TY - JOUR A1 - Krajinovic, K. A1 - Reimer, S. A1 - Kudlich, T. A1 - Germer, C. T. A1 - Wiegering, A. T1 - “Rendezvous technique” for intraluminal vacuum therapy of anastomotic leakage of the jejunum JF - Surgical Case Reports N2 - Background Anastomotic leakage (AL) is one of the most common and serious complications following visceral surgery. In recent years, endoluminal vacuum therapy has dramatically changed therapeutic options for AL, but its use has been limited to areas easily accessible by endoscope. Case presentation We describe the first use of endoluminal vacuum therapy in the small intestine employing a combined surgical and endoscopic “rendezvous technique” in which the surgeon assists the endoscopic placement of an endoluminal vacuum therapy sponge in the jejunum by means of a pullback string. This technique led to a completely closed AL after 27 days and 7 changes of the endosponge. Conclusion The combined surgical and endoscopic rendezvous technique can be useful in cases of otherwise difficult endosponge placement. KW - endosponge KW - anastomotic leakage Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147883 VL - 2 IS - 114 ER - TY - JOUR A1 - Shen, Yingjia A1 - Chalopin, Domitille A1 - Garcia, Tzintzuni A1 - Boswell, Mikki A1 - Boswell, William A1 - Shiryev, Sergey A. A1 - Agarwala, Richa A1 - Volff, Jean-Nicolas A1 - Postlethwait, John H. A1 - Schartl, Manfred A1 - Minx, Patrick A1 - Warren, Wesley C. A1 - Walter, Ronald B. T1 - X. couchianus and X. hellerii genome models provide genomic variation insight among Xiphophorus species JF - BMC Genomics N2 - Background Xiphophorus fishes are represented by 26 live-bearing species of tropical fish that express many attributes (e.g., viviparity, genetic and phenotypic variation, ecological adaptation, varied sexual developmental mechanisms, ability to produce fertile interspecies hybrids) that have made attractive research models for over 85 years. Use of various interspecies hybrids to investigate the genetics underlying spontaneous and induced tumorigenesis has resulted in the development and maintenance of pedigreed Xiphophorus lines specifically bred for research. The recent availability of the X. maculatus reference genome assembly now provides unprecedented opportunities for novel and exciting comparative research studies among Xiphophorus species. Results We present sequencing, assembly and annotation of two new genomes representing Xiphophorus couchianus and Xiphophorus hellerii. The final X. couchianus and X. hellerii assemblies have total sizes of 708 Mb and 734 Mb and correspond to 98 % and 102 % of the X. maculatus Jp 163 A genome size, respectively. The rates of single nucleotide change range from 1 per 52 bp to 1 per 69 bp among the three genomes and the impact of putatively damaging variants are presented. In addition, a survey of transposable elements allowed us to deduce an ancestral TE landscape, uncovered potential active TEs and document a recent burst of TEs during evolution of this genus. Conclusions Two new Xiphophorus genomes and their corresponding transcriptomes were efficiently assembled, the former using a novel guided assembly approach. Three assembled genome sequences within this single vertebrate order of new world live-bearing fishes will accelerate our understanding of relationship between environmental adaptation and genome evolution. In addition, these genome resources provide capability to determine allele specific gene regulation among interspecies hybrids produced by crossing any of the three species that are known to produce progeny predisposed to tumor development. KW - Xiphophorus KW - X. hellerii KW - Annotation KW - Single nucleotide change KW - Genome comparison KW - X. couchianus KW - Genome assembly KW - NGS Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164582 