TY - JOUR A1 - Coelho, Luis Pedro A1 - Alves, Renato A1 - Monteiro, Paulo A1 - Huerta-Cepas, Jaime A1 - Freitas, Ana Teresa A1 - Bork, Peer T1 - NG-meta-profiler: fast processing of metagenomes using NGLess, a domain-specific language JF - Microbiome N2 - Background Shotgun metagenomes contain a sample of all the genomic material in an environment, allowing for the characterization of a microbial community. In order to understand these communities, bioinformatics methods are crucial. A common first step in processing metagenomes is to compute abundance estimates of different taxonomic or functional groups from the raw sequencing data. Given the breadth of the field, computational solutions need to be flexible and extensible, enabling the combination of different tools into a larger pipeline. Results We present NGLess and NG-meta-profiler. NGLess is a domain specific language for describing next-generation sequence processing pipelines. It was developed with the goal of enabling user-friendly computational reproducibility. It provides built-in support for many common operations on sequencing data and is extensible with external tools with configuration files. Using this framework, we developed NG-meta-profiler, a fast profiler for metagenomes which performs sequence preprocessing, mapping to bundled databases, filtering of the mapping results, and profiling (taxonomic and functional). It is significantly faster than either MOCAT2 or htseq-count and (as it builds on NGLess) its results are perfectly reproducible. Conclusions NG-meta-profiler is a high-performance solution for metagenomics processing built on NGLess. It can be used as-is to execute standard analyses or serve as the starting point for customization in a perfectly reproducible fashion. NGLess and NG-meta-profiler are open source software (under the liberal MIT license) and can be downloaded from https://ngless.embl.de or installed through bioconda. KW - metagenomics KW - next-generation sequencing KW - domain-specific language Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223161 VL - 7 IS - 84 ER - TY - JOUR A1 - Breitenbach, Tim A1 - Lorenz, Kristina A1 - Dandekar, Thomas T1 - How to steer and control ERK and the ERK signaling cascade exemplified by looking at cardiac insufficiency JF - International Journal of Molecular Sciences N2 - Mathematical optimization framework allows the identification of certain nodes within a signaling network. In this work, we analyzed the complex extracellular-signal-regulated kinase 1 and 2 (ERK1/2) cascade in cardiomyocytes using the framework to find efficient adjustment screws for this cascade that is important for cardiomyocyte survival and maladaptive heart muscle growth. We modeled optimal pharmacological intervention points that are beneficial for the heart, but avoid the occurrence of a maladaptive ERK1/2 modification, the autophosphorylation of ERK at threonine 188 (ERK\(^{Thr188}\) phosphorylation), which causes cardiac hypertrophy. For this purpose, a network of a cardiomyocyte that was fitted to experimental data was equipped with external stimuli that model the pharmacological intervention points. Specifically, two situations were considered. In the first one, the cardiomyocyte was driven to a desired expression level with different treatment strategies. These strategies were quantified with respect to beneficial effects and maleficent side effects and then which one is the best treatment strategy was evaluated. In the second situation, it was shown how to model constitutively activated pathways and how to identify drug targets to obtain a desired activity level that is associated with a healthy state and in contrast to the maleficent expression pattern caused by the constitutively activated pathway. An implementation of the algorithms used for the calculations is also presented in this paper, which simplifies the application of the presented framework for drug targeting, optimal drug combinations and the systematic and automatic search for pharmacological intervention points. The codes were designed such that they can be combined with any mathematical model given by ordinary differential equations. KW - optimal pharmacological modulation KW - efficient intervention points KW - ERK signaling KW - optimal treatment strategies KW - optimal drug targeting KW - optimal drug combination Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285164 SN - 1422-0067 VL - 20 IS - 9 ER - TY - JOUR A1 - Bartel, Karin A1 - Pein, Helmut A1 - Popper, Bastian A1 - Schmitt, Sabine A1 - Janaki-Raman, Sudha A1 - Schulze, Almut A1 - Lengauer, Florian A1 - Koeberle, Andreas A1 - Werz, Oliver A1 - Zischka, Hans A1 - Müller, Rolf A1 - Vollmar, Angelika M. A1 - Schwarzenberg, Karin von T1 - Connecting lysosomes and mitochondria – a novel role for lipid metabolism in cancer cell death JF - Cell Communication and Signaling N2 - Background The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood. Methods LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements. Results Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells. Conclusion This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors. KW - lysosome KW - V-ATPase KW - mitochondria KW - fission KW - apoptosis KW - lipid metabolism KW - cardiolipin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221524 VL - 17 ER - TY - JOUR A1 - Thölken, Clemens A1 - Thamm, Markus A1 - Erbacher, Christoph A1 - Lechner, Marcus T1 - Sequence and structural properties of circular RNAs in the brain of nurse and forager honeybees (Apis mellifera) JF - BMC Genomics N2 - Background The honeybee (Apis mellifera) represents a model organism for social insects displaying behavioral plasticity. This is reflected by an age-dependent task allocation. The most protruding tasks are performed by young nurse bees and older forager bees that take care of the brood inside the hive and collect food from outside the hive, respectively. The molecular mechanism leading to the transition from nurse bees to foragers is currently under intense research. Circular RNAs, however, were not considered in this context so far. As of today, this group of non-coding RNAs was only known to exist in two other insects, Drosophila melanogaster and Bombyx mori. Here we complement the state of circular RNA research with the first characterization in a social insect. Results We identified numerous circular RNAs in the brain of A. mellifera nurse bees and forager bees using RNA-Seq with exonuclease enrichment. Presence and circularity were verified for the most abundant representatives. Back-splicing in honeybee occurs further towards the end of transcripts and in transcripts with a high number of exons. The occurrence of circularized exons is correlated with length and CpG-content of their flanking introns. The latter coincides with increased DNA-methylation in the respective loci. For two prominent circular RNAs the abundance in worker bee brains was quantified in TaqMan assays. In line with previous findings of circular RNAs in Drosophila, circAmrsmep2 accumulates with increasing age of the insect. In contrast, the levels of circAmrad appear age-independent and correlate with the bee's task. Its parental gene is related to amnesia-resistant memory. Conclusions We provide the first characterization of circRNAs in a social insect. Many of the RNAs identified here show homologies to circular RNAs found in Drosophila and Bombyx, indicating that circular RNAs are a common feature among insects. We find that exon circularization is correlated to DNA-methylation at the flanking introns. The levels of circAmrad suggest a task-dependent abundance that is decoupled from age. Moreover, a GO term analysis shows an enrichment of task-related functions. We conclude that circular RNAs could be relevant for task allocation in honeybee and should be investigated further in this context. KW - circRNA KW - circular transcriptome sequencing KW - honeybee KW - brain KW - neuronal KW - Methylation KW - CpG KW - alternative splicing KW - behavioral plasticity Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241302 VL - 20 ER - TY - JOUR A1 - Grebinyk, Anna A1 - Prylutska, Svitlana A1 - Grebinyk, Sergii A1 - Prylutskyy, Yuriy A1 - Ritter, Uwe A1 - Matyshevska, Olga A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - Complexation with C\(_{60}\) fullerene increases doxorubicin efficiency against leukemic cells in vitro JF - Nanoscale Research Letters N2 - Conventional anticancer chemotherapy is limited because of severe side effects as well as a quickly evolving multidrug resistance of the tumor cells. To address this problem, we have explored a C\(_{60}\) fullerene-based nanosized system as a carrier for anticancer drugs for an optimized drug delivery to leukemic cells.Here, we studied the physicochemical properties and anticancer activity of C\(_{60}\) fullerene noncovalent complexes with the commonly used anticancer drug doxorubicin. C\(_{60}\)-Doxorubicin complexes in a ratio 1:1 and 2:1 were characterized with UV/Vis spectrometry, dynamic light scattering, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The obtained analytical data indicated that the 140-nm complexes were stable and could be used for biological applications. In leukemic cell lines (CCRF-CEM, Jurkat, THP1 and Molt-16), the nanocomplexes revealed 3.5 higher cytotoxic potential in comparison with the free drug in a range of nanomolar concentrations. Also, the intracellular drug's level evidenced C\(_{60}\) fullerene considerable nanocarrier function.The results of this study indicated that C\(_{60}\) fullerene-based delivery nanocomplexes had a potential value for optimization of doxorubicin efficiency against leukemic cells. KW - C-60 fullerene KW - doxorubicin KW - noncovalent complex KW - leukemic cells KW - cytotoxicity KW - accumulation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228257 VL - 14 IS - 61 ER - TY - JOUR A1 - Ruiz, E. Josue A1 - Diefenbacher, Markus E. A1 - Nelson, Jessica K. A1 - Sancho, Rocio A1 - Pucci, Fabio A1 - Chakraborty, Atanu A1 - Moreno, Paula A1 - Annibaldi, Alessandro A1 - Liccardi, Gianmaria A1 - Encheva, Vesela A1 - Mitter, Richard A1 - Rosenfeldt, Mathias A1 - Snijders, Ambrosius P. A1 - Meier, Pascal A1 - Calzado, Marco A. A1 - Behrens, Axel T1 - LUBAC determines chemotherapy resistance in squamous cell lung cancer JF - Journal of Experimental Medicine N2 - Lung squamous cell carcinoma (LSCC) and adenocarcinoma (LADC) are the most common lung cancer subtypes. Molecular targeted treatments have improved LADC patient survival but are largely ineffective in LSCC. The tumor suppressor FBW7 is commonly mutated or down-regulated in human LSCC, and oncogenic KRasG12D activation combined with Fbxw7 inactivation in mice (KF model) caused both LSCC and LADC. Lineage-tracing experiments showed that CC10(+), but not basal, cells are the cells of origin of LSCC in KF mice. KF LSCC tumors recapitulated human LSCC resistance to cisplatin-based chemotherapy, and we identified LUBAC-mediated NF-kappa B signaling as a determinant of chemotherapy resistance in human and mouse. Inhibition of NF-kappa B activation using TAK1 or LUBAC inhibitors resensitized LSCC tumors to cisplatin, suggesting a future avenue for LSCC patient treatment. KW - Solid tumors Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227146 VL - 216 IS - 2 ER - TY - JOUR A1 - Villalobos, Alvaro S. A1 - Wiese, Jutta A1 - Imhoff, Johannes F. A1 - Dorador, Cristina A1 - Keller, Alexander A1 - Hentschel, Ute T1 - Systematic affiliation and genome analysis of Subtercola vilae DB165T with particular emphasis on cold adaptation of an isolate from a high-altitude cold volcano lake JF - Microorganisms N2 - Among the Microbacteriaceae the species of Subtercola and Agreia form closely associated clusters. Phylogenetic analysis demonstrated three major phylogenetic branches of these species. One of these branches contains the two psychrophilic species Subtercola frigoramans and Subtercola vilae, together with a larger number of isolates from various cold environments. Genomic evidence supports the separation of Agreia and Subtercola species. In order to gain insight into the ability of S. vilae to adapt to life in this extreme environment, we analyzed the genome with a particular focus on properties related to possible adaptation to a cold environment. General properties of the genome are presented, including carbon and energy metabolism, as well as secondary metabolite production. The repertoire of genes in the genome of S. vilae DB165\(^T\) linked to adaptations to the harsh conditions found in Llullaillaco Volcano Lake includes several mechanisms to transcribe proteins under low temperatures, such as a high number of tRNAs and cold shock proteins. In addition, S. vilae DB165\(^T\) is capable of producing a number of proteins to cope with oxidative stress, which is of particular relevance at low temperature environments, in which reactive oxygen species are more abundant. Most important, it obtains capacities to produce cryo-protectants, and to combat against ice crystal formation, it produces ice-binding proteins. Two new ice-binding proteins were identified which are unique to S. vilae DB165\(^T\). These results indicate that S. vilae has the capacity to employ different mechanisms to live under the extreme and cold conditions prevalent in Llullaillaco Volcano Lake. KW - cold adaptation KW - Subtercola vilae KW - genome analysis KW - systematic affiliation KW - Llullaillaco Volcano Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197394 SN - 2076-2607 VL - 7 IS - 4 ER - TY - JOUR A1 - Grebinyk, Anna A1 - Prylutska, Svitlana A1 - Chepurna, Oksana A1 - Grebinyk, Sergii A1 - Prylutskyy, Yuriy A1 - Ritter, Uwe A1 - Ohulchanskyy, Tymish Y. A1 - Matyshevska, Olga A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - Synergy of chemo- and photodynamic therapies with C\(_{60}\) Fullerene-Doxorubicin nanocomplex JF - Nanomaterials N2 - A nanosized drug complex was explored to improve the efficiency of cancer chemotherapy, complementing it with nanodelivery and photodynamic therapy. For this, nanomolar amounts of a non-covalent nanocomplex of Doxorubicin (Dox) with carbon nanoparticle C\(_{60}\) fullerene (C\(_{60}\)) were applied in 1:1 and 2:1 molar ratio, exploiting C\(_{60}\) both as a drug-carrier and as a photosensitizer. The fluorescence microscopy analysis of human leukemic CCRF-CEM cells, in vitro cancer model, treated with nanocomplexes showed Dox’s nuclear and C\(_{60}\)'s extranuclear localization. It gave an opportunity to realize a double hit strategy against cancer cells based on Dox's antiproliferative activity and C\(_{60}\)'s photoinduced pro-oxidant activity. When cells were treated with 2:1 C\(_{60}\)-Dox and irradiated at 405 nm the high cytotoxicity of photo-irradiated C\(_{60}\)-Dox enabled a nanomolar concentration of Dox and C\(_{60}\) to efficiently kill cancer cells in vitro. The high pro-oxidant and pro-apoptotic efficiency decreased IC\(_{50}\) 16, 9 and 7 × 10\(^3\)-fold, if compared with the action of Dox, non-irradiated nanocomplex, and C\(_{60}\)'s photodynamic effect, correspondingly. Hereafter, a strong synergy of therapy arising from the combination of C\(_{60}\)-mediated Dox delivery and C\(_{60}\) photoexcitation was revealed. Our data indicate that a combination of chemo- and photodynamic therapies with C\(_{60}\)-Dox nanoformulation provides a promising synergetic approach for cancer treatment. KW - photodynamic chemotherapy KW - synergistic effect KW - C\(_{60}\) fullerene KW - Doxorubicin KW - nanocomplex KW - leukemic cells KW - apoptosis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193140 SN - 2079-4991 VL - 9 IS - 11 ER - TY - JOUR A1 - Molinas-González, Carlos R. A1 - Castro, Jorge A1 - González-Megías, Adela A1 - Leverkus, Alexandro B. T1 - Effects of post-fire deadwood management on soil macroarthropod communities JF - Forests N2 - Dead wood comprises a vast amount of biological legacies that set the scene for ecological regeneration after wildfires, yet its removal is the most frequent management strategy worldwide. Soil-dwelling organisms are conspicuous, and they provide essential ecosystem functions, but their possible affection by different post-fire management strategies has so far been neglected. We analyzed the abundance, richness, and composition of belowground macroarthropod communities under two contrasting dead-wood management regimes after a large wildfire in the Sierra Nevada Natural and National Park (Southeast Spain). Two plots at different elevation were established, each containing three replicates of two experimental treatments: partial cut, where trees were cut and their branches lopped off and left over the ground, and salvage logging, where all the trees were cut, logs were piled, branches were mechanically masticated, and slash was spread on the ground. Ten years after the application of the treatments, soil cores were extracted from two types of microhabitat created by these treatments: bare-soil (in both treatments) and under-logs (in the partial cut treatment only). Soil macroarthropod assemblages were dominated by Hemiptera and Hymenoptera (mostly ants) and were more abundant and richer in the lowest plot. The differences between dead-wood treatments were most evident at the scale of management interventions: abundance and richness were lowest after salvage logging, even under similar microhabitats (bare-soil). However, there were no significant differences between microhabitat types on abundance and richness within the partial cut treatment. Higher abundance and richness in the partial cut treatment likely resulted from higher resource availability and higher plant diversity after natural regeneration. Our results suggest that belowground macroarthropod communities are sensitive to the manipulation of dead-wood legacies and that management through salvage logging could reduce soil macroarthropod recuperation compared to other treatments with less intense management even a decade after application. KW - forest fire KW - burnt-wood KW - species richness KW - soil fauna KW - post-fire management Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193079 SN - 1999-4907 VL - 10 IS - 11 ER - TY - JOUR A1 - Vey, Johannes A1 - Kapsner, Lorenz A. A1 - Fuchs, Maximilian A1 - Unberath, Philipp A1 - Veronesi, Giulia A1 - Kunz, Meik T1 - A toolbox for functional analysis and the systematic identification of diagnostic and prognostic gene expression signatures combining meta-analysis and machine learning JF - Cancers N2 - The identification of biomarker signatures is important for cancer diagnosis and prognosis. However, the detection of clinical reliable signatures is influenced by limited data availability, which may restrict statistical power. Moreover, methods for integration of large sample cohorts and signature identification are limited. We present a step-by-step computational protocol for functional gene expression analysis and the identification of diagnostic and prognostic signatures by combining meta-analysis with machine learning and survival analysis. The novelty of the toolbox lies in its all-in-one functionality, generic design, and modularity. It is exemplified for lung cancer, including a comprehensive evaluation using different validation strategies. However, the protocol is not restricted to specific disease types and can therefore be used by a broad community. The accompanying R package vignette runs in ~1 h and describes the workflow in detail for use by researchers with limited bioinformatics training. KW - bioinformatics tool KW - R package KW - machine learning KW - meta-analysis KW - biomarker signature KW - gene expression analysis KW - survival analysis KW - functional analysis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193240 SN - 2072-6694 VL - 11 IS - 10 ER - TY - JOUR A1 - Schmidt, Thomas S. B. A1 - Hayward, Matthew R. A1 - Coelho, Luiis P. A1 - Li, Simone S. A1 - Costea, Paul I. A1 - Voigt, Anita Y. A1 - Wirbel, Jakob A1 - Maistrenko, Oleksandr M. A1 - Alves, Renato J. C. A1 - Bergsten, Emma A1 - de Beaufort, Carine A1 - Sobhani, Iradj A1 - Heintz-Buschart, Anna A1 - Sunagawa, Shinichi A1 - Zeller, Georg A1 - Wilmes, Paul A1 - Bork, Peer T1 - Extensive transmission of microbes along the gastrointestinal tract JF - eLife N2 - The gastrointestinal tract is abundantly colonized by microbes, yet the translocation of oral species to the intestine is considered a rare aberrant event, and a hallmark of disease. By studying salivary and fecal microbial strain populations of 310 species in 470 individuals from five countries, we found that transmission to, and subsequent colonization of, the large intestine by oral microbes is common and extensive among healthy individuals. We found evidence for a vast majority of oral species to be transferable, with increased levels of transmission in colorectal cancer and rheumatoid arthritis patients and, more generally, for species described as opportunistic pathogens. This establishes the oral cavity as an endogenous reservoir for gut microbial strains, and oral-fecal transmission as an important process that shapes the gastrointestinal microbiome in health and disease. KW - Colonization KW - Annotation KW - Dynamics KW - Accurate KW - Strains KW - Barrier KW - Health KW - Acids KW - Research Article KW - Computational and Systems Biology KW - Microbiology and Infectious Disease KW - microbiome KW - gastrointestinal tract KW - colorectal cancer KW - rheumatoid arthritis KW - metagenomics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228954 VL - 8 ER - TY - JOUR A1 - Sauer, Markus A1 - Juranek, Stefan A. A1 - Marks, James A1 - De Magis, Alessio A1 - Kazemier, Hinke G A1 - Hilbig, Daniel A1 - Benhalevy, Daniel A1 - Wang, Xiantao A1 - Hafner, Markus A1 - Paeschke, Katrin T1 - DHX36 prevents the accumulation of translationally inactive mRNAs with G4-structures in untranslated regions JF - Nature Communications N2 - Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress. KW - RNA metabolism KW - Translation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227486 VL - 10 IS - 2421 ER - TY - JOUR A1 - Mayer, Alexander E. A1 - Löffler, Mona C. A1 - Loza Valdés, Angel E. A1 - Schmitz, Werner A1 - El-Merahbi, Rabih A1 - Trujillo-Viera, Jonathan A1 - Erk, Manuela A1 - Zhang, Thianzhou A1 - Braun, Ursula A1 - Heikenwalder, Mathias A1 - Leitges, Michael A1 - Schulze, Almut A1 - Sumara, Grzegorz T1 - The kinase PKD3 provides negative feedback on cholesterol and triglyceride synthesis by suppressing insulin signaling JF - Science Signaling N2 - Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity. KW - Protein kinase D3 (PKD3) KW - cholesterol KW - diacylglycerol (DAG) KW - liver KW - metabolism Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250025 ET - accepted manuscript ER - TY - JOUR A1 - Roth, Nicolas A1 - Doerfler, Inken A1 - Bässler, Claus A1 - Blaschke, Markus A1 - Bussler, Heinz A1 - Gossner, Martin M. A1 - Heideroth, Antje A1 - Thorn, Simon A1 - Weisser, Wolfgang W. A1 - Müller, Jörg T1 - Decadal effects of landscape-wide enrichment of dead wood on saproxylic organisms in beech forests of different historic management intensity JF - Diversity and Distributions N2 - Aim: European temperate forests have lost dead wood and the associated biodiversity owing to intensive management over centuries. Nowadays, some of these forests are being restored by enrichment with dead wood, but mostly only at stand scales. Here, we investigated effects of a seminal dead-wood enrichment strategy on saproxylic organisms at the landscape scale. Location: Temperate European beech forest in southern Germany. Methods: In a before-after control-impact design, we compared assemblages and gamma diversities of saproxylic organisms in strictly protected old-growth forest areas (reserves) and historically moderately and intensively managed forest areas before and a decade after starting a landscape-wide strategy of dead-wood enrichment. Results: Before enrichment with dead wood, the gamma diversity of saproxylic organisms in historically intensively managed forest stands was significantly lower than in reserves and historically moderately managed forest stands; this difference disappeared after 10 years of dead-wood enrichment. The species composition of beetles in forest stands of the three historical management intensities differed before the enrichment strategy, but a decade thereafter, the species compositions of previously intensively logged and forest reserve plots were similar. However, the differences in fungal species composition between historical management categories before and after 10 years of enrichment persisted. Main conclusions: Our results demonstrate that intentional enrichment of dead wood at the landscape scale is a powerful tool for rapidly restoring saproxylic beetle communities and for restoring wood-inhabiting fungal communities, which need longer than a decade for complete restoration. We propose that a strategy of area-wide active restoration combined with some permanent strict refuges is a promising means of promoting the biodiversity of age-long intensively managed Central European beech forests. KW - dead-wood enrichment KW - integrative management strategy KW - land sharing KW - lowland beech forests KW - saproxylic organisms Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227061 VL - 25 IS - 3 ER - TY - JOUR A1 - Kühnisch, Jirko A1 - Herbst, Christopher A1 - Al‐Wakeel‐Marquard, Nadya A1 - Dartsch, Josephine A1 - Holtgrewe, Manuel A1 - Baban, Anwar A1 - Mearini, Giulia A1 - Hardt, Juliane A1 - Kolokotronis, Konstantinos A1 - Gerull, Brenda A1 - Carrier, Lucie A1 - Beule, Dieter A1 - Schubert, Stephan A1 - Messroghli, Daniel A1 - Degener, Franziska A1 - Berger, Felix A1 - Klaassen, Sabine T1 - Targeted panel sequencing in pediatric primary cardiomyopathy supports a critical role of TNNI3 JF - Clinical Genetics N2 - The underlying genetic mechanisms and early pathological events of children with primary cardiomyopathy (CMP) are insufficiently characterized. In this study, we aimed to characterize the mutational spectrum of primary CMP in a large cohort of patients ≤18 years referred to a tertiary center. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel‐based next‐generation sequencing approach of 89 genes. At least one pathogenic or probably pathogenic variant was identified in 30/80 (38%) index patients. In all CMP subgroups, patients carried most frequently variants of interest in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. In MYH7, MYBPC3, and TNNI3, we identified 18 pathogenic/probably pathogenic variants (MYH7 n = 7, MYBPC3 n = 6, TNNI3 n = 5, including one homozygous (TNNI3 c.24+2T>A) truncating variant. