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 - Peck, Barrie A1 - Schug, Zachary T. A1 - Zhang, Qifeng A1 - Dankworth, Beatrice A1 - Jones, Dylan T. A1 - Smethurst, Elizabeth A1 - Patel, Rachana A1 - Mason, Susan A1 - Jian, Ming A1 - Saunders, Rebecca A1 - Howell, Michael A1 - Mitter, Richard A1 - Spencer-Dene, Bradley A1 - Stamp, Gordon A1 - McGarry, Lynn A1 - James, Daniel A1 - Shanks, Emma A1 - Aboagye, Eric O. A1 - Critchlow, Susan E. A1 - Leung, Hing Y. A1 - Harris, Adrian L. A1 - Wakelam, Michael J. O. A1 - Gottlieb, Eyal A1 - Schulze, Almut T1 - Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments JF - Cancer & Metabolism N2 - Background Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets. Results Using functional genomics, we identified stearoyl-CoA desaturase (SCD), an enzyme that controls synthesis of unsaturated fatty acids, as essential in breast and prostate cancer cells. SCD inhibition altered cellular lipid composition and impeded cell viability in the absence of exogenous lipids. SCD inhibition also altered cardiolipin composition, leading to the release of cytochrome C and induction of apoptosis. Furthermore, SCD was required for the generation of poly-unsaturated lipids in cancer cells grown in spheroid cultures, which resemble those found in tumour tissue. We also found that SCD mRNA and protein expression is elevated in human breast cancers and predicts poor survival in high-grade tumours. Finally, silencing of SCD in prostate orthografts efficiently blocked tumour growth and significantly increased animal survival. Conclusions Our data implicate lipid desaturation as an essential process for cancer cell survival and suggest that targeting SCD could efficiently limit tumour expansion, especially under the metabolically compromised conditions of the tumour microenvironment. KW - SCD KW - lipidomics KW - prostate cancer KW - breast cancer KW - lipid desaturation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145905 VL - 4 IS - 6 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 - 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 - THES A1 - Sickel, Wiebke T1 - High-throughput biodiversity assessment - Powers and limitations of meta-barcoding T1 - Hochdurchsatzerfassung von Biodiversität - Stärken und Grenzen von Meta-barcoding N2 - Traditional species identification based on morphological characters is laborious and requires expert knowledge. It is further complicated in the case of species assemblages or degraded and processed material. DNA-barcoding, species identification based on genetic data, has become a suitable alternative, yet species assemblages are still difficult to study. In the past decade meta-barcoding has widely been adopted for the study of species communities, due to technological advances in modern sequencing platforms and because manual separation of individual specimen is not required. Here, meta-barcoding is put into context and applied to the study of bee-collected pollen as well as bacterial communities. These studies provide the basis for a critical evaluation of the powers and limitations of meta-barcoding. Advantages identified include species identification without the need for expert knowledge as well as the high throughput of samples and sequences. In microbiology, meta-barcoding can facilitate directed cultivation of taxa of interest identified with meta-barcoding data. Disadvantages include insufficient species resolution due to short read lengths and incomplete reference databases, as well as limitations in abundance estimation of taxa and functional profiling. Despite these, meta-barcoding is a powerful method for the analysis of species communities and holds high potential especially for automated biomonitoring. N2 - Traditionelle Methoden der Identifizierung von Organismen anhand von morphologischen Merkmalen sind arbeits- und zeitaufwendig und benötigen Expertenkenntnisse der Morphologie. Weitere Probleme liegen in der Analyse von Artgemeinschaften und prozessiertem Material. DNA-barcoding, Artbestimmung anhand von genetischen Merkmalen, hat sich als Alternative herausgebildet, jedoch sind