VL - 17 ER - TY - JOUR A1 - Brunet, Frédéric G. A1 - Volff, Jean-Nicolas A1 - Schartl, Manfred T1 - Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates JF - Genome Biology Evolution N2 - The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling. KW - receptor tyrosine kinase KW - vertebrates KW - deuterostomes KW - whole genome duplications Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146988 VL - 8 IS - 15 ER - TY - JOUR A1 - Bemm, Felix A1 - Becker, Dirk A1 - Larisch, Christina A1 - Kreuzer, Ines A1 - Escalante-Perez, Maria A1 - Schulze, Waltraud X. A1 - Ankenbrand, Markus A1 - Van de Weyer, Anna-Lena A1 - Krol, Elzbieta A1 - Al-Rasheid, Khaled A. A1 - Mithöfer, Axel A1 - Weber, Andreas P. A1 - Schultz, Jörg A1 - Hedrich, Rainer T1 - Venus flytrap carnivorous lifestyle builds on herbivore defense strategies JF - Genome Research N2 - Although the concept of botanical carnivory has been known since Darwin's time, the molecular mechanisms that allow animal feeding remain unknown, primarily due to a complete lack of genomic information. Here, we show that the transcriptomic landscape of the Dionaea trap is dramatically shifted toward signal transduction and nutrient transport upon insect feeding, with touch hormone signaling and protein secretion prevailing. At the same time, a massive induction of general defense responses is accompanied by the repression of cell death-related genes/processes. We hypothesize that the carnivory syndrome of Dionaea evolved by exaptation of ancient defense pathways, replacing cell death with nutrient acquisition. KW - Dionaea-muscipula ellis KW - Plant utricularia-gibba KW - Programmed cell-death KW - Genomics data sets KW - RNA-SEQ data KW - Arabidopsis-thaliana KW - Jasmonate perception KW - Action potentials KW - Stress responses KW - Wonderful plants Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188799 VL - 26 IS - 6 ER - TY - JOUR A1 - Kaluza, Benjamin F. A1 - Wallace, Helen A1 - Heard, Tim A. A1 - Klein, Aelxandra-Maria A1 - Leonhardt, Sara D. T1 - Urban gardens promote bee foraging over natural habitats and plantations JF - Ecology and Evolution N2 - Increasing human land use for agriculture and housing leads to the loss of natural habitat and to widespread declines in wild bees. Bee foraging dynamics and fitness depend on the availability of resources in the surrounding landscape, but how precisely landscape related resource differences affect bee foraging patterns remains unclear. To investigate how landscape and its interaction with season and weather drive foraging and resource intake in social bees, we experimentally compared foraging activity, the allocation of foragers to different resources (pollen, nectar, and resin) and overall resource intake in the Australian stingless bee Tetragonula carbonaria (Apidae, Meliponini). Bee colonies were monitored in different seasons over two years. We compared foraging patterns and resource intake between the bees' natural habitat (forests) and two landscapes differently altered by humans (suburban gardens and agricultural macadamia plantations). We found foraging activity as well as pollen and nectar forager numbers to be highest in suburban gardens, intermediate in forests and low in plantations. Foraging patterns further differed between seasons, but seasonal variations strongly differed between landscapes. Sugar and pollen intake was low in plantations, but contrary with our predictions, it was even higher in gardens than in forests. In contrast, resin intake was similar across landscapes. Consequently, differences in resource availability between natural and altered landscapes strongly affect foraging patterns and thus resource intake in social bees. While agricultural monocultures largely reduce foraging success, suburban gardens