukleus delokalisiert ist. Interessanterweise ist außerdem die Proteinsyntheserate in mbmdRGG Mutanten erhöht, was möglicherweise in einer Erhöhung der TOR-Expression begründet ist. Somit könnte Mbm unter bestimmten Bedingungen eine verstärkende Funktion im TOR-Signalweg erfüllen, die durch seine eigene RGG-Box reguliert wird. Des Weiteren sind RGG-Boxen hinsichtlich ihrer Funktion häufig von der Methylierung durch Protein-Arginin-Methyltransferasen (in Drosophila: Darts – Drosophila arginine methyltransferases) abhängig. Mbm könnte innerhalb seiner RGG-Box symmetrisch dimethyliert sein, allerdings sind die Ergebnisse in dieser Hinsicht sehr zweifelhaft. Jedenfalls scheinen Dart1 und Dart5 nicht imstande zu sein, Mbm zu methylieren. Außerdem beinhaltet Mbm zwei Zink-Finger-Motive des C2HC-Typs, die in die Bindung von rRNA involviert sein könnten. Eine vorhergehende Studie konnte zeigen, dass die Mutation der Zink-Finger, mbmZnF, zwar nicht zu einer Veränderung der Neuroblastengröße führt, allerdings, dass MbmZnF ins Zytoplasma delokalisiert vorliegt. In der vorliegenden Studie wurden die mbmZnF Mutanten in die meisten Experimente mit einbezogen. Allerdings sind die Ergebnisse rätselhaft, da mbmZnF-mutierte Larven sogar eine geringere Überlebensrate zeigen als die mbm Nullmutanten und da MbmZnF eine stärkere Bindungsaffinität zu rRNA zeigt als wildtypisches Mbm. Dies weist auf eine unspezifische Interaktion zwischen MbmZnF und einem anderen Protein, RNA oder DNA hin, was einen dominant-negativen Effekt auslösen könnte, indem andere Interaktionspartner gestört werden. Somit gestaltet es sich schwierig, Schlussfolgerungen zur Funktion der Zink-Finger zu ziehen. Zusammengefasst liefert die vorliegende Studie weitere Anhaltspunkte, dass Mbm in der Neuroblastenproliferation sowie der Regulation der Ribosomenbiogenese involviert ist und dass Mbm seine RGG-Box benötigt, um seine Funktionen zu erfüllen. KW - Taufliege KW - Neuroblast KW - Gehirn KW - Entwicklung KW - Drosophila melanogaster KW - brain development KW - neuroblast proliferation KW - mushroom body miniature KW - Gehirnentwicklung KW - Neuroblastenproliferation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199674 ER - TY - THES A1 - Glaab, Sabine T1 - Green classroom at the wildlife park: Aspects of environmental, instructional and conceptual education of primary school children concerning the European wildcat. T1 - Das Grüne Klassenzimmer im Wildpark: Aspekte der Umweltbildung, Instruktion sowie Schülervorstellungen bei Grundschulkindern zum Thema Wildkatze. N2 - To foster sustainable environmentally friendly behavior in children it is important to provide an effective form of environmental education. In this context we studied three important factors: Attitude towards nature, environmental knowledge and advanced expert knowledge. Concerning attitude towards nature our first question was: “Is it possible to affect primary school children’s environmental values during a one-day visit at a wildlife park?” As a control, the program was also conducted in schools, leading to two different learning settings- wildlife park and school. Regarding environmental knowledge, in our second question we wanted to know, if our modified teaching approach “guided learning at workstations” (G) combining instructional and constructivist elements would lead to good cognitive learning results of primary school children. Additionally, we compared it to a stronger teacher-centered (T) as well as to a stronger student-centered (S) approach. The third question we asked was “Is it possible to convey fascinating expert knowledge on a more advanced subject to primary school children using conceptual change theory?” After gathering primary school children’s preconceptions, we defined different groups due to the heterogeneity of their pre-existing conceptions and the change in conceptions. Based on this research we designed a program along with an instrument to measure the impact of the conceptual change teaching method. After years of building a strong cooperation between the section Didactics of Biology at the Julius-Maximilians University Würzburg, the nearby schools and the wildlife park “Wild-Park Klaushof” near Bad Kissingen in northern Bavaria it was time to evaluate the environmental education programs prepared and applied by undergraduate university students. As a model species we chose the European wildcat (Felis silvestris silvestris) which represents endangered wildlife in Europe and the need for human interaction for the sake of preserving a species by restoring or recreating the habitat conditions needed while maintaining current infrastructure. Drawing from our own as well as teachers’ and university students’ experiences, we built, implemented and evaluated a hands-on program following several workstations between the wildcat enclosure and the wildlife park’s green classroom. The content of our intervention was presented as a problem-oriented lesson, where children were confronted with the need for human interaction in order to preserve the European wildcat. Not only on a theoretical basis, but very specific to their hometowns they were told where and when nature conservation groups met or where to donate money. 692 Bavarian third grade primary school children in 35 classes participated in the one-day intervention that took place between the months of april, 2014 and november, 2015 in the wildlife park or in their respective classrooms. The ages varied between 8 and 11 years with the mean age being 8.88 ± 0.56 years old. 48.6 % of them were boys, 51.4 % were girls. (1) To measure primary school children’s environmental attitudes a questionnaire on two major environmental values- preservation and utilization of nature- was administered in a pre, post- and retention test design. It was possible to affect primary school children’s environmental preservation values during our one-day program. This result could be found not only at the wildlife park but unexpectedly also in school, where we educated classes for control purposes. We also found this impact consistent in all used teaching approaches and were surprised to see the preservation values change in a way we did not expect from higher tendency towards preservation of nature to a lower one. We presume that children of this age group reflected on the contents of our intervention. This had an influence on their own values towards preservation which led to a more realistic marking behavior in the questionnaire. We therefore conclude that it is possible to affect primary school children’s environmental values with a one-day program on environmental content. (2) We were interested in conveying environmental knowledge about the European wildcat; its morphology, ecology and behavior. We designed and applied a knowledge questionnaire also in a pre-, post- and retention test design, to find out, whether different forms of instruction made a difference in learning success of primary school children. We used two approaches with a teacher in the role of a didactic leader- our modified guided approach (G) as well as a stronger teacher-centered one (T) with a higher focus on instruction. The third approach was presented as a strong student-centered learning at workstations (S) without a didactic leader we also called “free learning at workstations”. Overall, all children’s knowledge scores changed significantly from pre- to post-test and from pre- to retention test, indicating learning success. Differences could only be found between the posttest values of both approaches with a didactic leader (G, T) in comparison to the strong student-centered (S) form. It appears that these primary school children gained knowledge at the out of school learning setting regardless of the used teaching approach. On the subject of short-term differences, we discuss, that the difference in learning success might have been consistent from post to retention test if a consolidation phase had been added in the days following the program as should be common practice after a visit to an out-of- school learning setting but was not part of our intervention. When comparing both approaches with a didactic leader (G, T), we prefer our modified guided learning at workstations (G) since constructivist phases can be implemented without losses concerning learning success. Moreover, the (at least temporary) presence of a teacher in the role of a didactic leader ensures maintained discipline and counteracts off-task behavior. To make sure, different emotional states did not factor in our program, we measured children’s situational emotions directly after the morning intervention using a short scale that evaluated interest, wellbeing and boredom. We found, that these emotions remained consistent over both learning settings as well as different forms of instruction. While interest and wellbeing remained constantly high, boredom values remained low. We take this as a sign of high quality designing and conducting the intervention. (3) In the afternoon of the one-day intervention, children were given the opportunity to investigate the wildcat further, this time using the conceptual change theory in combination with a more complex and fascinating content: cats’ vision in dusk and dawn. Children were confronted with their preconceptions which had been sampled prior to the study and turned into three distinctive topics reflected in a special questionnaire. In a pre-, post and retention test design we included the most common alternative conceptions, the scientifically correct conceptions as well as other preconceptions. We gathered a high heterogeneity of preconceptions and defined three groups based on conceptual change literature: “Conceptual change”, “Synthetic Models” and “Conceptual Growth”. In addition to these we identified two more groups after our data analysis: “Knowledge” and “Non-addressed Concepts”. We found that instruction according to the conceptual change theory did not work with primary school children in our intervention. The conceptual change from the addressed alternative conceptions as well as from other preconceptions towards the scientifically correct conceptions was successfully achieved only on occasion. In our case and depending on the topic only one third to one fourth of the children actually held the addressed conception while the rest was not targeted by the instruction. Moreover, we conclude children holding other conceptions were rather confused than educated by the confrontation. We assume that children of this age group may be overchallenged by the conceptual change method. N2 - Bildung für nachhaltige Entwicklung soll unter anderem dazu führen, dass Kinder langfristig umweltfreundliches Verhalten zeigen. Um dies zu erreichen, sind verschiedene Faktoren nötig - in dieser Studie lag unser besonderes Augenmerk auf drei Punkten: den Umwelteinstellungen der Kinder, dem umweltrelevanten Wissen, besonders im Hinblick auf die Lebensbedingungen und den Schutz der europäischen Wildkatze sowie weiterführendem, komplexeren, biologischen Wissen. Zuerst fragten wir uns in Bezug auf die Umwelteinstellungen, ob es möglich ist, die Einstellungen der Grundschulkinder zum Thema „Erhaltung der Natur“ im Laufe nur eines Tages am Wildpark zu beeinflussen. Umweltwissen war der Bestandteil der zweiten Frage, wie Grundschulkinder am außerschulischen Lernort gute Lernerfolge erzielen können. Wir testeten unseren modifizierten Ansatz „Geführtes Lernen an Stationen“ (G), der instruktionale und konstruktivistische Elemente beinhaltet und verglichen ihn einerseits mit einem stärker lehrerzentrierten (T) sowie andererseits einem stark schülerzentrierten (S) Lernen an Stationen, das wir auch als „freies Lernen an Stationen“ bezeichneten. Die dritte Frage beschäftigte sich schließlich damit, ob es gelingen kann, faszinierendes, tiefergehendes Wissen mit Hilfe der „Conceptual Change Theorie“ an Grundschulkinder zu vermitteln. Hintergrund der didaktischen Arbeit mit Grundschülern am außerschulischen Lernort Wildpark ist die Kooperation zwischen der Fachgruppe Didaktik der Julius-Maximilians-Universität Würzburg mit dem „Wild-Park Klaushof“ bei Bad Kissingen. Im Rahmen dieser Zusammenarbeit stellt die Fachgruppe Didaktik Biologie angehende Biologielehrerinnen und -lehrer als Referenten von Führungen gemäß des „Geführten Lernen an Stationen“ zur Verfügung. Diese Führungen wurden inhaltlich und didaktisch ebenfalls von Lehramtsstudierenden in der Biologiedidaktik ausgearbeitet, meist im Rahmen der schriftlichen Hausarbeiten gegen Ende des Lehramtsstudiums. Die Führungen sind konstruktivistisch angelegt, bieten hohe Selbsttätigkeit der Schülerinnen und Schüler und folgen dem Prinzip des problemorientierten Unterrichts. Die Schülerinnen und Schüler arbeiten nicht völlig frei, es handelt sich aber auch nicht um einen rein lehrerzentrierten Vortrag, sondern eine Mischung aus beiden Formen, die wir als „Geführtes Lernen an Stationen“ (G) bezeichnen. In dieser Variante stellt der Referent die didaktische Leitung der Führung dar, der Impulse und Anleitungen gibt, immer für Fragen zur Verfügung steht, jedoch Anteile von Selbsttätigkeit ermuntert und begleitet. Im Zeitraum von April 2014 bis November 2015 nahmen 692 Grundschulkinder der dritten Klassen bayerischer Grundschulen in 35 Klassen an der Studie am Wild-Park Klaushof sowie in ihren eigenen Klassenzimmern in der Schule teil. Durchschnittlich waren die Kinder 8.88 ± 0.56 Jahre alt, das Alter variierte zwischen 8 und 11 Jahren. 