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of TNNI3 revealed that the TNNI3 protein is absent and associated with upregulation of the fetal isoform TNNI1. The present study further supports the clinical importance of sarcomeric mutation—not only in adult—but also in pediatric primary CMP. TNNI3 is the third most important disease gene in this cohort and complete loss of TNNI3 leads to severe pediatric CMP. KW - cardiomyopathy KW - genetics KW - pediatrics KW - sarcomere KW - TNNI3 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213958 VL - 96 IS - 6 SP - 549 EP - 559 ER - TY - JOUR A1 - Eisenreich, Wolfgang A1 - Rudel, Thomas A1 - Heesemann, Jürgen A1 - Goebel, Werner T1 - How viral and intracellular bacterial pathogens reprogram the metabolism of host cells to allow their intracellular replication JF - Frontiers in Cellular and Infection Microbiology N2 - Viruses and intracellular bacterial pathogens (IBPs) have in common the need of suitable host cells for efficient replication and proliferation during infection. In human infections, the cell types which both groups of pathogens are using as hosts are indeed quite similar and include phagocytic immune cells, especially monocytes/macrophages (MOs/MPs) and dendritic cells (DCs), as well as nonprofessional phagocytes, like epithelial cells, fibroblasts and endothelial cells. These terminally differentiated cells are normally in a metabolically quiescent state when they are encountered by these pathogens during infection. This metabolic state of the host cells does not meet the extensive need for nutrients required for efficient intracellular replication of viruses and especially IBPs which, in contrast to the viral pathogens, have to perform their own specific intracellular metabolism to survive and efficiently replicate in their host cell niches. For this goal, viruses and IBPs have to reprogram the host cell metabolism in a pathogen-specific manner to increase the supply of nutrients, energy, and metabolites which have to be provided to the pathogen to allow its replication. In viral infections, this appears to be often achieved by the interaction of specific viral factors with central metabolic regulators, including oncogenes and tumor suppressors, or by the introduction of virus-specific oncogenes. Less is so far known on the mechanisms leading to metabolic reprogramming of the host cell by IBPs. However, the still scant data suggest that similar mechanisms may also determine the reprogramming of the host cell metabolism in IBP infections. In this review, we summarize and compare the present knowledge on this important, yet still poorly understood aspect of pathogenesis of human viral and especially IBP infections. KW - metabolic adaptation KW - viruses KW - intracellular bacterial pathogens KW - metabolism of infected and uninfected host cells KW - reprogamming of host cell metabolism Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197188 SN - 2235-2988 VL - 9 ER - TY - JOUR A1 - Grebinyk, Anna A1 - Prylutska, Svitlana A1 - Buchelnikov, Anatoliy A1 - Tverdokhleb, Nina A1 - Grebinyk, Sergii A1 - Evstigneev, Maxim A1 - Matyshevska, Olga A1 - Cherepanov, Vsevolod A1 - Prylutskyy, Yuriy A1 - Yashchuk, Valeriy A1 - Naumovets, Anton A1 - Ritter, Uwe A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - C60 fullerene as an effective nanoplatform of alkaloid Berberine delivery into leukemic cells JF - Pharmaceutics N2 - A herbal alkaloid Berberine (Ber), used for centuries in Ayurvedic, Chinese, Middle-Eastern, and native American folk medicines, is nowadays proved to function as a safe anticancer agent. Yet, its poor water solubility, stability, and bioavailability hinder clinical application. In this study, we have explored a nanosized carbon nanoparticle—C60 fullerene (C60)—for optimized Ber delivery into leukemic cells. Water dispersions of noncovalent C60-Ber nanocomplexes in the 1:2, 1:1, and 2:1 molar ratios were prepared. UV–Vis spectroscopy, dynamic light scattering (DLS), and atomic force microscopy (AFM) evidenced a complexation of the Ber cation with the negatively charged C60 molecule. The computer simulation showed that π-stacking dominates in Ber and C\(_{60}\) binding in an aqueous solution. Complexation with C\(_{60}\) was found to promote Ber intracellular uptake. By increasing C\(_{60}\) concentration, the C\(_{60}\)-Ber nanocomplexes exhibited higher antiproliferative potential towards CCRF-CEM cells, in accordance with the following order: free Ber < 1:2 < 1:1 < 2:1 (the most toxic). The activation of caspase 3/7 and accumulation in the sub-G1 phase of CCRF-CEM cells treated with C\(_{60}\)-Ber nanocomplexes evidenced apoptosis induction. Thus, this study indicates that the fast and easy noncovalent complexation of alkaloid Ber with C\(_{60}\) improved its in vitro efficiency against cancer cells. KW - C60 fullerene KW - Berberine KW - noncovalent nanocomplex KW - UV–Vis KW - DLS and AFM measurements KW - drug release KW - leukemic cells KW - uptake KW - cytotoxicity KW - apoptosis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193216 SN - 1999-4923 VL - 11 IS - 11 ER - TY - JOUR A1 - Paponov, Ivan A. A1 - Dindas , Julian A1 - Król , Elżbieta A1 - Friz, Tatyana A1 - Budnyk, Vadym A1 - Teale, William A1 - Paponov, Martina A1 - Hedrich , Rainer A1 - Palme, Klaus T1 - Auxin-Induced plasma membrane depolarization is regulated by Auxin transport and not by AUXIN BINDING PROTEIN1 JF - Frontiers in Plant Science N2 - Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling. KW - auxin KW - ABP1 KW - plasma membrane depolarization KW - AUX1 KW - endocytosis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195914 SN - 1664-462X VL - 9 ER - TY - JOUR A1 - Gebert, Friederike A1 - Steffan-Dewenter, Ingolf A1 - Moretto, Philippe A1 - Peters, Marcell K. T1 - Climate rather than dung resources predict dung beetle abundance and diversity along elevational and land use gradients on Mt. Kilimanjaro JF - Journal of Biogeography N2 - Aim: While elevational gradients in species richness constitute some of the best depicted patterns in ecology, there is a large uncertainty concerning the role of food resource availability for the establishment of diversity gradients in insects. Here, we analysed the importance of climate, area, land use and food resources for determining diversity gradients of dung beetles along extensive elevation and land use gradients on Mt. Kilimanjaro, Tanzania. Location: Mt. Kilimanjaro, Tanzania. Taxon: Scarabaeidae (Coleoptera). Methods: Dung beetles were recorded with baited pitfall traps at 66 study plots along a 3.6 km elevational gradient. In order to quantify food resources for the dung beetle community in form of mammal defecation rates, we assessed mammalian diversity and biomass with camera traps. Using a multi‐model inference framework and path analysis, we tested the direct and indirect links between climate, area, land use and mammal defecation rates on the species richness and abundance of dung beetles. Results: We found that the species richness of dung beetles declined exponentially with increasing elevation. Human land use diminished the species richness of functional groups exhibiting complex behaviour but did not have a significant influence on total species richness. Path analysis suggested that climate, in particular temperature and to a lesser degree precipitation, were the most important predictors of dung beetle species richness while mammal defecation rate was not supported as a predictor variable. Main conclusions: Along broad climatic gradients, dung beetle diversity is mainly limited by climatic factors rather than by food resources. Our study points to a predominant role of temperature‐driven processes for the maintenance and origination of species diversity of ectothermic organisms, which will consequently be subject to ongoing climatic changes. KW - altitudinal gradients KW - diversity gradients KW - enercy-richness hypothesis KW - food resources KW - insect abundance KW - land use KW - Scarabaeidae KW - temperature‐richness hypothesis KW - temperature‐mediated resource exploitation hypothesis KW - species‐area hypothesis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204701 VL - 47 IS - 2 SP - 371 EP - 381 ER - TY - JOUR A1 - Hovestadt, Thomas A1 - Thomas, Jeremy A. A1 - Mitesser, Oliver A1 - Schönrogge, Karsten T1 - Multiple host use and the dynamics of host-switching in host-parasite systems JF - Insect Conservation and Diversity N2 - The link between multi‐host use and host switching in host–parasite interactions is a continuing area of debate. Lycaenid butterflies in the genus Maculinea, for example, exploit societies of different Myrmica ant species across their ranges, but there is only rare evidence that they simultaneously utilise multiple hosts at a local site, even where alternative hosts are present. We present a simple population‐genetic model accounting for the proportion of two alternative hosts and the fitness of parasite genotypes on each host. In agreement with standard models, we conclude that simultaneous host use is possible whenever fitness of heterozygotes on alternative hosts is not too low. We specifically focus on host‐shifting dynamics when the frequency of hosts changes. We find that (i) host shifting may proceed so rapidly that multiple host use is unlikely to be observed, (ii) back and forth transition in host use can exhibit a hysteresis loop, (iii) the parasites' host use may not be proportional to local host frequencies and be restricted to the rarer host under some conditions, and (iv) that a substantial decline in parasite abundance may typically precede a shift in host use. We conclude that focusing not just on possible equilibrium conditions but also considering the dynamics of host shifting in non‐equilibrium situations may provide added insights into host–parasite systems. KW - Host-parasite interaction KW - Maculinea butterfly KW - Myrmica ant non-equilibrium dynamics KW - population genetics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204747 VL - 12 IS - 6 ER - TY - JOUR A1 - Boetzl, Fabian A. A1 - Konle, Antonia A1 - Krauss, Jochen T1 - Aphid cards – useful model for assessing predation rates or bias prone nonsense? JF - Journal of Applied Entomology N2 - Predation on pest organisms is an essential ecosystem function supporting yields in modern agriculture. However, assessing predation rates is intricate, and they can rarely be linked directly to predator densities or functions. We tested whether sentinel prey aphid cards are useful tools to assess predation rates in the field. Therefore, we looked at aphid cards of different sizes on the ground level as well as within the vegetation. Additionally, by trapping ground‐dwelling predators, we examined whether obtained predation rates could be linked to predator densities and traits. Predation rates recorded with aphid cards were independent of aphid card size. However, predation rates on the ground level were three times higher than within the vegetation. We found both predatory carabid activity densities as well as community weighted mean body size to be good predictors for predation rates. Predation rates obtained from aphid cards are stable over card type and related to predator assemblages. Aphid cards, therefore, are a useful, efficient method for rapidly assessing the ecosystem function predation. Their use might especially be recommended for assessments on the ground level and when time and resource limitations rule out more elaborate sentinel prey methods using exclosures with living prey animals. KW - carabid beetles KW - ecosystem service KW - ground-dwelling predators KW - methods KW - natural pest control KW - sentinel prey Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204798 VL - 144 IS - 1-2 ER - TY - JOUR A1 - Figueiredo, Ludmilla A1 - Krauss, Jochen A1 - Steffan-Dewenter, Ingolf A1 - Cabral, Juliano Sarmento T1 - Understanding extinction debts: spatio-temporal scales, mechanisms and a roadmap for future research JF - Ecography N2 - Extinction debt refers to delayed species extinctions expected as a consequence of ecosystem perturbation. Quantifying such extinctions and investigating long‐term consequences of perturbations has proven challenging, because perturbations are not isolated and occur across various spatial and temporal scales, from local habitat losses to global warming. Additionally, the relative importance of eco‐evolutionary processes varies across scales, because levels of ecological organization, i.e. individuals, (meta)populations and (meta)communities, respond hierarchically to perturbations. To summarize our current knowledge of the scales and mechanisms influencing extinction debts, we reviewed recent empirical, theoretical and methodological studies addressing either the spatio–temporal scales of extinction debts or the eco‐evolutionary mechanisms delaying extinctions. Extinction debts were detected across a range of ecosystems and taxonomic groups, with estimates ranging from 9 to 90% of current species richness. The duration over which debts have been sustained varies from 5 to 570 yr, and projections of the total period required to settle a debt can extend to 1000 yr. Reported causes of delayed extinctions are 1) life‐history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions. Other potential factors that may extend survival time such as microevolutionary dynamics, or delayed extinctions of interaction partners, have rarely been analyzed. Therefore, we propose a roadmap for future research with three key avenues: 1) the microevolutionary dynamics of extinction processes, 2) the disjunctive loss of interacting species and 3) the impact of multiple regimes of perturbation on the payment of debts. For their ability to integrate processes occurring at different levels of ecological organization, we highlight mechanistic simulation models as tools to address these knowledge gaps and to deepen our understanding of extinction dynamics. KW - Anthropocene KW - biotic interaction KW - extinction dynamics KW - mechanistic modelling KW - time lag KW - transient dynamics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204859 VL - 42 IS - 12 ER - TY - JOUR A1 - Pauls, Dennis A1 - Hamarat, Yasmin A1 - Trufasu, Luisa A1 - Schendzielorz, Tim M. A1 - Gramlich, Gertrud A1 - Kahnt, Jörg A1 - Vanselow, Jens A1 - Schlosser, Andreas A1 - Wegener, Christian T1 - Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate JF - European Journal of Neuroscience N2 - Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well‐characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE ), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD ) in global neuropeptide processing and selected peptide‐regulated behaviours in Drosophila . We found that a deficiency in dCPD results in C‐terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD ‐encoding gene silver in the larva causes lethality, and leads to deficits in starvation‐induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide‐regulated behaviour in Drosophila . dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE. KW - direct muss spectrometric profiling KW - friut fly behaviour KW - M14 carboxypeptidasses KW - peptidomoics KW - protein processing Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204863 VL - 50 IS - 9 ER - TY - JOUR A1 - Requier, Fabrice A1 - Paillet, Yoan A1 - Laroche, Fabienne A1 - Rutschmann, Benjamin A1 - Zhang, Jie A1 - Lombardi, Fabio A1 - Svoboda, Miroslav A1 - Steffan-Dewenter, Ingolf T1 - Contribution of European forests to safeguard wild honeybee populations JF - Conservation Letters N2 - Abstract Recent studies reveal the use of tree cavities by wild honeybee colonies in European forests. This highlights the conservation potential of forests for a highly threatened component of the native entomofauna in Europe, but currently no estimate of potential wild honeybee population sizes exists. Here, we analyzed the tree cavity densities of 106 forest areas across Europe and inferred an expected population size of wild honeybees. Both forest and management types affected the density of tree cavities. Accordingly, we estimated that more than 80,000 wild honeybee colonies could be sustained in European forests. As expected, potential conservation hotspots were identified in unmanaged forests, and, surprisingly, also in other large forest areas across Europe. Our results contribute to the EU policy strategy to halt pollinator declines and reveal the potential of forest areas for the conservation of so far neglected wild honeybee populations in Europe. KW - Apis mellifera KW - Conservation KW - forest management KW - honeybees KW - native populations KW - protected forests KW - tree cavities KW - unmanaged broadleaved forests Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204407 VL - 13 IS - 2 ER - TY - JOUR A1 - Diers, J. A1 - Wagner, J. A1 - Baum, P. A1 - Lichthardt, S. A1 - Kastner, C. A1 - Matthes, N. A1 - Löb, S. A1 - Matthes, H. A1 - Germer, C.-T. A1 - Wiegering, A. T1 - Nationwide in-hospital mortality following colonic cancer resection according to hospital volume in Germany JF - BJS Open N2 - Background: Colonic cancer is the most common cancer of the gastrointestinal tract. The aim of this study was to determine mortality rates following colonic cancer resection and the effect of hospital caseload on in-hospital mortality in Germany. Methods: Patients admitted with a diagnosis of colonic cancer undergoing colonic resection from 2012 to 2015 were identifed from a nationwide registry using procedure codes. The outcome measure was in-hospital mortality. Hospitals were ranked according to their caseload for colonic cancer resection, and patients were categorized into five subgroups on the basis of hospital volume. Results: Some 129 196 colonic cancer resections were reviewed. The overall in-house mortality rate was 5⋅8 per cent, ranging from 6⋅9 per cent (1775 of 25 657 patients) in very low-volume hospitals to 4⋅8 per cent (1239 of 25 825) in very high-volume centres (P < 0⋅001). In multivariable logistic regression analysis the risk-adjusted odds ratio for in-house mortality was 0⋅75 (95 per cent c.i. 0⋅66 to 0⋅84) in very high-volume hospitals performing a mean of 85⋅0 interventions per year, compared with that in very low-volume hospitals performing a mean of only 12⋅7 interventions annually, after adjustment for sex, age, co-morbidity, emergency procedures, prolonged mechanical ventilation and transfusion. Conclusion: In Germany, patients undergoing colonic cancer resections in high-volume hospitals had with improved outcomes compared with patients treated in low-volume hospitals Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204385 VL - 3 IS - 5 ER - TY - JOUR A1 - Duque, Laura A1 - Poelman, Erik H. A1 - Steffan-Dewenter, Ingolf T1 - Plant-mediated effects of ozone on herbivores depend on exposure duration and temperature JF - Scientific Reports N2 - Abiotic stress by elevated tropospheric ozone and temperature can alter plants’ metabolism, growth, and nutritional value and modify the life cycle of their herbivores. We investigated how the duration of exposure of Sinapis arvensis plants to high ozone and temperature levels affect the life cycle of the large cabbage white, Pieris brassicae. Plants were exposed to ozone-clean (control) or ozone-enriched conditions (120 ppb) for either 1 or 5 days and were afterwards kept in a greenhouse with variable temperature conditions. When given the choice, P. brassicae butterflies laid 49% fewer eggs on ozone-exposed than on control plants when the exposure lasted for 5 days, but showed no preference when exposure lasted for 1 day. The caterpillars took longer to hatch on ozone-exposed plants and at lower ambient temperatures. The ozone treatment had a positive effect on the survival of the eggs. Ozone decreased the growth of caterpillars reared at higher temperatures on plants exposed for 5 days, but not on plants exposed for 1 day. Overall, longer exposure of the plants to ozone and higher temperatures affected the life cycle of the herbivore more strongly. With global warming, the indirect impacts of ozone on herbivores are likely to become more common. KW - Ecology KW - Environmental impact Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202805 VL - 9 ER - TY - JOUR A1 - Maierhofer, Anna A1 - Flunkert, Julia A1 - Oshima, Junko A1 - Martin, George M. A1 - Poot, Martin A1 - Nanda, Indrajit A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Haaf, Thomas T1 - Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes JF - Aging Cell N2 - Werner Syndrome (WS) is an adult‐onset segmental progeroid syndrome. Bisulfite pyrosequencing of repetitive DNA families revealed comparable blood DNA methylation levels between classical (18 WRN‐mutant) or atypical WS (3 LMNA‐mutant and 3 POLD1‐mutant) patients and age‐ and sex‐matched controls. WS was not associated with either age‐related accelerated global losses of ALU, LINE1, and α‐satellite DNA methylations or gains of rDNA methylation. Single CpG methylation was analyzed with Infinium MethylationEPIC arrays. In a correspondence analysis, atypical WS samples clustered together with the controls and were clearly separated from classical WS, consistent with distinct epigenetic pathologies. In classical WS, we identified 659 differentially methylated regions (DMRs) comprising 3,656 CpG sites and 613 RefSeq genes. The top DMR was located in the HOXA4 promoter. Additional DMR genes included LMNA, POLD1, and 132 genes which have been reported to be differentially expressed in WRN‐mutant/depleted cells. DMRs were enriched in genes with molecular functions linked to transcription factor activity and sequence‐specific DNA binding to promoters transcribed by RNA polymerase II. We propose that transcriptional misregulation of downstream genes by the absence of WRN protein contributes to the variable premature aging phenotypes of WS. There were no CpG sites showing significant differences in DNA methylation changes with age between WS patients and controls. Genes with both WS‐ and age‐related methylation changes exhibited a constant offset of methylation between WRN‐mutant patients and controls across the entire analyzed age range. WS‐specific epigenetic signatures occur early in life and do not simply reflect an acceleration of normal epigenetic aging processes. KW - (classical and atypical) Werner syndrome KW - bisulfite pyrosequencing KW - methylation array KW - premature aging KW - segmental progeria KW - transcription deficiency Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202733 VL - 18 ER - TY - JOUR A1 - Beer, Katharina A1 - Schenk, Mariela A1 - Helfrich-Förster, Charlotte A1 - Holzschuh, Andrea T1 - The circadian clock uses different environmental time cues to synchronize emergence and locomotion of the solitary bee Osmia bicornis JF - Scientific Reports N2 - Life on earth adapted to the daily reoccurring changes in environment by evolving an endogenous circadian clock. Although the circadian clock has a crucial impact on survival and behavior of solitary bees, many aspects of solitary bee clock mechanisms remain unknown. Our study is the first to show that the circadian clock governs emergence in Osmia bicornis, a bee species which overwinters as adult inside its cocoon. Therefore, its eclosion from the pupal case is separated by an interjacent diapause from its emergence in spring. We show that this bee species synchronizes its emergence to the morning. The daily rhythms of emergence are triggered by temperature cycles but not by light cycles. In contrast to this, the bee’s daily rhythms in locomotion are synchronized by light cycles. Thus, we show that the circadian clock of O. bicornis is set by either temperature or light, depending on what activity is timed. Light is a valuable cue for setting the circadian clock when bees have left the nest. However, for pre-emerged bees, temperature is the most important cue, which may represent an evolutionary adaptation of the circadian system to the cavity-nesting life style of O. bicornis. KW - Behavioural ecology KW - Evolutionary developmental biology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202721 VL - 9 ER - TY - JOUR A1 - Wagner, Fabienne A1 - Kunz, Tobias C. A1 - Chowdhury, Suvagata R. A1 - Thiede, Bernd A1 - Fraunholz, Martin A1 - Eger, Debora A1 - Kozjak-Pavlovic, Vera T1 - Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex JF - PLoS ONE N2 - Cristae architecture is important for the function of mitochondria, the organelles that play the central role in many cellular processes. The mitochondrial contact site and cristae organizing system (MICOS) together with the sorting and assembly machinery (SAM) forms the mitochondrial intermembrane space bridging complex (MIB), a large protein complex present in mammalian mitochondria that partakes in the formation and maintenance of cristae. We report here a new subunit of the mammalian MICOS/MIB complex, an armadillo repeat-containing protein 1 (ArmC1). ArmC1 localizes both to cytosol and mitochondria, where it associates with the outer mitochondrial membrane through its carboxy-terminus. ArmC1 interacts with other constituents of the MICOS/MIB complex and its amounts are reduced upon MICOS/MIB complex depletion. Mitochondria lacking ArmC1 do not show defects in cristae structure, respiration or protein content, but appear fragmented and with reduced motility. ArmC1 represents therefore a peripheral MICOS/MIB component that appears to play a role in mitochondrial distribution in the cell. KW - Mitochondria KW - Outer membrane proteins KW - HeLa cells KW - Immunoprecipitation KW - Cytosol KW - Small interfering RNAs KW - Confocal microscopy KW - Cell stainin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202670 VL - 14 IS - 10 ER - TY - JOUR A1 - Akhoon, Bashir A. A1 - Gupta, Shishir K. A1 - Tiwari, Sudeep A1 - Rathor, Laxmi A1 - Pant, Aakanksha A1 - Singh, Nivedita A1 - Gupta, Shailendra K. A1 - Dandekar, Thomas A1 - Pandey, Rakesh T1 - C. elegans protein interaction network analysis probes RNAi validated pro-longevity effect of nhr-6, a human homolog of tumor suppressor Nr4a1 JF - Scientific Reports N2 - Protein-protein interaction (PPI) studies are gaining momentum these days due to the plethora of various high-throughput experimental methods available for detecting PPIs. Proteins create complexes and networks by functioning in harmony with other proteins and here in silico network biology hold the promise to reveal new functionality of genes as it is very difficult and laborious to carry out experimental high-throughput