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 - Yanku, Yifat A1 - Bitman-Lotan, Eliya A1 - Zohar, Yaniv A1 - Kurant, Estee A1 - Zilke, Norman A1 - Eilers, Martin A1 - Orian, Amir T1 - Drosophila HUWE1 ubiquitin ligase regulates endoreplication and antagonizes JNK signaling during salivary gland development JF - Cells N2 - The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development. KW - HECT KW - HUWE1 KW - ubiquitin KW - salivary gland KW - endoreplication KW - JNK KW - dMyc KW - dmP53 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197630 SN - 2073-4409 VL - 7 IS - 10 ER - TY - JOUR A1 - Diao, Wenwen A1 - Mousset, Mathilde A1 - Horsburgh, Gavin J. A1 - Vermeulen, Cornelis J. A1 - Johannes, Frank A1 - van de Zande, Louis A1 - Ritchie, Michael G. A1 - Schmitt, Thomas A1 - Beukeboom, Leo W. T1 - Quantitative Trait Locus Analysis of Mating Behavior and Male Sex Pheromones in Nasonia Wasps JF - G3: Genes Genomes Genetics N2 - A major focus in speciation genetics is to identify the chromosomal regions and genes that reduce hybridization and gene flow. We investigated the genetic architecture of mating behavior in the parasitoid wasp species pair Nasonia giraulti and Nasonia oneida that exhibit strong prezygotic isolation. Behavioral analysis showed that N. oneida females had consistently higher latency times, and broke off the mating sequence more often in the mounting stage when confronted with N. giraulti males compared with males of their own species. N. oneida males produce a lower quantity of the long-range male sex pheromone (4R,5S)-5-hydroxy-4-decanolide (RS-HDL). Crosses between the two species yielded hybrid males with various pheromone quantities, and these males were used in mating trials with females of either species to measure female mate discrimination rates. A quantitative trait locus (QTL) analysis involving 475 recombinant hybrid males (F2), 2148 reciprocally backcrossed females (F3), and a linkage map of 52 equally spaced neutral single nucleotide polymorphism (SNP) markers plus SNPs in 40 candidate mating behavior genes revealed four QTL for male pheromone amount, depending on partner species. Our results demonstrate that the RS-HDL pheromone plays a role in the mating system of N. giraulti and N. oneida, but also that additional communication cues are involved in mate choice. No QTL were found for female mate discrimination, which points at a polygenic architecture of female choice with strong environmental influences. KW - Nasonia courtship KW - female choice KW - sex pheromone KW - QTL analysis KW - speciation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165412 VL - 6 IS - 6 ER - TY - JOUR A1 - Bencurova, Elena A1 - Gupta, Shishir K. A1 - Sarukhanyan, Edita A1 - Dandekar, Thomas T1 - Identification of antifungal targets based on computer modeling JF - Journal of Fungi N2 - Aspergillus fumigatus is a saprophytic, cosmopolitan fungus that attacks patients with a weak immune system. A rational solution against fungal infection aims to manipulate fungal metabolism or to block enzymes essential for Aspergillus survival. Here we discuss and compare different bioinformatics approaches to analyze possible targeting strategies on fungal-unique pathways. For instance, phylogenetic analysis reveals fungal targets, while domain analysis allows us to spot minor differences in protein composition between the host and fungi. Moreover, protein networks between host and fungi can be systematically compared by looking at orthologs and exploiting information from host–pathogen interaction databases. Further data—such as knowledge of a three-dimensional structure, gene expression data, or information from calculated metabolic fluxes—refine the search and rapidly put a focus on the best targets for antimycotics. We analyzed several of the best targets for application to structure-based drug design. Finally, we discuss general advantages and limitations in identification of unique fungal pathways and protein targets when applying bioinformatics tools. KW - Aspergillus KW - metabolic pathways KW - computational modelling KW - drug design Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197670 SN - 2309-608X VL - 4 IS - 3 ER - TY - JOUR A1 - Konte, Tilen A1 - Terpitz, Ulrich A1 - Plemenitaš, Ana T1 - Reconstruction of the High-Osmolarity Glycerol (HOG) Signaling Pathway from the Halophilic Fungus Wallemia ichthyophaga in Saccharomyces cerevisiae JF - Frontiers in Microbiology N2 - The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like other fungi, W. ichthyophaga detects changes in environmental salinity mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with protein modeling data, our study reveals conserved and non-conserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus. KW - signaling KW - protein-protein interaction KW - protein phosphorylation KW - mitogen activated protein kinase (MAPK) KW - high-osmolarity glycerol (HOG) KW - signaling pathway KW - Saccharomyces cerevisiae KW - halophilic fungus KW - Wallemia ichthyophaga Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165214 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 - Held, Martina A1 - Berz, Annuska A1 - Hensgen, Ronja A1 - Muenz, Thomas S. A1 - Scholl, Christina A1 - Rössler, Wolfgang A1 - Homberg, Uwe A1 - Pfeiffer, Keram T1 - Microglomerular Synaptic Complexes in the Sky-Compass Network of the Honeybee Connect Parallel Pathways from the Anterior Optic Tubercle to the Central Complex JF - Frontiers in Behavioral Neuroscience N2 - While the ability of honeybees to navigate relying on sky-compass information has been investigated in a large number of behavioral studies, the underlying neuronal system has so far received less attention. The sky-compass pathway has recently been described from its input region, the dorsal rim area (DRA) of the compound eye, to the anterior optic tubercle (AOTU). The aim of this study is to reveal the connection from the AOTU to the central complex (CX). For this purpose, we investigated the anatomy of large microglomerular synaptic complexes in the medial and lateral bulbs (MBUs/LBUs) of the lateral complex (LX). The synaptic complexes are formed by tubercle-lateral accessory lobe neuron 1 (TuLAL1) neurons of the AOTU and GABAergic tangential neurons of the central body’s (CB) lower division (TL neurons). Both TuLAL1 and TL neurons strongly resemble neurons forming these complexes in other insect species. We further investigated the ultrastructure of these synaptic complexes using transmission electron microscopy. We found that single large presynaptic terminals of TuLAL1 neurons enclose many small profiles (SPs) of TL neurons. The synaptic connections between these neurons are established by two types of synapses: divergent dyads and divergent tetrads. Our data support the assumption that these complexes are a highly conserved feature in the insect brain and play an important role in reliable signal transmission within the sky-compass pathway. KW - sky-compass orientation KW - insect brain KW - polarization vision KW - synaptic connections KW - anterior optic tubercle KW - central complex KW - honeybee Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165080 VL - 10 IS - 186 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 - Mena, Wilson A1 - Diegelmann, Sören A1 - Wegener, Christian A1 - Ewer, John T1 - Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators JF - eLife N2 - Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors. KW - neuropeptides Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165003 VL - 5 ER - TY - JOUR A1 - Drakulić, Sanja A1 - Feldhaar, Heike A1 - Lisičić, Duje A1 - Mioč, Mia A1 - Cizelj, Ivan A1 - Seiler, Michael A1 - Spatz, Theresa A1 - Rödel, Mark-Oliver T1 - Population-specific effects of developmental temperature on body condition and jumping performance of a widespread European frog JF - Ecology and Evolution N2 - All physiological processes of ectotherms depend on environmental temperature. Thus, adaptation of physiological mechanisms to the thermal environments is important for achieving optimal performance and fitness. The European Common Frog, Rana temporaria, is widely distributed across different thermal habitats. This makes it an exceptional model for studying the adaptations to different thermal conditions. We raised tadpoles from Germany and Croatia at two constant temperature treatments (15°C, 20°C), and under natural temperature fluctuations (in outdoor treatments), and tested how different developmental temperatures affected developmental traits, that is, length of larval development, morphometrics, and body condition, as well as jumping performance of metamorphs. Our results revealed population‐specific differences in developmental time, body condition, and jumping performance. Croatian frogs developed faster in all treatments, were heavier, in better body condition, and had longer hind limbs and better jumping abilities than German metamorphs. The populations further differed in thermal sensitivity of jumping performance. While metamorphs from Croatia increased their jumping performance with higher temperatures, German metamorphs reached their performance maximum at lower temperatures. These population‐specific differences in common environments indicate local genetic adaptation, with southern populations being better adapted to higher temperatures than those from north of the Alps. KW - Amphibians KW - ectotherms KW - physiological traits KW - plasticity KW - thermal adaptation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164960 VL - 6 IS - 10 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 - Jones, Julia C. A1 - Fruciano, Carmelo A1 - Keller, Anja A1 - Schartl, Manfred A1 - Meyer, Axel T1 - Evolution of the elaborate male intromittent organ of Xiphophorus fishes JF - Ecology and Evolution N2 - Internally fertilizing animals show a remarkable diversity in male genital morphology that is associated with sexual selection, and these traits are thought to be evolving particularly rapidly. Male fish in some internally fertilizing species have “gonopodia,” highly modified anal fins that are putatively important for sexual selection. However, our understanding of the evolution of genital diversity remains incomplete. Contrary to the prediction that male genital traits evolve more rapidly than other traits, here we show that gonopodial traits and other nongonopodial traits exhibit similar evolutionary rates of trait change and also follow similar evolutionary models in an iconic genus of poeciliid fish (Xiphophorus spp.). Furthermore, we find that both mating and nonmating natural selection mechanisms are unlikely to be driving the diverse Xiphophorus gonopodial morphology. Putative holdfast features of the male genital organ do not appear to be influenced by water flow, a candidate selective force in aquatic habitats. Additionally, interspecific divergence in gonopodial morphology is not significantly higher between sympatric species, than between allopatric species, suggesting that male genitals have not undergone reproductive character displacement. Slower rates of evolution in gonopodial traits compared with a subset of putatively sexually selected nongenital traits suggest that different selection mechanisms may be acting on the different trait types. Further investigations of this elaborate trait are imperative to determine whether it is ultimately an important driver of speciation. KW - Male intromittent organ KW - reproductive character displacement KW - sexual selection KW - species diversification KW - Xiphophorus fish Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164956 VL - 6 IS - 20 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 - Sarukhanyan, Edita A1 - Shityakov, Sergey A1 - Dandekar, Thomas T1 - Rational drug design of Axl tyrosine kinase type I inhibitors as promising candidates against cancer JF - Frontiers in Chemistry N2 - The high level of Axl tyrosine kinase expression in various cancer cell lines makes it an attractive target for the development of anti-cancer drugs. In this study, we carried out several sets of in silico screening for the ATP-competitive Axl kinase inhibitors based on different molecular docking protocols. The best drug-like candidates were identified, after parental structure modifications, by their highest affinity to the target protein. We found that our newly designed compound R5, a derivative of the R428 patented analog, is the most promising inhibitor of the Axl kinase according to the three molecular docking algorithms applied in the study. The molecular docking results are in agreement with the molecular dynamics simulations using the MM-PBSA/GBSA implicit solvation models, which confirm the high affinity of R5 toward the protein receptor. Additionally, the selectivity test against other kinases also reveals a high affinity of R5 toward ABL1 and Tyro3 kinases, emphasizing its promising potential for the treatment of malignant tumors. KW - Axl tyrosine kinase KW - anti-cancer drug-like molecules KW - rational drug design KW - molecular docking KW - molecular dynamics Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199505 SN - 2296-2646 VL - 7 IS - 920 ER - TY - THES A1 - Michel, Konstanze T1 - Die kardiale Bedeutung des Hormons C-Typ natriuretisches Peptid (CNP) und dessen Guanylylcyclase B (GC-B) Rezeptor T1 - The cardiac role of the C-type natriuretic peptide (CNP) and its guanylyl cyclase B (GC-B) receptor N2 - In der vorliegenden Dissertationsarbeit wurden die kardialen Effekte des C-Typ natriuretischen Peptids (CNP) an wildtypischen Mäusen (Studie 1) und an einem neuen genetischen Mausmodell, mit einer Kardiomyozyten-spezifischen Deletion des Guanylyl-Cyclase B (GC-B) Rezeptors (Studie 2) untersucht. In Studie 1 wurden die Wirkungen von exogenem, synthetischem CNP auf eine durch Druckbelastung-induzierte Herzinsuffizienz in wildtypischen Mäusen (C57Bl6 Hintergrund) untersucht. Dafür wurde CNP parallel zu einer operativen transversen Aortenkonstriktion (TAC) über osmotische Minipumpen in einer Dosierung von 50 ng/kg/min über 14 Tage appliziert. Die 14 Tage TAC führten zu einer ausgeprägten Linksherzhypertrophie. Diese wurde durch exogenes CNP auf zellulärer (verringerte Kardiomyozytenflächen) und molekularer (verringerte BNP mRNA Expression) Ebene signifikant gehemmt. Auch die durch TAC-induzierte linksventrikuläre Dilatation wurde durch exogenes CNP fast vollständig verhindert. Diese kardialen protektiven Effekte von CNP traten ohne eine wesentliche Veränderung des arteriellen Blutdrucks auf. Mögliche mechanistische Ursachen für die schützende Wirkung von CNP könnte die PKG-abhängige Phosphorylierung des sarkomerischen Proteins Titin sein. Eine gesteigerte Phosphorylierung von Titin an der elastischen N2B-Domäne verringert die Steifigkeit der Kardiomyozyten und verbessert somit deren Relaxationsfähigkeit (Hudson 2011). Die erhöhten linksventrikulären Volumina nach TAC (end-diastolische und end-systolische Volumina) wurden möglicherweise durch eine erhöhte Steifigkeit der Kardiomyozyten provoziert. Dies könnte durch den akuten IL-6 mRNA Anstieg nach TAC begünstigt werden, da Kruger et al. einen Zusammenhang zwischen passiver Steifigkeit der Kardiomyozyten und IL-6-Expression postulierten (Kotter 2016, Kruger 2009). Diese Veränderungen wurden durch exogenes CNP verhindert. Es ist wahrscheinlich, dass die CNP-induzierte Phosphorylierung von Titin an Serin 4080 in die Relaxationsfähigkeit der Kardiomyozyten und somit die diastolische Funktion des linken Ventrikels verbesserte. Aufgrund dieser Beobachtungen wurde in Studie 2 untersucht, ob auch endogenes CNP als parakrines Hormon im Herzen eine TAC-induzierte Herzhypertrophie und die kontraktile Funktion von Kardiomyozyten bei einer hypertensiven Herzerkrankung beeinflussen kann. Dafür wurde ein neues genetisches Mausmodell mit einer Kardiomyozyten-spezifischen Deletion des GC-B Rezeptors generiert (CM GC-B KO). Da vorangegangene Studien in unserer Arbeitsgruppe zeigten, dass die basale CNP-Expression im Herzen sehr gering ist, nach 3-tägiger TAC aber akut ansteigt und nach 14-tägiger TAC wieder abfällt, haben wir CM GC-B KO Mäuse und deren Geschwister-Kontrolltiere an beiden Zeitpunkten nach TAC untersucht. Die TAC führte Genotyp-unabhängig zu einem Anstieg der kardialen Nachlast nach 3 Tagen und weiter nach 14 Tagen. Diese Druckbelastung provozierte eine progressive, signifikante Linksherzhypertrophie. Allerdings reagierten die CM GC-B KO Mäuse im Vergleich zu den Kontrolltieren bereits nach 3-tägiger TAC mit einer ausgeprägten Kardiomyozyten-Hypertrophie. Zudem beobachteten wir nach 3-tägiger TAC in den Knockout-Mäusen eine Abnahme der Ejektionsfraktion und gleichzeitig eine signifikante Zunahme der beiden linksventrikulären Volumina (end-diastolische und end-systolische Volumen). Diese frühe linksventrikuläre Dilatation wurde in den Kontrolltieren nicht beobachtet. Daraus schlussfolgerten wir, dass endogenes kardiales CNP, dessen Expression zu frühen Zeitpunkten nach Druckbelastung ansteigt, das Herz vor kontraktiler Dysfunktion und Dilatation schützen kann. Um mögliche Mechanismen für die protektive Wirkung von endogenem CNP zu erklären, untersuchten wir die IL-6 mRNA Expression sowie die Titin-Phosphorylierung im Herzen. Der akute Anstieg der IL-6 mRNA Expression nach 3-tägiger TAC in den CM GC-B KO Mäusen korreliert mit der verminderten Phosphorylierung von Titin an der PGK-spezifischen Phosphorylierungsstelle (Serin 4080). Somit könnte der CNP/GC-B/cGMP-Signalweg zu einer Inhibition pro-inflammatorischer Gene beitragen, da der akute IL-6 mRNA Anstieg in den Kontrollen nicht beobachtet wurde. Auch die gesteigerte NOX4 Expression 3 Tage nach TAC, könnte zu der frühen dilatativen Kardiomyopathie in den Knockout-Mäusen beigetragen haben. Die verringerte STAT3 Aktivierung in den CM GC-B KO Mäusen würde laut Literatur zu vermehrter Apoptose führen, indem pro-apoptotische Gene wie Bcl oder Bax vermehrt transkribiert werden. Auch die erhöhte Cxcl-1 mRNA Expression in den Knockout-Mäusen deutet zusammen mit dem IL-6 Anstieg auf vermehrte Entzündungsreaktionen 3 Tage nach TAC hin. Zusammengenommen deuten die Ergebnisse dieser Dissertationsarbeit darauf hin, dass der CNP/GC-B/cGMP-Signalweg in frühen Stadien einer erhöhten kardialen Druckbelastung und der Entstehung einer dilatativen Kardiomyopathie entgegenwirken kann. Die Phosphorylierung des sarkomerischen Proteins Titin und die Hemmung der Expression pro-inflammatorischer Zytokine (speziell IL-6) könnten zu diesem protektiven Effekt beitragen. N2 - Here we investigated cardiac effects of c-type natriuretic peptide (CNP) in wildtype mice (study 1) and in a newly generated mouse model with cardiomyocyte-specific deletion of the guanylyl cyclase B (GC-B) receptor (study 2). In study 1 the effects of exogenous, synthetic CNP were investigated in wildtype mice (C57Bl6 background) after heart failure induced by pressure overload. Therefore, additionally to transverse aortic constriction (TAC) CNP (50 ng/kg/min) was administered via osmotic minipumps for 14 days. TAC provoked progressive heart hypertrophy. The TAC-induced hypertrophy was accompanied by enlarged cardiomyocyte areas and increased left ventricular mRNA levels of BNP. CNP significantly reduced cardiomyocyte areas and mRNA levels of BNP. Also, the TAC-induced left ventricular dilatation was almost prevented through synthetic CNP. These cardiac protective effects of CNP were accompanied by increased phosphorylation of the sarcomeric protein titin. Titin plays a critical role in maintaining the sarcomere structure and elasticity and thereby influences myocyte contraction and especially relaxation. The left ventricular end-diastolic and end-systolic volumes were significantly increased after TAC. The increased left-ventricular (end-diastolic and end-systolic) volumes after TAC are maybe provoked by an increased cardiomyocyte stiffness. The acute rise in IL-6 mRNA levels after TAC may favour these change, since Kruger et al postulate a relationship between passive cardiomyocyte stiffness and IL-6 expression. These changes were prevented by exogeneous CNP. Presumably, the CNP induced phosphorylation of titin on Serin 4080 within its elastic domain improves the cardiomyocyte relaxation and therefore the left-ventricular diastolic function. Because of these observations, we investigated in study 2, whether endogenous CNP as a paracrine hormone can affect TAC-induced hypertrophy and contractile function of cardiomyocytes. Therefore, we generated a new genetic mouse model with a cardiomyocyte specific deletion of the GC-B receptor (CM GC-B KO). Since previous studies in our group demonstrated that cardiac baseline expression levels of CNP are very low, but they markedly increased 3 days after TAC and decline to baseline levels 14 days after TAC, CM GC-B KO mice and control littermates were investigated at both timepoints after TAC. TAC resulted in increased cardiac afterload after 3 days, and more after 2 weeks, which was similar in both genotypes. Pressure overload provoked a progressive and significant cardiac hypertrophy, shown by the increased heart weight/body weight ratios, and were confirmed by histology. More severe myocyte hypertrophy was shown 3 days after TAC in CM GC-B KO mice, compared with control mice. Left ventricular contractile functions were also investigated by invasive catheterization in anesthetized mice. Notably, while in control mice subjected to TAC the LV ejection fraction was only mildly decreased, the KO mice reacted with a very marked and early decrease of the ejection fraction at three days after TAC, which tend to improve later on. Even more, the left ventricular end-diastolic and end-systolic volumes were markedly increased in the KO mice 3 days after TAC, indicating left ventricular