TY - JOUR A1 - Shannon, Graver A1 - Hein, Melanie T1 - Tumor cell response to bevacizumab single agent therapy in vitro JF - Cancer Cell International N2 - Background Angiogenesis represents a highly multi-factorial and multi-cellular complex (patho-) physiologic event involving endothelial cells, tumor cells in malignant conditions, as well as bone marrow derived cells and stromal cells. One main driver is vascular endothelial growth factor (VEGFA), which is known to interact with endothelial cells as a survival and mitogenic signal. The role of VEGFA on tumor cells and /or tumor stromal cell interaction is less clear. Condition specific (e.g. hypoxia) or tumor specific expression of VEGFA, VEGF receptors and co-receptors on tumor cells has been reported, in addition to the expression on the endothelium. This suggests a potential paracrine/autocrine loop that could affect changes specific to tumor cells. Methods We used the monoclonal antibody against VEGFA, bevacizumab, in various in vitro experiments using cell lines derived from different tumor entities (non small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC) and renal cell carcinoma (RCC)) in order to determine if potential VEGFA signaling could be blocked in tumor cells. The experiments were done under hypoxia, a major inducer of VEGFA and angiogenesis, in an attempt to mimic the physiological tumor condition. Known VEGFA induced endothelial biological responses such as proliferation, migration, survival and gene expression changes were evaluated. Results Our study was able to demonstrate expression of VEGF receptors on tumor cells as well as hypoxia regulated angiogenic gene expression. In addition, there was a cell line specific effect in tumor cells by VEGFA blockade with bevacizumab in terms of proliferation; however overall, there was a limited measurable consequence of bevacizumab therapy detected by migration and survival. Conclusion The present study showed in a variety of in vitro experiments with several tumor cell lines from different tumor origins, that by blocking VEGFA with bevacizumab, there was a limited autocrine or cell-autonomous function of VEGFA signaling in tumor cells, when evaluating VEGFA induced downstream outputs known in endothelial cells. KW - Bevacizumab KW - NCI-60 KW - Tumor angiogenesis KW - VEGFA KW - Hypoxia KW - In vitro Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97185 UR - http://www.cancerci.com/content/13/1/94 ER - TY - JOUR A1 - Schulze, Katja A1 - Tillich, Ulrich M. A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - PlanktoVision – an automated analysis system for the identification of phytoplankton JF - BMC Bioinformatics N2 - Background Phytoplankton communities are often used as a marker for the determination of fresh water quality. The routine analysis, however, is very time consuming and expensive as it is carried out manually by trained personnel. The goal of this work is to develop a system for an automated analysis. Results A novel open source system for the automated recognition of phytoplankton by the use of microscopy and image analysis was developed. It integrates the segmentation of the organisms from the background, the calculation of a large range of features, and a neural network for the classification of imaged organisms into different groups of plankton taxa. The analysis of samples containing 10 different taxa showed an average recognition rate of 94.7% and an average error rate of 5.5%. The presented system has a flexible framework which easily allows expanding it to include additional taxa in the future. Conclusions The implemented automated microscopy and the new open source image analysis system - PlanktoVision - showed classification results that were comparable or better than existing systems and the exclusion of non-plankton particles could be greatly improved. The software package is published as free software and is available to anyone to help make the analysis of water quality more reproducible and cost effective. KW - Bioinformatik Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96395 UR - http://www.biomedcentral.com/1471-2105/14/115 ER - TY - JOUR A1 - Schultz, Jörg A1 - Terhoeven, Niklas T1 - The bilaterian roots of cordon-bleu JF - BMC Research Notes N2 - Background The actin cytoskeleton is essential for many physiological processes of eukaryotic cells. The emergence of new actin fibers is initiated by actin nucleators. Whereas most of them are evolutionary old, the cordon-bleu actin nucleator is classified as vertebrate specific. Findings Using sensitive methods for sequence similarity detection, we identified homologs of cordon-bleu not only in non-vertebrate chordates but also in arthropods, molluscs, annelids and platyhelminthes. These genes contain only a single WH2 domain and therefore resemble more the vertebrate cordon-bleu related 1 protein than the three WH2 domain containing cordon-bleu. Furthermore, we identified a homolog of the N-terminal, ubiquitin like, cobl domain of cordon-bleu in the cnidarian Nematostella vectensis. Conclusion Our results suggest that the ur-form of the cordon-bleu protein family evolved already with the emergence of the bilateria by the combination of existing cobl and WH2 domains. Following a vertebrate specific gene-duplication, one copy gained two additional WH2 domains leading to the actin nucleating cordon-bleu. The function of the ur-form of the cordon-bleu protein family is so far unknown. The identification of a homolog in the model organism Drosophila melanogaster could facilitate its experimental characterization. KW - Actin nucleation KW - WH2 domain KW - Cobl domain KW - Gene duplication Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97161 UR - http://www.biomedcentral.com/1756-0500/6/393 ER - TY - JOUR A1 - Schultz, Jörg A1 - Keller, Daniela Barbara T1 - Connectivity, Not Frequency, Determines the Fate of a Morpheme JF - PLoS ONE N2 - Morphemes are the smallest meaningful parts of words and therefore represent a natural unit to study the evolution of words. To analyze the influence of language change on morphemes, we performed a large scale analysis of German and English vocabulary covering the last 200 years. Using a network approach from bioinformatics, we examined the historical dynamics of morphemes, the fixation of new morphemes and the emergence of words containing existing morphemes. We found that these processes are driven mainly by the number of different direct neighbors of a morpheme in words (connectivity, an equivalent to family size or type frequency) and not its frequency of usage (equivalent to token frequency). This contrasts words, whose survival is determined by their frequency of usage. We therefore identified features of morphemes which are not dictated by the statistical properties of words. As morphemes are also relevant for the mental representation of words, this result might enable establishing a link between an individual’s perception of language and historical language change. KW - confidence intervals KW - evolutionary biology KW - culture KW - language KW - lexicography KW - linguistic morphology KW - network analysis KW - psycholinguistics Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97039 ER - TY - JOUR A1 - Schul, Daniela A1 - Schmitt, Alexandra A1 - Regneri, Janine A1 - Schartl, Manfred A1 - Wagner, Toni Ulrich T1 - Bursted BMP Triggered Receptor Kinase Activity Drives Smad1 Mediated Long-Term Target Gene Oscillation in c2c12 Cells JF - PLoS ONE N2 - Bone Morphogenetic Proteins (BMPs) are important growth factors that regulate many cellular processes. During embryogenesis they act as morphogens and play a critical role during organ development. They influence cell fates via concentration-gradients in the embryos where cells transduce this extracellular information into gene expression profiles and cell fate decisions. How receiving cells decode and quantify BMP2/4 signals is hardly understood. There is little data on the quantitative relationships between signal input, transducing molecules, their states and location, and ultimately their ability to integrate graded systemic inputs and generate qualitative responses. Understanding this signaling network on a quantitative level should be considered a prerequisite for efficient pathway modulation, as the BMP pathway is a prime target for therapeutic invention. Hence, we quantified the spatial distribution of the main signal transducer of the BMP2/4 pathway in response to different types and levels of stimuli in c2c12 cells. We found that the subcellular localization of Smad1 is independent of ligand concentration. In contrast, Smad1 phosphorylation levels relate proportionally to BMP2 ligand concentrations and they are entirely located in the nucleus. Interestingly, we found that BMP2 stimulates target gene expression in non-linear, wave-like forms. Amplitudes showed a clear concentration-dependency, for sustained and transient stimulation. We found that even burst-stimulation triggers gene-expression wave-like modulations that are detectable for at least 30 h. Finally, we show here that target gene expression oscillations depend on receptor kinase activity, as the kinase drives further expression pulses without receptor reactivation and the target gene expression breaks off after inhibitor treatment in c2c12 cells. KW - gene expression KW - BMP signaling KW - SMAD signaling KW - genetic oscillators KW - cell fusion KW - DNA-binding proteins KW - luciferase KW - kinase inhibitors Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130131 VL - 8 IS - 4 ER - TY - THES A1 - Schul, Daniela T1 - Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway T1 - Raum-Zeitliche Untersuchung und quantitative Analyse des BMP-Signaltransduktionsweges N2 - Bone Morphogenetic Proteins (BMPs) are key regulators for a lot of diverse cellular processes. During embryonic development these proteins act as morphogens and play a crucial role particularly in organogenesis. BMPs have a direct impact on distinct cellular fates by means of concentration-gradients in the developing embryos. Using the diverse signaling input information within the embryo due to the gradient, the cells transduce the varying extracellular information into distinct gene expression profiles and cell fate decisions. Furthermore, BMP proteins bear important functions in adult organisms like tissue homeostasis or regeneration. In contrast to TGF-ß signaling, currently only little is known about how cells decode and quantify incoming BMP signals. There is poor knowledge about the quantitative relationships between signal input, transducing molecules, their states and location, and finally their ability to incorporate graded systemic inputs and produce qualitative responses. A key requirement for efficient pathway modulation is the complete comprehension of this signaling network on a quantitative level as the BMP signaling pathway, just like many other signaling pathways, is a major target for medicative interference. I therefore at first studied the subcellular distribution of Smad1, which is the main signal transducing protein of the BMP signaling pathway, in a quantitative manner and in response to various types and levels of stimuli in murine c2c12 cells. Results indicate that the subcellular localization of Smad1 is not dependent on the initial BMP input. Surprisingly, only the phospho-Smad1 level is proportionally associated to ligand concentration. Furthermore, the activated transducer proteins were entirely located in the nucleus. Besides the subcellular localization of Smad1, I have analyzed the gene expression profile induced by BMP signaling. Therefore, I examined two endogenous immediate early BMP targets as well as the expression of the stably transgenic Gaussia Luciferase. Interestingly, the results of these independent experimental setups and read-outs suggest oscillating target gene expression. The amplitudes of the oscillations showed a precise concentration-dependence for continuous and transient stimulation. Additionally, even short-time stimulation of 15’ activates oscillating gene-expression pulses that are detectable for at least 30h post-stimulation. Only treatment with a BMP type I receptor kinase inhibitor leads to the complete abolishment of the target gene expression. This indicated that target gene expression oscillations depend directly on BMP type I receptor kinase activity. N2 - Bone Morphogenetic Proteins (BMPs) stellen wichtige Regulatoren für eine Vielzahl von verschiedenen zellulären Prozessen dar. Während der Embryonalentwicklung agieren diese Proteine als Morphogene und spielen daher eine entscheidende Rolle für diesen Prozess, vor allem in der Organogenese. Durch Konzentrationsgradienten üben BMPs einen direkten Einfluss auf verschiedene zelluläre Schicksale im entwickelnden Embryo aus. Aufgrund dieser Gradienten gelangen vielfältige Signalinformationen zu den verschiedenen Zellen, welche die extrazelluläre Information in verschiedene Genexpressionsprofile und Zellschicksalsentscheidungen umwandeln. Darüber hinaus tragen BMPs wichtige Funktionen im erwachsenen Organismus, wie z.B. Gewebshomöostase oder -regeneration. Im Gegensatz zu dem verwandten TGF-ß Signaltransduktionsweg ist derzeit nur wenig über die zelluläre Übersetzung und Quantifizierung eingehender BMP-Signale bekannt. Es gibt wenige Kenntnisse über die quantitative Beziehung zwischen Signaleingang, Überträgerproteinen, ihren Zuständen sowie intrazellulären Positionen, und schließlich ihre Fähigkeit Signaleingänge systemisch zu integrieren und qualitative Antworten der Zelle zu produzieren. Eine wesentliche Voraussetzung für die effiziente Signaltransduktions-modulierung ist das vollständige Verständnis des Signalnetzwerkes auf einer quantitativen Ebene, da der BMP-Signalweg, wie auch viele andere Signalwege, ein wichtiges Ziel für medizinische Anwendungen und Medikamentenentwicklung ist. Daher untersuchte ich zunächst die subzelluläre Verteilung der wichtigsten Signalweiterleitungsproteine des BMP-Signalweges, der Smad1-Proteine, auf quantitativer Ebene und deren Reaktion auf verschiedene Stimulierungsarten und BMP-Konzentrationsstufen in murinen c2c12-Zellen. Die Ergebnisse zeigen, dass die subzelluläre Lokalisation von Smad1 unabhängig von der BMP-Konzentration ist und nur das phospho-Smad1 Level proportional zur Konzentration des Liganden steigt. Darüber hinaus befanden sich die aktiven Überträgerproteine nach Stimulierungvollständig im Zellkern. Neben der subzellulären Lokalisation von Smad1, habe ich das Genexpressionsprofil von BMP-Zielgenen analysiert. Ich untersuchte zwei endogene und frühe BMP-Zielgene sowie die Expression der stabil transgenen Gaussia Luciferase. Interessanterweise deuten die Ergebnisse dieser zwei unabhängigen Versuchsaufbauten und Detektionsmethoden auf eine oszillierende Expression der Zielgene hin. Die Amplituden der Schwingungen zeigten eine deutliche Konzentrationsabhängigkeit bei kontinuierlicher und transienter Stimulation. Außerdem aktiviert eine Kurzzeitstimulierung von 15 Minuten ebenfalls ein oszillierendes Genexpressionsprofil, welches für mindestens 30 Stunden nach der Stimulierung nachweisbar ist. Nur die Behandlung mit einem BMP Typ-I-Rezeptorkinaseinhibitor führt zur vollständigen Aufhebung der Zielgenexpression. Infolgedessen sind die Oszillationen der Zielgenexpression direkt von der Aktivität der BMP Typ-I-Rezeptorkinase abhängig. KW - Knochen-Morphogenese-Proteine KW - Signaltransduktion KW - BMP-Signaltransduktionsweg KW - Analyse KW - BMP signaling pathway KW - analysis Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-84224 ER - TY - JOUR A1 - Schubert, Maria A1 - Spahn, Martin A1 - Kneitz, Susanne A1 - Scholz, Claus Jürgen A1 - Joniau, Steven A1 - Stroebel, Philipp A1 - Riedmiller, Hubertus A1 - Kneitz, Burkhard T1 - Distinct microRNA Expression Profile in Prostate Cancer Patients with Early Clinical Failure and the Impact of let-7 as Prognostic Marker in High-Risk Prostate Cancer JF - PLoS ONE N2 - Background The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. Methodology and Principal Findings We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. Conclusion Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients. KW - biomarkers KW - gene expression KW - gene targeting KW - luciferase KW - MircoRNA KW - microarrays KW - oncogenes KW - prostate cancer Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96825 ER - TY - JOUR A1 - Scharmann, Mathias A1 - Thornham, Daniel G. A1 - Grafe, T. Ulmar A1 - Federle, Walter T1 - A Novel Type of Nutritional Ant-Plant Interaction: Ant Partners of Carnivorous Pitcher Plants Prevent Nutrient Export by Dipteran Pitcher Infauna JF - PLoS ONE N2 - Many plants combat herbivore and pathogen attack indirectly by attracting predators of their herbivores. Here we describe a novel type of insect-plant interaction where a carnivorous plant uses such an indirect defence to prevent nutrient loss to kleptoparasites. The ant Camponotus schmitzi is an obligate inhabitant of the carnivorous pitcher plant Nepenthes bicalcarata in Borneo. It has recently been suggested that this ant-plant interaction is a nutritional mutualism, but the detailed mechanisms and the origin of the ant-derived nutrient supply have remained unexplained. We confirm that N. bicalcarata host plant leaves naturally have an elevated \(^{15}N/^{14}N\) stable isotope abundance ratio (\(\delta ^{15}N\)) when colonised by C. schmitzi. This indicates that a higher proportion of the plants' nitrogen is insect-derived when C. schmitzi ants are present (ca. 100%, vs. 77% in uncolonised plants) and that more nitrogen is available to them. We demonstrated direct flux of nutrients from the ants to the host plant in a \(^{15}N\) pulse-chase experiment. As C. schmitzi ants only feed on nectar and pitcher contents of their host, the elevated foliar \(\delta ^{15}N\) cannot be explained by classic ant-feeding (myrmecotrophy) but must originate from a higher efficiency of the pitcher traps. We discovered that C. schmitzi ants not only increase the pitchers' capture efficiency by keeping the pitchers' trapping surfaces clean, but they also reduce nutrient loss from the pitchers by predating dipteran pitcher inhabitants (infauna). Consequently, nutrients the pitchers would have otherwise lost via emerging flies become available as ant colony waste. The plants' prey is therefore conserved by the ants. The interaction between C. schmitzi, N. bicalcarata and dipteran pitcher infauna represents a new type of mutualism where animals mitigate the damage by nutrient thieves to a plant. KW - community KW - alternative trapping strategies KW - leaf-litter utilization KW - nepenthes bicalcarata KW - camponotus schmitzi KW - food web KW - epiphytic fern KW - nitrogen KW - prey KW - rafflesiana Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130952 VL - 8 IS - 5 ER - TY - JOUR A1 - Rössler, Wolfgang A1 - Brill, Martin F. T1 - Parallel processing in the honeybee olfactory pathway: structure, function, and evolution JF - Journal of Comparative Physiology A N2 - Animals face highly complex and dynamic olfactory stimuli in their natural environments, which require fast and reliable olfactory processing. Parallel processing is a common principle of sensory systems supporting this task, for example in visual and auditory systems, but its role in olfaction remained unclear. Studies in the honeybee focused on a dual olfactory pathway. Two sets of projection neurons connect glomeruli in two antennal-lobe hemilobes via lateral and medial tracts in opposite sequence with the mushroom bodies and lateral horn. Comparative studies suggest that this dual-tract circuit represents a unique adaptation in Hymenoptera. Imaging studies indicate that glomeruli in both hemilobes receive redundant sensory input. Recent simultaneous multi-unit recordings from projection neurons of both tracts revealed widely overlapping response profiles strongly indicating parallel olfactory processing. Whereas lateral-tract neurons respond fast with broad (generalistic) profiles, medial-tract neurons are odorant specific and respond slower. In analogy to “what-” and “where” subsystems in visual pathways, this suggests two parallel olfactory subsystems providing “what-” (quality) and “when” (temporal) information. Temporal response properties may support across-tract coincidence coding in higher centers. Parallel olfactory processing likely enhances perception of complex odorant mixtures to decode the diverse and dynamic olfactory world of a social insect. KW - multi-unit recording KW - antennal lobe KW - glomeruli KW - projection neurons KW - mushroom bodies Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132548 VL - 199 ER - TY - JOUR A1 - Rybalka, Nataliya A1 - Wolf, Matthias A1 - Andersen, Robert A1 - Friedl, Thomas T1 - Congruence of chloroplast- and nuclear-encoded DNA sequence variations used to assess species boundaries in the soil microalga Heterococcus (Stramenopiles, Xanthophyceae) JF - BMC Evolutionary Biology N2 - Background: Heterococcus is a microalgal genus of Xanthophyceae (Stramenopiles) that is common and widespread in soils, especially from cold regions. Species are characterized by extensively branched filaments produced when grown on agarized culture medium. Despite the large number of species described exclusively using light microscopic morphology, the assessment of species diversity is hampered by extensive morphological