Artgemeinschaften nach wie vor schwierig zu analysieren. Im vergangenen Jahrzehnt wurde meta-barcoding zur Analyse von Artgemeinschaften entwickelt; insbesondere durch die Weiterentwicklung moderner Sequenziergeräte und da eine Auftrennung der Organismen innerhalb einer Gemeinschaft nicht mehr notwendig ist. In der vorliegenden Arbeit wurde zunächst ein Überblick über meta-barcoding erstellt. Die Methode wurde dann für die Analyse von Bienen-gesammeltem Pollen und Bakteriengemeinschaften angewandt. Diese Studien bilden eine gute Basis, um die Vor- und Nachteile von meta-barcoding kritisch zu bewerten. Vorteile beinhalten unter anderem, dass Organismen bestimmt werden können, ohne dass Expertenkenntnisse notwendig sind, sowie der hohe Durchsatz von Proben und Sequenzen. In der Mikrobiologie kann meta-barcoding eine gerichtete Kultivierung von Bakterien erleichtern, die durch meta-barcoding als Zielorganismen indentifiziert wurden. Nachteile finden sich in der manchmal noch unzureichenden Unterscheidung nah ver- wandter Arten aufgrund von kurzen Sequenzlängen und lückenhaften Referenzdatenbanken, sowie Einschränkungen in der Abschätzung von Abundanzen und Funktionen der Organismen innerhalb der Artgemeinschaft. Trotz dieser Problematiken ist meta-barcoding eine leistungsstarke Methode für die Analyse von Artgemeinschaften und ist besonders vielversprechend für automatisiertes Bio-Monitoring. KW - Bacterial community analysis KW - pollen analysis KW - Biodiversity assessment KW - Meta-barcoding KW - Biodiversität KW - DNS-Sequenz Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144573 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 - Kneitz, Susanne A1 - Mishra, Rasmi R. A1 - Chalopin, Domitille A1 - Postlethwait, John A1 - Warren, Wesley C. A1 - Walther, Ronald B. A1 - Schartl, Manfred T1 - Germ cell and tumor associated piRNAs in the medaka and \(Xiphophorus\) melanoma models JF - BMC Genomics N2 - Background A growing number of studies report an abnormal expression of Piwi-interacting RNAs (piRNAs) and the piRNA processing enzyme Piwi in many cancers. Whether this finding is an epiphenomenon of the chaotic molecular biology of the fast dividing, neoplastically transformed cells or is functionally relevant to tumorigenesisis is difficult to discern at present. To better understand the role of piRNAs in cancer development small laboratory fish models can make a valuable contribution. However, little is known about piRNAs in somatic and neoplastic tissues of fish. Results To identify piRNA clusters that might be involved in melanoma pathogenesis, we use several transgenic lines of medaka, and platyfish/swordtail hybrids, which develop various types of melanoma. In these tumors Piwi, is expressed at different levels, depending on tumor type. To quantify piRNA levels, whole piRNA populations of testes and melanomas of different histotypes were sequenced. Because no reference piRNA cluster set for medaka or Xiphophorus was yet available we developed a software pipeline to detect piRNA clusters in our samples and clusters were selected that were enriched in one or more samples. We found several loci to be overexpressed or down-regulated in different melanoma subtypes as compared to hyperpigmented skin. Furthermore, cluster analysis revealed a clear distinction between testes, low-grade and high-grade malignant melanoma in medaka. Conclusions Our data imply that dysregulation of piRNA expression may be associated with development of melanoma. Our results also reinforce the importance of fish as a suitable model system to study the role of piRNAs in tumorigenesis. KW - small RNA-sequencing KW - melanoma KW - piRNA KW - fish model Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146028 VL - 17 IS - 357 ER - TY - JOUR A1 - Widmann, Annekathrin A1 - Artinger, Marc A1 - Biesinger, Lukas A1 - Boepple, Kathrin A1 - Peters, Christina A1 - Schlechter, Jana A1 - Selcho, Mareike A1 - Thum, Andreas S. T1 - Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae JF - PLoS Genetics N2 - Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution. KW - genetic dissection KW - Drosophila KW - memory formation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166672 VL - 12 IS - 10 ER - TY - JOUR A1 - 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 - 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 - Markert, Sebastian Matthias A1 - Britz, Sebastian A1 - Proppert, Sven A1 - Lang, Marietta A1 - Witvliet, Daniel A1 - Mulcahy, Ben A1 - Sauer, Markus A1 - Zhen, Mei A1 - Bessereau, Jean-Louis A1 - Stigloher, Christian T1 - Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome JF - Neurophotonics N2 - Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses. KW - caenorhabditis elegans KW - localization micoscopy KW - fluorescent-probes KW - junction proteins KW - resolution limit KW - direct stochasticoptical reconstruction microscopy KW - structured illumination microscopy KW - correlative light and electron microscopy KW - gap junction KW - neural circuits KW - nervous-system KW - image data KW - reconstruction KW - innexins KW - super-resolution microscopy Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187292 VL - 3 IS - 4 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 - Jones, Julia C. A1 - Fruciano, Carmelo A1 - Keller, Anja A1 - Schartl, Manfred A1 - Meyer, Axel T1 - Evolution of the elaborate male intromittent organ of Xiphophorus fishes JF - Ecology and Evolution N2 - Internally fertilizing animals show a remarkable diversity in male genital morphology that is associated with sexual selection, and these traits are thought to be evolving particularly rapidly. Male fish in some internally fertilizing species have “gonopodia,” highly modified anal fins that are putatively important for sexual selection. However, our understanding of the evolution of genital diversity remains incomplete. Contrary to the prediction that male genital traits evolve more rapidly than other traits, here we show that gonopodial traits and other nongonopodial traits exhibit similar evolutionary rates of trait change and also follow similar evolutionary models in an iconic genus of poeciliid fish (Xiphophorus spp.). Furthermore, we find that both mating and nonmating natural selection mechanisms are unlikely to be driving the diverse Xiphophorus gonopodial morphology. Putative holdfast features of the male genital organ do not appear to be influenced by water flow, a candidate selective force in aquatic habitats. Additionally, interspecific divergence in gonopodial morphology is not significantly higher between sympatric species, than between allopatric species, suggesting that male genitals have not undergone reproductive character displacement. Slower rates of evolution in gonopodial traits compared with a subset of putatively sexually selected nongenital traits suggest that different selection mechanisms may be acting on the different trait types. Further investigations of this elaborate trait are imperative to determine whether it is ultimately an important driver of speciation. KW - Male intromittent organ KW - reproductive character displacement KW - sexual selection KW - species diversification KW - Xiphophorus fish Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164956 VL - 6 IS - 20 ER - TY - JOUR A1 - 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 - 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 - JOUR A1 - El Hajj, Nady A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Kraus, Theo F. J. A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - Schneider, Eberhard A1 - Haaf, Thomas T1 - Epigenetic dysregulation in the developing Down syndrome cortex JF - Epigenetics N2 - Using Illumina 450K arrays, 1.85% of all analyzed CpG sites were significantly hypermethylated and 0.31% hypomethylated in fetal Down syndrome (DS) cortex throughout the genome. The methylation changes on chromosome 21 appeared to be balanced between hypo- and hyper-methylation, whereas, consistent with prior reports, all other chromosomes showed 3-11times more hyper- than hypo-methylated sites. Reduced NRSF/REST expression due to upregulation of DYRK1A (on chromosome 21q22.13) and methylation of REST binding sites during early developmental stages may contribute to this