48,6 % der teilnehmenden Kinder waren Jungs, 51,4 % Mädchen. Im Vormittagsteil des Programms wurde im Rahmen einer problemorientierten Unterrichtseinheit gemeinsam mit den Schülerinnen und Schülern die Frage aufgeworfen, warum die europäische Wildkatze (Felis silvestris silvestris), eine Zeigerart für intakte Ökosysteme, nicht überall vorkommt, wo sie vorkommen könnte. Gemeinsam wurden Aspekte zu Morphologie, Ökologie und Verhalten der Wildkatze erarbeitet; die Frage konnte jedoch auch dann noch nicht beantwortet werden. Erst eine Verknüpfung der Verbreitungskarten und der gelernten Fakten führte zur Erkenntnis, dass die Wildkatze bestimmte Barrieren (Autobahnen, offene Wiesen- und Ackerflächen, bebaute Flächen etc.) nicht überwinden kann und hier der Eingriff des Menschen nötig ist. Nicht nur allgemein, sondern auch ganz konkret wurde der eigene Einsatz der Kinder, zum Beispiel im Rahmen der Mitarbeit in einer Naturschutz-Organisation oder einer Geldspende angeregt. (1) Zur Messung der Umwelteinstellungen verwendeten wir das 2-MEV Modell (two major environmental factors), das die Umwelteinstellungen in zwei Dimensionen darstellt, zum einen die Tendenz zur Erhaltung, zum anderen die Ausnutzungstendenz der Umwelt. Die Fragebögen wurden zu drei Testzeitpunkten ausgefüllt - einem Vortest ca. eine Woche vor dem Programm, einem Nachtest unmittelbar nach Beendigung des Programms und einem Behaltenstest etwa sechs bis acht Wochen nach dem Programm. Die Umwelt-Einstellungen konnten tatsächlich verändert werden, nicht nur am Wildpark, sondern auch in der Schule, wo Klassen das Programm zu Kontrollzwecken ebenfalls durchliefen. Auch blieb der Einfluss über alle verwendeten Lehrmethoden konsistent. Besonders überrascht waren wir von der Art der Änderung der Einstellungen zur Naturerhaltung. Statt sich wie erwartet von schwächerer Tendenz zur Erhaltung in Richtung stärkere Tendenz zur Naturerhaltung zu ändern, erfolgte die Änderung genau entgegengesetzt. Wir vermuten, dass die Kinder dieser Altersgruppe die Inhalte der Intervention reflektiert haben und dies einen Einfluss auf ihre Einstellungen zur Naturerhaltung hatte, was sich in einem realistischeren Ankreuzverhalten niederschlug. Zusammenfassend sehen wir es als möglich an, die Einstellungen zur Umwelt von Grundschulkindern mit einem Ein-Tagesprogramm zu verändern. (2) Auch für die Erhebung des Umweltwissens wählten wir die bereits erwähnten drei Testzeitpunkte für den Wissensfragebogen, der Fragen zur Morphologie, Ökologie und Verhalten der Wildkatze beinhaltete. Die Anzahl richtiger Antworten erhöhte sich vom Vor- zum Nachtest sowie vom Vor- zum Behaltenstest signifikant bei allen Schülerinnen und Schülern, es wurde also erfolgreich gelernt. Zwischen den einzelnen Führungsformen konnten wir signifikante Unterschiede nur kurzfristig vom Vor- zum Nachtest zwischen den beiden Methoden mit dem didaktischen Begleiter, also dem stärker lehrerzentrierten (T) und dem „Geführten Lernen an Stationen“ (G) einerseits und dem stark schülerzentrierten freien Lernen (S) andererseits erkennen. Der kurzfristige Wissenserwerb war mit didaktischem Begleiter (G, T) höher als ohne. Insgesamt konnte also ein Lernerfolg verzeichnet werden, unabhängig von der Führungsform. Allerdings vermuten wir, dass der kurzfristige Unterschied sich auch mittelfristig ausgewirkt hätte, wenn im Anschluss an den Besuch im Wildpark eine Nachbereitung stattgefunden hätte, was gewöhnlich zum Besuch des außerschulischen Lernorts gehören sollte, jedoch nicht Bestandteil dieser Untersuchung war. Vergleicht man die beiden Ansätze mit didaktischen Begleitern (G, T), bevorzugen wir nach wie vor unser „Geführtes Lernen an Stationen“ (G), da hier die Einbindung konstruktivistischer Phasen möglich ist. Darüber hinaus kann die (zumindest zeitweise) Anwesenheit eines Lehrers in der Rolle des didaktischen Begleiters sicherstellen, dass Disziplin gewahrt wird und Störungen vermieden werden. Um die situationalen Emotionen der Schülerinnen und Schüler mit einbeziehen zu können, beziehungsweise Effekte von situationalen Emotionen auf Umwelteinstellungen oder Wissenserwerb ausschließen zu können, wendeten wir zusätzlich eine Kurzskala zur Erfassung von Interesse, Langeweile und Wohlbefinden an. Diese Skala wurde nur einmalig angewendet, direkt im Anschluss an das Vormittagsprogramm. Wir konnten keine Unterschiede bei den erhobenen situationalen Emotionen finden - weder zwischen den Lernorten Schule und Wildpark noch zwischen den drei verschiedenen Führungsformen (G, T, S), überall zeigten sich hohe Werte für Interesse und Wohlbefinden sowie niedrige Werte für Langeweile. Dieses Ergebnis zeigt für uns die hohe didaktische Qualität der Entwicklung und Durchführung des Programms. (3) Am Nachmittag des Ein-Tages-Programms beschäftigten sich die Kinder weiter mit der Wildkatze, diesmal folgten wir einer anderen Methode der Wissensvermittlung, der „Conceptual Change Theorie“ in Kombination mit komplexerem und gleichzeitig faszinierendem Wissen zum Dämmerungssehen der Katze. Gemäß dem Prinzip der didaktischen Rekonstruktion wurden Wissensinhalte im Rahmen dieser Intervention nicht kontinuierlich erarbeitet wie im Vormittagsprogramm, sondern es fand eine Konfrontation der Schülerinnen und Schüler mit ihren eigenen Schülervorstellungen zum Thema Dämmerungssehen bei Mensch und Katze statt. Diese Vorstellungen wurden vorab in einem offenen Fragebogen erhoben und in drei Themenschwerpunkte gegliedert, die sich anschließend im Fragebogen zur Erhebung des Konzeptwechsels widerspiegelten. Auch dieser Fragebogen wurde zu den eingangs erwähnten drei Testzeitpunkten angewendet. Gemäß der Theorie erwarteten wir im Ankreuzverhalten drei Gruppen: „Conceptual Change“, „Synthetic Models“ sowie „Conceptual Growth“. Darüber hinaus fanden wir zwei weitere Gruppen „Knowledge“ und „Non-addressed Concepts“. Wir stellten fest, dass der Konzeptwechsel der Kinder von der wissenschaftlich nicht korrekten Schülervorstellung hin zur wissenschaftlich korrekten Vorstellung in unserer Intervention nicht gelang, nur punktuell kreuzten wenige Schülerinnen und Schüler das entsprechende Muster an. Auch der Wechsel in den anderen Gruppen hin zur wissenschaftlich korrekten Vorstellung funktionierte kaum. In unserem Fall hatten darüber hinaus je nach Thema nur ein Drittel bis ein Viertel der beteiligten Kinder überhaupt die adressierte Vorstellung, was unserer Meinung nach dazu führt, dass der Großteil der Kinder mit anderen Vorstellungen durch die Anwendung der „Conceptual Change Theorie“ eher verwirrt wurde. Wir vermuten, dass Grundschulkinder der dritten Klasse durch diese Form des Unterrichts überfordert sind. KW - Biologie KW - Education Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-169496 ER - TY - JOUR A1 - Doll, Julia A1 - Vona, Barbara A1 - Schnapp, Linda A1 - Rüschendorf, Franz A1 - Khan, Imran A1 - Khan, Saadullah A1 - Muhammad, Noor A1 - Alam Khan, Sher A1 - Nawaz, Hamed A1 - Khan, Ajmal A1 - Ahmad, Naseer A1 - Kolb, Susanne M. A1 - Kühlewein, Laura A1 - Labonne, Jonathan D. J. A1 - Layman, Lawrence C. A1 - Hofrichter, Michaela A. H. A1 - Röder, Tabea A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Graves, Tyler D. A1 - Kong, Il-Keun A1 - Nanda, Indrajit A1 - Kim, Hyung-Goo A1 - Haaf, Thomas T1 - Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families JF - Genes N2 - The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes GJB2, MYO7A, FGF3, CDC14A, SLITRK6, CDH23, and MYO15A, with an overall resolve rate of 61.9%. GJB2 and MYO7A were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes. KW - genetic diagnosis KW - consanguinity KW - genome-wide linkage analysis KW - hearing loss KW - Pakistan KW - exome sequencing Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219293 SN - 2073-4425 VL - 11 IS - 11 ER - TY - THES A1 - Lehmann, Julian T1 - Hochauflösende Fluoreszenzmikroskopie beleuchtet den Oligomerisierungsstatus pflanzlicher Membranproteine T1 - Super-resolution microscopy elucidates the stoichiometry of plant membrane proteins N2 - SLAC/SLAH Anionenkanäle, die zur Familie der langsamen Anionenkanäle gehören, repräsentieren Schlüsselproteine in der pflanzlichen Stressantwort. Neben ihrer Aufgabe in Stresssituationen, ist eine Untergruppe der Kanäle für die Beladung der Leitgefäße