TY - JOUR A1 - Ferero, Andrea A1 - Rivero, Olga A1 - Wäldchen, Sina A1 - Ku, Hsing-Ping A1 - Kiser, Dominik P. A1 - Gärtner, Yvonne A1 - Pennington, Laura S. A1 - Waider, Jonas A1 - Gaspar, Patricia A1 - Jansch, Charline A1 - Edenhofer, Frank A1 - Resink, Thérèse J. A1 - Blum, Robert A1 - Sauer, Markus A1 - Lesch, Klaus-Peter T1 - Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain JF - Frontiers in Cellular Neuroscience N2 - Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding, and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mice display increased cell densities in the DR at embryonic stages E13.5, E17.5, and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that Cdh13 deficiency affects the cell density of the developing DR and the posterior innervation of the prefrontal cortex (PFC), and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders. KW - serotonin KW - cadherin-13 (CDH13) KW - T-cadherin KW - neurodevelopment KW - psychiatric disorders KW - radial glia KW - dorsal raphe KW - prefrontal cortex Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170313 VL - 11 IS - 307 ER - TY - THES A1 - Faist, Hanna T1 - Bedeutung und Charakterisierung der bakteriellen Flora in Vitis vinifera mit und ohne Wurzelhalsgallen T1 - Significance and characterization of the bacterial community in Vitis vinifera with and without crown galls N2 - Am Rebstock werden in der Natur von Agrobacterium vitis, dem Auslöser Wurzelhalsgallenerkrankung, charakteristische Wurzelhalsgallentumore induziert. Virulente Vertreter der Gattung der Agrobacteria schleusen bakterielle DNA in das pflanzliche Genom ein, wodurch die Pflanze Tumore produziert. Die Wurzelhalsgallenerkrankung wird seit einem Jahrhundert als ein Beispiel der Pflanzen-Pathogen-Interaktion untersucht. Die Rolle der bakteriellen Flora im Zusammenhang mit der Wurzelhalsgallenerkrankung beim Rebstock wurde bisher kaum betrachtet. Um dieser Frage nachzugehen, habe ich die endophytische mikrobielle Zusammensetzung von Rebstöcken mit und ohne Wurzelhalsgalle analysiert. Es werden Proben von drei Zeitpunkten einer Wachstumsperiode (Frühling, Sommer und Herbst) und von den Organen der Rebstöcke (Wurzeln, Pfropfstelle und einjährige Triebe) sowie dem Boden in einer Weinanlage bei Himmelstadt in Unterfranken genommen. Die Bakterienflora dieser Umweltproben wird mit kultivierungsabhängigen (Isolierung von Bakterien) und kultivierungsunabhängigen (Hochdurchsatzsequenzierungen) Methoden untersucht. Zudem werden i) die Virulenz der verschiedenen Agrobacterium-Isolate in Tumorassays bestimmt, ii) synthetische Bakteriengemeinschaften von in vitro kultivierten Weinpflänzchen mit Wurzelhalsgallen analysiert, iii) die Genome von einem virulenten und einem nicht-virulenten Agrobacteria-Isolat aus der Wurzelhalsgalle verglichen, iv) erste Interaktionsstudien auf festen Nährmedien durchgeführt und v) virulente Agrobacteria mittels bildgebender Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) in Wurzelhalsgallen lokalisiert. Die Rebstöcke dieser Studie haben eine organspezifische Bakterienflora, die innerhalb einer Wachstumsperiode variiert. Nur die Bakterienflora der Pfropfstelle (mit oder ohne Wurzelhalsgalle) aber nicht die des Bodens, der Wurzeln, und der einjährigen Triebe unterscheidet sich strukturell zwischen gesunden und erkrankten Rebstöcken. Mikroskopisch konnten virulente Agrobacteria punktuell in Interzellularen, sklerenchymatischen Geweben und assoziiert mit Leitgefäßen nachgewiesen werden. Dadurch ist ausreichend Lebensraum vorhanden, der zusätzlich von tumorspezifischen Bakterien besiedelt werden kann. Im Gegensatz zur gesunden Pfropfstelle ist in der Wurzelhalsgalle eine saisonal stabile Kernmikroflora, bestehend aus Vertreter von A. vitis, Pseudomonas, Enterobacteriaceae, Agrobacterium tumefaciens, Gammaproteobacteria und Burkholderiales, vorhanden. Diese Bakterien werden überwiegend aus dem Boden rekrutiert und profitieren von der Nährstoffsituation in der Wurzelhalsgalle. Wurzelhalsgallen enthalten Opine, die nur von der transformierten Pflanzenzelle produziert werden. Interessanterweise hat in dieser Arbeit ein Agrobacterium-Isolat Gene, die zum Opinkatabolismus beitragen und ein Pseudomonas-Isolat kann Opine als einzige Kohlenstoffquelle nutzen. Trotzdem sind beide Isolate weder virulent noch verdrängen sie die virulenten A. vitis, die ebenso Opine nutzen, aus der Wurzelhalsgalle. In synthetischen Bakteriengemeinschaften an in vitro kultivierten Weinpflänzchen konnte gezeigt werden, dass diese und weitere tumorspezifischen Bakterien, neben A. vitis, nicht essentiell zur Entstehung der Wurzelhalsgalle nötig sind aber unterschiedliche Funktionen in der Wurzelhalsgalle übernehmen. Ein Serratia-Isolat hemmt das Wachstum von A. vitis auf festen Nährmedium, andere fördern oder hemmen das Wachstum der Wurzelhalsgalle. Nach Studien in der Literatur erhöhen weitere Bakterien die Resistenz des Rebstocks gegenüber biotischem und abiotischem Stress. Zusammengefasst identifizierten und isolierte ich in dieser Studie unter 150 unterschiedlichen Bakterien in der Wurzelhalsgalle jene Bakterien, die neben A. vitis von der neuen ökologischen Nische profitieren und somit wahrscheinlich Opportunisten mit unterschiedlichen Funktionen sind. In Folge von multiplen Interaktionen in der Wurzelhalsgalle entsteht ein ökologisches Gleichgewicht zwischen den opportunistischen Bakterien, der Wurzelhalsgalle und dem Rebstock, das den Fortbestand des Rebstocks mit Wurzelhalsgalle ermöglicht. N2 - In nature, Agrobacterium vitis is known for the ability to introduce bacterial DNA into the grapevine genome, thereby causing crown gall disease. This plant disease has been studied for a century as a model for plant-pathogen interaction, while the role of the plant microbiota in disease development is not well understood. My study contributes to the understanding of the microbial ecology in crown galls of grapevine, combining culture-dependent with culture-independent high-throughput sequencing techniques. I analysed the structure of the endophytic microbiota by collecting different samples (soil, roots, graft unions and canes) of diseased and non-diseased grapevines from one vine-yard in Franconia, Bavaria, Germany during one growing season (spring, summer, autumn). The characterization of the grapevine-associated bacterial microbiota was completed by (i) detecting the virulence of diverse agrobacterial isolates using a tumour growth assay with in vitro cultivated grapevine plantlets, (ii) microbial analysis of synthetic communities of in vitro cultivated grapevine plantlets with crown galls, (iii) genome sequencing of a virulent and a non-virulent agrobacterial isolate, (iv) in vitro interaction studies on solid medium with bacterial isolates and (v) localisation of virulent A. vitis using Fluorescence Lifetime Imaging Microscopy (FLIM) in tumour tissues. Grapevine plants of this study have an organ-specific bacterial community that varies during one growing season. Healthy and diseased grapevine plants differed in the struc-ture of the bacterial community only in the graft union (with or without a crown gall), but not in the soil, root and one-year old cane. Microscopy revealed that virulent Agrobacteria mainly accumulate in defined spots of sclerenchymatous tissue, intercellular space and tissues associated with vessels. Therefore, there is unoccupied living space in a crown gall, which can be additionally colonized by tumour-specific bacteria. A season-independent stable core bacteria exists