dilatation. Again, this acute dilatation seems to reverse or improve at later stages. Such changes were not observed in control mice. To clarify possible downstream signalling pathways mediating these protective effects of CNP, we investigated titin-phosphorylation and IL-6 expression after 3 days of TAC. The acute increased IL-6 mRNA expression correlates well with the diminished phosphorylation of titin (serin 4080) in CM GC-B KO mice. Also, the increased NOX4 expression 3 days after TAC, could be one of the reasons of the early dilatation in KO mice. The diminished phosphorylation of STAT3 in CM GC-B KO mice may induce apoptosis by transcription of Bcl or Bax genes. Also, the increased mRNA expression of Cxcl-1 and IL-6 after 3 days of TAC can lead to more inflammation the hearts of the KO mice. In summary, these results show, that the CNP/GC-B/cGMP pathway can prevent cardiac dilatation in early stages. The phosphorylation of titin and the inhibition of the pro-inflammatory cytokines can contribute to this protective effects of CNP in the heart. KW - Herzinsuffizienz KW - Peptide KW - Kardiomyopathie KW - C-Typ natriuretisches Peptid KW - Guanylylcyclase B Rezeptor KW - kardiale Hypertrophie KW - c-type natriuretic peptide KW - guanylyl cyclase B receptor KW - cardiac hypertrophy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200211 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 - Baur, Florentin A1 - Nietzer, Sarah L. A1 - Kunz, Meik A1 - Saal, Fabian A1 - Jeromin, Julian A1 - Matschos, Stephanie A1 - Linnebacher, Michael A1 - Walles, Heike A1 - Dandekar, Thomas A1 - Dandekar, Gudrun T1 - Connecting cancer pathways to tumor engines: a stratification tool for colorectal cancer combining human in vitro tissue models with boolean in silico models JF - Cancers N2 - To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification. KW - in silico simulation KW - 3D tissue models KW - colorectal cancer KW - BRAF mutation KW - targeted therapy KW - stratification Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193798 SN - 2072-6694 VL - 12 IS - 1 ER - TY - JOUR A1 - Gatto, Francesco A1 - Schulze, Almut A1 - Nielsen, Jens T1 - Systematic Analysis Reveals that Cancer Mutations Converge on Deregulated Metabolism of Arachidonate and Xenobiotics JF - Cell Reports N2 - Mutations are the basis of the clonal evolution of most cancers. Nevertheless, a systematic analysis of whether mutations are selected in cancer because they lead to the deregulation of specific biological processes independent of the type of cancer is still lacking. In this study, we correlated the genome and transcriptome of 1,082 tumors. We found that nine commonly mutated genes correlated with substantial changes in gene expression, which primarily converged on metabolism. Further network analyses circumscribed the convergence to a network of reactions, termed AraX, that involves the glutathione- and oxygen-mediated metabolism of arachidonic acid and xenobiotics. In an independent cohort of 4,462 samples, all nine mutated genes were consistently correlated with the deregulation of AraX. Among all of the metabolic pathways, AraX deregulation represented the strongest predictor of patient survival. These findings suggest that oncogenic mutations drive a selection process that converges on the deregulation of the AraX network. KW - Cancer genetics KW - Genetics research Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164814 VL - 16 IS - 3 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 - Klepsch, Maximilian Andreas T1 - Small RNA-binding complexes in Chlamydia trachomatis identified by Next-Generation Sequencing techniques T1 - Identifizierung von kleinen RNA-bindenden Komplexen in Chlamydia trachomatis mittels Hochdurchsatz- Sequenziertechniken N2 - Chlamydia infect millions worldwide and cause infertility and blinding trachoma. Chlamydia trachomatis (C. trachomatis) is an obligate intracellular gram-negative pathogen with a significantly reduced genome. This bacterium shares a unique biphasic lifecycle in which it alternates between the infectious, metabolically inert elementary bodies (EB) and the non-infections, metabolically active replicative reticular bodies (RB). One of the challenges of working with Chlamydia is its difficult genetic accessibility. In the present work, the high-throughput method TagRNA-seq was used to differentially label transcriptional start sites (TSS) and processing sites (PSS) to gain new insights into the transcriptional landscape of C. trachomatis in a coverage that has never been achieved before. Altogether, 679 TSSs and 1067 PSSs were detected indicating its high transcriptional activity and the need for transcriptional regulation. Furthermore, the analysis of the data revealed potentially new non-coding ribonucleic acids (ncRNA) and a map of transcriptional processing events. Using the upstream sequences, the previously identified σ66 binding motif was detected. In addition, Grad-seq for C. trachomatis was established to obtain a global interactome of the RNAs and proteins of this intracellular organism. The Grad-Seq data suggest that many of the newly annotated RNAs from the TagRNA-seq approach are present in complexes. Although Chlamydia lack the known RNA-binding proteins (RBPs), e.g. Hfq and ProQ, observations in this work reveal the presence of a previously unknown RBP. Interestingly, in the gradient analysis it was found that the σ66 factor forms a complex with the RNA polymerase (RNAP). On the other hand, the σ28 factor is unbound. This is in line with results from previous studies showing that most of the genes are under control of σ66. The ncRNA IhtA is known to function via direct base pairing to its target RNA of HctB, and by doing so is influencing the chromatin condensation in Chlamydia. This study confirmed that lhtA is in no complex. On the other hand, the ncRNA ctrR0332 was found to interact with the SNF2 protein ctl0077, a putative helicase. Both molecules co-sedimented in the gradient and were intact after an aptamer-based RNA pull-down. The SWI2/SNF2 class of proteins are nucleosome remodeling complexes. The prokaryotic RapA from E. coli functions as transcription regulator by stimulating the RNAP recycling. This view might imply that the small ncRNA (sRNA) ctrR0332 is part of the global regulation network in C. trachomatis controlling the transition between EBs and RBs via interaction with the SNF2 protein ctl0077. The present work is the first study describing a global interactome of RNAs and proteins in C. trachomatis providing the basis for future interaction studies in the field of this pathogen. N2 - Chlamydien verursachen jährlich Millionen Neuinfektionen weltweit und können zu Spätschäden wie Unfruchtbarkeit und Erblindung führen. Chlamydien sind obligat intrazelluläre, gram-negative Pathogene mit einem stark reduzierten Genom. Sie besitzen einen einzigartigen biphasischen Lebenszyklus, bei dem der Erreger zwischen den metabolisch inaktiven, infektiösen Elementarkörperchen (EBs) und den nicht infektiösen, metabolisch aktiven und replikativen Retikularkörperchen (RBs) alterniert. Eine Problemantik beim Arbeiten mit Chlamydien ist die Schwierigkeit der gezielten genetischen Manipulation des Pathogens. In der vorliegenden Arbeit wurde die Hochdurchsatz-Sequenziermethode TagRNA-Seq genutzt, um die transkriptionelle Organisation von Chlamydia trachomatis (C. trachomatis) zu analysieren und besser zu verstehen. Transkriptionelle Start Stellen (TSS) und Prozessierungsstellen (PSS) werden dabei unterschiedlich markiert, sodass eine zuverlässigere und genauere Auflösung erreicht wird als bisher durch in anderen Studien verwendete Methoden. Insgesamt konnten so 679 TSSs und 1067 PSSs detektiert werden. Es konnte gezeigt werden, dass das Transkriptom von C. trachomatis weitaus aktiver ist als bisher angenommen und eine Regulation auf transkriptioneller Ebene bedarf. Die Methode erlaubte zudem die Identifizierung von potenziell neuen nicht-kodierende RNAs sowie die Kartierung von transkriptionellen Prozessierungsereignissen. Unter Verwendung der 5’-upstreamliegenden Sequenzen konnte außerdem das in anderen Bakterien bereits bekannte σ66-Bindemotiv detektiert werden. In der vorliegenden Arbeit wurde zudem die Methode Grad-Seq in C. trachomatis etabliert, um ein globales Interaktom für RNAs (engl. ribonucleic acid) und Proteine des intrazellulären Organismus zu erstellen. Für viele der im TagRNA-Seq Ansatz identifizierten und neu annotierten RNAs konnte so eine Komplexbildung beobachtet werden. Dies deutet auf das Vorhandensein eines bislang unbekanntes RNA-Bindeprotein (RBP) hin, da Chlamydien keines der bekannten RBPs, z.B. Hfq oder ProQ, besitzen. Die Gradienten-Analyse ergab, dass der σ66-Faktor in einem Komplex mit der RNA-Polymerase (RNAP) vorliegt und dass der σ28-Faktor ungebunden ist. Diese Beobachtung entspricht den Ergebnissen vorheriger Studien, die zeigten das die meisten Gene durch σ66 kontrolliert werden. Die Daten bestätigen außerdem, dass IhtA, eine ncRNA (engl. non-coding ribonucleic acid), die über direkte Basenpaarbindung mit ihrem Ziel-RNA von hctB interagiert, nicht in einem Komplex vorliegt. Für die ncRNA ctrR0332 hingegen konnte das SNF2-Protein ctl0077 als Interaktionspartner identifiziert werden. Beide Moleküle co-sedimentieren im Gradienten und konnten mittels eines Aptamer-basierenden RNA Pull-Downs in intakter Form isoliert werden. Die Klasse der SWI2/SNF2-Proteine gehört zu den Nukleosomen-Remodeling-Komplexen. In Prokaryoten konnte für das in E. coli vorkommende RapA, welches ebenfalls zu den SWI2/SNF2-Proteinen zählt, die Funktion eines Transkriptionsregulators nachgewiesen werden, indem die RNAP-Wiederverwertung stimuliert wird. Dies könnte bedeuten, dass die ncRNA ctrR0332 ebenfalls Teil eines globalen Regulationsnetzwerks ist, welches durch Interaktion mit dem SNF2-Protein ctl0077 die Transition zwischen dem RB- und EB-Stadium reguliert. In der vorliegenden Arbeit konnte erstmals ein globales Interaktom von RNAs und Proteinen in C. trachomatis erstellt werden, welches als Grundlage für zukünftige Interaktionsstudien des Pathogens genutzt werden kann. KW - High throughput screening KW - Small RNA KW - Chlamydia trachomatis Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199741 ER - TY - THES A1 - Hartlieb, Heiko T1 - Functional analysis of Mushroom body miniature’s RGG-box and its role in neuroblast proliferation in Drosophila melanogaster T1 - Funktionelle Analyse der RGG-Box von Mushroom body miniature und deren Rolle in der Neuroblastenproliferation in Drosophila melanogaster N2 - Development of the central nervous system in Drosophila melanogaster relies on neural stem cells called neuroblasts. Neuroblasts divide asymmetrically to give rise to a new neuroblast as well as a small daughter cell which eventually generates neurons or glia cells. Between each division, neuroblasts have to re-grow to be able to divide again. In previous studies, it was shown that neuroblast proliferation, cell size and the number of progeny cells is negatively affected in larvae carrying a P-element induced disruption of the gene mushroom body miniature (mbm). This mbm null mutation called mbmSH1819 is homozygously lethal during pupation. It was furthermore shown that the nucleolar protein Mbm plays a role in the processing of ribosomal RNA (rRNA) as well as the translocation of ribosomal protein S6 (RpS6) in neuroblasts and that it is a transcriptional target of Myc. Therefore, it was suggested that Mbm might regulate neuroblast proliferation through a role in ribosome biogenesis. In the present study, it was attempted to further elucidate these proposed roles of Mbm and to identify the protein domains that are important for those functions. Mbm contains an arginine/glycine rich region in which a di-RG as well as a di-RGG motif could be found. Together, these two motifs were defined as Mbm’s RGG-box. RGG-boxes can be found in many proteins of different families and they can either promote or inhibit protein-RNA as well as protein-protein interactions. Therefore, Mbm’s RGG-box is a likely candidate for a domain involved in rRNA binding and RpS6 translocation. It could be shown by deletion of the RGG-box, that MbmdRGG is unable to fully rescue survivability and neuroblast cell size defects of the null mutation mbmSH1819. Furthermore, Mbm does indeed rely on its RGG-box for the binding of rRNA in vitro and in mbmdRGG as well as mbmSH1819 mutants RpS6 is partially delocalized. Mbm itself also seems to depend on the RGG-box for correct localization since MbmdRGG is partially delocalized to the nucleus. Interestingly, protein synthesis rates are increased in mbmdRGG mutants, possibly induced by an increase in TOR expression. Therefore, Mbm might possess a promoting function in TOR signaling in certain conditions, which is regulated by its RGG-box. Moreover, RGG-boxes often rely on methylation by protein arginine methyltransferases (in Drosophila: Darts – Drosophila arginine methyltransferases) to fulfill their functions. Mbm might be symmetrically dimethylated within its RGG-box, but the results are very equivocal. In any case, Dart1 and Dart5 do not seem to be capable of Mbm methylation. Additionally, Mbm contains two C2HC type zinc-finger motifs, which could be involved in rRNA binding. In an earlier study, it was shown that the mutation of the zinc-fingers, mbmZnF, does not lead to changes in neuroblast cell size, but that MbmZnF is delocalized to the cytoplasm. In the present study, mbmZnF mutants were included in most experiments. The results, however, are puzzling since mbmZnF mutant larvae exhibit an even lower viability than the mbm null mutants and MbmZnF shows stronger binding to rRNA than wild-type Mbm. This suggests an unspecific interaction of MbmZnF with either another protein, DNA or RNA, possibly leading to a dominant negative effect by disturbing other interaction partners. Therefore, it is difficult to draw conclusions about the zinc-fingers’ functions. In summary, this study provides further evidence that Mbm is involved in neuroblast proliferation as well as the regulation of ribosome biogenesis and that Mbm relies on its RGG-box to fulfill its functions. N2 - Die Entwicklung des zentralen Nervensystems von Drosophila melanogaster beruht auf neuronalen Stammzellen genannt Neuroblasten. Neuroblasten teilen sich asymmetrisch und bringen dabei sowohl einen neuen Neuroblasten als auch eine kleinere Tochterzelle hervor, die wiederum letztlich Neuronen oder Gliazellen generiert. Zwischen jeder Zellteilung müssen die Neuroblasten wieder auf ihre ursprüngliche Größe wachsen, sodass sie zur erneuten Teilung in der Lage sind. In vorhergehenden Studien konnte gezeigt werden, dass sowohl die Proliferation der Neuroblasten, deren Zellgröße als auch die Anzahl ihrer Tocherzellen reduziert ist in Larven, die eine P-Element-induzierte Unterbrechung des Gens mushroom body miniature (mbm) tragen. Diese mbm-Nullmutation, genannt mbmSH1819, ist homozygot letal während des Puppenstadiums. Es konnte außerdem gezeigt werden, dass das nucleoläre Protein Mbm eine Rolle in der Prozessierung ribosomaler RNA (rRNA), sowie der Translokation des ribosomalen Proteins S6 (RpS6) in Neuroblasten erfüllt und dass seine Transkription durch Myc reguliert wird. Daher wurde geschlussfolgert, dass Mbm die Proliferation von Neuroblasten durch eine Funktion in der Ribosomenbiogenese regulieren könnte. In der vorliegenden Studie wurde das Ziel verfolgt, weitere Hinweise auf diese möglichen Funktionen von Mbm zu finden und die Proteindomänen zu identifizieren, die dafür benötigt werden. Mbm beinhaltet einen Arginin/Glycin-reichen Abschnitt, der ein di-RG sowie ein di-RGG Motiv enthält. Diese beiden Motive wurden zusammen zu Mbms RGG-Box definiert. RGG-Boxen finden sich in vielen Proteinen verschiedener Familien und sie können sich sowohl verstärkend als auch inhibierend auf Protein-RNA- sowie Protein-Protein-Interaktionen auswirken. Somit stellt Mbms RGG-Box einen vielversprechenden Kandidaten dar für eine Proteindomäne, die in die rRNA-Bindung sowie die Translokation von RpS6 involviert ist. Es konnte gezeigt werden, dass Mbm mit deletierter RGG-Box (MbmdRGG) nicht in der Lage ist, die Überlebensfähigkeit und die Neuroblastengröße der Nullmutation mbmSH1819 vollständig zu retten. Des Weiteren benötigt Mbm die RGG-Box, um rRNA in vitro zu binden und in mbmdRGG sowie mbmSH1819 Mutanten konnte eine partielle Delokalisation von RpS6 beobachtet werden. Die korrekte Lokalisation von Mbm selbst scheint auch von der RGG-Box abzuhängen, da MbmdRGG teilweise in den Nukleus delokalisiert ist. Interessanterweise ist außerdem die Proteinsyntheserate in mbmdRGG Mutanten erhöht, was möglicherweise in einer Erhöhung der TOR-Expression begründet ist. Somit könnte Mbm unter bestimmten Bedingungen eine verstärkende Funktion im TOR-Signalweg erfüllen, die durch seine eigene RGG-Box reguliert wird. Des Weiteren sind RGG-Boxen hinsichtlich ihrer Funktion häufig von der Methylierung durch Protein-Arginin-Methyltransferasen (in Drosophila: Darts – Drosophila arginine methyltransferases) abhängig. Mbm könnte innerhalb seiner RGG-Box symmetrisch dimethyliert sein, allerdings sind die Ergebnisse in dieser Hinsicht sehr zweifelhaft. Jedenfalls scheinen Dart1 und Dart5 nicht imstande zu sein, Mbm zu methylieren. Außerdem beinhaltet Mbm zwei Zink-Finger-Motive des C2HC-Typs, die in die Bindung von rRNA involviert sein könnten. Eine vorhergehende Studie konnte zeigen, dass die Mutation der Zink-Finger, mbmZnF, zwar nicht zu einer Veränderung der Neuroblastengröße führt, allerdings, dass MbmZnF ins Zytoplasma delokalisiert vorliegt. In der vorliegenden Studie wurden die mbmZnF Mutanten in die meisten Experimente mit einbezogen. Allerdings sind die Ergebnisse rätselhaft, da mbmZnF-mutierte Larven sogar eine geringere Überlebensrate zeigen als die mbm Nullmutanten und da MbmZnF eine stärkere Bindungsaffinität zu rRNA zeigt als wildtypisches Mbm. Dies weist auf eine unspezifische Interaktion zwischen MbmZnF und einem anderen Protein, RNA oder DNA hin, was einen dominant-negativen Effekt auslösen könnte, indem andere Interaktionspartner gestört werden. Somit gestaltet es sich schwierig, Schlussfolgerungen zur Funktion der Zink-Finger zu ziehen. Zusammengefasst liefert die vorliegende Studie weitere Anhaltspunkte, dass Mbm in der Neuroblastenproliferation sowie der Regulation der Ribosomenbiogenese involviert ist und dass Mbm seine RGG-Box benötigt, um seine Funktionen zu erfüllen. KW - Taufliege KW - Neuroblast KW - Gehirn KW - Entwicklung KW - Drosophila melanogaster KW - brain development KW - neuroblast proliferation KW - mushroom body miniature KW - Gehirnentwicklung KW - Neuroblastenproliferation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199674 ER - TY - THES A1 - Kurz, Andreas T1 - Correlative live and fixed cell superresolution microscopy T1 - Korrelative hochauflösende Mikroskopie an lebenden und fixierten Zellen N2 - Over the last decade life sciences have made an enormous leap forward. The development of complex analytical instruments, in particular in fluorescence microscopy, has played a decisive role in this. Scientist can now rely on a wide range of imaging techniques that offer different advantages in terms of optical resolution, recording speed or living cell compatibility. With the help of these modern microscopy techniques, multi-protein complexes can be resolved, membrane receptors can be counted, cellular pathways analysed or the internalisation of receptors can be tracked. However, there is currently no universal technique for comprehensive experiment execution that includes dynamic process capture and super resolution imaging on the same target object. In this work, I built a microscope that combines two complementary imaging techniques and enables correlative experiments in living and fixed cells. With an image scanning based laser spot confocal microscope, fast dynamics in several colors with low photodamage of the cells can be recorded. This novel system also has an improved resolution of 170 nm and was thoroughly characterized in this work. The complementary technique is based on single molecule localization microscopy, which can achieve a structural resolution down to 20-30 nm. Furthermore I implemented a microfluidic pump that allows direct interaction with the sample placed on the microscope. Numerous processes such as living cell staining, living cell fixation, immunostaining and buffer exchange can be observed and performed directly on the same cell. Thus, dynamic processes of a cell can be frozen and the structures of interest can be stained and analysed with high-resolution microscopy. Furthermore, I have equipped the detection path of the single molecule technique with an adaptive optical element. With the help of a deformable mirror, imaging functions can be shaped and information on the 3D position of the individual molecules can be extracted. N2 - Im letzten Jahrzehnt hat der Bereich der Lebenswissenschaften einen enormen Sprung nach vorne gemacht. Maßgeblich dafür waren die Entwicklung von komplexen Analysegeräten insbesondere in der Fluoreszenz Mikroskopie. Die Anwender können nun auf eine Vielzahl von Bildgebungstechniken zurückgreifen die unterschiedliche Vorzüge hinsichtlich optischer Auflösung, Aufnahmegeschwindigkeit oder Lebend Zell Kompatibilität bieten. Mithilfe dieser modernen Mikroskopietechniken lassen sich beispielsweise Multiproteinkomplexe auflösen, Membranrezeptoren zählen, zelluläre Signalwege analysieren oder die Internalisierung von Rezeptoren verfolgen. Für eine umfassende Experimentdurchführung, die Erfassung dynamischer Prozesse sowie superhochauflösende Bildgebung an ein und demselben Zielobjekt beinhalten, gibt es derzeit keine einheitliche Technik. In dieser Arbeit habe ich ein