mit Nitrat und Chlorid in der Stele der Pflanzenwurzeln verantwortlich. Biophysikalische und pflanzenphysiologische Studien stellten heraus, dass vor Allem der Anionenkanal SLAH3 für die Beladung der Xylem Leitgefäße mit Nitrat und Chlorid verantwortlich ist. Ihm zur Seite gestellt werden noch die elektrisch inaktiven Homologe SLAH1 und SLAH4 in der Wurzel exprimiert. Sie steuern die Aktivität von SLAH3 durch die Assemblierung zu SLAH1/SLAH3 oder SLAH3/SLAH4 Heteromeren. Neben der Kontrolle durch Heteromerisierungsereignisse, werden SLAH3 Homomere sehr spezifisch und schnell durch zytosolische Ansäuerung aktiviert. Obwohl bereits die Kristallstruktur des bakteriellen Homologs HiTehA zu pflanzlichen SLAC/SLAH Anionenkanälen bekannt ist, welche HiTehA als Trimer charakterisiert, sind die Stöchiometrie und der Polymerisierungsgrad der pflanzlichen SLAC/SLAHs bisher noch unbekannt. Die Fluoreszenzmikroskopie umfasst viele etablierte Anwendungsmethoden, wie die konfokale Laserrastermikroskopie (CLSM), Techniken mit verbesserter Auflösung, wie die Mikroskopie mit strukturierter Beleuchtung (SIM) und hochauflösende Methoden, welche durch die Lokalisationsmikroskopie (z.B. dSTORM und PALM) oder die Expansionsmikroskopie (ExM) vertreten werden. Diese unterschiedlichen Mikroskopie-methoden ermöglichen neue Einblicke in die Organisation von Proteinen in biologischen Systemen, die bis auf die molekulare Ebene hinunterreichen. Insbesondere im Bereich der hochauflösenden Fluoreszenzmikroskopie sind im Gegensatz zu tierischen Frage-stellungen bisher jedoch nur wenige Untersuchungen in pflanzlichen Geweben durchgeführt worden. Die Lokalisationsmikroskopie ermöglicht die Quantifizierung einzelner Moleküle in nativen Systemen und lässt überdies Rückschlüsse auf den Polymerisierungsgrad von Proteinen zu. Da Poly- und Heteromerisierung von Proteinen oftmals mit der Funktionalität eines entsprechenden Proteins einhergeht, wie es bei den SLAC/SLAH Anionenkanälen der Fall ist, wurden in dieser Arbeit PALM Messungen zur Untersuchung des Polymerisierungsgrades und Interaktionsmuster der Anionenkanäle angewendet. Ferner wurden Expressionsmuster der SLAC/SLAHs untersucht und zudem Mikroskopieanwendungen im Pflanzengewebe etabliert und verbessert. In Bezug auf die Mikroskopieanwendungen konnten wir in Arabidopsis thaliana (At) Wurzeln die polare Verteilung von PIN Proteinen mittels SIM bestätigen und die gruppierte Verteilung in der Plasmamembran am Zellpol auflösen. In Wurzel-querschnitten war es möglich, Zellwände zu vermessen, den Aufbau der Pflanzenwurzel mit den verschiedenen Zelltypen zu rekonstruieren und diesen in Zusammenhang mit Zellwanddicken zu bringen. Anhand dieser Aufnahmen ließ sich die Auflösungsgrenze eines SIM-Mikroskops bestimmen, weshalb diese Probe als Modellstruktur für Auflösungsanalysen, zur Kontrolle für die korrekte Bildverarbeitung bei hochauflösender Bildgebung und andere Fragestellungen empfohlen werden kann. Für die Expansionsmikroskopie in pflanzlichen Proben konnten ein enzym- und ein denaturierungsbasiertes Präparationsprotokoll etabliert werden. Dabei wurden ganze At Setzlinge, Wurzelabschnitte und Blattstücke gefärbt, expandiert und mit zwei bis drei Mal verbesserter Auflösung bildlich dargestellt. In diesem Zusammenhang waren Aufnahmen ganzer Wurzel- und Blattproben mit beeindruckender Eindringtiefe und extrem geringem Hintergrundsignal möglich. Zudem wurden die Daten kritisch betrachtet, Probleme aufgezeigt, gewebespezifische Veränderungen dargestellt und limitierende Faktoren für die ExM in Pflanzenproben thematisiert. Im Fokus dieser Arbeit stand die Untersuchung der SLAC/SLAH Proteine. SLAH2 wird in den Wurzeln vornehmlich in Endodermis- und Perizykelzellen exprimiert, was anhand verschiedener At SLAH2 YFP Mutanten untersucht werden konnte. Dies unterstützt die Annahme, dass SLAH2 bei der Beladung der Leitgefäße mit Nitrat maßgeblich beteiligt ist. Es ist denkbar, dass SLAH2 ebenfalls eine wachstumsbeeinflussende Funktion über die Regulation von Nitratkonzentrationen zugeschrieben werden kann. Darauf deuten vor allem die verstärkte Expression von SLAH2 im Bereich der Seitenwurzeln und die heterogene Expression in der Elongations-, Differenzierungs- und meristematischen Zone hin. Die Membranständigkeit von SLAH4 konnte nachgewiesen werden und FRET FLIM Untersuchungen zeigten eine hohe Affinität von SLAH4 zu SLAH3, was die beiden Homologe als Interaktionspartner identifiziert. Für die Bestimmung des Oligomerisierungsgrades mittels PALM wurden die pflanzlichen Anionenkanäle in tierischen COS7-Zellen exprimiert. Die elektrophysiologische Funktionalität der mEOS2-SLAC/SLAH-Konstrukte wurde mit Hilfe von Patch-Clamp-Versuchen in COS7-Zellen überprüft. Um Expressionslevel, Membranständigkeit und die Verteilung über die Membran der SLAC/SLAHs zu verifizieren, wurden dSTORM-Aufnahmen herangezogen Schließlich ermöglichten PALM-Aufnahmen die Bestimmung des Polymerisierungs-grades der SLAC/SLAH Anionenkanäle, die stöchiometrischen Veränderungen bei Heteromerisierung von SLAH3 mit SLAH1 oder SLAH4 und auch der Einfluss einer zytosolischer Ansäuerung auf den Polymerisierungsgrad von SLAH3 Homomeren. Zudem weisen die Oligomerisierungsanalysen von SLAH3 Mutanten darauf hin, dass die Aminosäuren Histidin His330 und His454 entscheidend an der pH sensitiven Regulierung von SLAH3 beteiligt sind. Durch die erhobenen Daten konnten also entscheidende, neue Erkenntnisse über die Regulationsmechanismen von pflanzlichen Anionenkanälen auf molekularer Ebene gewonnen werden: Unter Standardbedingungen liegen SLAC1, SLAH2 und SLAH3 hauptsächlich als Dimer vor. Auf eine zytosolische Ansäuerung reagiert ausschließlich SLAH3 mit einer signifikanten stöchiometrischen Veränderung und liegt im aktiven Zustand vor Allem als Monomer vor. Der Oligomerisierungsgrad von SLAC1 und SLAH2 bleibt hingegen bei einer zytosolischen Ansäuerung unverändert. Ferner kommt es bei der Interaktion von SLAH3 mit SLAH1 oder SLAH4 zur Formierung eines Heterodimers, welches unbeeinflusst durch den zytosolischen pH bleibt. Im Gegensatz dazu bleiben die elektrisch inaktiven Untereinheiten SLAH1 und SLAH4 monomerisch und assemblieren ganz spezifisch nur mit SLAH3. Die hochauflösende Fluoreszenz-mikroskopie, insbesondere PALM erlaubt es also Heteromerisierungsereignisse und Änderungen im Poylmerisierungsgrad von Membranproteinen wie den SLAC/SLAHs auf molekularer Ebene zu untersuchen und lässt so Rückschlüsse auf physiologische Ereignisse zu. N2 - Anion channels of the slow anion channel family (SLAC/SLAH) are general master switches of plant stress responses. In addition a subgroup of channels load the vascular tissue in roots with nitrate and chloride. The activity of the main nitrate and chloride loading anion channel, SLAH3, is controlled by heteromerization with the electro-physiologically silent subunits SLAH1 and SLAH4 or alternatively by cytosolic acidification. Although the crystal structure of a bacterial homologue (HiTehA) of plant SLAC/SLAH anion channels is already known and suggests a trimeric structure, the stoichiometry and the multimerization level of the plant anion channel counterparts are still undiscovered. Fluorescence microscopy encompasses numerous well-established application methods