genome-wide excess of hypermethylated sites. Upregulation of DNMT3L (on chromosome 21q22.4) could lead to de novo methylation in neuroprogenitors, which then persists in the fetal DS brain where DNMT3A and DNMT3B become downregulated. The vast majority of differentially methylated promoters and genes was hypermethylated in DS and located outside chromosome 21, including the protocadherin gamma (PCDHG) cluster on chromosome 5q31, which is crucial for neural circuit formation in the developing brain. Bisulfite pyrosequencing and targeted RNA sequencing showed that several genes of PCDHG subfamilies A and B are hypermethylated and transcriptionally downregulated in fetal DS cortex. Decreased PCDHG expression is expected to reduce dendrite arborization and growth in cortical neurons. Since constitutive hypermethylation of PCDHG and other genes affects multiple tissues, including blood, it may provide useful biomarkers for DS brain development and pharmacologic targets for therapeutic interventions. KW - trisomy 21 KW - DNA methylation KW - Down syndrome KW - fetal brain development KW - frontal cortex KW - protocadherin gamma cluster Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191239 VL - 11 IS - 8 ER - TY - THES A1 - Schönwälder, Sina Maria Siglinde T1 - Entwicklung und Charakterisierung von Gelatine-basierten Hydrogelen und PLGA-basierten Janus-Partikeln T1 - Development and characterization of gelatin-based hydrogels and PLGA-based Janus particles N2 - Zusammenfassung In der Regenerativen Medizin sind polymerbasierte Biomaterialien von großer Bedeutung für die Entwicklung und Anwendung verbesserter bzw. neuer Therapien. Die Erforschung der Oberflächeneigenschaften von Biomaterialien, welche als Implantate eingesetzt werden, ist eine grundlegende Voraussetzung für deren erfolgreichen Einsatz. Die Protein-Oberflächen- Interaktion geschieht initial, sobald ein Implantat mit Körperflüssigkeiten oder mit Gewebe in Kontakt kommt, und trägt maßgeblich zur direkten Wechselwirkung von Implantat und umgebenden Zellen bei. Dieser Prozess wird in der vorliegenden Arbeit an Gelatine untersucht. Daher bestand ein Ziel darin, stabile, nanometerdünne Gelatineoberflächen herzustellen und darauf die Adsorption von humanen Plasmaproteinen und bakteriellen Proteinen zu analysieren. Die Abscheidung der Gelatinefilme in variabler Schichtdicke auf zuvor mit PPX-Amin modifizierten Oberflächen wurde unter Verwendung eines Rotationsbeschichters durchgeführt. Um stabile Hydrogelfilme zu erhalten, wurden die Amingruppen der disaggregierten Gelatinefibrillen untereinander und mit denen der Amin-Modifizierung durch ein biokompatibles Diisocyanat quervernetzt. Dieser Prozess lieferte einen reproduzierbaren und chemisch stabilen Gelatinefilm, welcher durch die substratunabhängige Amin-Modifizierung kovalent auf unterschiedlichste Oberflächen aufgebracht werden konnte. Die durch den Herstellungsprozess präzise eingestellte Schichtdicke (Nano- bzw. Mikrometermaßstab) wurde mittels Ellipsometrie und Rasterkraftmikroskopie ermittelt. Die ebenso bestimmte Rauheit war unabhängig von der Schichtdicke sehr gering. Gelatinefilme, die auf funktionalisierte und strukturierte Proben aufgebracht wurden, konnten durch Elektronenmikroskopie dargestellt werden. Mit Hilfe der Infrarot-Reflexions-Absorptions-Spektroskopie wurden die Gelatinefilme im Hinblick auf ihre Stabilität chemisch charakterisiert. Zur Quantifizierung der Adsorption humaner Plasmaproteine (Einzelproteinlösungen) und komplexer Proteingemische aus steril filtrierten Kulturüberständen des humanpathogenen Bakteriums Pseudomonas aeruginosa wurde die Quarzkristall-Mikrowaage mit Dissipationsüberwachung eingesetzt. Hiermit konnte nicht nur die adsorbierte Menge an Proteinen auf dem Gelatinehydrogel bzw. Referenzoberflächen (Gold, PPX-Amin, Titan), sondern auch die viskoelastischen Eigenschaften des adsorbierten Proteinfilms bestimmt werden. Allgemein adsorbierte auf dem Gelatinehydrogel eine geringere Proteinmasse im Vergleich zu den Referenzoberflächen. Circa ein Viertel der adsorbierten Proteine migrierte in die Poren des gequollenen Gels und veränderte