Mikroskop aufgebaut, das zwei komplementäre Bildgebungstechniken vereint und korrelative Experimente von lebend zu fixierten Zellen ermöglicht. Mit einem Image Scanning basierten Konfokal Mikroskop können schnelle Dynamiken in mehreren Farben mit geringer Photoschädigung der Zellen aufgenommen werden. Dieses neuartige System weist zudem eine Auflösungsverbesserung von 170 nm auf und wurde im Rahmen der Arbeit ausführlich charakterisiert. Die komplementäre Technik basiert auf der Einzel-Molekül Lokalisations Mikroskopie, mit der sich eine strukturelle Auflösung von bis zu 20 nm erreichen lässt. Desweiteren habe ich eine Mikrofluidpumpe implementiert, die eine direkte Interaktion mit der auf dem Mikroskop platzierten Probe erlaubt. Zahlreiche Prozesse wie Lebend-Zell Färbung, Lebend-Zell Fixierung, Immuno-Färbung und Puffertausch können damit direkt an der gleichen Zelle beobachtet und durchgeführt werden. So können dynamische Prozesse einer Zelle sozusagen eingefroren werden und die Strukturen von Interesse gefärbt und mit höchstauflösender Mikroskopie analysiert werden. Desweiteren habe ich den Detektionspfad der Einzel-Molekül Technik mit einem adaptiven optischen Element ausgestattet. Mithilfe eines deformierbaren Spiegels lässt sich so Abbildungsfunktion formen und Information zur 3D Position der einzelnen Moleküle gewinnen. KW - Einzelmolekülmikroskopie KW - Adaptive Optik KW - Image-Scanning Microscope KW - Correlative microscopy KW - Adaptive Optics Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199455 ER - TY - THES A1 - Beliu, Gerti T1 - Bioorthogonale Tetrazin-Farbstoffe für die Lebendzell-Markierung und hochaufgelöste Fluoreszenzmikroskopie T1 - Bioorthogonal tetrazine-dyes for live-cell labeling and super-resolution fluorescence microscopy N2 - Der genetische Code beschreibt die Ver- und Entschlüsselung der Erb-information für das universelle Prinzip der Proteinbiosynthese aus einzelnen Aminosäuren. Durch Erweiterung des genetischen Codes lassen sich unna-türliche Aminosäuren (uAA) mit einzigartigen biophysikalischen Eigenschaf-ten ortsspezifisch in Proteine einführen und ermöglichen die spezifische Ma-nipulation von Proteinen. Die Click-Reaktion zwischen der unnatürlichen Aminosäure TCO*-Lysin und Tetrazin besitzt eine außergewöhnliche Reaktionskinetik (≥800 M-1s-1) und ermöglicht eine spezifische und bioorthogonale Markierung von Bio- ¬molekülen unter physiologischen Bedingungen. Im Fokus dieser Arbeit stand zunächst die Markierung von Membran- ¬rezeptoren durch Click-Chemie in lebenden Zellen sowie die Untersuchung der Wechselwirkung 22 bekannter und neuartiger Tetrazin-Farbstoff- Konjugate. Darüber hinaus wurde die Anwendbarkeit von bioorthogonalen Click-Reaktionen für die hochauflösende Fluoreszenzmikroskopie untersucht. Durch Erweiterung des genetischen Codes in Proteine aus der Klasse der ionotropen Glutamatrezeptoren (iGluR), TNF-Rezeptoren oder Mikrotubu-li-assoziierten Proteinen (MAP) wurde ortspezifisch die unnatürliche Amino-säure TCO*-Lysin eingeführt und dadurch die Fluoreszenzmarkierung durch Tetrazin-Farbstoffe ermöglicht. Die direkte chemische Kopplung von TCO an Liganden wie Phalloidin und Docetaxel, welche spezifisch das Aktin-Zytoskelett bzw. Mikrotubuli-Filamente binden können, ermöglichte zudem die Click-Färbungen von fixierten und lebenden Zellen ohne genetische Ver-änderungen der Zielproteine. Des Weiteren wurden die spektroskopischen Eigenschaften von 22 Tetrazin-Farbstoffen, verteilt über den gesamten sichtbaren Wellenlängenbereich, untersucht. Ein charakteristisches Kennzeichen der Click-Reaktion mit Tet-razin-Farbstoffen ist dabei ihre Fluorogenität. Das Tetrazin fungiert nicht nur als reaktive Gruppe während der Click-Reaktion mit Alkenen, sondern führt in vielen Tetrazin-Farbstoff-Konjugaten zur Fluoreszenzlöschung. Während bei grün-absorbierenden Farbstoffe vor allem FRET-basierte Löschprozesse dominieren, konnte photoinduzierter Elektronentransfer (PET) vom angeregten Farbstoff zum Tetrazin als Hauptlöschmechanismus bei rot-absorbierenden Oxazin- und Rhodamin-Derivaten identifiziert werden. Die effiziente und spezifische Markierung aller untersuchten Tetrazin- Farbstoffe ermöglichte die Visualisierung von Aktin-Filamenten, Mikrotubuli und Membranrezeptoren sowohl durch konventionelle Fluoreszenzmikrosko-pie als auch durch hochauflösende Verfahren, wie z.B. dSTORM, auf Ein-zelmolekülebene. Die unterschiedliche Zellpermeabilität von Tetrazin-Farbstoffen kann dabei vorteilhaft für die spezifische intra- und extrazelluläre Markierung von Proteinen in fixierten und lebenden Zellen genutzt werden. N2 - The genetic code describes the encoding and decoding of genetic infor-mation for the universal principle of protein biosynthesis from individual amino acids. By expanding the genetic code, unnatural amino acids (uAA) with unique biophysical properties can be introduced site-specifically into pro-teins and enable the selective manipulation of proteins. The click reaction of the unnatural amino acid TCO*-lysine and tetrazine has an extraordinary reaction kinetic (≥800 M-1s-1) enabling the specific and bioorthogonal labeling of biomolecules under physiological conditions. The main focus of this work was the labeling of membrane receptors by click chemistry in living cells and the investigation of the interaction of 22 known and novel tetrazine dye conjugates. In addition, the applicability of bioorthogonal click reactions for high-resolution fluorescence microscopy was investigated. For this purpose, the unnatural amino acid TCO*-lysine was introduced site-specifically via genetic code expansion into proteins from the class of iono-tropic glutamate receptors (iGluR), TNF receptors or microtubule- associated proteins (MAP), thereby enabling fluorescence labeling with tetrazine dyes. The direct chemical coupling of TCO to ligands such as phalloidin and docetaxel, which can specifically bind the actin cytoskeleton or microtubule filaments, allowed click staining of fixed and living cells without genetic modifications of the target proteins. Furthermore, the spectroscopic properties of 22 tetrazine dyes spanning the entire visible wavelength range were investigated. A hallmark of the click reaction using tetrazine dyes is their fluorogenicity. Thus, the tetrazine not only functions as a reactive group during the click reaction with alkenes, but also leads to fluorescence quenching in many tetrazine-dye conjugates. While FRET-based quenching processes dominate in green-absorbing dyes, photoinduced electron transfer (PET) from excited dye to tetrazine has been identified as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. The efficient and specific labeling of all investigated tetrazine dyes facilitates the visualization of actin filaments, microtubules and membrane receptors by conventional fluorescence microscopy as well as by super-resolution microscopy techniques, e.g. dSTORM, also at single molecule level. The different cell permeability of tetrazine dyes can be used advantageously for the specific intra- and extracellular labeling of proteins in fixed and living cells. KW - Hochaufgelöste Fluoreszenzmikroskopie KW - Tetrazin Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189628 ER - TY - THES A1 - Becker, Mira Caroline T1 - Principles of olfactory-visual integration to form a common percept in honeybees T1 - Prinzipien der olfaktorisch-visuellen Integration des Lernverhaltens der Honigbienen N2 - The honeybee is a well studied and important organism in neuroethology. The possibility to train them with a classical conditioning paradigm and their miniature brain provide a perfect requisite to investigate the neuronal principles of learning and memory. Honeybees use visual and olfactory cues to detect flowers during their foraging trips. Hence, the reward association of a nectar source is a multi-modal construct, which has at least two major components - olfactory and visual cues. It is still an open question, how both sensory components are converged in the mushroom body, which represent the multi-modal integration centre of the honeybee brain. The main goal of this study, is to investigate the processing of multiple modalities and how a reward association is formed. This includes, how and wether both sensory modalities interfere during learning. Thus, in this study stimulation with UV, blue and green light was used to evoke distinct photoreceptor activities in the compound eye. Furthermore, three different odours (Geraniol, Citronellol and Farnesol) were used. These stimuli were tested in three different experimental series. The first experiment involved classical differential conditioning of the single modalities - odour and colour. Honeybees showed high learning performances in differentiating olfactory stimuli and also reliable responses for visual conditioning. Furthermore, a temporal discrepancy in the stimulus length for best learning in the olfatcoty and visual cues was found. In the second series, it was tested how multi-modal compounds are perceived. This includes, unique cues (configural processing) or the sum of the single components of a compound (elemen- tal processing). This was tested by combining single odour components with monochromatic light in a positive (PP) and negative patterning (NP) experiment. During PP, the olfactory- visual compound was rewarded, whereas the single components were unrewarded. In contrast, during NP the single components were reinforced, but the compound was not. In addition, the ability to distinguish between two different light stimuli presented as a part of an olfactory-visual compound with the same odour component during acquisition was tested. In a memory test, the light stimuli were presented again as a compound and in addition as the single components. The results revealed that bees used elemental processing with compounds containing green and blue light. In contrast, when UV light was presented the bees used configural processing. Finally, a third experiment was conducted at the neuronal level. Multi-unit recordings were established to provide a suitable method to analyse extrinsic neurons at the mushroom body output region, the so called ventral lobe of the pedunculus. Here, three different odours (Geran- iol, Farnesol and Citronellol), two colours (green and blue) and two combined stimuli (colour + odour) were chosen as stimuli, to search for possible variations in processing stimuli with different modalities. Two units could be detected that responded mainly to visual stimuli. N2 - Die Honigbiene ist ein gut untersuchter und wichtiger Organismus für die neuroethologische Forschung. Die Möglichkeit sie auf klassische Weise zu Konditionieren und ihr relativ kleines Gehirn macht sie zum idealen Untersuchungs-Gegenstand um die neuronalen Prinzipien des Lernens und der Gedächtnisbildung zu erforschen. Während des Furagierens nutzen Honigbi- enen beides: visuelle und olfaktorische Merkmale der Futterplanzen. Daher ist die Belohnungs- Assoziation mit der Nektar-Belohnung ein multi-modales Konstrukt, welches aus mindestens zwei Hauptkomponenten, den olfaktorischen und den visuellen Reizen, besteht. In dieser Arbeit soll untersucht werden, wie olfaktorische und visuelle Reize verarbeitet wer- den und wie sie im Pilzkörper, dem multi-modalen Integrationszentrum des Bienengehirnes, konvergieren. Wie beide sensorischen Modalitäten integriert werden um eine gemeingültige Belohnungs-Assoziation zu bilden, ist immer noch eine offene Frage. Weiterhin ist unklar ob und wie sie miteinander interferieren. Die hier dargestellten Studien nutzen Stimulationen mit UV, blauem und grünem Licht um unterschiedliche Photorezeptor Aktivitäten im Komplexauge auszulösen. Des Weiteren wurden drei verschiedene Duftkomponenten (Geraniol, Citronellol und Farnesol) verwendet. Diese Stimuli wurden in drei verschiedenen Experiment-Reihen gestestet. Das erste Experiment umfasste die klassische differentielle Konditionierung der Einzelmodalitäten (Duft und Farbe). Honigbienen zeigten eine hohe Lernfähigkeit bei der Unterscheidung zweier olfaktorischer Reize sowie eine solide Lern-Leistung während der Konditionierung mit Licht. Im zweiten Experiment wurde getestet, ob ein zusammengesetzter Reiz aus beiden Modalitäten als Summe der einzelnen Elemente (elementare Verarbeitung) oder als unikaler Reiz (konfigu- rale Verarbeitung) wahrgenommen wird. Hierbei wurde monochromatisches Licht und einzelne Duftkomponenten in positive patterning- (PP) und negative patterning-Experimenten (NP) getestet. Beim PP, wurde der zusammengesetzte Reiz belohnt, wohingegen die Einzelkom- ponenten unbelohnt blieben. Dagegen wurden beim NP nur die Einzelkomponenten belohnt, aber nicht ihre Kombination. Außerdem wurde der Frage nachgegangen, ob die Fähigkeit zur Differenzierung unterschiedlich ist, wenn zwei verschiedene Lichtreize teil einer olfaktorisch- visuellen Kombination sind, oder nicht. Interessanterweise zeigten die Verhaltensleistungen einen prominenten Fall von konfiguraler Verarbeitung, allerdings nur wenn UV-Licht ein El- ement der olfaktorisch-visuellen Zusammensetzung war. Die Ergebnisse der Experimente mit blauem oder grünem Licht hingegen, unterstützen die Theorie einer elementaren Verarbeitung. Abschließend wurde mittels elektrophysiologischer multi-unit-Aufnahmen eine passende Meth- ode etabliert, um die extrinsischen Neurone des Pilzkörpersausganges zu analysieren. Hierbei wurden drei verschiedene Düfte und zwei Farben sowie zwei Kombinationen aus Farbe und Duft getestet, um mögliche Variationen der multimodalen Reiz-Verarbeitung zu untersuchen. Zwei neuronale Einheiten (units) wurden gefunden, welche hauptsächlich auf Lichtreize antworteten. KW - honeybees KW - learning and behaviour KW - multi-modal stimuli Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199190 ER - TY - JOUR A1 - Shen, Yingjia A1 - Chalopin, Domitille A1 - Garcia, Tzintzuni A1 - Boswell, Mikki A1 - Boswell, William A1 - Shiryev, Sergey A. A1 - Agarwala, Richa A1 - Volff, Jean-Nicolas A1 - Postlethwait, John H. A1 - Schartl, Manfred A1 - Minx, Patrick A1 - Warren, Wesley C. A1 - Walter, Ronald B. T1 - X. couchianus and X. hellerii genome models provide genomic variation insight among Xiphophorus species JF - BMC Genomics N2 - Background Xiphophorus fishes are represented by 26 live-bearing species of tropical fish that express many attributes (e.g., viviparity, genetic and phenotypic variation, ecological adaptation, varied sexual developmental mechanisms, ability to produce fertile interspecies hybrids) that have made attractive research models for over 85 years. Use of various interspecies hybrids to investigate the genetics underlying spontaneous and induced tumorigenesis has resulted in the development and maintenance of pedigreed Xiphophorus lines specifically bred for research. The recent availability of the X. maculatus reference genome assembly now provides unprecedented opportunities for novel and exciting comparative research studies among Xiphophorus species. Results We present sequencing, assembly and annotation of two new genomes representing Xiphophorus couchianus and Xiphophorus hellerii. The final X. couchianus and X. hellerii assemblies have total sizes of 708 Mb and 734 Mb and correspond to 98 % and 102 % of the X. maculatus Jp 163 A genome size, respectively. The rates of single nucleotide change range from 1 per 52 bp to 1 per 69 bp among the three genomes and the impact of putatively damaging variants are presented. In addition, a survey of transposable elements allowed us to deduce an ancestral TE landscape, uncovered potential active TEs and document a recent burst of TEs during evolution of this genus. Conclusions Two new Xiphophorus genomes and their corresponding transcriptomes were efficiently assembled, the former using a novel guided assembly approach. Three assembled genome sequences within this single vertebrate order of new world live-bearing fishes will accelerate our understanding of relationship between environmental adaptation and genome evolution. In addition, these genome resources provide capability to determine allele specific gene regulation among interspecies hybrids produced by crossing any of the three species that are known to produce progeny predisposed to tumor development. KW - Xiphophorus KW - X. hellerii KW - Annotation KW - Single nucleotide change KW - Genome comparison KW - X. couchianus KW - Genome assembly KW - NGS Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164582 VL - 17 ER - TY - JOUR A1 - da Cruz, Irene A1 - Rodríguez-Casuriaga, Rosana A1 - Santiñaque, Frederico F. A1 - Farías, Joaquina A1 - Curti, Gianni A1 - Capoano, Carlos A. A1 - Folle, Gustavo A. A1 - Benavente, Ricardo A1 - Sotelo-Silveira, José Roberto A1 - Geisinger, Adriana T1 - Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage JF - BMC Genomics N2 - Background Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis. Results We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation. Conclusions This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals. KW - Spermatogenesis KW - Transcriptome KW - RNAseq KW - Flow cytometry Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164574 VL - 17 ER - TY - THES A1 - Lu, Yunzhi T1 - Kinetics of mouse and human muscle type nicotinic receptor channels T1 - Kinetik muriner und humaner nikotinischer Rezeptorkanäle vom Muskeltyp N2 - Acetylcholine (ACh) mediates transmission at vertebrate neuromuscular junctions and many other synapses. The postsynaptic ACh receptors at neuromuscular junctions are of the nicotinic subtype (nAChRs). They are among the best studied receptor channels and often serve as models or receptor prototypes. Despite a wealth of information on muscle type nAChRs so far little is known about species specific functional differences. In this work, mouse and human adult muscle type nAChRs are investigated. Cell attached recordings in the HEK293T heterologous expression system provided evidence that the ACh affinity of recombinant mouse and human adult muscle type nAChRs are different. To clarify this, I compared these receptors in outside-out patches employing a system for fast agonist application. Thus, the individual membrane patches with receptors can be exposed to various ligand concentrations. In response to 10 and 30 µM ACh normalized peak currents (î) were significantly larger and current rise-time (tr) shorter in human than in mouse receptors. Analyzing dose-response curves of î and tr and fitting them with a two-step equivalent binding-site kinetic mechanism revealed a two-fold higher ACh association rate constant in human compared to mouse receptors. Furthermore, human nAChRs were blocked faster in outside-out patches by superfusion of 300 nM α-Bungarotoxin (α-Bgtx) than mouse nAChRs. Finally, human nAChRs in outside-out patches showed higher affinity at 3 µM ACh than chimeric receptors consisting of mouse α- and human β-, γ- and ε-subunits. The higher affinity of human than mouse receptors for ACh and α-Bgtx is thus at least in part due to sequence difference in their α-subunits. N2 - Acetylcholin (ACh) vermittelt Erregungsübertragung an neuromuskulären synaptischen Kontakten (neuromuscular junction, NMJ) von Wirbeltieren und vielen anderen Synapsen. Die postsynaptischen ACh-Rezeptoren an der NMJ sind vom nikotinischen Subtyp (nAChRs). Als Teil der am besten erforschten Kanalrezeptoren dienen sie oft als Modelle oder auch Prototypen für Rezeptoren. Trotz einer Fülle an Informationen über nAChRs des Muskeltyps ist bis heute recht wenig über artenspezifischen funktionellen Unterschiede bekannt. Diese Studie befasst sich daher mit der Untersuchung von nAChRs des Muskeltyps in erwachsenen Mäusen und Menschen. Aufzeichnungen mit sogenannten Cell-attached Patches im heterologen Expressionssystem HEK293T-Zellen lieferten Beweise dafür, dass die ACh-Affinität von rekombinanten erwachsenen Maus- und menschlichen nAChRs vom Muskeltyp unterschiedlich sind. Um diesem nachzugehen, habe ich diese Rezeptoren in Outside-out Patches mit Hilfe eines schnellen Piezogetriebenen Applikationssystems verglichen. Dieses System bietet den Vorteil, dass einzelne Membran-Patches mit Rezeptoren unterschiedlichen Ligandenkonzentrationen ausgesetzt werden können. Als Reaktion auf 10 und 30 µM ACh waren die normalisierten Stromamplituden (î) und Stromanstiegszeiten (tr) der menschlichen Rezeptoren signifikant höher als die der Mausrezeptoren. Die Analyse der Dosis-Wirkungskurven von î und tr sowie die Anpassung eines quantitativen zweistufigen kinetischen Modells mit zwei äquivalenten Bindestellen an die Datensätze zeigten eine zweifach höhere Assoziationsrate für ACh bei menschlichen Rezeptoren, verglichen mit der von Mausrezeptoren. Zudem wurden menschliche nAChRs in Outside-Out-Patches schneller als Mausrezeptoren durch Superfusion mit 300 nM α-Bungarotoxin (α-Bgtx) blockiert, was für eine höhere Affinität auch für α-Bgtx spricht. Schließlich wiesen die menschlichen nAChRs in Outside-Out-Patches bei 3 µM ACh eine höhere Affinität als chimäre Rezeptoren aus Maus α- und menschlichen β-, γ- and ε-Untereinheiten auf. Die höhere Affinität der menschlichen Rezeptoren zu ACh und α-Bgtx im Vergleich zu Mausrezeptoren basiert somit zumindest in Teilen auf Sequenzdifferenzen ihrer α-Einheitenen. KW - nicotinic acetylcholine receptor KW - affinity KW - kinetic mechanism KW - Nicotinischer Acetylcholinrezeptor KW - Muskelzelle KW - Maus KW - Mensch Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192688 ER - TY - THES A1 - Neubert, Franziska T1 - Markierung postsynaptischer Proteine für die hochauflösende Fluoreszenzmikroskopie T1 - Labeling of postsynaptic proteins for super-resolution microscopy N2 - Das menschliche Gehirn ist ein Organ, das aufgrund seiner Komplexität und zellulären Diversität noch am wenigsten verstanden ist. Eine der Ursachen dafür