TY - JOUR A1 - Seren, Ümit A1 - Grimm, Dominik A1 - Fitz, Joffrey A1 - Weigel, Detlef A1 - Nordborg, Magnus A1 - Borgwardt, Karsten A1 - Korte, Arthur T1 - AraPheno: a public database for Arabidopsis thaliana phenotypes JF - Nucleic Acids Research N2 - Natural genetic variation makes it possible to discover evolutionary changes that have been maintained in a population because they are advantageous. To understand genotype–phenotype relationships and to investigate trait architecture, the existence of both high-resolution genotypic and phenotypic data is necessary. Arabidopsis thaliana is a prime model for these purposes. This herb naturally occurs across much of the Eurasian continent and North America. Thus, it is exposed to a wide range of environmental factors and has been subject to natural selection under distinct conditions. Full genome sequencing data for more than 1000 different natural inbred lines are available, and this has encouraged the distributed generation of many types of phenotypic data. To leverage these data for meta analyses, AraPheno (https://arapheno.1001genomes.org) provide a central repository of population-scale phenotypes for A. thaliana inbred lines. AraPheno includes various features to easily access, download and visualize the phenotypic data. This will facilitate a comparative analysis of the many different types of phenotypic data, which is the base to further enhance our understanding of the genotype–phenotype map. KW - phenotype KW - arabidopsis KW - genotype Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147909 VL - 45 IS - D1 ER - TY - JOUR A1 - Bargul, Joel L. A1 - Jung, Jamin A1 - McOdimba, Francis A. A1 - Omogo, Collins O. A1 - Adung'a, Vincent O. A1 - Krüger, Timothy A1 - Masiga, Daniel K. A1 - Engstler, Markus T1 - Species-Specific Adaptations of Trypanosome Morphology and Motility to the Mammalian Host JF - PLoS Pathogens N2 - African trypanosomes thrive in the bloodstream and tissue spaces of a wide range of mammalian hosts. Infections of cattle cause an enormous socio-economic burden in sub-Saharan Africa. A hallmark of the trypanosome lifestyle is the flagellate’s incessant motion. This work details the cell motility behavior of the four livestock-parasites Trypanosoma vivax, T. brucei, T. evansi and T. congolense. The trypanosomes feature distinct swimming patterns, speeds and flagellar wave frequencies, although the basic mechanism of flagellar propulsion is conserved, as is shown by extended single flagellar beat analyses. Three-dimensional analyses of the trypanosomes expose a high degree of dynamic pleomorphism, typified by the ‘cellular waveform’. This is a product of the flagellar oscillation, the chirality of the flagellum attachment and the stiffness of the trypanosome cell body. The waveforms are characteristic for each trypanosome species and are influenced by changes of the microenvironment, such as differences in viscosity and the presence of confining obstacles. The distinct cellular waveforms may be reflective of the actual anatomical niches the parasites populate within their mammalian host. T. vivax displays waveforms optimally aligned to the topology of the bloodstream, while the two subspecies T. brucei and T. evansi feature distinct cellular waveforms, both additionally adapted to motion in more confined environments such as tissue spaces. T. congolense reveals a small and stiff waveform, which makes these parasites weak swimmers and destined for cell adherence in low flow areas of the circulation. Thus, our experiments show that the differential dissemination and annidation of trypanosomes in their mammalian hosts may depend on the distinct swimming capabilities of the parasites. KW - swimming KW - viscosity KW - flagella KW - host-pathogen interactions KW - cell motility KW - blood KW - parasitic diseases KW - trypanosoma brucei gambiense Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146513 VL - 12 IS - 2 ER - TY - JOUR A1 - Rosenbaum, Corinna A1 - Schick, Martin Alexander A1 - Wollborn, Jakob A1 - Heider, Andreas A1 - Scholz, Claus-Jürgen A1 - Cecil, Alexander A1 - Niesler, Beate A1 - Hirrlinger, Johannes A1 - Walles, Heike A1 - Metzger, Marco T1 - Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo JF - PLoS One N2 - Background Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network. Methods and Results In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response. Conclusion and Significance Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine. KW - gene expression KW - gastrointestinal tract KW - inflammatory bowel disease KW - central nervous system KW - systemic inflammatory response syndrome KW - inflammation KW - astrocytes KW - cytokines Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146544 VL - 11 IS - 3 ER - TY - JOUR A1 - Chen, Jiangtian A1 - Reiher, Wencke A1 - Hermann-Luibl, Christiane A1 - Sellami, Azza A1 - Cognigni, Paola A1 - Kondo, Shu A1 - Helfrich-Förster, Charlotte A1 - Veenstra, Jan A. A1 - Wegener, Christian T1 - Allatostatin A Signalling in Drosophila Regulates Feeding and Sleep and Is Modulated by PDF JF - PLoS Genetics N2 - Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF. KW - neurons KW - neuroimaging KW - circadian rhythms KW - food consumption KW - sleep KW - biological locomotion KW - Drosophila melanogaster KW - signal peptides Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178170 VL - 12 IS - 9 ER - TY - JOUR A1 - Djuzenova, Cholpon S. A1 - Fiedler, Vanessa A1 - Katzer, Astrid A1 - Michel, Konstanze A1 - Deckert, Stefanie A1 - Zimmermann, Heiko A1 - Sukhorukov, Vladimir L. A1 - Flentje, Michael T1 - Dual PI3K-and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule JF - Oncotarget N2 - Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells. KW - cell cycle arrest KW - radiation sensitivity KW - histone γH2AX KW - DNA damage KW - colony survival Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177770 VL - 7 IS - 25 ER - TY - JOUR A1 - Endres, Marcel A1 - Kneitz, Susanne A1 - Orth, Martin F. A1 - Perera, Ruwan K. A1 - Zernecke, Alma A1 - Butt, Elke T1 - Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1) JF - Oncotarget N2 - The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines. By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines. In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown. Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression. The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies. KW - LASP1 KW - c-Fos KW - extracellular matrix KW - AP-1 KW - matrix metalloproteinases KW - breast cancer Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176920 VL - 7 IS - 39 ER - TY - THES A1 - Hackl, Thomas T1 - A draft genome for the Venus flytrap, Dionaea muscipula : Evaluation of assembly strategies for a complex Genome – Development of novel approaches and bioinformatics solutions T1 - Ein Genom für die Venus Fliegenfalle, Dionaea muscipula N2 - The Venus flytrap, \textit{Dionaea muscipula}, with its carnivorous life-style and its highly specialized snap-traps has fascinated biologist since the days of Charles Darwin. The goal of the \textit{D. muscipula} genome project is to gain comprehensive insights into the genomic landscape of this remarkable plant. The genome of the diploid Venus flytrap with an estimated size between 2.6 Gbp to 3.0 Gbp is comparatively large and comprises more than 70 % of repetitive regions. Sequencing and assembly of genomes of this scale are even with state-of-the-art technology and software challenging. Initial sequencing and assembly of the genome was performed by the BGI (Beijing Genomics Institute) in 2011 resulting in a 3.7 Gbp draft assembly. I started my work with thorough assessment of the delivered assembly and data. My analysis showed that the BGI assembly is highly fragmented and at the same time artificially inflated due to overassembly of repetitive sequences. Furthermore, it only comprises about on third of the expected genes in full-length, rendering it inadequate for downstream analysis. In the following I sought to optimize the sequencing and assembly strategy to obtain an assembly of higher completeness and contiguity by improving data quality and assembly procedure and by developing tailored bioinformatics tools. Issues with technical biases and high levels of heterogeneity in the original data set were solved by sequencing additional short read libraries from high quality non-polymorphic DNA samples. To address contiguity and heterozygosity I examined numerous alternative assembly software packages and strategies and eventually identified ALLPATHS-LG as the most suited program for assembling the data at hand. Moreover, by utilizing digital normalization to reduce repetitive reads, I was able to substantially reduce computational demands while at the same time significantly increasing contiguity of the assembly. To improve repeat resolution and scaffolding, I started to explore the novel PacBio long read sequencing technology. Raw PacBio reads exhibit high error rates of 15 % impeding their use for assembly. To overcome this issue, I developed the PacBio hybrid correction pipeline proovread (Hackl et al., 2014). proovread uses high coverage Illumina read data in an iterative mapping-based consensus procedure to identify and remove errors present in raw PacBio reads. In terms of sensitivity and accuracy, proovread outperforms existing software. In contrast to other correction programs, which are incapable of handling data sets of the size of D. muscipula project, proovread’s flexible design allows for the efficient distribution of work load on high-performance computing clusters, thus enabling the correction of the Venus flytrap PacBio data set. Next to the assembly process itself, also the assessment of the large de novo draft assemblies, particularly with respect to coverage by available sequencing data, is difficult. While typical evaluation procedures rely on computationally extensive mapping approaches, I developed and implemented a set of tools that utilize k-mer coverage and derived values to efficiently compute coverage landscapes of large-scale assemblies and in addition allow for automated visualization of the of the obtained information in comprehensive plots. Using the developed tools to analyze preliminary assemblies and by combining my findings regarding optimizations of the assembly process, I was ultimately able to generate a high quality draft assembly for D. muscipula. I further refined the assembly by removal of redundant contigs resulting from separate assembly of heterozygous regions and additional scaffolding and gapclosing using corrected PacBio data. The final draft assembly comprises 86 × 10 3 scaffolds and has a total size of 1.45 Gbp. The difference to the estimated genomes size is well explained by collapsed repeats. At the same time, the assembly exhibits high fractions full-length gene models, corroborating the interpretation that the obtained draft assembly provides a complete and comprehensive reference for further exploration of the fascinating biology of the Venus flytrap. N2 - Die Venus Fliegenfalle, D. muscipula fasziniert aufgrund ihres karnivoren Lebensstil und ihrer hochspezialisierten Fallen Biologen schon seit der Zeit von Charles Darwins. Das Ziel des D. muscipula Genomprojekts ist es, neue Einblicke in den genomischen Grundlagen dieser besonderen Pflanze zu gewinnen. Die diploide Venus Fliegenfalle verfügt mit eine geschätzten Größe von 2.6 bp bis 3Gbp über ein vergleichsweise großes Genom, das zudem zu über 70% aus repetitiven Regionen besteht. Sequenzierung und Assembly von Genomen dieser Größenordnung stellen selbst mit neusten technischen und informatischen Methoden eine große Herausforderung dar. Zum ersten mal sequenziert und assembliert wurde das Genom 2011 durch das BGI (Beijing Genomics Institute). Meine Arbeit am Genom der Fliegenfalle begann mit der Analyse des 3.7Gbp großen Assemblies, welches wir vom BGI erhalten haben. Mit meinen Untersuchungen könnte ich zeigen, dass das Assembly stark fragmentiert und gleichzeitig durch überrepräsentierte repetitive Sequenzen stark aufgebläht ist. Darüberhinaus beinhaltet es gerade ein mal eine drittel der erwarteten Gene in Volllänge, wodurch es für die weiter Analyse ungeeignet ist. In meiner weiteren Arbeit habe ich mich daher darauf konzentriert, unsere Sequenzierungsund Assemblierungsstrategie zu verfeinern um ein stärker zusammenhängendes und vollständigeres Assembly zu erhalten. Dafür war es notwendig die Qualität der Sequenzierdaten so wie den Assemblierungsprozess selbst zu optimieren, und Programme zu entwickeln, die eine Verbesserung der Daten und eine Analyse der Zwischenergebnisse ermöglichen. So wurden etwa zur neue Bibliotheken von nicht-polymorphen DNA-Proben sequenziert um die Heterogenität im Datensatz zu verringern. Um die Kontinuität der Assemblies zu verbessern und Probleme mit der Heterozygosität der Daten zu lösen habe ich eine Reihe verschiedener Assemblierungsprogramme getestet. Dabei zeigte sich, dass das Programm ALLPATHS-LG am besten geeignet ist für die Assemblierung von D. muscipula Daten. Durch den Einsatz von digitaler Normalisierung konnte ich den Bedarf an Computerressourcen für einzelne Assemblierungen deutlich reduzieren und gleichzeitig die Kontinuität der Assemblies deutlich erhöhen. Zur besseren Auflösung repetitiver Strukturen im Genom, habe ich auf eine neu entwickelte Sequenziertechnologie von PacBio zurückgegriffen, die deutlich länger Sequenzen erzeugt. Um die neuen Daten trotz ihrer hohen Fehlerrate von 15% für Assemblierungen nutzen zu können, entwickelte ich das Korrekturprogramm proovread (Hackl et al., 2014). proovread nutzt kurze Illumina Sequenzen mit hoher Sequenziertiefe um innerhalb eines iterativen Prozess Fehler in PacBio Daten ausfindig zu machen und zu korrigieren. Das Programm erreicht dabei eine bessere Genauigkeit und eine höhere Sensitivität als vergleichbare Software. Darüber hinaus erlaubt sein flexibles Design auch Datensätze in der Größenordung des Fliegenfallengenoms effizient auf großen Rechenclustern zu bearbeiten. Neben dem Assemblierungsprozess an sich, stellt auch die Analyse von Assemblies großer Genome eine Herausforderung dar. Klassische Methoden basieren oft auf der rechenintensiven Berechnung von Alignments zwischen Sequenzierdaten und Assembly. Um vergleichbare Analysen deutlich schneller generieren zu können, habe ich Programme entwickelt die auf der Auswertung von k-mer Häufigkeiten beruhen, und die gewonnenen Ergebnisse in übersichtlichen Graphiken darstellen. Durch Kombination der so gewonnenen Einblicke und der verschiedenen Erkenntnisse bezüglich der Optimierung es Assemblierungsprozesses, war es mir am Ende möglich, ein Assembly von hoher Qualität für das Genom der Venus Fliegenfalle zu rekonstruieren. Dieses habe ich weiter verfeinert, unter anderem durch das Entfernen heterozygoter Sequenzen und durch das Flicken von Lücken mit Hilfe von PacBio Daten. Das so erstelle Assembly besteht aus 86 × 103 Sequenzen und hat eine Gesamtgröße von 1.45Gbp. Der Unterschied zur erwarteten Genomgröße lässt sich dabei gut durch kollabierte repetitive Regionen erklären. Gleichzeitig untermauert ein hoher Anteil an Volllängengenen im Assembly die Interpretation, dass das vorliegende Assembly eine vollständiges und umfassendes Abbild der D. muscipula Genom zeigt, und dass es sich damit als gute Grundlage für weitere Untersuchungen zur Biologie dieser faszinierenden Pflanze eignet. KW - Venusfliegenfalle KW - genome assembly KW - repeats KW - heterozygosity KW - pacbio correction KW - Genom Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133149 ER - TY - THES A1 - Appelt-Menzel, Antje T1 - Etablierung und Qualifizierung eines humanen Blut-Hirn-Schranken-Modells unter Verwendung von induziert pluripotenten und multipotenten Stammzellen T1 - Establishment and qualification of a human blood-brain barrier model by use of human induced pluripotent stemm cells an multipotent stem cells N2 - Die Blut-Hirn-Schranke (BHS) stellt eine der dichtesten und wichtigsten Barrieren zwischen Blutzirkulation und Zentralnervensystem (ZNS) dar. Sie besteht aus spezialisierten Endothelzellen, welche die zerebralen Kapillaren auskleiden und durch sehr dichte Tight Junctions (TJs) miteinander verbunden sind. Weitere Komponenten der dynamischen Blut-Hirn-Schrankenbarriere stellen Perizyten, Astrozyten, Neurone und Mikrogliazellen dar, welche zusammen mit der extrazellulären Matrix der Basalmembran der Gehirnkapillaren und den zuvor genannten Endothelzellen ein komplexes regulatorisches System, die so genannte neurovaskuläre Einheit bilden (Hawkins und Davis 2005). Die Hauptfunktionen der BHS lassen sich in drei Untergruppen untergliedern, die physikalische, metabolische und Transport-Barriere (Neuhaus und Noe 2010). Hauptsächlich dient die BHS der Aufrechterhaltung der Homöostase des ZNS und dem Schutz vor neurotoxischen Substanzen sowie Pathogenen, wie Bakterien und Viren. Zudem ist sie auch für die Versorgung der Neuronen mit Nährstoffen und regulierenden Substanzen sowie den Efflux von Stoffwechselendprodukten des ZNS zurück ins Blut verantwortlich. Für die Entwicklung von Medikamenten zur Behandlung von neurodegenerativen Erkrankungen, wie Morbus Alzheimer, Morbus Parkinson und Multiple Sklerose oder Gehirntumoren, stellt die Dichtigkeit der BHS gegenüber Substanzen und die hohe metabolische Aktivität der Endothelzellen aber ein großes Problem dar. Viele Medikamente sind nicht in der Lage in ausreichender Konzentration die BHS zu überwinden, um an ihren Wirkort zu gelangen oder werden vor dem Transport metabolisiert und die Wirksamkeit dadurch eingeschränkt. Weiterhin spielen auch Defekte der BHS eine entscheidende Rolle in der Beeinflussung der Pathogenese vieler ZNS-Erkrankungen. Aufgrund des hohen Bedarfs an geeigneten Testsystemen in der Grundlagen- sowie präklinischen Forschung für Medikamentenentwicklung und Infektionsstudien wurden eine Vielzahl unterschiedlicher BHS-Modelle entwickelt. Neben in silico-, azellulären in vitro- und in vivo-Modellen sind auch zahlreiche zellbasierte Modelle der BHS entwickelt worden. Standardisierte Modelle auf Basis immortalisierter Zelllinien jedoch weisen nur eine inhomogene TJ-Expression auf und verfügen meist über eine geringe Barriereintegrität, erfasst über transendotheliale elektrische Widerstände (TEER) unter 150 · cm2 (Deli et al. 2005). Im Vergleich dazu wurden in Tierexperimenten TEER-Werte von mehr als 1500 · cm2 an der BHS gemessen (Butt et al. 1990; Crone und Olesen 1982). Die Verfügbarkeit humaner primärer BHS-Zellen ist sehr limitiert und ihr