can increase resource intake well above rates found in natural habitats of bees, indicating that human activities can both decrease and increase the availability of resources in a landscape and thus reduce or enhance bee fitness. KW - urbanization KW - anthropogenic activities KW - climate factors KW - meliponines KW - resource availability Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162713 VL - 6 IS - 5 ER - TY - JOUR A1 - Benoit, Joshua B. A1 - Adelman, Zach N. A1 - Reinhardt, Klaus A1 - Dolan, Amanda A1 - Poelchau, Monica A1 - Jennings, Emily C. A1 - Szuter, Elise M. A1 - Hagan, Richard W. A1 - Gujar, Hemant A1 - Shukla, Jayendra Nath A1 - Zhu, Fang A1 - Mohan, M. A1 - Nelson, David R. A1 - Rosendale, Andrew J. A1 - Derst, Christian A1 - Resnik, Valentina A1 - Wernig, Sebastian A1 - Menegazzi, Pamela A1 - Wegener, Christian A1 - Peschel, Nicolai A1 - Hendershot, Jacob M. A1 - Blenau, Wolfgang A1 - Predel, Reinhard A1 - Johnston, Paul R. A1 - Ioannidis, Panagiotis A1 - Waterhouse, Robert M. A1 - Nauen, Ralf A1 - Schorn, Corinna A1 - Ott, Mark-Christoph A1 - Maiwald, Frank A1 - Johnston, J. Spencer A1 - Gondhalekar, Ameya D. A1 - Scharf, Michael E. A1 - Raje, Kapil R. A1 - Hottel, Benjamin A. A1 - Armisén, David A1 - Crumière, Antonin Jean Johan A1 - Refki, Peter Nagui A1 - Santos, Maria Emilia A1 - Sghaier, Essia A1 - Viala, Sèverine A1 - Khila, Abderrahman A1 - Ahn, Seung-Joon A1 - Childers, Christopher A1 - Lee, Chien-Yueh A1 - Lin, Han A1 - Hughes, Daniel S.T. A1 - Duncan, Elizabeth J. A1 - Murali, Shwetha C. A1 - Qu, Jiaxin A1 - Dugan, Shannon A1 - Lee, Sandra L. A1 - Chao, Hsu A1 - Dinh, Huyen A1 - Han, Yi A1 - Doddapaneni, Harshavardhan A1 - Worley, Kim C. A1 - Muzny, Donna M. A1 - Wheeler, David A1 - Panfilio, Kristen A. A1 - Jentzsch, Iris M. Vargas A1 - Jentzsch, IMV A1 - Vargo, Edward L. A1 - Booth, Warren A1 - Friedrich, Markus A1 - Weirauch, Matthew T. A1 - Anderson, Michelle A.E. A1 - Jones, Jeffery W. A1 - Mittapalli, Omprakash A1 - Zhao, Chaoyang A1 - Zhou, Jing-Jiang A1 - Evans, Jay D. A1 - Attardo, Geoffrey M. A1 - Robertson, Hugh M. A1 - Zdobnov, Evgeny M. A1 - Ribeiro, Jose M.C. A1 - Gibbs, Richard A. A1 - Werren, John H. A1 - Palli, Subba R. A1 - Schal, Coby A1 - Richards, Stephen T1 - Unique features of a global human ectoparasite identified through sequencing of the bed bug genome JF - Nature Communications N2 - The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite. KW - human ectoparasite KW - bed bug KW - Cimex lectularius KW - genome Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166221 VL - 7 IS - 10165 ER - TY - JOUR A1 - da Cruz, Irene A1 - Rodríguez-Casuriaga, Rosana A1 - Santiñaque, Frederico F. A1 - Farías, Joaquina A1 - Curti, Gianni A1 - Capoano, Carlos A. A1 - Folle, Gustavo A. A1 - Benavente, Ricardo A1 - Sotelo-Silveira, José Roberto A1 - Geisinger, Adriana T1 - Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage JF - BMC Genomics N2 - Background Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis. Results We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation. Conclusions This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals. KW - Spermatogenesis KW - Transcriptome KW - RNAseq KW - Flow cytometry Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164574 VL - 17 ER - TY - THES A1 - Blättner, Sebastian T1 - The role of the non-ribosomal peptide synthetase AusAB and its product phevalin in intracellular virulence of Staphylococcus aureus T1 - Die Rolle der nicht-ribosomalen Peptidsynthetase AusAB und ihres Produktes Phevalin in der intrazellulären Virulenz von Staphylococcus aureus N2 - Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis. In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70% of the CA