in grapevine crown galls in contrast to healthy graft unions, consisting of OTUs assigned to A. vitis, Pseudomonas, Enterobacteriaceae, Agrobacterium tumefaciens, Gammaproteobacteria and Burkholderiales. These bacteria are predominantly recruited from the soil and most likely profit from special nutrients in the crown gall. The crown gall contains opines, exclusively produced by transformed plant cells. Curiously individual isolates of Agrobacteria and Pseudo-monas of this study that are non-virulent do not outcompete virulent A. vitis in the crown gall but harbour, like A. vitis, genes involved in octopin-catabolism or use opines in liquid cultures as a sole nutrient source. Although synthetic bacterial communities revealed that the tumour-specific bacteria are not required for crown gall induction us-ing in vitro grown grapevine plantlets, they may have different functions in crown gall persistence. A Serratia-isolate inhibits the growth of A. vitis on solid medium, others reduce or support crown gall development, while some, according to literature, increase resistance of the grapevine plant against biotic and abiotic stresses. Taken together, among the 150 bacteria found in the crown galls, I identified and isolated bacteria in addition to A. vitis that profit from the new ecological niche suggesting an opportunistic lifestyle with different ecological functions. An ecological equilibrium in a bacterial community that balances crown gall growth will support the existence of grapevine plants with a crown gall in vineyards. KW - Wurzelhalsgalle KW - DNA Barcoding KW - Agrobacterium vitis KW - Weinrebe KW - Angewandte Mikrobiologie KW - bakterielle Flora KW - Holobiont KW - Mikrobiota Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-154359 ER - TY - JOUR A1 - Ewald, Jan A1 - Bartl, Martin A1 - Dandekar, Thomas A1 - Kaleta, Christoph T1 - Optimality principles reveal a complex interplay of intermediate toxicity and kinetic efficiency in the regulation of prokaryotic metabolism JF - PLOS Computational Biology N2 - A precise and rapid adjustment of fluxes through metabolic pathways is crucial for organisms to prevail in changing environmental conditions. Based on this reasoning, many guiding principles that govern the evolution of metabolic networks and their regulation have been uncovered. To this end, methods from dynamic optimization are ideally suited since they allow to uncover optimality principles behind the regulation of metabolic networks. We used dynamic optimization to investigate the influence of toxic intermediates in connection with the efficiency of enzymes on the regulation of a linear metabolic pathway. Our results predict that transcriptional regulation favors the control of highly efficient enzymes with less toxic upstream intermediates to reduce accumulation of toxic downstream intermediates. We show that the derived optimality principles hold by the analysis of the interplay between intermediate toxicity and pathway regulation in the metabolic pathways of over 5000 sequenced prokaryotes. Moreover, using the lipopolysaccharide biosynthesis in Escherichia coli as an example, we show how knowledge about the relation of regulation, kinetic efficiency and intermediate toxicity can be used to identify drug targets, which control endogenous toxic metabolites and prevent microbial growth. Beyond prokaryotes, we discuss the potential of our findings for the development of antifungal drugs. KW - Enzyme regulation KW - Toxicity KW - Metabolic pathways KW - Enzymes KW - Transcriptional control KW - Enzyme kinetics KW - Enzyme metabolism KW - Predictive toxicology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180870 VL - 13 IS - 2 ER - TY - JOUR A1 - Dütting, Sebastian A1 - Gaits-Iacovoni, Frederique A1 - Stegner, David A1 - Popp, Michael A1 - Antkowiak, Adrien A1 - van Eeuwijk, Judith M.M. A1 - Nurden, Paquita A1 - Stritt, Simon A1 - Heib, Tobias A1 - Aurbach, Katja A1 - Angay, Oguzhan A1 - Cherpokova, Deya A1 - Heinz, Niels A1 - Baig, Ayesha A. A1 - Gorelashvili, Maximilian G. A1 - Gerner, Frank A1 - Heinze, Katrin G. A1 - Ware, Jerry A1 - Krohne, Georg A1 - Ruggeri, Zaverio M. A1 - Nurden, Alan T. A1 - Schulze, Harald A1 - Modlich, Ute A1 - Pleines, Irina A1 - Brakebusch, Cord A1 - Nieswandt, Bernhard T1 - A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis JF - Nature Communications N2 - Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V. KW - megakaryocytes KW - blood platelets KW - regulatory circuit downstream KW - glycoprotein Ib Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170797 VL - 8 IS - 15838 ER - TY - JOUR A1 - Drescher, Nora A1 - Klein, Alexandra-Maria A1 - Neumann, Peter A1 - Yañez, Orlando A1 - Leonhardt, Sara D. T1 - Inside Honeybee Hives: Impact of Natural Propolis on the Ectoparasitic Mite Varroa destructor and Viruses JF - Insects N2 - Social immunity is a key factor for honeybee health, including behavioral defense strategies such as the collective use of antimicrobial plant resins (propolis). While laboratory data repeatedly show significant propolis effects, field data are scarce, especially at the colony level. Here, we investigated whether propolis, as naturally deposited in the nests, can protect honeybees against ectoparasitic mites Varroa destructor and associated viruses, which are currently considered the most serious biological threat to European honeybee subspecies, Apis mellifera, globally. Propolis intake of 10 field colonies was manipulated by either reducing or adding freshly collected propolis. Mite infestations, titers of deformed wing virus (DWV) and sacbrood virus (SBV), resin intake, as well as colony strength were recorded monthly from July to September 2013. We additionally examined the effect of raw propolis volatiles on mite survival in laboratory assays. Our results showed no significant effects of adding or removing propolis on mite survival and infestation levels. However, in relation to V. destructor, DWV titers increased significantly less in colonies with added propolis than in propolis-removed colonies, whereas SBV titers were similar. Colonies with added propolis were also significantly stronger than propolis-removed colonies. These findings indicate that propolis may interfere with the dynamics of V. destructor-transmitted viruses, thereby further emphasizing the importance of propolis for honeybee health. KW - social immunity KW - Apis mellifera KW - deformed wing virus KW - plant-insect interactions KW - resin KW - sacbrood virus Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171164 VL - 8 IS - 1 ER - TY - JOUR A1 - Degen, Tobias A1 - Hovestadt, Thomas A1 - Mitesser, Oliver A1 - Hölker, Franz T1 - Altered sex-specific mortality and female mating success: ecological effects and evolutionary responses JF - Ecosphere N2 - Theory predicts that males and females should often join the mating pool at different times (sexual dimorphism in timing of emergence [SDT]) as the degree of SDT affects female mating success. We utilize an analytical model to explore (1) how important SDT is for female mating success, (2) how mating success might change if either sex's mortality (abruptly) increases, and (3) to what degree evolutionary responses in SDT may be able to mitigate the consequences of such mortality increase. Increasing male pre‐mating mortality has a non‐linear effect on the fraction of females mated: The effect is initially weak, but at some critical level a further increase in male mortality has a stronger effect than a similar