Einsatz nicht nur im Hinblick auf ethische Aspekte bedenklich. Humane Gehirnzellen können z. B. aus Biopsie- oder Autopsiematerial von Patienten mit Epilepsie oder Gehirntumoren isoliert werden. Allerdings besteht hier das Risiko, dass die isolierten Zellen krankheitsbedingt verändert sind, was die Eigenschaften der BHS-Modelle erheblich beeinflussen kann. Eine Alternative, die diese Probleme umgeht, ist die Verwendung von humanen induziert pluripotenten Stammzellen (hiPSCs), um standardisierte humane BHS-Modelle unter reproduzierbaren Bedingungen bereitzustellen. Im Rahmen dieser Arbeit ist es gelungen, hiPSCs in vitro nach etablierten und standardisierten Methoden in Endothelzellen der BHS, neurale Stammzellen (hiPS-NSCs) sowie Astrozyten (hiPS-A) zu differenzieren (Lippmann et al. 2012; Lippmann et al. 2014; Wilson et al. 2015; Yan et al. 2013;Reinhardt et al. 2013) und zum Aufbau der Modelle einzusetzen. Die Endothelzellen wurden mit Hilfe protein- und genbasierter Nachweismethoden auf das Vorhandensein von endothelzellspezifischen TJ-Markern sowie spezifischen Transportern untersucht und funktionell charakterisiert. Die Kryokonservierung der hiPS-EC-Progenitoren, die im Rahmen der vorliegenden Arbeit entwickelt wurde, ermöglicht eine größere räumliche und zeitliche Flexibilität beim Arbeiten mit den stammzellbasierten Modellen sowie das Anlegen standardisierter Zellbanken. Weiterhin wurden multipotente NSCs aus fetalen Gehirnbiopsien isoliert (fNSCs) und als Kontrollkulturen zu den hiPS-NSCs für den Aufbau von BHS-Modellen eingesetzt. Mit dem Ziel die in vivo-BHS bestmöglich zu imitieren und die Modelleigenschaften zu optimieren, wurde ein Set aus zehn unterschiedlichen BHS-Modellen basierend auf primären Zellen, hiPSCs und fNSCs analysiert. Der Aufbau der BHS-Modelle erfolgte unter Verwendung von Transwellsystemen. Durch die systematische Untersuchung des Einflusses der unterschiedlichen Zelltypen der neurovaskulären Einheit auf die Barriereintegrität und Genexpression des BHS-Endothels, konnten die Quadrupel-Kulturen mit Perizyten, Astrozyten und hiPS-NSCs als die Kultur mit den physiologischsten Eigenschaften identifiziert werden. Auf Grund der signifikant erhöhten TEER-Werte von bis zu 2500 · cm2 und einer um mindestens 1,5-fachen Steigerung der Genexpression BHSrelevanter Transporter und TJ-Moleküle gegenüber den Monokulturen, wurden diese Modelle für weiterführende Studien ausgewählt. Das Vorhandensein eines komplexen, in vivo-ähnlichen TJ-Netzwerkes, bestehend aus Occludin, Claudin 1, 3, 4 und 5, konnte mittels quantitativer Realtime-PCR, Western Blot sowie ultrastruktureller Analyse in der Gefrierbruch- und Raster-Elektronenmikroskopie nachgewiesen werden. Neben der Begrenzung der parazellulären Permeabilität, welche über die geringe Permeation von FITC-Dextran (4 kDa und 40 kDa), Fluoreszein und Lucifer Yellow nachgewiesen wurde, stellt die BHS ebenfalls eine Barriere für den transzellulären Transport von Substanzen dar. Eine Beurteilung der Modelle hinsichtlich der Qualifikation für die Nutzung im Wirkstoffscreening wurde mit Hilfe von Transportversuchen unter dem Einsatz von BHS-relevanten Referenzsubstanzen durchgeführt. Die Klassifikation der Testsubstanzen erfolgte analog ihrer Permeationsgeschwindigkeiten: Diazepam und Koffein gelten als schnell transportierte Wirkstoffe, Ibuprofen, Celecoxib und Diclofenac werden mit einer mittleren Geschwindigkeit über die BHS transportiert und Loratadin sowie Rhodamin 123 sind langsam permeierende Substanzen. Innerhalb der Versuche mit den Quadrupelkulturen wurde diese Reihenfolge bestätigt, lediglich für Koffein wurde ein signifikant niedrigerer Permeationskoeffizient verglichen mit der Monokultur erzielt. Der Einsatz der hiPSC-Technologie ermöglicht es zudem, aus einer Stammzelllinie große Mengen an humanen somatischen Zelltypen zu generieren und für gezielte Anwendungen bereitzustellen. Es konnte im Rahmen dieser Arbeit gezeigt werden, dass mit Hilfe eines eigens für diese Zwecke konstruierten Rührreaktorsystems eine reproduzierbare Expansion der hiPSCs unter definierten Bedingungen ermöglicht wurde. Basierend auf dieser Grundlage ist nun ein Hochdurchsatz-Screening von Medikamenten denkbar. Die in dieser Arbeit präsentierten Daten belegen die Etablierung eines stammzellbasierten in vitro- Quadrupelmodels der humanen BHS, welches über in vivo-ähnliche Eigenschaften verfügt. Die Anforderungen, die an humane BHS-Modelle gestellt werden, wie die Reproduzierbarkeit der Ergebnisse, eine angemessene Charakterisierung, welche die Untersuchung der Permeabilität von Referenzsubstanzen einschließt, die Analyse der Expression von BHS-relevanten Transportermolekülen sowie die solide und physiologische Morphologie der Zellen, wurden erfüllt. Das etablierte BHS-Modell kann in der Pharmaindustrie für die Entwicklung von Medikamenten eingesetzt werden. Ausreichend qualifizierte Modelle können hier in der präklinischen Forschung genutzt werden, um Toxizitäts- und Transportstudien an neu entwickelten Substanzen durchzuführen und eine bessere in vitro-in vivo-Korrelation der Ergebnisse zu ermöglichen oder Mechanismen zu entwickeln, um die BHS-Barriere gezielt zu überwinden. N2 - The blood-brain barrier (BBB) presents one of the tightest and most important barriers between the blood circulation and the central nervous system (CNS). The BBB consists of specialized endothelial cells, which line the cerebral capillaries and are connected through very dense tight junctions (TJs). Together with pericytes, astrocytes, neurons, microglial cells and the extracellular matrix of the basal membrane of the brain capillaries, they form a dynamic and complex regulatory system, the so-called neurovascular unit (Hawkins and Davis 2005). The main functions of the BBB can be divided into three subgroups, the physical-, metabolic- and transport-barrier (Neuhaus and Noe 2010). The BBB mainly serves to maintain the homeostasis of the CNS and for protection against neurotoxical substances and pathogens, such as bacteria and viruses. Moreover, the BBB ensures the supply of neurons with nutrients and regulatory substances. Furthermore, it is responsible for the efflux of CNS metabolism waste products. For the development of drugs applied for the treatment of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Multiple Sclerosis or even brain tumors, the tightness of the BBB models towards substances and the high metabolic activity of the endothelial cells pose a problem. Numerous drugs cannot overcome the BBB in sufficient enough concentration to reach the target location or they are metabolized before transportation and thus become less effective. Moreover, defects of the BBB play a decisive role in the manipulation of the pathogenesis of numerous CNS diseases. Due to the high demand for test systems in basic and preclinical research of drug development and infection studies, a range of different BBB models have been developed. Besides the in silico, acellular in vitro and in vivo models, numerous cell-based BBB models have been developed. However, standardized models based on immortalized cell lines show only inhomogeneous TJ expression and possess low barrier integrity which is detected through transendothelial electrical resistance (TEER) below 150 · cm2 (Deli et al. 2005). In comparison, the TEER values in animal tests reached more than 1500 · cm2 at the BBB (Butt et al. 1990; Crone and Olesen 1982). The availability of human primary BBB cells is highly limited. Moreover, using human primary BBB cells is an extremely serious matter, not only in respect of ethical aspects. Human brain cells can, for instance, be isolated from biopsy or autopsy material obtained from patients suffering epilepsy or brain cancer. However, there is the risk that the isolated cells are altered due to disease, which may significantly change the features of the BBB models. An alternative to avoid such problems and to provide standardized human BBB models by the use of reproducible conditions, is the application of human induced pluripotent stem cells (hiPSCs). In this context, it has been successful to differentiate hiPSCs in vitro – under established and reproducible methods – into endothelial cells of the BBB (hiPS-ECs), neural stem cells (hiPS-NSCs) as well as astrocytes (hiPS-A) (Lippmann et al. 2012; Lippmann et al. 2014; Wilson et al. 2015; Yan et al. 2013; Reinhardt et al. 2013) and to use them for model establishment. The endothelial cells were examined for the existence and the functionality of endothelial-specific markers as well as specific transporters by protein- and gene-based methods. Within this work, the croypreservation of hiPS-EC progenitors was established. This will allow an increase of the spatial and temporal flexibility while working with the stem cell based models as well as the establishment of standardized cell banks. Furthermore, multipotent NSCs, isolated from fetal brain biopsies (fNSCs), were used as a control population for hiPSC-NSCs and for BBB modelling. In order to imitate the in vivo BBB in the best possible way and to optimize model characteristics, a set of ten different BBB models based on primary cells, hiPSCs and fNSCs was analyzed. Model establishment was done by the use of transwell systems. By the systematically analysis of the influence of the different neurovascular unit cell types on barrier integrity and on endothelial cell gene expression, the quadruple culture with pericytes, astrocytes and hiPS-NSCs was identified demonstrating the most physiological properties. Due to the significant increase of TEER results up to 2500 · cm2 as well as the at least 1.5-fold increase in gene expression of BBB relevant transporter and TJ markers compared to the mono-cultures, this model was selected for further studies. The presence of a complex in vivo-like TJ network, based on occludin, claudin 1, 3, 4 and 5 was detected by quantitative reale time PCR, Western blot analyses as well as on ultrastructural level by freeze fracture electron microscopy and transmission electron microscopy. Beside the limitation of the paracellular permeability, proven by the low permeation of FITC dextran (4 kDa and 40 kDa), fluorescein and Lucifer yellow, the BBB represents also a barrier for transcellular transported substances. A model evaluation, to assess the models qualification to be used for drug screenings, was proven by transport studies based on BBB relevant reference substances. The classification of the test substances was made analog their permeation rates: diazepam and caffeine are classified as fast, ibuprofen, celecoxib and diclofenac as medium, and loratadine and rhodamine 123 as slow permeating substances. Within our tests, this ranking based on literature data could be confirmed by using the quadruple-culture models, only caffeine was transported with a significantly decreased permeation coefficient compared to the mono-cultures. Furthermore, the implementation of the hiPSC technology allows the generation of a large quantity of human somatic cell types form only one single stem cell line and their provision for specific applications. Within this work it was shown, that by the use of an in-house constructed stirred tank bio-reactor, providing defined culture conditions, a reproducible expansion of hiPSCs was enabled. On this basis, a high throughput drug screening might be possible. The data presented within this work demonstrate the establishment of a stem cell based in vitro quadruple-model of the human BBB with in vivo-like characteristics. All minimal requirements for human BBB modeling, including the reproducibility of the results, adequate characterization with regard on the permeability of reference components, expression of BBB transporters as well as the robust and physiological morphology are fulfilled. The established BBB model can be used in pharmaceutical drug development. In preclinical research adequate qualified models are asked for toxicity and transport studies with new developed substances in order to allow a better in vitro-in vivo correlation of the results. Moreover, the model can be used to develop mechanisms to selectively overcome the barrier. KW - Blut-Hirn-Schranke KW - Stammzelle KW - Zelldifferenzierung KW - In vitro KW - Endothelzelle KW - induziert pluripotente Stammzelle KW - multipotente Stammzelle KW - in vitro Modell KW - Neurovaskuläre Einheit KW - Neurale Stammzellen Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134646 ER - TY - THES A1 - Imes, Dennis T1 - Aufklärung der molekularen Struktur und Funktion des R-Typ Anionenkanals QUAC1 in Schließzellen T1 - Molecular structure and function analyses of the R-type anion channel QUAC1 in guard cells N2 - Zum Gasaustausch mit Ihrer Umgebung besitzen höhere Pflanzen stomatäre Komplexe. Die Turgor-getrieben Atmungsöffnungen in der Epidermis der Blätter werden von zwei Schließzellen umsäumt. Um bei Trockenheit einen exzessiven Verlust von Wasser zu verhindern, synthetisieren/importieren Schließzellen das Stresshormon ABA (Abszisinsäure), das über eine schnelle ABA-Signalkaskade plasmamembrangebundene Ionenkanäle steuert. Dabei wird der Stomaschluss durch die Aktivität von R-(rapid) und S-(slow)Typ Anionenkanälen initiiert. Obwohl die R- und S-Typ Anionenströme in Schließzellen seit Jahrzehnten bekannt waren, konnte erst kürzlich das Gen identifiziert werden, das für den S-Typ Anionenkanal (SLAC1, Slow activating Anion Channel 1) kodiert. Daraufhin wurde schnell der Zusammenhang zwischen dem Stresshormon ABA, der ABA-Signalkette und der Aktivität des SLAC1 Anionenkanals im heterologen Expressionssystem der X. laevis Oozyten als auch in Schließzellprotoplasten aufgeklärt. Es konnte gezeigt werden, dass ABA durch einen zytosolischen Rezeptor/Phosphatasekomplex (RCAR1/ABI1) erkannt wird und die Aktivität von kalziumabhängigen Kinasen (CPK-Familie) sowie kalziumunabhängigen Kinasen der SnRK2-Familie (OST1) steuert. In Anwesenheit von ABA phosphorylieren diese Kinasen SLAC1 und sorgen so für die Aktivierung von Anionenströmen und damit für die Initiierung des Stomaschlusses. Die genetische Herkunft der ABA-induzierten R-Typ Ströme in Schließzellen war zu Beginn der vorliegenden Arbeit noch nicht bekannt. R-Typ Ströme zeichnen sich durch eine strikte Spannungsabhängigkeit und sehr schnellen Aktivierungs- sowie Deaktivierungskinetiken aus. Die Charakterisierung von Verlustmutanten des Schließzell-exprimierten Gens ALMT12 (Aluminium-aktivierter Malattransporter 12) konnte in Zusammenarbeit mit der Arbeitsgruppe Martinoia (Zürich) erste Hinweise auf die Beteiligung dieses Gens an der Stomabewegung demonstrieren. Anschließende Patch-Clamp Untersuchungen an Schließzellprotoplasten aus Wildtyppflanzen und ALMT12-Verlustmutanten zeigten, dass ALMT12 für die Malat-aktivierte R-Typ Anionenstromkomponente verantwortlich ist. Deshalb wurde der Anionenkanal QUAC1 (Quickly activating Anion Channel 1) benannt - in Anlehnung an die Benennung des Anionenkanals SLAC1. Mit der Identifizierung von QUAC1 in planta war es nun meine Aufgabe, die elektrischen Eigenschaften von ALMT12/QUAC1 und dessen Aktivitätskontrolle durch die ABA-Signalkaskade im heterologen Expressionssystem der Xenopus Oozyten zu untersuchen. Protein-Protein Interaktionsstudien mit der Hilfe der Bimolekularen Fluoreszenz-Technik, sowie die Beobachtung von markant erhöhten QUAC1 Anionenströmen in Anwesenheit der SnRK2 Kinase OST1 und den Calcium-abhängigen Kinasen CPK2 und CPK20, ließen den Schluss zu, dass QUAC1, ebenso wie SLAC1, unter der Kontrolle des schnellen ABA-Signalwegs steht. Eine zusätzliche Expression des negativen Regulators ABI1 unterdrückte die aktivierenden Eigenschaften der QUAC1-aktivierenden Kinasen, was die Hypothese der Koregulation von S- und R-Typ Anionenkanälen durch die gleiche ABA-Signalkaskade weiter unterstützt. Zur weiteren Aufklärung der elektrischen Eigenschaften von QUAC1 wurden tiefgreifende elektrophysiologische Untersuchungen mit der Zwei-Elektroden-Spannungsklemmen Technik durchgeführt. Durch die Wahl von geschickten Spannungsprotokollen konnte sowohl die schnelle Aktivierungskinetik als auch die schnelle Deaktivierungskinetik von QUAC1 bestimmt und quantifiziert werden. Diese Stromantworten waren sehr ähnlich zu den R-Typ Strömen, die man von Patch-Clamp Untersuchungen an Schließzellprotoplasten kannte, was ein weiteres Indiz dafür war, dass es sich bei QUAC1 tatsächlich um eine Komponente des R-Typ Kanals aus Schließzellen handelt. Weiterführende Untersuchungen bezüglich der Spannungsabhängigkeit und der Selektivität von QUAC1 charakterisierten das Protein als einen Depolarisations-aktivierten Anionenkanal mit einer starken Präferenz für Dicarbonsäuren wie Malat und Fumarat. Zudem konnte auch eine Leitfähigkeit für Sulfat und Chlorid nachgewiesen werden. Interessanterweise erwies sich Malat nicht nur als ein permeierendes Ion, sondern auch als ein regulierendes Ion, welches das spannungsabhängige Schalten von QUAC1 maßgeblich beeinflusst. Extrazelluläres Malat verschob die Offenwahrscheinlichkeit von QUAC1 sehr stark zu negativeren Membranspannungen, so dass der Anionenkanal bereits bei typischen Ruhespannungen von Schließzellen (ca. -150 mV) aktiviert werden konnte. Eine Beladung von QUAC1-exprimierender Oozyten mit Malat bewirkte zum einen höhere Anioneneffluxströme, aber auch eine Verschiebung der spannungsabhängigen Offenwahrscheinlichkeit zu negativeren Membranpotentialen. Struktur-Funktionsanalysen sollten die umstrittene Topologie von ALMT-ähnlichen Proteinen beleuchten und die molekulare Herkunft der Phosphorylierungsaktivierung aufzeigen, sowie die Malatabhängigkeit und die starke Spannungsabhängigkeit von QUAC1 aufklären. Es zeigte sich jedoch schnell, dass Punktmutationen und Deletionen im C-Terminus von QUAC1 sehr häufig zu nicht-funktionellen Mutanten führten. Diese Tatsache weist darauf hin, dass es sich um einen hoch-strukturierten und funktionell sehr wichtigen Bereich des Anionenkanals handelt. Auch die Topologie des Anionenkanalproteins wird in der Literatur kontrovers diskutiert. Sowohl die Lage des N- und C-Terminus (extrazellulär oder intrazellulär), als auch die Anzahl der membrandurchspannenden Domänen war nicht abschließend geklärt. Deshalb wurde in einem Fluoreszenz-basiertem Ansatz die Lage der Termini bestimmt. Im Rahmen meiner Arbeit konnte somit eindeutig gezeigt werden, dass sich beide Termini im Zytosol der Zelle befinden. Auf Grundlage von Modellen aus der Literatur und meiner Topologiebestimmungen konnte schließlich ein erweitertes Modell zur Struktur von QUAC1 entwickelt werden. Dieses Modell kann in Zukunft als Ausgangspunkt für