genetic screens in living organisms. We demonstrate this approach by computationally screening C. elegans conserved homologs of already reported human tumor suppressor and aging associated genes. We select by this nhr-6, vab-3 and gst-23 as predicted longevity genes for RNAi screen. The RNAi results demonstrated the pro-longevity effect of these genes. Nuclear hormone receptor nhr-6 RNAi inhibition resulted in a C. elegans phenotype of 23.46% lifespan reduction. Moreover, we show that nhr-6 regulates oxidative stress resistance in worms and does not affect the feeding behavior of worms. These findings imply the potential of nhr-6 as a common therapeutic target for aging and cancer ailments, stressing the power of in silico PPI network analysis coupled with RNAi screens to describe gene function. KW - Computer modelling KW - Embryonic induction KW - RNAi Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202666 VL - 9 ER - TY - JOUR A1 - Kehrberger, Sandra A1 - Holzschuh, Andrea T1 - How does timing of flowering affect competition for pollinators, flower visitation and seed set in an early spring grassland plant? JF - Scientific Reports N2 - Knowledge on how the timing of flowering is related to plant fitness and species interactions is crucial to understand consequences of phenological shifts as they occur under climate change. Early flowering plants may face advantages of low competition for pollinators and disadvantages of low pollinator abundances and unfavourable weather conditions. However, it is unknown how this trade-off changes over the season and how the timing affects reproductive success. On eight grasslands we recorded intra-seasonal changes in pollinators, co-flowering plants, weather conditions, flower visitation rates, floral longevity and seed set of Pulsatilla vulgaris. Although bee abundances and the number of pollinator-suitable hours were low at the beginning of the season, early flowers of P. vulgaris received higher flower visitation rates and estimated total number of bee visits than later flowers, which was positively related to seed set. Flower visitation rates decreased over time and with increasing number of co-flowering plants, which competed with P. vulgaris for pollinators. Low interspecific competition for pollinators seems to be a major driver for early flowering dates. Thus, non-synchronous temporal shifts of co-flowering plants as they may occur under climate warming can be expected to strongly affect plant-pollinator interactions and the fitness of the involved plants. KW - ecology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202549 VL - 9 ER - TY - JOUR A1 - Heiby, Julia C. A1 - Goretzki, Benedikt A1 - Johnson, Christopher M. A1 - Hellmich, Ute A. A1 - Neuweiler, Hannes T1 - Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk JF - Nature Communications N2 - Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function. KW - Circular dichroism KW - Fluorescence spectroscopy KW - Protein folding KW - Solution-state NMR Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202539 VL - 10 ER - TY - JOUR A1 - Dechaud, Corentin A1 - Volff, Jean-Nicolas A1 - Schartl, Manfred A1 - Naville, Magali T1 - Sex and the TEs: transposable elements in sexual development and function in animals JF - Mobile DNA N2 - Transposable elements are endogenous DNA sequences able to integrate into and multiply within genomes. They constitute a major source of genetic innovations, as they can not only rearrange genomes but also spread ready-to-use regulatory sequences able to modify host gene expression, and even can give birth to new host genes. As their evolutionary success depends on their vertical transmission, transposable elements are intrinsically linked to reproduction. In organisms with sexual reproduction, this implies that transposable elements have to manifest their transpositional activity in germ cells or their progenitors. The control of sexual development and function can be very versatile, and several studies have demonstrated the implication of transposable elements in the evolution of sex. In this review, we report the functional and evolutionary relationships between transposable elements and sexual reproduction in animals. In particular, we highlight how transposable elements can influence expression of sexual development genes, and how, reciprocally, they are tightly controlled in gonads. We also review how transposable elements contribute to the organization, expression and evolution of sexual development genes and sex chromosomes. This underscores the intricate co-evolution between host functions and transposable elements, which regularly shift from a parasitic to a domesticated status useful to the host. KW - Transposable element KW - Sex determination KW - Sexual development and function KW - Germline KW - piRNA KW - Sex chromosome Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202510 VL - 10 ER - TY - JOUR A1 - Krebs, Markus A1 - Behrmann, Christoph A1 - Kalogirou, Charis A1 - Sokolakis, Ioannis A1 - Kneitz, Susanne A1 - Kruithof-de Julio, Marianna A1 - Zoni, Eugenio A1 - Rech, Anne A1 - Schilling, Bastian A1 - Kübler, Hubert A1 - Spahn, Martin A1 - Kneitz, Burkhard T1 - miR-221 Augments TRAIL-mediated apoptosis in prostate cancer cells by inducing endogenous TRAIL expression and targeting the functional repressors SOCS3 and PIK3R1 JF - BioMed Research International N2 - miR-221 is regarded as an oncogene in many malignancies, and miR-221-mediated resistance towards TRAIL was one of the first oncogenic roles shown for this small noncoding RNA. In contrast, miR-221 is downregulated in prostate cancer (PCa), thereby implying a tumour suppressive function. By using proliferation and apoptosis assays, we show a novel feature of miR-221 in PCa cells: instead of inducing TRAIL resistance, miR-221 sensitized cells towards TRAIL-induced proliferation inhibition and apoptosis induction. Partially responsible for this effect was the interferon-mediated gene signature, which among other things contained an endogenous overexpression of the TRAIL encoding gene TNFSF10. This TRAIL-friendly environment was provoked by downregulation of the established miR-221 target gene SOCS3. Moreover, we introduced PIK3R1 as a target gene of miR-221 in PCa cells. Proliferation assays showed that siRNA-mediated downregulation of SOCS3 and PIK3R1 mimicked the effect of miR-221 on TRAIL sensitivity. Finally, Western blotting experiments confirmed lower amounts of phospho-Akt after siRNA-mediated downregulation of PIK3R1 in PC3 cells. Our results further support the tumour suppressing role of miR-221 in PCa, since it sensitises PCa cells towards TRAIL by regulating the expression of the oncogenes SOCS3 and PIK3R1. Given the TRAIL-inhibiting effect of miR-221 in various cancer entities, our results suggest that the influence of miR-221 on TRAIL-mediated apoptosis is highly context- and entity-dependent. KW - Cancer Cell Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202480 VL - 2019 ER - TY - JOUR A1 - Otto, C. A1 - Schmidt, S. A1 - Kastner, C. A1 - Denk, S. A1 - Kettler, J. A1 - Müller, N. A1 - Germer, C.T. A1 - Wolf, E. A1 - Gallant, P. A1 - Wiegering, A. T1 - Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells JF - Neoplasia N2 - The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1. KW - Cancer Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202451 VL - 21 IS - 11 ER - TY - JOUR A1 - Schartl, Manfred A1 - Kneitz, Susanne A1 - Volkoff, Helene A1 - Adolfi, Mateus A1 - Schmidt, Cornelia A1 - Fischer, Petra A1 - Minx, Patrick A1 - Tomlinson, Chad A1 - Meyer, Axel A1 - Warren, Wesley C. T1 - The piranha genome provides molecular insight associated to its unique feeding behavior JF - Genome Biology and Evolution N2 - The piranha enjoys notoriety due to its infamous predatory behavior but much is still not understood about its evolutionary origins and the underlying molecular mechanisms for its unusual feeding biology. We sequenced and assembled the red-bellied piranha (Pygocentrus nattereri) genome to aid future phenotypic and genetic investigations. The assembled draft genome is similar to other related fishes in repeat composition and gene count. Our evaluation of genes under positive selection suggests candidates for adaptations of piranhas’ feeding behavior in neural functions, behavior, and regulation of energy metabolism. In the fasted brain, we find genes differentially expressed that are involved in lipid metabolism and appetite regulation as well as genes that may control the aggression/boldness behavior of hungry piranhas. Our first analysis of the piranha genome offers new insight and resources for the study of piranha biology and for feeding motivation and starvation in other organisms. KW - whole-genome sequencing KW - genome annotation KW - comparative genomics KW - RNA-seq transcriptome KW - energy homeostasis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202218 VL - 11 IS - 8 ER - TY - JOUR A1 - Jurowich, Christian A1 - Lichthardt, Sven A1 - Kastner, Caroline A1 - Haubitz, Imme A1 - Prock, Andre A1 - Filser, Jörg A1 - Germer, Christoph-Thomas A1 - Wiegering, Armin T1 - Laparoscopic versus open right hemicolectomy in colon carcinoma: A propensity score analysis of the DGAV StuDoQ|ColonCancer registry JF - PLoS ONE N2 - Objective To assess whether laparoscopy has any advantages over open resection for right-sided colon cancer. Summary background data Right hemicolectomy can be performed using either a conventional open or a minimally invasive laparoscopic technique. It is not clear whether these different access routes differ with regard to short-term postoperative outcomes. Methods Patients documented in the German Society for General and Visceral Surgery StuDoQ|ColonCancer registry who underwent right hemicolectomy were analyzed regarding early postoperative complications according to Clavien-Dindo (primary endpoint), operation (OP) time, length of postoperative hospital stay (LOS), MTL30 and number of lymph nodes retrieved (secondary endpoints). Results A total of 4.997 patients were identified as undergoing oncological right hemicolectomy without additional interventions. Of these, 4.062 (81.3%) underwent open, 935 (18.7%) laparoscopic surgery. Propensity score analysis showed a significantly shorter LOS (OR: 0.55 CI 95%0.47-.64) and a significantly longer OP time (OR2.32 CI 1.98–2.71) for the laparoscopic route. Risk factors for postoperative complications, anastomotic insufficiency, ileus, reoperation and positive MTL30 were higher ASA status, higher age and increasing BMI. The surgical access route (open / lap) had no influence on these factors, but the laparoscopic group did have markedly fewer lymph nodes retrieved. Conclusion The present registry-based analysis could detect no relevant advantages for the minimally invasive laparoscopic access route. Further oncological analyses are needed to clarify the extent to which the smaller lymph node harvest in the laparoscopic group is accompanied by a poorer oncological outcome. KW - Laparoscopy KW - Lymph nodes KW - Minimally invasive surgery KW - Surgical oncology KW - Oncology KW - Surgical and invasive medical procedures Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202184 VL - 14 IS - 6 ER - TY - JOUR A1 - Pattschull, Grit A1 - Walz, Susanne A1 - Gründl, Marco A1 - Schwab, Melissa A1 - Rühl, Eva A1 - Baluapuri, Apoorva A1 - Cindric-Vranesic, Anita A1 - Kneitz, Susanne A1 - Wolf, Elmar A1 - Ade, Carsten P. A1 - Rosenwald, Andreas A1 - von Eyss, Björn A1 - Gaubatz, Stefan T1 - The Myb-MuvB complex is required for YAP-dependent transcription of mitotic genes JF - Cell Reports N2 - YAP and TAZ, downstream effectors of the Hippo pathway, are important regulators of proliferation. Here, we show that the ability of YAP to activate mitotic gene expression is dependent on the Myb-MuvB (MMB) complex, a master regulator of genes expressed in the G2/M phase of the cell cycle. By carrying out genome-wide expression and binding analyses, we found that YAP promotes binding of the MMB subunit B-MYB to the promoters of mitotic target genes. YAP binds to B-MYB and stimulates B-MYB chromatin association through distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. The cooperation between YAP and B-MYB is critical for YAP-mediated entry into mitosis. Furthermore, the expression of genes coactivated by YAP and B-MYB is associated with poor survival of cancer patients. Our findings provide a molecular mechanism by which YAP and MMB regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways. KW - YAP KW - B-MYB KW - Myb-MuvB KW - mitotic genes KW - enhancer KW - transcription Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202039 VL - 27 IS - 12 ER - TY - JOUR A1 - Lyutova, Radostina A1 - Selcho, Mareike A1 - Pfeuffer, Maximilian A1 - Segebarth, Dennis A1 - Habenstein, Jens A1 - Rohwedder, Astrid A1 - Frantzmann, Felix A1 - Wegener, Christian A1 - Thum, Andreas S. A1 - Pauls, Dennis T1 - Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells JF - Nature Communications N2 - Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells we provide evidence that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, we argue that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. We further show that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. Our data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization. KW - Learning and memory KW - Neural circuits Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202161 VL - 10 ER - TY - JOUR A1 - Liedtke, Daniel A1 - Orth, Melanie A1 - Meissler, Michelle A1 - Geuer, Sinje A1 - Knaup, Sabine A1 - Köblitz, Isabell A1 - Klopocki, Eva T1 - ECM alterations in fndc3a (fibronectin domain containing protein 3A) deficient zebrafish cause temporal fin development and regeneration defects JF - Scientific Reports N2 - Fin development and regeneration are complex biological processes that are highly relevant in teleost fish. They share genetic factors, signaling pathways and cellular properties to coordinate formation of regularly shaped extremities. Especially correct tissue structure defined by extracellular matrix (ECM) formation is essential. Gene expression and protein localization studies demonstrated expression of fndc3a (fibronectin domain containing protein 3a) in both developing and regenerating caudal fins of zebrafish (Danio rerio). We established a hypomorphic fndc3a mutant line (fndc3a\(^{wue1/wue1}\)) via CRISPR/Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fin fold development. These developmental effects are mostly temporary, but result in a fraction of adults with permanent tail fin deformations. In addition, caudal fin regeneration in adult fndc3a\(^{wue1/wue1}\) mutants is hampered by interference with actinotrichia formation and epidermal cell organization. Investigation of the ECM implies that loss of epidermal tissue structure is a common cause for both of the observed defects. Our results thereby provide a molecular link between these developmental processes and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development and regeneration in zebrafish. KW - Extracellular matrix KW - Limb development KW - Self-renewal Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202141 VL - 9 ER - TY - JOUR A1 - Streinzer, Martin A1 - Chakravorty, Jharna A1 - Neumayer, Johann A1 - Megu, Karsing A1 - Narah, Jaya A1 - Schmitt, Thomas A1 - Bharti, Himender A1 - Spaethe, Johannes A1 - Brockmann, Axel T1 - Species composition and elevational distribution of bumble bees (Hymenoptera, Apidae, Bombus Latreille) in the East Himalaya, Arunachal Pradesh, India JF - ZooKeys N2 - The East Himalaya is one of the world’s most biodiverse ecosystems. However, very little is known about the abundance and distribution of many plant and animal taxa in this region. Bumble bees are a group of cold-adapted and high elevation insects that fulfil an important ecological and economical function as pollinators of wild and agricultural flowering plants and crops. The Himalayan mountain range provides ample suitable habitats for bumble bees. Systematic study of Himalayan bumble bees began a few decades ago and the main focus has centred on the western region, while the eastern part of the mountain range has received little attention and only a few species have been verified. During a three-year survey, more than 700 bumble bee specimens of 21 species were collected in Arunachal Pradesh, the largest of the north-eastern states of India. The material included a range of species that were previously known from a limited number of collected specimens, which highlights the unique character of the East Himalayan ecosystem. Our results are an important first step towards a future assessment of species distribution, threat, and conservation. Clear elevation patterns of species diversity were observed, which raise important questions about the functional adaptations that allow bumble bees to thrive in this particularly moist region in the East Himalaya. KW - Alpine habitats KW - Apidae KW - conservation KW - global change KW - insect collection KW - pollination Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201937 VL - 851 ER - TY - JOUR A1 - Thiem, Alexander A1 - Hesbacher, Sonja A1 - Kneitz, Hermann A1 - di Primio, Teresa A1 - Heppt, Markus V. A1 - Hermanns, Heike M. A1 - Goebeler, Matthias A1 - Meierjohann, Svenja A1 - Houben, Roland A1 - Schrama, David T1 - IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression JF - Journal of Experimental & Clinical Cancer Research N2 - Background Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma. Methods We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway. Results For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53\(^{L22Q,W23S}\), a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53\(^{L22Q,W23S}\) in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. Conclusions While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy. KW - Melanoma KW - PD-L1 KW - CD274 KW - p53 KW - TP53 KW - JAK2 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201016 VL - 38 ER - TY - JOUR A1 - Reuter, Isabel A1 - Jäckels, Jana A1 - Kneitz, Susanne A1 - Kuper, Jochen A1 - Lesch, Klaus-Peter A1 - Lillesaar, Christina T1 - Fgf3 is crucial for the generation of monoaminergic cerebrospinal fluid contacting cells in zebrafish JF - Biology Open N2 - In most vertebrates, including zebrafish, the hypothalamic serotonergic cerebrospinal fluid-contacting (CSF-c) cells constitute a prominent population. In contrast to the hindbrain serotonergic neurons, little is known about the development and function of these cells. Here, we identify fibroblast growth factor (Fgf)3 as the main Fgf ligand controlling the ontogeny of serotonergic CSF-c cells. We show that fgf3 positively regulates the number of serotonergic CSF-c cells, as well as a subset of dopaminergic and neuroendocrine cells in the posterior hypothalamus via control of proliferation and cell survival. Further, expression of the ETS-domain transcription factor etv5b is downregulated after fgf3 impairment. Previous findings identified etv5b as critical for the proliferation of serotonergic progenitors in the hypothalamus, and therefore we now suggest that Fgf3 acts via etv5b during early development to ultimately control the number of mature serotonergic CSF-c cells. Moreover, our analysis of the developing hypothalamic transcriptome shows that the expression of fgf3 is upregulated upon fgf3 loss-of-function, suggesting activation of a self-compensatory mechanism. Together, these results highlight Fgf3 in a novel context as part of a signalling pathway of critical importance for hypothalamic development. KW - Fgf-signalling KW - Serotonin KW - Dopamine KW - Hypothalamus KW - Central nervous system Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200749 VL - 8 ER - TY - JOUR A1 - Kehrberger, Sandra A1 - Holzschuh, Andrea T1 - Warmer temperatures advance flowering in a spring plant more strongly than emergence of two solitary spring bee species JF - PLoS ONE N2 - Climate warming has the potential to disrupt plant-pollinator interactions or to increase competition of co-flowering plants for pollinators, due to species-specific phenological responses to temperature. However, studies focusing on the effect of temperature on solitary bee emergence and the flowering onset of their food plants under natural conditions are still rare. We studied the effect of temperature on the phenology of the two spring bees Osmia cornuta and Osmia bicornis, by placing bee cocoons on eleven grasslands differing in mean site temperature. On seven grasslands, we additionally studied the effect of temperature on the phenology of the red-list plant Pulsatilla vulgaris, which was the first flowering plant, and of co-flowering plants with later flowering. With a warming of 0.1°C, the abundance-weighted mean emergence of O. cornuta males advanced by 0.4 days. Females of both species did not shift their emergence. Warmer temperatures advanced the abundance-weighted mean flowering of P. vulgaris by 1.3 days per 0.1°C increase, but did not shift flowering onset of co-flowering plants. Competition for pollinators between P. vulgaris and co-flowering plants does not increase within the studied temperature range. We demonstrate that temperature advances plant flowering more strongly than bee emergence suggesting an increased risk of pollinator limitation for the first flowers of P. vulgaris. KW - Flowering plants KW - Bees KW - Proteus vulgaris KW - Evolutionary emergence KW - Plants KW - Species delimitation KW - Flowers KW - Insect flight Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201165 VL - 14 IS - 6 ER - TY - JOUR A1 - Du, Kang A1 - Wuertz, Sven A1 - Adolfi, Mateus A1 - Kneitz, Susanne A1 - Stöck, Matthias A1 - Oliveira, Marcos A1 - Nóbrega, Rafael A1 - Ormanns, Jenny A1 - Kloas, Werner A1 - Feron, Romain A1 - Klopp, Christophe A1 - Parrinello, Hugues A1 - Journot, Laurent A1 - He, Shunping A1 - Postlethwait, John A1 - Meyer, Axel A1 - Guiguen, Yann A1 - Schartl, Manfred T1 - The genome of the arapaima (Arapaima gigas) provides insights into gigantism, fast growth and chromosomal sex determination system JF - Scientific Reports N2 - We have sequenced the genome of the largest freshwater fish species of the world, the arapaima. Analysis of gene family dynamics and signatures of positive selection identified genes involved in the specific adaptations and unique features of this iconic species, in particular it’s large size and fast growth. Genome sequences from both sexes combined with RAD-tag analyses from other males and females led to the isolation of male-specific scaffolds and supports an XY sex determination system in arapaima. Whole transcriptome sequencing showed that the product of the gland-like secretory organ on the head surface of males and females may not only provide nutritional fluid for sex-unbiased parental care, but that the organ itself has a more specific function in males, which engage more in parental care. KW - Genome KW - Genomics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201333 VL - 9 ER - TY - JOUR A1 - Matos, Isa A1 - Machado, Miguel P. A1 - Schartl, Manfred A1 - Coelho, Maria Manuela T1 - Allele-specific expression variation at different ploidy levels in Squalius alburnoides JF - Scientific Reports N2 - Allopolyploid plants are long known to be subject to a homoeolog expression bias of varying degree. The same phenomenon was only much later suspected to occur also in animals based on studies of single selected genes in an allopolyploid vertebrate, the Iberian fish Squalius alburnoides. Consequently, this species became a good model for understanding the evolution of gene expression regulation in polyploid vertebrates. Here, we analyzed for the first time genome-wide allele-specific expression data from diploid and triploid hybrids of S. alburnoides and compared homoeolog expression profiles of adult livers and of juveniles. Co-expression of alleles from both parental genomic types was observed for the majority of genes, but with marked homoeolog expression bias, suggesting homoeolog specific reshaping of expression level patterns in hybrids. Complete silencing of one allele was also observed irrespective of ploidy level, but not transcriptome wide as previously speculated. Instead, it was found only in a restricted number of genes, particularly ones with functions related to mitochondria and ribosomes. This leads us to hypothesize that allelic silencing may be a way to overcome intergenomic gene expression interaction conflicts, and that homoeolog expression bias may be an important mechanism in the achievement of sustainable genomic interactions, mandatory to the success of allopolyploid systems, as in S. alburnoides. KW - Gene expression analysis KW - Transcription KW - Transcriptomic Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200910 VL - 9 ER - TY - JOUR A1 - Drescher, Nora A1 - Klein, Alexandra-Maria A1 - Schmitt, Thomas A1 - Leonhardt, Sara Diana T1 - A clue on bee glue: New insight into the sources and factors driving resin intake in honeybees (Apis mellifera) JF - PLoS ONE N2 - Honeybees (Apis mellifera) are threatened by numerous pathogens and parasites. To prevent infections they apply cooperative behavioral defenses, such as allo-grooming and hygiene, or they use antimicrobial plant resin. Resin is a chemically complex and highly variable mixture of many bioactive compounds. Bees collect the sticky material from different plant species and use it for nest construction and protection. Despite its importance for colony health, comparatively little is known about the precise origins and variability in resin spectra collected by honeybees. To identify the botanical resin sources of A. mellifera in Western Europe we chemically compared resin loads of individual foragers and tree resins. We further examined the resin intake of 25 colonies from five different apiaries to assess the effect of location on variation in the spectra of collected resin. Across all colonies and apiaries, seven distinct resin types were categorized according to their color and chemical composition. Matches between bee-collected resin and tree resin indicated that bees used poplar (Populus balsamifera, P. x canadensis), birch (Betula alba), horse chestnut (Aesculus hippocastanum) and coniferous trees (either Picea abies or Pinus sylvestris) as resin sources. Our data reveal that honeybees collect a comparatively broad and variable spectrum of resin sources, thus assuring protection against a variety of antagonists sensitive to different resins and/or compounds. We further unravel distinct preferences for specific resins and resin chemotypes, indicating that honeybees selectively search for bioactive resin compounds. KW - Honey bees KW - Poplars KW - Trees KW - Forests KW - Chemical composition KW - Bees KW - Conifers KW - Phenols Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200935 VL - 14 IS - 2 ER - TY - JOUR A1 - Cecil, Alexander A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Dandekar, Thomas A1 - Szalay, Aladar A. T1 - Vaccinia virus injected human tumors: oncolytic virus efficiency predicted by antigen profiling analysis fitted boolean models JF - Bioengineered N2 - Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a promising approach for cancer therapy. Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a therapeutic potential in treating human prostate and hepatocellular carcinomas in xenografted mice. In this study, we describe the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus-injected human tumors. Antigen profiling data of vaccinia virus GLV-1h68-injected human xenografted mice were obtained, analyzed and used to calculate differences in the tumor growth signaling network by tumor type and gender. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, the T-killer cell mediated cell death, Interferon and Interleukin signaling networks. The in silico findings conform very well with in vivo findings of tumor growth. Similar to a previously published analysis of vaccinia virus-injected canine tumors, we were able to confirm the suitability of our boolean modeling for prediction of human tumor growth after virus infection in the current study as well. In summary, these findings indicate that our boolean models could be a useful tool for testing of the efficacy of VACV-mediated cancer therapy already before its use in human patients. KW - boolean modeling KW - oncolytic virus KW - human xenografted mouse models KW - cancer therapy Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200507 VL - 10 IS - 1 ER - TY - JOUR A1 - Doppler, Kathrin A1 - Schuster, Yasmin A1 - Appeltshauser, Luise A1 - Biko, Lydia A1 - Villmann, Carmen A1 - Weishaupt, Andreas A1 - Werner, Christian A1 - Sommer, Claudia T1 - Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model JF - Journal of Neuroinflammation N2 - Background: Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown. Methods: In the present study, we aimed to model acute autoantibody exposure by intraneural injection of IgG of patients with anti-contacin-1 autoantibodies to Lewis rats. Patient IgG obtained during acute onset of disease (IgG3 predominant) and IgG from the chronic phase of disease (IgG4 predominant) were studied in comparison. Results: Conduction blocks were measured in rats injected with the “acute” IgG more often than after injection of “chronic” IgG (83.3% versus 35%) and proved to be reversible within a week after injection. Impaired nerve conduction was accompanied by motor deficits in rats after injection of the “acute” IgG but only minor structural changes of the nodes. Paranodal complement deposition was detected after injection of the “acute IgG”. We did not detect any inflammatory infiltrates, arguing against an inflammatory cascade as cause of damage to the nerve. We also did not observe dispersion of paranodal proteins or sodium channels to the juxtaparanodes as seen in patients after chronic exposure to anti-contactin-1. Conclusions: Our data suggest that anti-contactin-1 IgG3 induces an acute conduction block that is most probably mediated by autoantibody binding and subsequent complement deposition and may account for acute onset of disease in these patients. This supports the notion of anti-contactin-1-associated neuropathy as a paranodopathy with the nodes of Ranvier as the site of pathogenesis. KW - complement deposition KW - paranodopathy KW - anti-contactin-1 KW - CIDP KW - passive transfer KW - autoantibody Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200476 VL - 16 IS - 73 ER - TY - JOUR A1 - Khayenko, Vladimir A1 - Maric, Hans Michael T1 - Targeting GABA\(_A\)R-associated proteins: new modulators, labels and concepts JF - Frontiers in Molecular Neuroscience N2 - γ-aminobutyric acid type A receptors (GABA\(_A\)Rs) are the major mediators of synaptic inhibition in the brain. Aberrant GABA\(_A\)R activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review article, we introduce a new strategy to modulate GABAeric signaling: targeting the intracellular protein interactors of GABA\(_A\)Rs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABA\(_A\)R subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABA\(_A\)R-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABA\(_A\)R numbers and clustering, modifying neuronal transmission. Interference with GABA\(_A\)R trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABA\(_A\)R to AP2 increase the surface concentration of GABA\(_A\)R clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with submicron resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABA\(_A\)R accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABA\(_A\)R modulators and following in vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs. KW - GABAA receptors KW - gephyrin KW - collybistin KW - protein-protein interaction (PPI) KW - super resolution microscopy KW - fluorescent probes KW - dimeric peptide KW - peptide inhibitor design Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201876 VL - 12 IS - 162 ER - TY - JOUR A1 - Derakhshani, Shaghayegh A1 - Kurz, Andreas A1 - Japtok, Lukasz A1 - Schumacher, Fabian A1 - Pilgram, Lisa A1 - Steinke, Maria A1 - Kleuser, Burkhard A1 - Sauer, Markus A1 - Schneider-Schaulies, Sibylle A1 - Avota, Elita T1 - Measles virus infection fosters dendritic cell motility in a 3D environment to enhance transmission to target cells in the respiratory epithelium JF - Frontiers in Immunology N2 - Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit. KW - dendritic cell KW - cell migration KW - measles virus KW - 3D tissue model KW - sphingosine-1-phosphate Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201818 VL - 10 IS - 1294 ER - TY - JOUR A1 - Lechermeier, Carina G. A1 - Zimmer, Frederic A1 - Lüffe, Teresa M. A1 - Lesch, Klaus-Peter A1 - Romanos, Marcel A1 - Lillesaar, Christina A1 - Drepper, Carsten T1 - Transcript analysis of zebrafish GLUT3 genes, slc2a3a and slc2a3b, define overlapping as well as distinct expression domains in the zebrafish (Danio rerio) central nervous system JF - Frontiers in Molecular Neuroscience N2 - The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for this function in vivo. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington’s and Alzheimer’s diseases. Furthermore, GLUT3 was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (Danio rerio) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish, slc2a3a and slc2a3b. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The slc2a3a transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the slc2a3b transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of slc2a3a is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The slc2a3b transcripts are present in overlapping as well as distinct regions compared to slc2a3a. Double in situ hybridizations were used to demonstrate that slc2a3a is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish slc2a3a and slc2a3b expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain disorders. KW - glucose transporter KW - nervous system KW - brain disorders KW - psychiatric disorders KW - brain development KW - GABA KW - GAD1 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201797 VL - 12 IS - 199 ER - TY - JOUR A1 - Kunz, Tobias C. A1 - Kozjak-Pavlovic, Vera T1 - Diverse facets of sphingolipid involvement in bacterial infections JF - Frontiers in Cell and Developmental Biology N2 - Sphingolipids are constituents of the cell membrane that perform various tasks as structural elements and signaling molecules, in addition to regulating many important cellular processes, such as apoptosis and autophagy. In recent years, it has become increasingly clear that sphingolipids and sphingolipid signaling play a vital role in infection processes. In many cases the attachment and uptake of pathogenic bacteria, as well as bacterial development and survival within the host cell depend on sphingolipids. In addition, sphingolipids can serve as antimicrobials, inhibiting bacterial growth and formation of biofilms. This review will give an overview of our current information about these various aspects of sphingolipid involvement in bacterial infections. KW - infection KW - pathogenic bacteria KW - sphingolipids KW - ceramide KW - autophagy Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201757 VL - 7 IS - 203 ER - TY - JOUR A1 - Voulgari‐Kokota, Anna A1 - Ankenbrand, Markus J. A1 - Grimmer, Gudrun A1 - Steffan‐Dewenter, Ingolf A1 - Keller, Alexander T1 - Linking pollen foraging of megachilid bees to their nest bacterial microbiota JF - Ecology and Evolution N2 - Solitary bees build their nests by modifying the interior of natural cavities, and they provision them with food by importing collected pollen. As a result, the microbiota of the solitary bee nests may be highly dependent on introduced materials. In order to investigate how the collected pollen is associated with the nest microbiota, we used metabarcoding of the ITS2 rDNA and the 16S rDNA to simultaneously characterize the pollen composition and the bacterial communities of 100 solitary bee nest chambers belonging to seven megachilid species. We found a weak correlation between bacterial and pollen alpha diversity and significant associations between the composition of pollen and that of the nest microbiota, contributing to the understanding of the link between foraging and bacteria acquisition for solitary bees. Since solitary bees cannot establish bacterial transmission routes through eusociality, this link could be essential for obtaining bacterial symbionts for this group of valuable pollinators. KW - foraging patterns KW - nest microbiota KW - plant–microbe–pollinator triangle KW - pollination network KW - solitary bees KW - wild bees Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201749 SN - 00 VL - 2019 IS - 9 ER - TY - JOUR A1 - Schlegel, Jan A1 - Peters, Simon A1 - Doose, Sören A1 - Schubert-Unkmeir, Alexandra A1 - Sauer, Markus T1 - Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites JF - Frontiers in Cell and Developmental Biology N2 - Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection. KW - Neisseria meningitidis KW - sphingolipids KW - gangliosides and lipid rafts KW - super-resolution microscopy KW - single-molecule tracking Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201639 VL - 7 IS - 194 ER - TY - JOUR A1 - Liu, Ruiqi A1 - Kinoshita, Masato A1 - Adolfi, Mateus C. A1 - Schartl, Manfred T1 - Analysis of the role of the Mc4r system in development, growth, and puberty of medaka JF - Frontiers in Endocrinology N2 - In mammals the melanocortin 4 receptor (Mc4r) signaling system has been mainly associated with the regulation of appetite and energy homeostasis. In fish of the genus Xiphophorus (platyfish and swordtails) puberty onset is genetically determined by a single locus, which encodes the mc4r. Wild populations of Xiphophorus are polymorphic for early and late-maturing individuals. Copy number variation of different mc4r alleles is responsible for the difference in puberty onset. To answer whether this is a special adaptation of the Mc4r signaling system in the lineage of Xiphophorus or a more widely conserved mechanism in teleosts, we studied the role of Mc4r in reproductive biology of medaka (Oryzias latipes), a close relative to Xiphophorus and a well-established model to study gonadal development. To understand the potential role of Mc4r in medaka, we characterized the major features of the Mc4r signaling system (mc4r, mrap2, pomc, agrp1). In medaka, all these genes are expressed before hatching. In adults, they are mainly expressed in the brain. The transcript of the receptor accessory protein mrap2 co-localizes with mc4r in the hypothalamus in adult brains indicating a conserved function of modulating Mc4r signaling. Comparing growth and puberty between wild-type and mc4r knockout medaka revealed that absence of Mc4r does not change puberty timing but significantly delays hatching. Embryonic development of knockout animals is retarded compared to wild-types. In conclusion, the Mc4r system in medaka is involved in regulation of growth rather than puberty. KW - medaka KW - Mc4r KW - knockout KW - puberty KW - growth Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201472 VL - 10 ER - TY - JOUR A1 - Panzer, Sabine A1 - Brych, Annika A1 - Batschauer, Alfred A1 - Terpitz, Ulrich T1 - Opsin 1 and Opsin 2 of the corn smut fungus ustilago maydis are green light-driven proton pumps JF - Frontiers in Microbiology N2 - In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis. KW - Ustilago maydis KW - patch-clamp KW - fungal rhodopsins KW - microbial rhodopsins KW - acetate KW - indole-3-acetic acid KW - structured illumination microscopy KW - sporidia Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201453 VL - 10 ER - TY - JOUR A1 - Srivastava, Mugdha A1 - Bencurova, Elena A1 - Gupta, Shishir K. A1 - Weiss, Esther A1 - Löffler, Jürgen A1 - Dandekar, Thomas T1 - Aspergillus fumigatus challenged by human dendritic cells: metabolic and regulatory pathway responses testify a tight battle JF - Frontiers in Cellular and Infection Microbiology N2 - Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other. KW - infection KW - dendritic cells KW - Aspergillus fumigalus KW - metabolic modelling KW - signalling Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201368 VL - 9 ER - TY - JOUR A1 - Djuzenova, Cholpon S. A1 - Fiedler, Vanessa A1 - Memmel, Simon A1 - Katzer, Astrid A1 - Sisario, Dmitri A1 - Brosch, Philippa K. A1 - Göhrung, Alexander A1 - Frister, Svenja A1 - Zimmermann, Heiko A1 - Flentje, Michael A1 - Sukhorukov, Vladimir L. T1 - Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells JF - BMC Cancer N2 - Background Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells. Methods Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition. Results We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy. Conclusions Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered. KW - DNA damage KW - glioblastoma multiforme KW - histone H2AX KW - irradiation KW - migration KW - mTOR KW - PTEN KW - p53 KW - radiation sensitivity KW - wound healing Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200290 VL - 19 ER - TY - JOUR A1 - Sbiera, Silviu A1 - Kunz, Meik A1 - Weigand, Isabel A1 - Deutschbein, Timo A1 - Dandekar, Thomas A1 - Fassnacht, Martin T1 - The new genetic landscape of Cushing’s disease: deubiquitinases in the spotlight JF - Cancers N2 - Cushing’s disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD’s genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5%) and USP48 (13.3%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5% and 7%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways. KW - Cushing’s disease KW - pathogenesis KW - somatic mutations KW - deubiquitinases Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193194 SN - 2072-6694 VL - 11 IS - 11 ER - TY - JOUR A1 - Horn, Melanie A1 - Mitesser, Oliver A1 - Hovestadt, Thomas A1 - Yoshii, Taishi A1 - Rieger, Dirk A1 - Helfrich-Förster, Charlotte T1 - The circadian clock improves fitness in the fruit fly, Drosophila melanogaster JF - Frontiers in Physiology N2 - It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. We raised each of the three classical Drosophila period mutants for >50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant’s natural cycle, i.e., a 19-h day in the case of pers mutants and a 29-h day for perl mutants. The arrhythmic per0 mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. We found that wildtype flies quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but perl mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast, pers and per0 mutants did not win against wildtype flies under the 19-h day and constant light, respectively. Our results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the period mutants and – as revealed for perl mutants – this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that perl and pers persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants. KW - competition KW - mutants KW - resonance theory KW - mating preference KW - fertility Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195738 SN - 1664-042X VL - 10 IS - 1374 ER - TY - JOUR A1 - Fathy, Moustafa A1 - Fawzy, Michael Atef A1 - Hintzsche, Henning A1 - Nikaido, Toshio A1 - Dandekar, Thomas A1 - Othman, Eman M. T1 - Eugenol exerts apoptotic effect and modulates the sensitivity of HeLa cells to cisplatin and radiation JF - Molecules N2 - Eugenol is a phytochemical present in different plant products, e.g., clove oil. Traditionally, it is used against a number of different disorders and it was suggested to have anticancer activity. In this study, the activity of eugenol was evaluated in a human cervical cancer (HeLa) cell line and cell proliferation was examined after treatment with various concentrations of eugenol and different treatment durations. Cytotoxicity was tested using lactate dehydrogenase (LDH) enzyme leakage. In order to assess eugenol’s potential to act synergistically with chemotherapy and radiotherapy, cell survival was calculated after eugenol treatment in combination with cisplatin and X-rays. To elucidate its mechanism of action, caspase-3 activity was analyzed and the expression of various genes and proteins was checked by RT-PCR and western blot analyses. Eugenol clearly decreased the proliferation rate and increased LDH release in a concentration- and time-dependent manner. It showed synergistic effects with cisplatin and X-rays. Eugenol increased caspase-3 activity and the expression of Bax, cytochrome c (Cyt-c), caspase-3, and caspase-9 and decreased the expression of B-cell lymphoma (Bcl)-2, cyclooxygenase-2 (Cox-2), and interleukin-1 beta (IL-1β) indicating that eugenol mainly induced cell death by apoptosis. In conclusion, eugenol showed antiproliferative and cytotoxic effects via apoptosis and also synergism with cisplatin and ionizing radiation in the human cervical cancer cell line. KW - eugenol KW - HeLa cells KW - cisplatin KW - radiation KW - apoptosis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193227 SN - 1420-3049 VL - 24 IS - 21 ER - TY - JOUR A1 - Mateos, Mariana A1 - Kang, Du A1 - Klopp, Christophe A1 - Parrinello, Hugues A1 - García-Olazábal, Mateo A1 - Schumer, Molly A1 - Jue, Nathaniel K. A1 - Guiguen, Yann A1 - Schartl, Manfred T1 - Draft genome assembly and annotation of the Gila Topminnow Poeciliopsis occidentalis JF - Frontiers in Ecology and Evolution N2 - No abstract available. KW - genome assembly KW - genome annotation KW - transposable elements KW - topminnow KW - mitochondrial genome Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190339 SN - 2296-701X VL - 7 IS - 404 ER - TY - JOUR A1 - Kunz, Tobias C. A1 - Götz, Ralph A1 - Sauer, Markus A1 - Rudel, Thomas T1 - Detection of chlamydia developmental forms and secreted effectors by expansion microscopy JF - Frontiers in Cellular and Infection Microbiology N2 - Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens. KW - expansion microscopy KW - chlamydia KW - secreted effectors KW - developmental forms KW - superresolution KW - imaging Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195716 SN - 2235-2988 VL - 9 IS - 276 ER - TY - JOUR A1 - Heydarian, Motaharehsadat A1 - Yang, Tao A1 - Schweinlin, Matthias A1 - Steinke, Maria A1 - Walles, Heike A1 - Rudel, Thomas A1 - Kozjak-Pavlovic, Vera T1 - Biomimetic human tissue model for long-term study of Neisseria gonorrhoeae infection JF - Frontiers in Microbiology N2 - Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions. KW - 3D tissue model KW - small intestinal submucosa scaffold KW - co-culture KW - infection KW - Neisseria gonorrhoeae Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197912 SN - 1664-302X VL - 10 IS - 1740 ER - TY - THES A1 - Griffoni, Chiara T1 - Towards advanced immunocompetent skin wound models for in vitro drug evaluation T1 - Auf dem Weg zu fortschrittlichen immunkompetenten Hautwundmodellen für die in vitro-Medikamentenbewertung N2 - Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models. N2 - Aktuelle präklinische Modelle zur Bewertung neuartiger Therapien für eine verbesserte Heilung um- fassen sowohl in vitro als auch in vivo Methoden. Ethische Bedenken im Zusammenhang mit der Ver- wendung von Tieren sowie die schlechte physiologische Übersetzung zwischen tierischer und mensch- licher Hautwundheilung bezeichnen In-vitro-Modelle jedoch als hochrelevante und vielversprechende Plattformen für die Heilungsforschung. Während die aktuellen in vitro 3D-Hautmodelle ein reifes Ge- webe mit heilenden Eigenschaften rekapitulieren, stellen sie dennoch eine Vereinfachung der in vivo- Bedingungen dar, bei denen beispielsweise die nach der Wundbildung entstehende Entzündungsreak- tion den Beitrag von Immunzellen beinhaltet. Makrophagen gehören zu den Hauptverursachern der Entzündungsreaktion und regulieren ihren Verlauf durch ihre Plastizität. Daher könnte ihre Implemen- tierung in die in vitro Haut die physiologische Relevanz der Modelle deutlich erhöhen. Da bisher kein volldickes, immunkompetentes Hautmodell mit Makrophagen berichtet wurde, wurden hier die für eine erfolgreiche Dreifach-Cokultur von Fibroblasten, Keratinozyten und Makrophagen notwendigen Parameter untersucht. Zuerst wurden die Zellquelle und die Kultur zeitlich festgelegt, aber auch eine Implementierungsstrategie für Makrophagen festgelegt. Die Implementierung von Makrophagen in das Hautmodell konzentrierte sich auf die Minimierung der Kultivierungszeit, um die Lebensfähigkeit und den Phänotyp der Immunzellen zu erhalten, da die Umgebung einen großen Einfluss auf die Zell- polarisation und Zytokinproduktion hat. Zu diesem Zweck wurde die Integration von Makrophagen in 3D-Gelen vor der Kombination mit Hautmodellen ausgewählt, um die in vivo-Umgebung besser nach- ahmen zu können. Eingebettet in Kollagenhydrogele zeigten Makrophagen eine homogene Zellvertei- lung im Gel, die die Zelllebensfähigkeit bewahrt, auf Reize reagiert und durch die Matrix wandert, die alle bei der Beteiligung von Makrophagen an der Entzündungsreaktion benötigt werden. Nachdem festgestellt worden war, wie Makrophagen in Hautmodelle eingeführt werden können, wurden ver- schiedene Kulturmedien hinsichtlich ihrer Auswirkungen auf Primärfibroblasten, Keratinozyten und Makrophagen untersucht, um eine geeignete Medienzusammensetzung für die Kultur immunkompe- tenter Haut zu identifizieren. Die vorliegende Arbeit bestätigte, dass jeder Zelltyp eine andere Supple- mentkombination zur Aufrechterhaltung der Funktionsmerkmale benötigt und zeigte erstmals, dass Medien, die eine reife Hautstruktur fördern und aufrechterhalten, negative Auswirkungen auf die pri- mären Makrophagen haben. Hautdifferenzierungsmedien wirkten sich negativ auf die Makrophagen in Bezug auf Lebensfähigkeit, Morphologie, Fähigkeit, auf pro- und antiinflammatorische Reize zu rea- gieren und durch ein Kollagengel zu wandern aus. Die Kombination aus verwundeten Hautäquivalen- ten und makrophagenhaltigen Gelen bestätigte, dass das Kulturmedium die Teilnahme der Makro- phage an der Entzündungsreaktion, die nach der Wunde auftritt, hemmt. Die beschriebene Makrophagen-Einschlussmethode zur immunkompetenten Hautbildung ist ein vielversprechender An- satz zur Generierung relevanterer Hautmodelle. Eine weitere Optimierung des Co-Kulturmediums wird es möglicherweise ermöglichen, eine physiologische Entzündungsreaktion nachzuahmen und die Aus- wirkungen neuartiger Medikamente zur verbesserten Heilung auf verbesserte In-vitro-Modelle zu be- werten. KW - skin model KW - macrophages KW - wound healing KW - immunocompetent skin KW - Haut KW - In vitro KW - Wundheilung Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192125 ER - TY - JOUR A1 - Yang, Manli A1 - Rajeeve, Karthika A1 - Rudel, Thomas A1 - Dandekar, Thomas T1 - Comprehensive Flux Modeling of Chlamydia trachomatis Proteome and qRT-PCR Data Indicate Biphasic Metabolic Differences Between Elementary Bodies and Reticulate Bodies During Infection JF - Frontiers in Microbiology N2 - Metabolic adaptation to the host cell is important for obligate intracellular pathogens such as Chlamydia trachomatis (Ct). Here we infer the flux differences for Ct from proteome and qRT-PCR data by comprehensive pathway modeling. We compare the comparatively inert infectious elementary body (EB) and the active replicative reticulate body (RB) systematically using a genome-scale metabolic model with 321 metabolites and 277 reactions. This did yield 84 extreme pathways based on a published proteomics dataset at three different time points of infection. Validation of predictions was done by quantitative RT-PCR of enzyme mRNA expression at three time points. Ct’s major active pathways are glycolysis, gluconeogenesis, glycerol-phospholipid (GPL) biosynthesis (support from host acetyl-CoA) and pentose phosphate pathway (PPP), while its incomplete TCA and fatty acid biosynthesis are less active. The modeled metabolic pathways are much more active in RB than in EB. Our in silico model suggests that EB and RB utilize folate to generate NAD(P)H using independent pathways. The only low metabolic flux inferred for EB involves mainly carbohydrate metabolism. RB utilizes energy -rich compounds to generate ATP in nucleic acid metabolism. Validation data for the modeling include proteomics experiments (model basis) as well as qRT-PCR confirmation of selected metabolic enzyme mRNA expression differences. The metabolic modeling is made fully available here. Its detailed insights and models on Ct metabolic adaptations during infection are a useful modeling basis for future studies. KW - metabolic modeling KW - metabolic flux KW - infection biology KW - elementary body KW - reticulate body KW - Chlamydia trachomatis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189434 SN - 1664-302X VL - 10 IS - 2350 ER - TY - THES A1 - Njovu, Henry Kenneth T1 - Patterns and drivers of herbivore diversity and invertebrate herbivory along elevational and land use gradients at Mt. Kilimanjaro, Tanzania T1 - Muster und Determinanten von Herbivorendiversität, von Herbivorieraten durch Invertebraten sowie die Diversität und Gesamtbiomasse von Säugetieren entlang von Höhen- und Landnutzungsgradienten am Kilimandscharo (Tansania) untersucht N2 - This thesis elucidates