increase in female mortality. Such a change is expected to impose selection for reduced SDT. Increasing mortality during the mating season has always a stronger effect on female mating success if the mortality affects the sex that emerges first. This bias results from the fact that enhancing mortality of the earlier emerging sex reduces female–male encounter rates. However, an evolutionary response in SDT may effectively mitigate such consequences. Further, if considered independently for females and males, the predicted evolutionary response in SDT could be quite dissimilar. The difference between female and male evolutionary response in SDT leads to marked differences in the fraction of fertilized females under certain conditions. Our model may provide general guidelines for improving harvesting of populations, conservation management of rare species under altered environmental conditions, or maintaining long‐term efficiency of pest‐control measures. KW - evolutionary response KW - sexual dimorphism in timing KW - sex-specific mortality KW - reproductive asynchrony KW - mating success Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170953 VL - 8 IS - 5 ER - TY - JOUR A1 - Danner, Nadja A1 - Keller, Alexander A1 - Härtel, Stephan A1 - Steffan-Dewenter, Ingolf T1 - Honey bee foraging ecology: Season but not landscape diversity shapes the amount and diversity of collected pollen JF - PLoS ONE N2 - The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes. KW - honey bees KW - pollen KW - season KW - foraging Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170424 VL - 12 IS - 8 ER - TY - THES A1 - Danner, Nadja T1 - Honey bee foraging in agricultural landscapes T1 - Sammelverhalten von Honigbienen in der Agrarlandschaft N2 - 1. Today honey bee colonies face a wide range of challenges in modern agricultural landscapes which entails the need for a comprehensive investigation of honey bees in a landscape context and the assessment of environmental risks. Within this dissertation the pollen foraging of honey bee colonies is studied in different agricultural landscapes to gain insight into the use of pollen resources and the influence of landscape structure across the season. General suggestions for landscape management to support honey bees and other pollinators are derived. 2. Decoding of waggle dances and a subsequent spatial foraging analysis are used as methods in Chapters 4 and 5 to study honey bee colonies in agricultural landscapes. The recently developed metabarcoding of mixed pollen samples was applied for the first time in honey bee foraging ecology and allowed for a detailed analysis of pollen, that was trapped from honey bees in front hive entrances (Chapter 6). 3. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach to light microscopy, which still is a tedious and error-prone task. In this study we assessed mixed pollen probes through next-generation sequencing and developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialized palynological expert knowledge. 4. During maize flowering, four observation hives were placed in and rotated between 11 landscapes covering a gradient in maize acreage. A higher foraging frequency on maize fields compared to other landuse types showed that maize is an intensively used pollen resource for honey bee colonies. Mean foraging distances were significantly shorter for maize pollen than for other pollen origins, indicating that effort is put into collecting a diverse pollen diet. The percentage of maize pollen foragers did not increase with maize acreage in the landscape and was not reduced by grassland area as an alternative pollen resource. Our findings allow estimating the distance-related exposure risk of honey bee colonies to pollen from surrounding maize fields treated with systemic insecticides. 5. It is unknown how an increasing area of mass-flowering crops like oilseed rape (OSR) or a decrease of semi-natural habitats (SNH) change the temporal and spatial availability of pollen resources for honey bee colonies, and thus foraging distances and frequency in different habitat types. Sixteen observation hives were placed in and rotated between 16 agricultural landscapes with independent gradients of OSR and SNH area within 2 km to analyze foraging distances and frequencies. SNH and OSR reduced foraging distance at different spatial scales and depending on season, with possible benefits for the performance of honey bee colonies. Frequency of pollen foragers per habitat type was equally high for SNH, grassland and OSR fields, but lower for other crops and forest. In landscapes with a small proportion of SNH a significantly higher density of pollen foragers on SNH was observed, indicating the limitation of pollen resources in simple agricultural landscapes and the importance of SNH. 6. Quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment we rotated 16 honey bee colonies across 16 agricultural landscapes (see also Chapter 5), used traps to get samples of collected pollen and observed the intra-colonial dance communication to gain information about foraging distances. Neither the amount of collected pollen nor pollen diversity were related to landscape diversity. The revealed increase of foraging distances with decreasing landscape diversity suggests that honey bees compensate for a lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. 7. Our results show the importance of diverse pollen resources for honey bee colonies in agricultural landscapes. Beside the risk of exposure to pesticides honey bees face the risk of nutritional deficiency with implications for their health. By modifying landscape composition and therefore availability of resources we are able to contribute to the wellbeing of honey bees. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes. N2 - 1. Honigbienen stehen heutzutage vor einer Vielzahl von Herausforderungen in der modernen Agrarlandschaft, was umfassende Untersuchungen von Honigbienen im Landschafskontext erforderlich macht. Im Rahmen dieser Arbeit wurde das Pollensammeln von Honigbienenvölkern in verschiedenen Agrarlandschaften studiert, um Einblick in die Nutzung von Pollenressourcen und auf den Einfluss der Landschaftsstruktur zu gewinnen. 2. Die Dekodierung von Schwänzeltänzen und eine anschließende räumliche Analyse des Sammelverhaltens werden als Methoden in den Kapiteln 4 und 5 eingesetzt, um Bienenvölker in Agrarlandschaften zu untersuchen. Das kürzlich entwickelte Metabarcoding von gemischten Pollenproben wurde zum ersten Mal in der Honigbienenökologie angewandt und ermöglichte eine detaillierte Analyse von Pollenproben, die per Pollenfallen vor den Stockeingängen gesammelt wurden (Kapitel 6). 3. Pollenbestimmung durch molekulare Sequenzierung und DNA Barcoding wurde als Alternative zur Lichtmikroskopie vorgeschlagen, die immer noch sehr mühsam und fehlerbehaftet ist. In dieser Studie bestimmten wir gemischte Pollenproben durch Next-Generation-Sequenzierung und entwickelten einen bioinformatischen Arbeitsablauf um diese Hochdurchsatz-Daten mit einer neu kreierten Referenzdatanbank zu analysieren. Um die Durchführbarkeit zu evaluieren verglichen wir Ergebnisse aus der klassischen Identifizierung via Lichtmikroskopie derselben Proben mit unseren Sequenzier-Ergebnissen. Häufigkeitsschätzungen auf Basis der Sequenzierdaten waren signifikant mit den gezählten Häufigkeiten via Lichtmikroskopie korreliert. Next-Generation-Sequenzierung stellt daher einen nützlichen und effizienten Arbeitsablauf dar, um Pollen auf dem Gattungs- und Artniveau zu bestimmen ohne spezielles palynologisches Expertenwissen zu benötigen. 