infections. The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur. S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient. In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae. The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria. N2 - Staphylococcus aureus ist ein weit verbreitetes kommensales Bakterium, welches zugleich einer der häufigsten Verursacher von Krankenhausinfektionen ist, und eine Reihe verschiedener Krankheiten, angefangen bei simplen Abszessen, bis hin zu schweren Erkrankungen wie Lungenentzündung, Osteomylitis und Sepsis verursachen kann. Das Risiko durch nosokomiale sowie epidemische S. aureus Infektionen ist in den vergangenen Jahren weiter gestiegen. Dazu beigetragen hat das Auftreten multiresistenter und hoch cytotoxischer Stämme, vor allem in den USA. Als Konsequenz hat die WHO S. aureus inzwischen als „Superbug“ tituliert und als globales Gesundheitsrisiko eingestuft. Bei CA-Infektionen dominieren die Isolate der Klassifizierung USA300 und USA400, wobei den Erstgenannten bis zu 70% aller in den USA registrierten CA-MRSA Infektionen der letzten Jahre zugesprochen werden. Lange Zeit wurde angenommen, dass S. aureus strikt extrazellulär im Infektionsbereich vorliegt und die cytotoxische Wirkung von z.B. α-Toxin für Wirtszelltod und Gewebeschädigungen verantwortlich ist. Erst vor kurzem wurde festgestellt, dass S. aureus auch durch fakultativ phagozytotische Zellen, wie Epithel- oder Endothelzellen, mittels zahlreicher Adhäsine aufgenommen wird. Die Aufnahme in die Zelle erfolgt zunächst in ein Phagoendosom, in dem die Pathogene durch antimikrobielle Mechanismen abgebaut würden. Um dies zu verhindern, verfügt S. aureus über Virulenzfaktoren, welche die endosomale Membran schädigen. Die Bakterien gelangen so in das Zellzytoplasma, wo sie sich vervielfältigen können, bevor die Wirtszelle schließlich getötet wird. Eine wichtige Funktion in diesem Vorgang konnte bereits in mehreren Studien den Phenol löslichen Modulinen (PSM) zugesprochen werden, Arbeiten unserer Gruppe deuten jedoch darauf hin, dass diese nicht alleine für den phagosomalen Ausbruch von S. aureus verantwortlich sind. In dieser Arbeit verwendete ich eine Transposon Mutantenbibliothek des S. aureus Stammes JE2 (USA300) in Verbindung mit automatisierter Fluoreszenzmikroskopie, um Gene zu identifizieren, die den phagosomalen Ausbruch von S. aureus beeinflussen. Unter den Mutanten, welche eine Minderung der Ausbruchsraten zeigten, fanden sich auch Mutanten in beiden Genen eines Operons, welches für die nicht-ribosomale Peptidsynthetase AusA/B codiert, die die beiden Dipeptide Phevalin und Tyrvalin produziert. Verminderte Ausbruchsraten konnten sowohl durch genetische Komplementation als auch mittels des Zusatzes synthetischen Phevalins wiederhergestellt werden. In Leukozyten verhindert Phevalin effizienten Calcium-Flux und die Aktivierung von Neutrophilen. Zudem fördert Phevalin die Cytotoxizität intrazellulärer Bakterien sowohl in Makrophagen, als auch Neutrophilen. Darüber hinaus konnten wir zeigen, dass die NRPS AusAB und ihre Produkte eine Rolle beim Überleben der Bakterien während einer Infektion im Tiermodell einnehmen. Die hier präsentierten Daten hinsichtlich des Einflusses von Phevalin auf Virulenz und der Interaktion zwischen Wirt und Pathogen lassen den Schluss zu, dass Phevalin direkt auf einen Wirtszellfaktor wirkt, um die Cytotoxicität intrazellulärer Bakterien zu stärken. KW - Staphylococcus aureus KW - MRSA KW - Virulenz KW - Intracellular virulence KW - Non-ribosomal peptide synthetase KW - USA300 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146662 ER - TY - THES A1 - Pasch, Elisabeth T1 - The role of SUN4 and related proteins in sperm head formation and fertility T1 - Die Rolle von SUN4 und verwandten Proteinen in der Spermienkopfformierung und Fertilität N2 - Spermiogenesis describes the differentiation