patterns and drivers of invertebrate herbivory, herbivore diversity, and community-level biomass along elevational and land use gradients at Mt. Kilimanjaro, Tanzania. Chapter I provides background information on the response and predictor variables, study system, and the study design. First, I give an overview of the elevational patterns of species diversity/richness and herbivory published in the literature. The overview illuminates existing debates on elevational patterns of species diversity/richness and herbivory. In connection to these patterns, I also introduce several hypotheses and mechanisms put forward to explain macroecological patterns of species richness. Furthermore, I explain the main variables used to test hypotheses. Finally, I describe the study system and the study design used. Chapter II explores the patterns of invertebrate herbivory and their underlying drivers along extensive elevational and land use gradients on the southern slopes of Mt. Kilimanjaro. I recorded standing leaf herbivory from leaf chewers, leaf miners and gall-inducing insects on 55 study sites located in natural and anthropogenic habitats distributed from 866 to 3060 meters above sea level (m asl) on Mt. Kilimanjaro. Standing leaf herbivory was related to climatic variables [mean annual temperature - (MAT) and mean annual precipitation - (MAP)], net primary productivity (NPP) and plant functional traits (leaf traits) [specific leaf area (SLA), carbon to nitrogen ratio (CN), and nitrogen to phosphorous ratio (NP)]. Results revealed an unimodal pattern of total leaf herbivory along the elevation gradient in natural habitats. Findings also revealed differences in the levels and patterns of herbivory among feeding guilds and between anthropogenic and natural habitats. Changes in NP and CN ratios which were closely linked to NPP were the strongest predictors of leaf herbivory. Our study uncovers the role of leaf nutrient stoichiometry and its linkages to climate in explaining the variation in leaf herbivory along climatic gradients. Chapter III presents patterns and unravels direct and indirect effects of resource (food) abundance (NPP), resource (food) diversity [Functional Dispersion (FDis)], resource quality (SLA, NP, and CN rations), and climate variables (MAT and MAP) on species diversity of phytophagous beetles. Data were collected from 65 study sites located in natural and anthropogenic habitats distributed from 866 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Sweep net and beating methods were used to collect a total of 3,186 phytophagous beetles representing 21 families and 304 morphospecies. Two groups, weevils (Curculionidae) and leaf beetles (Chrysomelidae) were the largest and most diverse families represented with 898 and 1566 individuals, respectively. Results revealed complex (bimodal) and dissimilar patterns of Chao1-estimated species richness (hereafter referred to as species diversity) along elevation and land use gradients. Results from path analysis showed that temperature and climate-mediated changes in NPP had a significant positive direct and indirect effect on species diversity of phytophagous beetles, respectively. The results also revealed that the effect of NPP (via beetles abundance and diversity of food resources) on species diversity is stronger than that of temperature. Since we found that factors affecting species diversity were intimately linked to climate, I concluded that predicted climatic changes over the coming decades will likely alter the species diversity patterns which we observe today. Chapter IV presents patterns and unravels the direct and indirect effects of climate, NPP and anthropogenic disturbances on species richness and community-level biomass of wild large mammals which represent endothermic organisms and the most important group of vertebrate herbivores. Data were collected from 66 study sites located in natural and anthropogenic habitats distributed from 870 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Mammals were collected using camera traps and used path analysis to disentangle the direct and indirect effects of climatic variables, NPP, land use, land area, levels of habitat protection and occurrence of domesticated mammals on the patterns of richness and community-level biomass of wild mammals, respectively. Results showed unimodal patterns for species richness and community-level biomass of wild mammals along elevation gradients and that the patterns differed depending on the type of feeding guild. Findings from path analysis showed that net primary productivity and levels of habitat protection had a strong direct effect on species richness and community-level biomass of wild mammals whereas temperature had an insignificant direct effect. Findings show the importance of climate-mediated food resources in determining patterns of species richness of large mammals. While temperature is among key predictors of species richness in several ectotherms, its direct influence in determining species richness of wild mammals was insignificant. Findings show the sensitivity of wild mammals to anthropogenic influences and underscore the importance of protected areas in conserving biodiversity. In conclusion, despite a multitude of data sets on species diversity and ecosystem functions along broad climatic gradients, there is little mechanistic understanding of the underlying causes. Findings obtained in the three studies illustrate their contribution to the scientific debates on the mechanisms underlying patterns of herbivory and diversity along elevation gradients. Results present strong evidence that plant functional traits play a key role in determining invertebrate herbivory and species diversity along elevation gradients and that, their strong interdependence with climate and anthropogenic activities will shape these patterns in future. Additionally, findings from path analysis demonstrated that herbivore diversity, community-level biomass, and herbivory are strongly influenced by climate (either directly or indirectly). Therefore, the predicted climatic changes are expected to dictate ecological patterns, biotic interactions, and energy and nutrient fluxes in terrestrial ecosystems in the coming decades with stronger impacts probably occurring in natural ecosystems. Furthermore, findings demonstrated the significance of land use effects in shaping ecological patterns. As anthropogenic pressure is advancing towards more pristine higher elevations, I advocate conservation measures which are responsive to and incorporate human dimensions to curb the situation. Although our findings emanate from observational studies which have to take several confounding factors into account, we have managed to demonstrate global change responses in real ecosystems and fully established organisms with a wide range of interactions which are unlikely to be captured in artificial experiments. Nonetheless, I recommend additional experimental studies addressing the effect of top-down control by natural enemies on herbivore diversity and invertebrate herbivory in order to deepen our understanding of the mechanisms driving macroecological patterns along elevation gradients.   N2 - In dieser Dissertation werden Muster und Determinanten von Herbivorendiversität, von Herbivorieraten durch Invertebraten sowie die Diversität und Gesamtbiomasse von Säugetieren entlang von Höhen- und Landnutzungsgradienten am Kilimandscharo (Tansania) untersucht. Kapitel I liefert Hintergrundinformationen zu den betrachteten Variablen, dem Untersuchungssystem und dem generellen Studiendesign: Zuerst fasse ich den aktuellen Kenntnisstand über die Muster des Artenreichtums und der Herbivorie entlang von Höhengradienten zusammen und erläutere in diesem Zusammenhang verschiedene Hypothesen, die zur Erklärung von Gradienten des Artenreichtum herangezogen werden. Ich erkläutere verschiedene Variablen, die zum Testen dieser Hypothesen erhoben wurden und stelle dar, wie diese den Artenreichtum, die Herbivorieraten und die Biomasse beeinflussen könnten. Anschließend beschreibe ich das Untersuchungssystem, sowie das generelle Design der Studie. In Kapitel II werden die Muster und Determinanten der Invertebratenherbivorie entlang von Höhen- und Landnutzungsgradienten an den südlichen Hängen des Kilimandscharos präsentiert. Auf insgesamt 55 Untersuchungsflächen, die sowohl natürliche als auch anthropogen genutzte Habitate am Kilimandscharo in Höhenlagen zwischen 866 und 3060 Meter über Normalnull (m ü. NN) umfassten, wurden die Herbivorieraten ektophager, minierender und gallbildener Insekten an Blättern erfasst. Die Blattherbivorie war sowohl mit klimatischen Variablen [Jahresmitteltemperatur und mittlere Jahresniederschlagsmenge], der Nettoprimärproduktivität (NPP) und mit funktionellen Blattmerkmalen von Pflanzen [spezifische Blattfläche (SLA), Kohlenstoff (C) / Stickstoff (N)-Verhältnis, sowie N / Phosphor (P)-Verhältnis] assoziiert. Die Gesamtherbivorie zeigte eine unimodale Verteilung über den Höhengradienten, wurde aber sowohl von der Herbivorengilde, als auch vom Habitattyp (natürlich versus anthropogen) beeinflusst. Das C/N-Verhältnis von Blättern war die stärkste Determinante der Blattherbivorie und wurde selbst stark durch die NPP bestimmt. Herbivorieraten sanken mit steigendem C/N-Verhältnis. Das C/N Verhältnis nahm mit steigender NPP zu.- Letztere konnte fast vollständig durch Änderungen der mittleren Jahrestemperatur (MAT) und des Jahresniederschlags (MAP) entlang des Höhengradienten erklärt werden. Damit zeigt unsere Studie, dass sich durch klimatische Faktoren und Energie, welche ihrerseits die Blattchemie beeinflussen und so Variationen in der Blattherbivorie entlang großer Klimagradienten ergeben. In Kapitel III werden die Muster im Artenreichtum phytophager Käfer entlang der Höhen- und Landnutzungsgradienten untersucht und die direkten und indirekten Effekte von klimatischen Faktoren (MAT, MAP), NPP und funktionellen Pflanzenmerkmalen (funktionelle Dispersion, SLA, C/N - und N/P - Verhältnisse) auf diese Muster analysiert. Die entsprechenden Daten wurden auf 65 Untersuchungsflächen, die sowohl natürliche als auch anthropogene Habitate entlang eines Höhengradienten am Kilimandscharo von 866 bis 4550 m ü. NN abdeckten, erhoben. Mittels Kescher wurden insgesamt 3186 phytophage Käfer aus 21 Familien gesammelt und in 304 Morphospezies eingeteilt. Der Artenreichtum phytophager Käfer zeigte eine komplexe, zweigipflige Verteilung entlang der Höhen- und Landnutzungsgradienten. Eine Pfadanalyse ergab, dass sowohl die MAT, als auch NPP positiven direkte bzw. indirekte Effekt auf die Artendiversität phytophager Käfer hatte. Die NPP war positiv mit der funktionellen Dispersion von Blattmerkmalen, ein Maß für die Diversität der Nahrungsressourcen, korreliert. Letztere hatte einen positiven Effekt auf die Diversität der Käfer. Die starken direkten und indirekten Effekte von Klima auf die Diversität und Abundanz von phytophagen Käfern, lassen vermuten dass der Klimawandel in den nächsten Dekaden großen Änderungen der Struktur von phytophagen Käfergemeinschaften bewirken wird. In Kapitel IV untersuchen wir den Effekt von Klima, NPP und anthropogener Störung auf den Artenreichtum und die Gesamtbiomasse von Großwild. Dazu wurden auf 66 Untersuchungsflächen, welche natürliche und anthropogene Habitate in Höhenstufen zwischen 870 und 4550m ü. NN umfassten, Daten zum Artenreichtum un der Abundanz von Großwild mittels Kamerafallen erfasst. Mittels einer Pfadanalyse wurden die direkten und indirekten Effekte von klimatischen Variablen, NPP, Landnutzung, Größe und Schutzstatus der Flächen, sowie der Präsenz von domestizierten Säugetieren auf den Artenreichtum und die Biomasse von Großwild untersucht. Artenreichtum und Gesamtbiomasse dieser endothermen Organismen zeigten eine unimodale Verteilung über den Höhengradienten. Verschiedene Nahrungsgilden zeigten unterschiedliche Muster. Es konnte gezeigt werden, dass NPP und der Schutzstatus der Fläche, aber nicht die Temperatur einen direkten, positiven Einfluss auf den Artenreichtum und die Gesamtbiomasse des Großwildes hatte. Die vom Klima abhängige Nahrungsressourcenverfügbarkeit ist also eine wichtige Determinante im Artenreichtum von Großwild. Die Temperatur hingegen, die den Artenreichtum verschiedener ektothermer Organismen entscheidend prägt, hatte keinen direkten Einfluss auf den Artenreichtum des Großwildes Dafür reagiert das Großwild besonders sensibel auf anthropogene Einflüsse, was wiederum die Wichtigkeit von Schutzgebieten unterstreicht. Obwohl die Muster im Artenreichtum und in Ökosystemfunktionen entlang großer klimatischer Gradienten bereits gut dokumentiert sind, ist das Wissen über die zu Grunde liegenden Prozesse nach wie vor unzureichend. Mit meinen drei Studien über die Muster und Determinanten der Herbivorendiversität, der Herbivorieraten und der Großwildbiomasse trage ich somit zur Verbesserung des mechanistischen Verständnisses solcher makroökologischer Muster bei. Wie die Pfadanalysen zeigten, wurden sowohl der Artenreichtum die Biomasse als auch ökologische Prozesse direkt oder indirekt vom Klima beeinflusst. Es ist somit zu erwarten, dass der vorhergesagte Klimawandel ökologische Muster, biotische Interaktionen, Energie- und Nährstoffkreisläufe in terrestrischen Ökosystemen wesentlich umstrukturieren wird, wobei natürliche Systeme wahrscheinlich besonders sensibel auf den Klimawandel reagieren werden. Meine Ergebnisse demonstrieren auch den Einfluss von Landnutzung auf Artenreichtum und ökologische Prozesse. Da der anthropogene Druck auf die natürlichen Ökosysteme des Kilimandscharos immer weiter zunimmt, sollten objektive Biodiversitätsmaße implementiert werden mit denen man Veränderungen in den Ökosystemen und in Ökosystemldienstleistungen schnell detektieren kann. Meine Ergebnisse basieren auf Beobachtungsdaten, die von bestimmten Nebenfaktoren im Feld beeinflusst werden können. Dennoch ist es mir gelungen mit korrelativen Methoden, Organismen in ihrem biotischen und abiotischen Interaktionsumfeld zu untersuchen – ein Szenario, welches in einem rein experimentellen Aufbau in dieser Form wahrscheinlich nicht geschaffen werden kann. Über weiterführende Experimente könnte jedoch zum Beispiel der Einfluss von Prädatoren auf die Herbivorendiversität und Herbivorieraten quantifiziert werden, welches unser Verständnis über die Determinanten makroökologischer Muster noch vertiefen würde.   KW - Species richness KW - Invertebrate herbivory KW - Leaf traits KW - drivers and patterns of diversity and herbivory KW - Patterns and drivers of invertebrate herbivory KW - Patterns and drivers of species diversity of phytophagous beetles KW - Patterns and drivers of species richness and community biomass of large mammals Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172544 ER - TY - THES A1 - Franke, Christian T1 - Advancing Single-Molecule Localization Microscopy: Quantitative Analyses and Photometric Three-Dimensional Imaging T1 - Weiterentwicklung von Einzel-Molekül Lokalisations-Mikroskopie: Quantitative Analysen und photometrische drei-dimensionale Bildgebung N2 - Since its first experimental implementation in 2005, single-molecule localization microscopy (SMLM) emerged as a versatile and powerful imaging tool for biological structures with nanometer resolution. By now, SMLM has compiled an extensive track-record of novel insights in sub- and inter- cellular organization.\\ Moreover, since all SMLM techniques rely on the analysis of emission patterns from isolated fluorophores, they inherently allocate molecular information $per$ $definitionem$.\\ Consequently, SMLM transitioned from its origin as pure high-resolution imaging instrument towards quantitative microscopy, where the key information medium is no longer the highly resolved image itself, but the raw localization data set.\\ The work presented in this thesis is part of the ongoing effort to translate those $per$ $se$ molecular information gained by SMLM imaging to insights into the structural organization of the targeted protein or even beyond. Although largely consistent in their objectives, the general distinction between global or segmentation clustering approaches on one side and particle averaging or meta-analyses techniques on the other is usually made.\\ During the course of my thesis, I designed, implemented and employed numerous quantitative approaches with varying degrees of complexity and fields of application.\\ \\ In my first major project, I analyzed the localization distribution of the integral protein gp210 of the nuclear pore complex (NPC) with an iterative \textit{k}-means algorithm. Relating the distinct localization statistics of separated gp210 domains to isolated fluorescent signals led, among others, to the conclusion that the anchoring ring of the NPC consists of 8 homo-dimers of gp210.\\ This is of particular significance, both because it answered a decades long standing question about the nature of the gp210 ring and it showcased the possibility to gain structural information well beyond the resolution capabilities of SMLM by crafty quantification approaches.\\ \\ The second major project reported comprises an extensive study of the synaptonemal complex (SNC) and linked cohesin complexes. Here, I employed a multi-level meta-analysis of the localization sets of various SNC proteins to facilitate the compilation of a novel model of the molecular organization of the major SNC components with so far unmatched extend and detail with isotropic three-dimensional resolution.\\ In a second venture, the two murine cohesin components SMC3 and STAG3 connected to the SNC were analyzed. Applying an adapted algorithm, considering the disperse nature of cohesins, led to the realization that there is an apparent polarization of those cohesin complexes in the SNC, as well as a possible sub-structure of STAG3 beyond the resolution capabilities of SMLM.\\ \\ Other minor projects connected to localization quantification included the study of plasma membrane glycans regarding their overall localization distribution and particular homogeneity as well as the investigation of two flotillin proteins in the membrane of bacteria, forming clusters of distinct shapes and sizes.\\ \\ Finally, a novel approach to three-dimensional SMLM is presented, employing the precise quantification of single molecule emitter intensities. This method, named TRABI, relies on the principles of aperture photometry which were improved for SMLM.\\ With TRABI it was shown, that widely used Gaussian fitting based localization software underestimates photon counts significantly. This mismatch was utilized as a $z$-dependent parameter, enabling the conversion of 2D SMLM data to a virtual 3D space. Furthermore it was demonstrated, that TRABI can be combined beneficially with a multi-plane detection scheme, resulting in superior performance regarding axial localization precision and resolution.\\ Additionally, TRABI has been subsequently employed to photometrically characterize a novel dye for SMLM, revealing superior photo-physical properties at the single-molecule level.\\ Following the conclusion of this thesis, the TRABI method and its applications remains subject of diverse ongoing research. N2 - Seit ihrer ersten experimentellen Umsetzung in 2005 hat sich die Einzel-Molekül Lokalisations-Mikroskopie (\textit{engl.} single-molecule localization microscopy (SMLM)) als vielseitig einsetzbares Verfahren in der biologischen Bildgebung etabliert, vor allem aufgrund ihres hohen Auflösungsvermögens im Nanometer Bereich. Bis heute wurde eine Reihe neuer Erkenntnisse bezüglich der sub- und inter- zellulären Organisation durch den Einsatz der SMLM erlangt.\\ Aufgrund der Tatsache, dass alle SMLM Techniken auf dem Prinzip basieren, isolierte Fluorophore zu detektieren und zu analysieren, beinhalten SMLM Daten $per$ $definitionem$ molekulare Informationen.\\ Folgerichtig entwickelte sich das Feld der SMLM vom reinen Bildgebungsinstrument mit Nanometer-Auflösung hin zu quantitativer Mikroskopie, bei welcher der Fokus nicht länger vornehmlich auf dem hochaufgelöstem Bild, sondern vielmehr auf den Lokalisationsdaten liegt.\\ Die vorliegende Arbeit ist als Teil der anhaltenden Bestrebungen zu sehen, aus den $per$ $se$ molekularen Informationen der SMLM weiterführende Erkenntnisse über die strukturelle Organisation der markierten Proteine zu gewinnen. Obwohl mit der gleichen prinzipiellen Zielsetzung versehen, unterscheiden sich hierbei globale oder Segmentierungs- Clusteranalysen von Lokalisations-Meta-Analysen oder so genannten \textit{particle averaging} Ansätzen.\\ Während meiner Doktorarbeit habe ich verschiedene Quantifizierungs Ansätze entworfen, implementiert und angewendet, mit unterschiedlichen Graden an Komplexität und Breite des Anwendungsgebietes.\\ \\ In meinem ersten wesentlichem Projekt analysierte ich mit einem iterativen \textit{k}-means Algorithmus die Lokalisationsverteilung des integralen Proteins gp210, welches Teil des Kernporenkomplexes ist (\textit{engl.} nuclear pore complex (NPC)). Durch den Vergleich der charakteristischen Lokalisations-Statistik von separierten gp210 Domänen mit isolierten Fluoreszenzmarkern konnte unter anderem festgestellt werden, dass der Verankerungsring des NPC aus acht gp210 Homodimeren bestehen muss.\\ Diese Erkenntnis beantwortet zum einen eine jahrzehntealte Frage nach der Zusammensetzung des gp210 Rings und zum anderen liefert sie ein Beispiel dafür, dass durch eine geschickte Analyse der Lokalisationsstatistik strukturelle Informationen erlangt werden können, die jenseits des räumlichen Auflösungsvermögens von SMLM liegen.\\ \\ Das zweite hier vorgestellte wesentliche Projekt beinhaltet eine umfassende Studie des Synaptonemalen Komplexes (\textit{engl.} synaptonemal complex (SNC)) und damit verbundenen Cohesin Komplexen. Um die molekulare Organisation des SNC zu untersuchen, implementierte ich eine multi-level Meta-Analyse der Lokalisationsdaten mehrerer SNC Komponenten. Aus dessen Ergebnissen konnte ein neues drei dimensionales molekulares Modell des SNC erstellt werden.\\ Nachfolgend wurden die beiden murinen Cohesine SMC3 und STAG3 mit adaptierter Methodik untersucht. Hierbei musste die starke intrinsische Dispersion der Cohesin-Signale berücksichtigt werden. Die Analyse ergab deutliche Hinweise auf eine Polarisation der Cohesine innerhalb des SNC. Zudem zeigte sich eine mögliche Substruktur in der Organisation von STAG3, die unterhalb der Auflösungsgrenze von SMLM liegt.\\ \\ Weitere Nebenprojekte im Zusammenhang mit quantitativer Lokalisationsanalyse umfassten die Untersuchung der Lokalisationsverteilung von Plasma-Membran Glykanen, sowie zweier Flotillin Proteine in den Membranen von Bakterien, welche Cluster unterschiedlicher Form und Größe aufzeigten.\\ \\ Schließlich wird ein neuartiger Ansatz für dreidimensionale SMLM vorge-stellt, die auf der genauen Bestimmung von Einzel-Molekül Intensitäten basiert. Diese Methode, genannt TRABI, stützt sich auf die Prinzipien der Apertur Photometrie, welche für die SMLM angepasst und verbessert wurden.\\ Mit TRABI konnte gezeigt werden, dass weit verbreitete Lokalisations-Software, die auf $Gaussian-Fitting$ basiert, die Photonenzahl von Emittern oftmals stark unterschätzt. Diese Diskrepanz kann als $z$-abhängiger Parameter verwendet werden um z.B. einen 2D SMLM Datenatz in einen virtuellen 3D Raum zu überführen. Außerdem wird gezeigt, dass TRABI vorteilhaft mit einem multi-plane Detektionsschema kombiniert werden kann und dabei höhere axiale Lokalisationsgenauigkeiten und Auflösungen er-reicht.\\ Zudem wurde TRABI eingesetzt, um einen neuen Fluoreszenzfarbstoff für SMLM zu charakterisieren und dessen verbesserte photo-physikalische Eigenschaften auf Einzel-Molekül Basis zu demonstrieren.