like confocal laser scanning microscopy (CLSM), high resolution techniques like structured illumination microscopy (SIM) and super resolution microscopy represented by single molecule localization microscopy (e.g. dSTORM and PALM) or recently upcoming methods like expansion microscopy (ExM). These different application methods open new fields of insight into the biological organization of proteins, even down to the molecular level. In comparison to faunal studies, very little floral enquiries have been conducted, especially in the super resolution-sector. Single-molecule localization microscopy enables individual molecules to be quantified in the native environment and therefore allows conclusions regarding protein stoichiometry. As protein stoichiometry often involves cellular function of the corresponding protein, we used PALM applications and single molecule counting strategies to analyze the stoichiometric distribution of anion channel complexes. Moreover, in this study, expression patterns of the SLAC/SLAH proteins were investigated and different microscopic applications on plant specific issues could be improved and established. Referring to microscopic applications, we confirmed the polar orientation of PIN proteins via SIM and succeeded in resolving the clustered distribution in the plasma membrane at the cellular pole. Besides we were also able to measure cellwall dimensions of root cross sections from Arabidopsis thaliana seedlings and therefore succeeded in concluding the root architecture, designating the various cell types within the root, comparing them with cellwall thickness and evaluating resolution limits of the SIM microscope. Due to these reasons, this specimen can be recommended as a model structure for resolution analyses, control measurements regarding tissue-intactness after image processing for super-resolution images, or further questions. We turned out to establish two different protocols for ExM-studies in plants. One is based on enzymatic digestion and the other one on denaturation. We were able to label, expand and image whole At-seedlings, root- and leaf segments and thereby improved the resolution 2 3 fold. In this regard we managed to comprehensively depict the intact structure of leaves and roots with impressive penetration depth and extremely low background. We also examined our data and identified tissue-specific changes, discuss problems and possible limits of ExM in plants. The major part of this work was the investigation of SLAC/SLAH proteins. The expression of SLAH2 in roots is mainly located in endodermal and pericycle cells which was observed in various At-SLAH2-YFP mutants. Thus, strengthening the hypothesis, that SLAH2 has a major role in loading the vascular tissue with nitrate. The heterogeneous expression levels of SLAH2 in the meristematic-, elongation- and differentiation zone and moreover the upregulation in areas of lateral root formation also suggests that SLAH2 has an effect on plant growth by regulating nitrate levels. SLAH4 is located in the plasma membrane and FRET FLIM measurements showed a high affinity to SLAH3, validating the two homologues as interaction partners. For PALM-stoichiometry analyses, the plant anion channels were expressed in mammalian COS7-cells, in order to avoid endogenous falsification of the stoichiometries, as well as impractical reasons of PALM imaging in plant tissue. Hence, checking the electrophysiological functionality of mEOS2-SLAC/SLAH constructs via patch-clamp measurements. dSTORM-measurements were used to verify expression levels, correct membrane-association and the distribution of the SLAC/SLAHs in COS7 cells. We determined the multimerization level of SLAC/SLAHs upon cytosolic acidification and monitor stoichiometric changes upon heteromerization of SLAH3 with SLAH1 and SLAH4. On the basis of our data the following valuable new insights into the regulation mechanisms of plant anion channels were revealed: under control conditions, SLAC1, SLAH2 and SLAH3 are mainly depicted as dimers. Upon cytosolic acidification with NaOAc the stoichiometries of SLAC1 and SLAH2 remained unchanged, whereas the amount of dimeric SLAH3 is significantly reduced and shifts to a mainly monomeric distribution. It could also be assessed that SLAH3 interacts with SLAH1 or SLAH4, thereby forming a heterodimer, which is barely separable by acidification. In contrast, for SLAH1 and SLAH4 no affinity was observed. Moreover, the stoichiometries of different SLAH3-mutants indicated a crucial role of the amino acids histidin His330 and His454 in the pH-sensitive regulation of SLAH3. Hence, super-resolution micrsocopy, especially PALM allows the quantification of polymerization- and heteromerization-levels of proteins like the SLAC/SLAH anion-channels on the molecular level and therefore enabling physiological conclusions. KW - Fluoreszenzmikroskopie KW - Membranproteine KW - Oligomerisation KW - Superresolution microscopy KW - SLAC/SLAH KW - PALM stoichiometry Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211762 ER - TY - JOUR A1 - Lichthardt, Sven A1 - Wagner, Johanna A1 - Löb, Stefan A1 - Matthes, Niels A1 - Kastner, Caroline A1 - Anger, Friedrich A1 - Germer, Christoph-Thomas A1 - Wiegering, Armin T1 - Pathological complete response due to a prolonged time interval between preoperative chemoradiation and surgery in locally advanced rectal cancer: analysis from the German StuDoQ|Rectalcarcinoma registry JF - BMC Cancer N2 - Background Preoperative chemoradiotherapy is the recommended standard of care for patients with local advanced rectal cancer. However, it remains unclear, whether a prolonged time interval to surgery results in an increased perioperative morbidity, reduced TME quality or better pathological response. Aim of this study was to determine the time interval for best pathological response and perioperative outcome compared to current recommended interval of 6 to 8 weeks. Methods This is a retrospective analysis of the German StuDoQ|Rectalcarcinoma registry. Patients were grouped for the time intervals of "less than 6 weeks", "6 to 8 weeks", "8 to 10 weeks" and "more than 10 weeks". Primary endpoint was pathological response, secondary endpoint TME quality and complications according to Clavien-Dindo classification. Results Due to our inclusion criteria (preoperative chemoradiation, surgery in curative intention, M0), 1.809 of 9.560 patients were suitable for analysis. We observed a trend for increased rates of pathological complete response (pCR: ypT0ypN0) and pathological good response (pGR: ypT0-1ypN0) for groups with a prolonged time interval which was not significant. Ultimately, it led to a steady state of pCR (16.5%) and pGR (22.6%) in "8 to 10" and "more than 10" weeks. We were not able to observe any differences between the subgroups in perioperative morbidity, proportion of rectal extirpation (for cancer of the lower third) or difference in TME quality. Conclusion A prolonged time interval between neoadjuvant chemoradiation can be performed, as the rate of pCR seems to be increased without influencing perioperative morbidity. KW - Rectal cancer KW - Surgery KW - Radiochemotherapy KW - Time interval Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229334 VL - 20 IS - 