weiterführende Struktur-Funktionsanalysen dienen. Diese Arbeit hat somit gezeigt, dass das Gen QUAC1 tatsächlich eine Komponente der R-Typ Ströme in Schließzellen kodiert. Ebenso wie SLAC1 steht der Malat-induzierte Anionenkanal QUAC1 unter der Kontrolle der schnellen ABA-Signalkaskade. In Zukunft bleibt zu klären, welche weiteren Gene für die R-Typ Kanalproteine in Schließzellen kodieren und welche strukturelle Grundlage für die besonderen Eigenschaften von QUAC1 hinsichtlich seiner schnellen Kinetiken, seiner Selektivität und Aktivierbarkeit durch Malat. N2 - Higher plants are able to exchange gases with their environment. This gas exchange is accomplished by the stomatal complex, which consist of two tugor-driven guard cells (GC) that surround a pore in the epidermis. Under drought conditions, guard cells produce and import the plant stress hormone abscisic acid (ABA). ABA is able to activate plasma membrane localized ion channels via the fast ABA-signal cascade, which leads to a closure of the stoma and thus minimizes the loss of water. The stomatal closure is initialized by the R-(rapid) and S-(slow) type anion channels. Although R- and S-type anion channels in guard cells have been known for over a decade, the gene which decodes the S-type anion channel SLAC1 (Slow activating Anion Channel 1) has only recently been identified. Consequently, the relationship between the plant hormone ABA, the ABA-signal-transduction-chain, and the activity of SLAC1 could be clarified in rapid succession in the heterologous expression system of X. laevis oocytes as well as in GC-protoplasts. It could be shown that ABA is recognized by a cytosolic receptor/phosphatase complex (RCAR/ABI1). This complex in turn regulates the activity of calcium dependent kinases of the CPK-family as well as the calcium independent kinases of the SnRK2-family (OST1). In the presence of ABA, these kinases activate SLAC1 by phosphorylation, and by this activate anion currents across the plasma membrane, ultimately leading to closure of the stomates. The genetic origin of the ABA induced R-type currents in guard cells was unknown at the beginning of this thesis. R-type currents are characterized by strong voltage-dependent behavior and fast activation- and deactivation-kinetics. In cooperation with the workgroup of Martinoia (Zürich), knock-out plants missing the guard cell gen ALMT12 (Aluminum activated Malate Transporter 12) were characterized. This work delivered the first hints that ALMT12 is involved in the stomatal movement. Subsequent patch-clamp studies on GC-protoplasts from WT and ALMT12 knock-out mutants revealed that ALMT12 is responsible for the malate-activated component of the R-type anion currents. Therefore, the anion-channel was named QUAC1 (Quick activating Anion Channel) in dependence on the naming of SLAC1. With the identification of QUAC1 in planta it was my duty to research the electrical properties of ALMT12/QUAC1 as well as the activation by the ABA-signal-transduction-chain in the heterologous expression system of X. laevis oocytes. Protein-protein interaction studies via bimolecular fluorescence complementation (BIFC) as well as significantly higher QUAC1 anion currents in the presence of the SnRK2 kinase OST1 and the calcium-dependent-kinases CPK2 and CPK20 led to the conclusion that QUAC1 is under the control of the fast ABA signaling pathway, as it was shown before for SLAC1. Furthermore expression of the negative regulator ABI1 inhibited the activating properties of the QUAC1-activating kinases. These findings support further the hypotheses of the simultaneous regulation of S- and R-type anion channels by the ABA-signaling pathway. To further elucidate the electrical properties of QUAC1, electrophysiological investigations were performed with the two-electrode-voltage-clamp technique (TEVC). In this way, the fast activation and deactivation of QUAC1 could be identified and quantified by carefully chosen voltage-clamp protocols. These current responses of QUAC1 closely resembled the R-type currents known from former patch-clamp studies from GC-protoplasts. This further supported the conclusion that QUAC1 is indeed a component of the R-type channels of guard cells. Additional investigations of the voltage-dependence and selectivity of QUAC1 characterized the protein as a depolarization-activated anion channel with strong preference for bicarbonate acids like malate and fumarate. Furthermore, a conductance for sulfate and chloride could also be shown. Interestingly, malate was not only able to permeate the channel, it was also able to alter the voltage-dependence of QUAC1. External malate strongly shifted the open probability of QUAC1 to negative membrane voltages. By this shift the anion channel could be activated at typical guard cell membrane potentials (approx. 150 mV). Loading of QUAC1 expressing oocytes with malate produced enhanced anion efflux currents and shift the voltage-dependent open probability to negative membrane potentials. Structure function analysis were performed to clarify the controversial topology of ALMT like proteins and the molecular origin of the phosphorylation activation. Furthermore, this should elucidate the origin of the malate dependence and the strong voltage dependence of QUAC1. It soon became evident that point mutations and deletions in the C-terminus of QUAC1 very often lead to nonfunctional mutants. This points toward a highly structured and functionally important region of the anion channel. In addition, the topology of the anion-channel-protein is controversially debated in literature. Neither the position of the C- and N-terminus (intra- or extracellular) nor the number of transmembrane domains has been conclusively established. Due to this, the position of the C- and N-termini were localized by a fluorescence based experiment. As part of this work, it could be shown explicitly that both termini reside in the cytosol of the cell. Based on models from the literature and my own topology studies, an enhanced structure model for QUAC1 could be generated. This model will serve as a starting point for future structure function analysis. This work has thus shown that the gene QUAC1 indeed encodes a component of the R-type currents in guard cells. Like SLAC1, the malate-induced anion channel QUAC1 is under the control of the fast ABA-signal-cascade. Future works must establish which further genes encode R-type channel proteins and which structural attributes are responsible for the special traits of QUAC1: its fast kinetics, its selectivity and its activation by malate. KW - Ackerschmalwand KW - Schließzelle KW - Anionentranslokator KW - Abscisinsäure KW - Struktur KW - Funktion KW - R-Typ KW - Anionenkanal KW - QUAC1 KW - TEVC Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136860 ER - TY - THES A1 - Pasch, Elisabeth T1 - The role of SUN4 and related proteins in sperm head formation and fertility T1 - Die Rolle von SUN4 und verwandten Proteinen in der Spermienkopfformierung und Fertilität N2 - Spermiogenesis describes the differentiation of haploid germ cells into motile, fertilization-competent spermatozoa. During this fundamental transition the species-specific sperm head is formed, which necessitates profound nuclear restructuring coincident with the assembly of sperm-specific structures and chromatin compaction. In the case of the mouse, it is characterized by reshaping of the early round spermatid nucleus into an elongated sickle-shaped sperm head. This tremendous shape change requires the transduction of cytoskeletal forces onto the nuclear envelope (NE) or even further into the nuclear interior. LINC (linkers of nucleoskeleton and cytoskeleton) complexes might be involved in this process, due to their general function in bridging the NE and thereby physically connecting the nucleus to the peripheral cytoskeleton. LINC complexes consist of inner nuclear membrane integral SUN-domain proteins and outer nuclear membrane KASH-domain counterparts. SUN- and KASH-domain proteins are directly connected to each other within the perinuclear space, and are thus capable of transferring forces across the NE. To date, these protein complexes are known for their essential functions in nuclear migration, anchoring and positioning of the nucleus, and even for chromosome movements and the maintenance of cell polarity and nuclear shape. In this study LINC complexes were investigated with regard to their potential role in sperm head formation, in order to gain further insight into the processes occurring during spermiogenesis. To this end, the behavior and function of the testis-specific SUN4 protein was studied. The SUN-domain protein SUN4, which had received limited characterization prior to this work, was found to be exclusively expressed in haploid stages during germ cell development. In these cell stages, it specifically localized to the posterior NE at regions decorated by the manchette, a spermatid-specific structure which was previously shown to be involved in nuclear shaping. Mice deficient for SUN4 exhibited severely disorganized manchette residues and gravely misshapen sperm heads. These defects resulted in a globozoospermia-like phenotype and male mice infertility. Therefore, SUN4 was not only found to be mandatory for the correct assembly and anchorage of the manchette, but also for the correct localization of SUN3 and Nesprin1, as well as of other NE components. Interaction studies revealed that SUN4 had the potential to interact with SUN3, Nesprin1, and itself, and as such is likely to build functional LINC complexes that anchor the manchette and transfer cytoskeletal forces onto the nucleus. Taken together, the severe impact of SUN4 deficiency on the nucleocytoplasmic junction during sperm development provided direct evidence for a crucial role of SUN4 and other LINC complex components in mammalian sperm head formation and fertility. N2 - Die Spermiogenese beschreibt die Differenzierung haploider Keimzellen zu beweglichen, fortpflanzungsfähigen Spermatozoen. Während dieses fundamentalen Entwicklungsabschnittes wird neben dem Aufbau von spermienspezifischen Strukturen und der Kompaktierung des Chromatins auch der speziesspezifische Spermienkopf geformt. Im Falle der Maus ist dies eine aktive Umformung des runden Zellkerns in einen gestreckten, sichelförmigen Spermienkopf. Eine derart gravierende Formveränderung erfordert eine Kraftweiterleitung aus dem Zytoskelett auf die Kernhülle und das Kerninnere. In diesem Zusammenhang könnten LINC (linkers of nucleoskeleton and cytoskeleton) Komplexe eine Rolle spielen, da ihre grundlegende Funktion darin besteht die Kernhülle zu überbrücken und somit den Kern mit dem peripheren Zytoskelett zu verbinden. LlNC Komplexe werden aus SUN und KASH Domänen Proteinen aufgebaut, welche in die innere beziehungsweise äußere Kernmembran eingelagert sind. Diese membranintegralen Proteine sind direkt miteinander verbunden, so dass sie einen Komplex bilden, der zur Kräfteübertragung geeignet ist. LINC Komplexe besitzen vielfältige Funktionen in Prozessen wie nuklearer Migration, Verankerung und Positionierung des Zellkerns, Chromosomenbewegungen und in der Aufrechterhaltung der Zellpolarität oder der Kernform. Um ein größeres Verständnis der Prozesse während der Spermiogenese zu gewinnen, wurden in dieser Studie die Funktionen von LINC Komplexen in der Spermiogenese und ihre spezifische Rolle bei der gerichteten Spermienkopf-strukturierung untersucht. Dabei wurde insbesondere das Verhalten und die Funktion des bisher wenig charakterisierten SUN Domänen Proteins SUN4 erforscht. Entsprechend der Ergebnisse dieser Studie ist SUN4 ein hodenspezifisches Protein, das ausschließlich in haploiden Keimzellen exprimiert wird. In diesen lokalisiert es in der posterioren Kernhülle, spezifisch in Regionen, an die sich die spermatidenspezifische Manschette anlagert. Dies ist eine Struktur, für die bereits gezeigt wurde, dass sie an der Verformung des Kerns beteiligt ist. SUN4 defiziente Mäuse zeigten ausschließlich Spermatiden mit stark desorganisierten Manschettenüberresten und einen gravierend verformten Spermienkopf. Insgesamt führten die Fehlbildungen zu einem globozoospermieartigen Phänotyp und männlicher Sterilität bei Mäusen. Dabei zeigte sich, dass SUN4 nicht nur zwingend erforderlich ist für den korrekten Aufbau und die Verankerung der Manschette, sondern auch für die korrekte Lokalisation von SUN3 und Nesprin1, wie auch für weitere Komponenten der posterioren Kernhülle. Interaktionsstudien zeigten, dass SUN4 sowohl mit SUN3 und Nesprin1 als auch mit sich selbst interagieren kann, vermutlich um funktionsfähige LINC Komplexe zu bilden, die die Manchette verankern und Kräfte aus dem Zytoskelett auf den Kern übertragen. Zusammenfassend zeigen die schwerwiegenden Auswirkungen auf die kernzytoplasmatische Verbindung während der Spermienentwicklung, die durch den Verlust von SUN4 entstanden, einen direkten Nachweis einer entscheidenden Rolle von SUN4 und anderen LINC-Komplex-Komponenten für die Spermienkopfentwicklung und Fertilität bei Säugetieren. KW - Maus KW - spermiogenesis KW - Fertilität KW - Spermatogenese KW - Kernhülle KW - Molekularbiologie KW - LINC complex KW - SUN domain proteins KW - sperm head formation KW - fertility KW - Spermiogenese KW - Spermienbildung KW - Kernproteine Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139092 ER - TY - THES A1 - Bertho, Sylvain T1 - Biochemical and molecular characterization of an original master sex determining gene in Salmonids T1 - Biochemische und molekulare Charakterisierung des Mastergens bei der Sex-bestimmung in Salmoniden N2 - Sexual development is a fundamental and versatile process that shapes animal morphology, physiology and behavior. The underlying developmental process is composed of the sex determination and the sex differentiation. Sex determination mechanisms are extremely labile among taxa. The initial triggers of the sex determination process are often genetics called sex determining genes. These genes are expressed in the bipotential gonad and tilt the balance to a developmental program allowing the differentiation of either a testis or an ovary. Fish represent a large and fascinating vertebrate group to study both sex determination and sex differentiation mechanisms. To date, among the known sex determining genes, three gene families namely sox, dmrt and TGF-β factors govern this developmental program. As exception to this rule, sdY “sexually dimorphic on the Y” does not belong to one of these families as it comes from the duplication / evolution of an ancestor gene related to immunity, i.e., the interferon related factor 9, irf9. sdY is the master sex determining gene in salmonids, a group of fishes that include species such as rainbow trout and Atlantic salmon. The present study was aimed to firstly characterize the features of SdY protein. Results indicate that SdY is predominantly localized in the cytoplasm tested in various fish and mammalian cell lines and confirmed by different methods. Predictive in silico analysis revealed that SdY is composed of a β-sandwich core surrounded by three α-helices as well specific characteristics conferring a putative protein-protein interaction site. Secondly, the study was aimed to understand how SdY could trigger testicular differentiation. SdY is a truncated divergent version of Irf9 that has a conserved protein-protein domain but lost the DNA interaction domain of its ancestor gene. It was then hypothesized that SdY could initiate testicular differentiation by protein-protein interactions. To evaluate this we first conducted a yeast-two-hybrid screen that revealed a high proportion of transcription factors including fox proteins. Using various biochemical and cellular methods we confirm an interaction between SdY and Foxl2, a major transcription factor involved in ovarian differentiation and identity maintenance. Interestingly, the interaction of SdY with Foxl2 leads to nuclear translocation of SdY from the cytoplasm. Furthermore, this SdY translocation mechanism was found to be specific to fish Foxl2 and to a lesser extend Foxl3 and not other Fox proteins or mammalian FoxL2. In addition, we found that this interaction allows the stabilization of SdY and prevents its degradation. Finally, to better decipher SdY action we used as a model a mutated version of SdY that was identified in XY females of Chinook salmon natural population. Results show that this mutation induces a local conformation defect obviously leading to a misfolded protein and a quick degradation. Moreover, the mutated version compromised the interaction with Foxl2 defining a minimal threshold to induce testicular differentiation. Altogether results from my thesis propose that SdY would trigger testicular differentiation in salmonids by preventing Foxl2 to promote ovarian differentiation. Further research should be now carried out on how this interaction of SdY and Foxl2 acts in-vivo. N2 - Le développement du sexe est un processus fondamental et versatile qui forme la morphologie, la physiologie et le comportement des animaux. Le processus de développement sous-jacent est composé de la détermination et de la différentiation du sexe. Les mécanismes de détermination du sexe sont extrêment labile parmi les taxons. Les signaux initiaux du processus de détermination du sexe sont souvent génétiques et nommés gènes de détermination du sexe. Ces gènes sont exprimés dans la gonade bipotente et font pencher l’équilibre vers un programme de développement permettant la formation soit d’un testicule soit d’un ovaire. Les poissons représentent un large et fascinant groupe de vertébrés pour étudier les processus de détermination et de différentiation du sexe. A l’heure actuelle, parmi les gènes de détermination connus, trois familles de gènes nommément sox, dmrt and les facteurs TGF-β gouvernent ce processus de développement. Comme exception à cette règle, sdY « sexually dimorphic on the Y » n’appartient à aucune de ces familles puisqu’il provient d’une duplication/évolution d’un gène ancestral de l’immunité, c’est-à-dire d’un facteur lié à l’interféron, irf9. sdY est le gène maître de la détermination du sexe chez les salmonidés, un groupe de poissons incluant des espèces tel que la truite arc-en-ciel et le saumon Altantique. L’étude présentée avait pour but de premièrement caractériser les propriétés de la protéine SdY. Les résultats indiquent que SdY est localisée de façon prédominante dans le cytoplasme testés dans diverses cellules de poissons et de mammifères et confirmé par des différentes méthodes. Une analyse in silico prédictive a révélé