plasticity. Results: Two independent types of molecular data, the chloroplast-encoded psbA/rbcL spacer complemented by rbcL gene and the internal transcribed spacer 2 of the nuclear rDNA cistron (ITS2), congruently recovered a robust phylogenetic structure. With ITS2 considerable sequence and secondary structure divergence existed among the eight species, but a combined sequence and secondary structure phylogenetic analysis confined to helix II of ITS2 corroborated relationships as inferred from the rbcL gene phylogeny. Intra-genomic divergence of ITS2 sequences was revealed in many strains. The 'monophyletic species concept', appropriate for microalgae without known sexual reproduction, revealed eight different species. Species boundaries established using the molecular-based monophyletic species concept were more conservative than the traditional morphological species concept. Within a species, almost identical chloroplast marker sequences (genotypes) were repeatedly recovered from strains of different origins. At least two species had widespread geographical distributions; however, within a given species, genotypes recovered from Antarctic strains were distinct from those in temperate habitats. Furthermore, the sequence diversity may correspond to adaptation to different types of habitats or climates. Conclusions: We established a method and a reference data base for the unambiguous identification of species of the common soil microalgal genus Heterococcus which uses DNA sequence variation in markers from plastid and nuclear genomes. The molecular data were more reliable and more conservative than morphological data. KW - xanthophyceae KW - psbA/rbcL spacer KW - ITS2 KW - tool KW - RBCL KW - alignment KW - evolution KW - chlorophyta KW - RNA secondary structure KW - terrestrial habitats KW - phylogenetic trees KW - mixed models KW - green algae KW - heterococcus KW - systematics KW - molecular phylogeny KW - species concept Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121848 SN - 1471-2148 VL - 13 IS - 39 ER - TY - JOUR A1 - Rudel, Thomas A1 - Prusty, Bhupesh K. A1 - Siegl, Christine A1 - Hauck, Petra A1 - Hain, Johannes A1 - Korhonen, Suvi J. A1 - Hiltunen-Back, Eija A1 - Poulakkainen, Mirja T1 - Chlamydia trachomatis Infection Induces Replication of Latent HHV-6 JF - PLoS ONE N2 - Human herpesvirus-6 (HHV-6) exists in latent form either as a nuclear episome or integrated into human chromosomes in more than 90% of healthy individuals without causing clinical symptoms. Immunosuppression and stress conditions can reactivate HHV-6 replication, associated with clinical complications and even death. We have previously shown that co-infection of Chlamydia trachomatis and HHV-6 promotes chlamydial persistence and increases viral uptake in an in vitro cell culture model. Here we investigated C. trachomatis-induced HHV-6 activation in cell lines and fresh blood samples from patients having Chromosomally integrated HHV-6 (CiHHV-6). We observed activation of latent HHV-6 DNA replication in CiHHV-6 cell lines and fresh blood cells without formation of viral particles. Interestingly, we detected HHV-6 DNA in blood as well as cervical swabs from C. trachomatis-infected women. Low virus titers correlated with high C. trachomatis load and vice versa, demonstrating a potentially significant interaction of these pathogens in blood cells and in the cervix of infected patients. Our data suggest a thus far underestimated interference of HHV-6 and C. trachomatis with a likely impact on the disease outcome as consequence of co-infection. KW - blood KW - chlamydia KW - chlamydia infection KW - chlamydia trachomatis KW - DNA replication KW - macrophages KW - polymerase chain reaction KW - viral load Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96731 ER - TY - JOUR A1 - Rudel, Thomas A1 - Mehlitz, Adrian T1 - Modulation of host signaling and cellular responses by Chlamydia JF - Cell Communication and Signaling N2 - Modulation of host cell signaling and cellular functions is key to intracellular survival of pathogenic bacteria. Intracellular growth has several advantages e.g. escape from the humoral immune response and access to a stable nutrient rich environment. Growth in such a preferred niche comes at the price of an ongoing competition between the bacteria and the host as well as other microbes that compete for the very same host resources. This requires specialization and constant evolution of dedicated systems for adhesion, invasion and accommodation. Interestingly, obligate intracellular bacteria of the order Chlamydiales have evolved an impressive degree of control over several important host cell functions. In this review we summarize how Chlamydia controls its host cell with a special focus on signal transduction and cellular modulation. KW - Chlamydia KW - Invasion KW - Inclusion KW - Type III secretion KW - Tarp KW - Inc KW - Signaling KW - Trafficking Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97225 UR - http://www.biosignaling.com/content/11/1/90 ER - TY - JOUR A1 - Rudel, Thomas A1 - Krohne, George A1 - Prusty, Bhupesh K. T1 - Reactivation of Chromosomally Integrated Human Herpesvirus-6 by Telomeric Circle Formation N2 - More than 95% of the human population is infected with human herpesvirus-6 (HHV-6) during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6). In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle) formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR). Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation. Author Summary: Human herpesviruses (HHVs) can reside in a lifelong non-infectious state displaying limited activity in their host and protected from immune responses. One possible way by which HHV-6 achieves this state is by integrating into the telomeric ends of human chromosomes, which are highly repetitive sequences that protect the ends of chromosomes from damage. Various stress conditions can reactivate latent HHV-6 thus increasing the severity of multiple human disorders. Recently, we have identified Chlamydia infection as a natural cause of latent HHV-6 reactivation. Here, we have sought to elucidate the molecular mechanism of HHV-6 reactivation. HHV-6 efficiently utilizes the well-organized telomere maintenance machinery of the host cell to exit from its inactive state and initiate replication to form new viral DNA. We provide experimental evidence that the shortening of telomeres, as a consequence of interference with telomere maintenance, triggers the release of the integrated virus from the chromosome. Our data provide a mechanistic basis to understand HHV-6 reactivation scenarios, which in light of the high prevalence of HHV-6 infection and the possibility of chromosomal integration of other common viruses like HHV-7 have important medical consequences for several million people worldwide. KW - chlamydia infection KW - circular DNA KW - telomeres KW - polymerase chain reaction KW - DNA electrophoresis KW - chromosomes KW - southern hybridization KW - DNA hybridization Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111380 ER - TY - JOUR A1 - Rudel, Thomas A1 - Faulstich, Michaela A1 - Böttcher, Jan-Peter A1 - Meyer, Thomas F. A1 - Fraunholz, Martin T1 - Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling JF - PLoS Pathogens N2 - Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection. KW - antibodies KW - bacterial pathogens KW - cell membranes KW - intracellular pathogens KW - neisseria gonorrhoeae KW - phosphates KW - phosphorylation KW - pili and fimbriae Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96679 ER - TY - JOUR A1 - Rodrigues, Lénia A1 - Popov, Nikita A1 - Kaye, Kenneth M. A1 - Simas, J. Pedro T1 - Stabilization of Myc through Heterotypic Poly-Ubiquitination by mLANA Is Critical for \(\gamma\)-Herpesvirus Lymphoproliferation JF - PLoS PATHOGENS N2 - Host colonization by lymphotropic \(\gamma\)-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of \(\gamma\)-cherpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic \(\gamma\)-herpesvirus MuHV- 4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B- cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological posttranslational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly- ubiquitination mediated via the viral E3 ubiquitin- ligase mLANA protein. \(EC_5S^{mLANA}\) modulates cellular control of Myc turnover by antagonizing \(SCF^{Fbw7}\) mediated proteasomal degradation of Myc, mimicking \(SCF^{\beta-TrCP}\). The findings here reported reveal that modulation of Myc is essential for \(\gamma\)-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease. KW - latency KW - murine gammaherpesvirus 68 KW - Epstein-Barr-virus KW - C-MYC KW - nuclear antigen KW - germinal center KW - B lymphocytes KW - protein KW - cells KW - beta-TRCP Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131227 VL - 9 IS - 8 ER - TY - JOUR A1 - Rinawati, Fitria A1 - Stein, Katharina A1 - Lindner, André T1 - Climate change impacts on biodiversity-the setting of a lingering global crisis JF - Diversity N2 - Climate change has created potential major threats to global biodiversity. The multiple components of climate change are projected to affect all pillars of biodiversity, from genes over species to biome level. Of particular concerns are "tipping points" where the exceedance of ecosystem thresholds will possibly lead to irreversible shifts of ecosystems and their functioning. As biodiversity underlies all goods and services provided by ecosystems that are crucial for human survival and wellbeing, this paper presents potential effects of climate change on biodiversity, its plausible impacts on human society as well as the setting in addressing a global crisis. Species affected by climate change may respond in three ways: change, move or die. Local species extinctions or a rapidly affected ecosystem as a whole respectively might move toward its particular "tipping point", thereby probably depriving its services to human society and ending up in a global crisis. Urgent and appropriate actions within various scenarios of climate change impacts on biodiversity, especially in tropical regions, are needed to be considered. Foremost a multisectoral approach on biodiversity issues with broader policies, stringent strategies and programs at international, national and local levels is essential to meet the challenges of climate change impacts on biodiversity. KW - biodiversity KW - climate change KW - ecosystem function KW - ecosystem service KW - tropical forest Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131866 VL - 5 IS - 1 ER - TY - JOUR A1 - Riedel, Alice A1 - Mofolo, Boitumelo A1 - Avota, Elita A1 - Schneider-Schaulies, Sibylle A1 - Meintjes, Ayton A1 - Mulder, Nicola A1 - Kneitz, Susanne T1 - Accumulation of Splice Variants and Transcripts in Response to PI3K Inhibition in T Cells JF - PLoS ONE N2 - Background Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA. Hypothesis Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing. Methods To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression. Results Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry. Conclusions PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression. KW - T cells KW - gene regulation KW - alternative splicing KW - measles virus KW - T cell receptors KW - reverse transcriptase-polymerase chain reaction KW - cell cycle and cell division KW - TCR signaling cascade Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130335 VL - 8 IS - 2 ER - TY - JOUR A1 - Riedel, Alice A1 - Mofolo, Boitumelo A1 - Avota, Elita A1 - Schneider-Schaulies, Sibylle A1 - Meintjes, Ayton A1 - Mulder, Nicola A1 - Kneitz, Susanne T1 - Accumulation of Splice Variants and Transcripts in Response to PI3K Inhibition in T Cells N2 - Background: Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA. Hypothesis: Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing. Methods: To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression. Results: Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry. Conclusions: PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression. KW - Biologie Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-77917 ER - TY - THES A1 - Reil, Michael T1 - Essentielle Rollen des LEM-Domänen Proteins MAN1 während der Organentwicklung von Xenopus laevis und überlappende Funktionen von Emerin T1 - Essential roles of LEM domaine protein MAN1 during organogenesis in Xenopus laevis and overlapping functions of emerin N2 - Mutationen in Genen, die für Kernhüllproteine codieren sind mit einer stetig zunehmenden Anzahl menschlicher Erkrankungen verbunden, die als Envelopathien bezeichnet werden. Erstaunlicherweise betrifft die Pathologie dieser Krankheiten spezifische Gewebe und Organe, obwohl entsprechende Proteine meist ubiquitär exprimiert werden. So führen beispielsweise Defekte in Emerin, einem Protein der inneren Kernhülle, zur X-chromosomalen Emery- Dreifuss Muskeldystrophie (EDMD). Diese Krankheit ist durch Muskelschwäche oder – schwund gekennzeichnet. Defekte im Kernhüllprotein MAN1 sind dagegen mit Krankheiten verbunden, die Knochen- und Hautgewebe betreffen. Interessanterweise besitzen beide Proteine eine evolutionär hoch konservierte Domäne, die sog. LEM-Domäne. LEM-Domänen Proteine können mit der Kernlamina interagieren, ebenso mit dem sog. Barrier-to- Autointegration Factor (BAF) sowie mit zahlreichen Transkriptionsfaktoren. Dennoch ist die funktionelle Rolle der LEM-Domänen Proteine bis dato nicht vollständig aufgeklärt. In der vorliegenden Studie sollten daher die Funktionen von MAN1 und Emerin während der Frühentwicklung von Xenopus laevis untersucht werden. Vorangehende Untersuchungen zeigten, dass Mikroinjektionen von XMAN1- Antikörpern in Zwei-Zell-Stadien befruchteter Eizellen zu einem Arrest der Zellteilung in der injizierten Blastomere führten. Da dabei eine Störung der Kernhüllbildung spekuliert wurde, sollte durch Antikörper-vermittelter Inhibition von XMAN1 die Bildung von in vitro Kernen im Xenopus Eiextrakt untersucht werden. Dabei wurden Kerne beobachtet, die dekondensiertes Chromatin zeigten, bei denen jedoch eine Fusion von Membranvesikeln zu einer durchgehenden Kernhülle nicht stattgefunden hatte. Frühere Charakterisierungen von MAN1 und Emerin zeigten unterschiedliche Expressionsmuster während der Entwicklung von X. laevis. Da XMAN1 ubiquitär exprimiert und Xemerin jedoch erstmals ab Stadium 41 nachweisbar ist, war es mittels Mikroinjektion von Xemerin möglich zu zeigen, dass es in der Lage ist den Arrest der Zellteilung zu verhindern. Es wurde daher die These aufgestellt, dass MAN1 und Emerin während der Frühentwicklung von Xenopus überlappende Funktionen besitzen. Um diese These zu prüfen, wurde zunächst unter Verwendung des Proximity Ligation Assays untersucht, ob beide Proteine miteinander interagieren können. Mit Hilfe dieser Methode konnte gezeigt werden, dass Interaktionen beider Proteine innerhalb der Kernhülle lokalisieren. Die Interaktionen blieben während der Mitose bestehen und waren erst wieder zum Ende der Mitose in der Kernhülle nachweisbar. Diese Resultate deuten daher darauf hin, dass XMAN1/Xemerin-Interaktionen während der ... N2 - Mutations in genes encoding for nuclear envelope proteins are linked to an increasing number of human diseases, called envelopathies. Interestingly, pathology of these diseases affects specific tissues and organs, even though the related proteins are expressed ubiquitous. Defects in the inner nuclear membrane protein emerin for example, are leading to X-linked Emery- Dreifuss muscular dystrophy (EDMD), characterized by muscle weakness or wasting. Conversely, defects in the nuclear envelope protein MAN1 are linked to bone and skin disorders. Both proteins share a highly conserved domain, called LEM-domain. LEM proteins are known to interact with the nuclear lamina, the so called Barrier-to-Autointegration Factor (BAF) and several transcription factors. Nevertheless, knowledge of the functional roles of LEM proteins is still unclear. For this reason, this study aimed to investigate the roles of MAN1 and emerin during early Xenopus laevis development and nuclear envelope assembly. ... KW - Organogenese KW - Emerin KW - Kernhülle KW - LEM domaine KW - emerin KW - MAN1 KW - nuclear envelope KW - organogenesis KW - LEM-Domänen KW - Organentwicklung Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85105 ER - TY - THES A1 - Regneri, Janine T1 - Transcriptional regulation of cancer genes in the Xiphophorus melanoma system T1 - Transkriptionelle Regulation von Krebsgenen im Xiphophorus-Melanommodell N2 - The Xiphophorus melanoma system is a useful animal model for the study of the genetic basis of tumor formation. The development of hereditary melanomas in interspecific hybrids of Xiphophorus is connected to pigment cell specific overexpression of the mutationally activated receptor tyrosine kinase Xmrk. In purebred fish the oncogenic function of xmrk is suppressed by the molecularly still unidentified locus R. The xmrk oncogene was generated by a gene duplication event from the Xiphophorus egfrb gene and thereby has acquired a new 5’ regulatory sequence, which has probably altered the transcriptional control of the oncogene. So far, the xmrk promoter region was still poorly characterized and the molecular mechanism by which R controls xmrk-induced melanoma formation in Xiphophorus still remained to be elucidated. To test the hypothesis that R controls melanoma development in Xiphophorus on the transcriptional level, the first aim of the thesis was to gain a deeper insight into the transcriptional regulation of the xmrk oncogene. To this end, a quantitative analysis of xmrk transcript levels in different Xiphophorus genotypes carrying either the highly tumorigenic xmrkB or the non-tumorigenic xmrkA allele was performed. I was able to demonstrate that expression of the tumorigenic xmrkB allele is strongly increased in malignant melanomas of R-free backcross hybrids compared to benign lesions, macromelanophore spots, and healthy skin. The expression level of the non-tumorigenic xmrkA allele, in contrast, is not influenced by the presence or absence of R. These findings strongly indicate that differential transcriptional regulation of the xmrk promoter triggers the tumorigenic potential of these xmrk alleles. To functionally characterize the xmrk promoter region, I established a luciferase assay using BAC clones containing the genomic regions where xmrk and egfrb are located for generation of reporter constructs. This approach showed for the first time a melanoma cell specific transcriptional activation of xmrkB by its flanking regions, thereby providing the first functional evidence that the xmrk oncogene is controlled by a pigment cell specific promoter region. Subsequent analysis of different deletion constructs of the xmrkB BAC reporter construct strongly indicated that the regulatory elements responsible for the tumor-inducing overexpression of xmrkB in melanoma cells are located within 67 kb upstream of the xmrk oncogene. Taken together, these data indicate that melanoma formation in Xiphophorus is regulated by a tight transcriptional control of the xmrk oncogene and that the R locus acts through this mechanism. As the identification of the R-encoded gene(s) is necessary to fully understand how melanoma formation in Xiphophorus is regulated, I furthermore searched for alternative R candidate genes in this study. To this end, three genes, which are located in the genomic region where R has been mapped, were evaluated for their potential to be a crucial constituent of the regulator locus R. Among these genes, I identified pdcd4a, the ortholog of the human tumor suppressor gene PDCD4, as promising new candidate, because this gene showed the expression pattern expected from the crucial tumor suppressor gene encoded at the R locus. N2 - Fische der Gattung Xiphophorus sind ein gut etabliertes Modellsystem zur Analyse der genetischen Grundlagen der Tumorentwicklung. Die Entwicklung hereditärer Melanome in bestimmten interspezifischen Xiphophorus-Hybriden wird durch die pigmentzellspezifische Überexpression des Onkogens xmrk ausgelöst. Dieses Gen codiert für eine durch Mutationen aktivierte Rezeptortyrosinkinase. In den reinerbigen Elterntieren wird die onkogene Funktion von xmrk durch den Regulator-Locus R unterdrückt, welcher jedoch auf molekularer Ebene noch nicht identifiziert wurde. Das Onkogen xmrk ist durch eine Genduplikation aus dem Protoonkogen egfrb entstanden und hat dabei eine neue regulatorische 5‘ Region erhalten, welche mit hoher Wahrscheinlichkeit die transkriptionelle Regulation des Onkogens verändert hat. Die Promotorregion von xmrk war allerdings bisher nur unzureichend charakterisiert und der molekulare Mechanismus, durch den der R-Locus die xmrk-induzierte Melanomentwicklung kontrolliert, war noch weitgehend unbekannt. Um zu analysieren, ob der R-Locus die Melanomentwicklung in Xiphophorus auf transkriptioneller Ebene kontrolliert, war das erste Ziel dieser Arbeit die transkriptionelle Regulation des xmrk Onkogens genauer zu untersuchen. Zu diesem Zweck habe ich eine quantitative Analyse der xmrk Expressionslevel in Geweben verschiedener Xiphophorus-Genotypen durchgeführt, welche entweder das stark tumorigene xmrkB oder das nicht tumorigene xmrkA Allel besitzen. Ich konnte zeigen, dass im Vergleich zu benignen Läsionen, Macromelanophoren und gesunder Haut, die Expression des