sind zahlreiche Herausforderungen in diversen neurobiologischen Bild-gebungsverfahren. Erst seit der Erfindung der hochauflösenden Fluoreszenz-mikroskopie ist es möglich, Strukturen unterhalb der Beugungsgrenze zu visua-lisieren und somit eine maximale Auflösung von bis zu 20 nm zu erreichen. Zusätzlich hängt die Fähigkeit, biologische Strukturen aufzulösen, von der Markierungs-größe und -dichte ab. Derzeit ist die häufigste Methode zur Proteinfärbung die indirekte Antikörperfärbung, bei der ein Fluorophor-markierter Sekundärantikörper an einen Epitop-spezifischen Primärantikörper bindet. Dabei kann der Abstand von Zielstruktur und Fluorophor bis zu 30 nm betragen, was eine Auflösungs-verminderung zur Folge haben kann. Aufgrund dessen wurden in dieser Arbeit alternative Markierungsmethoden getestet, um postsynaptische Proteine sicht-bar zu machen. Zunächst wurde der postsynaptische N-Methyl-D-Aspartat (NMDA)-Rezeptor mit Hilfe konventioneller indirekter Antikörperfärbung markiert. Hier war die NR1-Untereinheit des NMDA-Rezeptors von besonderem Interesse, da diese in der Autoimmunerkrankung Anti-NMDA-Rezeptor-Enzephalitis invol-viert ist. Patienten dieser seltenen Krankheit bilden Autoantikörper gegen die NR1-Untereinheit, wodurch ein schneller reversibler Verlust der NMDA-Rezeptoren auf der Postsynapse induziert wird. Wichtige Informationen können nicht mehr ausreichend weitergegeben werden, was psychiatrische und neurologi-sche Störungen zur Folge hat. In dieser Arbeit wurden sowohl kommerzielle NR1-Antikörper, als auch rekombinante monoklonale NR1-Antikörper von Patien-ten mit Anti-NMDA-Rezeptor-Enzephalitis getestet. In konfokalen und in hochaufgelösten SIM- (engl. structured illumination microscopy) und dSTORM- (engl. direct stochastic optical reconstruction microscopy) Messun-gen konnten kommerzielle NR1-Antikörper keine erfolgreichen Färbungen erzielen. Dagegen erwiesen sich die rekombinanten monoklonalen NR1-Patientenantikörper als sehr spezifisch, sowohl in primären Neuronen als auch im Hippocampus von murinen Gehirnschnitten und lieferten gute Kolokalisati-onen mit dem postsynaptischen Markerprotein Homer. Um die optische Auflösung zu verbessern, wurde eine neue Markierungs-methode mit sog. „Super-Binde-Peptiden“ (SBPs) getestet. SBPs sind modifi-zierte Peptide, die erhöhte Affinitäten und Spezifitäten aufweisen und mit ei-ner Größe von ~ 2,5 nm wesentlich kleiner als Antikörper sind. In dieser Arbeit bestätigte sich ein kleines hochspezifisches SPB, das an den Fluoreszenzfarb-stoff Tetra- methylrhodamin (TMR) gekoppelt ist, als effektiver Marker für das Ankerpro-tein Gephyrin. Gephyrin ist für die Lokalisation und Verankerung einiger post-synaptischer Rezeptoren zuständig, indem es sie mit dem Cytoskelett der Zelle verbindet. SIM-Messungen in primären Neuronen zeigten eine bessere Clus-terrepräsentation bei der Färbung von Gephyrin mit SBPs, als mit Antikörper-färbung. Zusätzlich wurden Kolokalisationsanalysen von Gephyrin zusammen mit dem inhibito-rischen präsynaptischen vesikulären GABA-Transporter VGAT durchgeführt. Eine weitere Färbemethode stellte die bioorthogonale Click-Färbung durch die Erweiterung des eukaryotischen genetischen Codes (engl. genetic code ex-pansion, GCE) dar. Dabei wurde eine unnatürliche, nicht-kanonische Amino-säure (engl. non-canonical amino acid, ncAA) ins Zielprotein eingebaut und in Kombination mit der Click-Chemie ortsspezifisch mit organischen Tetrazin-Farbstoff-Konjugaten angefärbt. Organische Fluorophore haben den Vorteil, dass sie mit einer Größe von 0,5 – 2 nm sehr klein sind und damit die natürli-chen Funktionen der Proteine in der Zelle kaum beeinflussen. In dieser Arbeit wurde zum ersten Mal gezeigt, dass der tetramere postsynaptische NMDA-Rezeptor durch die Amber-Supres-sionsmethode bioorthogonal angefärbt werden konnte. Aus sieben verschiede-nen Amber-Mutanten der NR1-Untereinheit stellte sich die Y392TAG-NR1-Mutante als diejenige mit der besten Proteinexpression, Färbeeffizienz und rezeptorfunktionalität heraus. Dies konnte durch Fluoreszenzmikroskopie- und Whole-Cell Patch-Clamp-Experimenten gezeigt werden. Die bioorthogo-nale Click-Färbung durch GCE eignete sich für die Färbung des NMDA-Rezeptors in verschiedenen Zelllinien, mit unterschiedlichen Tetrazin-Farbstoff-Konjugaten und für Lebendzellexperimente. In dSTORM-Messungen erwies sich das Tetrazin-Cy5-Farbstoff-Konjugat als ideal aufgrund seiner Grö-ße, Photostabilität, Helligkeit und seines geeigneten Blinkverhaltens, sodass eine homogene NMDA-Rezeptorverteilung auf der Zellmembran gezeigt wer-den konnte. NR1-Antikörperfärbungen wiesen dagegen starke Clusterbildun-gen auf. Die Ergebnisse konnten belegen, dass kleinere Farbstoffe eine deut-lich bessere Zugänglichkeit zu ihrem Zielprotein haben und somit besser für die hochauflösende Fluoreszenzmikroskopie geeignet sind. N2 - Due to its complexity and cellular diversity, the human brain is an organ which is poorly understood. In particular, there are numerous challenges in different neurobiological imaging processes. The advent of super-resolution fluorescence microscopy, where a maximal optical resolution of up to 20 nm is achievable, made it feasible to visualize structures beyond the diffraction limit. The ability to resolve biological structures is further dependent on the labeling size and density. Currently, indirect antibody staining is the most common method for protein labeling. Thereby, a fluorophore marked secondary anti-body binds an epitope specific primary antibody. Consequently, the off-distance between target structure and fluorophore can be up to 30 nm, which could provoke a decrease of resolution. As a result, alternative labeling methods were tested in this work to visualize postsynaptic proteins. Initially, labeling of the postsynaptic N-methyl-D-aspartate (NMDA) recep-tor was performed with conventional indirect antibody staining. Here, the NR1 subunit of the NMDA receptor was of special interest because it is involved in the autoimmune disease of Anti-NMDA receptor encephalitis. Patients of this rare disorder produce autoantibodies against the NR1 subunit, which induces a fast and reversible reduction of NMDA receptors on the postsynapse. Important synaptic information cannot be transferred sufficiently which results in psychiatrical and neurological deficiencies. In this work commercial NR1 antibodies as well as recombinant monoclonal NR1 antibodies from patients with Anti-NMDA receptor encephalitis were tested. In confocal and super-resolved SIM (structured illumination microscopy) and dSTORM (direct sto-chastic optical reconstruction microscopy) measurements the commercial NR1 antibodies could not obtain a successful staining. In contrast, recombinant monoclonal NR1 patient antibodies turn out to be very specific, both in primary neurons and in the hippocampus of murine brain slices. Additionally, they show perfect colocaliza-tion together with the postsynaptic marker protein Homer. To further improve the optical resolution, a new labeling method was tested with so called “super-binding peptides” (SBPs). SBPs are modified peptides with enhanced affinity and specificity. With a size of ~ 2.5 nm, they are much smaller than antibodies. In this work a small, highly specific SBP, coupled to the fluorescent dye tetramethylrhodamine (TMR), turned out to be an efficient marker for the postsynaptic anchor protein gephyrin. Gephyrin is responsible for the localization and anchoring of postsynaptic receptors by connecting them with the cytoskeleton of the cell. SIM measurements in primary neurons showed better cluster representation of gephyrin stained with SBPs than with antibody stain-ing. In addition, colocalization analysis of gephyrin together with the inhibitory presynaptic vesicular GABA transporter VGAT was performed. Another staining method was the bioorthogonal click chemistry by the eu-karyotic genetic code expansion (GCE). Thereby, an unnatural, non-canonical amino acid (ncAA) is incorporated into the target protein and click-labeled site- specifically with an organic tetrazine dye conjugate. Organic dyes are very small with a size of only 0.5 – 2 nm and barely influence the natural function of proteins within the cell, which is beneficial for super-resolution microscopy. In this work, tetrameric postsynaptic NMDA receptors were bioorthogonally la-beled via the amber suppression method for the first time. From a series of seven different amber mutants of the NR1 subunit, the Y392TAG mutant was the one with the best protein expression, labeling efficiency and receptor functionality, as shown by fluorescence microscopy and whole-cell patch clamp experiments. The bioortho-gonal click staining by GCE was suitable for the NMDA receptor stain-ing in different cell lines, with various tetrazine dye conjugates and for live-cell experiments. In dSTORM measurements the tetrazine-Cy5 dye conjugate was ideal because of its size, photostability, brightness and appropriate blinking be-havior. Accordingly, a homogenous NMDA receptor distribution on the cell membrane was observed. In contrast, NR1 antibody staining showed big cluster formation. The results show that small labels have a better accessibility to its target and are therefore much more suitable for super-resolution microscopy. KW - hochauflösende Fluoreszenzmikroskopie KW - Markierungen synaptischer Proteine Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192394 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 - JOUR A1 - Drescher, Nora A1 - Klein, Alexandra-Maria A1 - Neumann, Peter A1 - Yañez, Orlando A1 - Leonhardt, Sara D. T1 - Inside Honeybee Hives: Impact of Natural Propolis on the Ectoparasitic Mite Varroa destructor and Viruses JF - Insects N2 - Social immunity is a key factor for honeybee health, including behavioral defense strategies such as the collective use of antimicrobial plant resins (propolis). While laboratory data repeatedly show significant propolis effects, field data are scarce, especially at the colony level. Here, we investigated whether propolis, as naturally deposited in the nests, can protect honeybees against ectoparasitic mites Varroa destructor and associated viruses, which are currently considered the most serious biological threat to European honeybee subspecies, Apis mellifera, globally. Propolis intake of 10 field colonies was manipulated by either reducing or adding freshly collected propolis. Mite infestations, titers of deformed wing virus (DWV) and sacbrood virus (SBV), resin intake, as well as colony strength were recorded monthly from July to September 2013. We additionally examined the effect of raw propolis volatiles on mite survival in laboratory assays. Our results showed no significant effects of adding or removing propolis on mite survival and infestation levels. However, in relation to V. destructor, DWV titers increased significantly less in colonies with added propolis than in propolis-removed colonies, whereas SBV titers were similar. Colonies with added propolis were also significantly stronger than propolis-removed colonies. These findings indicate that propolis may interfere with the dynamics of V. destructor-transmitted viruses, thereby further emphasizing the importance of propolis for honeybee health. KW - social immunity KW - Apis mellifera KW - deformed wing virus KW - plant-insect interactions KW - resin KW - sacbrood virus Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171164 VL - 8 IS - 1 ER - TY - JOUR A1 - Kaluza, Benjamin F. A1 - Wallace, Helen A1 - Keller, Alexander A1 - Heard, Tim A. A1 - Jeffers, Bradley A1 - Drescher, Nora A1 - Blüthgen, Nico A1 - Leonhardt, Sara D. T1 - Generalist social bees maximize diversity intake in plant species-rich and resource-abundant environments JF - Ecosphere N2 - Numerous studies revealed a positive relationship between biodiversity and ecosystem functioning, suggesting that biodiverse environments may not only enhance ecosystem processes, but also benefit individual ecosystem members by, for example, providing a higher diversity of resources. Whether and how the number of available resources affects resource collection and subsequently consumers (e.g., through impacting functions associated with resources) have, however, been little investigated, although a better understanding of this relationship may help explain why the abundance and richness of many animal species typically decline with decreasing plant (resource) diversity. Using a social bee species as model (Tetragonula carbonaria), we investigated how plant species richness—recorded for study sites located in different habitats—and associated resource abundance affected the diversity and functionality (here defined as nutritional content and antimicrobial activity) of resources (i.e., pollen, nectar, and resin) collected by a generalist herbivorous consumer. The diversity of both pollen and resin collected strongly increased with increasing plant/tree species richness, while resource abundance was only positively correlated with resin diversity. These findings suggest that bees maximize resource diversity intake in (resource) diverse habitats. Collecting more diverse resources did, however, not increase their functionality, which appeared to be primarily driven by the surrounding (plant) source community in our study. In generalist herbivores, maximizing resource diversity intake may therefore primarily secure collection of sufficient amounts of resources across the entire foraging season, but it also ensures that the allocated resources meet all functional needs. Decreasing available resource diversity may thus impact consumers primarily by reduced resource abundance, but also by reduced resource functionality, particularly when resources of high functionality (e.g., from specific plant species) become scarce. KW - functional complementarity KW - functional redundancy KW - Meliponini KW - nutritional ecology KW - plant–insect interactions KW - pollinator decline Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171155 VL - 8 IS - 3 ER - TY - JOUR A1 - Fischer, Annette A1 - Harrison, Kelly S A1 - Ramirez, Yesid A1 - Auer, Daniela A1 - Chowdhury, Suvagata Roy A1 - Prusty, Bhupesh K A1 - Sauer, Florian A1 - Dimond, Zoe A1 - Kisker, Caroline A1 - Hefty, P Scott A1 - Rudel, Thomas T1 - Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense JF - eLife N2 - Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense. KW - cell-autonomous defense KW - Chlamydia trachomatis KW - deubiquitinase KW - Mcl-1 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171073 VL - 6 IS - e21465 ER - TY - JOUR A1 - Wiegering, Armin A1 - Matthes, Niels A1 - Mühling, Bettina A1 - Koospal, Monika A1 - Quenzer, Anne A1 - Peter, Stephanie A1 - Germer, Christoph-Thomas A1 - Linnebacher, Michael A1 - Otto, Christoph T1 - Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin JF - Neoplasia N2 - Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n = 9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n = 5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC\(_{50}\)< 3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents. KW - colorectal carcinoma KW - reactivating p53 and inducing tumor apoptosis (RITA) KW - chemotherapy KW - 5-fluorouracil KW - oxaliplatin Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171067 VL - 19 IS - 4 ER - TY - JOUR A1 - Degen, Tobias A1 - Hovestadt, Thomas A1 - Mitesser, Oliver A1 - Hölker, Franz T1 - Altered sex-specific mortality and female mating success: ecological effects and evolutionary responses JF - Ecosphere N2 - Theory predicts that males and females should often join the mating pool at different times (sexual dimorphism in timing of emergence [SDT]) as the degree of SDT affects female mating success. We utilize an analytical model to explore (1) how important SDT is for female mating success, (2) how mating success might change if either sex's mortality (abruptly) increases, and (3) to what degree evolutionary responses in SDT may be able to mitigate the consequences of such mortality increase. Increasing male pre‐mating mortality has a non‐linear effect on the fraction of females mated: The effect is initially weak, but at some critical level a further increase in male mortality has a stronger effect than a similar increase in female mortality. Such a change is expected to impose selection for reduced SDT. Increasing mortality during the mating season has always a stronger effect on female mating success if the mortality affects the sex that emerges first. This bias results from the fact that enhancing mortality of the earlier emerging sex reduces female–male encounter rates. However, an evolutionary response in SDT may effectively mitigate such consequences. Further, if considered independently for females and males, the predicted evolutionary response in SDT could be quite dissimilar. The difference between female and male evolutionary response in SDT leads to marked differences in the fraction of fertilized females under certain conditions. Our model may provide general guidelines for improving harvesting of populations, conservation management of rare species under altered environmental conditions, or maintaining long‐term efficiency of pest‐control measures. KW - evolutionary response KW - sexual dimorphism in timing KW - sex-specific mortality KW - reproductive asynchrony KW - mating success Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170953 VL - 8 IS - 5 ER - TY - JOUR A1 - Maierhofer, Anna A1 - Flunkert, Julia A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Schindler, Detlev A1 - Nanda, Indrajit A1 - Haaf, Thomas T1 - Analysis of global DNA methylation changes in primary human fibroblasts in the early phase following X-ray irradiation JF - PLoS ONE N2 - Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response. KW - DNA methylation KW - fibroblasts KW - methylation KW - alu elements KW - DNA damage KW - epigenetics KW - cancer treatment Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170895 VL - 12 IS - 5 ER - TY - JOUR A1 - Liu, Han A1 - Chen, Chunhai A1 - Gao, Zexia A1 - Min, Jiumeng A1 - Gu, Yongming A1 - Jian, Jianbo A1 - Jiang, Xiewu A1 - Cai, Huimin A1 - Ebersberger, Ingo A1 - Xu, Meng A1 - Zhang, Xinhui A1 - Chen, Jianwei A1 - Luo, Wei A1 - Chen, Boxiang A1 - Chen, Junhui A1 - Liu, Hong A1 - Li, Jiang A1 - Lai, Ruifang A1 - Bai, Mingzhou A1 - Wei, Jin A1 - Yi, Shaokui A1 - Wang, Huanling A1 - Cao, Xiaojuan A1 - Zhou, Xiaoyun A1 - Zhao, Yuhua A1 - Wei, Kaijian A1 - Yang, Ruibin A1 - Liu, Bingnan A1 - Zhao, Shancen A1 - Fang, Xiaodong A1 - Schartl, Manfred A1 - Qian, Xueqiao A1 - Wang, Weimin T1 - The draft genome of blunt snout bream (Megalobrama amblycephala) reveals the development of intermuscular bone and adaptation to herbivorous diet JF - GigaScience N2 - The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation. KW - Megalobrama amblycephala KW - whole genome KW - herbivorous diet KW - intermuscular bone KW - transcriptome KW - gut microflora Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170844 VL - 6 IS - 7 ER - TY - JOUR A1 - Selcho, Mareike A1 - Millán, Carola A1 - Palacios-Muñoz, Angelina A1 - Ruf, Franziska A1 - Ubillo, Lilian A1 - Chen, Jiangtian A1 - Bergmann, Gregor A1 - Ito, Chihiro A1 - Silva, Valeria A1 - Wegener, Christian A1 - Ewer, John T1 - Central and peripheral clocks are coupled by a neuropeptide pathway in Drosophila JF - Nature Communications N2 - Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals. KW - circadian clock KW - Drosophila KW - neuropeptide pathway KW - peripheral clocks KW - central clocks Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170831 VL - 8 IS - 15563 ER - TY - JOUR A1 - Nürnberger, Fabian A1 - Steffan-Dewenter, Ingolf A1 - Härtel, Stephan T1 - Combined effects of waggle dance communication and landscape heterogeneity on nectar and pollen uptake in honey bee colonies JF - PeerJ N2 - The instructive component of waggle dance communication has been shown to increase resource uptake of Apis mellifera colonies in highly heterogeneous resource environments, but an assessment of its relevance in temperate landscapes with different levels of resource heterogeneity is currently lacking. We hypothesized that the advertisement of resource locations via dance communication would be most relevant in highly heterogeneous landscapes with large spatial variation of floral resources. To test our hypothesis, we placed 24 Apis mellifera colonies with either disrupted or unimpaired instructive component of dance communication in eight Central European agricultural landscapes that differed in heterogeneity and resource availability. We monitored colony weight change and pollen harvest as measure of foraging success. Dance disruption did not significantly alter colony weight change, but decreased pollen harvest compared to the communicating colonies by 40%. There was no general effect of resource availability on nectar or pollen foraging success, but the effect of landscape heterogeneity on nectar uptake was stronger when resource availability was high. In contrast to our hypothesis, the effects of disrupted bee communication on nectar and pollen foraging success were not stronger in landscapes with heterogeneous compared to homogenous resource environments. Our results indicate that in temperate regions intra-colonial communication of resource locations benefits pollen foraging more than nectar foraging, irrespective of landscape heterogeneity. We conclude that the so far largely unexplored role of dance communication in pollen foraging requires further consideration as pollen is a crucial resource for colony development and health. KW - Apis mellifera KW - orientation KW - recruitment KW - landscape ecology KW - foraging behaviour KW - floral resource distribution Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170813 VL - 5 IS - e3441 ER - TY - JOUR A1 - Dütting, Sebastian A1 - Gaits-Iacovoni, Frederique A1 - Stegner, David A1 - Popp, Michael A1 - Antkowiak, Adrien A1 - van Eeuwijk, Judith M.M. A1 - Nurden, Paquita A1 - Stritt, Simon A1 - Heib, Tobias A1 - Aurbach, Katja A1 - Angay, Oguzhan A1 - Cherpokova, Deya A1 - Heinz, Niels A1 - Baig, Ayesha A. A1 - Gorelashvili, Maximilian G. A1 - Gerner, Frank A1 - Heinze, Katrin G. A1 - Ware, Jerry A1 - Krohne, Georg A1 - Ruggeri, Zaverio M. A1 - Nurden, Alan T. A1 - Schulze, Harald A1 - Modlich, Ute A1 - Pleines, Irina A1 - Brakebusch, Cord A1 - Nieswandt, Bernhard T1 - A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis JF - Nature Communications N2 - Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V. KW - megakaryocytes KW - blood platelets KW - regulatory circuit downstream KW - glycoprotein Ib Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170797 VL - 8 IS - 15838 ER - TY - JOUR A1 - Ruf, Franziska A1 - Fraunholz, Martin A1 - Öchsner, Konrad A1 - Kaderschabeck, Johann A1 - Wegener, Christian T1 - WEclMon - A simple and robust camera-based system to monitor Drosophila eclosion under optogenetic manipulation and natural conditions JF - PLoS ONE N2 - Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems. We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity. KW - chronobiology KW - infrared radiation KW - light pulses KW - molting KW - Drosophila melanogaster KW - optogenetics KW - eclosion Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170755 VL - 12 IS - 6 ER - TY - JOUR A1 - Memmel, Simon A1 - Sisario, Dmitri A1 - Zöller, Caren A1 - Fiedler, Vanessa A1 - Katzer, Astrid A1 - Heiden, Robin A1 - Becker, Nicholas A1 - Eing, Lorenz A1 - Ferreira, Fábio L.R. A1 - Zimmermann, Heiko A1 - Sauer, Markus A1 - Flentje, Michael A1 - Sukhorukov, Vladimir L. A1 - Djuzenova, Cholpon S. T1 - Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities JF - Oncotarget N2 - High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTOR-inhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell line-specific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM. KW - chemotherapy KW - glioblastoma multiforme KW - migration KW - treatment Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170719 VL - 8 IS - 28 ER - TY - JOUR A1 - Kollmannsberger, Philip A1 - Kerschnitzki, Michael A1 - Repp, Felix A1 - Wagermaier, Wolfgang A1 - Weinkamer, Richard A1 - Fratzl, Peter T1 - The small world of osteocytes: connectomics of the lacuno-canalicular network in bone JF - New Journal of Physics N2 - Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization. KW - bone KW - osteocytes KW - networks KW - biomaterials KW - mechanobiology KW - image analysis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170662 VL - 19 IS - 073019 ER - TY - JOUR A1 - Ziegler, Sabrina A1 - Weiss, Esther A1 - Schmitt, Anna-Lena A1 - Schlegel, Jan A1 - Burgert, Anne A1 - Terpitz, Ulrich A1 - Sauer, Markus A1 - Moretta, Lorenzo A1 - Sivori, Simona A1 - Leonhardt, Ines A1 - Kurzai, Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells JF - Scientific Reports N2 - Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response. KW - pattern recognition receptors KW - fungal infection KW - Aspergillus fumigatus KW - natural killer cells Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170637 VL - 7 IS - 6138 ER - TY - THES A1 - Kaltdorf [geb. Schuch], Kristin Verena T1 - Mikroskopie, Bildverarbeitung und Automatisierung der Analyse von Vesikeln in \(C.\) \(elegans\) und anderen biologischen Strukturen T1 - Microscopy, Image Processing and Automization of Analysis of Vesicles in \(C.\) \(elegans\) and other biological Structures N2 - Thema dieser Thesis ist die Analyse sekretorischer Vesikelpools auf Ultrastrukturebene in unterschiedlichen biologischen Systemen. Der erste und zweite Teil dieser Arbeit fokussiert sich auf die Analyse synaptischer Vesikelpools in neuromuskulären Endplatten (NME) im Modellorganismus Caenorhabditis elegans. Dazu wurde Hochdruckgefrierung und Gefriersubstitution angewandt, um eine unverzügliche Immobilisation der Nematoden und somit eine Fixierung im nahezu nativen Zustand zu gewährleisten. Anschließend wurden dreidimensionale Aufnahmen der NME mittels Elektronentomographie erstellt. Im ersten Teil dieser Arbeit wurden junge adulte, wildtypische C. elegans Hermaphroditen mit Septin-Mutanten verglichen. Um eine umfassende Analyse mit hoher Stichprobenzahl zu ermöglichen und eine automatisierte Lösung für ähnliche Untersuchungen von Vesikelpools bereit zu stellen wurde eine Software namens 3D ART VeSElecT zur automatisierten Vesikelpoolanalyse entwickelt. Die Software besteht aus zwei Makros für ImageJ, eines für die Registrierung der Vesikel und eines zur Charakterisierung. Diese Trennung in zwei separate Schritte ermöglicht einen manuellen Verbesserungsschritt zum Entfernen falsch positiver Vesikel. Durch einen Vergleich mit manuell ausgewerteten Daten neuromuskulärer Endplatten von larvalen Stadien des Modellorganismus Zebrafisch (Danio rerio) konnte erfolgreich die Funktionalität der Software bewiesen werden. Die Analyse der neuromuskulären Endplatten in C. elegans ergab kleinere synaptische Vesikel und dichtere Vesikelpools in den Septin-Mutanten verglichen mit Wildtypen. Im zweiten Teil der Arbeit wurden neuromuskulärer Endplatten junger adulter C. elegans Hermaphroditen mit Dauerlarven verglichen. Das Dauerlarvenstadium ist ein spezielles Stadium, welches durch widrige Umweltbedingungen induziert wird und in dem C. elegans über mehrere Monate ohne Nahrungsaufnahme überleben kann. Da hier der Vergleich der Abundanz zweier Vesikelarten, der „clear-core“-Vesikel (CCV) und der „dense-core“-Vesikel (DCV), im Fokus stand wurde eine Erweiterung von 3D ART VeSElecT entwickelt, die einen „Machine-Learning“-Algorithmus zur automatisierten Klassifikation der Vesikel integriert. Durch die Analyse konnten kleinere Vesikel, eine erhöhte Anzahl von „dense-core“-Vesikeln, sowie eine veränderte Lokalisation der DCV in Dauerlarven festgestellt werden. Im dritten Teil dieser Arbeit wurde untersucht ob die für synaptische Vesikelpools konzipierte Software auch zur Analyse sekretorischer Vesikel in Thrombozyten geeignet ist. Dazu wurden zweidimensionale und dreidimensionale Aufnahmen am Transmissionselektronenmikroskop erstellt und verglichen. Die Untersuchung ergab, dass hierfür eine neue Methodik entwickelt werden muss, die zwar auf den vorherigen Arbeiten prinzipiell aufbauen kann, aber den besonderen Herausforderungen der Bilderkennung sekretorischer Vesikel aus Thrombozyten gerecht werden muss. N2 - Subject of this thesis was the analysis of the ultrastructure of vesicle pools in various biological systems. The first and second part of this thesis is focused on the analysis of synaptic vesicle pools in neuromuscular junctions in the model organism Caenorhabditis elegans. In order to get access of synaptic vesicle pools in their near-to native state high-pressure freezing and freeze substitution was performed. Subsequently three-dimensional imaging of neuromuscular junctions using electron tomography was performed. In the first part young adult wild-type C. elegans hermaphrodites and septin mutants were compared. To enable extensive analysis and to provide an automated solution for comparable studies, a software called 3D ART VeSElecT for automated vesicle pool analysis, was developed. The software is designed as two macros for ImageJ, one for registration of vesicles and one for characterization. This separation allows for a manual revision step in between to erase false positive particles. Through comparison with manually evaluated data of neuromuscular junctions of larval stages of the model organism zebrafish (Danio rerio), functionality of the software was successfully proved. As a result, analysis of C. elegans neuromuscular junctions revealed smaller synaptic vesicles and more densely packed vesicle pools in septin mutants compared to wild-types. In the second part of this thesis NMJs of young adult C. elegans hermaphrodites were compared with dauer larvae. The dauer larva is a special state that is induced by adverse environmental conditions and enables C. elegans to survive several months without any foot uptake. Aiming for an automated analysis of the ratio of two vesicle types, clear core vesicles (CCVs) and dense core vesicles (DCVs), an extension for 3D ART VeSElecT was developed, integrating a machine-learning classifier. As a result, smaller vesicles and an increased amount of dense core vesicles in dauer larvae were found. In the third part of this thesis the developed software, designed for the analysis of synaptic vesicle pools, was checked for its suitability to recognize secretory vesicles in thrombocytes. Therefore, two-dimensional and three-dimensional transmission electron microscopic images were prepared and compared. The investigation has shown that a new methodology has to be developed which, although able to build on the previous work in principle, must meet the special challenges of image recognition of secretory vesicles from platelets. KW - Mikroskopie KW - Bildverarbeitung KW - Registrierung KW - Synaptische Vesikel KW - Bildanalyse KW - Automatisierung der Analyse KW - Automated Image Analysis KW - Caenorhabditis elegans KW - Electron Microscopy KW - Elektronenmikroskopie KW - Caenorhabditis elegans KW - automatisierte Bildanalyse Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-160621 ER - TY - JOUR A1 - Wanzek, Katharina A1 - Schwindt, Eike A1 - Capra, John A. A1 - Paeschke, Katrin T1 - Mms1 binds to G-rich regions in Saccharomyces cerevisiae and influences replication and genome stability JF - Nucleic Acids Research N2 - The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity. KW - replication KW - regulation KW - genome integrity KW - Saccharomyces cerevisiae Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170577 VL - 45 IS - 13 ER - TY - JOUR A1 - Scholz, Nicole A1 - Guan, Chonglin A1 - Nieberler, Matthias A1 - Grotmeyer, Alexander A1 - Maiellaro, Isabella A1 - Gao, Shiqiang A1 - Beck, Sebastian A1 - Pawlak, Matthias A1 - Sauer, Markus A1 - Asan, Esther A1 - Rothemund, Sven A1 - Winkler, Jana A1 - Prömel, Simone A1 - Nagel, Georg A1 - Langenhan, Tobias A1 - Kittel, Robert J T1 - Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons JF - eLife N2 - Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response. KW - Latrophilin KW - adhesion GPCR KW - dCIRL KW - sensory physiology KW - metabotropic signalling KW - mechanotransduction Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170520 VL - 6 IS - e28360 ER - TY - JOUR A1 - Rössler, Wolfgang A1 - Spaethe, Johannes A1 - Groh, Claudia T1 - Pitfalls of using confocal-microscopy based automated quantification of synaptic complexes in honeybee mushroom bodies (response to Peng and Yang 2016) JF - Scientific Reports N2 - A recent study by Peng and Yang in Scientific Reports using confocal-microscopy based automated quantification of anti-synapsin labeled microglomeruli in the mushroom bodies of honeybee brains reports potentially incorrect numbers of microglomerular densities. Whereas several previous studies using visually supervised or automated counts from confocal images and analyses of serial 3D electron-microscopy data reported consistent numbers of synaptic complexes per volume, Peng and Yang revealed extremely low numbers differing by a factor of 18 or more from those obtained in visually supervised counts, and by a factor 22–180 from numbers in two other studies using automated counts. This extreme discrepancy is especially disturbing as close comparison of raw confocal images of anti-synapsin labeled whole-mount brain preparations are highly similar across these studies. We conclude that these discrepancies may reside in potential misapplication of confocal imaging followed by erroneous use of automated image analysis software. Consequently, the reported microglomerular densities during maturation and after manipulation by insecticides require validation by application of appropriate confocal imaging methods and analyses tools that rely on skilled observers. We suggest several improvements towards more reliable or standardized automated or semi-automated synapse counts in whole mount preparations of insect brains. KW - confocal-microscopy based automated quantification KW - mushroom bodies KW - honeybees KW - brain Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170451 VL - 7 IS - 9786 ER - TY - JOUR A1 - Danner, Nadja A1 - Keller, Alexander A1 - Härtel, Stephan A1 - Steffan-Dewenter, Ingolf T1 - Honey bee foraging ecology: Season but not landscape diversity shapes the amount and diversity of collected pollen JF - PLoS ONE N2 - The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes. KW - honey bees KW - pollen KW - season KW - foraging Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170424 VL - 12 IS - 8 ER - TY - JOUR A1 - Klein-Hessling, Stefan A1 - Muhammad, Khalid A1 - Klein, Matthias A1 - Pusch, Tobias A1 - Rudolf, Ronald A1 - Flöter, Jessica A1 - Qureischi, Musga A1 - Beilhack, Andreas A1 - Vaeth, Martin A1 - Kummerow, Carsten A1 - Backes, Christian A1 - Schoppmeyer, Rouven A1 - Hahn, Ulrike A1 - Hoth, Markus A1 - Bopp, Tobias A1 - Berberich-Siebelt, Friederike A1 - Patra, Amiya A1 - Avots, Andris A1 - Müller, Nora A1 - Schulze, Almut A1 - Serfling, Edgar T1 - NFATc1 controls the cytotoxicity of CD8\(^{+}\) T cells JF - Nature Communications N2 - Cytotoxic T lymphocytes are effector CD8\(^{+}\) T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1\(^{-/-}\) cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1\(^{-/-}\) CD8\(^{+}\) T cells, including Tbx21, Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1\(^{-/-}\), but not Nfatc2\(^{-/-}\) CD8\(^{+}\) T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions. KW - cytotoxic T cells KW - lymphocyte activation KW - signal transduction KW - gene regulation KW - immune cells KW - NFATc1 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170353 VL - 8 IS - 511 ER - TY - JOUR A1 - Ferero, Andrea A1 - Rivero, Olga A1 - Wäldchen, Sina A1 - Ku, Hsing-Ping A1 - Kiser, Dominik P. A1 - Gärtner, Yvonne A1 - Pennington, Laura S. A1 - Waider, Jonas A1 - Gaspar, Patricia A1 - Jansch, Charline A1 - Edenhofer, Frank A1 - Resink, Thérèse J. A1 - Blum, Robert A1 - Sauer, Markus A1 - Lesch, Klaus-Peter T1 - Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain JF - Frontiers in Cellular Neuroscience N2 - Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding, and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mice display increased cell densities in the DR at embryonic stages E13.5, E17.5, and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that Cdh13 deficiency affects the cell density of the developing DR and the posterior innervation of the prefrontal cortex (PFC), and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders. KW - serotonin KW - cadherin-13 (CDH13) KW - T-cadherin KW - neurodevelopment KW - psychiatric disorders KW - radial glia KW - dorsal raphe KW - prefrontal cortex Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170313 VL - 11 IS - 307 ER - TY - JOUR A1 - Steijven, Karin A1 - Spaethe, Johannes A1 - Steffan-Dewenter, Ingolf A1 - Härtel, Stephan T1 - Learning performance and brain structure of artificially-reared honey bees fed with different quantities of food JF - PeerJ N2 - Background Artificial rearing of honey bee larvae is an established method which enables to fully standardize the rearing environment and to manipulate the supplied diet to the brood. However, there are no studies which compare learning performance or neuroanatomic differences of artificially-reared (in-lab) bees in comparison with their in-hive reared counterparts. Methods Here we tested how different quantities of food during larval development affect body size, brain morphology and learning ability of adult honey bees. We used in-lab rearing to be able to manipulate the total quantity of food consumed during larval development. After hatching, a subset of the bees was taken for which we made 3D reconstructions of the brains using confocal laser-scanning microscopy. Learning ability and memory formation of the remaining bees was tested in a differential olfactory conditioning experiment. Finally, we evaluated how bees reared with different quantities of artificial diet compared to in-hive reared bees. Results Thorax and head size of in-lab reared honey bees, when fed the standard diet of 160 µl or less, were slightly smaller than hive bees. The brain structure analyses showed that artificially reared bees had smaller mushroom body (MB) lateral calyces than their in-hive counterparts, independently of the quantity of food they received. However, they showed the same total brain size and the same associative learning ability as in-hive reared bees. In terms of mid-term memory, but not early long-term memory, they performed even better than the in-hive control. Discussion We have demonstrated that bees that are reared artificially (according to the Aupinel protocol) and kept in lab-conditions perform the same or even better than their in-hive sisters in an olfactory conditioning experiment even though their lateral calyces were consistently smaller at emergence. The applied combination of experimental manipulation during the larval phase plus subsequent behavioral and neuro-anatomic analyses is a powerful tool for basic and applied honey bee research. KW - nutrition KW - cognition KW - neuroanatomy KW - differential olfactory conditioning KW - mushroom bodies KW - proboscis extension reflex KW - confocal laser scanning microscopy KW - Apis mellifera KW - brain development KW - morphometry Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170137 VL - 5 IS - e3858 ER - TY - JOUR A1 - Beer, Katharina A1 - Joschinski, Jens A1 - Sastre, Alazne Arrazola A1 - Krauss, Jochen A1 - Helfrich-Förster, Charlotte T1 - A damping circadian clock drives weak oscillations in metabolism and locomotor activity of aphids (Acyrthosiphon pisum) JF - Scientific Reports N2 - Timing seasonal events, like reproduction or diapause, is crucial for the survival of many species. Global change causes phenologies worldwide to shift, which requires a mechanistic explanation of seasonal time measurement. Day length (photoperiod) is a reliable indicator of winter arrival, but it remains unclear how exactly species measure day length. A reference for time of day could be provided by a circadian clock, by an hourglass clock, or, as some newer models suggest, by a damped circadian clock. However, damping of clock outputs has so far been rarely observed. To study putative clock outputs of Acyrthosiphon pisum aphids, we raised individual nymphs on coloured artificial diet, and measured rhythms in metabolic activity in light-dark illumination cycles of 16:08 hours (LD) and constant conditions (DD). In addition, we kept individuals in a novel monitoring setup and measured locomotor activity. We found that A. pisum is day-active in LD, potentially with a bimodal distribution. In constant darkness rhythmicity of locomotor behaviour persisted in some individuals, but patterns were mostly complex with several predominant periods. Metabolic activity, on the other hand, damped quickly. A damped circadian clock, potentially driven by multiple oscillator populations, is the most likely explanation of our results. KW - circadian mechanisms KW - behavioural ecology KW - damped circadian clock KW - Acyrthosiphon pisum Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170020 VL - 7 IS - 14906 ER - TY - JOUR A1 - Chen, Yi-chun A1 - Mishra, Dushyant A1 - Gläß, Sebastian A1 - Gerber, Bertram T1 - Behavioral Evidence for Enhanced Processing of the Minor Component of Binary Odor Mixtures in Larval Drosophila JF - Frontiers in Psychology N2 - A fundamental problem in deciding between mutually exclusive options is that the decision needs to be categorical although the properties of the options often differ but in grade. We developed an experimental handle to study this aspect of behavior organization. Larval Drosophila were trained such that in one set of animals odor A was rewarded, but odor B was not (A+/B), whereas a second set of animals was trained reciprocally (A/B+). We then measured the preference of the larvae either for A, or for B, or for “morphed” mixtures of A and B, that is for mixtures differing in the ratio of the two components. As expected, the larvae showed higher preference when only the previously rewarded odor was presented than when only the previously unrewarded odor was presented. For mixtures of A and B that differed in the ratio of the two components, the major component dominated preference behavior—but it dominated less than expected from a linear relationship between mixture ratio and preference behavior. This suggests that a minor component can have an enhanced impact in a mixture, relative to such a linear expectation. The current paradigm may prove useful in understanding how nervous systems generate discrete outputs in the face of inputs that differ only gradually. KW - learning KW - memory KW - perception KW - compound conditioning KW - decision-making KW - Drosophila Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170011 VL - 8 IS - 1923 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 - THES A1 - Dirks, Johannes T1 - Charakterisierung der Wechselwirkung zwischen N-Myc und Aurora-A im MYCN-amplifizierten Neuroblastom T1 - Characterization of the Interaction between N-Myc and Aurora-A in MYCN amplified Neuroblastomas N2 - Im Neuroblastom ist die Amplifikation des MYCN-Gens, eines Mitglieds der MYC-Onkogenfamilie, mit einer ungünstigen Prognose assoziiert. Der von dem Gen kodierte Transkriptionsfaktor N-Myc ist für die Proliferation der MYCN-amplifizierten