4. Während der Maisblüte wurden vier Beobachtungsstöcke in 11 Landschaften mit einem Maisflächengradienten platziert und zwischen diesen rotiert. Maisfelder wurden intensiver genutzt als Flächen anderer Landnutzungstypen. Die mittleren Sammeldistanzen waren signifikant niedriger für Maispollen als Pollen anderer Herkunft, was darauf hinweist, dass Aufwand in das Sammeln einer diversen Pollendiät gesetzt wird. Der Anteil an Maispollensammlerinnen stieg nicht mit der Maisanbaufläche in der Landschaft und wurde nicht durch Grünlandfläche als alternative Pollenressource reduziert. Unsere Ergebnisse ermöglichen die Schätzung des entfernungsbezogenen Expositionsrisikos von Honigbienenvölker auf Pollen aus den umliegenden Maisfeldern, die mit systemischen Insektiziden behandelt werden. 5. Es ist nicht bekannt, wie eine Zunahme von Massentrachten wie Raps (OSR) oder eine Abnahme von halbnatürlichen Habitaten (SNH) die zeitliche und räumliche Verfügbarkeit von Pollenressourcen für die Honigbienen, und damit Sammeldistanzen und -frequenzen in verschiedenen Lebensraumtypen verändert. Sechzehn Beobachtungsstöcke wurden in 16 Agrarlandschaften mit unabhängigen Gradienten an OSR- und SNH-Fläche innerhalb von 2 km platziert und regelmäßig rotiert, um Sammeldistanzen und -frequenzen zu analysieren. SNH und OSR reduzierten die Sammeldistanzen auf verschiedenen räumlichen Skalen und je nach Saison, mit möglichen Vorteilen für die Leistungsfähigkeit von Bienenvölkern. Die Häufigkeit der Pollensammler pro Habitattyp war gleich hoch für SNH, Grünland und OSR, aber niedriger für andere Kulturen und Wald. In Landschaften mit einem kleinen Anteil von SNH wurde eine deutlich höhere Dichte von Pollensammlerinnen auf SNH beobachtet, was auf die Begrenzung der Pollenressourcen in einfachen Agrarlandschaften und die Bedeutung von SNH hinweist. 6. Menge und Diversität des gesammelten Pollens können das Wachstum und die Gesundheit von Honigbienenvölkern beeinflussen, aber es ist wenig über den Einfluss der Landschaftsstruktur auf die Pollendiät bekannt. In einem Feldexperiment rotierten wir 16 Honigbienenkolonien über 16 Agrarlandschaften (siehe auch Kapitel 5), nutzten Pollenfallen um Proben des gesammelten Pollens zu nehmen und beobachteten die intrakoloniale Tanzkommunikation, um Informationen über die Sammeldistanzen zu erhalten. Weder Pollenmenge noch -diversität waren von der Landschaftsdiversität abhängig. Der offenbarte Anstieg von Sammeldistanzen mit abnehmender Landschaftsdiversität legt nahe, dass Honigbienen durch die Erweiterung des Pollensammelbereichs eine niedrigere Landschaftsdiversität kompensieren, um Pollenmenge und -diversität zu erhalten. 7. Unsere Ergebnisse zeigen die Bedeutung eines diversen Pollenangebots für Bienenvölker in der Agrarlandschaft. Neben dem Risiko einer Exposition gegenüber Pestiziden, stehen Bienenvölker vor der Gefahr von Mangelernährung mit Auswirkungen auf ihre Gesundheit. Durch eine Änderung der Landschaftzusammensetzung und damit der Verfügbarkeit von Ressourcen können wir zum Wohlergehen der Honigbienen beitragen. Agrarumweltmaßnahmen mit dem Ziel Bestäuber zu unterstützen, sollten sich auf mögliche räumliche und zeitliche Lücken in der Pollenverfügbarkeit und Vielfalt in der Agrarlandschaft konzentrieren. KW - Apis mellifera KW - Zea mays KW - Resource Use KW - Exposure Risk KW - Oilseed Rape KW - foraging distances KW - Sammeldistanzen KW - semi-natural habitat KW - halbnatürliche Habitate KW - next-generation sequencing KW - pollen KW - Pollen KW - Next-Generation Sequenzierung KW - Landschaftsstruktur KW - landscape structure Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139322 ER - TY - JOUR A1 - Dainese, Matteo A1 - Schneider, Gudrun A1 - Krauss, Jochen A1 - Steffan-Dewenter, Ingolf T1 - Complementarity among natural enemies enhances pest suppression JF - Scientific Reports N2 - Natural enemies have been shown to be effective agents for controlling insect pests in crops. However, it remains unclear how different natural enemy guilds contribute to the regulation of pests and how this might be modulated by landscape context. In a field exclusion experiment in oilseed rape (OSR), we found that parasitoids and ground-dwelling predators acted in a complementary way to suppress pollen beetles, suggesting that pest control by multiple enemies attacking a pest during different periods of its occurrence in the field improves biological control efficacy. The density of pollen beetle significantly decreased with an increased proportion of non-crop habitats in the landscape. Parasitism had a strong effect on pollen beetle numbers in landscapes with a low or intermediate proportion of non-crop habitats, but not in complex landscapes. Our results underline the importance of different natural enemy guilds to pest regulation in crops, and demonstrate how biological control can be strengthened by complementarity among natural enemies. The optimization of natural pest control by adoption of specific management practices at local and landscape scales, such as establishing non-crop areas, low-impact tillage, and temporal crop rotation, could significantly reduce dependence on pesticides and foster yield stability through ecological intensification in agriculture. KW - ecosystem services KW - agroecology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158621 VL - 7 ER - TY - JOUR A1 - Costea, Paul I. A1 - Coelho, Louis Pedro A1 - Sunagawa, Shinichi A1 - Munch, Robin A1 - Huerta-Cepas, Jaime A1 - Forslund, Kristoffer A1 - Hildebrand, Falk A1 - Kushugulova, Almagul A1 - Zeller, Georg A1 - Bork, Peer T1 - Subspecies in the global human gut microbiome JF - Molecular Systems Biology N2 - Population genomics of prokaryotes has been studied in depth in only a small number of primarily pathogenic bacteria, as genome sequences of isolates of diverse origin are lacking for most species. Here, we conducted a large‐scale survey of population structure in prevalent human gut microbial species, sampled from their natural environment, with a culture‐independent metagenomic approach. We examined the variation landscape of 71 species in 2,144 human fecal metagenomes and found that in 44 of these, accounting for 72% of the total assigned microbial abundance, single‐nucleotide variation clearly indicates the existence of sub‐populations (here termed subspecies). A single subspecies (per species) usually dominates within each host, as expected from ecological theory. At the global scale, geographic distributions of subspecies differ between phyla, with Firmicutes subspecies being significantly more geographically restricted. To investigate the functional significance of the delineated subspecies, we identified genes that consistently distinguish them in a manner that is independent of reference genomes. We further associated these subspecies‐specific genes with properties of the microbial community and the host. For example, two of the three Eubacterium rectale subspecies consistently harbor an accessory pro‐inflammatory flagellum operon that is associated with lower gut community diversity, higher host BMI, and higher blood fasting insulin levels. Using an additional 676 human oral samples, we further demonstrate the existence of niche specialized subspecies in the different parts of the oral cavity. Taken together, we provide evidence for subspecies in the majority of abundant gut prokaryotes, leading to a better functional and ecological