que SdY est composé d’un core β-sandwich entouré par trois hélices-α ainsi que des caractéristiques lui conférant un site d’interaction protéine-protéine. Deuxièment, l’étude avait pour but de comprendre comment SdY pouvait entraîner la différentiation testiculaire. SdY est une version tronquée divergente de Irf9 qui a conservé le domaine protéine-protéine mais a perdu le domaine d’interaction à l’ADN présent dans le gène ancestral. Il a été proposé que SdY entraîne la différentiation testiculaire par interaction(s) protéine-protéine. Afin d’évaluer cette hypothèse, un crible double-hybride en système levure a révélé une forte proportion de facteurs de transcription incluant les protéines fox. En utilisant de nombreuses méthodes au niveau cellulaire et biochimique, nous avons confirmé une interaction entre SdY et Foxl2, un facteur majeur impliqué dans la différentiation ovarienne et gardien de son identité. De façon intéressante, l’interaction de SdY avec Foxl2 conduit à une translocation nucléaire de SdY à partir du cytoplasme. De plus, le mécanisme de translocation de SdY est spécifique à la protéine Foxl2 et dans une moindre mesure à Foxl3 parmi les protéines Fox de poissons ou bien des protéines FoxL2 de mammifères. Puis, nous avons montré que cette interaction permet la stabilisation de SdY et empêche sa dégradation. Enfin, pour mieux décrypter l’action de SdY, nous avons utilisé comme modèle une version mutée qui a été identifiée dans une population naturelle de saumon Chinook avec des individus XY femelles. Les résultats montrent que la mutation induit un défaut de conformation local menant à une protéine mal-repliée et à sa dégradation. De plus, la version mutée compromet l’interaction avec Foxl2 définissant un seuil minimal d’induction de la différentiation testiculaire. Les résultats de ma thèse pris dans leur ensemble proposent que SdY pourrait entraîner la différentiation testiculaire chez les salmonidés en empêchant Foxl2 d’induire la différentiation ovarienne. Les recherches doivent se poursuivre dans le but de comprendre comment l’interaction SdY avec Foxl2 fonctionne in vivo. N2 - Sexuelle Entwicklung ist ein grundlegender und vielfältiger Prozess, der die Morphologie, Physiologie und das Verhalten von Tieren gestaltet. Der zugrundeliegende Entwicklungsprozess besteht aus der Geschlechtsbestimmung und der Geschlechtsdifferenzierung. Die Mechanismen der Geschlechtsbestimmung sind sehr instabil zwischen verschiedenen Arten. Die Auslöser des Prozesses der Geschlechtsbestimmung sind oft genetischen Ursprungs wie geschlechtsbestimmende Gene. Diese Gene werden in den bipotentialen Gonaden exprimiert und steuern die Balance eines entwicklungsgemäßen Programms, das die Differenzierung zum Testis oder Ovar erlaubt. Fische repräsentieren eine umfangreiche und faszinierende Gruppe von Vertebraten, um die Mechanismen der Geschlechtsbestimmung und –differenzierung zu untersuchen. Bislang ist bekannt, dass –unter den bekannten geschlechtsbestimmenden Genen- die drei Gen-Familien sox, dmrt und die TGFß-Faktoren dieses Entwicklungsprogramm steuern. Als Ausnahme von dieser Regel ist sdY „sexually dimorphic on the Y“ keiner dieser Familien zugehörig da es von der Duplikation / Evolution eines Vorgänger-Gens, das mit Immunität wie z.B. interferon related factor9, irf9, in Verbindung steht, herrührt. sdY ist das Mastergen der Geschlechtsbestimmung in Salmoniden, die als Gruppe von Fischen Arten wie die Regenbogenforelle und den Atlantischen Lachs umfassen. Das Ziel der vorliegenden Arbeit war es zunächst die Eigenschaften des SdY Proteins zu charakterisieren. Die Ergebnisse zeigen, dass SdY vor allem im Zytoplasma lokalisiert ist. Dies wurde in verschiedenen Fischen und Säugetier Zelllinien untersucht und mit Hilfe verschiedener Methoden bestätigt. Prädiktive in silico Analysen zeigten, dass SdY aus einem ß-sandwich Kern besteht, der von drei α-Helices umgeben ist sowie spezifischen Eigenschaften für eine putative Protein-Protein Interaktion Stelle. Das zweite Ziel der vorliegenden Arbeit war es, zu verstehen, wie SdY die testikuläre Differenzierung auslösen könnte. SdY ist eine verkürzte, divergente Version von Irf9, das eine konservierte Protein-Protein Domäne aufweist, jedoch seine DNA Interaktion Domäne a seines Vorläufer Gens verloren hat. Daher wurde angenommen, dass SdY die testikuläre Differenzierung durch Protein-Protein Interaktion initiieren könnte. Um diese Hypothese zu bestätigen führten wir zuerst einen Yeast Two-Hybrid Screen durch, der einen hohen Anteil an Transkriptionsfaktoren darunter fox Proteine zeigte. Unter Einsatz verschiedener biochemischer und zellulärer Methoden bestätigten wir eine Interaktion zwischen SdY und Foxl2, einem wesentlichen Transkriptionsfaktor, der in die Differenzierung und die Erhaltung der Identität der Ovarien involviert ist. Interessanterweise führt die Interaktion von SdY mit Foxl2 zu einer nukleären Translokation von SdY aus dem Zytoplasma. Außerdem wurde festgestellt, dass dieser SdY Translokations-Mechanismus für das Fisch Foxl2 und in einem geringerem Maße für Foxl3 spezifisch ist aber nicht für andere Fox Proteine oder Säuger FoxL2. Des Weiteren haben wir herausgefunden, dass diese Interaktion die Stabilisierung von SdY ermöglicht und sein Abbau verhindert. Zuletzt haben wir ein Modell einer mutierten Version von SdY benutzt, die in XY Weibchen der natürlichen Population der Königslachse identifiziert wurde, um die Wirkung von SdY besser zu entschlüsseln. Die Ergebnisse zeigen, dass diese Mutation einen lokalen Konformationsdefekt verursacht, der zu fehlgefalteten Proteinen und einem raschen Abbau führt. Darüber hinaus beeinträchtigt die mutierte Version die Interaktion mit FoxL2 und definiert einen minimalen Grenzwert, um die testikuläre Differenzierung zu induzieren. Insgesamt deuten die Ergebnisse meiner Dissertation darauf hin, dass SdY die testikuläre Differenzierung in Salmoniden auslöst, indem es verhindert, dass Foxl2 die Differenzierung der Ovarien fördert. In Zukunft soll erforscht werden, wie sich die Interaktion von SdY und Foxl2 in-vivo auswirkt. KW - Fish Sex determination KW - gonad development KW - SdY KW - salmonids KW - Lachsartige KW - Geschlechtsdifferenzierung KW - Molekulargenetik Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139130 ER - TY - THES A1 - Costea, Paul Igor T1 - Stratification and variation of the human gut microbiota T1 - Stratifikation und Variation des menschlichen Darmmikrobioms N2 - The microbial communities that live inside the human gastrointestinal tract -the human gut microbiome- are important for host health and wellbeing. Characterizing this new “organ”, made up of as many cells as the human body itself, has recently become possible through technological advances. Metagenomics, the high-throughput sequencing of DNA directly from microbial communities, enables us to take genomic snapshots of thousands of microbes living together in this complex ecosystem, without the need for isolating and growing them. Quantifying the composition of the human gut microbiome allows us to investigate its properties and connect it to host physiology and disease. The wealth of such connections was unexpected and is probably still underestimated. Due to the fact that most of our dietary as well as medicinal intake affects the microbiome and that the microbiome itself interacts with our immune system through a multitude of pathways, many mechanisms have been proposed to explain the observed correlations, though most have yet to be understood in depth. An obvious prerequisite to characterizing the microbiome and its interactions with the host is the accurate quantification of its composition, i.e. determining which microbes are present and in what numbers they occur. Historically, standard practices have existed for sample handling, DNA extraction and data analysis for many years. However, these were generally developed for single microbe cultures and it is not always feasible to implement them in large scale metagenomic studies. Partly because of this and partly because of the excitement that new technology brings about, the first metagenomic studies each took the liberty to define their own approach and protocols. From early meta-analysis of these studies it became clear that the differences in sample handling, as well as differences in computational approaches, made comparisons across studies very difficult. This restricts our ability to cross-validate findings of individual studies and to pool samples from larger cohorts. To address the pressing need for standardization, we undertook an extensive comparison of 21 different DNA extraction methods as well as a series of other sample manipulations that affect quantification. We developed a number of criteria for determining the measurement quality in the absence of a mock community and used these to propose best practices for sampling, DNA extraction and library preparation. If these were to be accepted as standards in the field, it would greatly improve comparability across studies, which would dramatically increase the power of our inferences and our ability to draw general conclusions about the microbiome. Most metagenomics studies involve comparisons between microbial communities, for example between fecal samples from cases and controls. A multitude of approaches have been proposed to calculate community dissimilarities (beta diversity) and they are often combined with various preprocessing techniques. Direct metagenomics quantification usually counts sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to technology-inherent differences in sampling depth, normalizing counts is necessary, for instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by subsampling. To derive a single value for community (dis-)similarity, multiple distance measures have been proposed. Although it is theoretically difficult to benchmark these approaches, we developed a biologically motivated framework in which distance measures can be evaluated. This highlights the importance of data transformations and their impact on the measured distances. Building on our experience with accurate abundance estimation and data preprocessing techniques, we can now try and understand some of the basic properties of microbial communities. In 2011, it was proposed that the space of genus level variation of the human gut microbial community is structured into three basic types, termed enterotypes. These were described in a multi-country cohort, so as to be independent of geography, age and other host properties. Operationally defined through a clustering approach, they are “densely populated areas in a multidimensional space of community composition”(source) and were proposed as a general stratifier for the human population. Later studies that applied this concept to other datasets raised concerns about the optimum number of clusters and robustness of the clustering approach. This heralded a long standing debate about the existence of structure and the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in the context of the vastly increased amounts of available data. We propose a refined framework in which the different types should be thought of as weak attractors in compositional space and we try to implement an approach to determining which attractor a sample is closest to. To this end, we train a classifier on a reference dataset to assign membership to new samples. This way, enterotypes assignment is no longer dataset dependent and effects due to biased sampling are minimized. Using a model in which we assume the existence of three enterotypes characterized by the same driver genera, as originally postulated, we show the relevance of this stratification and propose it to be used in a clinical setting as a potential marker for disease development. Moreover, we believe that these attractors underline different rules of community assembly and we recommend they be accounted for when analyzing gut microbiome samples. While enterotypes describe structure in the community at genus level, metagenomic sequencing can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis methodology for this level of resolution has only recently been developed and little exploration has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the landscape of genomic variation seems highly structured even beyond species resolution, indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several cases, these subspecies exhibit geo-stratification, with some subspecies only found in the Chinese population. Generally however, they present only minor dispersion limitations and are seen across most of our study populations. Within one individual, one subspecies is commonly found to dominate and only rarely are several subspecies observed to co-occur in the same ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable over periods of more than three years. When interrogating their functional properties we find many differences, with specific ones appearing relevant to the host. For example, we identify a subspecies of E. rectale that is lacking the flagellum operon and find its presence to be significantly associated with lower body mass index and lower insulin resistance of their hosts; it also correlates with higher microbial community diversity. These associations could not be seen at the species level (where multiple subspecies are convoluted), which illustrates the importance of this increased resolution for a more comprehensive understanding of microbial interactions within the microbiome and with the host. Taken together, our results provide a rigorous basis for performing comparative metagenomics of the human gut, encompassing recommendations for both experimental sample processing and computational analysis. We furthermore refine the concept of community stratification into enterotypes, develop a reference-based approach for enterotype assignment and provide compelling evidence for their relevance. Lastly, by harnessing the full resolution of metagenomics, we discover a highly structured genomic variation landscape below the microbial species level and identify common subspecies of the human gut microbiome. By developing these high-precision metagenomics analysis tools, we thus hope to contribute to a greatly improved understanding of the properties and dynamics of the human gut microbiome. N2 - Die mikrobiellen Gemeinschaften innerhalb des menschlichen Darmtrakts – das menschliche Darm-Mikrobiom - sind wichtig für das Wohlbefinden und die Gesundheit des Wirts. Die Charakterisierung dieses neuen “Organs”, welches aus ähnlich vielen Zellen besteht wie der menschliche Körper, ist in jüngster Zeit durch technologische Fortschritte möglich geworden. Die Metagenomik, die direkte Hochdurchsatz-Sequenzierung mikrobieller DNA, ermöglicht die Aufnahme “genomischer Schnappschüsse” tausender verschiedener, in einem komplexen Ökosystem zusammenlebender Bakterien, ohne dafür auf deren Isolierung und Wachstum angewiesen zu sein. Die Quantifizierung des menschlichen Mikrobioms erlaubt es uns, seine Eigenschaften zu untersuchen und Verbindungen zu Wirtsphysiologie und -krankheiten zu knüpfen. Der Reichtum dieser Informationen ist unerwartet hoch und wahrscheinlich noch immer unterbewertet. Aufgrund der Tatsache, dass der Großteil unserer Ernährung und unseres Medikamentenkonsums unser Mikrobiom, welches wiederum selbst über verschiedene Arten mit unserem Immunsystem interagiert, beeinflusst, wurden viele Mechanismen vorgeschlagen, um die beobachteten Korrelationen zu erklären. Die meisten davon sind jedoch noch nicht vollständig verstanden. Eine offensichtliche Komponente zur Charakterisierung des Mikrobioms und dessen Interaktionen mit dem Wirt ist eine akkurate Quantifizierung seiner genauen Zusammensetzung, womit sowohl die Anwesenheit von bestimmten Bakterien als auch deren Anzahl gemeint ist. Obwohl etablierte Standardprozeduren zur Probenbehandlung, DNA- Extrahierung und Datenanalyse existieren, sind sie nicht immer für metagenomische Studien anwendbar, da sie für isolierte Bakterienkulturen entwickelt worden. Deswegen und auch wegen der Begeisterung, die neuartige Technologien mit sich bringen, nahmen sich die ersten metagenomischen Studien jeweils die Freiheit, ihre eigenen Protokolle und Herangehensweisen zu definieren. Die Metaanalyse dieser Studien zeigte, dass Unterschiede sowohl in der Probenbehandlung als auch in der statistischen Auswertung den Vergleich zwischen Studien sehr schwierig machen. Das wiederum beschneidet unsere Fähigkeit, Entdeckungen zu bestätigen und Daten über Studien hinweg zu kombinieren. Um die zwingend notwendige Standardisierung voranzutreiben haben wir einen umfassenden Vergleich von 21 verschiedenen DNA-Extraktionsmethoden sowie verschiedener weiterer Probenbehandlungen, welche Quantifizierungen beeinflussen, vorgenommen. Wir haben eine Reihe von Kriterien entwickelt, um die Messqualität in Abwesenheit von Mock-Kontrollen zu bestimmen und schlagen anhand dieser Methoden für Probenbeschaffung, DNA-Extraktion und Library- Generierung optimale Verfahren vor. Wenn diese als Standard akzeptiert werden, würde das eine stark verbesserte Vergleichbarkeit zwischen Studien ermöglichen und damit sowohl einen extremen Zuwachs an statistischer Power als auch unserer Fähigkeit, generelle Schlüsse über das Mikrobiom zu ziehen, zur Folge haben. Die meisten metagenomischen Studien teilen ihre Datensätze auf um Vergleiche anzustellen, z.B. zwischen Stuhlproben gesunder und erkrankter Menschen. Eine Vielzahl verschiedener Ansätze, welche wiederum oft mit verschiedenen Datenvorbehandlungen kombiniert werden, wurden vorgeschlagen, um Dissimilarität zwischen Gemeinschaften (Beta-Diversität) zu berechnen. Um metagenomische Daten auf Spezies-, Genus- und höheren Ebenen zu quantifizieren werden üblicherweise reads auf Referenzgenome bestimmter taxonomischer Einheiten aligniert und gezählt. Aufgrund technologieabhängiger Unterschiede in Sequenziertiefe müssen reads normalisiert werden, z.B. indem man alle counts durch die Gesamtanzahl der counts einer Sequenzierung teilt (total sum scaling), oder durch subsampling. Für die Messung der Gemeinschafts(dis)similarität wurden viele Distanzmaße vorgeschlagen. Da es schwierig ist diese Ansätze theoretisch zu vergleichen, haben wir ein biologisch motiviertes Konzept entwickelt, mit dem man Distanzmaße evaluieren kann. Dies unterstreicht die Wichtigkeit der Datentransformation und dessen Einwirkung auf Distanzmaße. Aufbauend auf unserer Erfahrung mit Häufigkeitsabschätzungen und Techniken zur Datenvorbehandlung können wir nun versuchen, grundlegende Eigenschaften mikrobieller Gemeinschaften zu verstehen. 