dessen viskoelastische Eigenschaften. Durch anschließende MALDI-ToF/MS- und MS/MS-Analyse konnten die bakteriellen Proteine auf den untersuchten Oberflächen identifiziert und untereinander verglichen werden. Hierbei zeigten sich nur geringfügige Unterschiede in der Proteinzusammensetzung. Zudem wurde eine Sekundärionenmassenspektrometrie mit Flugzeitanalyse an reinen Gelatinefilmen und an mit humanen Plasmaproteinen beladenen Gelatinefilmen durchgeführt. Durch eine anschließende multivariante Datenanalyse konnte zwischen den untersuchten Proben eindeutig differenziert werden. Dieser Ansatz ermöglicht es, die Adsorption von unterschiedlichen Proteinen auf proteinbasierten Oberflächen markierungsfrei zu untersuchen und kann zur Aufklärung der in vivo-Situation beitragen. Darüber hinaus bietet dieser Untersuchungsansatz neue Perspektiven für die Gestaltung und das schnelle und effiziente Screening von unterschiedlichen Proteinzusammensetzungen. Biomaterialien können jedoch nicht nur als Implantate oder Implantatbeschichtungen eingesetzt werden. Im Bereich des drug delivery und der Depotarzneimittel sind biologisch abbaubare Polymere, aufgrund ihrer variablen Eigenschaften, von großem Interesse. Die Behandlung von bakteriellen und fungalen Pneumonien stellt insbesondere bei Menschen mit Vorerkrankungen wie Cystische Fibrose oder primäre Ziliendyskinesie eine große Herausforderung dar. Oral oder intravenös applizierte Wirkstoffe erreichen die Erreger aufgrund der erhöhten Zähigkeit des Bronchialsekretes oft nicht in ausreichender Konzentration. Daher besteht ein weiteres Ziel der vorliegenden Arbeit darin, mittels electrohydrodynamic cojetting mikrometergroße, inhalierbare, wirkstoffbeladene Partikel mit zwei Kompartimenten (Janus-Partikel) herzustellen und deren Eignung für die therapeutische Anwendung bei Lungeninfektionen zu untersuchen. Durch das in dieser Arbeit entwickelte Lösungsmittelsystem können Janus-Partikel aus biologisch abbaubaren Co-Polymeren der Polymilchsäure (Poly(lactid-co-glycolid), PLGA) hergestellt und mit verschiedenen Wirkstoffen beladen werden. Darunter befinden sich ein Antibiotikum (Aztreonam, AZT), ein Antimykotikum (Itraconazol, ICZ), ein Mukolytikum (Acetylcystein, ACC) und ein Antiphlogistikum (Ibuprofen, IBU). Die Freisetzung der eingelagerten Wirkstoffe, mit Ausnahme von ICZ, konnte unter physiologischen Bedingungen mittels Dialyse und anschließender Hochleistungsflüssigkeitschromatographie gemessen werden. Die Freisetzungsrate wird von der Kettenlänge des Polymers beeinflusst, wobei eine kürzere Kettenlänge zu einer schnelleren Freisetzung führt. Das in die Partikel eingelagerte Antimykotikum zeigte in vitro eine gute Wirksamkeit gegen Aspergillus nidulans. Durch das Einlagern von ICZ in die Partikel ist es möglich diesen schlecht wasserlöslichen Wirkstoff in eine für Patienten zugängliche und wirksame Applikationsform zu bringen. In Interaktion mit P. aeruginosa erzielten die mit Antibiotikum beladenen Partikel in vitro bessere Ergebnisse als der Wirkstoff in Lösung, was sich in einem in vivo-Infektionsmodell mit der Wachsmotte Galleria mellonella bestätigte. AZT-beladene Partikel hatten gegenüber einer identischen Wirkstoffmenge in Lösung eine 27,5% bessere Überlebensrate der Wachsmotten zur Folge. Des Weiteren hatten die Partikel keinen messbaren negativen Einfluss auf die Wachsmotten. Dreidimensionale Atemwegsschleimhautmodelle, hergestellt mit Methoden des Tissue Engineerings, bildeten die Basis für Untersuchungen der Partikel in Interaktion mit humanen Atemwegszellen. Die Untersuchung von Apoptose- und Entzündungsmarkern im Überstand der 3D-Modelle zeigte diesbezüglich keinen negativen Einfluss der Partikel auf die humanen Zellen. Diese gut charakterisierten und standardisierten in vitro-Testsysteme machen es möglich, Medikamentenuntersuchungen an menschlichen Zellen durchzuführen. Hinsichtlich der histologischen Architektur und funktionellen Eigenschaften der 3D-Modelle konnte eine hohe in vitro-/in vivo-Korrelation zu menschlichem Gewebe festgestellt werden. Humane Mucine auf den 3D-Modellen dienten zur Untersuchung der schleimlösenden