understanding of the human gut microbiome in conjunction with its host. KW - biology KW - genetic variation KW - metagenomics KW - microbiome KW - population structure KW - prokaryotic subspecies Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172674 VL - 13 IS - 12 ER - TY - THES A1 - Contar Adolfi, Mateus T1 - Sex determination and meiosis in medaka: The role of retinoic acid T1 - Geschlechtsbestimmung und Meiose in Medaka: Die Rolle der Retinsäure N2 - Sex determination (SD) is a complex and diverse developmental process that leads to the decision whether the bipotential gonad anlage will become a testis or an ovary. This mechanism is regulated by gene cascades, networks and/or chromosomal systems, and can be influenced by fluctuations of extrinsic factors like temperature, exposure to hormones and pollution. Within vertebrates, the group of fish show the widest variety of sex determination mechanism. This whole diversity of processes and mechanisms converges to the formation of two different gametes, the eggs and the sperm, the first bigger and static, and the second smaller and motile. Meiosis is crucial for the formation of both types of gametes, and the timing of meiosis entry is one of the first recognizable differences between male and female in vertebrates. The germ cells go into meiosis first in female than in male, and in mammals, this event has been shown to be regulated by retinoic acid (RA). This small polar molecule induces in the germ cells the expression of the pre-meiotic marker Stra8 (stimulated by retinoic acid gene 8), which is necessary for meiosis initiation. Interestingly, genome analyzes have shown that the majority of fish (including medaka) lack the stra8 gene, adding a question mark to the role of RA in meiosis induction in this group. Since a role of RA in entry of meiosis and sexual development of fish is still far from being understood, I investigated in medaka (Oryzias latipes) a possible signaling function of RA during the SD period in embryos and in reproductively active gonads of adults. I generated a transgenic medaka line that reports responsiveness to RA in vivo. With this tool, I compared RA responsiveness with the expression of the main gene involved in the synthesis of RA. My results show that there is a de-correlation between the action of RA with its source. In adults, expression of the RA metabolizing enzymes show sexually dimorphic RA levels, with aldh1a2 levels being higher in testis, and cyp26a1 stronger in female gonad. In ovary, the responsiveness is restricted to the early meiotic oocytes. In testis, RA is acting directly in the pre-meiotic cells, but also in Sertoli and Leydig cells. Treatment experiments on testis organ culture showed that RA pathway activation leads to a decrease in meiosis markers expression levels. During the development, RA responsiveness in the germ cells was observed in both sexes much earlier than the first female meiosis entry. Treatments with RA-synthesis inhibitor show a decrease in meiosis markers expression levels only after the sex differentiation period in female. Expression analyzes of embryos treated with exogenous RA showed induction of dmrt1a at the gonad levels and an increase of amh levels. Both genes are not only involved in male formation, but also in the regulation of germ cell proliferation and differentiation. RA is important in meiosis induction and gametogenesis in adult medaka. However, there is no evidence for a similar role of RA in initiating the first meiosis in female germ cells at the SD stage. Moreover, contrary to common expectation, RA seems to induce sex related genes that are involved indirectly in meiosis inhibition. In this thesis, I showed for the first time that RA can be involved in both induction and inhibition of meiosis entry, depending on the sex and the developmental stage in a stra8-independent model organism. N2 - Geschlechtsbestimmung ist ein komplexer und vielfältiger Entwicklungsprozess, der zu der Entscheidung führt, ob sich aus der bipotenten Gonadenanlage Hoden oder Ovarien entwickeln. Dieser Mechanismus ist durch Genkaskaden, Netzwerke und/oder chromosomale Systeme reguliert, kann aber auch durch Fluktuation äußerer Faktoren wie beispielsweise Temperatur, durch Hormonexposition oder durch Umweltverschmutzung beeinflusst werden. Innerhalb der Wirbeltiere zeigen Fische die größte Vielfalt in Bezug auf die Mechanismen der Geschlechtsbestimmung. Die unterschiedlichen Mechanismen der Geschlechtsbestimmung konvergieren in der Entstehung von der beiden unterschiedlichen Geschlechtszellen, der Eizelle und des Spermiums. Die Eizelle ist groß und statisch, das Spermium hingegen ist kleiner und beweglich. Die entscheidende Rolle für die Entstehung der Geschlechtzellen spielt die Meiose. Der Zeitpunkt, an dem zum ersten Mal in der Entwicklung die Meiose einsetzt, ist der erste erkennbare Unterschied in der Gonadenentwicklung zwischen männlichen und weiblichen Wirbeltieren. Die Meiose der Keimzellen beginnt bei Weibchen früher als bei Männchen. Bei Säugetieren reguliert Retinsäure (RA) diesen Prozess. Dieses kleine polare Molekül induziert die Expression des Prä-Meiose-Markers Stra8 (stimulated by retinoic acid gene 8) in den Keimzellen, welcher für den Eintritt in die Meiose essentiell ist. Interessanterweise haben Genomanalyzen gezeigt, dass das stra8 Gen in Medaka sowie in den meisten anderen Fischarten nicht vorhanden ist. Dies stellt eine vergleichbare Rolle von RA für die Induktion der Meiose wie bei Säugetieren in diesen Fischen in Frage. Da die Rolle von RA für den Eintritt in die Meiose sowie für die Geschlechtsentwicklung in Fischen bisher nur unzureichend untersucht und verstanden ist, habe ich bei Medaka (Oryzias latipes) eine mögliche Funktion von RA für die Geschlechtsdetermination in Embryonen sowie in Gonaden geschlechtsreifer Tiere untersucht. Ich habe im Rahmen dieser Arbet eine transgene Medakalinie generiert, die in vivo eine RA induzierte Genexpression durch ein GFP Reportergen anzeigt. Mit Hilfe dieser Linie wurde die transkriptionsreulierende Aktivität von RAmit der Expression der wichtigsten Gene, die in die RA Synthese involviert sind, verglichen. Meine Ergebnisse zeigen eine Diskrepanz zwischen dem Wirkungs- und Syntheseort von RA. Die RA metabolisierenden Enzyme zeigten eine geschlechtsdimorphe Expression in adulten Medakas, mit einer höheren aldh1a2 Expression im Hoden sowie einer stärkeren cyp26a1 Expression in weiblichen Gonaden. Im Ovar sind lediglich frühe meiotische Eizellen RA-sensitiv. Im Hoden wirkt RA direkt in prä-meiotische Zellen, aber auch in Sertoli und Leydig Zellen. Stimulations-Experimente an Hoden Organkulturen ergaben, dass eine Aktivierung des RA Signalwegs zu einer Abnahme des Expressionslevels von Meiose-Markern führt. Während der Embryonalentwicklung konnte in den Keimzellen beider Geschlechter eine transkriptions-induziernende Aktivität von RA bereits zu einem Zeitpunkt beobachtet werden, der deutlich vor dem ersten Meiose Eintritt in Weibchen liegt. Behandlungen mit einem RA-Synthese Inhibitor zeigten lediglich nach der Geschlechtsdifferenzierung in Weibchen eine verminderte Expression der Meiose-Marker. Expressionsanalysen von Embryonen, die mit exogener RA behandelt wurden, ergaben eine Induktion von dmrt1a in den Gonaden und ein Anstieg von amh. Beide Gene sind sowohl in die männliche Geschlechtsentwicklung involviert, als auch in die Regulation der Keimzellproliferation und –differenzierung. Zusammen ergaben meine Untersuchungen, dass RA für die Induktion