of haploid germ cells into motile, fertilization-competent spermatozoa. During this fundamental transition the species-specific sperm head is formed, which necessitates profound nuclear restructuring coincident with the assembly of sperm-specific structures and chromatin compaction. In the case of the mouse, it is characterized by reshaping of the early round spermatid nucleus into an elongated sickle-shaped sperm head. This tremendous shape change requires the transduction of cytoskeletal forces onto the nuclear envelope (NE) or even further into the nuclear interior. LINC (linkers of nucleoskeleton and cytoskeleton) complexes might be involved in this process, due to their general function in bridging the NE and thereby physically connecting the nucleus to the peripheral cytoskeleton. LINC complexes consist of inner nuclear membrane integral SUN-domain proteins and outer nuclear membrane KASH-domain counterparts. SUN- and KASH-domain proteins are directly connected to each other within the perinuclear space, and are thus capable of transferring forces across the NE. To date, these protein complexes are known for their essential functions in nuclear migration, anchoring and positioning of the nucleus, and even for chromosome movements and the maintenance of cell polarity and nuclear shape. In this study LINC complexes were investigated with regard to their potential role in sperm head formation, in order to gain further insight into the processes occurring during spermiogenesis. To this end, the behavior and function of the testis-specific SUN4 protein was studied. The SUN-domain protein SUN4, which had received limited characterization prior to this work, was found to be exclusively expressed in haploid stages during germ cell development. In these cell stages, it specifically localized to the posterior NE at regions decorated by the manchette, a spermatid-specific structure which was previously shown to be involved in nuclear shaping. Mice deficient for SUN4 exhibited severely disorganized manchette residues and gravely misshapen sperm heads. These defects resulted in a globozoospermia-like phenotype and male mice infertility. Therefore, SUN4 was not only found to be mandatory for the correct assembly and anchorage of the manchette, but also for the correct localization of SUN3 and Nesprin1, as well as of other NE components. Interaction studies revealed that SUN4 had the potential to interact with SUN3, Nesprin1, and itself, and as such is likely to build functional LINC complexes that anchor the manchette and transfer cytoskeletal forces onto the nucleus. Taken together, the severe impact of SUN4 deficiency on the nucleocytoplasmic junction during sperm development provided direct evidence for a crucial role of SUN4 and other LINC complex components in mammalian sperm head formation and fertility. N2 - Die Spermiogenese beschreibt die Differenzierung haploider Keimzellen zu beweglichen, fortpflanzungsfähigen Spermatozoen. Während dieses fundamentalen Entwicklungsabschnittes wird neben dem Aufbau von spermienspezifischen Strukturen und der Kompaktierung des Chromatins auch der speziesspezifische Spermienkopf geformt. Im Falle der Maus ist dies eine aktive Umformung des runden Zellkerns in einen gestreckten, sichelförmigen Spermienkopf. Eine derart gravierende Formveränderung erfordert eine Kraftweiterleitung aus dem Zytoskelett auf die Kernhülle und das Kerninnere. In diesem Zusammenhang könnten LINC (linkers of nucleoskeleton and cytoskeleton) Komplexe eine Rolle spielen, da ihre grundlegende Funktion darin besteht die Kernhülle zu überbrücken und somit den Kern mit dem peripheren Zytoskelett zu verbinden. LlNC Komplexe werden aus SUN und KASH Domänen Proteinen aufgebaut, welche in die innere beziehungsweise äußere Kernmembran eingelagert sind. Diese