2011 wurde vorgeschlagen, dass sich die Variation auf Genusebene im menschlichen Darm auf drei grundlegende Typen beschränkt, welche Enterotypen getauft wurden. Diese wurden in Datensätzen verschiedener Länder als unabhängig von Herkunft, Alter und anderer Wirtseigenschaften beschrieben. Die Enterotypen sind durch einen Cluster-Ansatz als „dicht besiedelte Bereiche in einem multidimensionalen Raum der Gemeinschaftszusammensetzung“ definiert und wurden als grundlegende Stratifikatoren für die menschlichen Population vorgeschlagen. Spätere Studien, welche dieses Konzept auf andere Datensätze anwandten, erhoben Zweifel bezüglich der optimalen Anzahl an Clustern und an der generellen Robustheit des Ansatzes. Dies leitete erneut eine langanhaltende Debate über die Existenz von Strukturen und die besten Wege, diese zu bestimmen und einzufangen, ein. Hier überdenken wir, in Anbetracht der stark gestiegenen Anzahl an verfügbaren Daten, das Enterotypen-Konzept. Wir schlagen ein überarbeitetes Konzept vor, in welchem die verschiedenen Enterotypen als schwache Attraktoren im multidimensionalen Raum verstanden werden und implementieren einen Ansatz zur Berechnung des Attraktors, der dem Datensatz am ähnlichsten ist. Dafür trainieren wir einen Klassifizierer auf einen Referenz- Datensatz, um neue Datensätze zuzuordnen. Damit ist Enterotypisierung nicht mehr datensatzabhängig und der Effekt von sampling bias ist minimiert. Indem wir ein Modell nutzen für das wir die Existenz dreier Enterotypen (definiert durch die selben Genera wie ursprünglich postuliert) annehmen, zeigen wir die Relevanz dieser Stratifikation und schlagen es in einem klinischen Zusammenhang als potentiellen Marker für Krankheitsfortschritt vor. Außerdem glauben wir, dass diese Attraktoren verschiedene Regeln mikrobieller Zusammensetzung widerspiegeln und schlagen vor, sie bei der Analyse von mikrobiellen Daten zu berücksichtigen. Während Enterotypen Struktur in der Gemeinschaft auf Genusebene beschreiben, kann metagenomische Sequenzierung prinzipiell Auflösung auf Nukleotidebene erreichen, womit single nucleotide polymorphisms (SNPs) und andere genomische Variationen im Darm- Mikrobiom identifiziert werden können. Analysemethoden für dieses Auflösungsniveau wurden erst kürzlich entwickelt und bis heute wurden diese erst wenig erforscht. Wir zeigen, dass die Landschaft an genomischer Variation von SNPs in einer großen, multinationalen Kohorte sogar über die Speziesebene hinaus geht und hochgradig strukturiert ist, was das Vorkommen klar abgrenzbarer Subspezies unter Darmmikroben suggeriert. In mehreren Fällen zeigen diese Subspezies geographische Stratifikation, wobei einige Subspezies nur in chinesischen Populationen vorkommen. Im Allgemein zeigen Sie jedoch nur eine geringfügige Beschränkung der Dispersion und sind in der Mehrzahl der Populationen vorhanden. Innerhalb eines Individuums dominiert häufig eine bestimmte Subspezies, nur selten dominieren verschieden gemeinsam im gleichen Ökosystem. Eine Analyse von Zeitreihenexperimenten deutet darauf hin, dass die dominante Subspezies über Zeiträume von mehr als drei Jahren stabil bleibt. Wenn man ihre funktionalen Eigenschaften untersucht findet man viele Unterschiede, von denen bestimmte relevant für den Wirt erscheinen. Zum Beispiel identifizieren wir eine Subspezies von E. rectale, welcher das Flagellum-Operon fehlt, die signifikant assoziiert ist mit geringerem BMI und geringerer Insulinresistenz ihres Wirts; sie korreliert zudem mit höherer mikrobieller Diversität. Diese Assoziationen konnten auf Speziesebene nicht gesehen werden (auf der mehrere Subspezies überlagert sind), was die Wichtigkeit dieser erhöhten Auflösung für ein umfassenderes Verständnis mikrobieller Interaktionen innerhalb des Mikrobioms und mit dem Wirt illustriert. Zusammenfassend bieten unsere Ergebnisse eine präzise Grundlage für vergleichende Metagenomik des menschlichen Darms, einschließlich Empfehlungen über experimentelles Sampling und statistische Analysen. Weiterhin verfeinern wir das Konzept der Enterotypen- Stratifikation in Gemeinschaften, entwickeln referenzbasierte Ansätze für Enterotypen- Zuordnung und bieten überzeugende Beweise für ihre Relevanz. Indem wir die volle Auflösung metagenomischer Sequenzierungen nutzen entdecken wir eine Landschaft hochgradig strukturierter genomischer Variation unterhalb der Speziesebene und identifizieren gemeinsame Subspezies des menschlichen Darm-Mikrobioms. Durch die Entwicklung dieser hochpräzisen metagenomischen Untersuchungsansätze tragen wir zu einem verbesserten KW - metagenomics KW - microbiology KW - Mensch KW - Darmflora KW - Metagenom Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139649 ER - TY - THES A1 - Eck, Saskia T1 - The impact of thermogenetic depolarizations of specific clock neurons on Drosophila melanogaster's circadian clock T1 - Der Einfluss thermogenetischer Depolarisationen spezifischer Uhrneurone auf Drosophila melanogasters circadiane Uhr N2 - The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms’ behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly’s circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock’s interneuronal communication. Drosophila melanogaster’s circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly’s locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature. Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs). The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs & l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day. To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner. However, the molecular link between clock neuron depolarization and shifts in the molecular clock’s cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock. Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways. N2 - Die Rotation der Erde um ihre eigene Achse hat periodisch verändernde Umweltbedingungen, wie beispielsweise Veränderungen in den Lichtverhältnissen und der Temperatur, zur Folge. Um das Verhalten, die Physiologie und den Metabolismus eines Organismus an stets wiederkehrende Veränderungen anzupassen, haben sich endogene/circadiane Uhren entwickelt, die es dem Organismus erlauben diese Umweltbedingungen zu antizipieren. In der Chronobiologie, einem wissenschaftlichen Fachbereich, der sich mit der Untersuchung der zugrunde liegenden Mechanismen der Inneren Uhr befasst, dient die Taufliege Drosophila melanogaster als nützlicher Modellorganismus. Die Innere Uhr der Taufliege ist anatomisch eher einfach organisiert, weist trotz alledem jedoch Homologien zum Säugersystem auf. Auch im Rahmen dieser Doktorarbeit diente die Taufliege daher dazu grundlegende Netzwerkeigenschaften der circadianen Uhr zu untersuchen. Die Innere Uhr von Drosophila melanogaster besteht aus ungefähr 150 Uhrneuronen, die sich im zentralen Nervensystem der Fliege befinden. Diese Uhrneurone können, bezüglich ihrer anatomischen Position im Gehirn in die Gruppe der dorsalen Neurone (DN1, DN2, DN3), sowie in die der lateralen Neurone untergliedert werden (LPN, LNd, s-LNv, l-LNv). Funktionell werden diese Uhrneuronengruppen als Morgen- und Abendoszillatoren (M- und E-Oszillatoren) klassifiziert, da sie für unterschiedliche Verhaltensanteile in der Laufaktivität der Fliege unter Licht-Dunkel-Verhältnissen (LD) verantwortlich sind. Die s-LNv stellen dabei die Morgenoszillatoren (M-Oszillatoren) dar und werden als Hauptschrittmacher betrachtet, da sie die Geschwindigkeit der Uhr unter konstanten Bedingungen (Dauerdunkel; DD) bestimmen. Die Gruppe der Abendoszillatoren (EOszillatoren) besteht aus den LNd, einigen DN1 und der fünften s-LNv (5th s-LNv) und ist für die richtige Terminierung der Abendaktivität in LD zuständig. All diese Uhrneurone sind funktionell nicht unabhängig voneinander, sondern bilden komplexe neuronale Verschaltungen untereinander aus, die durch einen hohen Grad an Plastizität bezüglich ihrer Reaktion auf unterschiedliche Umweltparameter (Zeitgeber), wie Licht oder Temperatur, gekennzeichnet sind. Obwohl bereits vieles hinsichtlich der Funktion und der Bedeutung einiger Gruppen von Uhrneuronen bekannt ist, ist das genaue Zusammenspiel unter ihnen immer noch recht unklar. Um die Mechanismen, die in den Kommunikationsprozessen zwischen verschiedenen Uhrneuronen involviert sind, zu untersuchen, machten wir Gebrauch von dTrpA1, einem thermosensitiven Kationenkanal, der es durch die Applizierung von Temperaturpulsen (TP) über 29°C ermöglicht, Neuronen in der intakten und sich frei bewegenden Fliege zeitlich begrenzt und zellspezifisch zu depolarisieren. Mithilfe verschiedener Uhr-spezifischer GAL4-Treiberlinien und der Verabreichung von TP zu verschiedenen Zeitpunkten des circadianen Zyklus in DD, war es uns möglich Rückschlüsse auf die Eigenschaften der Inneren Uhr anhand von Phasen-Verschiebungsexperimenten zu ziehen. Die hervorgerufenen Phasenverschiebungen im Laufverhalten, die durch die Aktivierung spezieller Uhrneuronen hervorgerufen wurden, wurden dabei als Phasen Responz Kurve (engl. phase response curve; PRC) dargestellt. Die Depolarisierung aller Uhrneurone verschob die Phase der Aktivität am stärksten, insbesondere in der Phasen-Verzögerungszone der PRC. Wurden ausschließlich die M-Oszillatoren zusammen mit den l-LNv (PDF+ Neurone: s-LNv & l-LNv) depolarisiert, wurden ebenso Phasenverschiebungen nach vorne, wie auch nach hinten hervorgerufen, jedoch reichten die Verschiebungen nach vorne deutlich in den subjektiven Tag hinein. Daraus schlussfolgerten wir, dass Licht inhibitorischen Einfluss in diesem Bereich der PRC haben muss, da typische Licht-PRCs nicht derart ausgeprägte Vorverschiebungen aufweisen. Aufgrund des vollständigen Lichtausschlusses in den PRC-Versuchen dieser Doktorarbeit fehlt jedoch dieser Licht-vermittelte inhibitorische Einfluss zu den PDF+ Neuronen und führt daher zur zeitlich stark ausgeprägten Phasen-Vorverschiebungszone. Diese Ergebnisse lassen daher vermuten, dass ein inhibitorisch wirkender Licht-vermittelter Eingang zu den PDF+ Neuronen von den photorezeptiven Organen (Hofbauer-Buchner Äuglein, Photorezeptoren der Komplexaugen, Ocellen) oder von anderen Uhrneuronen existieren muss, der die Phasen-Vorverschiebungen während des subjektiven Tages unterdrückt. Um Kenntnis über den molekularen Status der Uhr in der Verzögerungs- und Phasen-Vorverschiebungszone zu erlangen, wurden Färbungen gegen das Protein Period (PER), eines der zentralen Bestandteile der Inneren Uhr und gegen das Neuropeptid Pigment Dispersing Factor (PDF) angefertigt. Der zeitliche Verlauf im Auf- und Abbau des PER Proteins spiegelte die Phasenverschiebungen im Verhalten der Experimentalfliegen wider, wohingegen die Kontrollen weitestgehend unauffällig blieben. Zudem waren nur diejenigen Neurone von einer unmittelbaren Verschiebung der PER Protein Oszillation betroffen, die depolarisiert wurden, was auf einen schnellen Zell-autonomen Prozess schließen lässt. Die molekulare Verknüpfung, die zwischen der Depolarisation der Uhrneuronen und der Verschiebung der molekularen Uhr-Oszillation fungiert, ist immer noch unbekannt. Diesem Thema wurde nachgegangen, indem CREB (engl. cAMP responsive element binding protein) in den großen ventrolateralen Neuronen (l-LNv) quantifiziert wurde, da diese Neuronen unerwarteterweise und am wirksamsten auf die artifizielle Depolarisation mit einer starken PER-Akkumulation reagiert haben. In vorherigen Arbeiten wurde bereits angenommen, dass CREB in die circadiane Rhythmik involviert sei, indem es an Regulationssequenzen des period Gens bindet (Belvin et al., 1999) und somit dessen Transkription aktiviert. Wir konnten zeigen, dass die Menge an CREB Protein in den l-LNv circadian reguliert wird, da diese am Ende der subjektiven Nacht im Vergleich zum Ende des subjektiven Tages deutlich erhöht ist. Dieser Effekt konnte durch die artifizielle Depolarisation, aber unabhängig von deren Zeitpunkt, weiter verstärkt werden. Zudem deuten die Ergebnisse darauf hin, dass die Akkumulation des CREB Proteins mit dem Zeitpunkt des Anstiegs des PER Proteins in den l-LNv koinzidiert. Das lässt die Vermutung zu, dass CREB als molekulare Verbindung zwischen dem elektrischen neuronalen Status und der molekularen Uhr dienen kann. Zusammenfassend lässt sich sagen, dass die zeitlich begrenzte Depolarisation mithilfe von dTrpA1 signifikante Phasenverschiebungen im Verhalten wie auch auf der Proteinebene hervorrufen kann. Eine artifizielle Depolarisation zu Beginn der subjektiven Nacht verursacht Phasenverschiebungen nach hinten, wohingegen eine Depolarisation zum Ende der subjektiven Nacht Phasenverschiebungen nach vorne zur Folge hat. Die Aktivierung aller Uhrneurone brachte eine PRC hervor, die weitestgehend einer Licht-PRC gleicht. Die Depolarisierung der PDF+ Zellen hingegen ergab eine PRC, die sich insbesondere bezüglich der ausgeprägten Phasen-Vorverschiebungszone von einer Licht-vermittelten PRC unterscheidet. Die Innere Uhr scheint somit die Fähigkeit zu besitzen, exzitatorische und inhibitorische Eingänge in komplexer Art und Weise zu verarbeiten. Obwohl der in dieser Doktorarbeit gewählte experimentelle Ansatz hochgradig artifiziell ist, war es uns gerade durch den Ausschluss von Licht möglich, neue Schlussfolgerungen bezüglich der Kommunikation innerhalb des Netzwerks und dessen Lichtinformations-Eingänge zu ziehen. KW - Chronobiologie KW - Circadian clock KW - Tagesrhythmus KW - Taufliege Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137118 ER - TY - THES A1 - König, Sebastian T1 - Spatially selective visual attention in Drosophila melanogaster T1 - Räumlich selektive visuelle Aufmerksamkeit in Drosophila melanogaster N2 - Finding the right behavior at the right time is one of the major tasks of brains. In a natural scenery there is often an abundance of stimuli present and the brain has to separate the relevant from the irrelevant ones. Selective visual attention (SVA) is a property of higher visual systems that achieves this separation, as it allows to ‘[…] focus on one source of sensory input to the exclusion of others’ (Luck and Mangun, 1996). There are probably several forms of SVA depending upon the criteria used for the separation, such as salience, color, location in space, novelty, or motion. Many studies have investigated SVA in humans and non-human primates. However, complex functions like attention were initially not expected to be already implemented in the brains of simple organisms like Drosophila. After a first demonstration of selective attention in the fly (Wolf and Heisenberg, 1980), it took some time until other studies included attentional mechanisms in their argumentation to explain certain behaviors of Drosophila. However, their definition and characterization of attention differed and often was ambiguous. Here, one particular form, spatially selective visual attention in the fly Drosophila is investigated. It has been shown earlier that the fly spontaneously may restrict its behavioral responses in stationary flight to the visual stimuli on one side of the visual field. On the basis of experiments of Sareen et al., (2011) it has been conjectured that the fly has a focus of attention (FoA) and that the fly responds to the visual stimuli within this area of the visual field. Whether the FoA is the adequate concept for this spatial property of SVA in the fly needs to be further discussed and is a subject also of the present study. At this stage, the concept will be used in the description of the new results expanding the characterization of SVA. This study continued the investigation of SVA during tethered flight with variable but controlled visual input and an automated primary data evaluation. This standardized paradigm allowed for analysis of wild-type behavior as well as for a comparison of several mutant and pharmacologically manipulated strains to the wild-type. Some properties of human SVA like the occurrence of externally as well as internally caused shifts of attention were found in Drosophila and it could be shown, that SVA in the fly can be externally guided and has an attention span. Additionally, a neurotransmitter and proteins, which play a significant role in SVA were discovered. Based on this, the genetic tools available for Drosophila provided the means to a first examination of cells and circuits involved in SVA. Finally, the free walk behavior of flies that had been shown to have compromised SVA was characterized. The results suggested that the observed phenotypes of SVA were not behavior specific. Covert shifts of the FoA were investigated. The FoA can be externally guided by visual cues to one or the other side of the visual field and even after the cue has disappeared it remains there for <4s. An intriguing finding of this study is the fact, that the quality of the cue determines whether it is attractive or repellent. For example a cue can be changed from being repellent (negative) to being attractive (positive) by changing its oscillation amplitude from 4° to 2°. Testing the effectiveness of cues in the upper and lower visual field separately, revealed that the perception of a cue by the fly is not exclusively based on a sum of its specifications. Because positive cueing did not have an after-effect in each of the two half-fields alone, but did so if the cue was shown in both, the fly seems to evaluate the cue for each combination of parameters specifically. Whether this evaluation of the cue changed on a trial-to-trial basis or if the cue in some cases failed to shift the FoA can at this point not be determined. Looking at the responses of the fly to the displacement of a black vertical stripe showed that they can be categorized as no responses, syn-directional responses (following the direction of motion of the stripe) and anti-directional responses (in the opposite direction of the motion of the stripe). The yaw-torque patterns of the latter bared similarities with spontaneous body saccades and they most likely represented escape attempts of the fly. Syn-directional responses, however, were genuine object responses, distinguishable by a longer latency until they were elicited and a larger amplitude. These properties as well as the distribution of response polarities were not influenced by the presence or absence of a cue. When two stripes were displaced simultaneously in opposite directions the rate of no responses increased in comparison to the displacement of a single stripe. If one of the stripes was cued, both, the responses towards and away from the side of cue resembled the syn-directional responses. Significant progress was made with the elucidation of