tumorigenen xmrkB Allels in den malignen Melanomen der R-defizienten Rückkreuzungshybride stark erhöht ist. Das Expressionslevel des xmrkA Allels wird hingegen nicht durch den R-Locus beeinflusst. Dieses Ergebnis deutet darauf hin, dass eine differenzielle transkriptionelle Regulierung des xmrk Promotors für die Unterschiede im onkogenen Potential dieser Allele verantwortlich ist. Um die xmrk Promotorregion funktional zu charakterisieren, habe ich in der hier vorliegenden Studie einen Luciferase-Assay etabliert, für den BAC-Klone, welche die xmrk- oder egfrb-Region enthalten, zur Herstellung von Reporterkonstrukten verwendet wurden. Mit Hilfe dieses Ansatzes konnte ich zum ersten Mal eine melanomzellspezifische Aktivierung des xmrkB Gens durch seine regulatorischen Regionen zeigen. Dies liefert den ersten funktionalen Beweis, dass das xmrk Onkogen tatsächlich durch einen pigmentzellspezifischen Promotor kontrolliert wird. Durch die nachfolgende Analyse einer Deletionsserie des xmrkB Reporterkonstrukts konnte gezeigt werden, dass die regulatorischen Elemente, welche die starke Überexpression von xmrk in Melanomzellen steuern, in den proximalen 67 kb der xmrk 5‘ Region lokalisiert sind. Zusammengefasst deuten diese Ergebnisse darauf hin, dass die Melanomentwicklung in Xiphophorus durch eine strikte transkriptionelle Kontrolle des xmrk Onkogens reguliert wird und dass der Regulator-Locus R seine tumorsuppressive Funktion über diesen Mechanismus ausübt. Da die Identifizierung des R-Locus-Gens entscheidend ist, um die Melanomentwicklung in Xiphophorus vollständig zu verstehen, habe ich im zweiten Teil dieser Arbeit drei Gene, welche in derselben genomischen Region liegen in der R lokalisiert wurde, genauer untersucht, um zu testen, ob es sich bei einem dieser Gene um eine entscheidende tumorsuppressive Komponente des R-Locus handelt. Von diesen Genen wurde pdcd4a, welches das Ortholog zum humanen Tumorsuppressorgen PDCD4 ist, als vielversprechendes neues Kandidatengen identifiziert, da das Expressionsmuster von pdcd4a mit dem zu erwartenden Expressionsmuster des am R-Locus codierten Tumorsuppressorgens übereinstimmt. KW - Melanom KW - Schwertkärpfling KW - Chromatophor KW - Epidermaler Wachstumsfaktor KW - Onkogen KW - Tumorsuppressorgen KW - Hybrid KW - transkriptionelle Regulation KW - melanoma KW - Xiphophorus KW - xmrk KW - transcriptional control KW - pigment cell KW - Hautkrebs KW - Epidermaler Wachstumsfaktor-Rezeptor Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82319 ER - TY - JOUR A1 - Pielström, Steffen A1 - Roces, Flavio T1 - Sequential Soil Transport and Its Influence on the Spatial Organisation of Collective Digging in Leaf-Cutting Ants JF - PLoS ONE N2 - The Chaco leaf-cutting ant Atta vollenweideri (Forel) inhabits large and deep subterranean nests composed of a large number of fungus and refuse chambers. The ants dispose of the excavated soil by forming small pellets that are carried to the surface. For ants in general, the organisation of underground soil transport during nest building remains completely unknown. In the laboratory, we investigated how soil pellets are formed and transported, and whether their occurrence influences the spatial organisation of collective digging. Similar to leaf transport, we discovered size matching between soil pellet mass and carrier mass. Workers observed while digging excavated pellets at a rate of 26 per hour. Each excavator deposited its pellets in an individual cluster, independently of the preferred deposition sites of other excavators. Soil pellets were transported sequentially over 2 m, and the transport involved up to 12 workers belonging to three functionally distinct groups: excavators, several short-distance carriers that dropped the collected pellets after a few centimetres, and long-distance, last carriers that reached the final deposition site. When initiating a new excavation, the proportion of long-distance carriers increased from 18% to 45% within the first five hours, and remained unchanged over more than 20 hours. Accumulated, freshly-excavated pellets significantly influenced the workers' decision where to start digging in a choice experiment. Thus, pellets temporarily accumulated as a result of their sequential transport provide cues that spatially organise collective nest excavation. KW - animal behavior KW - ants KW - confidence interval KW - decision making KW - foraging KW - fungal structure KW - fungi KW - hormone transport Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96275 ER - TY - THES A1 - Pielström, Steffen T1 - On the Role of Local Information in the Spatial Organisation of Collective Nest Digging in the Leaf-Cutting Ant Atta vollenweideri (Forel, 1893) T1 - Über die Rolle lokaler Information in der räumlichen Organisation des kollektiven Nestgrabeverhaltens bei der Blattschneiderameise Atta vollenweideri (Forel, 1893) N2 - Many ant species excavate underground nests. One of the most impressive examples is the Chaco leaf-cutting ant Atta vollenweideri from the Gran Chaco region in South America. The nests excavated by the workers of that species are among the largest insect-built structures on the planet. They are ecavated over years possibly involving millions of working individuals. However, the mechanisms underlying the organisation of collective nest digging in ants remain largely unknown. Considering the sheer dimensions of the nest in comparison to the size and presumably limited perceptual and cognitive abilities of the single worker, the assumption can be made that organising mechanisms are mostly based on responses of individuals to local stimuli within their perceptual range. Among these local stimuli that guide nest digging we can expect environmental variables, stimuli that relate to the requirements of the colony, and stimuli related to the spatial coordination of collective effort. The present thesis investigates the role of local stimuli from these three categories in the organisation of collective digging behaviour in the Chaco leaf-cutting ant. It describes experiments on (1) how workers respond in the context of digging to differences in soil moisture, which comprises an important environmental variable; (2) how available nest space influences nest enlargement; (3) and how the spatial coordination of excavating workers is implemented by responding to stimuli arising from nest mates while engaged in digging behaviour. The experiments on soil water content show that workers prefer to dig in moist materials that allow for fast excavation and transport rates. Accordingly, an unequal distribution of water in the soil around a nest can influence how the nest shape develops. On the other hand, results also indicate that workers strongly avoid excavating in extremely moist materials. Regarding the abundant occurrence of flooding events in the Gran Chaco region, the latter can be interpreted as an adaptation to avoid water inflow into the nest. In the experiments on the effect of nest space, the ants excavated less when presented with larger nests. When a large amount of space was suddenly added to the nest during the digging process, excavation rates decreased according to the new volume. These observations confirm the hypothesis that digging activity is regulated according to space requirements, possibly because crowding conditions inside the nest influence excavation behaviour. However, observations also indicate an intrinsic decrease of digging motivation with time. Moreover, excavation rates correlate with nest size only when comparing nests of similar shape. Distributing a similar nest volume to three smaller chambers, instead of one, resulted in drastically decreased digging rates. A possible explanation for that observation lies in the distribution of workers inside the nest that may vary according to nest geometry: a different distribution of individuals can lead to in different local crowding conditions in similar nest volumes. Furthermore, two different stimuli are described that are used in the spatial coordination of collective digging effort. First, fresh soil pellets deposited close to the digging site on their way from the surface increase the probability that arriving workers join excavation efforts at the same site. The deposition of pellets on the way is a consequence of sequential task partitioning during soil transport. The pellets are carried in transport chains that closely resemble the modalities of leaf transport observed at the surface. Second, workers stridulate while digging. The short-ranged vibrational signals produced thereby also attract nest mates to excavate at the same location. Accordingly, two mutually complementing mechanisms are described that allow to concentrate excavators at one location. In both cases, a local stimulus that is generated by current close-by excavation activity increases the probability of the stimulus receiver to dig close to other excavators. In an environment otherwise poor in digging stimuli, these mechanisms can be especially important to give collective digging efforts a common direction. As a consequence it can be argued that the spatial organisation of collective digging is based on choice copying. Individuals copy nest mate decisions on where to excavate by responding to local stimuli provided by nest mate digging activity. Taken together, responses to local stimuli can determine the direction of nest growth, aid in preventing the inflow of surface water into the nest, guide the adjustment of nest size to colony requirements and spatially coordinate collective digging efforts. Even though it cannot be ruled out that digging responses based e.g. on spatial memory or long-term experience exist, the results presented here clearly demonstrate that responses to local information account for many important aspects of nest development. N2 - Viele Ameisenarten graben unterirdische Nester. Eines der wohl eindrucksvollsten Beispiele ist die Chaco-Blattschneiderameise Atta vollenweideri aus der Gran Chaco Region in Südamerika. Die Nester dieser Art gehören zu den größten von Insekten gebauten Strukturen auf unserem Planeten, und an ihrer Konstruktion sind vermutlich Millionen von Individuen über mehrere Jahre beteiligt. Die Mechanismen, die der Organisation des kollektiven Nestgrabeverhaltens zu Grunde liegen, sind weitgehend unbekannt. Berücksichtigt man allerdings allein die Dimensionen des gebauten Nestes im Vergleich zur Größe, und den vermutlich begrenzten Sinnes- und Kognitionsleistungen der einzelnen Arbeiterin, so liegt die Vermutung nahe, daß den Organisationsmechanismen überwiegend Reaktionen auf lokale Reize innerhalb der Wahrnehmungsreichweite zu Grunde liegen. Zu diesen lokalen Reizen gehören vermutlich Umweltvariablen, Reize, die mit den Bedürfnissen der Kolonie zusammenhängen, und Reize, die der räumlichen Koordination gemeinsamer Bemühungen dienen. Die vorliegende Dissertation untersucht die Rolle lokaler Reize in der Organisation des kollektiven Grabeverhaltens bei Atta vollenweideri auf diesen drei Ebenen. Sie beschreibt Experimente (1) zur Reaktion grabender Arbeiterinnen auf Unterschiede in der Bodenfeuchte, die in diesem Zusammenhang eine wichtige Umweltvariable darstellt, (2) zum Einfluß der verfügbaren Nestgröße auf die Nesterweiterung und (3) zur räumlichen Koordination grabender Arbeiterinnen durch Reize, die von anderen grabenden Arbeiterinnen ausgehen. Die Versuche zur Bodenfeuchte zeigen eine Präferenz für feuchte Materialen, die hohe Grabe- und Transportraten ermöglichen. Demzufolge kann die Verteilung des Wassers im Boden um ein Nest erheblichen Einfluß auf die Entwicklung der Nestform haben. Andererseits zeigen die Ergebnisse aber auch, daß Arbeiterinnen vermeiden, in extrem nassen Materialen zu graben. In Anbetracht der regelmäßigen Überflutungen in der Gran Chaco Region lässt sich diese Ablehnung als eine Adaptation interpretieren, die hilft, Wassereinbrüche ins Nest zu vermeiden. In den Experimenten zum Einfluß der Nestgröße gruben die Ameisen weniger, wenn ihnen ein größeres Nest zur Verfügung stand. Wenn das Nest im Laufe des Grabeprozesses künstlich schlagartig vergrößert wurde, passte sich die Graberate dem neuen Volumen an. Diese Beobachtungen bestätigen die Hypothese, daß die Grabeaktivität abhängig vom Platzbedarf reguliert wird, vermutlich, weil die Individuendichte im Nest das Grabeverhalten beeinflusst. Andererseits zeigen die Beobachtungen aber auch eine zeitabhängige, intrinsische Abnahme der Motivation zu graben. Zudem korreliert die Graberate nur dann mit der Nestgröße, wenn Nester von vergleichbarer Form in Betracht gezogen werden. Die Verteilung eines ähnlichen Nestvolumens auf drei kleinere Kammern statt einer größeren führte zu deutlich niedrigeren Graberaten. Eine mögliche Erklärung für diese Beobachtung liegt in der Verteilung der Arbeiterinnen im Nestinnern, die möglicherweise mit der Nestgeometrie variiert. Unterschiede in der Verteilung der Individuen können bei gleichem Nestvolumen unterschiedliche lokale Individuendichten zur Folge haben. Des weiteren werden zwei verschiedene Stimuli beschrieben, die zur räumlichen Koordination des kollektiven Grabeverhaltens genutzt werden. Zum einen erhöht sich die Wahrscheinlichkeit, daß eine ankommende Arbeiterin sich den Grabearbeiten an einer bestimmten Stelle anschließt, wenn auf dem Weg dorthin, nahe der betreffenden Stelle, frisch ausgegrabene Bodenpartikel, sogenannte pellets, abgelegt wurden. Das passiert in Folge der sequentiellen Arbeitsteilung beim Transportieren der Erde, denn der Transport der pellets erfolgt in Arbeitsketten, ähnlich wie der Transport von Blattfragmenten an der Oberfläche. Zum anderen stridulieren die Arbeiterinnen beim Graben und erzeugen dadurch Vibrationssignale von kurzer Reichweite, die ebenfalls Nestgenossinnen zum Graben an derselben Stelle motivieren. Es werden also zwei sich gegenseitig ergänzende Mechanismen beschrieben, die es erlauben, grabende Arbeiterinnen an einer bestimmten Stelle zu konzentrieren. In beiden Fällen erhöht ein Reiz, der durch aktuelle Grabeaktivität in unmittelbarer Nähe generiert wird, die Wahrscheinlichkeit, daß der Reizempfänger in der Nähe anderer grabender Arbeiterinnen zu Graben beginnt. Besonders in einem ansonsten, in Bezug auf das Grabeverhalten reizarmen Umfeld können derartige Mechanismen wichtig sein, um den kollektiven Grabebemühungen eine gemeinsame Richtung zu geben. Dementsprechend lässt sich argumentieren, daß die räumliche Organisation des Grabeverhaltens auf der Nachahmung von Entscheidungen basiert. Individuen kopieren die Entscheidungen ihrer Nestgenossinnen, wo gegraben wird, indem sie auf Reize reagieren, die von der Grabeaktivität anderer Arbeiterinnen ausgehen. KW - Blattschneiderameisen KW - Nestbau KW - Verhalten KW - Organisation KW - Stridulation KW - Transport KW - Boden KW - leaf-cutting ants KW - nest building KW - organisation KW - stridulation KW - transport Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-79118 ER - TY - JOUR A1 - Pahl, Mario A1 - Si, Aung A1 - Zhang, Shaowu T1 - Numerical cognition in bees and other insects JF - Frontiers in Comparative Psychology N2 - The ability to perceive the number of objects has been known to exist in vertebrates for a few decades, but recent behavioral investigations have demonstrated that several invertebrate species can also be placed on the continuum of numerical abilities shared with birds, mammals, and reptiles. In this review article, we present the main experimental studies that have examined the ability of insects to use numerical information. These studies have made use of a wide range of methodologies, and for this reason it is striking that a common finding is the inability of the tested animals to discriminate numerical quantities greater than four. Furthermore, the finding that bees can not only transfer learnt numerical discrimination to novel objects, but also to novel numerosities, is strongly suggestive of a true, albeit limited, ability to count. Later in the review, we evaluate the available evidence to narrow down the possible mechanisms that the animals might be using to solve the number-based experimental tasks presented to them. We conclude by suggesting avenues of further research that take into account variables such as the animals’ age and experience, as well as complementary cognitive systems such as attention and the time sense. KW - bee KW - insect KW - counting KW - learning KW - memory KW - Biene KW - numerical cognition KW - quantity discrimination Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-95935 UR - http://journal.frontiersin.org/Journal/10.3389/fpsyg.2013.00162/full ER - TY - THES A1 - Ott, Christine Kornelia T1 - Diverse Aspects of the Sorting and Assembly Machinery in Human Mitochondria T1 - Diverse Aspekte der Sortierungs- und Assemblierungsmaschinerie in humanen Mitochondrien N2 - Mitochondria are organelles of endosymbiotic origin, which play many important roles in eukaryotic cells. Mitochondria are surrounded by two membranes and, considering that most of the mitochondrial proteins are produced in the cytosol, possess import machineries, which transport mitochondria-targeted proteins to their designated location. A special class of outer mitochondrial membrane (OMM) proteins, the β-barrel proteins, require the sorting and assembly machinery (SAM) for their OMM integration. Both mitochondrial β-barrel proteins and the central component of the SAM complex, Sam50, have homologs in gram-negative bacteria. In yeast mitochondria, bacterial β-barrel proteins can be imported and assembled into the OMM. Our group demonstrated that this, however, is not the case for human mitochondria, which import only neisserial β barrel proteins, but not those of Escherichia coli and Salmonella enterica. As a part of this study, I could demonstrate that β-barrel proteins such as Omp85 and PorB of different Neisseria species are targeted to human mitochondria. Interestingly, only proteins belonging to the neisserial Omp85 family were integrated into the OMM, whereas PorB was imported into mitochondria but not assembled. By exchanging parts of homologous neisserial Omp85 and E. coli BamA and, similarly, of neisserial PorB and E. coli OmpC, it could be demonstrated in this work that the mitochondrial import signal of bacterial β barrel proteins cannot be limited to one short linear sequence, but rather secondary structure and protein charge seem to play an important role, as well as specific residues in the last β-strand of Omp85. Omp85 possesses five conserved POTRA domains in its amino-terminal part. This work additionally demonstrated that in human mitochondria, at least two POTRA domains of Omp85 are necessary for membrane integration and functionality of Omp85. In the second part of this work, the influence of Sam50 on the mitochondrial cristae structure was investigated. This work contributed to a study performed by our group in which it was confirmed that Sam50 is present in a high molecular weight complex together with mitofilin, CHCHD3, CHCHD6, DnaJC11, metaxin 1 and metaxin 2. This connection between the inner and outer mitochondrial membrane was shown to be crucial for the maintenance of the mitochondrial cristae structure. In addition, a role of Sam50 in respiratory complex assembly, suggested by a SILAC experiment conducted in our group, could be confirmed by in vitro import studies. An influence of Sam50 not only on respiratory complexes but also on the recently described respiratory complex assembly factor TTC19 was demonstrated. It was shown that TTC19 not only plays a role in complex III assembly as published, but also influences the assembly of respiratory complex IV. Thus, in this part of the work a connection between the OMM protein Sam50 and maintenance of cristae structure, respiratory complex assembly and an assembly factor could be established. N2 - Mitochondrien sind Zellorganellen endosymbiotischen Ursprungs, die viele wichtige Funktionen in eukaryotischen Zellen haben. Mitochondrien sind von zwei Membranen umgeben, und da die meisten Mitochondrienproteine im Cytosol hergestellt werden, besitzen sie Importmaschinerien, die die für die Mitochondrien bestimmten Proteine zu ihrem jeweiligen Zielort transportieren. Eine besondere Klasse von Proteinen der äußeren Mitochondrienmembran (ÄMM), die β-Fassproteine, benötigen die Sortierungs- und Assemblierungsmaschinerie (SAM) für ihre Integration in die ÄMM. Sowohl mitochondriale β-Fassproteine als auch die zentrale Komponente des SAM-Komplexes, Sam50, haben Homologe in gramnegativen Bakterien. In Hefemitochondrien können bakterielle β Fassproteine importiert und in der ÄMM assembliert werden. Unsere Gruppe hat gezeigt, dass dies jedoch nicht auf humane Mitochondrien zutrifft, die nur neisserielle β-Fassproteine importieren, nicht aber diejenigen von Escherichia coli und Salmonella enterica. Im Rahmen dieser Studie konnte ich zeigen, dass β Fassproteine verschiedener Neisserienarten, wie Omp85 und PorB, in humane Mitochondrien aufgenommen werden. Interessanterweise wurden nur Proteine der neisseriellen Omp85-Familie in die ÄMM eingebaut, während PorB zwar importiert, jedoch nicht assembliert wurde. Durch das Austauschen von Teilen von homologem neisseriellen Omp85 und E.coli BamA und ebenso von neisseriellem PorB und E. coli OmpC konnte in dieser Arbeit gezeigt werden, dass das mitochondriale Importsignal bakterieller β-Fassproteine nicht auf eine kurze lineare Sequenz eingegrenzt werden kann, sondern