1 ER - TY - THES A1 - Seibold, Marcel T1 - Funktionelle Charakterisierung des Ras family small GTP binding protein RAL im Multiplen Myelom T1 - Functional characterization of the Ras family small GTP binding protein RAL in multiple myeloma N2 - Die monoklonale Proliferation maligner Plasmazellen im Knochenmark ist charakteristisch für das multiple Myelom (MM) und kann bei Erkrankten zu Störungen in der Hämatopoese sowie zu Knochenläsionen und Niereninsuffizienz führen. Die Weiterentwicklung und der Einsatz neuer Therapieoptionen konnten das Überleben von MM-Patienten zwar erheblich verbessern, jedoch gilt diese Krankheit weiterhin als unheilbar. Onkogene Mutationen und das Knochenmarkmikromilieu führen in MM-Zellen zur Entstehung eines onkogenen Signalnetzwerks, das das Wachstum und Überleben der Zellen aufrechterhält. Mutationen der GTPase RAS treten bei bis zu 50 % der MM-Patienten auf und tragen zum Überleben von MM-Zellen bei. Trotz der Häufigkeit und Bedeutsamkeit von onkogenem RAS, auch in anderen Tumorentitäten, ist die GTPase nach wie vor therapeutisch nicht angreifbar. Die GTPase RAL aus der Familie der RAS-GTPasen wird als Downstream-Effektor von RAS angesehen, der damit ebenfalls zur Aufrechterhaltung des Tumorzellüberlebens beitragen könnte. In einigen Tumorentitäten konnte bisher gezeigt werden, dass eine Überexpression von RAL in den Tumorzellen vorliegt und die Proliferation und Apoptose von Tumorzellen durch RAL beeinflusst wird. Daher stellte sich die Frage, ob RAL im MM ebenfalls das Überleben von Tumorzellen beeinflusst und ob eine direkte Verbindung zwischen onkogenem RAS und RAL besteht. In dieser Arbeit wurde die funktionelle Rolle von RAL sowie dessen Zusammenhang mit onkogenem RAS im MM untersucht. Hierbei konnte eine Überexpression von RAL in MM-Zellen im Vergleich zu MGUS oder normalen Plasmazellen beobachtet werden. In Knockdown-Analysen wurde gezeigt, dass RAL überlebensnotwendig für MM-Zellen ist. Dabei wurde in Western Blot-Analysen festgestellt, dass diese Überlebenseffekte unabhängig von MAPK/ERK-Signaling vermittelt werden. Es konnte teilweise jedoch eine Abhängigkeit von der AKT-Aktivität beobachtet werden. Da RAL-Knockdown Einfluss auf das Überleben von MM-Zellen hat, wurde eine pharmakologische Inhibition von RAL durch den Inhibitor RBC8 untersucht. RBC8 zeigte in höheren Dosen nur bei einem Teil der MM-Zelllinien eine Wirkung auf das Zellüberleben sowie auf die RAL-Aktivierung. Die Weiterentwicklung potenter RAL-Inhibitoren ist daher für eine klinische Translation einer RAL-Inhibition von großer Bedeutung. Zur Untersuchung des Zusammenhangs zwischen onkogenem RAS und der RAL-Aktivierung wurden RAL-Pulldown-Analysen nach Knockdown von onkogenem RAS durchgeführt. In diesen Experimenten wurde keine Abhängigkeit der RAL-Aktivierung von onkogenem RAS festgestellt. Darüber hinaus zeigten Genexpressionsanalysen nach RAS- bzw. RAL-Knockdown unterschiedliche Genexpressionsprofile. In Massenspektrometrie-Analysen wurden mögliche Effektoren, die mit RAL an der Beeinflussung des Zellüberlebens beteiligt sein könnten, untersucht. Hierbei wurden die Komponenten des Exozyst-Komplexes EXO84 und SEC5 als Interaktionspartner von RAL identifiziert. Nachdem gezeigt wurde, dass RAL ausschlaggebend für das Überleben von MM-Zellen ist, wurde eine Kombination von RAL-Knockdown mit klinisch relevanten Wirkstoffen analysiert. Diese zeigte bei der Kombination mit PI3K oder AKT-Inhibitoren verstärkte Effekte auf das Zellüberleben der MM-Zellen. Zusammenfassend wurde die Bedeutung von RAL für das Überleben von Tumorzellen im MM gezeigt und RAL als potentielles therapeutisches Target im MM beschrieben, welches unabhängig von onkogenem RAS reguliert wird. N2 - Multiple myeloma (MM) is a hematologic neoplasia which is characterized by monoclonal proliferation of malignant plasma cells in the bone marrow leading to hematopoetic failure, bone lesions and renal failure. Although continuous development of existing therapeutics and new therapeutic options vastly improved MM patient survival, MM still remains an incurable disease. Oncogenic mutations and the bone marrow microenvironment contribute to a signaling network which sustains MM cell proliferation and survival. Within this network mutations of the RAS oncogene account for up to 50 % of MM patients. Despite its prevalence and importance not only in MM, RAS still remains undruggable. The GTPase-family Member RAL is considered as a RAS effector which might also influence maintainance of tumor cell survival. In several tumor entities RAL is overexpressed in tumor cells and influences proliferation and apoptosis. Therefore, in MM RAL might also be controlled by oncogenic RAS and mediate cell survival of tumor cells. In this work, RAL’s functional role as well as the potential interconnection with oncogenic RAS was investigated. In MM cells RAL is ovexpressed compared to non-malignant MGUS or plasma cells. Knockdown analyses showed that RAL is essential for MM cell survival. These survival effects are transferred independently of MAPK/ERK signaling as shown by Western Blot analysis. However, to some extent RAL influenced MM cell survival dependently of AKT activity. Because RAL knockdown had a significant effect on MM cell survival a pharmacological inhibition was tested using the inhibitor RBC8. In a portion of MM cell lines RBC8 exerts effects on cell survival. But the effects of RBC8 on RAL activation were only visible at higher concentrations as shown by pulldown assays. Thus, subsequent development of potent RAL inhibitors is of major importance for clinical translation. To investigate whether RAL is directly activated by oncogenic RAS, RAL pulldown assays were performed after knockdown of oncogenic RAS. Strikingly, there was no direct connection between the presence of oncogenic RAS and RAL activation. Furthermore, gene expression profiles after RAS or RAL knockdown showed differing expression signatures. Potential effectors of RAL which might also influence MM cell survival were investigated in mass spectrometric analyses where the exocyst complex components EXO84 and SEC5 were identified as RAL interaction partners. Since RAL is of importance for MM cell survival, RAL knockdown was combined with clinically relevant agents. There was an enhanced induction of apoptosis upon combination of PI3K or AKT inhibitors with RAL knockdown. Taken together, the influence of RAL as a crucial mediator of MM cell survival was shown in this work. Therefore, RAL represents a potential therapeutic target which is regulated independently of oncogenic RAS. KW - Kleine GTP-bindende Proteine KW - Signaltransduktion KW - Plasmozytom KW - RAL KW - Multiples Myelom KW - Zellüberleben KW - Knochenmark Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-208003 ER - TY - THES A1 - Spindler, Marie-Christin T1 - Molecular architecture of meiotic multiprotein complexes T1 - Molekulare Architektur meiotischer Multiproteinkomplexe N2 - Sexually reproducing organisms depend on meiosis for the generation of haploid, genetically diverse gametes to maintain genome stability and the potential to adapt to changing environments. Haploidization is achieved through two successive rounds of cell division after a single initial pre-meiotic DNA