\\ Auch nach Abschluss dieser Arbeit ist die TRABI Methode und deren Anwendung weiterhin Gegenstand diverser Forschungen. KW - Einzelmolekülmikroskopie KW - Quantitative Mikroskopie KW - dSTORM Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-156355 ER - TY - THES A1 - Pahlavan, Pirasteh T1 - Integrated Systems Biology Analysis; Exemplified on Potyvirus and Geminivirus interaction with \(Nicotiana\) \(benthamiana\) T1 - Integrierte Systeme Biologie Analyse, Beispiel für Potyvirus und Geminivirus Interaktion mit \(Nicotiana\) \(benthamiana\) N2 - Viral infections induce a significant impact on various functional categories of biological processes in the host. The understanding of this complex modification of the infected host immune system requires a global and detailed overview on the infection process. Therefore it is essential to apply a powerful approach which identifies the involved components conferring the capacity to recognize and respond to specific pathogens, which in general are defeated in so-called compatible virus-plant infections. Comparative and integrated systems biology of plant-virus interaction progression may open a novel framework for a systemic picture on the modulation of plant immunity during different infections and understanding pathogenesis mechanisms. In this thesis these approaches were applied to study plant-virus infections during two main viral pathogens of cassava: Cassava brown streak virus and African cassava mosaic virus. Here, the infection process was reconstructed by a combination of omics data-based analyses and metabolic network modelling, to understand the major metabolic pathways and elements underlying viral infection responses in different time series, as well as the flux activity distribution to gain more insights into the metabolic flow and mechanism of regulation; this resulted in simultaneous investigations on a broad spectrum of changes in several levels including the gene expression, primary metabolites, and enzymatic flux associated with the characteristic disease development process induced in Nicotiana benthamiana plants due to infection with CBSV or ACMV. Firstly, the transcriptome dynamics of the infected plant was analysed by using mRNA-sequencing, in order to investigate the differential expression profile according the symptom developmental stage. The spreading pattern and different levels of biological functions of these genes were analysed associated with the infection stage and virus entity. A next step was the Real-Time expression modification of selected key pathway genes followed by their linear regression model. Subsequently, the functional loss of regulatory genes which trigger R-mediated resistance was observed. Substantial differences were observed between infected mutants/transgenic lines and wild-types and characterized in detail. In addition, we detected a massive localized accumulation of ROS and quantified the scavenging genes expression in the infected wild-type plants relative to mock infected controls. Moreover, we found coordinated regulated metabolites in response to viral infection measured by using LC-MS/MS and HPLC-UV-MS. This includes the profile of the phytohormones, carbohydrates, amino acids, and phenolics at different time points of infection with the RNA and DNA viruses. This was influenced by differentially regulated enzymatic activities along the salicylate, jasmonate, and chorismate biosynthesis, glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways, as well as photosynthesis, photorespiration, transporting, amino acid and fatty acid biosynthesis. We calculated the flux redistribution considering a gradient of modulation for enzymes along different infection stages, ranging from pre-symptoms towards infection stability. Collectively, our reverse-engineering study consisting of the generation of experimental data and modelling supports the general insight with comparative and integrated systems biology into a model plant-virus interaction system. We refine the cross talk between transcriptome modification, metabolites modulation and enzymatic flux redistribution during compatible infection progression. The results highlight the global alteration in a susceptible host, correlation between symptoms severity and the alteration level. In addition we identify the detailed corresponding general and specific responses to RNA and DNA viruses at different stages of infection. To sum up, all the findings in this study strengthen the necessity of considering the timing of treatment, which greatly affects plant defence against viral infection, and might result in more efficient or combined targeting of a wider range of plant pathogens. N2 - Virale Infektionen haben einen signifikanten Einfluss auf verschiedene funktionelle Eigenschaften und biologische Prozesse im Wirt. Das Verständnis dieser komplexen Modifikation des infizierten Wirtsimmunsystems benötigt eine globale und detaillierte Einsicht in den Infektionsprozess. Diese erfordert einen leistungsfähigen Ansatz zur Identifizierung der beteiligten Komponenten, welche eine Pathogen-Erkennung und Antwort vermitteln bzw. eine kompatible Virus-Pflanze-Infektion voraussetzen. Die Anwendung der vergleichenden und integrierten Systembiologie zur Untersuchung dieser Pflanzen-Virus-Interaktionen im Infektionsverlauf kann eine neue Grundlage zum systematischen Verständnis der Modulation des Immunsystems der Pflanze und der Pathogen-Mechanismen während verschiedener Infektionen eröffnen. In dieser Arbeit wenden wir diese Ansätze an, um Pflanzen-Virus-Infektionen der zwei häufigsten viralen Pathogenen von Maniok zu untersuchen, den Cassava brown streak virus (CBSV) und den African cassava mosaic virus (ACMV). Dazu rekonstruieren wir den Infektionsprozess durch die Kombination von „omics“ basierten Datenanalysen und metabolischen Netzwerkmodellen um die wichtigen Elemente des viralen Infektionsprozesses zu verschieden Zeitpunkten aufzuklären. Metabolische Flussanalysen geben Einblick in metabolische Umsätze und deren Regulierung. Diese simultanen Untersuchungen erfassen ein breites Spektrum der Virus-vermittelten Veränderungen im Wirt über mehrere „omics“ Ebenen, einschließlich Geneexpression, Primärmetabolite und enzymatischer Aktivitäten, die mit dem charakteristischen Krankheitsentwicklungsprozess assoziiert sind, der in Nicotiana benthamiana Pflanzen aufgrund einer Infektion mit CBSV oder ACMV induziert wurde. Zuerst wurde die Dynamik des Transkriptoms infizierter Pflanzen mittels mRNA-Sequenzierung analysiert um das differentielle Expressionsprofil nach dem Symptomentwicklungsstadium zu untersuchen. Die Expressionsmuster und die biologischen Funktionen dieser Gene wurden im Hinblick auf die Infektionsstufe und den Virus Einheiten aufgelöst. Ein nächster Schritt war die Echtzeit-Expressionsmodifikation ausgewählter Schlüsselprozess-Gene, gefolgt von der Umsetzung im linearen Regressionsmodell. Anschließend wurde der funktionelle Verlust von regulatorischen Genen ermittelt, welche eine R-vermittelte Resistenz auslösen können. Es wurden erhebliche Unterschiede zwischen infizierten Mutanten / transgenen Linien und Wild-typen beobachtet und im Detail charakterisiert. Darüber hinaus entdeckten wir eine massive lokalisierte Akkumulation von reaktiven Sauerstoffspezies und quantifizierten die Expression von Abbauproteinen in den infizierten Wildtyp-Pflanzen relativ zu Mock-infizierten Kontrollen. Darüber hinaus fanden wir koordinierte regulierte Metaboliten als Reaktion auf eine virale Infektion, gemessen unter Verwendung von LC-MS / MS und HPLC-UV-MS Techniken. Dazu gehören die Analyse der Profile von Phytohormonen, Kohlenhydraten, Aminosäuren, und Phenolika zu verschiedenen Zeitpunkten der Infektion mit den RNA und DNA-Viren. Diese wurden beeinflusst durch die differentielle regulierten enzymatischen Aktivitäten entlang der Salicylat-, Jasmonat- und Chorismat-Biosynthese, der Glykolyse, Tricarbonsäure und Pentose-Phosphat-Umsetzung, der Photosynthese und Photorespiration, des Transportes und der Aminosäure sowie Fettsäure-Biosynthese. Wir berechneten die Umverteilung des metabolischen Flusses unter Berücksichtigung einer ansteigenden Beeinflussung von Enzymen in den verschiedeneren Infektionsstadien, die von Prä-Symptomen zur Infektionsstabilität reichen. Zusammengefasst beinhaltet unsere Reverse-Engineering-Studie die Generierung von experimentellen Daten und deren Modellierung mittels vergleichender und integrierter Systembiologie zum Einblick in das Modell-Pflanzen-Virus-Interaktionssystem. Wir lösten die Interaktion zwischen Transkriptom-Modifikation, Metabolitenmodulation und die Umverteilung des metabolischen Flusses während des kompatiblen Infektionsprozesses auf. Das Ergebnis zeigt die globalen Veränderungen in einem anfälligen Wirt auf, sowie die Korrelation zwischen Symptomschwere und der Stärke dieser Veränderungen. Darüber hinaus identifizieren wir im Detail die entsprechenden allgemeinen und spezifischen Reaktionen auf RNA und DNA-Viren in den verschiedenen Stadien der Infektion. Zusammenfassend lässt sich feststellen, dass die Erkenntnisse aus dieser Studie die Notwendigkeit aufzeigen, den zeitlichen Ablauf bei einer Pflanzenschutzbehandlung zu berücksichtigen, welche die pflanzliche Abwehr gegen eine Virusinfektion stark beeinflusst; und insgesamt zu einer effizienteren oder kombinierten Anwendung gegen ein breiteres Spektrum von Pflanzenpathogenen führen könnte. KW - RNA-seq KW - virus KW - next generation sequencing KW - transcriptome KW - fluxosome KW - metabolite profiling Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-153412 N1 - I also provided some supplementary data in digital version, which are available on-request from the dean's office. ER - TY - THES A1 - Romanov, Natalie T1 - Characterizing Variation of Protein Complexes and Functional Modules on a Temporal Scale and across Individuals T1 - Charakterisierung der Variation von Proteinkomplexen und funktionellen Modulen im zeitlichen Kontext und zwischen Individuen N2 - A fundamental question in current biology concerns the translational mechanisms leading from genetic variability to phenotypes. Technologies have evolved to the extent that they can efficiently and economically determine an individual’s genomic composition, while at the same time big data on clinical profiles and diagnostics have substantially accumulated. Genome-wide association studies linking genomic loci to certain traits, however, remain limited in their capacity to explain the cellular mechanisms that underlie the given association. For most associations, gene expression has been blamed; yet given that transcript and protein abundance oftentimes do not correlate, that finding does not necessarily decrypt the underlying mechanism. Thus, the integration of further information is crucial to establish a model that could prove more accurate in predicting genotypic effects on the human organism. In this work we describe the so-called proteotype as a feature of the cell that could provide a substantial link between genotype and phenotype. Rather than looking at the proteome as a set of independent molecules, we demonstrate a consistent modular architecture of the proteome that is driven by molecular cooperativity. Functional modules, especially protein complexes, can be further interrogated for differences between individuals and tackled as imprints of genetic and environmental variability. We also show that subtle stoichiometric changes of protein modules could have broader effects on the cellular system, such as the transport of specific molecular cargos. The presented work also delineates to what extent temporal events and processes influence the stoichiometry of protein complexes and functional modules. The re-wiring of the glycolytic pathway for example is illustrated as a potential cause for an increased Warburg effect during the ageing of the human bone marrow. On top of analyzing protein abundances we also interrogate proteome dynamics in terms of stability and solubility transitions during the short temporal progression of the cell cycle. One of our main observations in the thesis encompass the delineation of protein complexes into respective sub-complexes according to distinct stability patterns during the cell cycle. This has never been demonstrated before, and is functionally relevant for our understanding of the dis- and assembly of large protein modules. The insights presented in this work imply that the proteome is more than the sum of its parts, and primarily driven by variability in entire protein ensembles and their cooperative nature. Analyzing protein complexes and functional modules as molecular reflections of genetic and environmental variations could indeed prove to be a stepping stone in closing the gap between genotype and phenotype and customizing clinical treatments in the future. N2 - Eine fundamentale Frage in der heutigen biologischen Forschung ist durch welche Mechanismen eine gebenene genetische Variation sich in einem Phänotyp äußert. Etliche Technologien können heutzutage effizient und ökonomisch die genomische Komposition eines Individuals mit beispielloser Genaugikeit aufschlüsseln. Gleichzeitig gibt es wesentliche Erfolge und Bemühungen, große Datenmengen von Patienten zu sammeln, sowohl klinische Profile, als auch Diagnosen. Es gibt bereits mehrere genomweite Assoziationsstudien, die auf spezifische genomische Loci hinweisen, die womöglich einem bestimmenten phänotypischen Merkmalen zugrunde liegen. Obwohl für die meisten genetischen Assoziationen, eine veränderte Genexpression oftmals als Ursache diskutiert wird, ist dies wahrscheinlich nur ein Teil des zugrundeliegenden Mechanismus. Wir können dies annehmen, da RNA-Transkripte nicht unbedingt mit ihrem Protein-Produkt korrelieren aufgrund von post-transkriptioneller und translationeller Regulation. Um dementsprechend ein Modell zu etablieren, das die genotypischen Effekte auf den human Organismus akkurat vorhersagen kann, ist eine Integration von mehreren zellulären Informationsschichten notwendig. In der folgenden Arbeit beschreiben wir den sogenannten Proteotyp als ein zelluläres Merkmal, das eine substanzielle Verknüpfung zwischen dem Genotyp und dem Phänotyp eines Individuums schaffen könnte. Statt das Proteom als ein Set unabhängiger Moleküle zu betrachten, zeigen wir eine konsistent moduläre Architektur des Proteoms auf, das durch die molekulare Kooperativität zustande kommt. Funktionelle Module, v.a. Proteinkomplexe, können weiters auf Unterschiede zwischen Individuen untersucht werden, sowie deren Variabilität aufgrund genetischer oder umweltbedingter Ursachen. Wir demonstrieren u.a. auch, dass leichte stöchiometrische Veränderungen in solchen Modulen zu weitläufigen Effekten im zellulären Haushalt führen können, z.B. im Transport von spezifischen Molekülen. Die vorgestellte Arbeit beschreibt allerdings auch inwieweit temporäre Ereignisse und Prozesse die Stöchiometrie von Proteinkomplexen und funktionellen Modulen beeinflussen. Wir zeigen z.B. auf, dass eine Veränderung in der glycolytischen Enzym-Stöchiometrie die Ursache für den Warburgeffekt in gealterten Zellen des humanen Knochenmarks darstellen könnte. Neben der Analyse von Protein-Abundanzen untersucht die vorliegende Arbeit Proteomdynamik auch in Hinblick auf Stabilitäts- und Löslichkeitsveränderungen von Proteine in kürzeren Zeitabläufen wie den Zellzyklus. Wir können dabei feststellen, dass Untereinheiten von größeren Proteinkomplexen verschiedene Stabilitätsmuster aufweisen. Dies ist durchaus eine neue Erkennis, die weittragende Folgen für unser Verständnis des Ab- und Aufbauprozesses von Proteinkomplexen haben könnte. Die Einblicke, die aus dieser Arbeit gewonnen werden können, implizieren in jedem Falle, dass das Proteom mehr als die Summe der Einzelteile darstellt, und hauptsächlich durch die Variabilität von gesamten Proteinensembls und deren Kooperativität bestimmt wird. Proteinkomplexe und funktionelle Module sollten daher als molekulare Reflektionen von genetisch- und umweltbedingter Variation betrachtet werden. Solch ein Perspektivenwechsel könnte damit die Möglichkeit bieten eine mechanistische Verknüpfung von Genotyp und Phänotyp zu gewährleisten, und ein Fundament für zukünftige individuell angepasste klinische Behandlungen darstellen. KW - Proteotype KW - Proteomics Analysis of Complexes Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168139 ER - TY - INPR A1 - Dandekar, Thomas T1 - Biological heuristics applied to cosmology suggests a condensation nucleus as start of our universe and inflation cosmology replaced by a period of rapid Weiss domain-like crystal growth N2 - Cosmology often uses intricate formulas and mathematics to derive new theories and concepts. We do something different in this paper: We look at biological processes and derive from these heuristics so that the revised cosmology agrees with astronomical observations but does also agree with standard biological observations. We show that we then have to replace any type of singularity at the start of the universe by a condensation nucleus and that the very early period of the universe usually assumed to be inflation has to be replaced by a period of rapid crystal growth as in Weiss magnetization domains. Impressively, these minor modifications agree well with astronomical observations including removing the strong inflation perturbations which were never observed in the recent BICEP2 experiments. Furthermore, looking at biological principles suggests that such a new theory with a condensation nucleus at start and a first rapid phase of magnetization-like growth of the ordered, physical laws obeying lattice we live in is in fact the only convincing theory of the early phases of our universe that also is compatible with current observations. We show in detail in the following that such a process of crystal creation, breaking of new crystal seeds and ultimate evaporation of the present crystal readily leads over several generations to an evolution and selection of better, more stable and more self-organizing crystals. Moreover, this explains the “fine-tuning” question why our universe is fine-tuned to favor life: Our Universe is so self-organizing to have enough offspring and the detailed physics involved is at the same time highly favorable for all self-organizing processes including life. This biological theory contrasts with current standard inflation cosmologies. The latter do not perform well in explaining any phenomena of sophisticated structure creation or self-organization. As proteins can only thermodynamically fold by increasing the entropy in the solution around them we suggest for cosmology a condensation nucleus for a universe can form only in a “chaotic ocean” of string-soup or quantum foam if the entropy outside of the nucleus rapidly increases. We derive an interaction potential for 1 to n-dimensional strings or quantum-foams and show that they allow only 1D, 2D, 4D or octonion interactions. The latter is the richest structure and agrees to the E8 symmetry fundamental to particle physics and also compatible with the ten dimensional string theory E8 which is part of the M-theory. Interestingly, any other interactions of other dimensionality can be ruled out using Hurwitz compositional theorem. Crystallization explains also extremely well why we have only one macroscopic reality and where the worldlines of alternative trajectories exist: They are in other planes of the crystal and for energy reasons they crystallize mostly at the same time, yielding a beautiful and stable crystal. This explains decoherence and allows to determine the size of Planck´s quantum h (very small as separation of crystal layers by energy is extremely strong). Ultimate dissolution of real crystals suggests an explanation for dark energy agreeing with estimates for the “big rip”. The halo distribution of dark matter favoring galaxy formation is readily explained by a crystal seed starting with unit cells made of normal and dark matter. That we have only matter and not antimatter can be explained as there may be right handed mattercrystals and left-handed antimatter crystals. Similarly, real crystals are never perfect and we argue that exactly such irregularities allow formation of galaxies, clusters and superclusters. Finally, heuristics from genetics suggest to look for a systems perspective to derive correct vacuum and Higgs Boson energies. KW - heuristics KW - inflation KW - cosmology KW - crystallization KW - crystal growth KW - E8 symmetry KW - Hurwitz theorem KW - evolution KW - Lee Smolin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-183945 ER - TY - THES A1 - Breitenbach, Tim T1 - A mathematical optimal control based approach to pharmacological modulation with regulatory networks and external stimuli T1 - Ein auf mathematischer Optimalkontrolle basierender Ansatz für pharmakologische Modulation mit regulatorischen Netzwerken und externen Stimuli N2 - In this work models for molecular networks consisting of ordinary differential equations are extended by terms that include the interaction of the corresponding molecular network with the environment that the molecular network is embedded in. These terms model the effects of the external stimuli on the molecular network. The usability of this extension is demonstrated with a model of a circadian clock that is extended with certain terms and reproduces data from several experiments at the same time. Once the model including external stimuli is set up, a framework is developed in order to calculate external stimuli that have a predefined desired effect on the molecular network. For this purpose the task of finding appropriate external stimuli is formulated as a mathematical optimal control problem for which in order to solve it a lot of mathematical methods are available. Several methods are discussed and worked out in order to calculate a solution for the corresponding optimal control problem. The application of the framework to find pharmacological intervention points or effective drug combinations is pointed out and discussed. Furthermore the framework is related to existing network analysis tools and their combination for network analysis in order to find dedicated external stimuli is discussed. The total framework is verified with biological examples by comparing the calculated results with data from literature. For this purpose platelet aggregation is investigated based on a corresponding gene regulatory network and associated receptors are detected. Furthermore a transition from one to another type of T-helper cell is analyzed in a tumor setting where missing agents are calculated to induce the corresponding switch in vitro. Next a gene regulatory network of a myocardiocyte is investigated where it is shown how the presented framework can be used to compare different treatment strategies with respect to their beneficial effects and side effects quantitatively. Moreover a constitutively activated signaling pathway, which thus causes maleficent effects, is modeled and intervention points with corresponding treatment strategies are determined that steer the gene regulatory network from a pathological expression pattern to physiological one again. N2 - In dieser Arbeit werden Modelle für molekulare Netzwerke bestehend aus gewöhnlichen Differentialgleichungen durch Terme erweitert, die die Wechselwirkung zwischen dem entsprechenden molekularen Netzwerk und der Umgebung berücksichtigen, in die das molekulare Netzwerk eingebettet ist. Diese Terme modellieren die Effekte von externen Stimuli auf das molekulare Netzwerk. Die Nutzbarkeit dieser Erweiterung wird mit einem Modell der circadianen Uhr demonstriert, das mit gewissen Termen erweitert wird und Daten von mehreren verschiedenen Experimenten zugleich reproduziert. Sobald das Modell einschließlich der externen Stimuli aufgestellt ist, wird eine Grundstruktur entwickelt um externe Stimuli zu berechnen, die einen gewünschten vordefinierte Effekt auf das molekulare Netzwerk haben. Zu diesem Zweck wird die Aufgabe, geeignete externe Stimuli zu finden, als ein mathematisches optimales Steuerungsproblem formuliert, für welches, um es zu lösen, viele mathematische Methoden zur Verfügung stehen. Verschiedene Methoden werden diskutiert und ausgearbeitet um eine Lösung für das entsprechende optimale Steuerungsproblem zu berechnen. Auf die Anwendung dieser Grundstruktur pharmakologische Interventionspunkte oder effektive Wirkstoffkombinationen zu finden, wird hingewiesen und diese diskutiert. Weiterhin wird diese Grundstruktur in Bezug zu existierenden Netzwerkanalysewerkzeugen gesetzt und ihre Kombination für die Netzwerkanalyse diskutiert um zweckbestimmte externe Stimuli zu finden. Die gesamte Grundstruktur wird mit biologischen Beispielen verifiziert, indem man die berechneten Ergebnisse mit Daten aus der Literatur vergleicht. Zu diesem Zweck wird die Blutplättchenaggregation untersucht basierend auf einem entsprechenden genregulatorischen Netzwerk und damit assoziierte Rezeptoren werden detektiert. Weiterhin wird ein Wechsel von einem T-Helfer Zelltyp in einen anderen in einer Tumorumgebung analysiert, wobei fehlende Agenzien berechnet werden um den entsprechenden Wechsel in vitro zu induzieren. Als nächstes wird ein genregulatorisches Netzwerk eines Myokardiozyten untersucht, wobei gezeigt wird wie die präsentierte Grundstruktur genutzt werden kann um verschiedene Behandlungsstrategien in Bezug auf ihre nutzbringenden Wirkungen und Nebenwirkungen quantitativ zu vergleichen. Darüber hinaus wird ein konstitutiv aktivierter Signalweg, der deshalb unerwünschte Effekte verursacht, modelliert und Interventionspunkte mit entsprechenden Behandlungsstrategien werden bestimmt, die das genregulatorische Netzwerk wieder von einem pathologischen Expressionsmuster zu einem physiologischen steuern. KW - Bioinformatik KW - systematic drug targeting KW - optimal drug combination KW - disease modelling KW - external stimuli KW - intervention point analyzing KW - Molekülsystem KW - Reiz Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-174368 ER - TY - THES A1 - Kaymak, Irem T1 - Identification of metabolic liabilities in 3D models of cancer T1 - Identifikation metabolischer Abhängigkeiten in 3D Tumormodellen N2 - Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments. N2 - In soliden Tumoren führt die ineffiziente Bildung von Blutgefäßen (Vaskularisierung) zu einem Nährstoff- und Sauerstoffgradienten im gesamten Tumor, welches eine spezifische Tumormikroumgebung schafft. Um diese Tumorumgebung nachzuahmen, wurde ein spezielles multi-zelluläres Tumorsphäroid (SPH) Zellkultursystem verwendet. Da Tp53 wichtige regulatorische Funktionen im Tumormetabolismus hat, wurde zur Generierung von Sphäroiden p53 isogene Darmkrebs-Zelllinen HCT116 (p53 +/+ und p53 -/-) verwendet. Zunächst wurden die Sphäroide mittels RNA Sequenzierung und Metabolomik charakterisiert, um die Genexpression und metabolischen Eigenschaften in verschiedenen Zellkulturbedingungen zu vergleichen. Diese Analyse hat gezeigt, dass gewisse Genexpressionsmuster in Tumoren wie beispielsweise Proliferations- und Hypoxia verwandte Gene in Sphäroiden übereinstimmen, nicht jedoch in Monolayer-Kulturen. Vergleicht man die zwei unterschiedlichen Genotypen miteinander, so sind Gene, die in der Cholesterinhomöostase involviert sind, in p53 defizienten Zellen induziert, nicht jedoch in p53 wildtypischen Zellen. Dieser Unterschied ist in Sphäroiden vorhanden, nicht jedoch in Monolayer-Kulturen. Verlust von p53 führt über die Aktivierung des Transkriptionsfaktors SREBP2 zur Induktion von Enzymen des Mevalonat-Synthesewegs und zudem zu einer neuen metabolischen Vernetzung, die die Generierung von Ubichinon (Coenzym Q10) unterstützt. Eine ausreichende Ubichinon-Versorgung ist wichtig, um den mitochondrialen Elektronentransport und die Pyrimidin-Biosynthese in p53-defizienten Krebszellen unter metabolischen Stressbedingungen zu unterstützen. Darüber hinaus induziert die Inhibition des Mevalonat-Synthesewegs durch Statine in p53-defizienten Darmkrebszellen, die Sauerstoff und Nährstoffmangel ausgesetzt sind, selektiv oxidativen Stress und Apoptose. Verursacht wird dies durch einen Mangel an Ubichinon, welches für den Elektronentransfer der Dihydroorotatdehydrogenase, einem essentiellen Enzym der Pyrimidinnukleotid-Biosynthese, notwendig ist. Gabe von exogenen Nukleosiden entlastete die Nachfrage an Elektronentransfer und stellte die Lebensfähigkeit von p53-defizienten Krebszellen unter metabolischem Stress wieder her. Darüber hinaus konnte gezeigt werden, dass der Mevalonat-Syntheseweg auch für die Synthese von Ubichinon für die Pyrimidinnukleotid-Biosynthese unerlässlich ist, um das Wachstum von Darmtumor-Organoiden zu unterstützen. Zusammengenommen interstreichen diese Ergebnisse die Bedeutung des Mevalonat-Syntheseweg in Krebszellen und liefern den molekularen Mechanismus für die erhöhte Empfindlichkeit von Tumorzellen gegenüber der Hemmung des mitochondrialen Elektronentransfers in einer Tumor-ähnlichen Stoffwechselumgebung. KW - p53 KW - cancer KW - CoQ10 KW - Tumor KW - Modell KW - Stoffwechsel Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181544 ER - TY - THES A1 - Grimm, Johannes T1 - Autocrine and paracrine effects of BRAF inhibitor induced senescence in melanoma T1 - Autokrine und parakrine Effekte BRAF-Inhibitor-induzierter Seneszenz im Melanom N2 - The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels. In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic. N2 - Die Zulassung der spezifischen BRAFV600E Inhibitoren Vemurafenib und Dabrafenib im Jahr 2011 war der erste wirksame Schritt nach Jahrzehnten der Stagnation in der Behandlung des metastasierenden Melanoms. Allerdings zeigte sich, dass trotz erhöhter Gesamtüberlebensrate und gestiegenem progressionsfreien Überleben komplette Remissionen selten waren. Wir konnten in vorangegangenen Versuchen zeigen, dass eine Behandlung BRAFV600E-mutierter Melanom Zelllinien mit Vemurafenib mit der Induktion von Seneszenz-assoziierten Merkmalen einhergeht. Da bekannt ist, dass seneszente Zellen, darunter auch Melanom Zellen, ein sogenanntes Sekretom ausbilden können, welches andere Zellen beeinflussen kann, war die Identifizierung und Charakterisierung von Vemurafenib-induzierten sezernierten Faktoren das Ziel meiner Arbeit. Initiale Versuche zeigten, dass konditionierter Überstand von Vemurafenib behandelten Zellen das Wachstum naiver Zelllinien erhöhen kann. Ich konnte in weiteren Versuchen zeigen, dass sich die Transkription und Expression des Cytokins CCL2, der Matrixmetalloprotease MMP2 und des Wachstumsfaktors FGF1 nach Vemurafenib Behandlung erhöht. Darüber hinaus konnte ich interessanterweise auch eine gesteigerte Transkription anderer FGF Liganden (FGF7, FGF17) feststellen, was meinen Fokus auf die Analyse von FGFR abhängigen Prozessen als Antwort auf die BRAF Inhibition gelenkt hat. Es zeigte sich, dass sich Melanomzellen mittels Zugabe von FGF1 besser gegen die Vemurafenib-induzierte MEK/ERK1/2 Hemmung behaupten können. Darüber hinaus konnte durch den Einsatz eines spezifischen FGFR Inhibitors die Viabilität von Melanomzellen unter Vemurafenib Behandlung vermindert werden. Auch der stimulierende Effekt des Vemurafenib konditionierten Überstandes konnte dadurch teilweise aufgehoben werden. Die Induktion des BRAF Inhibitor assoziierten Sekretoms ist auf einen aktiven PI3K/AKT Signalweg angewiesen. So führt eine gleichzeitige Hemmung des MEK/ERK1/2 und PI3K/AKT Signalwegs zu einer verminderten Seneszenzinduktion und einer niedrigeren Transkription der Seneszenz-assoziierten Gene. Zudem konnte ich feststellen, dass auch eine gemeinsame Hemmung von BRAF und MEK Seneszenz und das damit einhergehende Sekretom unter Beteiligung von CCL2, MMP2 und den FGFs induziert. Zusammenfassend zeigen meine Daten, dass BRAFV600E-mutierte Melanomzellen nach Vemurafenib Behandlung ein Sekretom ausbilden, welches potentiell wachstumsfördernde und matrix-modellierende Faktoren beinhaltet. Dies ist abhängig vom PI3K/AKT Signalweg und charakterisiert durch die Sekretion von FGF1, CCL2 und MMP2. Klinische Relevanz erlangen diese Erkenntnisse durch die Möglichkeit, diese Faktoren im Rahmen einer Kombinationstherapie, z.B. mit einem spezifischen FGFR Inhibitor, zu inaktivieren. KW - Melanoma KW - Inhibitor KW - Melanom Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181161 ER - TY - THES A1 - Letschert, Sebastian T1 - Quantitative Analysis of Membrane Components using Super-Resolution Microscopy T1 - Quantitative Analyse von Membrankomponenten mittels hochauflösender Fluoreszenzmikroskopie N2 - The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules. An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed. Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and –lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates. In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60–1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed. N2 - Die Plasmamembran gehört zu den am meisten untersuchten, gleichzeitig aber auch zu den komplexesten, vielfältigsten und am wenigsten verstandenen biologischen Strukturen. Ihre Funktion wird nicht nur durch die molekulare Zusammensetzung bestimmt, sondern auch durch die räumliche Anordnung ihrer Bestandteile. Selbst nach Jahrzehnten intensiver Forschung und der Veröffentlichung dutzender Membranmodelle und Theorien bleibt die genaue strukturelle Organisation der Plasmamembran ein Rätsel. Moderne Bildgebungsverfahren wie etwa die hochauflösende Fluoreszenzmikroskopie gehören mittlerweile zu den effizientesten Techniken der Lebenswissenschaften und werden immer öfter verwendet, um die räumliche Anordnung als auch die Anzahl von Biomolekülen in fixierten und lebenden Zellen zu studieren. Im Rahmen dieser Arbeit wurde die hochauflösende Mikroskopie-Methode dSTORM (direct stochastic optical reconstruction microscopy) angewendet, um die räumliche Verteilung von Membranmolekülen mit annähernd molekularer Auflösung zu untersuchen. Schwerpunkte dieser Arbeit sind dabei verschiedene Präparations- und Färbemethoden für die mikroskopische Untersuchung von Zellmembranen sowie lokalisationsbasierte quantitative Analysemethoden von Membranmolekülen. Eine Voraussetzung für die räumliche als auch quantitative Analyse von Membranmolekülen ist die Vermeidung von Photoschalt-Artefakten in rekonstruierten Lokalisationsmikroskopie-Bildern. Um dies genauer zu demonstrieren, wurden die Auswirkungen von Anregungsintensität, Markierungsdichte und verändertem Photoschalten auf die räumliche Verteilung von Proteinen der Plasma- und Mitochondrienmembran in dSTORM-Bildern analysiert. Es wird gezeigt, dass eine dicht markierte Plasmamembran in Kombination mit ungeeigneten Photoschaltraten zu artifiziellen Clustern in der Membran führt. Es sind vor Allem oft die Projektionen dreidimensionaler Membranstrukturen wie etwa Mikrovilli und Vesikel dafür verantwortlich, dass lokale Unterschiede in der Lokalisationsdichte entstehen, wodurch unter Umständen Bildartefakte generiert werden können. Darüber hinaus werden alternative Mikroskopie-Methoden und Möglichkeiten, Artefakte in Einzelmolekül-Lokalisationsmikroskopie-Bildern zu verhindern, präsentiert und ausführlich diskutiert. Ein weiteres zentrales Thema dieser Arbeit ist die räumliche Anordnung von glykosylierten Membranmolekülen. Es wird demonstriert, wie ein bioorthogonales chemisches Reportersystem bestehend aus modifizierten Monosacchariden und organischen Fluorophoren für die spezifische Markierung von Membran-assoziierten Glykoproteinen und –lipiden eingesetzt werden kann. Mittels dSTORM wird gezeigt, dass die Verteilung von Glykanen in der Plasmamembran unterschiedlicher Zelllinien homogen und frei von Clustern ist. Des Weiteren zeigt eine quantitative Analyse, dass sich in etwa fünf Millionen Glykane auf einer einzigen Zelle befinden. Die Ergebnisse demonstrieren, dass die Kombination aus metabolisch markierten Zielmolekülen, Click-Chemie und Einzelmolekül-Lokalisationsmikroskopie effizient genutzt werden kann, um Glykokonjugate auf Zelloberflächen zu untersuchen. In einem dritten Projekt wurde dSTORM zur Untersuchung von Rezeptormolekülen auf Krebszellen verwendet. Die Expression dieser Oberflächenproteine ist so gering, dass sich nur wenige Moleküle auf einer Zelle befinden, die jedoch als Zielmoleküle in der personalisierten Immuntherapie dienen könnten. Dafür wurden primäre Tumorzellen aus dem Knochenmark von Patienten, die am Multiplen Myelom erkrankt sind, auf die Expression des CD19-Oberflächenproteins als potentielles Ziel für CAR-modifizierte T-Zellen (chimeric antigen receptor) untersucht. Es wird gezeigt, dass sich, abhängig vom untersuchten Patienten, auf einer Zelle 60 bis 1600 CD19-Moleküle befinden. Funktionale in-vitro-Experimente demonstrieren, dass weniger als 100 CD19 Moleküle ausreichen, um CD19-CAR-T-Zellen zu aktivieren. Diese Ergebnisse werden mit Durchflusszytometrie-Daten verglichen und die wichtige Rolle von Lebendzellfärbung und geeigneten Kontrollexperimenten wird diskutiert. KW - Fluoreszenzmikroskopie KW - Quantifizierung KW - Polysaccharide KW - Immuntherapie KW - Antigen CD19 KW - super-resolution fluorescence microscopy KW - dSTORM KW - click chemistry KW - plasma membrane organization KW - localization microscopy KW - artifacts KW - Hochauflösende Fluoreszenzmikroskopie KW - Plasmamembranorganisation KW - Click Chemie KW - Glykane Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162139 ER - TY - THES A1 - Simon, Katja T1 - Identifying the role of Myb-MuvB in gene expression and proliferation of lung cancer cells T1 - Identifizierung der Rolle des Myb-MuvB in der Genexpression und der Proliferation von Lungenkrebszellen N2 - The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009). This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo. In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker. N2 - Der evolutionär konservierte Myb-MuvB (MMB) Multiproteinkomplex ist ein transkriptionaler Meisterregulator der mitotischen Genexpression. Die MMB Untereinheiten B-MYB, FOXM1 und ihre Zielgene sind oft überexprimiert in verschiedenen Krebsarten. Die erhöhte Expression dieser Gene korreliert mit einem fortgeschrittenen Tumorstadium und einer schlechten Prognose für Patienten. Außerdem wurde berichtet, dass Signalwege, die die Mitosemaschinerie betreffen, reizvoll sind als mögliches Target für die Behandlung von Ras mutierten Krebsarten (Lao et al., 2009). Dies weißt auf darauf hin, dass der MMB Komplex an der Tumorentstehung beteiligt sein könnte, indem er die Überexpression mitotischer Gene fördert und damit ein geeignetes Target zur Behandlung von Krebs darstellen könnte. Obwohl der MMB biochemisch eingehend untersucht wurde, ist die Beteiligung des MMB an der Tumorgenese weitestgehend unbekannt speziell in vivo. In dieser Doktorarbeit wurde anhand eines NSCLC Mausmodells gezeigt, dass der MMB für die Lungentumorgenese in vivo erforderlich ist. Erhöhte Level von B-MYB, NUSAP1 oder CENPF in fortgeschrittenen Tumoren und im Gegenzug niedrigen Leveln in Grad 1 und 2 Tumoren unterstreichen die mögliche Beteiligung des MMB an der Lungentumorgenese und das onkogene Potential von B-MYB. Die Tumorwachstum-fördernde Funktion von B-MYB wurde veranschaulicht durch eine geringere Anzahl an KI-67 positiven Zellen in vivo und einem signifikant hohen Beeinträchtigung der Proliferation nach dem Verlust von B-MYB in vitro. Defekte in der Zytokinese und ein abnormales Zellzyklusprofil nach dem Verlust von B-MYB heben den Einfluss von B-Myb auf die Proliferation von Lungenkrebszelllinien hervor. Die unvollständige Rekombination von B-Myb in murinen Lungentumoren und den daraus hergestellten primären Tumorzelllinien veranschaulichen den Selektionsdruck auf den kompletten Verlust von B-MYB und zeigen zusätzlich, dass B-MYB ein für den Tumor essentielles Gen ist. Im letzten Teil der Doktorarbeit konnte die Beteiligung des MMB auf die Proliferation auf Lungenkrebszellen gezeigt werden durch den Verlust von B-MYB durch RNAi-Interferenz (RNAi). Detektion erhöhter B-Myb Level in humanen Adenokarzinomen und eine verminderte Proliferation, Zytokinese-Defekte und ein abnormales Zellzyklusprofil nach B-MYB Verlust in humanen Lungenkrebszelllinien unterstreichen das Potential von B-MYB als klinischer Marker zu fungieren. KW - Lungenkrebs KW - MMB KW - B-MYB KW - K-RAS KW - lung cancer KW - Mitose KW - Nicht-kleinzelliges Bronchialkarzinom Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-161814 ER - TY - JOUR A1 - Wagner, Johanna A1 - Eiken, Barbara A1 - Haubitz, Imme A1 - Lichthardt, Sven A1 - Matthes, Niels A1 - Löb, Stefan A1 - Klein, Ingo A1 - Germer, Christoph-Thomas A1 - Wiegering, Armin T1 - Suprapubic bladder drainage and epidural catheters following abdominal surgery—a risk for urinary tract infections? JF - PLoS ONE N2 - Background Epidural catheters are state of the art for postoperative analgesic in abdominal surgery. Due to neurolysis it can lead to postoperative urinary tract retention (POUR), which leads to prolonged bladder catheterization, which has an increased risk for urinary tract infections (UTI). Our aim was to identify the current perioperative management of urinary catheters and, second, to identify the optimal time of suprapubic bladder catheter removal in regard to the removal of the epidural catheter. Methods We sent a questionnaire to 102 German hospitals and analyzed the 83 received answers to evaluate the current handling of bladder drainage and epidural catheters. Then, we conducted a retrospective study including 501 patients, who received an epidural and suprapubic catheter after abdominal surgery at the University Hospital Würzburg. We divided the patients into three groups according to the point in time of suprapubic bladder drainage removal in regard to the removal of the epidural catheter and analyzed the onset of a UTI. Results Our survey showed that in almost all hospitals (98.8%), patients received an epidural catheter and a bladder drainage after abdominal surgery. The point in time of urinary catheter removal was equally distributed between before, simultaneously and after the removal of the epidural catheter (respectively: ~28–29%). The retrospective study showed a catheter-associated UTI in 6.7%. Women were affected significantly more often than men (10,7% versus 2,5%, p<0.001). There was a non-significant trend to more UTIs when the suprapubic catheter was removed after the epidural catheter (before: 5.7%, after: 8.4%). Conclusion The point in time of suprapubic bladder drainage removal in relation to the removal of the epidural catheter does not seem to correlate with the rate of UTIs. The current handling in Germany is inhomogeneous, so further studies to standardize treatment are recommended. KW - catheters KW - epidural block KW - bladder KW - urinary tract infections KW - abdominal surgery KW - catheterization KW - surgical and invasive medical procedures KW - rectum Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177731 VL - 14 IS - 1 ER - TY - JOUR A1 - Goos, Carina A1 - Dejung, Mario A1 - Wehman, Ann M. A1 - M-Natus, Elisabeth A1 - Schmidt, Johannes A1 - Sunter, Jack A1 - Engstler, Markus A1 - Butter, Falk A1 - Kramer, Susanne T1 - Trypanosomes can initiate nuclear export co-transcriptionally JF - Nucleic Acids Research N2 - The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes. KW - molecular biology KW - nuclear export KW - trypanosomes KW - mRNA KW - nuclear envelope Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177709 VL - 47 IS - 1 ER - TY - THES A1 - Fleischmann, Pauline Nikola T1 - Starting foraging life: Early calibration and daily use of the navigational system in \(Cataglyphis\) ants T1 - Start in den Außendienst: Zur anfänglichen Kalibrierung und alltäglichen Nutzung des Navigationssystem in \(Cataglyphis\)-Ameisen N2 - Cataglyphis ants are famous for their navigational abilities. They live in hostile habitats where they forage as solitary scavengers covering distances of more than hundred thousand times their body lengths. To return to their nest with a prey item – mainly other dead insects that did not survive the heat – Cataglyphis ants constantly keep track of their directions and distances travelled. The navigational strategy is called path integration, and it enables an ant to return to the nest in a straight line using its home vector. Cataglyphis ants mainly rely on celestial compass cues, like the position of the sun or the UV polarization pattern, to determine directions, and they use an idiothetic step counter and optic flow to measure distances. In addition, they acquire information about visual, olfactory and tactile landmarks, and the wind direction to increase their chances of returning to the nest safe and sound. Cataglyphis’ navigational performance becomes even more impressive if one considers their life style. Most time of their lives, the ants stay underground and perform tasks within the colony. When they start their foraging careers outside the nest, they have to calibrate their compass systems and acquire all information necessary for navigation during subsequent foraging. This navigational toolkit is not instantaneously available, but has to be filled with experience. For that reason, Cataglyphis ants perform a striking behavior for up to three days before actually foraging. These so-called learning walks are crucial for the success as foragers later on. In the present thesis, both the ontogeny and the fine-structure of learning walks has been investigated. Here I show with displacement experiments that Cataglyphis ants need enough space and enough time to perform learning walks. Spatially restricted novices, i. e. naïve ants, could not find back to the nest when tested as foragers later on. Furthermore, ants have to perform several learning walks over 1-3 days to gain landmark information for successful homing as foragers. An increasing number of feeder visits also increases the importance of landmark information, whereas in the beginning ants fully rely on their path-integration vector. Learning walks are well-structured. High-speed video analysis revealed that Cataglyphis ants include species-specific rotational elements in their learning walks. Greek Cataglyphis ants (C. noda and C. aenescens) inhabiting a cluttered pine forest perform voltes, small walked circles, and pirouettes, tight turns about the body axis with frequent stopping phases. During the longest stopping phases, the ants gaze back to their nest entrance. The Tunisian Cataglyphis fortis ants inhabiting featureless saltpans only perform voltes without directed gazes. The function of voltes has not yet been revealed. In contrast, the fine structure of pirouettes suggests that the ants take snapshots of the panorama towards their homing direction to memorize the nest’s surroundings. The most likely hypothesis was that Cataglyphis ants align the gaze directions using their path integrator, which gets directional input from celestial cues during foraging. To test this hypothesis, a manipulation experiment was performed changing the celestial cues above the nest entrance (no sun, no natural polarization pattern, no UV light). The accurately directed gazes to the nest entrance offer an easily quantifiable readout suitable to ask the ants where they expect their nest entrance. Unexpectedly, all novices performing learning walks under artificial sky conditions looked back to the nest entrance. This was especially surprising, because neuronal changes in the mushroom bodies and the central complex receiving visual input could only be induced with the natural sky when comparing test animals with interior workers. The behavioral findings indicated that Cataglyphis ants use another directional reference system to align their gaze directions during the longest stopping phases of learning walk pirouettes. One possibility was the earth’s magnetic field. Indeed, already disarraying the geomagnetic field at the nest entrance with an electromagnetic flat coil indicated that the ants use magnetic information to align their looks back to the nest entrance. To investigate this finding further, ants were confronted with a controlled magnetic field using a Helmholtz coil. Elimination of the horizontal field component led to undirected gaze directions like the disarray did. Rotating the magnetic field about 90°, 180° or -90° shifted the ants’ gaze directions in a predictable manner. Therefore, the earth’s magnetic field is a necessary and sufficient reference system for aligning nest-centered gazes during learning-walk pirouettes. Whether it is additionally used for other navigational purposes, e. g. for calibrating the solar ephemeris, remains to be tested. Maybe the voltes performed by all Cataglyphis ant species investigated so far can help to answer this question.. N2 - Cataglyphis-Ameisen sind für ihre Navigationsfähigkeiten berühmt. Sie bewohnen lebens- feindliche Regionen in denen sie einzeln und über weite Strecken Futter suchen müssen. Um mit Beute (meist ein totes Insekt, das die große Hitze nicht überlebt hat) zu ihrem Nest zurückzukehren, bedienen sie sich einer Navigationsstrategie, die als Wegintegration beze- ichnet wird. Dabei müssen die Ameisen die zurückgelegten Distanzen messen und jeden Richtungswechsel registrieren, um schließlich in gerader Linie nachhause zurückkehren zu können. Als Kompass nutzen sie Himmelsinformationen, wie den Stand der Sonne oder das UV-Polarisationsmuster, und für die Distanzmessung verwenden sie einen inneren Schrittzäh- ler sowie optischen Fluss. Außerdem nutzen sie alle weiteren Informationen, die hilfreich sein könnten, um sicher zum Nest zurückzukehren. Dazu gehören visuelle, olfaktorische und taktile Landmarken sowie die Richtung des Windes. Die Navigationsleistungen von Cataglyphis-Ameisen sind insbesondere dann bemerkenswert, wenn man sich bewusst macht, dass sie die meiste Zeit ihres Lebens unter der Erde verbringen. Dort übernehmen sie Auf- gaben im Nest bis sie dann schließlich alt genug sind, um draußen Futter zu suchen. Dann müssen sie ihre Kompasssysteme kalibrieren und alle Informationen lernen, die sie für eine erfolgreiche Futtersuche brauchen. Dieses sogenannte Navigations-Toolkit steht den Ameisen nicht automatisch zur Verfügung, vielmehr müssen sie es mit eigener Erfahrung füllen. Dafür nutzen sie die ersten ein bis drei Tage außerhalb des Nestes. Während dieser Zeit suchen sie kein Futter, sondern vollführen sogenannte Lernläufe. Lernläufe sind unabdingbar, um später als Fourageur erfolgreich zu sein. In der vorliegenden Doktorarbeit wurde sowohl die zeitliche und räumliche Entwicklung der Lernläufe als auch deren Feinstruktur untersucht. Mit Versetzungsexperimenten konnte ich zeigen, dass Ameisen genügend Zeit und Raum brauchen, um Lernläufe durchzuführen. Wurden Neulinge während ihrer Lernläufe räumlich eingeschränkt, so konnten sie nicht zum Nest zurückfinden, wenn sie als erfahrene Fourageure getestet wurden. Außerdem brauchen die Ameisen ein bis drei Tage Zeit, um ein Landmarkenpanorama zu erlernen, das sie dann später erfolgreich zur Landmarkenorientierung nutzen können. Eine größere Anzahl an Besuchen am Futterplatz erhöht die Wichtigkeit von Landmarkeninformation für die Ameisen, die anfangs nur ihren Wegintegrator nutzen. Lernläufe weisen eine beeindruckende Struktur auf. Mit High-Speed-Videoaufnahmen konnte gezeigt werden, dass Cataglyphis-Ameisen artspezifische Drehungen während der Lernläufe vollführen. Die griechischen Cataglyphis-Ameisen (C. noda und C. aenescens) leben in einem Pinienwald, der ihnen ein vielfältiges und landmarkenreiches Panorama bietet. Ihre Lernläufe beinhalten zwei Drehungsformen, nämlich sogenannte Volten (kleine gelaufene Kreise) und Pirouetten (enge Drehungen um die eigene Körperachse mit häufigen Stoppphasen). Während der längsten Stoppphase einer Pirouette schauen die Ameisen zurück in die Richtung ihres Nesteingangs, obwohl sie ihn nicht direkt sehen können. Die tunesischen Cataglyphis-Ameisen (C. fortis ) leben auf einem landmarkenarmen Salzsee. Sie vollführen nur Volten und machen keine Pirouetten während ihrer Lernläufe. Die Funktion von Volten ist noch unbekannt, wohingegen die Feinstruktur der Pirouetten die Vermutung nahelegt, dass die Ameisen sogenannte Schnappschüsse von der Umgebung ihres Nestes machen, um dorthin zurückkehren zu können. Es schien wahrscheinlich, dass die Ameisen ihren Wegintegrator nutzen, um ihre Blickrich- tungen zum Nest auszurichten. Während der Futtersuche bekommt der Wegintegrator seine Richtungsinformationen vom Himmelskompass. Daher wurde ein Experiment geplant und durchgeführt bei dem die Himmelsinformationen über dem Nesteingang manipuliert wurden (keine Sicht auf die Sonne, kein natürliches Polarisationsmuster oder kein UV-Licht). Die nest- zentrierten Blickrichtungen der Ameisen ermöglichen es relativ einfach zu überprüfen, ob die Ameisen die Position des Nesteingangs kennen. Überraschenderweise schauten die Ameisen unter allen Bedingungen weiterhin zurück zum Nesteingang. Dies war insbesondere be- merkenswert, da die Himmelsmanipulation neuronale Veränderungen in den Pilzkörpern und dem Zentralkomplex (das sind Regionen im Gehirn der Ameisen, die visuelle Informationen verarbeiten) bewirkten bzw. diese verhinderten. Nur unter natürlichen Bedingungen, also bei freiem Blick auf die Sonne, gab es Unterschiede auf neuronaler Ebene zwischen den Testtieren und den Innendiensttieren, die als Kontrolle dienten. Die Ergebnisse des Verhaltensversuchs deuteten darauf hin, dass die Ameisen ein anderes direktionales Referenzsystem nutzen, um ihre Blickrichtungen zu kontrollieren. Eine Möglichkeit war das Erdmagnetfeld. Tatsächlich zeigte schon die experimentelle Streuung des Magnetfelds am Nesteingang mittels einer elektromagnetischen Flachspule, dass die Ameisen tatsächlich Magnetinformationen nutzen, um ihre Blicke auszurichten. Die Blickrichtungen während der längsten Stoppphasen waren nicht mehr zum Nesteingang gerichtet. Um dies genauer zu untersuchen wurden die Ameisen mit dem kontrollierten Magnetfeld einer Helmholtzspule konfrontiert. Die Eliminierung der Horizontalkomponente des Magnetfelds bewirkte wiederum, dass die Ameisen nicht zum Nesteingang zurückschauten. Wurde die Horizontalkomponente jedoch um 90◦, 180◦ oder -90◦ gedreht, so folgten die Blickrichtungen der Ameisen dieser Drehung voraussagbar im selben Winkel. Dies zeigt, dass das Erdmagnetfeld tatsächlich das Referenzsystem für die Ausrichtungen der Blicke während der Lernlaufpirouetten darstellt. Ob es auch noch an- deren Navigationszwecken, wie beispielsweise der Kalibrierung der solaren Ephemeris dient, muss zukünftig überprüft werden. Vielleicht können die Volten, die alle bisher untersuchten KW - Cataglyphis KW - Learning walk Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159951 ER - TY - THES A1 - Mekala, SubbaRao T1 - Generation of cardiomyocytes from vessel wall-resident stem cells T1 - Erzeugung von Kardiomyozyten aus Gefäßwand-residenten Stammzellen N2 - Myocardial infarction (MI) is a major cause of health problems and is among the leading deadly ending diseases. Accordingly, regenerating functional myocardial tissue and/or cardiac repair by stem cells is one of the most desired aims worldwide. Indeed, the human heart serves as an ideal target for regenerative intervention, because the capacity of the adult myocardium to restore itself after injury or infarct is limited. Thus, identifying new sources of tissue resident adult stem or progenitor cells with cardiovascular potential would help to establish more sophisticated therapies in order to either prevent cardiac failure or to achieve a functional repair. Ongoing research worldwide in this field is focusing on a) induced pluripotent stem (iPS) cells, b) embryonic stem (ES) cells and c) adult stem cells (e. g. mesenchymal stem cells) as well as cardiac fibroblasts or myofibroblasts. However, thus far, these efforts did not result in therapeutic strategies that were transferable into the clinical management of MI and heart failure. Hence, identifying endogenous and more cardiac-related sources of stem cells capable of differentiating into mature cardiomyocytes would open promising new therapeutic opportunities. The working hypothesis of this thesis is that the vascular wall serves as a niche for