Neuroblastomzelllinien notwendig und seine Depletion oder Destabilisierung führen zum Proliferationsarrest (Otto et al., 2009). Da N-Myc auf Proteinebene durch die Interaktion mit der mitotischen Kinase Aurora-A stabilisiert wird, bewirkt deren Depletion oder die Hemmung der Interaktion der beiden Proteine mittels spezieller Aurora- A-Inhibitoren (z.B. MLN8054 und MLN8237) ebenso eine Hemmung der Proliferation – in vitro und in vivo (Brockmann et al., 2013). Bisher ist jedoch unklar, über welchen Mechanismus Aurora-A die Stabilisierung von N-Myc erreicht, die Kinaseaktivität spielt hierbei jedoch keine Rolle (Otto et al., 2009). Eine Möglichkeit stellt die Rekrutierung von Usps dar, die das angehängte Ubiquitinsignal so modifizieren, dass die Erkennung und der Abbau des Proteins durch das Proteasom verringert werden. In der vorliegenden Arbeit wurde die Wirkung von Usp7 und Usp11 auf die Stabilität von N-Myc untersucht. Für beide konnte in Immunpräzipitationen die Interaktion mit N-Myc gezeigt werden. Ebenso erhöhten beide Proteasen in Überexpressionsexperimenten die vorhandene Menge an NMyc. Die Depletion von Usp7 mittels shRNAs führte in IMR-32 zu einem Arrest in der G1-Phase und zur Differenzierung der Zellen. Gleichzeitig wurden stark erniedrigte mRNA- und Proteinmengen von N-Myc und Aurora-A nachgewiesen. Es konnte jedoch nicht eindeutig gezeigt werden, ob die beobachteten zellulären Effekte durch eine vermehrte proteasomale Degradation von N-Myc begründet sind oder ob dabei die veränderte Regulation weiterer Zielproteine von Usp7 eine Rolle spielt. Die Depletion von Usp11 mit shRNAs bewirkte eine Abnahme der N-Myc-Mengen auf posttranslationaler Ebene. Somit stellen beide Usps vielversprechende Angriffspunkte einer gezielten Therapie in MYCN-amplifizierten Neuroblastomen dar und sollten deshalb Gegenstand weiterführender Untersuchungen sein. Über welche Proteindomäne in N-Myc die Interaktion mit Aurora-A stattfindet ist nicht bekannt. Eine mögliche Pseudosubstratbindungssequenz in Myc-Box I (Idee Richard Bayliss, University of Leicester) wurde in der vorliegenden Arbeit untersucht. Durch Mutation dieser Sequenz sollte die Bindung von Aurora-A unmöglich gemacht werden. Allerdings wurde die erwartete Abnahme der Stärke der Interaktion von Aurora-A und N-Myc durch die Mutation ebensowenig beobachtet wie eine verringerte Stabilität. Die Regulation der Phosphorylierung von N-Myc im Verlauf des Zellzyklus wurde durch die Mutation beeinträchtigt. Wie diese Veränderung exakt zu begründen ist bedarf weiterer Experimente N2 - Neuroblastomas with an amplification of the MYCN-gene, a member of the MYC-oncogene family, are associated with a poor prognosis. The transcription factor encoded by this gene, N-Myc, is essential for the proliferation of MYCN-amplified neuroblastoma cell lines and its depletion or destabilization leads to an arrest of proliferation (Otto et al., 2009). Since N-Myc is stabilized by the interaction with the mitotic kinase Aurora-A, the depletion of the kinase or the inhibition of the interaction with N-Myc using a special class of Aurora-A inhibitors (e.g. MLN8054 and MLN8237) inhibits proliferation – in vitro and in vivo (Brockmann et al., 2013). Up to date it is not known by which mechanism Aurora-A is able to stabilize N-Myc preventing it from Fbxw7-mediated proteasomal degradation, interestingly the Aurora-A kinase activity is not necessary (Otto et al., 2009). One possible explanation is the recruitment of Usps, which modify the attached ubiquitin signal and therefore reduce the recognition and degradation of the protein by the proteasome. In this thesis the influence of Usp7 and Usp11 on N-Myc stability was studied. For both the interaction with N-Myc was shown in immune precipitations. Furthermore the overexpression of both proteases increased the amount of N-Myc protein in transfection experiments. The depletion of Usp7 via shRNAs caused the arrest of IMR-32 cells in G1-phase and the differentiation of the cells. Simultaneously strongly reduced amounts of N-Myc and Aurora-A mRNA and proteins were observed. However it could not be shown that the observed effects were mediated by an increased proteasomal degradation of N-Myc and not via the changed regulation of other targets of Usp7. The depletion of Usp11 led to a decrease of the N-Myc amounts, whereas mRNA-levels were unaffected. Thus both Usps are promising targets for a targeted therapy of MYCN-amplified neuroblastomas and the underlying mechanism should be the object of further research. Furthermore the N-Myc domain binding to Aurora-A still remains to be idientified. A possible pseudosubstrate binding site in Myc-Box I (idea of Richard Bayliss, University of Leicester) was investigated in this thesis. To inhibit the possible binding of Aurora-A to this site, two lysines in Myc-Box I were mutated to glutamate (KK51EE). Nonetheless neither the expected decrease of the intensity of interaction of N-Myc and Aurora-A was observed nor was a decrease of the stability of N-Myc. The regulation of the phosphorylation of N-Myc during the cell cycle was changed through the mutagenesis however. It must be clarified in further experiments, what the reasons for this change are. KW - Neuroblastom KW - N-Myc KW - Aurora-A Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186600 ER - TY - JOUR A1 - Stein, Katharina A1 - Coulibaly, Drissa A1 - Stenchly, Kathrin A1 - Goetze, Dethardt A1 - Porembski, Stefan A1 - Lindner, André A1 - Konaté, Souleymane A1 - Linsenmair, Eduard K. T1 - Bee pollination increases yield quantity and quality of cash crops in Burkina Faso, West Africa JF - Scientific Reports N2 - Mutualistic biotic interactions as among flowering plants and their animal pollinators are a key component of biodiversity. Pollination, especially by insects, is a key element in ecosystem functioning, and hence constitutes an ecosystem service of global importance. Not only sexual reproduction of plants is ensured, but also yields are stabilized and genetic variability of crops is maintained, counteracting inbreeding depression and facilitating system resilience. While experiencing rapid environmental change, there is an increased demand for food and income security, especially in sub-Saharan communities, which are highly dependent on small scale agriculture. By combining exclusion experiments, pollinator surveys and field manipulations, this study for the first time quantifies the contribution of bee pollinators to smallholders’ production of the major cash crops, cotton and sesame, in Burkina Faso. Pollination by honeybees and wild bees significantly increased yield quantity and quality on average up to 62%, while exclusion of pollinators caused an average yield gap of 37% in cotton and 59% in sesame. Self-pollination revealed inbreeding depression effects on fruit set and low germination rates in the F1-generation. Our results highlight potential negative consequences of any pollinator decline, provoking risks to agriculture and compromising crop yields in sub-Saharan West Africa. KW - bees KW - pollination KW - Burkina Faso KW - cash crops KW - cotton KW - sesame Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-169914 VL - 7 IS - 17691 ER - TY - JOUR A1 - De Palma, Adriana A1 - Abrahamczyk, Stefan A1 - Aizen, Marcelo A. A1 - Albrecht, Matthias A1 - Basset, Yves A1 - Bates, Adam A1 - Blake, Robin J. A1 - Boutin, Céline A1 - Bugter, Rob A1 - Connop, Stuart A1 - Cruz-López, Leopoldo A1 - Cunningham, Saul A. A1 - Darvill, Ben A1 - Diekötter, Tim A1 - Dorn, Silvia A1 - Downing, Nicola A1 - Entling, Martin H. A1 - Farwig, Nina A1 - Felicioli, Antonio A1 - Fonte, Steven J. A1 - Fowler, Robert A1 - Franzen, Markus Franzén A1 - Goulson, Dave A1 - Grass, Ingo A1 - Hanley, Mick E. A1 - Hendrix, Stephen D. A1 - Herrmann, Farina A1 - Herzog, Felix A1 - Holzschuh, Andrea A1 - Jauker, Birgit A1 - Kessler, Michael A1 - Knight, M. E. A1 - Kruess, Andreas A1 - Lavelle, Patrick A1 - Le Féon, Violette A1 - Lentini, Pia A1 - Malone, Louise A. A1 - Marshall, Jon A1 - Martínez Pachón, Eliana A1 - McFrederick, Quinn S. A1 - Morales, Carolina L. A1 - Mudri-Stojnic, Sonja A1 - Nates-Parra, Guiomar A1 - Nilsson, Sven G. A1 - Öckinger, Erik A1 - Osgathorpe, Lynne A1 - Parra-H, Alejandro A1 - Peres, Carlos A. A1 - Persson, Anna S. A1 - Petanidou, Theodora A1 - Poveda, Katja A1 - Power, Eileen F. A1 - Quaranta, Marino A1 - Quintero, Carolina A1 - Rader, Romina A1 - Richards, Miriam H. A1 - Roulston, T’ai A1 - Rousseau, Laurent A1 - Sadler, Jonathan P. A1 - Samnegård, Ulrika A1 - Schellhorn, Nancy A. A1 - Schüepp, Christof A1 - Schweiger, Oliver A1 - Smith-Pardo, Allan H. A1 - Steffan-Dewenter, Ingolf A1 - Stout, Jane C. A1 - Tonietto, Rebecca K. A1 - Tscharntke, Teja A1 - Tylianakis, Jason M. A1 - Verboven, Hans A. F. A1 - Vergara, Carlos H. A1 - Verhulst, Jort A1 - Westphal, Catrin A1 - Yoon, Hyung Joo A1 - Purvis, Andy T1 - Predicting bee community responses to land-use changes: Effects of geographic and taxonomic biases JF - Scientific Reports N2 - Land-use change and intensification threaten bee populations worldwide, imperilling pollination services. Global models are needed to better characterise, project, and mitigate bees' responses to these human impacts. The available data are, however, geographically and taxonomically unrepresentative; most data are from North America and Western Europe, overrepresenting bumblebees and raising concerns that model results may not be generalizable to other regions and taxa. To assess whether the geographic and taxonomic biases of data could undermine effectiveness of models for conservation policy, we have collated from the published literature a global dataset of bee diversity at sites facing land-use change and intensification, and assess whether bee responses to these pressures vary across 11 regions (Western, Northern, Eastern and Southern Europe; North, Central and South America; Australia and New Zealand; South East Asia; Middle and Southern Africa) and between bumblebees and other bees. Our analyses highlight strong regionally-based responses of total abundance, species richness and Simpson's diversity to land use, caused by variation in the sensitivity of species and potentially in the nature of threats. These results suggest that global extrapolation of models based on geographically and taxonomically restricted data may underestimate the true uncertainty, increasing the risk of ecological surprises. KW - bee community KW - land-use change KW - intensification KW - geographic biases KW - taxonomic biases KW - global dataset Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167642 VL - 6 ER - TY - JOUR A1 - Deeleman-Reinhold, Christa L. A1 - Miller, Jeremy A1 - Floren, Andreas T1 - Depreissia decipiens, an enigmatic canopy spider from Borneo revisited (Araneae, Salticidae), with remarks on the distribution and diversity of canopy spiders in Sabah, Borneo JF - ZooKeys N2 - Depreissia is a little known genus comprising two hymenopteran-mimicking species, one found in Central Africa and one in the north of Borneo. The male of D. decipiens is redescribed, the female is described for the first time. The carapace is elongated, dorsally flattened and rhombus-shaped, the rear of the thorax laterally depressed and transformed, with a pair of deep pits; the pedicel is almost as long as the abdomen. The male palp is unusual, characterized by the transverse deeply split membranous tegulum separating a ventral part which bears a sclerotized tegular apophysis and a large dagger-like retrodirected median apophysis. The female epigyne consists of one pair of large adjacent spermathecae and very long copulatory ducts arising posteriorly and rising laterally alongside the spermathecae continuing in several vertical and horizontal coils over the anterior surface. Relationships within the Salticidae are discussed and an affinity with the Cocalodinae is suggested. Arguments are provided for a hypothesis that D. decipiens is not ant-mimicking as was previously believed, but is a mimic of polistinine wasps. The species was found in the canopy in the Kinabalu area only, in primary and old secondary rainforest at 200–700 m.a.s.l. Overlap of canopy-dwelling spider species with those in the understorey are discussed and examples of species richness and endemism in the canopy are highlighted. Canopy fogging is a very efficient method of collecting for most arthropods. The canopy fauna adds an extra dimension to the known biodiversity of the tropical rainforest. In southeast Asia, canopy research has been neglected, inhibiting evaluation of comparative results of this canopy project with that from other regions. More use of fogging as a collecting method would greatly improve insight into the actual species richness and species distribution in general. KW - depreissia decipiens KW - jumping spiders KW - canopy spiders KW - taxonomy KW - biodiversity KW - ant-mimicking spiders KW - wasp-mimicking KW - Mt. Kinabalu KW - rainforest KW - Cocalodinae KW - Polistine wasps KW - endemism Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168342 VL - 556 ER - TY - JOUR A1 - Schlinkert, Hella A1 - Ludwig, Martin A1 - Batáry, Péter A1 - Holzschuh, Andrea A1 - Kovács-Hostyánszki, Anikó A1 - Tscharntke, Teja A1 - Fischer, Christina T1 - Forest specialist and generalist small mammals in forest edges and hedges JF - Wildlife Biology N2 - Agricultural intensification often leads to fragmentation of natural habitats, such as forests, and thereby negatively affects forest specialist species. However, human introduced habitats, such as hedges, may counteract negative effects of forest fragmentation and increase dispersal, particularly of forest specialists. We studied effects of habitat type (forest edge versus hedge) and hedge isolation from forests (connected versus isolated hedge) in agricultural landscapes on abundance, species richness and community composition of mice, voles and shrews in forest edges and hedges. Simultaneously to these effects of forest edge/hedge type we analysed impacts of habitat structure, namely percentage of bare ground and forest edge/hedge width, on abundance, species richness and community composition of small mammals. Total abundance and forest specialist abundance (both driven by the most abundant species Myodes glareolus, bank vole) were higher in forest edges than in hedges, while hedge isolation had no effect. In contrast, abundance of habitat generalists was higher in isolated compared to connected hedges, with no effect of habitat type (forest edge versus hedge). Species richness as well as abundance of the most abundant habitat generalist Sorex araneus (common shrew), were not affected by habitat type or hedge isolation. Decreasing percentage of bare ground and increasing forest edge/hedge width was associated with increased abundance of forest specialists, while habitat structure was unrelated to species richness or abundance of any other group. Community composition was driven by forest specialists, which exceeded habitat generalist abundance in forest edges and connected hedges, while abundances were similar to each other in isolated hedges. Our results show that small mammal forest specialists prefer forest edges as habitats over hedges, while habitat generalists are able to use unoccupied ecological niches in isolated hedges. Consequently even isolated hedges can be marginal habitats for forest specialists and habitat generalists and thereby may increase regional farmland biodiversity. KW - forest specialists KW - forest fragmentation KW - forest hedges KW - forest edges Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168333 VL - 22 IS - 3 ER - TY - JOUR A1 - Biscotti, Maria Assunta A1 - Gerdol, Marco A1 - Canapa, Adriana A1 - Forconi, Mariko A1 - Olmo, Ettore A1 - Pallavicini, Alberto A1 - Barucca, Marco A1 - Schartl, Manfred T1 - The Lungfish Transcriptome: A Glimpse into Molecular Evolution Events at the Transition from Water to Land JF - Scientific Reports N2 - Lungfish and coelacanths are the only living sarcopterygian fish. The phylogenetic relationship of lungfish to the last common ancestor of tetrapods and their close morphological similarity to their fossil ancestors make this species uniquely interesting. However their genome size, the largest among vertebrates, is hampering the generation of a whole genome sequence. To provide a partial solution to the problem, a high-coverage lungfish reference transcriptome was generated and assembled. The present findings indicate that lungfish, not coelacanths, are the closest relatives to land-adapted vertebrates. Whereas protein-coding genes evolve at a very slow rate, possibly reflecting a “living fossil” status, transposable elements appear to be active and show high diversity, suggesting a role for them in the remarkable expansion of the lungfish genome. Analyses of single genes and gene families documented changes connected to the water to land transition and demonstrated the value of the lungfish reference transcriptome for comparative studies of vertebrate evolution. KW - lungfish KW - transcriptome KW - genome KW - sarcopterygian fish Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167753 VL - 6 IS - 21571 ER - TY - JOUR A1 - Pfeiffer, Susanne A1 - Krüger, Jacqueline A1 - Maierhofer, Anna A1 - Böttcher, Yvonne A1 - Klöting, Nora A1 - El Hajj, Nady A1 - Schleinitz, Dorit A1 - Schön, Michael R. A1 - Dietrich, Arne A1 - Fasshauer, Mathias A1 - Lohmann, Tobias A1 - Dreßler, Miriam A1 - Stumvoll, Michael A1 - Haaf, Thomas A1 - Blüher, Matthias A1 - Kovacs, Peter T1 - Hypoxia-inducible factor 3A gene expression and methylation in adipose tissue is related to adipose tissue dysfunction JF - Scientific Reports N2 - Recently, a genome-wide analysis identified DNA methylation of the HIF3A (hypoxia-inducible factor 3A) as strongest correlate of BMI. Here we tested the hypothesis that HIF3A mRNA expression and CpG-sites methylation in adipose tissue (AT) and genetic variants in HIF3A are related to parameters of AT distribution and function. In paired samples of subcutaneous AT (SAT) and visceral AT (VAT) from 603 individuals, we measured HIF3A mRNA expression and analyzed its correlation with obesity and related traits. In subgroups of individuals, we investigated the effects on HIF3A genetic variants on its AT expression (N = 603) and methylation of CpG-sites (N = 87). HIF3A expression was significantly higher in SAT compared to VAT and correlated with obesity and parameters of AT dysfunction (including CRP and leucocytes count). HIF3A methylation at cg22891070 was significantly higher in VAT compared to SAT and correlated with BMI, abdominal SAT and VAT area. Rs8102595 showed a nominal significant association with AT HIF3A methylation levels as well as with obesity and fat distribution. HIF3A expression and methylation in AT are fat depot specific, related to obesity and AT dysfunction. Our data support the hypothesis that HIF pathways may play an important role in the development of AT dysfunction in obesity. KW - gene expression KW - adipose KW - hypoxia-inducible factor 3A KW - adipose tissue dysfunction KW - obesity Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167662 VL - 6 IS - 27969 ER - TY - JOUR A1 - Jahn, Martin T. A1 - Markert, Sebastian M. A1 - Ryu, Taewoo A1 - Ravasi, Timothy A1 - Stigloher, Christian A1 - Hentschel, Ute A1 - Moitinho-Silva, Lucas T1 - Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling JF - Scientific Reports N2 - Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology. KW - high resolution visualisation KW - transcriptional profiling KW - FISH-CLEM KW - cell compartmentation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167513 VL - 6 IS - 35860 ER - TY - JOUR A1 - Weisschuh, Nicole A1 - Mayer, Anja K. A1 - Strom, Tim M. A1 - Kohl, Susanne A1 - Glöckle, Nicola A1 - Schubach, Max A1 - Andreasson, Sten A1 - Bernd, Antje A1 - Birch, David G. A1 - Hamel, Christian P. A1 - Heckenlively, John R. A1 - Jacobson, Samuel G. A1 - Kamme, Christina A1 - Kellner, Ulrich A1 - Kunstmann, Erdmute A1 - Maffei, Pietro A1 - Reiff, Charlotte M. A1 - Rohrschneider, Klaus A1 - Rosenberg, Thomas A1 - Rudolph, Günther A1 - Vámos, Rita A1 - Varsányi, Balázs A1 - Weleber, Richard G. A1 - Wissinger, Bernd T1 - Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing JF - PLoS ONE N2 - Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes. KW - mutation detection KW - retinal dystrophies KW - next generation sequencing Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167398 VL - 11 IS - 1 ER - TY - JOUR A1 - Thormann, Birthe A1 - Ahrens, Dirk A1 - Armijos, Diego Marín A1 - Peters, Marcell K. A1 - Wagner, Thomas A1 - Wägele, Johann W. T1 - Exploring the Leaf Beetle Fauna (Coleoptera: Chrysomelidae) of an Ecuadorian Mountain Forest Using DNA Barcoding JF - PLoS ONE N2 - Background Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates. Methodology/Principal Findings Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284–289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469–481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation. Conclusions/Significance Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing. KW - leaf beetle KW - Coleoptera: Chrysomelidae KW - Podocarpus National Park KW - DNA-based species delimitation KW - biodiversity Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167253 VL - 11 IS - 2 ER - TY - JOUR A1 - Hölldobler, Bert T1 - Queen Specific Exocrine Glands in Legionary Ants and Their Possible Function in Sexual Selection JF - PLoS ONE N2 - The colonies of army ants and some other legionary ant species have single, permanently wingless queens with massive post petioles and large gasters. Such highly modified queens are called dichthadiigynes. This paper presents the unusually rich exocrine gland endowment of dichthadiigynes, which is not