Wirkung von ACC-beladenen Partikeln. Standen diese in räumlichem Kontakt zu den Mucinen, wurde deren Zähigkeit durch das freigesetzte ACC herabgesetzt, was qualitativ mittels histologischen Methoden bestätigt werden konnte. Die in dieser Arbeit entwickelten Herstellungsprotokolle dienen als Grundlage und können für die Synthese ähnlicher Systeme, basierend auf anderen Polymeren und Wirkstoffen, modifiziert werden. Gelatine und PLGA erwiesen sich als vielseitig einsetzbare Werkstoffe und bieten eine breite Anwendungsvielfalt in der Regenerativen Medizin, was die erzielten Resultate bekräftigen. N2 - In the field of regenerative medicine, polymer-based biomaterials are of great importance for the development and application of improved or new therapies. The research on the surface properties of biomaterials, which are used as implants, is essential for their successful use. The protein-surface interaction is the initial step and occurs when an implant comes into contact with bodily fluids or tissues and significantly increases direct interaction of the implant and the surrounding cells. This thesis investigates these processes on gelatin. Accordingly, one of the project’s major goals was to produce stable nanometer-thin gelatin surfaces and analyze the adsorption of human plasma and bacterial proteins. The deposition of gelatin films and the assortment of layer thicknesses on PPX-amine modified surfaces were carried out using a spin coater. To gain hydrogel films with reproducible properties, the amine groups of the disaggregated gelatin fibrils were cross- linked with each other and with those of the amine modification by a biocompatible diisocyanate. The result was a reproducible and chemically stable gelatin film, which could be applied to a wide variety of surfaces through the substrate-independent amine modification. The manufacturing process precisely adjusted the layer thickness to the nano- or micrometer scale which could be determined applying ellipsometry and atomic- force microscopy. The roughness was very low regardless of the layer thickness. Gelatin films applied to the functionalized and patterned samples could be visualized by electron microscopy. With the help of infrared reflection absorption spectroscopy, the gelatin films were chemically characterized in terms of stability. The adsorption of human plasma proteins (single protein solutions) as well as the complex protein mixtures of sterile filtered supernatants belonging to Pseudomonas aeruginosa, a human pathogenic bacterium, were quantified by quartz crystal microbalance with dissipation monitoring. Both the adsorbed amount of proteins on the gelatin hydrogel or reference surfaces (gold, PPX-amine, titanium) and the viscoelastic properties of the adsorbed protein film were determined. In general, there was less protein mass adsorbed on the gelatin hydrogel compared to the reference surfaces. About a quarter of the adsorbed proteins migrated into the pores of the swollen gel and changed its viscoelastic properties. Subsequent MALDI-ToF/MS and MS/MS analysis were used to identify and compare the adsorbed bacterial proteins on the investigated surfaces. Only slight differences were found in the adsorbed protein composition. A secondary ion mass spectrometry with time-of-flight analysis was performed on pure gelatin films and gelatin films loaded with human plasma proteins. By subsequent multivariate data analysis, it was possible to clearly differentiate between the examined samples. Not only does this approach enable us to screen the adsorption of different proteins on protein-based surfaces without labeling, but it also contributes to the elucidation of the in vivo-situation. ach provides new perspectives regarding the design and efficient screening of different protein compositions. ... KW - PLGA KW - Partikel KW - Gelatine KW - Polylactid-co-Glycolid KW - Hydrogel KW - Tissue Engineering Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142636 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 - 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 - 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 -