cardiogenic stem cells. In recent years, various groups have identified different types of progenitors or mesenchymal stem cell-like cells in the adventitia and sub-endothelial zone of the adult vessel wall, the so called vessel wall-resident stem cells (VW-SCs). Considering the fact that heart muscle tissue contains blood vessels in very high density, the physiological relevance of VW-SCs for the myocardium can as yet only be assumed. The aim of the present work is to study whether a subset of VW-SCs might have the capacity to differentiate into cardiomyocyte-like cells. This assumption was challenged using adult mouse aorta-derived cells cultivated in different media and treated with selected factors. The presented results reveal the generation of spontaneously beating cardiomyocyte-like cells using specific media conditions without any genetic manipulation. The cells reproducibly started beating at culture days 8-10. Further analyses revealed that in contrast to several publications reporting the Sca-1+ cells as cardiac progenitors the Sca-1- fraction of aortic wall-derived VW-SCs reproducibly delivered beating cells in culture. Similar to mature cardiomyocytes the beating cells developed sarcomeric structures indicated by the typical cross striated staining pattern upon immunofluorescence analysis detecting α-sarcomeric actinin (α-SRA) and electron microscopic analysis. These analyses also showed the formation of sarcoplasmic reticulum which serves as calcium store. Correspondingly, the aortic wall-derived beating cardiomyocyte-like cells (Ao-bCMs) exhibited calcium oscillations. This differentiation seems to be dependent on an inflammatory microenvironment since depletion of VW-SC-derived macrophages by treatment with clodronate liposomes in vitro stopped the generation of Ao bCMs. These locally generated F4/80+ macrophages exhibit high levels of VEGF (vascular endothelial growth factor). To a great majority, VW-SCs were found to be positive for VEGFR-2 and blocking this receptor also stopped the generation VW-SC-derived beating cells in vitro. Furthermore, the treatment of aortic wall-derived cells with the ß-receptor agonist isoproterenol or the antagonist propranolol resulted in a significant increase or decrease of beating frequency. Finally, fluorescently labeled aortic wall-derived cells were implanted into the developing chick embryo heart field where they became positive for α-SRA two days after implantation. The current data strongly suggest that VW-SCs resident in the vascular adventitia deliver both progenitors for an inflammatory microenvironment and beating cells. The present study identifies that the Sca-1- rather than Sca-1+ fraction of mouse aortic wall-derived cells harbors VW-SCs differentiating into cardiomyocyte-like cells and reveals an essential role of VW-SCs-derived inflammatory macrophages and VEGF-signaling in this process. Furthermore, this study demonstrates the cardiogenic capacity of aortic VW-SCs in vivo using a chimeric chick embryonic model. N2 - Der Myokardinfarkt (MI) ist einer der Hauptgründe für gesundheitliche Probleme und zählt zu einer der am häufigsten tödlich verlaufenden Krankheiten weltweit. Daher ist es nicht verwunderlich, dass die Regeneration von funktionellem Myokardgewebe und/oder die kardiale Reparatur durch Stammzellen eines der weltweit am meisten angestrebten Ziele darstellt. Das adulte menschliche Herz stellt aufgrund seiner äußerst eingeschränkten endogenen Regenerationskapazität, die bei weitem nicht ausreicht, das geschädigte Gewebe zu erneuern, ein ideales Zielorgan für regenerative Therapieverfahren dar. Folglich könnte die Identifizierung neuer Quellen adulter Stamm- oder Vorläuferzellen mit kardiovaskulärem Differenzierungspotential dabei helfen, verfeinerte Therapien zu entwickeln, um entweder kardiale Fehlfunktionen zu verhindern oder eine deutlich verbesserte myokardiale Reparatur zu erreichen. Die aktuelle weltweite Forschung auf diesem Gebiet fokussiert sich auf: a) induzierte pluripotente Stammzellen (iPS), b) embryonale Stammzellen (ES) und c) adulte Stammzellen, wie z. B. mesenchymale Stammzellen, kardiale Fibroblasten und Mesangioblasten sowie Myofibroblasten. Bisher haben jedoch alle Bemühungen noch zu keinem Durchbruch geführt, so dass die teilweise vielversprechenden experimentellen Ergebnisse nicht in die klinische Therapie des MI und der kardialen Defekte mittels Stammzellen transferiert werden können. Abgesehen davon, ob und wie stark so ein endogenes herzeigenes Potential wäre, würde die Identifizierung neuer endogener Stammzellen mit kardiogenem Potential, die genaue Charakterisierung ihrer Nischen und der Mechanismen ihrer Differenzierung einen Meilenstein in der kardioregenerativen Stammzelltherapie darstellen. Die Arbeitshypothese der hier vorgelegten Dissertation besagt, dass die Gefäßwand als Nische solcher Zellen dienen könnte. Innerhalb der letzten Jahre konnte die Adventitia und die subendotheliale Zone der adulten Gefäßwand als Nische für unterschiedliche Typen von Vorläuferzellen und multipotenten Stammzellen, die sogenannten Gefäßwand-residenten Stammzellen (VW-SCs) identifiziert werden. In Anbetracht der Tatsache, dass die Blutgefäße aufgrund ihrer hohen Dichte im Herzen eine essentielle stromale Komponente des Herzgewebes darstellen, kann die mögliche klinische Relevanz von VW-SCs für das Myokardium im Moment nur erahnt werden. Ausgehend von der Annahme, dass eine Subpopulation dieser VW-SCs die Fähigkeit besitzt, sich in Kardiomyozyten-ähnliche Zellen zu differenzieren, sollte im Rahmen dieser Dissertationsarbeit das myokardiale Potential der Gefäßwand-residenten Stammzellen aus der Aorta adulter Mäuse studiert werden, indem die Zellen unter unterschiedlichen definierten Bedingungen kultiviert und dann sowohl morphologisch als auch funktionell charakterisiert werden. Erstaunlicherweise zeigten die ersten Ergebnisse die Generierung spontan schlagender Kardiomyozyten-ähnlicher Zellen, nur durch Verwendung eines speziellen Nährmediums und ohne jegliche genetische Manipulation. Die im Rahmen dieser Arbeit durchgeführten Analysen belegen zudem, dass die Kardiomyozyten-ähnlichen Zellen reproduzierbar nach ca. 9-11 Tagen in der Kultur anfangen, spontan zu schlagen. In immunzytochemischen Analysen zeigten die schlagenden Zellen ein quergestreiftes Färbemuster für α sarkomeres Actinin. Passend dazu wiesen diese spontan schlagenden Zellen, wie reife Kardiomyozyten, Sarkomerstrukturen mit Komponenten des sarkoplasmatischen Retikulums in elektronenmikroskopischen Analysen auf. Sie zeigten dementsprechend eine mit dem spontanen Schlag assoziierte Kalzium-Oszillation. Erstaunlicherweise zeigten die hier vorgelegten Befunde erstmalig, dass es nicht die Sca-1+ (stem cell antigen-1) Zellen waren, denen seit Jahren eine kardiomyozytäre Kapazität zugeschrieben wird, sondern es waren die Sca-1- Zellen der Mausaorta, die sich zu den spontan schlagenden Zellen differenzierten. Des Weiteren scheint diese Differenzierung von einer endogen generierten inflammatorischen Mikroumgebung abhängig zu sein. Die hier vorgelegten Ergebnisse legen daher den Schluss nahe, dass die VW-SCs in der vaskulären Adventitia sowohl die inflammatorische Mikroumgebung als auch die spontan schlagenden Kardiomyozyten-ähnlichen Zellen bereitstellten. So entstanden in der Kultur aortaler Zellen unter anderem auch Makrophagen, die hohe Mengen des Gefäßwachstumsfaktors VEGF (Vascular Endothelial Growth Factor) aufweisen. Wurden die Makrophagen in der Zellkultur durch Zugabe von Clodtronat-Liposomen depletiert, so wurde damit auch die Generierung spontan schlagender Zellen aus den aortalen VW-SCs unterbunden. Um zu testen, ob und inwieweit dieser Einfluss der Makrophagen auf die Entstehung spontan schlagender Zellen aus den VW-SCs auf den VEGF zurückzuführen ist, wurden kultivierte Zellen der Mausaorta mit dem VEGF-Rezeptor-2-Blocker (E7080) behandelt. Auch diese Behandlung resultierte wie bei der Depletion von Makrophagen darin, dass keine spontan schlagenden Zellen entstanden. Um die von VW-SCs generierten spontan schlagenden Zellen funktionell zu charakterisieren, wurden die kultivierten Zellen der Mausaorta mit Isoproterenol (ß-Sympathomimetikum) und Propranolol (ß-Blocker) behandelt. Eine signifikante Steigerung der Schlagfrequenz unter Isoproterenol und eine Reduzierung bei Zugabe von Propranolol unterstreichen ebenfalls die Kardiomyozyten-ähnliche Eigenschaft der spontan schlagenden Zellen. Schließlich wurden die aus der Mausaorta isolierten Zellen Fluoreszenz-markiert und dann in das kardiale Feld des sich entwickelnden Hühnerembryos (am fünften Tag der Entwicklung) implantiert. Zwei Tage später wurden die Herzen entnommen. Immunfärbungen zeigten, dass ein Teil der implantierten Zellen auch unter diesen in vivo-Bedingungen für α-sarkomeres Actinin positiv wurde und somit einen kardiomyozytären Phänotyp aufwies. KW - vessel wall resident stem cells KW - cardiomyocytes KW - Herzmuskelzelle KW - Stammzelle Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146046 N1 - My PhD research work has been published in Circ Res. 2018 Aug 31;123(6):686-699. ER - TY - THES A1 - Hieke, Marie T1 - Synaptic arrangements and potential communication partners of \(Drosophila’s\) PDF-containing clock neurons within the accessory medulla T1 - Synaptische Konstellationen und potentielle Kommunikationspartner von \(Drosophila’s\) PDF-enthaltenden Uhrneuronen innerhalb der akzessorischen Medulla N2 - Endogenous clocks regulate physiological as well as behavioral rhythms within all organisms. They are well investigated in D. melanogaster on a molecular as well as anatomical level. The neuronal clock network within the brain represents the center for rhythmic activity control. One neuronal clock subgroup, the pigment dispersing factor (PDF) neurons, stands out for its importance in regulating rhythmic behavior. These neurons express the neuropeptide PDF (pigment dispersing factor). A small neuropil at the medulla’s edge, the accessory medulla (AME), is of special interest, as it has been determined as the main center for clock control. It is not only highly innervated by the PDF neurons but also by terminals of all other clock neuron subgroups. Furthermore, terminals of the photoreceptors provide light information to the AME. Many different types of neurons converge within the AME and afterward spread to their next target. Thereby the AME is supplied with information from a variety of brain regions. Among these neurons are the aminergic ones whose receptors’ are expressed in the PDF neurons. The present study sheds light onto putative synaptic partners and anatomical arrangements within the neuronal clock network, especially within the AME, as such knowledge is a prerequisite to understand circadian behavior. The aminergic neurons’ conspicuous vicinity to the PDF neurons suggests synaptic communication among them. Thus, based on former anatomical studies regarding this issue detailed light microscopic studies have been performed. Double immunolabellings, analyses of the spatial relation of pre- and postsynaptic sites of the individual neuron populations with respect to each other and the identification of putative synaptic partners using GRASP reenforce the hypothesis of synaptic interactions within the AME between dopaminergic/ serotonergic neurons and the PDF neurons. To shed light on the synaptic partners I performed first steps in array tomography, as it allows terrific informative analyses of fluorescent signals on an ultrastructural level. Therefore, I tested different ways of sample preparation in order to achieve and optimize fluorescent signals on 100 nm thin tissue sections and I made overlays with electron microscopic images. Furthermore, I made assumptions about synaptic modulations within the neuronal clock network via glial cells. I detected their cell bodies in close vicinity to the AME and PDFcontaining clock neurons. It has already been shown that glial cells modulate the release of PDF from s-LNvs’ terminals within the dorsal brain. On an anatomical level this modulation appears to exist also within the AME, as synaptic contacts that involve PDF-positive dendritic terminals are embedded into glial fibers. Intriguingly, these postsynaptic PDF fibers are often VIIAbstract part of dyadic or even multiple-contact sites in opposite to prolonged presynaptic active zonesimplicating complex neuronal interactions within the AME. To unravel possible mechanisms of such synaptic arrangements, I tried to localize the ABC transporter White. Its presence within glial cells would indicate a recycling mechanism of transmitted amines which allows their fast re-provision. Taken together, synapses accompanied by glial cells appear to be a common arrangement within the AME to regulate circadian behavior. The complexity of mechanisms that contribute in modulation of circadian information is reflected by the complex diversity of synaptic arrangements that involves obviously several types of neuron populations N2 - Endogene Uhren steuern sowohl physiologische als auch verhaltensbedingte Rhythmen bei allen Organismen. In D. melanogaster sind sie nicht nur auf molekularer sondern auch auf anatomischer Ebene bereits gut erforscht. Das neuronale Uhrnetzwerk im Gehirn stellt das Zentrum der Steuerung der rhythmischen Aktivität dar. Eine Uhrneuronengruppe sticht allein schon durch ihre besonderen anatomischen Eigenschaften hervor. Diese Neurone exprimieren das Neuropeptid PDF (pigment dispersing factor), welches zudem besonderen Einfluss auf die Lokomotionsaktivität der Fliege hat. Ein kleines Neuropil am Rande der Medulla, die akzessorische Medulla (AME) ist von besonderem Interesse, da neben seiner intensiven Innervation durch die PDF-Neurone auch Terminale aller anderen Uhrneuronengruppen zu finden sind. Zudem wird sie durch Terminale der Photorezeptoren mit Informatonen über die Lichtverhätnisse versorgt. Die AME erreichen des Weiteren Informationen aus vielen anderen Hirnregionen. Eine Vielzahl von Neuronentypen laufen in ihr zusammen, um sich anschließend wieder in verschiedenste Hirnareale zu verteilen. So wird die AME auch durchzogen von Fasern mit aminergem Inhalt, dessen Rezeptoren wiederum auf den PDF-Neuronen zu finden sind. Die vorliegende Arbeit gibt Aufschluss über vermutliche synaptische Partner und anatomische Anordnungen innerhalb des neuronalen Uhrnetzwerkes, insbesondere innerhalb der AME. Solch Wissen stellt eine Grundvoraussetzung dar, um zirkadianes Verhalten verstehen zu können. Die auffällige Nähe der aminergen Neurone zu den PDF Neuronen lässt eine synaptische Interaktion zwischen ihnen vermuten. Deshalb wurden basierend auf vorangegangen Studien detailiertere Untersuchungen dieser Thematik durchgeführt. So wird die Hypothese über synaptische Interaktionen innerhalb der AME zwischen dopaminergen/ serotonergen Neuronen und den PDF Neuronen bestärkt mittels Doppelimmunofärbungen, gegenüberstellende Analysen über die räumlichen Nähe von prä- und postsynaptischen Stellen der jeweiligen Neuronenpopulationen und durch die Identifikation vermutlicher synaptischer Partner unter Verwendung von GRASP. Zur möglichen Identifikation der synaptischen Partner unternahm ich erste Schritte in der Array Tomographie, welche hochinformative Analysen von fluoreszierenden Signalen auf einem ultrastrukturellen Level ermöglicht. Dazu testete ich verschieden Wege der Gewebepräparation, um Flureszenzsignale zu erhalten bzw. zu optimieren und bildete erste Überlagerungen der Fluoreszenz- und Elektronenmikrskopbilder. Die Auswertung der elektronenmikroskopischen Bilder erlaubten Mutmaßungen über mö- gliche synaptische Modulationen innerhalb des neuronalen Uhrnetzwerkes durch Gliazellen. Ihre Zellkörper fand ich in unmittelbarer Nähe zu den PDF Neuronen. Im dorsalen Hirn wurden neuronale Modulationen an den kleinen PDF Neuronen durch Gliazellen bereits festgestellt. Auf anatomischer Ebene scheint diese Modulation auch innerhalb der AME zu erfolgen, da synaptische Kontakte, welche PDF-positive Dendriten involvieren, von Gliafasern umgeben sind. Interessanterweise sind diese postsynaptischen PDF Fasern dabei oftmals Teil dyadischer oder sogar multipler Kontakte, die sich gegenüber einer ausgedehnten aktiven Zone befinden. Um mögliche Mechanismen solcher synaptischer Anordnungen zu erklären, versuchte ich den ABC Transporter White im Hirn von Drosophila zu lokalisieren. Seine Präsenz in Gliazellen würde auf einen Recyclingmechanismus hindeuten, welcher eine schnelle Wiederbereitstellung des Transmiters ermöglichen würde. Zusammengefasst scheinen Synapsen mit postsynaptischen PDF-Neuronen in Begleitung von Gliazellen, ein gebräuchliches synaptisches Arrangement innerhalb der AME dazustellen. Diese komplexe Diversität der synaptischen Anordnung reflektiert die komplexen Mechanismen, welche der Verarbeitung der zirkadianen Informationen zugrunde liegen KW - Taufliege KW - Chronobiologie KW - Endogene Rhythmik KW - PDF neurons KW - glia cells KW - circadian clock KW - accessory medulla KW - sleep KW - aminergic neurons KW - synapses KW - Gliazelle KW - Aminerge Nervenzelle KW - Pigmentdispergierender Faktor KW - Drosophila melanogaster Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175988 ER - TY - THES A1 - Horn, Jessica T1 - Molecular and functional characterization of the long non-coding RNA SSR42 in \(Staphylococcus\) \(aureus\) T1 - Molekulare und funktionelle Charakterisierung der langen nicht-kodierenden RNA SSR42 in \(Staphylococcus\) \(aureus\) N2 - Staphylococcus aureus asymptomatically colonizes the skin and anterior nares of 20-30% of the healthy human population. As an opportunistic human pathogen it elicits a variety of infections ranging from skin and soft tissue infections to highly severe manifestations such as pneumonia, endocarditis and osteomyelitis. Due to the emergence of multi resistant strains, treatment of staphylococcal infections becomes more and more challenging and the WHO therefore classified S. aureus as a “superbug”. The variety of diseases triggered by S. aureus is the result of a versatile expression of a large set of virulence factors. The most prominent virulence factor is the cytotoxic and haemolytic pore-forming α-toxin whose expression is mediated by a complex regulatory network involving two-component systems such as the agr quorum-sensing system, accessory transcriptional regulators and alternative sigma-factors. However, the intricate regulatory network is not yet understood in its entirety. Recently, a transposon mutation screen identified the AraC-family transcriptional regulator ‘Repressor of surface proteins’ (Rsp) to regulate haemolysis, cytotoxicity and the expression of various virulence associated factors. Deletion of rsp was accompanied by a complete loss of transcription of a 1232 nt long non-coding RNA, SSR42. This doctoral thesis focuses on the molecular and functional characterization of SSR42. By analysing the transcriptome and proteome of mutants in either SSR42 or both SSR42 and rsp, as well as by complementation of SSR42 in trans, the ncRNA was identified as the main effector of Rsp-mediated virulence. Mutants in SSR42 exhibited strong effects on transcriptional and translational level when compared to wild-type bacteria. These changes resulted in phenotypic alterations such as strongly reduced haemolytic activity and cytotoxicity towards epithelial cells as well as reduced virulence in a murine infection model. Deletion of SSR42 further promoted the formation of small colony variants (SCV) during long term infection of endothelial cells and demonstrated the importance of this molecule for intracellular bacteria. The impact of this ncRNA on staphylococcal haemolysis was revealed to be executed by modulation of sae mRNA stability and by applying mutational studies functional domains within SSR42 were identified. Moreover, various stressors modulated the transcription of SSR42 and antibiotic challenge resulted in SSR42-dependently increased haemolysis and cytotoxicity. Transcription of SSR42 itself was found under control of various important global regulators including AgrA, SaeS, CodY and σB, thereby illustrating a central position in S. aureus virulence gene regulation. The present study thus demonstrates SSR42 as a global virulence regulatory RNA which is important for haemolysis, disease progression and adaption of S. aureus to intracellular conditions via formation of SCVs. N2 - Staphylococcus aureus kolonisiert asymptomatisch als Kommensal die Haut und Nasenschleimhäute von circa 20-30% der gesunden Weltbevölkerung. Als opportunistisches Humanpathogen löst S. aureus dagegen eine Reihe von Krankheiten aus, die von leichten Hautinfektionen und Abszessen bis hin zu schwerwiegenden und lebensbedrohlichen Krankheitsformen wie Pneumonie, Endokarditis und Osteomyelitis reichen können. Die Behandlung von Staphylokokken-Infektionen stellt aufgrund der Entstehung multi-resistenter Stämme vermehrt eine Herausforderung dar, weshalb S. aureus von der WHO als „superbug“ klassifiziert wurde. Die Vielzahl an möglichen Krankheitsformen sind das Ergebnis der anpassungsfähigen und koordinierten Expression einer Vielzahl von Virulenzfaktoren. Der dabei wohl bedeutendste und am besten charakterisierte Virulenzfaktor ist das porenbildende α-toxin, dessen zytotoxische und hämolytische Aktivität für eine Reihe diverser Krankheiten verantwortlich ist. Die Expression dieses Toxins wird durch ein komplexes, bis jetzt noch nicht komplett verstandenes, regulatorisches Netzwerk gesteuert, das sowohl Zwei-Komponentensysteme wie das agr Quorum-sensing System, diverse akzessorische transkriptionelle Regulatoren sowie alternative Sigmafaktoren beinhaltet. Kürzlich wurde in einem Transposon-Mutanten-Screen der AraC-Familie transkriptionelle Regulator „Repressor of surface proteins” (Rsp) identifiziert, der die Expression diverser Virulenz-assoziierter Faktoren beeinflusste. Eine Deletion von rsp ging, neben reduzierter Hämolyse und Zytotoxizität, auch mit dem kompletten Verlust der Transkription einer 1232 nt langen nicht-kodierenden RNA, SSR42, einher. Diese Doktorarbeit befasst sich mit der molekularen und funktionellen Charakterisierung dieser nicht-kodierenden RNA. Mittels Transkriptom- und Proteomanalysen wurden eine SSR42 Deletionsmutante sowie eine Doppelmutante in SSR42 und rsp charakterisiert und SSR42 als Hauptfaktor der Rsp-vermittelten Virulenzregulation identifiziert. Neben weitreichenden Veränderungen auf trans-kriptioneller und translationaler Ebene wiesen Mutanten in SSR42 eine stark reduzierte hämolytische und zytotoxische Aktivität sowie verringerte Virulenz in einem murinen Infektionsmodell auf. Eine Deletion von SSR42 begünstigte weiterhin die Bildung von sog. „small colony variants“ während Langzeit-Infektionen von Endothelzellen und demonstrierte die Bedeutung dieser nicht-codierenden RNA für intrazelluläre Staphylokokken. Die regulatorische Wirkung von SSR42 auf die hämolytische Aktivität von S. aureus wurde in dieser Arbeit aufgeklärt. Dabei konnte ein stabilisierender Einfluss der nicht-kodierenden RNA auf sae mRNA nachgewiesen werden. Weiterhin wurde SSR42 durch Mutagenese-Studien auf molekularer Ebene charakterisiert, wobei funktionelle und stabilisierende Domänen identifiziert wurden. Ebenso wurden in dieser Arbeit diverse Stressoren und Antibiotika erfasst, die eine modulatorische Wirkung auf die Transkription von SSR42 ausüben. Neben einer Erhöhung der Transkription von SSR42 resultierte eine Behandlung von S. aureus mit sub-inhibitorischen Konzentrationen von Antibiotika in einer drastischen, SSR42-abhängigen, Steigerung der hämolytischen und zytotoxischen Aktivität. Mithilfe von Promotoraktivitätsstudien wurde der Einfluss diverser Regulatoren wie AgrA, SaeS, CodY und σB auf die transkriptionellen Regulation von SSR42 identifiziert und SSR42 somit eine zentrale Rolle in der Regulation von Virulenzgenen verliehen. SSR42 wurde demnach als ein neuartiger globaler Regulator identifiziert, der eine wichtige Rolle für Hämolyse, den Krankheitsverlauf sowie bei der Anpassung an intrazelluläre Bedingungen, über die Bildung von „small colony variants“, spielt. KW - Staphylococcus aureus KW - Non-coding RNA KW - SSR42 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175778 ER - TY - THES A1 - Schubert, Frank Klaus T1 - The circadian clock network of \(Drosophila\) \(melanogaster\) T1 - Das Uhrneuronennetzwerk von \(Drosophila\) \(melanogaster\) N2 - All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism. The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons. To further study the functional hierarchy within the clock network and to monitor the “ticking clock“ over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells. N2 - Alle lebenden Organismen benötigen Mechanismen zur Zeitmessung, um sich auf periodisch wiederkehrende Umweltveränderungen einstellen zu können. Zirkadiane Uhren verleihen die Fähigkeit, tages- und jahreszeitliche Veränderungen vorauszuahnen und sich an diese anzupassen. Die Eigenschaften des zirkadianen Systems, als auch dessen molekularer Mechanismus scheinen über sämtliche Taxa konserviert zu sein. Daher bietet es sich an, die leicht handhabbare Taufliege Drosophila melanogaster als Modellorganismus zu benutzen. Das relativ kleine Gehirn (ca. 135.000 Neurone) und die herausragende genetische Zugänglichkeit der Fliege prädestinieren sie dazu, das zirkadiane System in einem komplexen, aber dennoch überschaubaren Kontext zu untersuchen. Die vergangenen 50 Jahre chronobiologischer Forschung an Drosophila führten zu einem tiefgreifenden Verständnis der molekularen Mechanismen, die für tageszeitliche Rhythmizität verantwortlich sind. Anhand zahlreicher histologischer Untersuchungen wurde die neuronale Grundlage, das Uhrneuronennetzwerk im zentralen Nervensystem, beschrieben. Nichtsdestotrotz, gibt es noch immer keine detaillierte neuroanatomische und physiologische Charakterisierung der Uhrneurone auf Einzelzellebene. Daher war das Ziel der vorliegenden Arbeit die umfangreiche Beschreibung der Einzelzellanatomie ausgewählter Uhrneurone sowie die Identifikation mutmaßlicher post- und präsynaptischer Verzweigungen. Darüber hinaus war es mir möglich, eine Methode zur Messung von Biolumineszenzrhythmen in explantierten lebenden Gehirnen zu etablieren. Mit einem Lumineszenzmikroskop können die Proteinoszillationen einzelner Uhrneurone über die Dauer mehrerer zirkadianer Zyklen aufgezeichnet werden, wodurch neue funktionale Studien ermöglicht werden. KW - Taufliege KW - Chronobiologie KW - Tagesrhythmus KW - Neuroanatomie KW - Drosophila melanogaster KW - circadian rhythms KW - single cell anatomy Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157136 ER -