found in queens of other ant species. It has been suggested these kinds of glands produce secretions that attract and maintain worker retinues around queens, especially during migration. However, large worker retinues also occur in non-legionary species whose queens do not have such an exuberance of exocrine glands. We argue and present evidence in support of our previously proposed hypothesis that the enormous outfit of exocrine glands found in dichthadiigynes is due to sexual selection mediated by workers as the main selecting agents KW - exocrine glands KW - dichthadiigynes KW - legionary ants KW - queens KW - sexual selection KW - army ants Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167057 VL - 11 IS - 3 ER - TY - JOUR A1 - Vogtmann, Emily A1 - Hua, Xing A1 - Zeller, Georg A1 - Sunagawa, Shinichi A1 - Voigt, Anita Y. A1 - Hercog, Rajna A1 - Goedert, James J. A1 - Shi, Jianxin A1 - Bork, Peer A1 - Sinha, Rashmi T1 - Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing JF - PLoS ONE N2 - Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing. KW - colorectal cancer KW - gut microbiota KW - whole-genome shotgun sequencing Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166904 VL - 11 IS - 5 ER - TY - THES A1 - Memmel, Simon T1 - Automatisierte Algorithmen zur Analyse der Migration und der strahleninduzierten DNA-Schäden humaner Glioblastomzellen nach kombinierter PI3K/mTOR/Hsp90-Inhibierung T1 - Automated algorithms for the analysis of cell migration and radiation induced DNA-damage in human glioblastoma cells after combined PI3K/mTOR/Hsp90 inhibition N2 - Das hohe invasive Potential und die starke Resistenz gegen Radio-/Chemotherapie von Glioblastoma multiforme (GBM) Zellen machen sie zu dem tödlichsten Tumor ihrer Art. Es ist deshalb von großem Interesse die Grundlagen, welche der Migrationsfähigkeit und DNA Reparatur zu Grunde liegen, besser zu verstehen. Im ersten Teil dieser Arbeit wurden zwei Algorithmen zur automatischen Analyse der Migration in der Einzelzellverfolgung und im Wundheilungsassay modifiziert. Die Auswertung der Daten konnte automatisch und somit schnell, effektiv und mit geringerem Arbeitsaufwand durchgeführt werden. Mit Hilfe dieser automatischen Algorithmen wurde die Migrationsfähigkeit von zwei GBM-Zelllinien (DK-MG und SNB19) untersucht. Zusätzlich wurde die konfokale Laserscanning- sowie die hochauflösende dSTORM-Fluoreszenzmikroskopie verwendet um die, der Zellbewegung zu Grunde liegende, Struktur des F Aktin und der fokalen Adhäsionskinase (FAK) aufzulösen und darzustellen. Unter Anwendung dieser genannten Methoden sind die Effekte des dualen PI3K/mTOR Inhibitors PI-103 alleine und in Kombination mit dem Hsp90 Inhibitor NVP AUY922 mit und ohne Bestrahlung auf die Bewegung untersucht worden. Es konnte festgestellt werden, dass sich beide Zelllinien deutlich in ihrem migratorischem Potential in vitro unterscheiden und zudem auch markante Unterschiede in ihrer Morphologie aufweisen. Die weniger invasiven DK MG-Zellen besitzen eine polarisierte Zellstruktur, wohingegen SNB19-Zellen sich durch multipolare ungerichtete Bewegung auszeichneten. Zudem wurde die Migration, durch PI3K/mTOR Inhibition mit PI-103 bei den DK-MG-Zellen (p53 wt, PTEN wt), sehr effektiv unterdrückt. Wohingegen sich die SNB19-Zellen (p53 mut, PTEN mut) resistent gegen diesen Inhibitor zeigten. Hsp90 Inhibition offenbarte in beiden Zelllinien einen starken inhibitorischen Effekt auf die Migration der Zellen sowie die Reorganisierung des F Aktinskelettes. In der zweiten Hälfte dieser Arbeit wurde ein Augenmerk auf die DNA-DSB-Reparatur der GBM Zellen nach ionisierender Strahlung gelegt. Zunächst wurde eine automatische Analysesoftware „FocAn-3D“ entwickelt, mit dessen Hilfe die DNA Doppelstrangbruchreparaturkinetik untersucht werden sollte. Diese Software ermöglicht es die gesamten Zellkerne mit ihren γH2AX-Foci in 3D-cLSM-Aufnahmen zu untersuchen. Es konnte somit eine Verbesserung der Genauigkeit in der Auszählung der γH2AX-Foci erreicht werden, welche 2D beschränkter Software verwehrt bleibt. Mit FocAn-3D konnte der gesamte Verlauf der Induktions- und Abbauphase der γH2AX-Foci in DK MG- und SNB19-Zellen mit einem mathematischen Modell ausgewertet und dargestellt werden. Des Weiteren wurde die Nanometerstruktur von γH2AX- und pDNA-PKcs-Foci mittels hochauflösender dSTORM-Mikroskopie untersucht. Konventionelle Mikroskopiemethoden, begrenzt durch das Beugungslimit und einer Auflösung von ~200 nm, konnten die Nanometerstruktur (<100 nm) der Reparaturfoci bisher nicht darstellen. Mit Hilfe der beugungsunbegrenzten dSTORM-Mikroskopie war es möglich in DK MG- und SNB19-Zellen die Nanometerstruktur genannten Reparaturproteine in den Foci mit einer Auflösung von bis zu ~20 nm darzustellen. γH2AX-Foci zeigten sich als eine Verteilung aus einzelnen Untereinheiten („Nanofoci“) mit einem Durchmesser von ~45 nm. Dies lässt die Vermutung zu, dass es sich hier um die elementare Substruktur der Foci und somit der γH2AX enthaltenen Nukleosome handelt. DNA-PK-Foci wiesen hingegen eine diffusere Verteilung auf. Die in dieser Arbeit ermittelten Unterschiede im Migrationsverhalten der Zellen rechtfertigen eine weitere präklinische Untersuchung der verwendeten Inhibitoren als potentielle Zelltherapeutika für die Behandlung von GBM. Zudem konnte sich dSTORM als machtvolles Hilfsmittel, sowohl zur Analyse der Migration zugrundeliegenden Zytoskelettstruktur und der Effekte der Hsp90 Inhibierung, als auch, der Nanostruktur der DNA-DSB-Reparaturfoci herausstellen. Es ist anzunehmen, dass beugungsunbegrenzte Mikroskopiemethoden sich als bedeutende Werkzeuge in der medizinischen und biologischen Erforschung der DNA-Reparaturmechanismen herausstellen werden. Das in dieser Arbeit entwickelte ImageJ Plugin „FocAn-3D“ bewies sich ebenfalls als ein vielversprechendes Werkzeug für die Analyse der Reparaturkinetik. Mit Hilfe von „FocAn-3D“ sollte es somit möglich sein u.a. den Einfluss gezielter Inhibition auf den zeitlichen Verlauf der Induktion und des Abbaus der DNA-Reparaturmaschinerie genauer zu studieren. N2 - The high invasive Potential and increased resistance to radio- and chemotherapy of glioblastoma multiforme (GBM) tumor cells make it the most lethal of all primary brain tumors. It is therefore of great interest to gain a better understanding of the mechanisms facilitating the migration and DNA repair. In the first part of this study, two algorithms for single cell tracking and wound healing assays were modified to increase effectiveness and speed of the automatic data analysis. The migratory capacity of the two GBM cell lines, DK MG and SNB19, were analyzed using these automatic algorithms. In addition, employing confocal microscopy and high resolution dSTORM imaging, the underlying F actin/FAK structure was resolved and studied. Together, these automatic algorithms enabled me to elucidate the effects of the dual PI3K/mTOR inhibitor PI 103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, focal adhesions and F-actin cytoskeleton of DK-MG and SNB19 cells. Both cell lines differ markedly in their migratory capacity in vitro and display distinctive differences in their morphology. The less invasive DK-MG cells retained their polarized structure, while SNB19 cells demonstrate multipolar morphology with random migration. The PI3K/mTOR Inhibition using PI-103 suppressed migration of the PTEN wt and p53 wt DK-MG cells but not of the PTEN mut and p53 mut SNB19 cells. In contrast, Hsp90 inhibition using NVP-AUY922 exerted a strong inhibitory effect on the migration in both cell lines as well as massive morphological changes and reorganization of the F-actin cytoskeleton. The second part of this study was designed to gain further insights in the DNA double strand break (DSB) repair of both GBM cell lines. The DNA DSB repair kinetics were analyzed using the novel software “FocAn-3D”. The software enables the 3D analysis of foci in entire nuclei using cLSM-imaging. This in turn results in increased accuracy of the foci counts, compared to approaches restricted to 2D. Using the new software approach, I was able to determine the whole γH2AX-foci induction and decay process and apply a well described mathematical model for the γH2AX-foci repair kinetics. Additionally, diffraction unlimited microscopy (dSTORM) was applied to resolve the nanometer scale of the foci forming repair proteins γH2AX and DNA-PK. Although conventional microscopy is able to reveal the repair foci as diffuse spots, the underlying protein distribution is well beyond the diffraction limit of ~200 nm. In this study, using the diffraction unlimited dSTORM microscopy with a lateral resolution of ~20 nm, it was possible to resolve the nanometer scale of both γH2AX and DNA-PK. γH2AX foci appeared not as diffuse spots, but rather as a distribution of distinct subunits (“nanofoci”). In contrast DNA-PK mostly showed a more diffuse distribution. The nanofoci diameter was about ~45 nm and it can be concluded that these clusters represent the elementary structural subunits of repair foci, the γH2AX-containing nucleosomes. Using the newly developed or modified algorithms for the analysis of cell migration, I was able to show a cell line specific response of the PI3K/mTOR inhibition on the cell migration. This warrants further preclinical trials for its potential as an anti-migratory agent in the treatment of GBM. In addition, dSTORM emerged as a powerful tool for the analysis of the cytoskeletal structure, underlying the cells migration capacity and the effects of Hsp90 inhibition. Also, dSTORM was able to unravel the elementary nanostructure of the DSB repair foci. This means diffraction unlimited single-molecule localization nanoscopy methods will likely emerge as powerful tools for the analysis of targeted inhibition on the DSB repair mechanisms. In addition, the newly developed software “FocAn-3D” showed promising results in the analysis of Foci kinetics. Consequently, it should enable the future study of targeted inhibition and its effects on foci induction and decay processes of the DNA repair. KW - Glioblastom KW - Zellmigration KW - DNS-Schädigung KW - Algorithmus KW - Automatisierung KW - PI3K/mTOR inhibierung KW - yH2AX-Foci KW - Dnaschaden KW - DNS-Doppelstrangbruch Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-185710 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 - JOUR A1 - Heurich, Marco A1 - Zeis, Klara A1 - Küchenhoff, Helmut A1 - Müller, Jörg A1 - Belotti, Elisa A1 - Bufka, Luděk A1 - Woelfing, Benno T1 - Selective Predation of a Stalking Predator on Ungulate Prey JF - PLoS ONE N2 - Prey selection is a key factor shaping animal populations and evolutionary dynamics. An optimal forager should target prey that offers the highest benefits in terms of energy content at the lowest costs. Predators are therefore expected to select for prey of optimal size. Stalking predators do not pursue their prey long, which may lead to a more random choice of prey individuals. Due to difficulties in assessing the composition of available prey populations, data on prey selection of stalking carnivores are still scarce. We show how the stalking predator Eurasian lynx (Lynx lynx) selects prey individuals based on species identity, age, sex and individual behaviour. To address the difficulties in assessing prey population structure, we confirm inferred selection patterns by using two independent data sets: (1) data of 387 documented kills of radio-collared lynx were compared to the prey population structure retrieved from systematic camera trapping using Manly’s standardized selection ratio alpha and (2) data on 120 radio-collared roe deer were analysed using a Cox proportional hazards model. Among the larger red deer prey, lynx selected against adult males—the largest and potentially most dangerous prey individuals. In roe deer lynx preyed selectively on males and did not select for a specific age class. Activity during high risk periods reduced the risk of falling victim to a lynx attack. Our results suggest that the stalking predator lynx actively selects for size, while prey behaviour induces selection by encounter and stalking success rates. KW - stalking predators KW - prey selection KW - Lynx lynx Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166827 VL - 11 IS - 8 ER - TY - JOUR A1 - Dreschers, Stephan A1 - Saupp, Peter A1 - Hornef, Mathias A1 - Prehn, Andrea A1 - Platen, Christopher A1 - Morschhäuser, Joachim A1 - Orlikowsky, Thorsten W. T1 - Reduced PICD in Monocytes Mounts Altered Neonate Immune Response to Candida albicans JF - PLoS ONE N2 - Background Invasive fungal infections with Candida albicans (C. albicans) occur frequently in extremely low birthweight (ELBW) infants and are associated with poor outcome. Phagocytosis of C.albicans initializes apoptosis in monocytes (phagocytosis induced cell death, PICD). PICD is reduced in neonatal cord blood monocytes (CBMO). Hypothesis Phagocytosis of C. albicans causes PICD which differs between neonatal monocytes (CBMO) and adult peripheral blood monocytes (PBMO) due to lower stimulation of TLR-mediated immune responses. Methods The ability to phagocytose C. albicans, expression of TLRs, the induction of apoptosis (assessment of sub-G1 and nick-strand breaks) were analyzed by FACS. TLR signalling was induced by agonists such as lipopolysaccharide (LPS), Pam3Cys, FSL-1 and Zymosan and blocked (neutralizing TLR2 antibodies and MYD88 inhibitor). Results Phagocytic indices of PBMO and CBMO were similar. Following stimulation with agonists and C. albicans induced up-regulation of TLR2 and consecutive phosphorylation of MAP kinase P38 and expression of TNF-α, which were stronger on PBMO compared to CBMO (p < 0.005). Downstream, TLR2 signalling initiated caspase-3-dependent PICD which was found reduced in CBMO (p < 0.05 vs PBMO). Conclusion Our data suggest direct involvement of TLR2-signalling in C. albicans-induced PICD in monocytes and an alteration of this pathway in CBMO. KW - Candida albicans KW - monocytes KW - immune response KW - PICD Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166778 VL - 11 IS - 11 ER - TY - JOUR A1 - Xu, Li A1 - He, Jianzheng A1 - Kaiser, Andrea A1 - Gräber, Nikolas A1 - Schläger, Laura A1 - Ritze, Yvonne A1 - Scholz, Henrike T1 - A Single Pair of Serotonergic Neurons Counteracts Serotonergic Inhibition of Ethanol Attraction in Drosophila JF - PLoS ONE N2 - Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. Here, we dissect the function of the key regulator of serotonin signaling—the serotonin transporter–in innate olfactory attraction to ethanol in Drosophila melanogaster. We generated a mutated version of the serotonin transporter that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. We identified four serotonergic neurons that inhibit the olfactory attraction to ethanol and two additional neurons that counteract this inhibition by strengthening olfactory information. Our results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior. KW - attraction KW - ethanol KW - Drosophila melanogaster KW - serotonin transporter Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166762 VL - 11 IS - 12 ER - TY - JOUR A1 - Künstner, Axel A1 - Hoffmann, Margarete A1 - Fraser, Bonnie A. A1 - Kottler, Verena A. A1 - Sharma, Eshita A1 - Weigel, Detlef A1 - Dreyer, Christine T1 - The Genome of the Trinidadian Guppy, Poecilia reticulata, and Variation in the Guanapo Population JF - PLoS ONE N2 - For over a century, the live bearing guppy, Poecilia reticulata, has been used to study sexual selection as well as local adaptation. Natural guppy populations differ in many traits that are of intuitively adaptive significance such as ornamentation, age at maturity, brood size and body shape. Water depth, light supply, food resources and predation regime shape these traits, and barrier waterfalls often separate contrasting environments in the same river. We have assembled and annotated the genome of an inbred single female from a high-predation site in the Guanapo drainage. The final assembly comprises 731.6 Mb with a scaffold N50 of 5.3 MB. Scaffolds were mapped to linkage groups, placing 95% of the genome assembly on the 22 autosomes and the X-chromosome. To investigate genetic variation in the population used for the genome assembly, we sequenced 10 wild caught male individuals. The identified 5 million SNPs correspond to an average nucleotide diversity (π) of 0.0025. The genome assembly and SNP map provide a rich resource for investigating adaptation to different predation regimes. In addition, comparisons with the genomes of other Poeciliid species, which differ greatly in mechanisms of sex determination and maternal resource allocation, as well as comparisons to other teleost genera can begin to reveal how live bearing evolved in teleost fish. KW - Trinidadian guppy KW - Poecilia reticulata KW - genetics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166755 VL - 11 IS - 12 ER - TY - THES A1 - Sauer, Mark T1 - Die microRNA-26 Familie kontrolliert über den REST-Komplex ein für die Neurogenese essentielles regulatorisches RNA Netzwerk T1 - The microRNA-26 family controls a regulatory RNA network which is essential for neurogenesis via the REST-complex N2 - In einem sich entwickelnden multizellulären Organismus ist die räumlich-zeitliche Regulation der Genexpression von entscheidender Bedeutung für die Bildung, Identität und Funktion von Zellen. Der REST (repressor element silencing transcription factor) Komplex spielt bei der neuronalen Differenzierung und bei der Aufrechterhaltung des neuronalen Status eine essentielle Rolle, indem er in nicht neuronalen Zellen und neuralen Vorläufern die Expression neuronaler Gene unterdrückt, in deren Promotorregion eine RE1 (repressor element 1) Erkennungssequenz vorhanden ist. Während der neuronalen Differenzierung wird der REST-Komplex schrittweise inaktiviert, was zur Einleitung eines neuronalen Genexpression-Programms führt. Es wird daher angenommen, dass die Inhibierung des REST-Komplexes ein essentieller Vorgang der Neurogenese ist. Wichtige Bestandteile für die transkriptionell repressive Funktion des REST-Komplexes sind kleine Phosphatasen (CTDSP = C-terminal domain small phosphatases), welche die Polymerase-II-Aktivität an Zielgenen inhibieren. Im Zebrafisch wurde gezeigt, dass ctdsp2 durch die miR-26b negativ reguliert wird. Alle miR-26 Familienmitglieder sind in Vertebraten evolutionär konserviert und in Introns von Ctdsp Genen kodiert. Sie sind in der Lage, die Expression ihres eigenen Wirtsgens mittels einer autoregulatorischen Rückkopplungsschleife zu regulieren. Im Rahmen dieser Dissertation wurde als Modellsystem für die Neurogenese ein neurales Differenzierungssystem, welches auf murinen, embryonalen Stammzellen (ESCs) aufbaut, eingesetzt. Zur funktionellen Analyse der miR-26 Familie wurden mit Hilfe der CRISPR/Cas9-Methode verschiedene miR-26 Knockout (KO) ESC-Linien hergestellt. Hierbei wurden die Sequenzen der einzelnen Familienmitglieder und der gesamten miR-26 Familie im Genom von Wildtyp (Wt) ESCs deletiert. Diese miR-26-defizienten ESCLinien behielten ihre Pluripotenz und zeigten keinen Phänotyp hinsichtlich Proliferation, Morphologie und Identität der Zellen während der Differenzierung bis zum neuralen Vorläuferzellstadium (NPCs, engl.: neural progenitor cells). Jedoch führte die Deletion sowohl der gesamten miR-26 Familie als auch einzelner Mitglieder bei der terminalen Differenzierung zu einem spezifischen Entwicklungsstillstand im NPC Stadium und infolgedessen zu einer starken Reduktion der Anzahl von Neuronen und Astroglia. Die Transkriptom-Analyse der differenzierten miR-26-KO ESCs mittels RNA-Seq zeigte, dass die Expression von Genen die mit der Neurogenese und der neuronalen Differenzierung, aber auch der Gliogenese assoziert sind, herunterreguliert war. Die Abwesenheit der miR-26 Familie führte außerdem zu einer selektiven Reduzierung bestimmter miRNAs (REST-miRs), die einerseits die Expression von REST-Komplex Komponenten unterdrücken können, und andererseits selbst unter dessen transkriptioneller Kontrolle stehen. Zu diesem REST-miR Netzwerk gehören einige miRNAs (miR-9, miR-124, miR-132 und miR-218), die wichtige Funktionen bei verschiedenen Prozessen der neuronalen Entwicklung haben. Weiterhin führte der miR-26-KO zu einer Derepression der Proteinlevel von REST und CTDSP2 während der terminalen Differenzierung. Funktionelle Analysen mit miRNA mimics zeigten, dass erhöhte miR-26 Level zu einer Hochregulation von REST-miRs führen. Weitere Experimente, die darauf zielten, die Hierarchie des REST-miR Netwerks aufzuklären zeigten, dass die miR-26 Familie stromaufwärts die REST-miR Expression reguliert. Zusammengefasst weisen die in dieser Arbeit gezeigten Daten darauf hin, dass die miR-26 Familie als Initiator der schrittweisen Inaktivierung des REST-Komplexes eine zentrale Rolle bei der Differenzierung von neuralen Vorläuferzellen zu postmitotischen Neuronen spielt. N2 - The spatio-temporal control of gene expression in a developing multicellular organism is a key determinant for the formation, cellular identity and function of cells. The REST (repressor element silencing transcription factor) complex plays a crucial role in the process of neuronal differentiation and the maintenance of the neuronal status by suppressing neuronal genes which contain a RE1 (repressor element 1) recognition sequence within their promotor region in non-neuronal cells or in neural progenitors. During neuronal differentiation, the REST complex is gradually inactivated, leading to the initiation of a neuronal gene expression program. It is therefore assumed that the regulation of the REST complex is an essential component for the initiation of neurogenesis. Critical effector proteins of the REST complex are small phosphatases (CTDSPs = C-terminal domain small