replication. Meiosis I segregates the homologous chromosomes, followed by the segregation of the sister chromatids in meiosis II. Genetic diversity is achieved through the process of recombination that de-scribes the exchange of genetic material between the maternal and paternal homolog. Recombination and the initial steps of haploidization are executed already early on in prophase I. Both essential processes depend on a variety of multiprotein complexes, such as the linker of nucleo- and cytoplasm (LINC) complex and the synaptonemal complex (SC). The structure of multiprotein complexes is adjusted according to their function, environment, and the forces they are subjected to. Coiled-coil domains typical in load-bearing proteins characterize the meiotic mechanotransducing LINC complexes. SCs resemble ladder-like structures that are highly conserved amongst eukaryotes, while the primary sequence of the proteins that form the complex display very little if any sequence homology. Despite the apparent significance of the structure to their function, little quantitative and topological data existed on the LINC complexes and the SC within their morphological context prior to the present work. Here, the molecular architecture of the meiotic telomere attachment site where LINC complexes reside and the SC have been analyzed in depth, mainly on the basis of electron microscope tomography derived 3D models complemented by super-resolution light microscopic acquisitions of the respective protein components. N2 - Sich sexuell fortpflanzende Organismen sind auf die Meiose angewiesen, um haploide, genetisch vielfältige Keimzellen zu erzeugen, die die Stabilität des Genoms und die Fähigkeit zur Anpassung an sich verändernde Umgebungen erhalten. Die Haploidisierung wird durch zwei aufeinanderfolgende Runden der Zellteilung nach einer einzigen anfänglichen prä-meiotischen DNA Replikation erreicht. In der Meiose I werden die homologen Chromosomen getrennt, gefolgt von der Trennung der Schwesterchromatiden während der Meiose II. Genetische Diversität wird durch den Prozess der Rekombination erreicht, der den Austausch von genetischem Material zwischen den mütterlichen und väterlichen Homologen beschreibt. Die Rekombination und die ersten Schritte der Haploidisierung werden bereits früh in der Prophase I durchgeführt. Beide essentiellen Prozesse hängen von einer Vielzahl von Multiproteinkomplexen ab, wie z.B. dem Linker of Nucleo- and Cytoplasm (LINC)-Komplex und dem synaptonemalen Komplex (SC). Die Struktur von Multiproteinkomplexen wird je nach ihrer Funktion, ihrer Umgebung und den Kräften, denen sie ausgesetzt sind, angepasst. Coiled-coil-Domänen, die für tragende Proteine typisch sind, charakterisieren die meiotischen, mechanotransduzierenden LINC-Komplexe. SCs ähneln leiterähnlichen Strukturen, die unter Eukaryonten hoch konserviert sind, während die Primärsequenz der Proteine, die den Komplex bilden, sehr wenig bis gar keine Sequenzhomologie aufweist. Trotz der offensichtlichen Bedeutung der Struktur für ihre Funktion gab es vor der vorliegenden Arbeit nur wenige quantitative und topologische Daten über die LINC Komplexe und den SC in ihrem morphologischen Kontext. Hier wurde die molekulare Architektur der Telomeranheftungsstellen, an denen sich die LINC-Komplexe befinden, und die des SCs eingehend analysiert, hauptsächlich auf der Grundlage von auf der Elektronenmikroskop-Tomographie basierenden 3D-Modellen, ergänzt durch hochauflösende lichtmikroskopische Aufnahmen der jeweiligen Proteinkomponenten. KW - Meiose KW - Meiosis Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212105 ER - TY - THES A1 - Lu, Yunzhi T1 - Kinetics of mouse and human muscle type nicotinic receptor channels T1 - Kinetik muriner und humaner nikotinischer Rezeptorkanäle vom Muskeltyp N2 - Acetylcholine (ACh) mediates transmission at vertebrate neuromuscular junctions and many other synapses. The postsynaptic ACh receptors at neuromuscular junctions are of the nicotinic subtype (nAChRs). They are among the best studied receptor channels and often serve as models or receptor prototypes. Despite a wealth of information on muscle type nAChRs so far little is known about species specific functional differences. In this work, mouse and human adult muscle type nAChRs are investigated. Cell attached recordings in the HEK293T heterologous expression system provided evidence that the ACh affinity of recombinant mouse and human adult muscle type nAChRs are different. To clarify this, I compared these receptors in outside-out patches employing a system for fast agonist application. Thus, the individual membrane patches with receptors can be exposed to various ligand concentrations. In response to 10 and 30 µM ACh normalized peak currents (î) were significantly larger and current rise-time (tr) shorter in human than in mouse receptors. Analyzing dose-response curves of î and tr and fitting them with a two-step equivalent binding-site kinetic mechanism revealed a two-fold higher ACh association rate constant in human compared to mouse receptors. Furthermore, human nAChRs were blocked faster in outside-out patches by superfusion of 300 nM α-Bungarotoxin (α-Bgtx) than mouse nAChRs. Finally, human nAChRs in outside-out patches showed higher affinity at 3 µM ACh than chimeric receptors consisting of mouse α- and human β-, γ- and ε-subunits. The higher affinity of human than mouse receptors for ACh and α-Bgtx is thus at least in part due to sequence difference in their α-subunits. N2 - Acetylcholin (ACh) vermittelt Erregungsübertragung an neuromuskulären synaptischen Kontakten (neuromuscular junction, NMJ) von Wirbeltieren und vielen anderen Synapsen. Die postsynaptischen ACh-Rezeptoren an der NMJ sind vom nikotinischen Subtyp (nAChRs). Als Teil der am besten erforschten Kanalrezeptoren dienen sie oft als Modelle oder auch Prototypen für Rezeptoren. Trotz einer Fülle an Informationen über nAChRs des Muskeltyps ist bis heute recht wenig über artenspezifischen funktionellen Unterschiede bekannt. Diese Studie befasst sich daher mit der Untersuchung von nAChRs des Muskeltyps in erwachsenen Mäusen und Menschen. Aufzeichnungen mit sogenannten Cell-attached Patches im heterologen Expressionssystem HEK293T-Zellen lieferten Beweise dafür, dass die ACh-Affinität von rekombinanten erwachsenen Maus- und menschlichen nAChRs vom Muskeltyp unterschiedlich sind. Um diesem nachzugehen, habe ich diese Rezeptoren in Outside-out Patches mit Hilfe eines schnellen Piezogetriebenen Applikationssystems verglichen. Dieses System bietet den Vorteil, dass einzelne Membran-Patches mit Rezeptoren unterschiedlichen Ligandenkonzentrationen ausgesetzt werden können. Als Reaktion auf 10 und 30 µM ACh waren die normalisierten Stromamplituden (î) und Stromanstiegszeiten (tr) der menschlichen Rezeptoren signifikant höher als die der Mausrezeptoren. Die Analyse der Dosis-Wirkungskurven von î und tr sowie die Anpassung eines quantitativen zweistufigen kinetischen Modells mit zwei äquivalenten Bindestellen an die Datensätze zeigten eine zweifach höhere Assoziationsrate für ACh bei menschlichen Rezeptoren, verglichen mit der von Mausrezeptoren. Zudem wurden menschliche nAChRs in Outside-Out-Patches schneller als Mausrezeptoren durch Superfusion mit 300 