the neuronal underpinnings of SVA. Ablation of the mushroom bodies (MB) demonstrated their requirement for SVA. Furthermore, it was shown that dopamine signaling has to be balanced between too much and too little. Either inhibiting the synthesis of dopamine or its re-uptake at the synapse via the dDAT impaired the flies’ susceptibility to cueing. Using the Gal4/UAS system, cell specific expression or knockdown of the dDAT was used to scrutinize the role of MB sub-compartments in SVA. The αβ-lobes turned out to be necessary and sufficient to maintain SVA. The Gal4-line c708a labels only a subset of Kenyon cells (KC) within the αβ-lobes, αβposterior. These cells stand out, because of (A) the mesh-like arrangement of their fibers within the lobes and (B) the fact that unlike the other KCs they bypass the calyx and thereby the main source of olfactory input to the MBs, forming connections only in the posterior accessory calyx (Tanaka et al., 2008). This structure receives no or only marginal olfactory input, suggesting for it a role in tasks other than olfaction. This study shows their requirement in a visual task by demonstrating that they are necessary to uphold SVA. Restoring dDAT function in these approximately only 90 cells was probably insufficient to lower the dopamine concentration at the relevant synapses and hence a rescue failed. Alternatively, the processes mediating SVA at the αβ-lobes might require an interplay between all of their KCs. In conclusion, the results provide an initial point for future research to fully understand the localization of and circuitry required for SVA in the brain. In the experiments described so far, attention has been externally guided. However, flies are also able to internally shift their FoA without any cues from the outside world. In a set of 60 consecutive simultaneous displacements of two stripes, they were more likely to produce a response with the same polarity as the preceding one than a random polarity selection predicted. This suggested a dwelling of the FoA on one side of the visual field. Assuming that each response was influenced by the previous one in a way that the probability to repeat the response polarity was increased by a certain factor (dwelling factor, df), a random selection of response type including a df was computed. Implementation of the df removed the difference between observed probability of polarity repetition and the one suggested by random selection. When the interval between displacements was iteratively increased to 5s, no significant df could be detected anymore for pauses longer than 4s. In conclusion, Drosophila has an attention span of approximately 4s. Flies with a mutation in the radish gene expressed no after-effect of cueing and had a shortened attention span of about 1s. The dDAT inhibitor methylphenidate is able to rescue the first, but does not affect the latter phenotype. Probably, radish is differently involved in the two mechanisms. This study showed, that endogenous (covert) shifts of spatially selective visual attention in the fly Drosophila can be internally and externally guided. The variables determining the quality of a cue turned out to be multifaceted and a more systematic approach is needed for a better understanding of what property or feature of the cue changes the way it is evaluated by the fly. A first step has been made to demonstrate that SVA is a fundamental process and compromising it can influence the characteristics of other behaviors like walking. The existence of an attention span, the dependence of SVA on dopamine as well as the susceptibility to pharmacological manipulations, which in humans are used to treat respective diseases, point towards striking similarities between SVA in humans and Drosophila. N2 - Eine der Hauptaufgaben eines Gehirns ist es, das richtige Verhalten zur richtigen Zeit zu finden. In einer natürlichen Umgebung gibt es eine Vielzahl visueller Reize, die das Gehirn unterteilen muss in solche, die irrelevant und solche, die bedeutsam sind. Selektive visuelle Aufmerksamkeit (SVA) ist eine Eigenschaft hoch entwickelter visueller Systeme, die diese Unterteilung erzielt, indem sie es erlaubt „[…] eine Quelle sensorischen Inputs zu fokussieren und dabei andere auszuschließen“ (Luck and Mangun, 1996). In Abhängigkeit der Kriterien (z.B. Salienz, Farbe, Lage im Raum, Neuartigkeit oder Bewegung), die für die Aufteilung herangezogen werden, existieren wahrscheinlich mehrere Formen von SVA. Viele Studien haben sich mit SVA in Menschen und in Primaten beschäftigt, ohne jedoch zu erwarten, dass eine komplexe Funktion wie Aufmerksamkeit bereits in den Gehirnen von einfachen Organismen wie Drosophila implementiert zu finden. Erst einige Zeit nachdem selektive Aufmerksamkeit ein erstes Mal in der Fliege gezeigt worden war (Wolf, Heisenberg, 1980) begannen auch andere Studien Aufmerksamkeit in ihrer Argumentation als Erklärung für bestimmte Verhaltensweisen von Drosophila heranzuziehen. Definition und Charakterisierung des Begriffes Aufmerksamkeit waren jedoch oft mehrdeutig und unterschieden sich von Studie zu Studie. In dieser Arbeit wird eine ganz bestimmte Form von Aufmerksamkeit – räumlich selektive visuelle Aufmerksamkeit - anhand der Fliege Drosophila untersucht. Es wurde bereits gezeigt, dass die Fliege im stationären Flug ihre Verhaltensantworten spontan auf visuelle Reize einer Seite des visuellen Feldes beschränken kann. Basierend auf Experimenten von Sareen et al. (2011) wurde vermutet, dass die Fliege einen Aufmerksamkeitsfokus (FoA) besitzt und auf Reize, die innerhalb dieses Teils des visuellen Feldes liegen antwortet. Ob der FoA ein angemessenes Konzept für diese räumliche Eigenschaft von SVA in der Fliege ist, steht zur Debatte und ist auch ein Thema dieser Studie. Vorerst soll dieses Konzept jedoch für die Beschreibung der Ergebnisse, die die Charakterisierung von SVA vorantreiben, genutzt werden. Die vorliegende Arbeit führt die Untersuchung von SVA mit variablem aber kontrolliertem visuellem Input im stationären Flug fort und nutzt dazu eine automatisierte Datenerfassung. Dieses standardisierte Paradigma ermöglicht eine Analyse von Verhalten im Wildtyp aber auch einen Vergleich mit verschiedenen mutanten und pharmakologisch manipulierten Fliegenstämmen. Einige im Menschen auftretende Eigenschaften von SVA wurden auch in Drosophila gefunden. Dazu zählt das Auftreten von extern und intern verursachten Aufmerksamkeitsverlagerungen. Es konnte gezeigt werden, dass SVA in der Fliege extern gelenkt werden kann und eine Aufmerksamkeitsspanne aufweist. Zusätzlich wurden ein Neurotransmitter und einige Proteine entdeckt, die eine wichtige Rolle in SVA einnehmen. Darauf basierend ermöglichten es die verfügbaren genetischen Werkzeuge mit einer ersten Untersuchung der an SVA beteiligten Zellen und Netzwerke zu beginnen. Des Weiteren wurde das Laufverhalten von Fliegen, die Einschränkungen in SVA aufwiesen charakterisiert. Die Ergebnisse lassen vermuten, dass die beobachteten Phänotypen von SVA nicht verhaltensspezifisch sind. Als nächstes wurden interne Bewegungen des Aufmerksamkeitskegels (FoA) betrachtet. Der FoA kann durch visuelle Reize von außerhalb zu der einen oder der anderen Seite des visuellen Feldes gelenkt werden. Er verweilt dort für >4s nachdem der lenkende Reiz verschwunden ist. Es ist ein spannender Befund dieser Arbeit, dass dieser Reiz in Abhängigkeit seiner Beschaffenheit abstoßend oder anziehend sein kann. So kann ein abstoßender (negativer) Reiz auf einmal anziehend (positiv) werden, wenn seine Oszillationsamplitude von 4° auf 2° reduziert wird. Eine Überprüfung der Wirksamkeit von Aufmerksamkeitslenkung durch Reize im oberen und unteren Teil des visuellen Feldes ergab, dass die Wahrnehmung eines Reizes durch die Fliege sich nicht ausschließlich aus der Summe seiner Spezifikationen ergibt. Da positive Aufmerksamkeitslenkung in keinem der beiden Halbfelder einen Nacheffekt hatte, ein solcher aber bei der Präsentation von Reizen in beiden Felder gleichzeitig auftrat, kann vermutet werden, dass die Fliege den Reiz für jede Kombination von Parametern spezifisch bewertet. Ob sich diese Bewertung in jedem einzelnen Durchgang änderte oder ob der Reiz in manchen Fällen den FoA nicht auf eine Seite lenkte kann mit dem jetzigen Kenntnisstand nicht bestimmt werden. Betrachtet man die Antworten der Fliege auf eine Versetzung eines schwarzen vertikalen Streifens, so zeigt sich eine mögliche Unterteilung in die Kategorien „keine Antwort“, „syn-direktionale Antwort“ (der Bewegungsrichtung des Streifens folgend) und „anti-direktionale Antwort“ (entgegengesetzt zur Bewegungsrichtung des Streifens). Die Drehmomentmuster der letzteren Kategorie wiesen starke Ähnlichkeit zu spontanen Körpersakkaden auf und es handelte sich bei ihnen sehr wahrscheinlich um Fluchtversuche der Fliege. Syn-direktionale Antworten waren hingegen reine Objekt-Bewegungsantworten, erkennbar an einer längeren Latenz bis zu ihrer Auslösung und einer größeren Amplitude. Diese Eigenschaften und auch die Verteilung der Antworten auf die beiden Kategorien wurden durch die An- oder Abwesenheit eines vorhergehenden Reizes nicht beeinflusst. Wurden zwei Streifen gleichzeitig gegenläufig versetzt, so blieben die Antworten im Vergleich zur Versetzung eines einzelnen Streifens häufiger aus. Wurde der FoA zuvor auf eine Seite gelenkt, so entsprachen die Drehmomentmuster der Antworten auf diese Seite und auch die der Antworten auf die andere Seite denen der syn-direktionalen Antworten. Die Aufklärung der SVA zu Grunde liegenden neuronalen Strukturen konnte bedeutend vorangetrieben werden. Eine Ablation der Pilzkörper (MB) zeigte, dass diese für SVA benötigt werden. Außerdem konnte gezeigt werden, dass die von Dopamin übermittelte Signalstärke weder zu stark, noch zu schwach sein darf. Wurde die Synthese von Dopamin inhibiert oder seine Wiederaufnahme aus dem synaptischen Spalt mittels dDAT blockiert, führte dies dazu, dass die Aufmerksamkeit dieser Fliegen nicht mehr extern gelenkt werden konnte. Mithilfe des Gal4/UAS-Systems und zellspezifischer Expression oder Unterdrückung der Bildung von dDAT wurde die Rolle einzelner Strukturen der Pilzkörper in SVA genauer untersucht. Es zeigte sich, dass die αβ-Loben sowohl ausreichend als auch notwendig sind, um SVA nachhaltig zu lenken. Die Gal4-Linie c708a markiert einen Teil der Kenyonzellen (KC) innerhalb der αβ-Loben, αβposterior. Diese Zellen sind besonders, da (A) ihre Fasern innerhalb der Loben eine netzartige Anordnung aufweisen und (B) da sie anders als die anderen KCs nicht mit der Kalyx, der größten Quelle olfaktorischen Inputs in die MBs, verknüpft sind, sondern nur in der posterioren akzessorischen Kalyx Verbindungen ausbilden (Tanaka et al., 2008). Diese Struktur erhält keinen oder zumindest nur marginalen olfaktorischen Input und es ist anzunehmen, dass sie eher an Aufgaben aus anderen sensorischen Modalitäten beteiligt ist. In dieser Arbeit wird die Beteiligung dieser Zellen an einem visuellen Task gezeigt, genauer ihre Notwendigkeit für einen Nacheffekt der Lenkung von SVA. Eine Wiederherstellung der Funktion von dDAT in diesen ca. 90 Zellen war erfolglos, da die geringe Anzahl möglicherweise nicht ausreichte, um die Konzentration von Dopamin an den relevanten Synapsen zu senken. Es ist jedoch auch möglich, dass die Prozesse, die SVA über die αβ-Loben vermitteln ein Zusammenspiel aller dortigen KCs erfordern. Zusammen bilden die gesammelten Ergebnisse einen Ausgangspunkt für zukünftige Bestrebungen, die für SVA erforderlichen neuronalen Strukturen und deren Verortung komplett zu verstehen. In den bisher beschriebenen Experimenten wurde die Aufmerksamkeit extern gelenkt. Fliegen können ihren FoA aber auch ganz ohne äußerliche Reize intern verlagern. In einer Reihe von 60 aufeinanderfolgenden gleichzeitigen Versetzungen zweier Streifen zeigte sich, dass die Fliegen häufiger Antworten mit der gleichen Polarität wie die vorausgegangene produzierten, als dies eine zufällige Auswahl der Polarität vorhersagte. Dies ließ vermuten, dass der FoA auf einer Seite des visuellen Feldes verweilt. Es wurde angenommen, dass jede Antwort von der vorhergehenden beeinflusst wird, sodass die Wahrscheinlichkeit die Polarität dieser Antwort zu wiederholen um einen gewissen Faktor erhöht wird (dwelling factor, df). Deswegen wurde eine zufällige Verteilung der Antwortpolaritäten unter Berücksichtigung des df berechnet. Dadurch verschwand der Unterschied zwischen der beobachteten Wiederholungswahrscheinlichkeit einer Antwortpolarität und derer einer rein zufälligen Wahl der Antwort. Als das Intervall zwischen den einzelnen Versetzungen schrittweise auf 5s erhöht wurde, konnte bereits bei Pausen über 4s kein signifikanter df mehr festgestellt werden. Als Schlussfolgerung ergibt sich, dass Drosophila eine Aufmerksamkeitsspanne von etwa 4s besitzt. Fliegen mit einer Mutation im radish Gen zeigten keine anhaltende Lenkung von SVA und hatten zudem eine verkürzte Aufmerksamkeitsspanne von ungefähr 1s. Der dDAT-Inhibitor Methylphenidat beseitigte den zuerst erwähnten Phänotyp, verlängerte jedoch nicht die Aufmerksamkeitsspanne. Es ist anzunehmen, dass radish auf unterschiedliche Art und Weise an beiden Mechanismen beteiligt ist. Im Zuge dieser Arbeit wurde gezeigt, dass endogene (covert) Verlagerungen von räumlich selektiver visueller Aufmerksamkeit in der Fliege Drosophila intern und extern gelenkt werden können. Vielfältige Variablen bestimmen die Beschaffenheit eines Reizes. Es bedarf eines systematischeren Ansatzes, um die Eigenschaften eines Reizes genauer zu verstehen, die dessen Wahrnehmung durch die Fliege verändern. Es konnte bereits grundlegend gezeigt werden, dass SVA ein fundamentaler Prozess ist, dessen Fehlfunktion auch die Eigenschaften anderer Verhaltensweisen wie z.B. Laufen beeinflusst. Die Existenz einer Aufmerksamkeitsspanne, die Abhängigkeit von SVA von Dopamin sowie deren Zugänglichkeit für pharmakologische Manipulationen, deren Nutzen für den Menschen in der Behandlung aufmerksamkeitsbezogener Erkrankungen liegt, deuten auf starke Ähnlichkeiten zwischen SVA in Menschen und in Drosophila hin. KW - Taufliege KW - Visueller Reiz KW - visuell KW - Selektive Wahrnehmung KW - Aufmerksamkeit KW - Drosophila Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134452 ER - TY - THES A1 - Beck, Katherina T1 - Einfluss von RSK auf die Aktivität von ERK, den axonalen Transport und die synaptische Funktion in Motoneuronen von \(Drosophila\) \(melanogaster\) T1 - RSK2 alters ERK activity, axonal transport and synaptic function in motoneurons of \(Drosophila\) \(melanogaster\) N2 - In dieser Arbeit sollte die Funktion von RSK in Motoneuronen von Drosophila untersucht werden. Mutationen im RSK2-Gen verursachen das Coffin-Lowry-Syndrom (CLS), das durch mentale Retardierung charakterisiert ist. RSK2 ist hauptsächlich in Regionen des Gehirns exprimiert, in denen Lernen und Gedächtnisbildung stattfinden. In Mäusen und Drosophila, die als Modellorganismen für CLS dienen, konnten auf makroskopischer Ebene keine Veränderungen in den Hirnstrukturen gefunden werden, dennoch wurden in verschiedenen Verhaltensstudien Defekte im Lernen und der Gedächtnisbildung beobachtet. Die synaptische Plastizität und die einhergehenden Veränderungen in den Eigenschaften der Synapse sind fundamental für adaptives Verhalten. Zur Analyse der synaptischen Plastizität eignet sich das neuromuskuläre System von Drosophila als Modell wegen des stereotypen Innervierungsmusters und der Verwendung ionotroper Glutamatrezeptoren, deren Untereinheiten homolog sind zu den Untereinheiten der Glutamatrezeptoren des AMPA-Typs aus Säugern, die wesentlich für die Bildung von LTP im Hippocampus sind. Zunächst konnte gezeigt werden, dass RSK in den Motoneuronen von Drosophila an der präsynaptischen Seite lokalisiert ist, wodurch RSK eine Synapsen-spezifische Funktion ausüben könnte. Morphologische Untersuchungen der Struktur der neuromuskulären Synapsen konnten aufzeigen, dass durch den Verlust von RSK die Größe der neuromuskulären Synapse, der Boutons sowie der Aktiven Zonen und Glutamatrezeptorfelder reduziert ist. Obwohl mehr Boutons gebildet werden, sind weniger Aktive Zonen und Glutamatrezeptorfelder in der neuromuskulären Synapse enthalten. RSK reguliert die synaptische Transmission, indem es die postsynaptische Sensitivität, nicht aber die Freisetzung der Neurotransmitter an der präsynaptischen Seite beeinflusst, obwohl in immunhistochemischen Analysen eine postsynaptische Lokalisierung von RSK nicht nachgewiesen werden konnte. RSK ist demnach an der Regulation der synaptischen Plastizität glutamaterger Synapsen beteiligt. Durch immunhistochemische Untersuchungen konnte erstmals gezeigt werden, dass aktiviertes ERK an der präsynaptischen Seite lokalisiert ist und diese synaptische Lokalisierung von RSK reguliert wird. Darüber hinaus konnte in dieser Arbeit nachgewiesen werden, dass durch den Verlust von RSK hyperaktiviertes ERK in den Zellkörpern der Motoneurone vorliegt. RSK wird durch den ERK/MAPK-Signalweg aktiviert und übernimmt eine Funktion sowohl als Effektorkinase als auch in der Negativregulation des Signalwegs. Demnach dient RSK in den Zellkörpern der Motoneurone als Negativregulator des ERK/MAPK-Signalwegs. Darüber hinaus könnte RSK die Verteilung von aktivem ERK in den Subkompartimenten der Motoneurone regulieren. Da in vorangegangenen Studien gezeigt werden konnte, dass ERK an der Regulation der synaptischen Plastizität beteiligt ist, indem es die Insertion der AMPA-Rezeptoren zur Bildung der LTP reguliert, sollte in dieser Arbeit aufgeklärt werden, ob der Einfluss von RSK auf die synaptische Plastizität durch seine Funktion als Negativregulator von ERK zustande kommt. Untersuchungen der genetischen Interaktion von rsk und rolled, dem Homolog von ERK in Drosophila, zeigten, dass die durch den Verlust von RSK beobachtete reduzierte Gesamtzahl der Aktiven Zonen und Glutamatrezeptorfelder der neuromuskulären Synapse auf die Funktion von RSK als Negativregulator von ERK zurückzuführen ist. Die Größe der neuromuskulären Synapse sowie die Größe der Aktiven Zonen und Glutamatrezeptorfelder beeinflusst RSK allerdings durch seine Funktion als Effektorkinase des ERK/MAPK-Signalwegs. Studien des axonalen Transports von Mitochondrien zeigten, dass dieser in vielen neuropathologischen Erkrankungen beeinträchtigt ist. Die durchgeführten Untersuchungen des axonalen Transports in Motoneuronen konnten eine neue Funktion von RSK in der Regulation des axonalen Transports aufdecken. In den Axonen der Motoneurone von RSK-Nullmutanten wurden BRP- und CSP-Agglomerate nachgewiesen. RSK könnte an