dass die Sekundärstruktur und die Ladung des Proteins eine wichtige Rolle zu spielen scheinen, sowie im Fall von Omp85 einige bestimmte Aminosäurereste des letzten β-Stranges. Omp85 besitzt fünf konservierte POTRA-Domänen in seiner aminoterminalen Hälfte. In dieser Arbeit wurde zudem demonstriert, dass in humanen Mitochondrien mindestens zwei POTRA-Domänen von Omp85 für die Membranintegration und Funktionalität von Omp85 vorhanden sein müssen. Im zweiten Teil dieser Arbeit wurde der Einfluss von Sam50 auf die mitochondriale Cristaestruktur untersucht. Diese Arbeit hat zu einer von unserer Gruppe durchgeführten Studie beigetragen, in der bestätigt werden konnte, dass Sam50 in einem hochmolekularen Komplex mit Mitofilin, CHCHD3, CHCHD6, DnaJC11, Metaxin 1 und Metaxin 2 vorliegt. Es wurde gezeigt, dass diese Verbindung zwischen der inneren und äußeren Mitochondrienmembran unverzichtbar für die Aufrechterhaltung der mitochondrialen Cristaestruktur ist. Zudem konnte eine Rolle von Sam50 bei der Assemblierung von Atmungskettenkomplexen, die durch ein in unserem Labor durchgeführtes SILAC-Experiment nahegelegt worden war, durch in-vitro-Importstudien bestätigt werden. Weiterhin wurde ein Einfluss von Sam50 nicht nur auf Atmungskettenkomplexe, sondern auch auf einen vor kurzem beschriebenen Assemblierungsfaktor der Atmungskette, TTC19, demonstriert. Es wurde gezeigt, dass TTC19 nicht nur, wie veröffentlicht, eine Rolle bei der Assemblierung des Atmungskettenkomplexes III spielt, sondern auch die Assemblierung des Atmungskettenkomplexes IV beeinflusst. In diesem Teil der Arbeit konnte folglich eine Verbindung zwischen dem ÄMM-Protein Sam50 und der Organisation der Cristaestruktur, der Atmungskettenassemblierung und einem Assemblierungsfaktor nachgewiesen werden. KW - Mitochondrien KW - Mensch KW - Molekularbiologie KW - mitochondria KW - Import KW - Omp85 KW - beta-barrel-proteins KW - cristae KW - beta-Fassproteine KW - Cristaestruktur Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85462 ER - TY - JOUR A1 - Ngwa, Che Julius A1 - Scheuermayer, Matthias A1 - Mair, Gunnar Rudolf A1 - Kern, Selina A1 - Brügl, Thomas A1 - Wirth, Christine Clara A1 - Aminake, Makoah Nigel A1 - Wiesner, Jochen A1 - Fischer, Rainer A1 - Vilcinskas, Andreas A1 - Pradel, Gabriele T1 - Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito JF - BMC Genomics N2 - Background: The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector. Results: To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy. Conclusions: The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector. KW - parasitophorous vacuole KW - sexual development KW - gametocyte KW - transcriptome KW - signal peptide peptidase KW - host cell interface KW - alpha-tubulin-II KW - life-cycle KW - protein kinases KW - in-vitro KW - erythroyte invation KW - blocking antibodies KW - malaria KW - plasmodium falciparum KW - gametogenesis KW - mosquito KW - transmission Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121905 SN - 1471-2164 VL - 14 IS - 256 ER - TY - JOUR A1 - Muranyi, Walter A1 - Malkusch, Sebastian A1 - Müller, Barbara A1 - Heilemann, Mike A1 - Kräusslich, Hans-Georg T1 - Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail JF - PLoS Pathogens N2 - The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env\((\Delta CT)\) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse. KW - ENV KW - fluorescent-probes KW - type-1 matrix KW - glycoprotein incorporation KW - GP41 cytoplasmic tail KW - human immunodeficiency virus KW - cellular proteins KW - plasma membrane KW - virions KW - particles Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131235 VL - 9 IS - 2 ER - TY - JOUR A1 - Mueller, Thomas D. A1 - Fiebig, Juliane E. A1 - Weidauer, Stella E. A1 - Qiu, Li-Yan A1 - Bauer, Markus A1 - Schmieder, Peter A1 - Beerbaum, Monika A1 - Zhang, Jin-Li A1 - Oschkinat, Hartmut A1 - Sebald, Walter T1 - The Clip-Segment of the von Willebrand Domain 1 of the BMP Modulator Protein Crossveinless 2 Is Preformed JF - Molecules N2 - Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop. KW - bone morphogenetic proteins KW - TGF-β superfamily KW - BMP antagonist KW - protein-protein recognition KW - NMR spectroscopy KW - von Willebrand type C domain Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97196 ER - TY - JOUR A1 - Moll, Karin A1 - Roces, Flavio A1 - Federle, Walter T1 - How Load-Carrying Ants Avoid Falling Over: Mechanical Stability during Foraging in Atta vollenweideri Grass-Cutting Ants JF - PLoS ONE N2 - Background: Foraging workers of grass-cutting ants (Atta vollenweideri) regularly carry grass fragments larger than their Fragment length has been shown to influence the ants' running speed and thereby the colony's food intake rate. We investigated whether and how grass-cutting ants maintain stability when carrying fragments of two different lengths but identical mass. Principal Findings: Ants carried all fragments in an upright, backwards-tilted position, but held long fragments more vertically than short ones. All carrying ants used an alternating tripod gait, where mechanical stability was increased by overlapping stance phases of consecutive steps. The overlap was greatest for ants carrying long fragments, resulting in more legs contacting the ground simultaneously. For all ants, the projection of the total centre of mass (ant and fragment) was often outside the supporting tripod, i.e. the three feet that would be in stance for a non-overlapping tripod gait. Stability was only achieved through additional legs in ground contact. Tripod stability (quantified as the minimum distance of the centre of mass to the edge of the supporting tripod) was significantly smaller for ants with long fragments. Here, tripod stability was lowest at the beginning of each step, when the center of mass was near the posterior margin of the supporting tripod. By contrast, tripod stability was lowest at the end of each step for ants carrying short fragments. Consistently, ants with long fragments mainly fell backwards, whereas ants carrying short fragments mainly fell forwards or to the side. Assuming that transporting ants adjust neither the fragment angle nor the gait, they would be less stable and more likely to fall over. Conclusions: In grass-cutting ants, the need to maintain static stability when carrying long grass fragments has led to multiple kinematic adjustments at the expense of a reduced material transport rate. KW - selection KW - tissue transport KW - stepping patterns KW - size determination KW - leaf-cutter ants KW - locomotion KW - distance KW - formicidae KW - strategies KW - cephalotes Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131211 VL - 8 IS - 1 ER - TY - THES A1 - Mischnik, Marcel T1 - Systembiologische Analyse der ADP- und Prostaglandin-vermittelten Signaltransduktion humaner Thrombozyten T1 - Systems biological analysis of ADP and prostaglandin mediated signal transduction in human thrombocytes N2 - Thrombozyten (Blutplättchen) sind die Vermittler der zellulären Hämostase. Ihre Fähigkeit zu Aggregieren und sich an das umgebende Gewebe verletzter Blutgefässe anzulagern, wird durch ein komplexes intrazelluläres Signaltransduktionsnetzwerk bestimmt, das sowohl aktivierende, als auch inhibierende Subnetzwerke beinhaltet. Das Verständnis dieser Prozesse ist von hoher medizinischer Bedeutung. Im Rahmen dieser Arbeit wurde die thrombozytäre Signaltransduktion sowohl mittels eines Boole'schen, als auch verschiedener dynamischer Modelle analysiert. Die Boole'sche Modellierung führte zu interessanten Erkenntnissen über das Zusammenwirken einzelner Subnetzwerke bei der Vermittlung irreversibler Plättchenaktivierung und zeigte Mechanismen der Interaktion mit dem hemmenden Prostaglandinsystem auf. Das Modell beinhaltet unter Anderem wichtige Systemkomponenten wie Calciumsignalgebung, Aktivierung von Schlüsselkinasen wie Src und PKC, Integrin-vermitteltes outside-in sowie inside-out Signalgebung und autokrine ADP- und Thromboxan-Produktion. Unter Verwendung dieses Boole'schen Ansatzes wurde weiterhin das System-eigene Schwellenwertverhalten analysiert. Dabei stellte sich eine umgekehrt proportionale Abhängigkeit des relativen aktivierenden Reizes, der notwendig ist um den Schwellenwert zu überschreiten, vom absoluten hemmenden Input heraus. Das System adaptiert demnach an höhere Prostaglandinkonzentrationen durch eine Erhöhung der Sensitivität für Aktivatoren wie dem van-Willebrandt-Faktor und Kollagen, und ermöglicht somit auch unter lokal hemmenden Bedingungen eine Plättchen-vermittelte Hämostase. Der nächste Schritt bestand in der Implementierung eines Differentialgleichungs-basierten Modells der thrombozytären Prostaglandin-Signaltransduktion, um einen detaillierten Überblick über die Dynamik des inhibierenden Netzwerkteils zu erhalten. Die kinetischen Parameter dieses Modells wurden teilweise der Literatur entnommen. Der andere Teil wurde anhand einer umfassenden Kombination dosis- und zeitabhängiger cAMP und phospho-VASP Messdaten geschätzt. Der Prozess beinhaltete mehrere Iterationen aus Modellvorhersagen einerseits und experimentellem Design andererseits. Das Modell liefert die quantitativen Effekte der Prostaglandinrezeptoren IP, DP1, EP3 und EP4 und des ADP-Rezeptors P2Y12 auf die zugrunde liegende Signalkaskade. EP4 zeigt den stärksten Effekt in der aktivierenden Fraktion, wohingegen EP3 einen stärkeren inhibitorischen Effekt ausübt, als der durch Clopidogrel hemmbare ADP-Rezeptor P2Y12. Weiterhin wurden die Eigenschaften des negativen feedback-loops der PKA auf den cAMP-Spiegel untersucht, und eine direkte Beeinflussung der Adenylatzyklase durch die PKA festgestellt, in Form einer Reduzierung der maximalen katalytischen Geschwindigkeit. Die Identifizierbarkeit der geschätzten Parameter wurde mittels profile-Likelihood-Schätzung untersucht. In einem dritten Schritt wurde ein sowohl die aktivierenden, als auch die hemmenden Netzwerkteile umfassendes dynamisches Modell implementiert. Die Topologie dieses Modells wurde in Anlehnung an die des Boole'schen Modells auf der Basis von a priori Wissen festgelegt. Die Modellparameter wurden anhand von Western-Blot, Calcium- und Aggregationsmessungen geschätzt. Auch hier wurde die Identifizierbarkeit der Modellparameter durch profile-likelihood-Schätzung überprüft. Die bei niedrigen Ligandenkonzentrationen auftretende Reversibilität der Plättchen-Aggregation konnte mittels dieses Modells reproduziert werden. Jedoch zeigte sich bei mittleren ADP-Konzentrationen ein Fließgleichgewicht in einem teilweise aktivierten Zustand, und damit kein bistabiles Schwellenwertverhalten. Inwiefern dieses Verhalten durch einen Umgebungs-basierteren Mechanismus des Alles-Oder-Nichts-Verhaltens begründet wird, bei dem der Übergang von reversibler zu irreversibler Aggregation mehr durch parakrine Effekte des gesammten Thrombus bestimmt wird, als durch spezifische Signaltransduktionseigenschaften der einzelnen Zelle, müssen zukünftige Experimente zeigen. Insgesamt geben die erstellten Modelle interessante Einblicke in die Funktionsweise der Thrombozyten und ermöglichen die Simulation von pharmakologischen und genetischen Einflüssen, wie Rezeptormodulationen und knock-outs. Sie geben damit Implikationen zur Entstehung und Behandlung pathophysiologischer Zustände, und wertvolle Denkanstöße für die weitere Forschung. N2 - Platelets represent the key-players in mammalian wound-healing. Their ability to aggregate and attach to the surrounding tissue of damaged blood vessels is thereby mediated by a complex signal -transduction network that comprises both activatory and inhibitory components. Due to its medical relevance and the lack of profound understanding, the network constitutes a convenient target for modeling. In a first step, a Boolean implementation of platelet signal transduction, comprising both activating and inhibiting networks components was established. This led to important information, on how the function of different subnetworks coalesce to fully activate the platelet and to promote irreversible aggregation. These include calcium signalling, activation of key-kinases like Akt, Src and PKC, Integrin outside-in as well as inside-out signalling and autocrin ADP and thromboxane production. In addition, using this data-free approach, the systems inherent threshold behaviour was analysed. The model revealed, that the relative activating strength, that transgresses the threshold, decreases with elevating PGI inputs and is also dependent on auto- and parakrin effects. Thus, the system adapts for higher prostaglandin-concentrations by increasing its sensitivity for activators like vWF and collagen, and thereby commits thrombocyte-dependent active processes such as wound healing even if subsequently blood prostaglandin-levels are higher in later time points. Secondly, an ordinary-differential-equation based model of platelet prostaglandin signalling was established, to get a detailed view of the dynamics governing the inhibiting network part. The kinetic parameters of the model were partly taken from literature and in part estimated along a comprehensive combination of time-course and dose-response measurements of cAMP and phosphorylated VASP. The process involved an iterative cycle between model predictions and experimental design. The model delivered the quantitative effects of the prostaglandin receptors IP, DP1, EP3, EP4 and the ADP receptor P2Y12 on the underlying signalling cascade. EP4 showed the strongest effect in the activating fraction, whereas EP3 turned out to exert a greater inhibiting impact than the commonly established pharmacological target P2Y12. Furthermore, the nature of the double-negative feedback loop constituted by PKA was examined, which disclosed a direct influence of PKA on adenylate cyclase, reducing its maximum catalytic speed. The identifiablility of all kinetic parameters was analysed via profile likelihood estimation. Finally, a dynamical model comprising both activating ADP-signalling through P2Y1 and P2Y12 receptors, and the inhibiting prostaglandin-pathway was implemented. The topology of this larger model was established on the basis of a priori knowledge. Model parameters were fitted along time-resolved Western-Blot, calcium and aggregation measurements. The identifiability of the model parameters was again check by means of profile likelihood estimation. Reversibility of platelet activation at low ADP concentrations could be reproduced by this model. However, at medium concentrations the system appears to assume a steady-state in a partly activated condition, thus not providing for a bistable threshold behaviour. If this behaviour is based on a more enviroment-based character of the observed point-of-no-return behaviour, in which the transition from reversible to irreversible aggregation is rather due to parakrin effects evoked by the entire cell array, than due to specific network properties present in the single cell, has to be investigated by further experiments. All in all, the models show interesting properties of platelet signal transduction, and give valuable implications for medical treatment and future research. KW - Thrombozyt KW - Modellierung KW - ADP KW - Prostaglandine KW - Signaltransduktion KW - Thrombozyten KW - Modellierung KW - ADP KW - Prostaglandine KW - platelets KW - ADP KW - prostaglandin KW - modeling KW - boolean KW - ODE Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78807 ER - TY - JOUR A1 - Meuche, Ivonne A1 - Brusa, Oscar A1 - Linsenmair, K. Eduard A1 - Keller, Alexander A1 - Pröhl, Heike T1 - Only distance matters - non-choosy females in a poison frog population JF - Frontiers in Zoology N2 - Background: Females have often been shown to exhibit preferences for certain male traits. However, little is known about behavioural rules females use when searching for mates in their natural habitat. We investigated mate sampling tactics and related costs in the territorial strawberry poison frog (Oophaga pumilio) possessing a lek-like mating system, where both sequential and simultaneous sampling might occur. We continuously monitored the sampling pattern and behaviour of females during the complete period between two successive matings. Results: We found no evidence that females compared males by visiting them. Instead females mated with the closest calling male irrespective of his acoustic and physical traits, and territory size. Playback experiments in the natural home ranges of receptive females revealed that tested females preferred the nearest speaker and did not discriminate between low and high call rates or dominant frequencies. Conclusions: Our results suggest that females of O. pumilio prefer the closest calling male in the studied population. We hypothesize that the sampling tactic in this population is affected by 1) a strongly female biased sex ratio and 2) a low variance in traits of available males due to strong male-male competition, preventing low quality males from defending a territory and mating. KW - operational sex ratio KW - sequential mate choice KW - gray tree frogs KW - treefrogs hyla-gratiosa KW - male mating success KW - Bocas-del-Toro KW - dendrobates pumilio KW - oophaga pumilio KW - pied flycatchers KW - sampling behavior Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122617 SN - 1742-9994 VL - 10 IS - 29 ER - TY - JOUR A1 - Menzel, Florian A1 - Blüthgen, Nico A1 - Tolasch, Till A1 - Conrad, Jürgen A1 - Beifuss, Uwe A1 - Beuerle, Till A1 - Schmitt, Thomas T1 - Crematoenones - a novel substance class exhibited by ants functions as appeasement signal JF - Frontiers in Zoology N2 - Background: Parasitic, commensalistic, and mutualistic guests in social insect colonies often circumvent their hosts' nestmate recognition system to be accepted. These tolerance strategies include chemical mimicry and chemical insignificance. While tolerance strategies have been studied intensively in social parasites, little is known about these mechanisms in non-parasitic interactions. Here, we describe a strategy used in a parabiotic association, i.e. two mutualistic ant species that regularly share a common nest although they have overlapping food niches. One of them, Crematogaster modiglianii, produces an array of cuticular compounds which represent a substance class undescribed in nature so far. They occur in high abundances, which suggests an important function in the ant's association with its partner Camponotus rufifemur. Results: We elucidated the structure of one of the main compounds from cuticular extracts using gas chromatography, mass spectrometry, chemical derivatizations and nuclear magnetic resonance spectroscopy (NMR). The compound consists of two fused six-membered rings with two alkyl groups, one of which carries a keto functionality. To our knowledge, this is the first report on the identification of this substance class in nature. We suggest naming the compound crematoenone. In behavioural assays, crematoenones reduced interspecific aggression. Camponotus showed less aggression to allospecific cuticular hydrocarbons when combined with crematoenones. Thus, they function as appeasement substances. However, although the crematoenone composition was highly colony-specific, interspecific recognition was mediated by cuticular hydrocarbons, and not by crematoenones. Conclusions: Crematenones enable Crematogaster to evade Camponotus aggression, and thus reduce potential costs from competition with Camponotus. Hence, they seem to be a key factor in the parabiosis, and help Crematogaster to gain a net benefit from the association and thus maintain a mutualistic association over evolutionary time. To our knowledge, putative appeasement substances have been reported only once so far, and never between non-parasitic species. Since most organisms associated with social insects need to overcome their nestmate recognition system, we hypothesize that appeasement substances might play an important role in the evolution and maintenance of other mutualistic associations as well, by allowing organisms to reduce costs from antagonistic behaviour of other species. KW - cuticular hydrocarbons KW - appeasement substance KW - bees KW - ecology KW - parasitism KW - alkyloctahydronaphthalene KW - usurpation KW - venom KW - pheromone KW - crematogaster KW - parabiotic ants KW - Dufours gland KW - polyergus rufescens KW - formicidae KW - interspecific aggression KW - nestmate recognition cues KW - parabiotic association Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122595 SN - 1742-9994 VL - 10 IS - 32 ER - TY - THES A1 - Melzer, Juliane T1 - Die Funktion der p21-aktivierten Kinase Mbt in Neuroblasten während der Entwicklung des zentralen Nervensystems von Drosophila melanogaster T1 - The function of the p21-activated kinase Mbt in neuroblasts during the development of the central nervous system of Drosophila melanogaster N2 - p21-aktivierte Kinasen regulieren zahlreiche zelluläre Prozesse, die während der Entwicklung, aber auch beispielsweise bei der Krebsentstehung, von zentraler Bedeutung sind. Mbt, das einzige Typ II PAK-Protein von Drosophila melanogaster, spielt eine Rolle bei der Gehirnentwicklung. Eine Nullmutation von mbt, mbtP1, bildet kleinere Gehirne mit stark verkleinerten Pilzkörpern aus. In dieser Arbeit wurde die Funktion von Mbt in Neuroblasten untersucht. Mbt wurde als Teil des apikalen Proteinkomplexes in Neuroblasten des Zentralhirns nachgewiesen. Die apikale Lokalisation von Mbt ist Zellzyklus-abhängig