phosphatases), which reduces the polymerase II activity on target genes. In zebrafish it was shown that the REST complex-associated phosphatase ctdsp2 is negatively regulated by miR-26b. All miR-26 family members are evolutionarily conserved in vertebrates and located in introns of Ctdsp genes. Furthermore the miR-26 family members repress their own host genes through an intrinsic autoregulatory negative feedback loop. In this study, a murine embryonic stem cell (ESC) -based neural differentiation paradigm was used as a model system for neurogenesis. To analyze the function of the miR-26 family, the CRISPR/Cas9 technology was employed to generate various miR-26 knockout (KO) ESC lines, with deletions of individual family members and the entire miR-26 family in the genome of ESCs. These miR-26-deficient ESCs retained their pluripotency and did not show altered proliferation, morphology, or cell identity during neural differentiation up to the neural progenitor cell (NPC) stage. However, deletion of the entire miR-26 family as well as of single members disrupted the terminal differentiation and led to a specific developmental arrest at the NPC stage and consequently a strong reduction of neuron and astroglia cell frequencies. Global gene expression analyses in differentiated miR-26-KO ESCs further revealed that genes, which are associated with neurogenesis, neuronal differentiation, but also gliogenesis, were downregulated. The absence of the miR-26 familiy resulted in the selective reduction of a specific set of miRNAs (REST-miRs), which on the one hand suppress the expression of REST complex components and on the other hand are themselves under the transcriptional control of the REST complex. Among others, several miRNAs (miR-9, miR-124, miR-132 and miR-218), which play an important role in various processes of neuronal development, belong to this REST-miR network. Moreover, the miR-26-KO led to the derepression of REST and CTDSP2 protein levels during terminal differentiation. Functional analyses with miRNA mimics showed that increased miR-26 levels resulted in an upregulation of REST-miRs. Further experiments aimed at elucidating the hierarchy of REST-miR regulation revealed that the miR-26 family act upstream to regulate RESTmiR expression and presumably has an initial function in the regulation of this network. Taken together, the data presented in this work suggest that the miR-26 family act as an initiator for the stepwise inactivation of the REST complex during neural differentiation. Therefore, these findings are consistent with the notion that the miR-26 family represents a central regulator for neural progenitor cell differentiation into postmitotic neurons. KW - Neurogenese KW - miR-26 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-184008 ER - TY - JOUR A1 - Vendelova, Emilia A1 - de Lima, Jeferson Camargo A1 - Lorenzatto, Karina Rodrigues A1 - Monteiro, Karina Mariante A1 - Mueller, Thomas A1 - Veepaschit, Jyotishman A1 - Grimm, Clemens A1 - Brehm, Klaus A1 - Hrčková, Gabriela A1 - Lutz, Manfred B. A1 - Ferreira, Henrique B. A1 - Nono, Justin Komguep T1 - Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions JF - PLoS Neglected Tropical Diseases N2 - Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts. KW - proteomic analysis KW - excretory-secretory KW - Mesocestoides corti Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166742 VL - 10 IS - 10 ER - TY - JOUR A1 - Widmann, Annekathrin A1 - Artinger, Marc A1 - Biesinger, Lukas A1 - Boepple, Kathrin A1 - Peters, Christina A1 - Schlechter, Jana A1 - Selcho, Mareike A1 - Thum, Andreas S. T1 - Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae JF - PLoS Genetics N2 - Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution. KW - genetic dissection KW - Drosophila KW - memory formation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166672 VL - 12 IS - 10 ER - TY - JOUR A1 - Schwarz, Roland F. A1 - Tamuri, Asif U. A1 - Kultys, Marek A1 - King, James A1 - Godwin, James A1 - Florescu, Ana M. A1 - Schultz, Jörg A1 - Goldman, Nick T1 - ALVIS: interactive non-aggregative visualization and explorative analysis of multiple sequence alignments JF - Nucleic Acids Research N2 - Sequence Logos and its variants are the most commonly used method for visualization of multiple sequence alignments (MSAs) and sequence motifs. They provide consensus-based summaries of the sequences in the alignment. Consequently, individual sequences cannot be identified in the visualization and covariant sites are not easily discernible. We recently proposed Sequence Bundles, a motif visualization technique that maintains a one-to-one relationship between sequences and their graphical representation and visualizes covariant sites. We here present Alvis, an open-source platform for the joint explorative analysis of MSAs and phylogenetic trees, employing Sequence Bundles as its main visualization method. Alvis combines the power of the visualization method with an interactive toolkit allowing detection of covariant sites, annotation of trees with synapomorphies and homoplasies, and motif detection. It also offers numerical analysis functionality, such as dimension reduction and classification. Alvis is user-friendly, highly customizable and can export results in publication-quality figures. It is available as a full-featured standalone version (http://www.bitbucket.org/rfs/alvis) and its Sequence Bundles visualization module is further available as a web application (http://science-practice.com/projects/sequence-bundles). KW - visualization KW - multiple sequence alignments KW - phylogenetic trees KW - Alvis Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166374 VL - 44 IS - 8 ER - TY - JOUR A1 - Letunic, Ivica A1 - Bork, Peer T1 - Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees JF - Nucleic Acids Research N2 - Interactive Tree Of Life (http://itol.embl.de) is a web-based tool for the display, manipulation and annotation of phylogenetic trees. It is freely available and open to everyone. The current version was completely redesigned and rewritten, utilizing current web technologies for speedy and streamlined processing. Numerous new features were introduced and several new data types are now supported. Trees with up to 100,000 leaves can now be efficiently displayed. Full interactive control over precise positioning of various annotation features and an unlimited number of datasets allow the easy creation of complex tree visualizations. iTOL 3 is the first tool which supports direct visualization of the recently proposed phylogenetic placements format. Finally, iTOL's account system has been redesigned to simplify the management of trees in user-defined workspaces and projects, as it is heavily used and currently handles already more than 500,000 trees from more than 10,000 individual users. KW - Interactive Tree Of Life (iTOL) KW - phylogenetic trees KW - visualization KW - tool Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166181 VL - 44 IS - W1 ER - TY - JOUR A1 - Benoit, Joshua B. A1 - Adelman, Zach N. A1 - Reinhardt, Klaus A1 - Dolan, Amanda A1 - Poelchau, Monica A1 - Jennings, Emily C. A1 - Szuter, Elise M. A1 - Hagan, Richard W. A1 - Gujar, Hemant A1 - Shukla, Jayendra Nath A1 - Zhu, Fang A1 - Mohan, M. A1 - Nelson, David R. A1 - Rosendale, Andrew J. A1 - Derst, Christian A1 - Resnik, Valentina A1 - Wernig, Sebastian A1 - Menegazzi, Pamela A1 - Wegener, Christian A1 - Peschel, Nicolai A1 - Hendershot, Jacob M. A1 - Blenau, Wolfgang A1 - Predel, Reinhard A1 - Johnston, Paul R. A1 - Ioannidis, Panagiotis A1 - Waterhouse, Robert M. A1 - Nauen, Ralf A1 - Schorn, Corinna A1 - Ott, Mark-Christoph A1 - Maiwald, Frank A1 - Johnston, J. Spencer A1 - Gondhalekar, Ameya D. A1 - Scharf, Michael E. A1 - Raje, Kapil R. A1 - Hottel, Benjamin A. A1 - Armisén, David A1 - Crumière, Antonin Jean Johan A1 - Refki, Peter Nagui A1 - Santos, Maria Emilia A1 - Sghaier, Essia A1 - Viala, Sèverine A1 - Khila, Abderrahman A1 - Ahn, Seung-Joon A1 - Childers, Christopher A1 - Lee, Chien-Yueh A1 - Lin, Han A1 - Hughes, Daniel S.T. A1 - Duncan, Elizabeth J. A1 - Murali, Shwetha C. A1 - Qu, Jiaxin A1 - Dugan, Shannon A1 - Lee, Sandra L. A1 - Chao, Hsu A1 - Dinh, Huyen A1 - Han, Yi A1 - Doddapaneni, Harshavardhan A1 - Worley, Kim C. A1 - Muzny, Donna M. A1 - Wheeler, David A1 - Panfilio, Kristen A. A1 - Jentzsch, Iris M. Vargas A1 - Jentzsch, IMV A1 - Vargo, Edward L. A1 - Booth, Warren A1 - Friedrich, Markus A1 - Weirauch, Matthew T. A1 - Anderson, Michelle A.E. A1 - Jones, Jeffery W. A1 - Mittapalli, Omprakash A1 - Zhao, Chaoyang A1 - Zhou, Jing-Jiang A1 - Evans, Jay D. A1 - Attardo, Geoffrey M. A1 - Robertson, Hugh M. A1 - Zdobnov, Evgeny M. A1 - Ribeiro, Jose M.C. A1 - Gibbs, Richard A. A1 - Werren, John H. A1 - Palli, Subba R. A1 - Schal, Coby A1 - Richards, Stephen T1 - Unique features of a global human ectoparasite identified through sequencing of the bed bug genome JF - Nature Communications N2 - The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite. KW - human ectoparasite KW - bed bug KW - Cimex lectularius KW - genome Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166221 VL - 7 IS - 10165 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 - Wilde, Sabrina T1 - Einsatz von mechanistischen Biomarkern zur Charakterisierung und Bewertung von \(in\) \(vitro\) Genotoxinen T1 - Use of mechanistic biomarkers for the characterization and evaluation of \(in\) \(vitro\) genotoxins N2 - Die verfügbaren in vitro Genotoxizitätstests weisen hinsichtlich ihrer Spezifität und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschränkungen auf. Um diese Mängel zu überwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Prüfung auf Genotoxizität in der Arzneimittelentwicklung beitragen. 1. Etablierung und Bewertung einer neuen in vitro Genotoxizitätsmethode (MultiFlow Methode) Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbrüche), phosphorylierten H3 (S10) (mitotische Zellen), nukleären Protein p53 (Genotoxizität) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und ergänzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre Fähigkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen“ gegenüber der „alten“ Methode führte zu einer verbesserten Sensitivität von 95% gegenüber 90%, Spezifität von 90% gegenüber 72% und einer MoA-Klassifizierungsrate von 85% gegenüber 45% (Aneugen vs. Klastogen). 2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen Die Analyse 67 ausgewählter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen können. Die Kombination der höchstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 ermöglichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivität, Spezifität und Prädiktivität von 86%, 83% und 85%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizität mit TP53I3, Klastogenität mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann. Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen für BubR1 (S670) und pH3 (S28) mit Aneugenität, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenität, p53 (K373) mit Genotoxizität und Nrf2 (S40) mit oxidativem Stress identifiziert. Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinveränderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivitäten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden können und daher eine bessere Risikobewertung von Wirkstoffkandidaten ermöglichen. N2 - Available in vitro genotoxicity tests have limitations regarding their specificity and mode of action (MoA) information. To overcome these shortages, two objectives were pursued in this work to develop and establish new in vitro tools for genotoxicity testing. 1. Establishment and evaluation of a novel in vitro genotoxicity method (MultiFlow method) The MultiFlow method is based on DNA damage-related protein responses of γH2AX (DNA double-strand breaks), phosphorylated H3 (S10) (mitotic cells), nuclear protein p53 (genotoxicity) and cleaved PARP1 (apoptosis) in TK6 cells. In total, 31 model substances were studied flow cytometrically in the MultiFlow assay - and also with the well-established micronucleus test (MicroFlow MNT) - for their ability to classify across MoA groups: aneugens, clastogens and non-genotoxicants. The performance of the new method resulted in an improved sensitivity of 95% to 90%, specificity of 90% to 72% and a MoA classification rate of 85% to 45% (aneugen vs. clastogen). 2. Identification of mechanistic biomarkers for the characterization of genotoxicants The analysis of 67 selected DNA-damage associated genes using the QuantiGene Plex method showed that a combinaten of genes can contribute to MoA classification. The combination of the highest-ranked markers (BIK, KIF20A, TP53I3, DDB2 and OGG1) highlighted the best identification rate of model substances. The synergistic statistic tool correctly categorized 16 of 16 substances into aneugens, clastogens and non-genotoxicants. By using leave-one out cross validation, the model was evaluated and achieved a sensitivity, specificity and predictivity of 86%, 83%, 85% respectively. Follow-up with qPCR was conducted and revealed associations with TP53I3 for genotoxicity, ATR and RAD17 for clastogenicity and NFE2L2 for oxidative stress. By investigating posttranslational modifications using high-content imaging, associations for BubR1 (S670) and pH3 (S28) with aneugenicity, 53BP1 (S1778) and FANCD2 (S1404) with clastogenicity, p53 (K373) with genotoxicity and Nrf2 (S40) with oxidative stress were found to be further useful for MoA identification. This work demonstrates that genotoxicants and non-genotoxicants induce different gene- and protein expression changes in the TK6 cells that can be used to classify the MoA groups (aneugen/clastogen/non-genotoxicant/reactive oxygen species), thus enabling better risk assessment of potential drug candidates. KW - Genotoxizität KW - Genotoxicitiy KW - Klastogene KW - Aneugene KW - Biomarker KW - Klassifizierung KW - clastogens KW - aneugens KW - biomarker KW - classification Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-182782 ER - TY - THES A1 - Diegmann [geb. Weißbach], Susann T1 - Identifizierung des Mutationsspektrums und Charakterisierung relevanter Mutationen im Multiplen Myelom T1 - Identification of Mutation Spectrum and Characterization of relevant Mutations in Multiple Myeloma N2 - Das Multiple Myelom (MM) ist eine maligne B-Zell-Erkrankung, welche von einer großen Heterogenität auf der biologischen und klinischen Ebene sowie in der Therapieantwort geprägt ist. Durch die biologische Interpretation von whole exome sequencing (WES)-Daten der Tumor- und Normalproben von fünf MM-Patienten und sechs MM-Zelllinien (ZL) sowie dem Einbezug von publizierten next generation sequencing (NGS)-Daten von 38 MM-Patienten konnten in dieser Dissertation sowohl somatische tumorrelevante Mutationen identifiziert als auch ein MM-spezifisches Signaltransduktionsnetzwerk definiert werden. Interessanterweise wurde in fast 100 % der MM-Patienten mindestens eine Mutation und in ~50 % der MM-Patienten sogar mehr als eine Mutation innerhalb dieses Netzwerkes beobachtet, was auf eine inter- und intra-individuelle Signalweg-Redundanz hinweist, die für die individuelle Therapieentscheidung möglicherweise von Bedeutung sein könnte. Außerdem konnte bestätigt werden, dass identische, positionsspezifische und genspezifische Mutationen im MM selten wiederholt auftreten. Als häufig mutierte Gene im MM konnten KRAS, NRAS, LRP1B, FAM46C, WHSC1, ALOX12B, DIS3 und PKHD1 identifiziert werden. Interessanterweise wurde die DIS3-Mutation in der MM-ZL OPM2 gemeinsam mit einer copy neutral loss of heterozygosity (CNLOH) im DIS3-Lokus detektiert, und in der MM-ZL AMO1 wurde eine noch nicht näher charakterisierte KRAS-Mutation in Exon 4 in Verbindung mit einem copy number (CN)-Zugewinn und einer erhöhten KRAS-Genexpression gefunden. DIS3 ist ein enzymatisch aktiver Teil des humanen RNA-Exosom-Komplexes und KRAS ein zentrales Protein im RTK-Signalweg, wodurch genetische Aberrationen in diesen Genen möglicherweise in der Entstehung oder Progression des MMs eine zentrale Rolle spielen. Daher wurde die gesamte coding sequence (CDS) der Gene DIS3 und KRAS an Tumorproben eines einheitlich behandelten Patientensets der DSMM-XI-Studie mit einem Amplikon-Tiefen-Sequenzierungsansatz untersucht. Das Patientenset bestand aus 81 MM-Patienten mit verfügbaren zytogenetischen und klinischen Daten. Dies ergab Aufschluss über die Verteilung der Mutationen innerhalb der Gene und dem Vorkommen der Mutationen in Haupt- und Nebenklonen des Tumors. Des Weiteren wurde die Assoziation der Mutationen mit weiteren klassischen zytogenetischen Alterationen (z.B. Deletion von Chr 13q14, t(4;14)-Translokation) untersucht und der Einfluss der Mutationen in Haupt- und Nebenklonen auf den klinischen Verlauf und die Therapieantwort bestimmt. Besonders hervorzuheben war dabei die Entdeckung von sieben neuen Mutationen sowie drei zuvor unbeschriebenen hot spot-Mutationen an den Aminosäure (AS)-Positionen p.D488, p.E665 und p.R780 in DIS3. Es wurde des Weiteren die Assoziation von DIS3-Mutationen mit einer Chr 13q14-Deletion und mit IGH-Translokationen bestätigt. Interessanterweise wurde ein niedrigeres medianes overall survival (OS) für MM-Patienten mit einer DIS3-Mutation sowie auch eine schlechtere Therapieantwort für MM-Patienten mit einer DIS3-Mutation im Nebenklon im Vergleich zum Hauptklon beobachtet. In KRAS konnten die bereits publizierten Mutationen bestätigt und keine Auswirkungen der KRAS-Mutationen in Haupt- oder Nebenklon auf den klinischen Verlauf oder die Therapieantwort erkannt werden. Erste siRNA vermittelte knockdown-Experimente von KRAS und Überexpressionsexperimente von KRAS-Wildtyp (WT) und der KRAS-Mutationen p.G12A, p.A146T und p.A146V mittels lentiviraler Transfektion zeigten eine Abhängigkeit der Phosphorylierung von MEK1/2 und ERK1/2 von dem KRAS-Mutationsstatus. Zusammenfassend liefert die vorliegende Dissertation einen detaillierten Einblick in die molekularen Strukturen des MMs, vor allem im Hinblick auf die Rolle von DIS3 und KRAS bei der Tumorentwicklung und dem klinischen Verlauf. N2 - Multiple Myeloma (MM) is a malignant B-cell neoplasm that is characterized by a great heterogeneity on the biological and clinical level as well as by a heterogeneous response to therapeutic approaches. Biological interpretation of whole exome sequencing (WES) data of tumor and normal samples of five MM patients and six MM cell lines (CL), as well as the inclusion of published next generation sequencing (NGS) data of 38 MM patients, identified somatic tumor relevant mutations as well as a signal transduction network that was commonly affected in MM. Interestingly, almost 100 % of the MM patients harbored one mutation and ~50 % of the MM patients harbored more than one mutation in different genes of this defined network, which predicted an inter- and intra-individual pathway redundancy that might be of particular importance for individual therapeutic approaches. Furthermore, it was confirmed that the recurrent occurrence of point-specific mutations and even gene specific mutations are rare events in MM. KRAS, NRAS, LRP1B, FAM46C, WHSC1, ALOX12B, DIS3 and PKHD1 were among the most recurrently mutated genes in MM. Of note, one of the DIS3 mutations was accompanied by a copy neutral loss of heterozygosity (CNLOH) in the CL OPM2 and a so far undefined exon 4 mutation in KRAS was associated with an increased copy number (CN) and gene expression level of KRAS in the CL AMO1. DIS3 is one of the active parts of the human RNA exosome complex and KRAS is a central protein in the RTK pathway leading to the hypothesis that one or more genetic abberations within these genes may play an important role in the development and progression of MM. To further investigate this hypothesis the whole coding sequence (CDS) of DIS3 and KRAS of tumor samples of a uniquely treated patient set of the DSMM XI was sequenced using an amplicon deep sequencing approach. The study included 81 MM patients for whom cytogenetic and clinical data were available. This approach revealed information about the mutational landscape within DIS3 and KRAS and the occurrence of mutations in major and minor clones. In addition, we were able to investigate the association of the DIS3 and KRAS mutations with additional cytogenetic alterations (such as deletion of chr 13q14, translocation t(4;14)) and we studied the impact of mutations in major and minor clones on the clinical outcome and response to therapy. In particular, we discovered seven unknown mutations and three previously undescribed hot spot mutations at amino acid positions p.D488, p.E665 and p.R780 in DIS3. An association of DIS3 mutations with deletion of chr 13q14 and IGH-translocations, that was described previously, was confirmed. Interestingly, a trend towards a lower median overall survival of MM patients with a DIS3 mutation was observed. Patients with a DIS3 mutation in the minor clone also showed a worse response to therapy as compared to patients with a mutation in the major clone. Published mutations in KRAS were confirmed. Moreover, we revealed no impact of these mutations (in major or minor clones) on the clinical outcome or response to therapy. First siRNA mediated knockdown experiments on KRAS and lentivirus mediated overexpression of KRAS WT and mutated KRAS (p.G12A, p.A146T and p.A146V) showed that the phosphorylation status of MEK1/2 and ERK1/2 is dependent on the mutation status of KRAS. In summary, this present doctoral thesis allowed more detailed insights into the molecular structure of MM, specifically with regard to the role of DIS3 and KRAS in tumor development and outcome. KW - Plasmozytom KW - Multiples Myelom Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114800 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 -