der Meiose und der Gametogenese in adulten Medakas wichtig ist. Allerdings gibt es keinen Hinweis für eine ähnliche Rolle 11 von RA bei der Initiierung der ersten Meiose in weiblichen Keimzellen während der Geschlechtsdetermination. Im Gegensatz zur bisher beschriebenen Situation, scheint darüber hinaus RA die Expression geschlechtsspezifischer Gene zu induzieren, die indirekt in die Inhibition der Meiose involviert sind. In der vorliegenden Arbeit konnte in einem stra8-unabhängigen Modelorganismus das erste Mal gezeigt werden, dass RA – abhängig vom Geschlecht und vom Stadium der Entwicklung - sowohl in die Induktion als auch in die Inhibition des Meiose-Eintritts involviert ist. KW - Japankärpfling KW - Meiose KW - Sex determination KW - Meiosis KW - Retinoic acid KW - Medaka KW - Geschlechtsdifferenzierung KW - Retinoesäure KW - Geschlechtsbestimmung Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136335 ER - TY - JOUR A1 - Chen, Yi-chun A1 - Mishra, Dushyant A1 - Gläß, Sebastian A1 - Gerber, Bertram T1 - Behavioral Evidence for Enhanced Processing of the Minor Component of Binary Odor Mixtures in Larval Drosophila JF - Frontiers in Psychology N2 - A fundamental problem in deciding between mutually exclusive options is that the decision needs to be categorical although the properties of the options often differ but in grade. We developed an experimental handle to study this aspect of behavior organization. Larval Drosophila were trained such that in one set of animals odor A was rewarded, but odor B was not (A+/B), whereas a second set of animals was trained reciprocally (A/B+). We then measured the preference of the larvae either for A, or for B, or for “morphed” mixtures of A and B, that is for mixtures differing in the ratio of the two components. As expected, the larvae showed higher preference when only the previously rewarded odor was presented than when only the previously unrewarded odor was presented. For mixtures of A and B that differed in the ratio of the two components, the major component dominated preference behavior—but it dominated less than expected from a linear relationship between mixture ratio and preference behavior. This suggests that a minor component can have an enhanced impact in a mixture, relative to such a linear expectation. The current paradigm may prove useful in understanding how nervous systems generate discrete outputs in the face of inputs that differ only gradually. KW - learning KW - memory KW - perception KW - compound conditioning KW - decision-making KW - Drosophila Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170011 VL - 8 IS - 1923 ER - TY - THES A1 - Brühlmann, David T1 - Tailoring Recombinant Protein Quality by Rational Media Design T1 - Der Einfluss von Zellkulturmedien auf Qualitätsattribute von rekombinanten Proteinen N2 - Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression. At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor. Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing. N2 - Mehr als die Hälfte der Biotherapeutika werden heutzutage aufgrund korrekter Proteinfaltung und korrektem Zusammenbau in tierischen Zelllinien hergestellt, welche zudem die Fähigkeit besitzen, verschiedene posttranslationale Modifikationen zu bewerkstelligen, hergestellt. Der ausgeprägte Aufschwung von Biosimilars hat den Entwicklungsschwerpunkt von Zellkulturverfahren verlagert. Dabei hat die Modulierung der Qualitätsattribute von rekombinanten Proteinen bereits in frühen Entwicklungsstadien eine wichtige Bedeutung erlangt. Die Qualitätsattribute beeinflussen die klinische Wirksamkeit und die In-Vivo-Sicherheit direkt. Somit ist die Regulierung der posttranslationalen Modifikationen, einschließlich der Glykosylierung (mannosereiche, fukosylierte, galaktosylierte und sialylierte Glykane), der Ladungsvarianten, sowie die Bildung von Aggregaten und niedermolekularen Spezien, für effiziente Rezeptorbindung und das Auslösen der gewünschten Immunantwort in Patienten entscheidend. Im Rahmen der Biosimilarentwicklung werden Methoden zur Anpassung der Produktqualität innerhalb des Potentials der Wirtszelle gesucht, um sie möglichst genau dem Referenzarzneimittel anzugleichen. Die Umgebung, in der die Zelle verweilt, beeinflusst ihren Metabolismus und das resultierende Produktqualitätsprofil. Dabei spielen Medien eine zentrale Rolle in der Zellkultur. Im Rahmen dieser Doktorarbeit wurden durch Adjustierung von ausgewählten Medienbestandteilen und Ergänzung mit einer Vielfalt von Stoffen diverse Stoffwechselwege, Enzymaktivitäten und in einigen Fällen das Genexpressionsniveau von kultivierten Chinesischen Hamster-Ovarialzellen (CHO) verändert. Die Ergänzung von Zellkulturmedium mit Raffinose, ein Trisaccharid, führte zu einer Erhöhung des mannosereichen Glykosylierungsmusters in zwei unterschiedlichen CHO-Zelllinien. Raffinose begünstigte hauptsächlich Mannose-5-Spezien. Gleichzeitig wurde die Zellkulturleistung beeinträchtigt und zudem intrazelluläre Nukleotidkonzentrationen sowie das Expressionsniveau von Glykosylierungsgenen verändert. Ergänzung mit mehreren Inhibitoren der Galaktosyltransferase, insbesondere fluorierte Galaktosenachbildungen (Alpha- und Beta-2F-Peracetyl-Galaktose), verringerte stetig die Produktion von galaktosylierten monoklonalen Antikörpern (mAb). Durch gezielte Zugabe im Verlauf der Kultur, statt bereits am Anfang, wurde die Inhibition weiter erhöht, und dabei die Einwirkung auf das Zellwachstum und die Produktivität beschränkt. Ein Hochdurchsatz-Screening in 96-Deep-Well-Platten zeigte, dass Spermin und L-Ornithin auch das Ausmaß der Galaktosylierung reduzierte. Andererseits zeigten erste Nachforschungen anhand eines Screenings einer Auswahl von potenziellen Disulfidbrücken-Reduktionsmittel, dass wahrscheinlich begünstigte Reduktion das inhärente Niedermolekular-Speziesniveau des firmeneigenen Zellkulturplattformverfahrens verursacht. Die Hypothese wurde durch die Beigabe von Cystein und N-Acetylcystein bekräftigt. Diese Stoffe begünstigten die Fragmentierung, wohingegen sie bei höherer Proteinexpression abnahm. Mit dem Ziel die Entwicklungseffizienz zu steigern, wurde daraufhin zur Identifikation von qualitätsverändernden Stoffen und Bestimmung der optimalen Konzentrationsbereichen eine rationale Versuchsanordnungsmethode entwickelt. Dazu wurde eine Kombination von Hochdurchsatz-Fed-Batch-Tests und multivariater Datenanalyse herbeigezogen. Siebzehn Mediumergänzungsstoffe wurden in fünf parallelen 96-Deep-Well-Platten-Experimenten getestet. Das Auswahlverfahren von erfolgsversprechenden Modulatoren fürs Nachfolgeexperiment in Schüttelröhrchen umfasste drei Schritte: Hauptkomponentenanalyse, quantitative Evaluierung der Leistung der Modulatoren hinsichtlich der Biosimilaritätsspezifikationen und die Auswahl in Anlehnung an eine hierarchische Entscheidungsreihenfolge mit Hilfe eines Entscheidungsbaums. Die Methode führte in nur zwei Versuchsreihen zu einer erheblichen Annäherung an das gewünschte Glykosylierungsprofil. Anschließende Entwicklungsschritte (Validierung und Transfer in die großtechnische Anlage) erforden eine rigorose Kontrolle der Produktqualität. Demzufolge konnte dank der Non-Targeted Metabolomics Analyse von vier verschiedenen Herstellungsverfahren einer mAb exprimierenden CHO-Zelllinie weitere mechanistische Kenntnisse der zugrunde liegenden Vorgängen gewonnen werden. Univariate Analysen der intra- und extrazellulären Stoffwechselprodukte und die zeitliche Glykosylierungsprofile lieferten einen Einblick in verschiedene