und wird über Bindung an Cdc42 reguliert. Sie ist essentiell für die Funktion von Mbt in Neuroblasten. Trotz apikaler Mbt-Lokalisation in Neuroblasten zeigte die mbt Nullmutante keine Defekte des basalen Mechanismus der asymmetrischen Zellteilung. Mud zeigte geringfügige Lokalisationsveränderungen, die auf einen möglichen Einfluss von Mbt hinweisen. Obwohl PAKs zentrale Regulatoren des Zytoskeletts sind, zeigte die mbtP1 Mutante keine offensichtlichen Veränderungen des Aktin- und Tubulin-Zytoskeletts. Armadillo, ein Aktin-assoziiertes Mbt-Substrat, zeigte ebenfalls keine Lokalisationsveränderung in Neuroblasten. Mbt steuert jedoch die apikale Anreicherung von Cno, einem weiteren Aktin-assoziierten Protein, in Neuroblasten. Darüber hinaus beeinflusst Mbt die Zellgröße von Neuroblasten, sowie deren Proliferationspotenzial und Überleben. mbtP1 Neuroblasten sind kleiner als wildtypische Neuroblasten, haben ein geringeres Proliferationsvermögen und eine geringere Überlebenswahrscheinlichkeit. Der Zelltod von Neuroblasten ist jedoch ein sekundärer Effekt. Daher kann eine Blockierung von Apoptose den adulten Pilzkörperphänotyp nicht retten. Signalwege, die Zellgröße und Proliferation regulieren, wurden auf eine Beteiligung von Mbt hin analysiert. mbtP1 induzierte leichte Effekte im Insulin-Signalweg und die Delokalisation eines nukleolären Proteins. Eine genetische Interaktion von mbtP1 mit Mutationen in Genen des klassischen MAPK-Signalweges identifzierte mbt als Positivregulator dieses Signalweges im Auge. Ein ähnlicher, schwächerer Effekt wurde auch bzgl. der Proliferation und Größe von Neuroblasten beobachtet. Eine 2D-Gelanalyse von Larvengehirnen identifizierte Bic und Hsp83 als mögliche von Mbt regulierte Proteine. Diese Arbeit charakterisiert eine bisher unbekannte Funktion der p21-aktivierten Kinase Mbt in neuronalen Stammzellen und liefert damit Ansatzpunkte für eine detaillierte Aufklärung der Funktionsmechanismen von Typ II PAKs bei der Regulation von Zellproliferation und Überleben N2 - p21-activated kinases regulate numerous cellular processes central not only during development, but also for example for cancer pathogenesis. Mbt, the only type II PAK in Drosophila, regulates brain development. The mbt null mutant mbtP1 exhibits reduced brain size, with the mushroom bodies showing the most pronounced reduction. In this work, the function of Mbt in neuroblasts was investigated. Mbt was identified as a component of the apical protein complex in central brain neuroblasts. The apical localization of Mbt was cell cycle dependent and regulated by binding to Cdc42, which is essential for Mbt function in neuroblasts. Despite apical localization of Mbt, the mbtP1 null allel showed no defects in the basic mechanism of asymmetric cell division in larval neuroblasts. However, Mud showed minor localization changes indicating a possible influence of Mbt. Even though PAKs are well-known regulators of the cytoskeleton, no obvious changes in the actin and tubulin cytoskeleton were observed in mbtP1 neuroblasts. The localization of Armadillo, an actin-associated Mbt substrate, was also undisturbed throughout the cell cycle. Mbt controls the apical enrichment of Cno, another actin-associated protein. Moreover, Mbt influences neuroblast cell size, proliferation potential and survival. mbtP1 neuroblasts were smaller than wildtype neuroblasts and showed reduced proliferation activity and survival. However, the apoptotic loss of mbtP1 neuroblasts is a secondary effect. Thus, the adult mushroom body phenotype cannot be rescued by blocking apoptosis. Signalling pathways known to regulate growth and proliferation were analyzed with respect to a possible participation of Mbt. mbtP1 induced slight effects in the insulin pathway and strongly influenced the localization of an unknown nucleolar protein. Genetic interactions of mbtP1 with mutations in genes involved in the classical MAPK pathway identified mbt as a positive regulator of the MAPK pathway. A similar effect was also observed with respect to neuroblast proliferation and size. A 2D gel analysis of larval brains identified Bic and Hsp83 as candidate proteins, that might be regulated by Mbt. This work characterizes a novel function of the p21-activated kinase Mbt in neural stem cells. It provides starting points for a detailed analysis of the mechanisms of type II PAK functions in the control of cell growth, proliferation and survival. KW - Taufliege KW - Auge KW - Ontogenie KW - Pilzkörper KW - Molekularbiologie KW - p21-aktivierten Kinase KW - PAK KW - Neuroblast KW - Pilzkörper KW - Drosophila melanogaster KW - p21 activated kinase KW - PAK KW - neuroblast KW - mushroombody KW - Drosophila melanogaster Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85619 ER - TY - THES A1 - Ljaschenko, Dmitrij T1 - Hebbian plasticity at neuromuscular synapses of Drosophila T1 - Hebbsche Plastizität an den neuromuskulären Synapsen in Drosophila melanogaster N2 - Synaptic plasticity determines the development of functional neural circuits. It is widely accepted as the mechanism behind learning and memory. Among different forms of synaptic plasticity, Hebbian plasticity describes an activity-induced change in synaptic strength, caused by correlated pre- and postsynaptic activity. Additionally, Hebbian plasticity is characterised by input specificity, which means it takes place only at synapses, which participate in activity. Because of its correlative nature, Hebbian plasticity suggests itself as a mechanism behind associative learning. Although it is commonly assumed that synaptic plasticity is closely linked to synaptic activity during development, the mechanistic understanding of this coupling is far from complete. In the present study channelrhodopsin-2 was used to evoke activity in vivo, at the glutamatergic Drosophila neuromuscular junction. Remarkably, correlated pre- and postsynaptic stimulation led to increased incorporation of GluR-IIA-type glutamate receptors into postsynaptic receptor fields, thus boosting postsynaptic sensitivity. This phenomenon is input-specific. Conversely, GluR-IIA was rapidly removed from synapses at which neurotransmitter release failed to evoke substantial postsynaptic depolarisation. This mechanism might be responsible to tame uncontrolled receptor field growth. Combining these results with developmental GluR-IIA dynamics leads to a comprehensive physiological concept, where Hebbian plasticity guides growth of postsynaptic receptor fields and sparse transmitter release stabilises receptor fields by preventing overgrowth. Additionally, a novel mechanism of retrograde signaling was discovered, where direct postsynaptic channelrhodopsin-2 based stimulation, without involvement of presynaptic neurotransmitter release, leads to presynaptic depression. This phenomenon is reminiscent of a known retrograde homeostatic mechanism, of inverted polarity, where neurotransmitter release is upregulated, upon reduction of postsynaptic sensitivity. N2 - Das Phänomen der synaptischen Plastizität bestimmt die Entwicklung funktionaler neuronaler Schaltkreise. Die meisten Neurowissenschaftler betrachten synaptische Plastizität als die neuronal Grundlage von Lernen und Gedächtnis. Es gibt viele Ausprägungsarten synaptischer Plastizität, eine davon ist die sogenannte Hebb’sche Plastizität. Diese ist definiert durch eine aktivitätsinduzierte, langanhaltende Veränderung der Stärke einer synaptischen Verbindung, verursacht durch korrelative Aktivierung der Prä- und der Postsynapse. Zusätzlich ist die Ausbreitung der Hebb’sche Plastizität synapsenspezifisch, d.h. nur die Synapsen, die an der korrelativen Aktivierung teilnehmen, erfahren auch die Veränderung. Das Wachstumssignal breitet sich also nicht auf benachbarte Synapsen aus. Der korrelative Wesenszug der Hebb’schen Plastizität macht sie zu einem naheliegenden zellulären Mechanismus assoziativen Lernens. Es wird angenommen, dass synaptische Aktivität und synaptische Plastizität während der Entwicklung neuronaler Schaltkreise eng gekoppelt sind. Das mechanistische Verständnis dieser Kopplung ist jedoch weitgehend unverstanden. In der vorliegenden Arbeit wurde das lichtaktivierbare Kanalrhodopsin-2 verwendet, um Aktivität an der glutamatergen neuromuskulären Synapse in der lebenden, sich frei bewegenden, Drosophila melanogaster Larve auszulösen. Wenn die Prä- und die Postsynapse korrelativ aktiviert wurden, führte dies zur verstärkten Integration von Glutamatrezeptoren des GluR-IIA Typs in die postsynaptischen Rezeptorfelder, was in einer Erhöhung der postsynaptischer Empfindlichkeit mündete. Dieses Platizitätsphänomen wurde als synapsenspezifisch identifiziert und damit als Hebb’sch. Im Gegenzug, wurde der gleiche Rezeptortyp entfernt, wenn Neurotransmitterfreisetzung nicht zu einer erheblichen Depolarisation der Postsynapse führte. Dieser Mechanismus könnte für die Kontrolle des Rezeptorfeldwachstums verantwortlich sein. Es wurde ein physiologisches Modell erarbeitet, bei dem Hebb’sche Plastizität das Wachstum postsynaptischer Rezeptorfelder während der Entwicklung leitet und sporadische, nicht synchronisierte Neurotransmitterfreisetzung die Rezeptorfeldgröße stabilisiert, indem sie das Wachstum Dieser begrenzt. Zusätzlich wurde eine neue Modalität der synaptischen Plastizität an der neuromuskulären Synapse entdeckt: Ein retrograder Signalweg wird aktiviert wenn die postsynaptische Seite, unter Umgehung der Präsynapse, direkt, lichtinduziert aktiviert wird. Dieser Signalweg führt zur präsynaptischen Depression. Das Phänomen erinnert stark an einen bereits bekannten retrograden homöostatischen Mechanismus, reziproker Polarität, bei dem Neurotransmitter Freisetzung hochreguliert wird, wenn die Empfindlichkeit der Postsynapse verringert wird. KW - Synapse KW - Hebbian plasticity KW - synapse KW - Drosophila KW - Plastizität KW - Hebbsche Lernregel KW - Taufliege Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-90465 ER - TY - THES A1 - Link, Jana T1 - The role of meiotic nuclear envelope components in chromosome dynamics and meiotic progression T1 - Die Rolle meiotischer Kernhüllenkomponenten für Chromosomendynamiken und den meiotischen Ablauf N2 - Meiosis is the specialised cell division which produces haploid germ cells, capable of developing into fertile gametes, from diploid progenitor cells. During meiosis, chromosomes undergo strictly regulated and strongly conserved dynamic processes, at the beginning of which the telomeres are actively tethered and intimately attached to the nuclear envelope (NE). The attached telomeres are then moved within the NE through cytoskeletal forces to cluster within a restricted region, forming the highly conserved bouquet stage. Subsequently, the bouquet is released simultaneously to the completion of the synaptonemal complex assembly tightly linking homologous chromosome pairs together. In combination these processes are essential for the successful completion of meiosis. Because the meiotic NE serves as a platform for telomere attachment and movement it can be assumed to be critically involved in these events crucial for fertility. However, the precise roles of many meiotic NE proteins in the attachment and movement of telomeres still remain elusive. Therefore, it was the aim of this thesis to investigate the functions of two mammalian meiotic NE components in telomere attachment and dynamics. The first part of this thesis is concerned with the meiosis-specific lamin C2. Lamin C2 is the only A-type lamin expressed during meiosis and has in previous studies shown to feature altered meiosis-specific properties, clearly distinguishing it from somatic lamins. Because lamin C2 is enriched at sites of telomere attachment, exhibits a high mobility within the nuclear lamina and influences NE integrity, it has been postulated that it may locally increase NE flexibility to allow efficient meiotic telomere movement. Therefore, possible functions of lamin C2 in the movement of attached telomeres were investigated in this thesis by studying the bouquet formation and release of pubertal mice specifically lacking lamin C2. This revealed that lamin C2 deficient mice show a delayed bouquet release, leading to severe defects in the synaptic pairing of homologous chromosomes, which in turn results in infertility of the males. Therefore, the efficient repositioning of attached meiotic telomeres, facilitated by lamin C2, seems essential for completing meiosis. The second part of this thesis focuses on the protein complex responsible for the attachment of meiotic telomeres to the NE and their coupling to the cytoskeleton. The so-called LINC complex is composed of SUN domain proteins in the inner nuclear membrane interacting with KASH domain proteins of the outer nuclear membrane. In previous studies it had been shown that SUN1, SUN2 and KASH5 localise to the attached meiotic telomeres. Regarding the meiotic role of SUN2, however, contradicting results have recently been discussed, showing the need for further investigations. Using an available SUN1 deficient mouse strain, this thesis was able to show that SUN2 is sufficient for telomere attachment per se although telomere attachment is impaired in SUN1 deficient mice leading to infertility. It is also demonstrated that SUN2 forms a functional LINC complex together with KASH5 to mediate this telomere attachment. This LINC complex in the absence of SUN1 is able to move attached telomeres into a bouquet-like cluster formation. Therefore, this demonstrates that SUN2 is involved in the functional attachment and movement of meiotic telomeres. In summary, this thesis has shown SUN2 and the meiotic nuclear lamina to be directly involved in or essential for the highly conserved attachment and movement of telomeres, making them critical for a successful meiosis. The meiotic NE is therefore in this thesis demonstrated to be a determinant of mammalian fertility. N2 - Die Meiose, eine spezialisierte Zellteilung, produziert aus diploiden Vorläuferzellen haploide Keimzellen, welche sich zu befruchtungsfähigen Gameten entwickeln können. Chromosomen durchlaufen während der Meiose stark regulierte, evolutionär hochkonservierte Bewegungen. Zunächst werden die Telomere aktiv und stabil an der Kernhülle verankert. Angeheftete Telomere werden durch das Cytoskelett entlang der Kernhülle bewegt um sich in einer begrenzten Region anzureichern und das chromosomale Bouquet bilden. Das Bouquet wird durch gerichtete Telomerbewegungen anschließend wieder aufgelöst während die finalen Schritte des Zusammenbaus des Synaptonemalkomplexes stattfinden. Diese Prozesse sind in ihrer Summe essentiell für den erfolgreichen Ablauf der Meiose. Da die meiotische Kernhülle als Plattform für die Anheftung und Bewegung der Telomere dient, kann angenommen werden, dass sie in diese für die Fertilität kritischen Prozesse involviert ist. Die genaue Funktion von Kernhüllenproteinen in der Anheftung und Bewegung meiotischer Telomere ist trotzdem zu großen Teilen noch unverstanden. Deshalb war es Ziel dieser Arbeit zwei Komponenten der meiotische Kernhülle und deren Rolle in Telomeranheftung und –dynamik zu untersuchen. Der erste Teil dieser Arbeit beschäftigt sich mit dem meiosespezifischen Lamin C2, welches das einzige während der Meiose exprimierte A-typ Lamin ist. Weil Lamin C2 in Bereichen der Telomeranheftung angereichter ist, eine hohe Mobilität in der Kernhülle aufweist und Kernhülleneigenschaften verändern kann, wurde postuliert dass es lokal die Flexibilität der Kernhülle steigern könnte um leichtere Bewegungen der Telomere zu ermöglichen. Demzufolge sollte in dieser Arbeit eine mögliche Rolle von Lamin C2 in der effizienten Bewegung angehefteter Telomere anhand von jungen Lamin C2-defizienten Mäuse untersucht werden. Dies ergab, dass Lamin C2-defiziente Mäuse eine verlangsamte Auflösung des meiotische Bouquets zeigten, was Defekte in der Homologenpaarung verursacht und letztlich zur Infertilität der Männchen führt. Schlussendlich, scheint die effiziente Bewegung angehefteter Telomere, ermöglicht durch Lamin C2, damit essentiell für einen erfolgreichen Ablauf der Meiose. Der zweite Teil dieser Arbeit ist auf einen Proteinkomplex fokussiert welcher für die Anheftung meiotische Telomere und deren Verbindung zum Cytoskelett verantwortlich ist. Dieser LINC complex besteht aus SUN-domänenproteinen der inneren Kernmembran welche mit KASH-domänenproteinen der äußeren Kernmembran interagieren. Aus früheren Studien ist bekannt, dass SUN1, SUN2 und KASH5 an angehefteten Telomeren lokalisieren. Die meiotische Funktion von SUN2, jedoch, wird aktuell anhand widersprüchlicher Ergebnisse diskutiert. Durch die Verwendung SUN1-defizienter Mäuse konnte diese Arbeit zeigen, dass obwohl die partiell unvollständige Telomeranheftung in der Abwesenheit von SUN1 zu Infertilität führt, SUN2 dennoch für Telomeranheftung an sich ausreichend ist. Um diese Anheftung zu vermitteln, bildet SUN2 einen funktionellen LINC complex mit KASH5 welcher angeheftete Telomere in der Abwesenheit von SUN1 in bouquet-ähnliche Konformationen führt. Demzufolge demonstriert diese Arbeit, dass SUN2 an der funktionellen Telomeranheftung und –bewegung beteiligt ist. Zusammenfassend hat diese Arbeit gezeigt, dass SUN2 und die meiotische Lamina in die hochkonservierte Anheftung und Bewegung von Telomeren direkt involviert oder für sie essentiell sind, und somit unabdingbar für eine erfolgreiche Meiose. Damit definiert diese Arbeit die meiotische Kernhülle als eine Determinante für die Fertilität von Säugern. KW - Meiose KW - Kernhülle KW - Meiosis KW - Nuclear envelope Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-83540 ER - TY - THES A1 - Leingärtner, Annette T1 - Combined effects of climate change and extreme events on plants, arthropods and their interactions T1 - Kombinierte Effekte von Klimawandel und Extremereignissen auf Pflanzen, Arthropoden und ihre Interaktionen N2 - I. Global climate change directly and indirectly influences biotic and abiotic components of ecosystems. Changes in abiotic ecosystem components caused by climate change comprise temperature increases, precipitation changes and more frequently occurring extreme events. Mediated by these abiotic changes, biotic ecosystem components including all living organisms will also change. Expected changes of plants and animals are advanced phenologies and range shifts towards higher latitudes and altitudes which presumably induce changes in species interactions and composition. Altitudinal gradients provide an optimal opportunity for climate change studies, because they serve as natural experiments due to fast changing climatic conditions within short distances. In this dissertation two different approaches were conducted to reveal species and community responses to climate change. First, species richness and community trait analyses along an altitudinal gradient in the Bavarian Alps (chapters II, III) and second, climate change manipulation experiments under different climatic contexts (chapters IV, V, IV). II. We performed biodiversity surveys of butterfly and diurnal moth species on 34 grassland sites along an altitudinal gradient in the National Park Berchtesgaden. Additionally, we analysed the dominance structure of life-history traits in butterfly assemblages along altitude. Species richness of butterflies and diurnal moths decreased with increasing altitude. The dominance of certain life-history-traits changed along the altitudinal gradient with a higher proportion of larger-winged species and species with higher egg numbers towards higher altitudes. However, the mean egg maturation time, population density and geographic distribution within butterfly assemblages decreased with increasing altitude. Our results indicate that butterfly assemblages were mainly shaped by environmental filtering. We conclude that butterfly assemblages at higher altitudes will presumably lack adaptive capacity to future climatic conditions, because of specific trait combinations. III. In addition to butterfly and diurnal moth species richness we also studied plant species richness in combination with pollination type analyses along the altitudinal gradient. The management type of the alpine grasslands was also integrated in the analyses to detect combined effects of climate and management on plant diversity and pollination type. Plant species richness was highest at intermediate altitudes, whereby the management type influenced the plant diversity with more plant species at grazed compared to mown or non-managed grasslands. The pollination type was affected by both the changing climate along the gradient and the management type. These results suggest that extensive grazing can maintain high plant diversity along the whole altitudinal gradient. With ongoing climate change the diversity peak of plants may shift upwards, which can cause a decrease in biodiversity due to reduced grassland area but also changes in species composition and adaptive potential of pollination types. IV. We set up manipulation experiments on 15 grassland sites along the altitudinal gradient in order to determine the combined effects of extreme climatic events (extreme drought, advanced and delayed snowmelt) and elevation on the nutritional quality and herbivory rates of alpine plants. The leaf CN (carbon to nitrogen) ratio and the plant damage through herbivores were not significantly affected by the simulated extreme events. However, elevation influenced the CN ratios and herbivory