der Regulation des axonalen Transports von präsynaptischem Material beteiligt sein. Durch den Verlust von RSK wurden weniger Mitochondrien in anterograder Richtung entlang dem Axon transportiert, dafür verweilten mehr Mitochondrien in stationären Phasen. Diese Ergebnisse zeigen, dass auch der anterograde Transport von Mitochondrien durch den Verlust von RSK beeinträchtigt ist. N2 - In this thesis the function RSK in motoneurons of Drosophila has been analyzed. Mutations in the RSK2-gene cause the Coffin-Lowry-Syndrome (CLS) which is characterized by mental retardation. RSK2 is predominantly expressed in regions of the brain where learning and formation of the memory take place. Even no obvious changes in brain structures could be observed at macroscopic level in mouse and Drosophila which serve as an animal model for CLS. However deficits in various learning tasks could be observed due to the loss of the RSK function. Synaptic plasticity and the following changes in synaptic properties are fundamental for adaptive behaviors. The neuromuscular system of Drosophila suits as a model for studies of the synaptic plasticity because of the stereotypic innervation pattern and the use of ionotropic glutamate receptors which subunits are homologous to the subunits of the mammalian AMPA-type of glutamate receptors which are essential for the formation of LTP in the hippocampus. This study shows that RSK is located at the presynaptic site of the motoneurons of Drosophila which indicates a synapse-specific function of RSK. The structural analysis of the neuromuscular junction (NMJ) show that the loss of RSK causes a reduction in size of the NMJ, boutons, active zones and glutamate receptor fields. More boutons were found at the NMJ, but less active zones and glutamate receptor fields were established. The localization of RSK at the postsynaptic side could not be detected in this study although RSK regulates the synaptic transmission by affecting the postsynaptic sensitivity but not the presynaptic neurotransmitter release. Hence RSK could take part in the regulation of synaptic plasticity. Immunohistochemical analysis could depict a novel function of RSK in the synapse-specific localization of ERK. Further this study show that due to the loss of RSK more activated ERK is located in den cell bodies of the motoneurons. RSK functions as a negative regulator of the ERK/MAPK signaling in the somata of motoneurons. Additionally, RSK could regulate the distribution of ERK in the different subcompartments of the motoneurons. Previous studies show ERK as a regulator of synaptic plasticity by influencing the insertion of AMPA receptors into the postsynaptic membrane during LTP. RSK is activated by the ERK/MAPK signaling and functions not only as an effector kinase but also as a negative regulator of this pathway. If the effect of RSK on synaptic plasticity is due to its function as a negative regulator of ERK should be clarified in this work. Analysis of the genetic interactions of rsk and rolled, the Drosophila homologue of mammalian ERK, show that the reduced number of active zones and glutamate receptor fields found at the NMJ of RSK null mutants is caused by the function of RSK as a negative regulator of ERK. In turn RSK affects the size of the NMJ, also the size of the active zones and glutamate receptor fields by its function as an effector kinase of the ERK/MAPK signaling. Several studies have shown that the axonal transport of mitochondria is affected in many neuropathological diseases. This work could uncover a novel function of RSK in the regulation of the axonal transport in motoneurons. The loss of RSK causes the formation of agglomerates of the presynaptic proteins BRP and CSP. Therefore RSK takes part in the regulation of the transport of presynaptic material. In absence of RSK less mitochondria are transported in anterograde direction and more mitochondria are pausing. This results implicate a function of RSK in regulating the anterograde transport of mitochondria. KW - Taufliege KW - RSK KW - axonaler Transport KW - synaptische Funktion KW - ERK KW - Motoneuron KW - Motoneuron KW - Genmutation KW - Drosophila Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130717 ER - TY - JOUR A1 - Bemm, Felix A1 - Becker, Dirk A1 - Larisch, Christina A1 - Kreuzer, Ines A1 - Escalante-Perez, Maria A1 - Schulze, Waltraud X. A1 - Ankenbrand, Markus A1 - Van de Weyer, Anna-Lena A1 - Krol, Elzbieta A1 - Al-Rasheid, Khaled A. A1 - Mithöfer, Axel A1 - Weber, Andreas P. A1 - Schultz, Jörg A1 - Hedrich, Rainer T1 - Venus flytrap carnivorous lifestyle builds on herbivore defense strategies JF - Genome Research N2 - Although the concept of botanical carnivory has been known since Darwin's time, the molecular mechanisms that allow animal feeding remain unknown, primarily due to a complete lack of genomic information. Here, we show that the transcriptomic landscape of the Dionaea trap is dramatically shifted toward signal transduction and nutrient transport upon insect feeding, with touch hormone signaling and protein secretion prevailing. At the same time, a massive induction of general defense responses is accompanied by the repression of cell death-related genes/processes. We hypothesize that the carnivory syndrome of Dionaea evolved by exaptation of ancient defense pathways, replacing cell death with nutrient acquisition. KW - Dionaea-muscipula ellis KW - Plant utricularia-gibba KW - Programmed cell-death KW - Genomics data sets KW - RNA-SEQ data KW - Arabidopsis-thaliana KW - Jasmonate perception KW - Action potentials KW - Stress responses KW - Wonderful plants Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188799 VL - 26 IS - 6 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 - JOUR A1 - Hacker, Ulrich T. A1 - Escalona-Espinosa, Laura A1 - Consalvo, Nicola A1 - Goede, Valentin A1 - Schiffmann, Lars A1 - Scherer, Stefan J. A1 - Hedge, Priti A1 - Van Cutsem, Eric A1 - Coutelle, Oliver A1 - Büning, Hildegard T1 - Evaluation of Angiopoietin-2 as a biomarker in gastric cancer: results from the randomised phase III AVAGAST trial JF - British Journal of Cancer N2 - Background: In the phase III AVAGAST trial, the addition of bevacizumab to chemotherapy improved progression-free survival (PFS) but not overall survival (OS) in patients with advanced gastric cancer. We studied the role of Angiopoietin-2 (Ang-2), a key driver of tumour angiogenesis, metastasis and resistance to antiangiogenic treatment, as a biomarker. Methods: Previously untreated, advanced gastric cancer patients were randomly assigned to receive bevacizumab (n = 387) or placebo (n = 387) in combination with chemotherapy. Plasma collected at baseline and at progression was analysed by ELISA. The role of Ang-2 as a prognostic and a predictive biomarker of bevacizumab efficacy was studied using a Cox proportional hazards model. Logistic regression analysis was applied for correlations with metastasis. Results: Median baseline plasma Ang-2 levels were lower in Asian (2143 pg ml\(^-\)\(^1\)) vs non-Asian patients (3193 pg ml\(^-\)\(^1\)), P<0.0001. Baseline plasma Ang-2 was identified as an independent prognostic marker for OS but did not predict bevacizumab efficacy alone or in combination with baseline VEGF. Baseline plasma Ang-2 correlated with the frequency of liver metastasis (LM) at any time: Odds ratio per 1000 pg ml\(^-\)\(^1\) increase: 1.19; 95% CI 1.10-1.29; P<0.0001 (non-Asians) and 1.37; 95% CI 1.13-1.64; P = 0.0010 (Asians). Conclusions: Baseline plasma Ang-2 is a novel prognostic biomarker for OS in advanced gastric cancer strongly associated with LM. Differences in Ang-2 mediated vascular response may, in part, account for outcome differences between Asian and non-Asian patients; however, data have to be further validated. Ang-2 is a promising drug target in gastric cancer. KW - gastric cancer KW - angiogenesis KW - Angiopoietin-2 KW - bevacizumab KW - liver metastasis KW - biomarker Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189578 VL - 114 IS - 8 ER - TY - JOUR A1 - Ahmed, Zeeshan A1 - Zeeshan, Saman A1 - Dandekar, Thomas T1 - Mining biomedical images towards valuable information retrieval in biomedical and life sciences JF - Database - The Journal of Biological Databases and Curation N2 - Biomedical images are helpful sources for the scientists and practitioners in drawing significant hypotheses, exemplifying approaches and describing experimental results in published biomedical literature. In last decades, there has been an enormous increase in the amount of heterogeneous biomedical image production and publication, which results in a need for bioimaging platforms for feature extraction and analysis of text and content in biomedical images to take advantage in implementing effective information retrieval systems. In this review, we summarize technologies related to data mining of figures. We describe and compare the potential of different approaches in terms of their developmental aspects, used methodologies, produced results, achieved accuracies and limitations. Our comparative conclusions include current challenges for bioimaging software with selective image mining, embedded text extraction and processing of complex natural language queries. KW - humans KW - software KW - image processing KW - animals KW - computer-assisted KW - data mining/methods KW - natural language processing Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162697 VL - 2016 ER - TY - JOUR A1 - Bert, Bettina A1 - Chmielewska, Justyna A1 - Bergmann, Sven A1 - Busch, Maximilian A1 - Driever, Wolfgang A1 - Finger-Baier, Karin A1 - Hößler, Johanna A1 - Köhler, Almut A1 - Leich, Nora A1 - Misgeld, Thomas A1 - Nöldner, Torsten A1 - Reiher, Annegret A1 - Schartl, Manfred A1 - Seebach-Sproedt, Anja A1 - Thumberger, Thomas A1 - Schönfelder, Gilbert A1 - Grune, Barbara T1 - Considerations for a European animal welfare standard to evaluate adverse phenotypes in teleost fish JF - The EMBO Journal N2 - No abstract available. KW - Danio-rerio KW - Zebrafish KW - Pain Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188783 VL - 35 IS - 11 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 - Chagtai, Tasnim A1 - Zill, Christina A1 - Dainese, Linda A1 - Wegert, Jenny A1 - Savola, Suvi A1 - Popov, Sergey A1 - Mifsud, William A1 - Vujanic, Gordan A1 - Sebire, Neil A1 - Le Bouc, Yves A1 - Ambros, Peter F. A1 - Kager, Leo A1 - O`Sullivan, Maureen J. A1 - Blaise, Annick A1 - Bergeron, Christophe A1 - Holmquist Mengelbier, Linda A1 - Gisselsson, David A1 - Kool, Marcel A1 - Tytgat, Godelieve A.M. A1 - van den Heuvel-Eibrink, Marry M. A1 - Graf, Norbert A1 - van Tinteren, Harm A1 - Coulomb, Aurore A1 - Gessler, Manfred A1 - Williams, Richard Dafydd A1 - Pritchard-Jones, Kathy T1 - Gain of 1q As a Prognostic Biomarker in Wilms Tumors (WTs) Treated With Preoperative Chemotherapy in the International Society of Paediatric Oncology (SIOP) WT 2001 Trial: a SIOP Renal Tumours Biology Consortium Study JF - Journal of Clinical Oncology N2 - Purpose Wilms tumor (WT) is the most common pediatric renal tumor. Treatment planning under International Society of Paediatric Oncology (SIOP) protocols is based on staging and histologic assessment of response to preoperative chemotherapy. Despite high overall survival (OS), many relapses occur in patients without specific risk factors, and many successfully treated patients are exposed to treatments with significant risks of late effects. To investigate whether molecular biomarkers could improve risk stratification, we assessed 1q status and other potential copy number biomarkers in a large WT series. Materials and Methods WT nephrectomy samples from 586 SIOP WT 2001 patients were analyzed using a multiplex ligation-dependent probe amplification (MLPA) assay that measured the copy number of 1q and other regions of interest. Results One hundred sixty-seven (28%) of 586 WTs had 1q gain. Five-year event-free survival (EFS) was 75.0% in patients with 1q gain (95% CI, 68.5% to 82.0%) and 88.2% in patients without gain (95% CI, 85.0% to 91.4%). OS was 88.4% with gain (95% CI, 83.5% to 93.6%) and 94.4% without gain (95% CI, 92.1% to 96.7%). In univariable analysis, 1q gain was associated with poorer EFS (P<.001; hazard ratio, 2.33) and OS (P=.01; hazard ratio, 2.16). The association of 1q gain with poorer EFS retained significance in multivariable analysis adjusted for 1p and 16q loss, sex, stage, age, and histologic risk group. Gain of 1q remained associated with poorer EFS in tumor subsets limited to either intermediate-risk localized disease or nonanaplastic localized disease. Other notable aberrations associated with poorer EFS included MYCN gain and TP53 loss. Conclusion Gain of 1q is a potentially valuable prognostic biomarker in WT, in addition to histologic response to preoperative chemotherapy and tumor stage. KW - Poor-prognosis KW - Mutations KW - Gene KW - Drosha KW - MYCN KW - Mechanisms KW - Reveals KW - Event KW - Relapse KW - Locus Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187478 VL - 34 IS - 26 ER - TY - JOUR A1 - Lorenzin, Francesca A1 - Benary, Uwe A1 - Baluapuri, Apoorva A1 - Walz, Susanne A1 - Jung, Lisa Anna A1 - von Eyss, Björn A1 - Kisker, Caroline A1 - Wolf, Jana A1 - Eilers, Martin A1 - Wolf, Elmar T1 - Different promoter affinities account for specificity in MYC-dependent gene regulation JF - eLife N2 - Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells. KW - MYC KW - promoter affinity KW - human KW - mathematical modeling KW - mouse KW - ChIP-sequencing KW - MIZ1 KW - cancer biology KW - cell biology KW - WDR5 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162913 VL - 5 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 - 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 - 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 - 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 - 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 - Vaze, Koustubh M. A1 - Helfrich-Förster, Charlotte T1 - Drosophila ezoana uses an hour-glass or highly damped circadian clock for measuring night length and inducing diapause JF - Physiological Entomology N2 - Insects inhabiting the temperate zones measure seasonal changes in day or night length to enter the overwintering diapause. Diapause induction occurs after the duration of the night exceeds a critical night length (CNL). Our understanding of the time measurement mechanisms is continuously evolving subsequent to Bünning’s proposal that circadian systems play the clock role in photoperiodic time measurement (Bünning, 1936). Initially, the photoperiodic clocks were considered to be either based on circadian oscillators or on simple hour-glasses, depending on ‘positive’ or ‘negative’ responses in Nanda–Hamner and Bünsow experiments (Nanda & Hammer, 1958; Bünsow, 1960). However, there are also species whose responses can be regarded as neither ‘positive’, nor as ‘negative’, such as the Northern Drosophila species Drosophila ezoana, which is investigated in the present study. In addition, modelling efforts show that the ‘positive’ and ‘negative’ Nanda–Hamner responses can also be provoked by circadian oscillators that are damped to different degrees: animals with highly sustained circadian clocks will respond ‘positive’ and those with heavily damped circadian clocks will respond ‘negative’. In the present study, an experimental assay is proposed that characterizes the photoperiodic oscillators by determining the effects of non-24-h light/dark cycles (T-cycles) on critical night length. It is predicted that there is (i) a change in the critical night length as a function of T-cycle period in sustained-oscillator-based clocks and (ii) a fxed night-length measurement (i.e. no change in critical night length) in damped-oscillator-based clocks. Drosophila ezoana flies show a critical night length of approximately 7 h irrespective of T-cycle period, suggesting a damped-oscillator-based photoperiodic clock. The conclusion is strengthened by activity recordings revealing that the activity rhythm of D. ezoana flies also dampens in constant darkness. KW - photoperiodic time mesurement KW - wyeomyia smithii KW - protophormia terraenovae KW - immunoreactive neurons KW - geographical variation KW - reproductive diapause KW - rhythmic components KW - locomotor activity KW - circadian clock KW - damped-oscillator-model of photoperiodic clock KW - diapause KW - Drosophila KW - hour-glass KW - pitcher-plant mosquito KW - bug riptortus-pedestris KW - Nanda-Hamner KW - photoperiodism Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204278 VL - 41 IS - 4 ER - TY - JOUR A1 - Beer, Katharina A1 - Steffan-Dewenter, Ingolf A1 - Härtel, Stephan A1 - Helfrich-Förster, Charlotte T1 - A new device for monitoring individual activity rhythms of honey bees reveals critical effects of the social environment on behavior JF - Journal of Comparative Physiology A N2 - Chronobiological studies of individual activity rhythms in social insects can be constrained by the artificial isolation of individuals from their social context. We present a new experimental set-up that simultaneously measures the temperature rhythm in a queen-less but brood raising mini colony and the walking activity rhythms of singly kept honey bees that have indirect social contact with it. Our approach enables monitoring of individual bees in the social context of a mini colony under controlled laboratory conditions. In a pilot experiment, we show that social contact with the mini colony improves the survival of monitored young individuals and affects locomotor activity patterns of young and old bees. When exposed to conflicting Zeitgebers consisting of a light-dark (LD) cycle that is phase-delayed with respect to the mini colony rhythm, rhythms of young and old bees are socially synchronized with the mini colony rhythm, whereas isolated bees synchronize to the LD cycle. We conclude that the social environment is a stronger Zeitgeber than the LD cycle and that our new experimental set-up is well suited for studying the mechanisms of social entrainment in honey bees. KW - Social entrainment KW - Foragers KW - Nurses KW - Locomotor activity KW - Temperature rhythms Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188030 VL - 202 IS - 8 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 - Viera, Jonathan Trujillo A1 - El-Merahbi, Rabih A1 - Nieswandt, Bernhard A1 - Stegner, David A1 - Sumara, Grzegorz T1 - Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight JF - PLoS ONE N2 - Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1\(^{-/-}\) and