Stoffwechselwege. Die Vielzahl von Parametern führte dazu, nach dem Prinzip der Hauptkomponentenanalyse vorzugehen, und dann anhand der Partial Least Squares (PLS)-Projektion auf latente Strukturen ein multivariates Modell zu erstellen, das die extrazellulären Daten mit den individuellen Glykosylierungsprofilen korreliert. Das PLS Beobachtungsmodell stellte sich als verlässlich heraus und zeigte seinen außerordentlichen Nutzen zur Regulierung der Glykanen in der Routineherstellung, insbesondere in der Großanlage. Anstatt sich auf Glykosylierungsresultate nach dem Ende der Produktion zu verlassen, kann die Glykosylierung, basierend auf den Niveaus der extrazellulären Stoffwechselprodukte im Bioreaktor, in Echtzeit vorausgesagt werden. Schließlich können im Rahmen des Arzneigenehmigungsverfahrens Gesundheitsbehörden verlangen, die Glykanunterschiede zwischen dem Biosimilar und dem Referenzarzneimittel zu untersuchen. Damit der biologische Test die Unterschiede nachweisen kann, muss eine erweiterte Palette von Glykanvarianten hergestellt werden. Die entwickelte Glykosylierungsmodulierungsbibliothek ermöglichte, extreme Varianten für mannosereiche, afukosylierte, galaktosylierte und sialylierte Glykane von mAb und einem Antikörperfusionsmolekül mit drei N-Glykosylierungsstellen zu generieren. Für erhöhte Variantenvielfalt wurde die enzymatische Glykoengineering Technologie für die Galaktosylierung und Sialylierung untersucht. Die Glykanvarianten erzeugten signifikante Antworten in der jeweiligen In-Vitro-Bestimmung der biologischen Aktivität. Die Ergebnisse unterstreichen das immense Potential von Zellkulturmediumoptimierung zur Anpassung der Produktqualität. Ergänzung des Mediums und der Nährstofflösung brachte reproduzierbare und beträchtliche Veränderungen der Produktqualität von mAb und eines Fusionsantikörpers hervor. Zusätzlich zu den intermediären Modulierungsbereichen, die mehr als ausreichend den Anforderungen für die Entwicklung von neuen biologischen Wirkstoffen und Biosimilars genügen, ermöglichte die Mediumergänzung auf schnelle und einfache Art und Weise selbst extreme Glykanvarianten zu bilden, die für die Bestimmung der biologischen Aktivität geeignet waren. KW - CHO cell culture KW - product qualitymodulation KW - media design KW - metabolism KW - glycosylation KW - high throughput KW - Zellkultur KW - CHO-Zelle KW - Produktivität KW - Nährboden KW - Stoffwechsel Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147345 ER - TY - THES A1 - Borst, Andreas T1 - Apoptosis & senescence: cell fate determination in inhibitor-treated melanoma cells T1 - Apoptose & Seneszenz: Bestimmung der Zell-spezifischen Reaktion von Melanomzellen auf Inhibitoren N2 - Neoplasms of the skin represent the most frequent tumors worldwide; fortunately, most of them are benign or semi-malignant and well treatable. However, the two most aggressive and deadly forms of malignant skin-neoplasms are melanoma and Merkel cell carcinoma (MCC), being responsible for more than 90% of skin-cancer related deaths. The last decade has yielded enormous progress in melanoma therapy with the advent of targeted therapies, like BRAF or MEK inhibitors, and immune-stimulating therapies, using checkpoint antibodies targeting CTLA- 4, PD-1 or PD-L1. Very recent studies suggest that also MCC patients benefit from a treatment with checkpoint antibodies. Nevertheless, in an advanced metastatic stage, a cure for both of these aggressive malignancies is still hard to achieve: while only a subset of patients experience durable benefit from the immune-based therapies, the widely applicable targeted therapies struggle with development of resistances that inevitably occur in most patients, and finally lead to their death. The four articles included in this thesis addressed current questions concerning therapy and carcinogenesis of melanoma and MCC. Moreover, they are discussed in the light of the up-to-date research regarding targeted and immune-based therapies. In article I we demonstrated that besides apoptosis, MAPK pathway inhibition in BRAF-mutated melanoma cells also induces senescence, a permanent cell cycle arrest. These cells may provide a source for relapse, as even permanently arrested cancer cells can contribute to a pro-tumorigenic milieu. To identify molecular factors determining the differential response, we established M14 melanoma cell line derived single cell clones that either undergo cell death or arrest when treated with BRAF/MEK inhibitors. Using these single cell clones, we demonstrated in article IV that downregulation of the pro-apoptotic BH3-only protein BIK via epigenetic silencing is involved in apoptosis deficiency, which can be overcome by HDAC inhibitors. These observations provide a possible explanation for the lack of a complete and durable response to MAPK inhibitor treatment in melanoma patients, and suggest the application of HDAC inhibitors as a complimentary therapy to MAPK pathway inhibition. Concerning MCC, we scrutinized the interactions between the Merkel cell polyomavirus’ (MCV) T antigens (TA) and the tumor suppressors p53 and Rb in article II and III, respectively. In article III, we demonstrated that the cell cycle master regulator Rb is the crucial target of MCV large T (LT), while it - in contrast to other polyomavirus LTs - exhibits much lower affinity to the related proteins p107 and p130. Knockdown of MCV LT led to proliferation arrest in MCC cells, which can be rescued by knockdown of Rb, but not by knockdown of p107 and p130. Contrary to Rb, restriction of p53 in MCC seems to be independent of the MCV TAs, as we demonstrated in article II. In conclusion, the presented thesis has revealed new molecular details, regarding the response of melanoma cells towards an important treatment modality and the mechanisms of viral carcinogenesis in MCC. N2 - Die häufigsten Tumore weltweit sind Neoplasien der Haut; glücklicherweise sind die meisten dieser benigne oder semi-maligne und gut behandelbar. Die beiden aggressivsten und tödlichsten Formen bösartiger Hauttumoren sind das Melanom und das Merkelzell-Karzinom (MCC), welche verantwortlich für über 90% aller durch Hauttumore verursachten Todesfälle sind. Im letzten Jahrzehnt gab es jedoch erstaunliche Fortschritte in der Therapie des malignen Melanoms, was vor allem durch das Aufkommen der zielgerichteten Therapien wie den BRAF oder MEK Inhibitoren und den immunstimulierenden Therapien, welche Checkpoint-Antikörper gegen CTLA-4, PD-1 oder PD-L1 verwenden, bedingt ist. Neueste Studien legen nahe, dass auch MCC Patienten von diesen Checkpoint-Antikörpern profitieren können. In fortgeschrittenen, metastasierten Stadien ist jedoch für beide Malignitäten eine Heilung immer noch sehr schwer erreichbar: nur eine kleine Gruppe der Patienten erreichen einen dauerhaften Nutzen durch die Immuntherapien, während die breit anwendbaren zielgerichteten Therapien mit der Entwicklung von Resistenzen zu kämpfen haben, welche unausweichlich in den meisten Patienten entstehen und letztendlich zu deren Tod führen. Die vier dieser Dissertation beigefügten Publikationen adressierten aktuelle Fragestellungen bezüglich Therapie und Karzinogenese des Melanoms und des MCCs. Des Weiteren werden diese im Licht des heutigen Forschungsstandes diskutiert, im Besonderen mit Blick auf die zielgerichteten und immunbasierten Therapien. In Publikation I zeigten wir, dass Inhibition des MAPK Signalwegs in BRAF-mutierten Melanom-Zellen neben Apoptose auch zu Seneszenz, einem permanenten Zellzyklusarrest, führen kann. Diese