rates of alpine plants with contrasting responses between plant guilds. Furthermore, we found differences in nitrogen concentrations and herbivory rates between grasses, legumes and forbs, whereas legumes had the highest nitrogen concentrations and were damaged most. Additionally, CN ratios and herbivory rates increased during the growing season, indicating a decrease of food plant quality during the growing season. Contrasting altitudinal responses of grasses, legumes and forbs presumably can change the dominance structure among these plant guilds with ongoing climate change. V. In this study we analysed the phenological responses of grassland species to an extreme drought event, advanced and delayed snowmelt along the altitudinal gradient. Advanced snowmelt caused an advanced beginning of flowering, whereas this effect was more pronounced at higher than at lower altitudes. Extreme drought and delayed snowmelt had rather low effects on the flower phenology and the responses did not differ between higher and lower sites. The strongest effect influencing flower phenology was altitude, with a declining effect through the season. The length of flowering duration was not significantly influenced by treatments. Our data suggest that plant species at higher altitudes may be more affected by changes in snowmelt timing in contrast to lowland species, as at higher altitudes more severe changes are expected. However, the risk of extreme drought events on flowering phenology seems to be low. VI. We established soil-emergence traps on the advanced snowmelt and control treatment plots in order to detect possible changes in abundances and emergence phenologies of five arthropod orders due to elevation and treatment. Additionally, we analysed the responses of Coleoptera species richness to elevation and treatment. We found that the abundance and species richness of Coleoptera increased with elevation as well as the abundance of Diptera. However, the abundance of Hemiptera decreased with elevation and the abundances of Araneae and Hymenoptera showed no elevational patterns. The advanced snowmelt treatment increased the abundances of Araneae and Hymenoptera. The emergence of soil-hibernating arthropods was delayed up to seven weeks at higher elevations, whereas advanced snowmelt did not influence the emergence phenology of arthropods immediately after snowmelt. With climate change earlier snowmelt will occur more often, which especially will affect soil-hibernating arthropods in alpine regions and may cause desynchronisations between species interactions. VII. In conclusion, we showed that alpine ecosystems are sensitive towards changing climate conditions and extreme events and that many alpine species in the Bavarian Alps are endangered. Many alpine species could exist under warmer climatic conditions, however they are expected to be outcompeted by more competitive lowland species. Furthermore, host-parasite or predator-prey interactions can be disrupted due to different responses of certain guilds to climate change. Understanding and predicting the complex dynamics and potential risks of future climate change remains a great challenge and therefore further studies analysing species and community responses to climate change are needed. N2 - I. Der globale Klimawandel beeinflusst direkt und indirekt biotische und abiotische Komponenten der Ökosysteme. Durch Klimawandel verursachte Veränderungen in den abiotischen Komponenten der Ökosysteme umfassen Temperaturanstiege, Veränderungen im Niederschlag und häufiger auftretende Extremereignisse. Als Folge dieser abiotischen Veränderungen, werden sich auch die biotischen Komponenten der Ökosysteme, die alle lebenden Organismen einschließen, verändern. Voraussichtliche Veränderungen bei Pflanzen und Tieren sind vorverlegte Phänologien und Verbreitungsverschiebungen in Richtung höherer Breitengrade und Höhenlagen, was möglicherweise Veränderungen von Interaktionen zwischen Arten und in der Artzusammensetzung verursacht. Höhengradienten bieten durch sich schnell verändernde klimatische Bedingungen innerhalb kurzer Distanzen eine optimale Möglichkeit für Klimawandelstudien im Freiland. In dieser Dissertation wurden zwei unterschiedliche Versuchsansätze genutzt, um die Reaktionen von Arten und Artengemeinschaften auf den Klimawandel zu untersuchen: erstens Analysen zum Artenreichtum und zu Merkmalen innerhalb von Artengemeinschaften entlang eines Höhengradienten in den Bayerischen Alpen (Kapitel II, III) und zweitens Manipulationsexperimente zur Simulation von Klimawandel bei unterschiedlichen klimatischen Bedingungen (Kapitel IV, V, VI). II. Wir haben Biodiversitätsaufnahmen von Schmetterlings- und tagaktiven Nachtfalterarten entlang eines Höhengradienten im Nationalpark Berchtesgaden durchgeführt. Zusätzlich haben wir die Dominanzstruktur von Life-History-Merkmalen in Schmetterlingsgesellschaften entlang des Höhengradienten analysiert. Der Artenreichtum von Schmetterlingen und tagaktiven Nachtfaltern nahm mit zunehmender Höhe ab. Die Dominanz von bestimmten Life-History-Merkmalen veränderte sich entlang des Höhengradienten. Zum Beispiel fanden wir einen höheren Anteil an Arten mit größeren Flügeln und eine größere Anzahl an Eiern in höheren Lagen. Die mittlere Eireifezeit, Populationsdichte und geographische Verbreitung von Schmetterlingsgesellschaften nahm mit steigender Höhe ab. Unsere Ergebnisse deuten darauf hin, dass Schmetterlingsgesellschaften hauptsächlich durch den Filtereffekt der Umwelt geformt werden. Wir schlussfolgern, dass sich bestimmte Merkmalskombinationen von Schmetterlingsgesellschaften in höheren Lagen möglicherweise ungünstig auf die Anpassungskapazität an zukünftige klimatische Veränderungen auswirken. III. Zusätzlich zum Artenreichtum von Schmetterlingen und tagaktiven Nachtfaltern haben wir auch den Artenreichtum von Pflanzen in Kombination mit Analysen zu Bestäubungstypen entlang des Höhengradienten untersucht. Die Bewirtschaftungsform der alpinen Grasländer wurde in die Analysen integriert, um kombinierte Auswirkungen von Klima und Bewirtschaftungsform auf die Pflanzendiversität und den Bestäubungstyp zu erfassen. Der Artenreichtum von Pflanzen war auf mittleren Höhen am größten, wobei die Bewirtschaftungsform die Pflanzendiversität beeinflusste. Es kamen mehr Pflanzenarten auf beweideten im Vergleich zu gemähten oder nicht bewirtschafteten Wiesen vor. Der Bestäubungstyp wurde sowohl durch das sich verändernde Klima entlang des Gradienten als auch durch die Bewirtschaftungsform beeinflusst. Unsere Ergebnisse lassen vermuten, dass extensive Beweidung eine hohe Pflanzendiversität entlang des gesamten Höhengradienten erhalten kann. Mit fortschreitendem Klimawandel könnte sich der Bereich mit höchster Pflanzendiversität nach oben verschieben, was zu einem Biodiversitätsverlust durch eine Abnahme an Grasflächen führen könnte, aber auch zu Veränderungen in der Artenzusammensetzung und dem Anpassungspotential von Bestäubungstypen. IV. Wir simulierten Extremereignisse (extreme Dürre, frühere und spätere Schneeschmelze) auf 15 Grasflächen entlang des Höhengradienten, um kombinierte Effekte von extremen klimatischen Ereignissen und Höhenlage auf die Futterqualität und den Blattfraß von alpinen Pflanzen zu untersuchen. Das Verhältnis von Kohlenstoff zu Stickstoff (CN) in Blättern und die Fraßschäden durch Pflanzenfresser wurden durch die simulierten Extremereignisse nicht signifikant beeinflusst. Dagegen beeinflusste die Höhenlage das CN-Verhältnis und die Herbivorieraten von alpinen Pflanzen mit entgegengesetzten Reaktionen unter den Pflanzengruppen. Des Weiteren haben wir Unterschiede in den Stickstoffkonzentrationen und Herbivorieraten zwischen Gräsern, Leguminosen und krautigen Pflanzen gefunden, wobei die Leguminosen die höchsten Stickstoffkonzentrationen aufwiesen und am stärksten angefressen waren. Zusätzlich stiegen die CN-Verhältnisse und die Fraßschäden während der Vegetationsperiode an, was auf eine Abnahme der Futterqualität im Verlauf der Vegetationsperiode hindeutet. Entgegengesetzte Muster von Gräsern, Leguminosen und krautigen Pflanzen über die Höhe können möglicherweise die Dominanzstruktur zwischen diesen Pflanzengruppen mit fortschreitendem Klimawandel verändern. V. In dieser Studie haben wir die phänologischen Reaktionen von Graslandarten auf ein extremes Dürreereignis, eine frühere und eine spätere Schneeschmelze entlang des Höhengradienten, analysiert. Die frühere Schneeschmelze bewirkte einen früheren Blühbeginn, wobei dieser Effekt auf höheren Lagen ausgeprägter war als auf tieferen Lagen. Extreme Dürre und spätere Schneeschmelze hatten eher geringe Auswirkungen auf die Blühphänologie und die Auswirkungen unterschieden sich nicht zwischen höher und tiefer gelegenen Flächen. Am stärksten würde die Blühphänologie von der Höhenlage beeinflusst wobei sich der Effekt im Verlauf der Vegetationsperiode verringerte. Die Länge der Blühdauer wurde durch die Simulationen nicht signifikant beeinflusst. Unsere Ergebnisse deuten darauf hin, dass Pflanzenarten in höheren Lagen stärker durch Veränderungen des Zeitpunktes der Schneeschmelze beeinflusst werden als Tieflandarten, weil in höheren Lagen stärkere Veränderungen erwartet werden. Das Risiko von extremer Dürre für die Blühphänologie scheint aber gering zu sein. VI. Wir untersuchten Effekte der Höhenlage und früherer Schneeschmelze auf Häufigkeiten und Schlupfphänologien fünf verschiedener Arthropodenordnungen. Dazu installierten wir Bodenphotoeklektoren auf Flächen mit früherer Schneeschmelze und Kontrollflächen. Außerdem analysierten wir die Auswirkungen der Höhenlage und der früheren Schneeschmelze auf den Artenreichtum von Coleoptera. Wir stellten fest, dass die Abundanz und der Artenreichtum von Coleoptera sowie die Abundanz der Diptera mit steigender Höhenlage zunahmen, während die Abundanz der Hemiptera mit steigender Höhe abnahm. Araneae und Hymenoptera zeigten keine Abundanzmuster entlang des Höhengradienten. Eine simulierte frühere Schneeschmelze ließ die Abundanz der Araneae und Hymenoptera ansteigen. Arthropoden, die im Boden überwinterten, schlüpften in höheren Lagen bis zu sieben Wochen später. Eine frühere Schneeschmelze beeinflusste die Schlupfphänologie der Arthropoden unmittelbar nach der Schneeschmelze jedoch nicht. Aufgrund des Klimawandels wird eine frühere Schneeschmelze häufiger auftreten, was vor allem Auswirkungen auf bodenüberwinternde Arthropoden in der Alpenregion haben kann und zu Desynchronisationen mit interagierenden Arten führen kann. VII. Abschließend lässt sich sagen, dass alpine Ökosysteme sensibel auf klimatische Veränderungen und Extremereignisse reagieren und dass viele alpine Arten in den Bayerischen Alpen gefährdet sind. Viele alpine Arten könnten unter wärmeren klimatischen Bedingungen existieren, aber vermutlich werden sie von konkurrenzstärkeren Tieflandarten verdrängt. Des Weiteren können Wirt-Parasit oder Räuber-Beute Interaktionen durch unterschiedliche Reaktionen von bestimmten Gruppen auf Klimawandel gestört werden. Es bleibt eine große Herausforderung die komplexen Dynamiken und möglichen Gefahren des zukünftigen Klimawandels zu verstehen und vorherzusagen. Wir empfehlen weitere Studien, die die Auswirkungen des Klimawandels auf Arten und Artengesellschaften untersuchen. KW - Insekten KW - Klimaänderung KW - Pflanzen KW - Höhenstufe KW - climate change KW - insects KW - altitudinal gradient KW - extreme events KW - Klimawandel KW - Insekten KW - Höhengradient KW - Extremereignisse Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-87758 ER - TY - JOUR A1 - Kozjak‑Pavlovic, Vera A1 - Ott, Christine A1 - Utech, Mandy A1 - Goetz, Monika A1 - Rudel, Thomas T1 - Requirements for the import of neisserial Omp85 into the outer membrane of human mitochondria JF - Bioscience Reports N2 - β-Barrel proteins are present only in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria. Fungal mitochondria were shown to readily import and assemble bacterial β-barrel proteins, but human mitochondria exhibit certain selectivity. Whereas enterobacterial β-barrel proteins are not imported, neisserial ones are. Of those, solely neisserial Omp85 is integrated into the outer membrane of mitochondria. In this study, we wanted to identify the signal that targets neisserial β-barrel proteins to mitochondria. We exchanged parts of neisserial Omp85 and PorB with their Escherichia coli homologues BamA and OmpC. For PorB, we could show that its C-terminal quarter can direct OmpC to mitochondria. In the case of Omp85, we could identify several amino acids of the C-terminal β-sorting signal as crucial for mitochondrial targeting. Additionally, we found that at least two POTRA (polypeptide-transport associated) domains and not only the β-sorting signal of Omp85 are needed for its membrane integration and function in human mitochondria. We conclude that the signal that directs neisserial β-barrel proteins to mitochondria is not conserved between these proteins. Furthermore, a linear mitochondrial targeting signal probably does not exist. It is possible that the secondary structure of β-barrel proteins plays a role in directing these proteins to mitochondria. KW - β-barrel KW - mitochondrion KW - Omp85 KW - PorB KW - POTRA domain Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96381 ER - TY - JOUR A1 - Kneitz, Burkhard A1 - Kalogirou, Charis A1 - Spahn, Martin A1 - Krebs, Markus A1 - Joniau, Steven A1 - Lerut, Evelyne A1 - Burger, Maximilian A1 - Scholz, Claus-Jürgen A1 - Kneitz, Susanne A1 - Riedmiller, Hubertus T1 - MiR-205 Is Progressively Down-Regulated in Lymph Node Metastasis but Fails as a Prognostic Biomarker in High-Risk Prostate Cancer JF - International Journal of Molecular Sciences N2 - The treatment of high-risk prostate cancer (HRPCa) is a tremendous challenge for uro-oncologists. The identification of predictive moleculobiological markers allowing risk assessment of lymph node metastasis and systemic progression is essential in establishing effective treatment. In the current study, we investigate the prognostic potential of miR-205 in HRPCa study and validation cohorts, setting defined clinical endpoints for both. We demonstrate miR-205 to be significantly down-regulated in over 70% of the HRPCa samples analysed and that reconstitution of miR-205 causes inhibition of proliferation and invasiveness in prostate cancer (PCa) cell lines. Additionally, miR-205 is increasingly down-regulated in lymph node metastases compared to the primary tumour indicating that miR-205 plays a role in migration of PCa cells from the original location into extraprostatic tissue. Nevertheless, down-regulation of miR-205 in primary PCa was not correlated to the synchronous presence of metastasis and failed to predict the outcome for HRPCa patients. Moreover, we found a tendency for miR-205 up-regulation to correlate with an adverse outcome of PCa patients suggesting a pivotal role of miR-205 in tumourigenesis. Overall, we showed that miR-205 is involved in the development and metastasis of PCa, but failed to work as a useful clinical biomarker in HRPCa. These findings might have implications for the use of miR-205 as a prognostic or therapeutic target in HRPCa. KW - high-risk prostate cancer KW - microRNA KW - miR-205 KW - prognosis KW - biomarker Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97321 ER - TY - JOUR A1 - Klamp, Tobias A1 - Camps, Marta A1 - Nieto, Benjamin A1 - Guasch, Francesc A1 - Ranasinghe, Rohan T. A1 - Wiedemann, Jens A1 - Petrášek, Zdeněk A1 - Schwille, Petra A1 - Klenerman, David A1 - Sauer, Markus T1 - Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay JF - Scientific Reports N2 - There is an urgent need for rapid and highly sensitive detection of pathogen-derivedDNAin a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp targetDNA fragments of Micrococcus luteus in small sample volumes of 20 mL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of,5 pMand a linear detection range spanning three orders of magnitude. KW - laboratory techniques and procedures KW - diseases KW - infectious diseases KW - assay systems Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130500 VL - 3 IS - 1852 ER - TY - JOUR A1 - Keller, Alexander A1 - Grimmer, Gudrun A1 - Steffan-Dewenter, Ingolf T1 - Diverse Microbiota Identified in Whole Intact Nest Chambers of the Red Mason Bee Osmia bicornis (Linnaeus 1758) JF - PLoS One N2 - Microbial activity is known to have profound impact on bee ecology and physiology, both by beneficial and pathogenic effects. Most information about such associations is available for colony-building organisms, and especially the honey bee. There, active manipulations through worker bees result in a restricted diversity of microbes present within the colony environment. Microbial diversity in solitary bee nests remains unstudied, although their larvae face a very different situation compared with social bees by growing up in isolated compartments. Here, we assessed the microbiota present in nests and pre-adults of Osmia bicornis, the red mason bee, by culture-independent pyrosequencing. We found high bacterial diversity not comparable with honey bee colonies. We identified a variety of bacteria potentially with positive or negative interactions for bee larvae. However, most of the other diverse bacteria present in the nests seem to originate from environmental sources through incorporated nest building material and stored pollen. This diversity of microorganisms may cause severe larval mortality and require specific physiological or symbiotic adaptations against microbial threats. They may however also profit from such a diverse environment through gain of mutualistic partners. We conclude that further studies of microbiota interaction in solitary bees will improve the understanding of fitness components and populations dynamics. KW - bacteria KW - bacterial pathogens KW - bees KW - gut bacteria KW - honey bees KW - larvae KW - Pollen KW - Polymerase chain reaction Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97305 ER - TY - JOUR A1 - Kato, Hiroki A1 - Lu, Qiping A1 - Rapaport, Doron A1 - Kozjak-Pavlovic, Vera T1 - Tom70 Is Essential for PINK1 Import into Mitochondria JF - PLoS ONE N2 - PTEN induced kinase 1 (PINK1) is a serine/threonine kinase in the outer membrane of mitochondria (OMM), and known as a responsible gene of Parkinson's disease (PD). The precursor of PINK1 is synthesized in the cytosol and then imported into the mitochondria via the translocase of the OMM (TOM) complex. However, a large part of PINK1 import mechanism remains unclear. In this study, we examined using cell-free system the mechanism by which PINK1 is targeted to and assembled into mitochondria. Surprisingly, the main component of the import channel, Tom40 was not necessary for PINK1 import. Furthermore, we revealed that the import receptor Tom70 is essential for PINK1 import. In addition, we observed that although PINK1 has predicted mitochondrial targeting signal, it was not processed by the mitochondrial processing peptidase. Thus, our results suggest that PINK1 is imported into mitochondria by a unique pathway that is independent of the TOM core complex but crucially depends on the import receptor Tom70. KW - binding KW - outer-membrane proteins KW - Parkinsons diesease KW - intracellular membranes KW - quality control KW - pathway KW - recruitment KW - biogenesis KW - mechanisms KW - complex Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131061 VL - 8 IS - 3 ER - TY - JOUR A1 - Karl, Stefan A1 - Dandekar, Thomas T1 - Jimena: Efficient computing and system state identification for genetic regulatory networks JF - BMC Bioinformatics N2 - Background: Boolean networks capture switching behavior of many naturally occurring regulatory networks. For semi-quantitative modeling, interpolation between ON and OFF states is necessary. The high degree polynomial interpolation of Boolean genetic regulatory networks (GRNs) in cellular processes such as apoptosis or proliferation allows for the modeling of a wider range of node interactions than continuous activator-inhibitor models, but suffers from scaling problems for networks which contain nodes with more than ~10 inputs. Many GRNs from literature or new gene expression experiments exceed those limitations and a new approach was developed. Results: (i) As a part of our new GRN simulation framework Jimena we introduce and setup Boolean-tree-based data structures; (ii) corresponding algorithms greatly expedite the calculation of the polynomial interpolation in almost all cases, thereby expanding the range of networks which can be simulated by this model in reasonable time. (iii) Stable states for discrete models are efficiently counted and identified using binary decision diagrams. As application example, we show how system states can now be sampled efficiently in small up to large scale hormone disease networks (Arabidopsis thaliana development and immunity, pathogen Pseudomonas syringae and modulation by cytokinins and plant hormones). Conclusions: Jimena simulates currently available GRNs about 10-100 times faster than the previous implementation of the polynomial interpolation model and even greater gains are achieved for large scale-free networks. This speed-up also facilitates a much more thorough sampling of continuous state spaces which may lead to the identification of new stable states. Mutants of large networks can be constructed and analyzed very quickly enabling new insights into