Pld2\(^{-/-}\) mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes. KW - enzyme regulation KW - insulin resistance KW - body weight KW - mouse models KW - bioenergetics KW - insulin KW - hypothalamus KW - adipose tissue Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179729 VL - 11 IS - 6 ER - TY - JOUR A1 - Goméz-H, Laura A1 - Felipe-Medina, Natalia A1 - Sánchez-Martín, Manuel A1 - Davies, Owen R. A1 - Ramos, Isabel A1 - García-Tuñón, Ignacio A1 - de Rooij, Dirk G. A1 - Dereli, Ihsan A1 - Tóth, Attila A1 - Barbero, José Luis A1 - Benavente, Ricardo A1 - Llano, Elena A1 - Pendas, Alberto M. T1 - C14ORF39/SIX6OS1 is a constituent of the synaptonemal complex and is essential for mouse fertility JF - Nature Communications N2 - Meiotic recombination generates crossovers between homologous chromosomes that are essential for genome haploidization. The synaptonemal complex is a ‘zipper’-like protein assembly that synapses homologue pairs together and provides the structural framework for processing recombination sites into crossovers. Humans show individual differences in the number of crossovers generated across the genome. Recently, an anonymous gene variant in C14ORF39/SIX6OS1 was identified that influences the recombination rate in humans. Here we show that C14ORF39/SIX6OS1 encodes a component of the central element of the synaptonemal complex. Yeast two-hybrid analysis reveals that SIX6OS1 interacts with the well-established protein synaptonemal complex central element 1 (SYCE1). Mice lacking SIX6OS1 are defective in chromosome synapsis at meiotic prophase I, which provokes an arrest at the pachytene-like stage and results in infertility. In accordance with its role as a modifier of the human recombination rate, SIX6OS1 is essential for the appropriate processing of intermediate recombination nodules before crossover formation. KW - Chromosomes KW - Meiosis KW - Spermatogenesis Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165907 VL - 7 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 - 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 - 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 - Hartel, Andreas J.W. A1 - Glogger, Marius A1 - Jones, Nicola G. A1 - Abuillan, Wasim A1 - Batram, Christopher A1 - Hermann, Anne A1 - Fenz, Susanne F. A1 - Tanaka, Motomu A1 - Engstler, Markus T1 - N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold JF - Nature Communications N2 - The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed. KW - parasitology KW - cellular imaging KW - membrane biophysics KW - single-molecule biophysics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171368 VL - 7 ER - TY - JOUR A1 - Peters, Marcell K. A1 - Hemp, Andreas A1 - Appelhans, Tim A1 - Behler, Christina A1 - Classen, Alice A1 - Detsch, Florian A1 - Ensslin, Andreas A1 - Ferger, Stefan W. A1 - Frederiksen, Sara B. A1 - Gebert, Frederike A1 - Haas, Michael A1 - Helbig-Bonitz, Maria A1 - Hemp, Claudia A1 - Kindeketa, William J. A1 - Mwangomo, Ephraim A1 - Ngereza, Christine A1 - Otte, Insa A1 - Röder, Juliane A1 - Rutten, Gemma A1 - Costa, David Schellenberger A1 - Tardanico, Joseph A1 - Zancolli, Giulia A1 - Deckert, Jürgen A1 - Eardley, Connal D. A1 - Peters, Ralph S. A1 - Rödel, Mark-Oliver A1 - Schleuning, Matthias A1 - Ssymank, Axel A1 - Kakengi, Victor A1 - Zhang, Jie A1 - Böhning-Gaese, Katrin A1 - Brandl, Roland A1 - Kalko, Elisabeth K.V. A1 - Kleyer, Michael A1 - Nauss, Thomas A1 - Tschapka, Marco A1 - Fischer, Markus A1 - Steffan-Dewenter, Ingolf T1 - Predictors of elevational biodiversity gradients change from single taxa to the multi-taxa community level JF - Nature Communications N2 - The factors determining gradients of biodiversity are a fundamental yet unresolved topic in ecology. While diversity gradients have been analysed for numerous single taxa, progress towards general explanatory models has been hampered by limitations in the phylogenetic coverage of past studies. By parallel sampling of 25 major plant and animal taxa along a 3.7 km elevational gradient on Mt. Kilimanjaro, we quantify cross-taxon consensus in diversity gradients and evaluate predictors of diversity from single taxa to a multi-taxa community level. While single taxa show complex distribution patterns and respond to different environmental factors, scaling up diversity to the community level leads to an unambiguous support for temperature as the main predictor of species richness in both plants and animals. Our findings illuminate the influence of taxonomic coverage for models of diversity gradients and point to the importance of temperature for diversification and species coexistence in plant and animal communities. KW - community ecology KW - macroecology KW - tropical ecology KW - biodiversity Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-169374 VL - 7 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 - Senthilan, Pingkalai R. A1 - Helfrich-Förster, Charlotte T1 - Rhodopsin 7-The unusual Rhodopsin in Drosophila JF - PeerJ N2 - Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1–Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a “vertebrate-melanopsin-type”–cluster, and Rh3, Rh4 and Rh5 form an “insect-type”-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins. KW - vision KW - Drosophila KW - Opsins KW - Rhodopsins KW - phototransduction Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177998 VL - 4 ER - TY - JOUR A1 - Feldbauer, Katrin A1 - Schlegel, Jan A1 - Weissbecker, Juliane A1 - Sauer, Frank A1 - Wood, Phillip G. A1 - Bamberg, Ernst A1 - Terpitz, Ulrich T1 - Optochemokine Tandem for Light-Control of Intracellular Ca\(^{2+}\) JF - PLoS ONE N2 - An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca\(^{2+}\)-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca\(^{2+}\) by tandem endosomes into the cytosol via CatCh was visualized using the Ca\(^{2+}\)-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca\(^{2+}\) in response to light. KW - capacitance KW - endosomes KW - cell membranes KW - membrane proteins KW - intracellular membranes KW - vesicles KW - confocal laser microscopy KW - cytosol Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178921 VL - 11 IS - 10 ER - TY - JOUR A1 - Yadav, Preeti A1 - Selvaraj, Bhuvaneish T. A1 - Bender, Florian L. P. A1 - Behringer, Marcus A1 - Moradi, Mehri A1 - Sivadasan, Rajeeve A1 - Dombert, Benjamin A1 - Blum, Robert A1 - Asan, Esther A1 - Sauer, Markus A1 - Julien, Jean-Pierre A1 - Sendtner, Michael T1 - Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling JF - Acta Neuropathologica N2 - In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features. KW - Amyotrophic-lateral-sclerosis KW - Transgenic mice KW - Mouse model KW - Alzheimers disease KW - Neurofilament KW - Progressive motor neuronopathy KW - Axonal transport KW - Intermediate filaments KW - Motoneuron disease KW - Lacking neurofilaments KW - Missense mutation KW - Axon degeneration KW - Microtubules KW - Stathmin KW - Stat3 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188234 VL - 132 IS - 1 ER - TY - JOUR A1 - Fischer, Robin A1 - Helfrich-Förster, Charlotte A1 - Peschel, Nicolai T1 - GSK-3 Beta Does Not Stabilize Cryptochrome in the Circadian Clock of Drosophila JF - PLoS ONE N2 - Cryptochrome (CRY) is the primary photoreceptor of Drosophila’s circadian clock. It resets the circadian clock by promoting light-induced degradation of the clock protein Timeless (TIM) in the proteasome. Under constant light, the clock stops because TIM is absent, and the flies become arrhythmic. In addition to TIM degradation, light also induces CRY degradation. This depends on the interaction of CRY with several proteins such as the E3 ubiquitin ligases Jetlag (JET) and Ramshackle (BRWD3). However, CRY can seemingly also be stabilized by interaction with the kinase Shaggy (SGG), the GSK-3 beta fly orthologue. Consequently, flies with SGG overexpression in certain dorsal clock neurons are reported to remain rhythmic under constant light. We were interested in the interaction between CRY, Ramshackle and SGG and started to perform protein interaction studies in S2 cells. To our surprise, we were not able to replicate the results, that SGG overexpression does stabilize CRY, neither in S2 cells nor in the relevant clock neurons. SGG rather does the contrary. Furthermore, flies with SGG overexpression in the dorsal clock neurons became arrhythmic as did wild-type flies. Nevertheless, we could reproduce the published interaction of SGG with TIM, since flies with SGG overexpression in the lateral clock neurons shortened their free-running period. We conclude that SGG does not directly interact with CRY but rather with TIM. Furthermore we could demonstrate, that an unspecific antibody explains the observed stabilization effects on CRY. KW - neurons KW - RNA interference KW - hyperexpression techniques KW - circadian rhythms KW - Drosophila melanogaster KW - animal behavior KW - phosphorylation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180370 VL - 11 IS - 1 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 - Holzschuh, Andrea A1 - Dainese, Matteo A1 - Gonzalez-Varo, Juan P. A1 - Mudri-Stojnic, Sonja A1 - Riedinger, Verena A1 - Rundlöf, Maj A1 - Scheper, Jeroen A1 - Wickens, Jennifer B. A1 - Wickens, Victoria J. A1 - Bommarco, Riccardo A1 - Kleijn, David A1 - Potts, Simon G. A1 - Roberts, Stuart P. M. A1 - Smith, Henrik G. A1 - Vilà, Montserrat A1 - Vujic, Ante A1 - Steffan-Dewenter, Ingolf T1 - Mass-flowering crops dilute pollinator abundance in agricultural landscapes across Europe JF - Ecology Letters N2 - Mass-flowering crops (MFCs) are increasingly cultivated and might influence pollinator communities in MFC fields and nearby semi-natural habitats (SNHs). Across six European regions and 2 years, we assessed how landscape-scale cover of MFCs affected pollinator densities in 408 MFC fields and adjacent SNHs. In MFC fields, densities of bumblebees, solitary bees, managed honeybees and hoverflies were negatively related to the cover of MFCs in the landscape. In SNHs, densities of bumblebees declined with increasing cover of MFCs but densities of honeybees increased. The densities of all pollinators were generally unrelated to the cover of SNHs in the landscape. Although MFC fields apparently attracted pollinators from SNHs, in landscapes with large areas of MFCs they became diluted. The resulting lower densities might negatively affect yields of pollinator- dependent crops and the reproductive success of wild plants. An expansion of MFCs needs to be accompanied by pollinator-supporting practices in agricultural landscapes. KW - wild plant pollination KW - Colony growth KW - Densities KW - Context KW - crop pollination KW - Oilseed rape KW - Nesting resources KW - Bee abundance KW - Yield KW - Richness KW - Habitats KW - Agricultural intensification KW - agri-environment schemes KW - biofuels KW - ecosystem services KW - field boundaries KW - landscape compositionv KW - non-crop habitats KW - semi-natural habitats KW - spillover Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187356 VL - 19 IS - 10 ER - TY - JOUR A1 - Wölfling, Mirko A1 - Becker, Mira C. A1 - Uhl, Britta A1 - Traub, Anja A1 - Fiedler, Konrad T1 - How differences in the settling behaviour of moths (Lepidoptera) may contribute to sampling bias when using automated light traps JF - European Journal of Entomology N2 - Quantitative community-wide moth surveys frequently employ flight-interception traps equipped with UV-light emitting sources as attractants. It has long been known that moth species differ in their responsiveness to light traps. We studied how the settling behaviour of moths at a light trap may further contribute to sampling bias. We observed the behaviour of 1426 moths at a light tower. Moths were classified as either, settling and remaining still after arrival, or continually moving on the gauze for extended periods of time. Moths that did not move after settling may not end up in the sampling container of the light trap and therefore are under-represented in automated trap samples relative to their true proportions in the community. Our analyses revealed highly significant behavioural differences between moths that differed in body size. Small moths were more likely to remain stationary after settling. As a corollary, representatives of three taxa, which in Europe are predominantly small species (Nolidae, Geometridae: Eupitheciini, Erebidae: Lithosiini), usually settled down immediately, whereas most other moths remained active on or flying around the trap for some time. Moth behaviour was also modulated by ambient temperature. At high temperatures, they were less likely to settle down immediately, but this behavioural difference was most strongly apparent among medium-sized moths. These results indicate the likely extent of the sampling bias when analysing and interpreting automated light-trap samples. Furthermore, to control for temperature modulated sampling bias temperature should always be recorded when sampling moths using flight-interception traps. KW - Lepidoptera KW - moths KW - biodiversity assessment KW - sampling method KW - light-trapping KW - sampling bias Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191154 VL - 113 ER - TY - JOUR A1 - Kramer, Susanne A1 - Piper, Sophie A1 - Estevez, Antonio A1 - Carrington, Mark T1 - Polycistronic trypanosome mRNAs are a target for the exosome JF - Molecular and Biochemical Parasitology N2 - Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNA5 from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5'-3' exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNA5. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. KW - Trypanosoma brucei KW - Exosome KW - NMD KW - Polycistronic mRNA KW - trans-splicing KW - Trypanosomes Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191350 VL - 205 IS - 1-2 ER - TY - JOUR A1 - Meder, Lydia A1 - König, Katharina A1 - Ozretić, Luka A1 - Schultheis, Anne M. A1 - Ueckeroth, Frank A1 - Ade, Carsten P. A1 - Albus, Kerstin A1 - Boehm, Diana A1 - Rommerscheidt-Fuss, Ursula A1 - Florin, Alexandra A1 - Buhl, Theresa A1 - Hartmann, Wolfgang A1 - Wolf, Jürgen A1 - Merkelbach-Bruse, Sabine A1 - Eilers, Martin A1 - Perner, Sven A1 - Heukamp, Lukas C. A1 - Buettner, Reinhard T1 - NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas JF - International Journal of Cancer N2 - Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionaly, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well. KW - lung cancer KW - small cell lung cancer KW - achaete-scute homolog 1 KW - neurogenic locus notch homolog KW - retinoblastoma protein Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190853 VL - 138 IS - 4 ER - TY - JOUR A1 - Singh, Krishna P. A1 - Verma, Neeraj A1 - Akhoon, Bashir A . A1 - Bhatt, Vishal A1 - Gupta, Shishir K. A1 - Gupta, Shailendra K. A1 - Smita, Suchi T1 - Sequence-based approach for rapid identification of cross-clade CD8+ T-cell vaccine candidates from all high-risk HPV strains JF - 3 Biotech N2 - Human papilloma virus (HPV) is the primary etiological agent responsible for cervical cancer in women. Although in total 16 high-risk HPV strains have been identified so far. Currently available commercial vaccines are designed by targeting mainly HPV16 and HPV18 viral strains as these are the most common strains associated with cervical cancer. Because of the high level of antigenic specificity of HPV capsid antigens, the currently available vaccines are not suitable to provide cross-protection from all other high-risk HPV strains. Due to increasing reports of cervical cancer cases from other HPV high-risk strains other than HPV16 and 18, it is crucial to design vaccine that generate reasonable CD8+ T-cell responses for possibly all the high-risk strains. With this aim, we have developed a computational workflow to identify conserved cross-clade CD8+ T-cell HPV vaccine candidates by considering E1, E2, E6 and E7 proteins from all the high-risk HPV strains. We have identified a set of 14 immunogenic conserved peptide fragments that are supposed to provide protection against infection from any of the high-risk HPV strains across globe. KW - HPV KW - Epitope KW - Cytotoxic KW - T lymphocytes KW - Cervical cancer KW - Vaccine Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191056 VL - 6 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 - Kupper, Maria A1 - Stigloher, Christian A1 - Feldhaar, Heike A1 - Gross, Roy T1 - Distribution of the obligate endosymbiont Blochmannia floridanus and expression analysis of putative immune genes in ovaries of the carpenter ant Camponotus floridanus JF - Arthropod Structure & Development N2 - The bacterial endosymbiont Blochmannia floridanus of the carpenter ant Camponotus floridanus contributes to its hosts' ontogeny via nutritional upgrading during metamorphosis. This primary endosymbiosis is essential for both partners and vertical transmission of the endosymbionts is guaranteed by bacterial infestation of oocytes. Here we present a detailed analysis of the presence and localisation of B. floridanus in the ants' ovaries obtained by FISH and TEM analyses. The most apical part of the germarium harbouring germ-line stem cells (GSCs) is not infected by the bacteria. The bacteria are detectable for the first time in lower parts of the germarium when cystocytes undergo the 4th and 5th division and B. floridanus infects somatic cells lying under the basal lamina surrounding the ovarioles. With the beginning of cystocyte differentiation, the endosymbionts are exclusively transported from follicle cells into the growing oocytes. This infestation of the oocytes by bacteria very likely involves exocytosis endocytosis processes between follicle cells and the oocytes. Nurse cells were never found to harbour the endosymbionts. Furthermore we present first gene expression data in C floridanus ovaries. These data indicate a modulation of immune gene expression which may facilitate tolerance towards the endosymbionts and thus may contribute to their transovarial transmission. KW - Ecologically important traits KW - Bacterial symbionts KW - Arthropods KW - Peptidoglycan recognition KW - Transovarial transmission KW - Horizontal transfer KW - Insect hosts KW - Microorganisms KW - Reproduction KW - Hymenoptera KW - Primary endosymbiont KW - Oogenesis KW - Insects Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187482 VL - 45 IS - 5 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 - 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 - 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 - 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 -