nM α-Bungarotoxin (α-Bgtx) blockiert, was für eine höhere Affinität auch für α-Bgtx spricht. Schließlich wiesen die menschlichen nAChRs in Outside-Out-Patches bei 3 µM ACh eine höhere Affinität als chimäre Rezeptoren aus Maus α- und menschlichen β-, γ- and ε-Untereinheiten auf. Die höhere Affinität der menschlichen Rezeptoren zu ACh und α-Bgtx im Vergleich zu Mausrezeptoren basiert somit zumindest in Teilen auf Sequenzdifferenzen ihrer α-Einheitenen. KW - nicotinic acetylcholine receptor KW - affinity KW - kinetic mechanism KW - Nicotinischer Acetylcholinrezeptor KW - Muskelzelle KW - Maus KW - Mensch Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192688 ER - TY - THES A1 - Becker, Mira Caroline T1 - Principles of olfactory-visual integration to form a common percept in honeybees T1 - Prinzipien der olfaktorisch-visuellen Integration des Lernverhaltens der Honigbienen N2 - The honeybee is a well studied and important organism in neuroethology. The possibility to train them with a classical conditioning paradigm and their miniature brain provide a perfect requisite to investigate the neuronal principles of learning and memory. Honeybees use visual and olfactory cues to detect flowers during their foraging trips. Hence, the reward association of a nectar source is a multi-modal construct, which has at least two major components - olfactory and visual cues. It is still an open question, how both sensory components are converged in the mushroom body, which represent the multi-modal integration centre of the honeybee brain. The main goal of this study, is to investigate the processing of multiple modalities and how a reward association is formed. This includes, how and wether both sensory modalities interfere during learning. Thus, in this study stimulation with UV, blue and green light was used to evoke distinct photoreceptor activities in the compound eye. Furthermore, three different odours (Geraniol, Citronellol and Farnesol) were used. These stimuli were tested in three different experimental series. The first experiment involved classical differential conditioning of the single modalities - odour and colour. Honeybees showed high learning performances in differentiating olfactory stimuli and also reliable responses for visual conditioning. Furthermore, a temporal discrepancy in the stimulus length for best learning in the olfatcoty and visual cues was found. In the second series, it was tested how multi-modal compounds are perceived. This includes, unique cues (configural processing) or the sum of the single components of a compound (elemen- tal processing). This was tested by combining single odour components with monochromatic light in a positive (PP) and negative patterning (NP) experiment. During PP, the olfactory- visual compound was rewarded, whereas the single components were unrewarded. In contrast, during NP the single components were reinforced, but the compound was not. In addition, the ability to distinguish between two different light stimuli presented as a part of an olfactory-visual compound with the same odour component during acquisition was tested. In a memory test, the light stimuli were presented again as a compound and in addition as the single components. The results revealed that bees used elemental processing with compounds containing green and blue light. In contrast, when UV light was presented the bees used configural processing. Finally, a third experiment was conducted at the neuronal level. Multi-unit recordings were established to provide a suitable method to analyse extrinsic neurons at the mushroom body output region, the so called ventral lobe of the pedunculus. Here, three different odours (Geran- iol, Farnesol and Citronellol), two colours (green and blue) and two combined stimuli (colour + odour) were chosen as stimuli, to search for possible variations in processing stimuli with different modalities. Two units could be detected that responded mainly to visual stimuli. N2 - Die Honigbiene ist ein gut untersuchter und wichtiger Organismus für die neuroethologische Forschung. Die Möglichkeit sie auf klassische Weise zu Konditionieren und ihr relativ kleines Gehirn macht sie zum idealen Untersuchungs-Gegenstand um die neuronalen Prinzipien des Lernens und der Gedächtnisbildung zu erforschen. Während des Furagierens nutzen Honigbi- enen beides: visuelle und olfaktorische Merkmale der Futterplanzen. Daher ist die Belohnungs- Assoziation mit der Nektar-Belohnung ein multi-modales Konstrukt, welches aus mindestens zwei Hauptkomponenten, den olfaktorischen und den visuellen Reizen, besteht. In dieser Arbeit soll untersucht werden, wie olfaktorische und visuelle Reize verarbeitet wer- den und wie sie im Pilzkörper, dem multi-modalen Integrationszentrum des Bienengehirnes, konvergieren. Wie beide sensorischen Modalitäten integriert werden um eine gemeingültige Belohnungs-Assoziation zu bilden, ist immer noch eine offene Frage. Weiterhin ist unklar ob und wie sie miteinander interferieren. Die hier dargestellten Studien nutzen Stimulationen mit UV, blauem und grünem Licht um unterschiedliche Photorezeptor Aktivitäten im Komplexauge auszulösen. Des Weiteren wurden drei verschiedene Duftkomponenten (Geraniol, Citronellol und Farnesol) verwendet. Diese Stimuli wurden in drei verschiedenen Experiment-Reihen gestestet. Das erste Experiment umfasste die klassische differentielle Konditionierung der Einzelmodalitäten (Duft und Farbe). Honigbienen zeigten eine hohe Lernfähigkeit bei der Unterscheidung zweier olfaktorischer Reize sowie eine solide Lern-Leistung während der Konditionierung mit Licht. Im zweiten Experiment wurde getestet, ob ein zusammengesetzter Reiz aus beiden Modalitäten als Summe der einzelnen Elemente (elementare Verarbeitung) oder als unikaler Reiz (konfigu- rale Verarbeitung) wahrgenommen wird. Hierbei wurde monochromatisches Licht und einzelne Duftkomponenten in positive patterning- (PP) und negative patterning-Experimenten (NP) getestet. Beim PP, wurde der zusammengesetzte Reiz belohnt, wohingegen die Einzelkom- ponenten unbelohnt blieben. Dagegen wurden beim NP nur die Einzelkomponenten belohnt, aber nicht ihre Kombination. Außerdem wurde der Frage nachgegangen, ob die Fähigkeit zur Differenzierung unterschiedlich ist, wenn zwei verschiedene Lichtreize teil einer olfaktorisch- visuellen Kombination sind, oder nicht. Interessanterweise zeigten die Verhaltensleistungen einen prominenten Fall von konfiguraler Verarbeitung, allerdings nur wenn UV-Licht ein El- ement der olfaktorisch-visuellen Zusammensetzung war. Die Ergebnisse der Experimente mit blauem oder grünem Licht hingegen, unterstützen die Theorie einer elementaren Verarbeitung. Abschließend wurde mittels elektrophysiologischer multi-unit-Aufnahmen eine passende Meth- ode etabliert, um die extrinsischen Neurone des Pilzkörpersausganges zu analysieren. Hierbei wurden drei verschiedene Düfte und zwei Farben sowie zwei Kombinationen aus Farbe und Duft getestet, um mögliche Variationen der multimodalen Reiz-Verarbeitung zu untersuchen. Zwei neuronale Einheiten (units) wurden gefunden, welche hauptsächlich auf Lichtreize antworteten. KW - honeybees KW - learning and behaviour KW - multi-modal stimuli Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199190 ER -