network robustness and behavior. KW - Boolean function KW - genetic regulatory network KW - interpolation KW - stable state KW - binary decision diagram KW - Boolean tree Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128671 VL - 14 ER - TY - JOUR A1 - Kang, Ji Hyoun A1 - Schartl, Manfred A1 - Walter, Ronald B. A1 - Meyer, Axel T1 - Comprehensive phylogenetic analysis of all species of swordtails and platies (Pisces: Genus Xiphophorus) uncovers a hybrid origin of a swordtail fish, Xiphophorus monticolus, and demonstrates that the sexually selected sword originated in the ancestral lineage of the genus, but was lost again secondarily JF - BMC Evolutionary Biology N2 - Background: Males in some species of the genus Xiphophorus, small freshwater fishes from Meso-America, have an extended caudal fin, or sword - hence their common name "swordtails". Longer swords are preferred by females from both sworded and - surprisingly also, non-sworded (platyfish) species that belong to the same genus. Swordtails have been studied widely as models in research on sexual selection. Specifically, the pre-existing bias hypothesis was interpreted to best explain the observed bias of females in presumed ancestral lineages of swordless species that show a preference for assumed derived males with swords over their conspecific swordless males. However, many of the phylogenetic relationships within this genus still remained unresolved. Here we construct a comprehensive molecular phylogeny of all 26 known Xiphophorus species, including the four recently described species (X. kallmani, X. mayae, X. mixei and X. monticolus). We use two mitochondrial and six new nuclear markers in an effort to increase the understanding of the evolutionary relationships among the species in this genus. Based on the phylogeny, the evolutionary history and character state evolution of the sword was reconstructed and found to have originated in the common ancestral lineage of the genus Xiphophorus and that it was lost again secondarily. Results: We estimated the evolutionary relationships among all known species of the genus Xiphophorus based on the largest set of DNA markers so far. The phylogeny indicates that one of the newly described swordtail species, Xiphophorus monticolus, is likely to have arisen through hybridization since it is placed with the southern platyfish in the mitochondrial phylogeny, but with the southern swordtails in the nuclear phylogeny. Such discordance between these two types of markers is a strong indication for a hybrid origin. Additionally, by using a maximum likelihood approach the possession of the sexually selected sword trait is shown to be the most likely ancestral state for the genus Xiphophorus. Further, we provide a well supported estimation of the phylogenetic relationships between the previously unresolved northern swordtail groups. Conclusions: This comprehensive molecular phylogeny of the entire genus Xiphophorus provides evidence that a second swordtail species, X. monticolus, arose through hybridization. Previously, we demonstrated that X. clemenciae, another southern swordtail species, arose via hybridization. These findings highlight the potential key role of hybridization in the evolution of this genus and suggest the need for further investigations into how hybridization contributes to speciation more generally. KW - parten-offspring conflict KW - introgressive hybridization KW - mitochondrial DNA KW - molecular phylogeny KW - likelihood approach KW - tree selection KW - preexisting bias KW - adaptive radiation KW - evolution KW - poeciliidae Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121853 SN - 1471-2148 VL - 13 IS - 25 ER - TY - THES A1 - Huang, Ting T1 - Vaccinia Virus-mediated Therapy of Solid Tumor Xenografts: Intra-tumoral Delivery of Therapeutic Antibodies T1 - Vaccini-Virus-vermittelte Therapie solider Tumoren: Intra-tumoraler Transport therapeutischer Antikörper N2 - Over the past 30 years, much effort and financial support have been invested in the fight against cancer, yet cancer still represents the leading cause of death in the world. Conventional therapies for treatment of cancer are predominantly directed against tumor cells. Recently however, new treatments options have paid more attention to exploiting the advantage of targeting the tumor stroma instead. Vaccinia virus (VACV) has played an important role in human medicine since the 18th century as a vaccination against smallpox. In our laboratory, the recombinant, replication-competent vaccinia virus, GLV-1h68, was shown to enter, colonize and destroy cancer cells both in cell culture, and in vivo, in xenograft models (Zhang, Yu et al. 2007). In addition, combined therapy of GLV-1h68 and anti-VEGF immunotherapy significantly enhanced antitumor therapy in vivo (Frentzen, Yu et al. 2009). In this study, we constructed several new recombinant VACVs carrying genes encoding different antibodies against fibroblast activation protein (FAP) in stroma (GLV-1h282), nanobody against the extracellular domain of epidermal growth factor receptor (EGFR, GLV-1h442) or antibodies targeting both vascular endothelial growth factor (VEGF) and EGFR (GLV-1h444) or targeting both VEGF and FAP (GLV-1h446). The expression of the recombinant proteins was first verified using protein analytical methods, SDS-gel electrophoresis, Western blot analysis, immunoprecipitation (IP) assays and ELISA assays. The proteins were detected after infection of the cells with the different VACVs and the recombinant proteins purified by affinity adsorption. The purified antibodies were shown to specifically bind to their respective antigens. Secondly, the infection and replication capability of all the virus strains was analyzed in cell culture using several human tumor cell lines (A549, FaDu or DU145), revealing that all the new recombinant VACVs were able to infect cancer cells with comparable efficiency to the parental viruses from which they were derived. Thirdly, the antitumor efficacy of the new recombinant VACVs was evaluated in vivo using several human cancer xenograft models in mice. In A549 and DU145 xenografts, the new recombinant VACVs exhibited an enhanced therapeutic efficacy compared to GLV-1h68 with no change in toxicity in mice. In the FaDu xenograft, treatment with GLV-1h282 (anti-FAP) significantly slowed down the speed of tumor growth compared to GLV-1h68. Additionally, treatment with the recombinant VACVs expressed the various antibodies achieved comparable or superior therapeutic effects compared to treatment with a combination of GLV-1h68 and the commercial therapeutic antibodies, Avastin, Erbitux or both. Next, the virus distribution in tumors and organs of treated mice was evaluated. For most of the viruses, the virus titer in tumors was not signficantly diffferent than GLV-1h68. However, for animals treated with GLV-1h282, the virus titer in tumors was significantly higher than with GLV-1h68. This may be the reason for enhanced antitumor efficacy of GLV-1h282 in vivo. Lastly, the underlying mechanisms of therapeutic antibody-enhanced antitumor effects were investigated by immunohistochemistry. Blood vessels density and cell proliferation in tumors were suppressed after treatment with the antibody-encoded VACVs. The results indicated that the suppression of angiogenesis or cell proliferation in tumors may cause the observed therapeutic effect. In conclusion, the results of the studies presented here support the hypothesis that the treatment of solid tumors with a combination of oncolytic virotherapy and immunotherapy has an additive effect over each treatment alone. Moreover, expression of the immunotherapeutic antibody by the oncolytic VACV locally in the tumor enhances the antitumor effect over systemic treatment with the same antibody. Combined, these results indicate that therapy with oncolytic VACVs expressing-therapeutic antibodies may be a promising approach for the treatment of cancer. N2 - In den letzten 30 Jahren wurde viel Aufwand und finanzielle Unterstützung in den Kampf gegen Krebs investiert, doch das Resultat ist limitiert, da Krebs immer noch die zweithöchste Todesursache in der Welt darstellt. Zusätzlich zu gegenwärtig verwendeten Therapien, die vorwiegend gegen Tumorzellen gerichtet sind, wird neuen Therapien mehr Aufmerksamkeit gewidmet, die stattdessen direkt auf das Tumorstroma zielen. Onkolytische Vaccinia Viren haben seit dem 18ten Jahrhundert als Impfstoff gegen Pocken in der Humanmedizin eine wichtige Rolle gespielt. In unserem Labor hat das rekombinante, replikationskompetente Vaccinia Virus GLV-1h68 gezeigt, dass es in Zellkultur und in Xenograft Modellen in Krebszellen eindringen sowie diese kolonisieren und zerstören kann (Zhang, Yu et al. 2007). Zusätzlich verbessert die kombinierte Therapie von GLV-1h68 und anti-VEGF Immunotherapy signifikant die Antitumortherapie in vivo (Frentzen, Yu et al. 2009). In dieser Studie haben wir mehrere neue rekombinante VACVs konstruiert, die die Gene für verschiedene Antikörper gegen das Fibroblasten Aktivierungs Protein (FAP) im Stroma (GLV-1h282) oder einen Nanobody gegen die extrazelluläre Domäne des Epidermalen Wachstumsfaktor (EGFR; GLV-1h442) kodieren. Ausserdem wurden Viren konstruiert, die eine Ko-Expression von Antikörpern gegen sowohl vaskulären Endothelwachstumsfaktor (VEGF) als auch EGFR (GLV-1h444) oder gegen sowohl VEGF als auch FAP (GLV-1h446) erlauben. Zunächst wurden SDS-Gelelektrophorese, Western Blot Analyse, Immunprezipitation (IP) und ELISA Assays durchgeführt, um die Expression der rekombinanten Proteine in Zellen mit proteinanalytischen Methoden zu untersuchen. Die Proteine waren nach Infektion der Zellen mit den verschiedenen VACVs nachweisbar und wurden mittles des FLAG Tags mit einem IP Kit aufgereinigt. Es konnte gezeigt werden, dass die aufgereinigten Antikörper spezifisch an ihr jeweiliges Antigen binden. Zweitens wurde die Infektion und Replikationsfähigkeit aller Virusstämme in Zellkultur untersucht (A549, FaDu oder DU145) und mit ihrem jeweiligen Ausgangsstamm GLV-1h68, GLV-1h164, GLV-1h282 oder GLV-1h442 verglichen. Die Ergebnisse zeigten, dass alle neuen rekombinanten VACVs Zellen mit vergleichbarer Effizienz infizieren konnten wie ihre Ausgangsstämme. Drittens, um die Antitumoreffizienz der neuen rekombinanten Stämme in vivo zu testen, wurden verschiedene humane Tumor Xenotransplantat-tragende Nacktmäuse mit verschiedenen VACVs behandelt. In A549 und DU145 Xenotransplantaten zeigten die neuen rekombinanten VACVs erhöhte therapeutische Effizienz verglichen mit dem Ausgangsstamm GLV-1h68, ohne Veränderung der Toxizität in Mäusen. Im FaDu Xenotransplantat verursachte die Behandlung mit GLV-1h68 keine Tumorregression, wohingegen die Behandlung mit GLV-1h282 (anti-FAP) die Geschwindigkeit des Tumorwachstums signifikant verlangsamte sowie das Überleben verlängerte. Zusätzlich haben wir herausgefunden, dass die Behandlung mit Antikörpern, die mittels Virus geliefert wurden, einen identischen oder sogar erhöhten inhibitorischen Effekt erzielen können, wie in einer Kombinationstherapie von GLV-1h68 und kommerziell erhältlichen Antikörpern, wie Avastin, Erbitux oder beidem. Um die virale Verteilung in vivo zu untersuchen, wurden Tumore und Organe von Mäusen seziert und homogenisiert, gefolgt von Titration der Virusmenge. Die Virus-Titer in Tumoren waren signifikant höher in Tieren, die mit GLV-1h282 behandelt wurden als solche, die mit GLV-1h68 behandelt wurden. Dies mag den Grund für die erhöhte Antitumoreffizienz von GLV-1h282 in vivo darstellen. Die Virus-Titer in allen anderen Gruppen zeigten keinen signifikanten Unterschied. Um den Mechanismus der durch therapeutische Antikörper erhöhten Antitumortherapie zu untersuchen, wurde Immunohistochemie durchgeführt. Nach Behandlung mit den Antikörper-kodierenden VACVs waren die Blutgefäβdichte und Zellproliferation in Tumoren reduziert, nachgewiesen durch die jeweilige CD31 and Ki67 Färbung. Die Resultate deuteten an, dass die Suppression der Angiogenese oder der Zellproliferation in Tumoren den beobachteten Effekt verursachen könnte. Zusammenfassend zeigen die hier präsentierten Daten dass die Kombination der Behandlung von onkolytischer Virotherapie mit Immunotherapie durch Virus-gelieferte Antikörper einen vielversprechenden Ansatz für Krebstherapie darstellt. KW - Vaccinia-Virus KW - therapeutic antibody KW - oncolytic virus KW - Krebs KW - Therapie KW - Antikörper KW - Tumor Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-91327 ER - TY - JOUR A1 - Holzschuh, Andrea A1 - Dormann, Carsten F. A1 - Tscharntke, Teja A1 - Steffan-Dewenter, Ingolf T1 - Mass-flowering crops enhance wild bee abundance JF - Oecologia N2 - Although agricultural habitats can provide enormous amounts of food resources for pollinator species, links between agricultural and (semi-)natural habitats through dispersal and foraging movements have hardly been studied. In 67 study sites, we assessed the interactions between mass-flowering oilseed rape fields and semi-natural grasslands at different spatial scales, and their effects on the number of brood cells of a solitary cavity-nesting bee. The probability that the bee Osmia bicornis colonized trap nests in oilseed rape fields increased from 12 to 59 % when grassland was nearby, compared to fields isolated from grassland. In grasslands, the number of brood cells of O. bicornis in trap nests was 55 % higher when adjacent to oilseed rape compared to isolated grasslands. The percentage of oilseed rape pollen in the larval food was higher in oilseed rape fields and grasslands adjacent to oilseed rape than in isolated grasslands. In both oilseed rape fields and grasslands, the number of brood cells was positively correlated with the percentage of oilseed rape pollen in the larval food. We show that mass-flowering agricultural habitats—even when they are intensively managed—can strongly enhance the abundance of a solitary bee species nesting in nearby semi-natural habitats. Our results suggest that positive effects of agricultural habitats have been underestimated and might be very common (at least) for generalist species in landscapes consisting of a mixture of agricultural and semi-natural habitats. These effects might also have—so far overlooked—implications for interspecific competition and mutualistic interactions in semi-natural habitats. KW - spillover KW - trap nests KW - pollen KW - oilseed rape KW - canola Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126852 VL - 172 IS - 2 ER - TY - JOUR A1 - Holzschuh, Andrea A1 - Dormann, Carsten F. A1 - Tscharntke, Teja A1 - Steffan-Dewenter, Ingolf T1 - Mass-flowering crops enhance wild bee abundance JF - Oecologia N2 - Although agricultural habitats can provide enormous amounts of food resources for pollinator species, links between agricultural and (semi-)natural habitats through dispersal and foraging movements have hardly been studied. In 67 study sites, we assessed the interactions between mass-flowering oilseed rape fields and semi-natural grasslands at different spatial scales, and their effects on the number of brood cells of a solitary cavity-nesting bee. The probability that the bee Osmia bicornis colonized trap nests in oilseed rape fields increased from 12 to 59 % when grassland was nearby, compared to fields isolated from grassland. In grasslands, the number of brood cells of O. bicornis in trap nests was 55 % higher when adjacent to oilseed rape compared to isolated grasslands. The percentage of oilseed rape pollen in the larval food was higher in oilseed rape fields and grasslands adjacent to oilseed rape than in isolated grasslands. In both oilseed rape fields and grasslands, the number of brood cells was positively correlated with the percentage of oilseed rape pollen in the larval food. We show that mass-flowering agricultural habitats—even when they are intensively managed—can strongly enhance the abundance of a solitary bee species nesting in nearby semi-natural habitats. Our results suggest that positive effects of agricultural habitats have been underestimated and might be very common (at least) for generalist species in landscapes consisting of a mixture of agricultural and semi-natural habitats. These effects might also have—so far overlooked—implications for interspecific competition and mutualistic interactions in semi-natural habitats. KW - spillover KW - pollen KW - oilseed rape KW - canola KW - trap nests Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132149 VL - 172 IS - 2 ER - TY - THES A1 - Hoiß, Bernhard T1 - Effects of climate change, extreme events and management on plants, pollinators and mutualistic interaction networks T1 - Auswirkungen von Klimawandel, Extremereignissen und Management auf Pflanzen, Bestäuber und mutualistische Netzwerke N2 - I. Climate change comprises average temperatures rise, changes in the distribution of precipitation and an increased amount and intensity of extreme climatic events in the last decades. Considering these serious changes in the abiotic environment it seems obvious that ecosystems also change. Flora and fauna have to adapt to the fast changing conditions, migrate or go extinct. This might result in shifts in biodiversity, species composition, species interactions and in ecosystem functioning and services. Mountains play an important role in the research of these climate impacts. They are hotspots of biodiversity and can be used as powerful natural experiments as they provide, within short distances, the opportunity to research changes in the ecosystem induced by different climatic contexts. In this dissertation two approaches were pursued: i) surveys of biodiversity, trait dominance and assembly rules in communities depending on the climatic context and different management regimes were conducted (chapters II and III) and ii) the effects of experimental climate treatments on essential ecosystem features along the altitudinal gradient were assessed (chapters IV, V and VI). II. We studied the relative importance of management, an altitudinal climatic gradient and their interactions for plant species richness and the dominance of pollination types in 34 alpine grasslands. Species richness peaked at intermediate temperatures and was higher in grazed grasslands compared to non-managed grasslands. We found the climatic context and also management to influence the distribution and dominance structures of wind- and insect-pollinated plants. Our results indicate that extensive grazing maintains high plant diversity over the full subalpine gradient. Rising temperatures may cause an upward shift of the diversity peak of plants and may also result in changed species composition and adaptive potential of pollination types. III. On the same alpine grasslands we studied the impact of the climatic context along an altitudinal gradient on species richness and community assembly in bee communities. Species richness and abundance declined linearly with increasing altitude. Bee species were more closely related at high altitudes than at low altitudes. The proportion of social and ground-nesting species, as well as mean body size and altitudinal range of bees, increased with increasing altitude, whereas the mean geographic distribution decreased. Our results suggest that community assembly at high altitudes is dominated by environmental filtering effects, while the relative importance of competition increases at low altitudes. We conclude that ongoing climate change poses a threat for alpine specialists with adaptations to cool environments but low competitive capacities. IV. We determined the impacts of short-term climate events on flower phenology and assessed whether those impacts differed between lower and higher altitudes. For that we simulated advanced and delayed snowmelt as well as drought events in a multi site experiment along an altitudinal gradient. Flower phenology was strongly affected by altitude, however, this effect declined through the season. The manipulative treatments caused only few changes in flowering phenology. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but altitude did not influence the effect of the other treatments. The length of flowering duration was not significantly influenced by treatments. Our data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps. V. Changes in the structure of plant-pollinator networks were assessed along an altitudinal gradient combined with the experimental simulation of potential consequences of climate change: extreme drought events, advanced and delayed snowmelt. We found a trend of decreasing specialisation and therefore increasing complexity in networks with increasing altitude. After advanced snowmelt or drought networks were more specialised especially at higher altitudes compared to control plots. Our results show that changes in the network structures after climate manipulations depend on the climatic context and reveal an increasing susceptibility of plant-pollinator networks with increasing altitude. VI. The aim of this study was to determine the combined effects of extreme climatic events and altitude on leaf CN (carbon to nitrogen) ratios and herbivory rates in different plant guilds. We found no overall effect of climate manipulations (extreme drought events, advanced and delayed snowmelt) on leaf CN ratios and herbivory rates. However, plant guilds differed in CN ratios and herbivory rates and responded differently to altitude. CN ratios of forbs (legume and non-legume) decreased with altitude, whereas CN ratios of grasses increased with altitude. Further, CN ratios and herbivory rates increased during the growing season, indicating a decrease of food plant quality during the growing season. Insect herbivory rates were driven by food plant quality. Contrasting altitudinal responses of forbs versus grasses give reason to expect changed dominance structures among plant guilds with ongoing climate change. VII. This dissertation contributes to the understanding of factors that determine the composition and biotic interactions of communities in different climates. The results presented indicate that warmer climates will not only change species richness but also the assembly-rules for plant and bee communities depending on the species' functional traits. Our investigations provide insights in the resilience of different ecosystem features and processes towards climate change and how this resilience depends on the environmental context. It seems that mutualistic interactions are more susceptible to short-term climate events than flowering phenology and antagonistic interactions such as herbivory. However, to draw more general conclusions more empirical data is needed. N2 - I. Das Klima ändert sich: die Durchschnittstemperaturen steigen, die Niederschlagsverteilung ändert sich und sowohl die Anzahl als auch die Intensität von klimatischen Extremereignissen hat in den letzten Jahrzehnten zugenommen. In Anbetracht dieser beträchtlichen Veränderungen in der abiotischen Umwelt scheint es offensichtlich, dass sich auch die Ökosysteme verändern. Flora und Fauna müssen sich an die sich schnell verändernden Bedingungen anpassen, wandern oder sie sterben aus. Dies kann zu Veränderungen in der Biodiversität, der Artzusammensetzung, den Ökosystemfunktionen sowie von Ökosystemdienstleistungen führen. Gebirge spielen eine wichtige Rolle in der Erforschung dieser Klimafolgen. Sie sind Biodiversitäts-Hotspots und können als großräumige natürliche Experimente genutzt werden, da sie die Möglichkeit bieten, innerhalb kurzer Distanzen Veränderungen im Ökosystem unter verschiedenen klimatischen Bedingungen zu untersuchen. In dieser Dissertation wurden zwei Ansätze verfolgt: i) Es wurden Untersuchungen zur Abhängigkeit von Biodiversität, der Dominanz von funktionalen Merkmalen sowie den Gesetzmäßigkeiten in der Zusammensetzung von Artengemeinschaften vom klimatischen Kontext sowie verschiedenen Management Regimen durchgeführt. ii) Die Effekte von Klimaexperimenten auf essentielle Ökosystemeigenschaften, biotische Interaktionen und Nahrungsnetze entlang eines Höhengradienten wurden untersucht. II. Die relative Bedeutung von Höhenlage, Bewirtschaftungsform sowie ihre Interaktionen für den Artenreichtum von Pflanzen und die Dominanz von Bestäubungstypen wurden in 34 alpinen Wiesen untersucht. Der Artenreichtum erreichte bei mittleren Temperaturen ein Maximum und war auf beweideten Flächen höher als auf nicht bewirtschafteten Wiesen. Wir stellten außerdem fest, dass sowohl der klimatische Kontext als auch die Bewirtschaftungsform die Verteilung und Dominanzstrukturen von wind- und insektenbestäubten Pflanzen beeinflusste. Unsere Ergebnisse zeigen, dass extensive Beweidung eine hohe Artenvielfalt über den gesamten subalpinen Gradienten erhält. Steigende Temperaturen könnten eine Verschiebung des Bereiches mit maximaler Artenvielfalt nach oben sowie veränderte Zusammensetzungen von Artengemeinschaften und Veränderungen in der Bedeutung von Bestäubungstypen als Anpassung verursachen. III. Auf den selben alpinen Wiesen untersuchten wir den Einfluss der klimatischen Gegebenheiten entlang des Höhengradienten auf die Artenzahl und die Gesetzmäßigkeiten in der Zusammensetzung von Wildbienen Artengemeinschaften. Die Artenzahl und Abundanz nahm mit zunehmender Höhe linear ab. Die Bienenarten in höheren Lagen waren näher miteinander verwandt als in niedrigen Lagen. Der Anteil sozialer, im Boden nistender Arten sowie die mittlere Körpergröße und Höhenverbreitung der Bienen nahm mit zunehmender Höhe zu, wohingegen die mittlere geographische Verbreitung der Arten abnahm. Unsere Ergebnisse legen nahe, dass die Zusammensetzung von Artengemeinschaften in höheren Lagen primär vom Filtereffekt der Umwelt bestimmt wird, wohingegen der Einfluss von Konkurrenz in niedrigen Lagen an Bedeutung gewinnt. Wir folgern, dass der fortschreitende Klimawandel eine Gefährdung für alpine Spezialisten darstellt, die zwar Anpassungen an kühle Bedingungen aber oft eine nur geringe Konkurrenzfähigkeit aufweisen. IV. Wir untersuchten die Auswirkung von kurzzeitigen klimatischen Ereignissen auf die Blütenphänologie und analysierten, ob sich diese Auswirkungen zwischen hohen und tiefen Lagen unterscheiden. Dazu simulierten wir verfrühte und verspätete Schneeschmelze sowie Dürreereignisse in Experimenten auf multiplen Standorten entlang eines Höhengradienten. Die Blütenphänologie wurde von der Höhenlage stark beeinflusst, dieser Effekt nahm im Laufe der Saison allerdings ab. Die Manipulationen zeitigten nur wenige Effekte auf die Blühphänologie. Die Auswirkungen von verfrühter Schneeschmelze waren auf hohen Flächen signifikant höher als in niedrigen Lagen, es wurden jedoch keine Unterschiede für die anderen Behandlungen zwischen den Höhenstufen gefunden. Die Blühdauer wurde durch die Behandlungen nicht beeinflusst. Unsere Daten zeigen ein relativ geringes Risiko für die Blütenphänologie durch Dürreereignisse in den bayerischen Alpen auf. V. Veränderungen in der Struktur von Pflanzen-Bestäuber Netzwerken wurden entlang eines Höhengradienten in Kombination mit der experimentellen Simulation von potentiellen Konsequenzen des Klimawandels (extreme Dürre, verfrühte und verspätete Schneeschmelze) untersucht. Wir fanden einen Trend hin zu einem abnehmenden Spezialisierungsgrad und daher einer Zunahme der Komplexität in Netzwerken mit zunehmender Höhe. Die Netzwerke nach verfrühter Schneeschmelze und nach Dürre waren, insbesondere in höheren Lagen, stärker spezialisiert als in den Kontrollflächen. Unsere Ergebnisse zeigen, dass Veränderungen in den Netzwerkstrukturen nach Klimamanipulationen vom klimatischen Zusammenhang abhängen und zeigen auf, dass die Anfälligkeit von Pflanzen-Bestäuber Netzwerken mit der Höhe zunimmt. VI. Das Ziel dieser Studie war es die kombinierten Auswirkungen von kurzzeitigen klimatischen Ereignissen und Meereshöhe auf das CN (Kohlenstoff zu Stickstoff) Verhältnis in Blättern und den Blattfraß in verschiedenen Pflanzengruppen zu untersuchen. Wir fanden keinen Gesamteffekt der Klimamanipulationen (extremes Dürreereignis, verfrühte und verspätete Schneeschmelze) auf das CN Verhältnis und die Herbivorieraten. Die Pflanzengruppen unterschieden sich jedoch in ihrer Reaktion auf die Meereshöhe hinsichtlich ihres CN Verhältnisses und des Blattfraßes. Das CN Verhältnis in Gräsern nahm mit der Höhe zu, wohingegen das CN Verhältnis in den restlichen krautigen Pflanzen mit zunehmender Höhe abnahm. Außerdem nahmen CN Verhältnis und die Herbivorierate im Laufe der Saison zu, was auf eine Abnahme der Futterqualität im Saisonverlauf hindeutet. Die Herbivorieraten wurden von der Futterqualität der Pflanzen bestimmt. Die gegensätzlichen Muster von Gräsern und anderen krautigen Pflanzen über die Höhe lassen veränderte Dominanzstrukturen zwischen Pflanzengruppen mit fortschreitendem Klimawandel zu erwarten. VII. Diese Dissertation leistet einen Beitrag zur Identifikation von Gesetzmäßigkeiten in der Zusammensetzung von Artengemeinschaften unter unterschiedlichen klimatischen Bedingungen. Die präsentierten Ergebnisse weisen darauf hin, dass ein wärmeres Klima nicht nur den Artenreichtum, sondern auch diese Gesetzmäßigkeiten für Pflanzen- und Bienenvergesellschaftungen in Abhängigkeit von den funktionellen Merkmalen der Arten verändern wird. Unsere Untersuchungen liefern Erkenntnisse über die Stabilität verschiedener Ökosystemaspekte und -prozesse gegenüber dem Klimawandel und wie diese Stabilität vom Umweltkontext abhängt. Es scheint, dass mutualistische Interaktionen anfälliger sind für kurzfristige Klimaereignisse als die Phänologie von Blüten oder antagonistische Interaktionen wie die Herbivorie. Um allgemeinere Rückschlüsse ziehen zu können bedarf es jedoch dringend weiterer empirischer Daten. KW - Klimaänderung KW - Alpen KW - Biodiversität KW - Bestäubungsökologie KW - Mutualismus KW - climate change KW - land use KW - altitudinal gradient KW - elevation KW - life history traits KW - bees KW - vascular plants KW - alpine ecosystems KW - environmental filtering KW - Klimawandel KW - Extremereignisse KW - Management KW - Gefäßpflanzen KW - mutualistische Netzwerke KW - Pflanzen-Bestäuber-Interaktionen KW - Höhengradient Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-87919 ER - TY - THES A1 - Hillebrand, Frank T1 - Der Einfluss des PI3-Kinase Signalwegs auf die Regulation des alternativen HIV-1 prä-mRNA Spleißens T1 - The influence of the PI3-kinase pathway on the regulation of of alternative HIV-1 pre-mRNA splicing N2 - In der vorliegenden Arbeit wurden ausgehend von HIV-1 basierten Minigenkonstrukten und der proviralen NL4-3 DNA die Einflüsse der PI3K Signalwegmodulation auf das alternative Spleißen der HIV-1 prä-mRNA sowie auf die Virus Replikation untersucht. Mittels RT-PCR Analysen konnte gezeigt werden, dass die PI3K Inhibition im Falle der HIV-1 basierten Minigenkonstrukte in einer erhöhten Abundanz ungespleißter bzw. intronhaltiger mRNAs resultierte, während im Kontext des Virus die Induktion alternativer Tat Transkriptvarianten nachgewiesen werden konnte. Als Folge der Inhibition des PI3K Signalwegs kam es zu einem vermehrten Einschluss der HIV-1 Leader Exone2/2b und 3. Da der Einschluss dieser Exone durch die hnRNP A/B- und F/H-abhängigen Silencer Elemente ESSV und GI2-1 negativ reguliert wird, wurde vermutet, dass die PI3K Inhibition mit der Funktionalität dieser spleißregulatorischen Aktivität interferiert. Unterstützt wurde diese Hypothese durch Replikationsexperimente mit ESSV und GI2-1 Mutanten in Gegenwart und Abwesenheit des PI3K-Inhibitors. Zusätzlich wurde auch der Einfluss des Inhibitors unter Überexpressionsbedingungen von hnRNP H auf das alternative HIV-1 Spleißen analysiert. In dieser Arbeit konnte ebenfalls gezeigt werden, dass die PI3K Inhibition ein verändertes hnRNP H Spleißmuster bedingt sowie die SR-Protein Phosphorylierung und Expression beeinflusst. Des Weiteren war es im Verlauf der vorliegenden Arbeit möglich, eine Interferenz der PI3K Modulation mit der Virus Replikation nachzuweisen. Die Überexpression der aktivierten Akt-Kinase lies hier nur eine sehr geringe Virus Produktion zu während die PI3K Inhibition diese auf ca. die Hälfte reduzierte. Weiterführende Experimente zeigten, dass die Überexpression der aktivierten Akt-Kinase den nuklearen Export Rev-abhängiger HIV-1 mRNAs zu blockieren scheint. Darüber hinaus beeinflusste die PI3K Inhibition neben dem alternativen HIV-1 Spleißen auch die virale Transkription sowie die zelluläre Translation. Zusammen könnten diese Effekte die reduzierte virale Replikation erklären. Der PI3K Signalweg spielt somit eine zentrale Rolle bei dem alternativen HIV-1 Spleißen und der viralen Replikation und bietet so die Möglichkeit der Entwicklung neuer Ansätze einer antiviralen Therapie.   N2 - In this thesis outgoing from HIV-1 based minigenes and the proviral NL4-3 DNA the influences of the PI3K signaling modulation on the alternative HIV-1 pre-mRNA splicing and also the viral replication were investigated. By performing RT-PCR analysis it could be shown that in the case of the minigene experiments the PI3K inhibition displayed an increased amount of unspliced or intron containing mRNAs, while the production of alternative Tat variants was demonstrated in the context of the virus. As a result of the PI3K inhibition an increased inclusion of the HIV-1 leader exons2/2b and 3 was observed. Because the inclusion of these exons is negatively regulated by the hnRNP H/F- and hnRNP A/B-dependent silencere elements ESSV and GI2-1, it was suggested that the PI3K inhibition interferes with the functionality of this splicing regulatory activity. Replication experiments either with GI2-1 or ESSV mutants in the presence or absence of the PI3K-Inhibitior supported this hypothesis. In addition, the influence of the inhibitor on the alternative HIV-1 splicing was analyzed under hnRNP H overexpression conditions. Furthermore, it was shown that the hnRNP H splicing pattern as well as the SR-protein phosphorylation and expression were altered as a consequence of the PI3K inhibition. During this thesis an interference of the PI3K modulation with the viral replication was also shown. The overexpression of the activated Akt kinase nearly prevented viral production while the PI3K inhibition reduced viral production by half. In further experiments it was shown that the overexpression of the activated Akt kinase seems to block the nuclear export of Rev-dependent HIV-1 mRNAs. In addition, beside the effect on the viral splicing pattern the PI3K inhibition also showed an influence on the viral transcription and the cellular translation suggesting that the sum of all these effects could contribute to the reduced virus production. These findings demonstrate that the PI3K signaling pathway has indeed a central influence on the alternative HIV-1 splicing as well as on the viral replication and may offer a new approach for antiviral therapy. KW - RNS-Spleißen KW - HIV KW - Phosphatidylinositolkinase KW - PI3K/Akt pathway KW - HIV-1 KW - alternative splicing KW - Phosphatidylinositol-3-Kinase KW - PI3K/Akt Signalweg KW - alternatives Spleißen Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76914 ER - TY - THES A1 - Heß, Michael T1 - Vaccinia virus-encoded bacterial beta-glucuronidase as a diagnostic biomarker for oncolytic virotherapy T1 - Vaccinia Virus-codierte bakterielle Beta-Glucuronidase als diagnostischer Biomarker in der onkolytischen Virotherapie N2 - Oncolytic virotherapy represents a promising approach to revolutionize cancer therapy. Several preclinical and clinical trials display the safety of oncolytic viruses as wells as their efficiency against solid tumors. The development of complementary diagnosis and monitoring concepts as well as the optimization of anti-tumor activity are key points of current virotherapy research. Within the framework of this thesis, the diagnostic and therapeutic prospects of beta-glucuronidase expressed by the oncolytic vaccinia virus strain GLV-1h68 were evaluated. In this regard, a beta-glucuronidase-based, therapy-accompanying biomarker test was established which is currently under clinical validation. By using fluorescent substrates, the activity of virally expressed beta-glucuronidase could be detected and quantified. Thereby conclusions about the replication kinetics of oncolytic viruses in animal models and virus-induced cancer cell lysis could be drawn. These findings finally led to the elaboration and establishment of a versatile biomarker assay which allows statements regarding the replication of oncolytic viruses in mice based on serum samples. Besides the analysis of retrospective conditions, this test is able to serve as therapy-accompanying monitoring tool for virotherapy approaches with beta-glucuronidase-expressing viruses. The newly developed assay also served as complement to routinely used plaque assays as well as reference for virally expressed anti-angiogenic antibodies in additional preclinical studies. Further validation of this biomarker test is currently taking place in the context of clinical trials with GL-ONC1 (clinical grade GLV-1h68) and has already shown promising preliminary results. It was furthermore demonstrated that fluorogenic substrates in combination with beta-glucuronidase expressed by oncolytic viruses facilitated the optical detection of solid tumors in preclinical models. In addition to diagnostic purposes, virus-encoded enzymes could also be combined with prodrugs resulting in an improved therapeutic outcome of oncolytic virotherapy. In further studies, the visualization of virus-induced immune reactions as well as the establishment of innovative concepts to improve the therapeutic outcome of oncolytic virotherapy could be accomplished. In conclusion, the results of this thesis provide crucial findings about the influence of virally expressed beta-glucuronidase on various diagnostic concepts in the context of oncolytic virotherapy. In addition, innovative monitoring and therapeutic strategies could be established. Our preclinical findings have important clinical influence, particularly by the development of a therapy-associated biomarker assay which is currently used in different clinical trials. N2 - Onkolytische Viren stellen einen vielversprechenden Therapieansatz dar, der die Behandlung von Krebserkrankungen revolutionieren könnte. Intensive präklinische und klinische Studien zeigen sowohl die körperliche Verträglichkeit von onkolytischen Viren, als auch deren Wirksamkeit gegenüber soliden Tumoren. Die Entwicklung von therapiebegleitenden Diagnose- und Monitoringkonzepten sowie eine Optimierung der Antitumorwirkung onkolytischer Viren stellen Eckpunkte der aktuellen Forschung auf dem Gebiet der Virotherapie dar. Im Rahmen dieser Arbeit wurde untersucht, welche diagnostischen und therapeutischen Möglichkeiten die virale Expression von beta-Glucuronidase durch den onkolytischen Vaccinia-Virus-Stamm GLV-1h68 eröffnet. In diesem Zusammenhang wurde ein, auf beta-Glucuronidase basierender, therapiebegleitender Biomarkertest entwickelt, dessen klinische Validierung derzeit stattfindet. Mit Hilfe von fluorogenen Substraten konnte die Aktivität viral exprimierter beta-Glucuronidase detektiert und quantifiziert werden. Dies lies direkte Rückschlüsse auf das Replikationsverhalten von onkolytischen Viren im Tiermodell zu und ermöglichte zudem Aussagen über die Zelllyse Virus-infizierter Krebszellen. Diese Erkenntnisse führten letztendlich zur Ausarbeitung und Etablierung eines vielseitig anwendbaren Biomarker-Assays, der es ermöglicht anhand von Blutproben Aussagen über das Replikationsverhalten onkolytischer Viren in Mäusen zu machen. Neben retrospektiven Analysen erlaubt dieser Test auch ein therapiebegleitendes Monitoring der onkolytischen Virotherapie mit beta-Glucuronidase-exprimierenden Viren. In weiteren präklinischen Untersuchungen diente der entwickelte Assay zudem als Ergänzung zum viralen Plaque Assays sowie als Referenz für Virus-exprimierte anti-angiogene Antikörper. Eine fortführende Validierung dieses neuartigen Biomarkertests findet derzeit im Rahmen humaner Studien mit der klinischen Formulierung von GLV-1h68, GL-ONC1, statt und zeigte bereits erste positive Resultate. Weiterhin konnte im Rahmen dieser Arbeit gezeigt werden, dass die Expression von beta-Glucuronidase durch onkolytische Viren in Verbindung mit fluoreszierenden Substraten eine optische Detektion von Karzinomen im präklinischen Tiermodell ermöglicht. Neben diagnostischen Zwecken, konnten Virus-kodierte Enzyme in Kombination mit Prodrugs genutzt werden, um den Therapieerfolg der onkolytischen Virotherapie zu verbessern. In zusätzlichen Studien konnten zudem Methoden zur Visualisierung der Virus-induzierten Immunantwort sowie neuartige Konzepte zur Therapieverbesserung etabliert werden. Zusammenfassend liefern die Ergebnisse der vorliegenden Arbeit wichtige Erkenntnisse über den Einfluss Virus-exprimierter beta-Glucuronidase auf unterschiedliche Diagnosekonzepte im Rahmen der onkolytischen Virotherapie. Daneben konnten entscheidende Erkenntnisse über den möglichen Einsatz neuer Monitoring- und Therapieansätze erzielt werden. Insbesondere durch die Entwicklung eines therapiebegleitenden Biomarkertests haben diese Resultate erheblichen Einfluss auf die weitere klinische Anwendung von onkolytischen Vaccinia-Viren. KW - Vaccinia-Virus KW - Glucuronidase KW - Krebs KW - cancer KW - oncolytic virus KW - biomarker KW - beta-glucuronidase Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86789 ER - TY - JOUR A1 - Herpin, Amaury A1 - Adolfi, Mateus C. A1 - Nicol, Barbara A1 - Hinzmann, Maria A1 - Schmidt, Cornelia A1 - Klughammer, Johanna A1 - Engel, Mareen A1 - Tanaka, Minoru A1 - Guiguen, Yann A1 - Schartl, Manfred T1 - Divergent Expression Regulation of Gonad Development Genes in Medaka Shows Incomplete Conservation of the Downstream Regulatory Network of Vertebrate Sex Determination JF - Molecular Biology and Evolution N2 - Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of “master sex-determining genes” at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought. KW - gonadal development KW - divergent expression regulation KW - gene regulatory network evolution KW - adaptive evolution Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132262 VL - 30 IS - 10 ER - TY - JOUR A1 - Hendriksma, Harmen P. A1 - Küting, Meike A1 - Härtel, Stephan A1 - Näther, Astrid A1 - Dohrmann, Anja B. A1 - Steffan-Dewenter, Ingolf A1 - Tebbe, Christoph C. T1 - Effect of Stacked Insecticidal Cry Proteins from Maize Pollen on Nurse Bees (Apis mellifera carnica) and Their Gut Bacteria JF - PLoS ONE N2 - Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees. KW - communities KW - 16S ribosomal-RNA KW - T-RFLP analysis KW - honey bees KW - bacillus thuringiensis KW - risk assessment KW - multivariate analyses KW - worker honeybees KW - corn pollen KW - larvae Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131025 VL - 8 IS - 3 ER -