Zellen können der Ursprung der Resistenzbildung sein, da auch permanent arretierte Krebszellen zu einem Tumor-fördernden Milieu beitragen können. Um molekulare Faktoren zu identifizieren, die für diese unterschiedliche Behandlungsreaktion ursächlich sind, haben wir Einzelzellklone aus der M14 Melanom-Zelllinie etabliert, welche entweder mit Zelltod oder Arrest auf die BRAF/MEK Inhibitor Behandlung reagieren. Mit Hilfe dieser Klone zeigten wir in Publikation IV, dass die Herunterregulierung des pro-apoptotischen BH3-only Proteins BIK durch einen epigenetischen Mechanismus zur Apoptose-Resistenz dieser Zellen führt, was durch den Einsatz von HDAC-Inhibitoren umgangen werden kann. Diese Beobachtungen bieten eine mögliche Erklärung für das Ausbleiben eines vollständigen und dauerhaften Ansprechens auf die MAPK-Inhibitor Behandlung der Melanom-Patienten, und legen den Einsatz von HDAC-Inhibitoren als komplementäre Therapieoption nahe. Beim MCC haben wir jeweils die Interaktion zwischen den Merkelzell-Polyomavirus (MCV) T Antigenen (TA) und den Tumor-Suppressoren p53 und Rb in Publikation II und III näher betrachtet. In Publikation III haben wir gezeigt, dass das zentrale, Zellzyklus-regulierende Protein Rb das vorrangige Ziel des MCV large T Antigens (LT) ist, während es - im Gegensatz zu anderen Polyomavirus-LTs - viel weniger Affinität zu den verwandten Proteinen p107 und p 130 aufweist. Der Knockdown des MCV LT führte zu Proliferationsarrest in MCC Zellen, welcher durch Knockdown von Rb aufgehoben werden konnte, nicht jedoch durch Knockdown von p107 und p130. Die Restriktion von p53 scheint im Gegensatz zu Rb im MCC unabhängig von den MCV TAs zu sein, wie wir in Publikation II gezeigt haben. Zusammenfassend gibt diese Dissertation Aufschluss über neue molekulare Zusammenhänge bezüglich der Reaktion von Melanom-Zellen gegenüber einer wichtigen Behandlungsmöglichkeit und den Mechanismen der viralen Karzinogenese des MCC. KW - Melanom KW - Apoptosis KW - MAP-Kinase KW - Senescence KW - BRAF inhibition Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-155085 ER - TY - JOUR A1 - Beer, Katharina A1 - Joschinski, Jens A1 - Sastre, Alazne Arrazola A1 - Krauss, Jochen A1 - Helfrich-Förster, Charlotte T1 - A damping circadian clock drives weak oscillations in metabolism and locomotor activity of aphids (Acyrthosiphon pisum) JF - Scientific Reports N2 - Timing seasonal events, like reproduction or diapause, is crucial for the survival of many species. Global change causes phenologies worldwide to shift, which requires a mechanistic explanation of seasonal time measurement. Day length (photoperiod) is a reliable indicator of winter arrival, but it remains unclear how exactly species measure day length. A reference for time of day could be provided by a circadian clock, by an hourglass clock, or, as some newer models suggest, by a damped circadian clock. However, damping of clock outputs has so far been rarely observed. To study putative clock outputs of Acyrthosiphon pisum aphids, we raised individual nymphs on coloured artificial diet, and measured rhythms in metabolic activity in light-dark illumination cycles of 16:08 hours (LD) and constant conditions (DD). In addition, we kept individuals in a novel monitoring setup and measured locomotor activity. We found that A. pisum is day-active in LD, potentially with a bimodal distribution. In constant darkness rhythmicity of locomotor behaviour persisted in some individuals, but patterns were mostly complex with several predominant periods. Metabolic activity, on the other hand, damped quickly. A damped circadian clock, potentially driven by multiple oscillator populations, is the most likely explanation of our results. KW - circadian mechanisms KW - behavioural ecology KW - damped circadian clock KW - Acyrthosiphon pisum Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170020 VL - 7 IS - 14906 ER - TY - JOUR A1 - Becam, Jérôme A1 - Walter, Tim A1 - Burgert, Anne A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Seibel, Jürgen A1 - Schubert-Unkmeir, Alexandra T1 - Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria JF - Scientific Reports N2 - Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C\(_{6}\) and long-chain C\(_{16}\)-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C\(_{6}\)-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω–azido-C\(_{6}\)-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C\(_{6}\)-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae. KW - ceramide analogs KW - Neisseria KW - ceramide Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159367 VL - 7 ER - TY - JOUR A1 - Arenas, Andrés A1 - Roces, Flavio T1 - Avoidance of plants unsuitable for the symbiotic fungus in leaf-cutting ants: Learning can take place entirely at the colony dump JF - PLoS ONE N2 - Plants initially accepted by foraging leaf-cutting ants are later avoided if they prove unsuitable for their symbiotic fungus. Plant avoidance is mediated by the waste produced in the fungus garden soon after the incorporation of the unsuitable leaves, as foragers can learn plant odors and cues from the damaged fungus that are both present in the recently produced waste particles. We asked whether avoidance learning of plants unsuitable for the symbiotic fungus can take place entirely at the colony dump. In order to investigate whether cues available in the waste chamber induce plant avoidance in naïve subcolonies, we exchanged the waste produced by subcolonies fed either fungicide-treated privet leaves or untreated leaves and measured the acceptance of untreated privet leaves before and after the exchange of waste. Second, we evaluated whether foragers could perceive the avoidance cues directly at the dump by quantifying the visits of labeled foragers to the waste chamber. Finally, we asked whether foragers learn to specifically avoid untreated leaves of a plant after a confinement over 3 hours in the dump of subcolonies that were previously fed fungicide-treated leaves of that species. After the exchange of the waste chambers, workers from subcolonies that had access to waste from fungicide-treated privet leaves learned to avoid that plant. One-third of the labeled foragers visited the dump. Furthermore, naïve foragers learned to avoid a specific, previously unsuitable plant if exposed solely to cues of the dump during confinement. We suggest that cues at the dump enable foragers to predict the unsuitable effects of plants even if they had never been experienced in the fungus garden. KW - leaves KW - ants KW - fungi KW - foraging KW - animal sociality KW - social systems KW - learning KW - symbiosis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157559 VL - 12 IS - 3 ER - TY - JOUR A1 - Ampattu, Biju Joseph A1 - Hagmann, Laura A1 - Liang, Chunguang A1 - Dittrich, Marcus A1 - Schlüter, Andreas A1 - Blom, Jochen A1 - Krol, Elizaveta A1 - Goesmann, Alexander A1 - Becker, Anke A1 - Dandekar, Thomas A1 - Müller, Tobias A1 - Schoen, Christoph T1 - Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence JF - BMC Genomics N2 - Background: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. Results: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. Conclusions: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis. KW - neisseria meningitidis KW - MITE KW - virulenceregulatory evolution KW - systems biology KW - metabolism KW - cryptic KW - genetic variation KW - stringent response KW - relA Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157534 VL - 18 IS - 282 ER -