membranintegralen Proteine sind direkt miteinander verbunden, so dass sie einen Komplex bilden, der zur Kräfteübertragung geeignet ist. LINC Komplexe besitzen vielfältige Funktionen in Prozessen wie nuklearer Migration, Verankerung und Positionierung des Zellkerns, Chromosomenbewegungen und in der Aufrechterhaltung der Zellpolarität oder der Kernform. Um ein größeres Verständnis der Prozesse während der Spermiogenese zu gewinnen, wurden in dieser Studie die Funktionen von LINC Komplexen in der Spermiogenese und ihre spezifische Rolle bei der gerichteten Spermienkopf-strukturierung untersucht. Dabei wurde insbesondere das Verhalten und die Funktion des bisher wenig charakterisierten SUN Domänen Proteins SUN4 erforscht. Entsprechend der Ergebnisse dieser Studie ist SUN4 ein hodenspezifisches Protein, das ausschließlich in haploiden Keimzellen exprimiert wird. In diesen lokalisiert es in der posterioren Kernhülle, spezifisch in Regionen, an die sich die spermatidenspezifische Manschette anlagert. Dies ist eine Struktur, für die bereits gezeigt wurde, dass sie an der Verformung des Kerns beteiligt ist. SUN4 defiziente Mäuse zeigten ausschließlich Spermatiden mit stark desorganisierten Manschettenüberresten und einen gravierend verformten Spermienkopf. Insgesamt führten die Fehlbildungen zu einem globozoospermieartigen Phänotyp und männlicher Sterilität bei Mäusen. Dabei zeigte sich, dass SUN4 nicht nur zwingend erforderlich ist für den korrekten Aufbau und die Verankerung der Manschette, sondern auch für die korrekte Lokalisation von SUN3 und Nesprin1, wie auch für weitere Komponenten der posterioren Kernhülle. Interaktionsstudien zeigten, dass SUN4 sowohl mit SUN3 und Nesprin1 als auch mit sich selbst interagieren kann, vermutlich um funktionsfähige LINC Komplexe zu bilden, die die Manchette verankern und Kräfte aus dem Zytoskelett auf den Kern übertragen. Zusammenfassend zeigen die schwerwiegenden Auswirkungen auf die kernzytoplasmatische Verbindung während der Spermienentwicklung, die durch den Verlust von SUN4 entstanden, einen direkten Nachweis einer entscheidenden Rolle von SUN4 und anderen LINC-Komplex-Komponenten für die Spermienkopfentwicklung und Fertilität bei Säugetieren. KW - Maus KW - spermiogenesis KW - Fertilität KW - Spermatogenese KW - Kernhülle KW - Molekularbiologie KW - LINC complex KW - SUN domain proteins KW - sperm head formation KW - fertility KW - Spermiogenese KW - Spermienbildung KW - Kernproteine Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139092 ER - TY - JOUR A1 - Othman, Eman M. A1 - Naseem, Muhammed A1 - Awad, Eman A1 - Dandekar, Thomas A1 - Stopper, Helga T1 - The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells JF - PLoS One N2 - Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing. KW - DNA damage KW - apoptosis KW - oxidative stress KW - fluorescence recovery after photobleaching KW - lymphocytes KW - antioxidants KW - cell staining KW - cytokinins Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147983 VL - 11 IS - 12 ER - TY - JOUR A1 - Biscotti, Maria Assunta A1 - Gerdol, Marco A1 - Canapa, Adriana A1 - Forconi, Mariko A1 - Olmo, Ettore A1 - Pallavicini, Alberto A1 - Barucca, Marco A1 - Schartl, Manfred T1 - The Lungfish Transcriptome: A Glimpse into Molecular Evolution Events at the Transition from Water to Land JF - Scientific Reports N2 - Lungfish and coelacanths are the only living sarcopterygian fish. The phylogenetic relationship of lungfish to the last common ancestor of tetrapods and their close morphological similarity to their fossil ancestors make this species uniquely interesting. However their genome size, the largest among vertebrates, is hampering the generation of a whole genome sequence. To provide a partial solution to the problem, a high-coverage lungfish reference transcriptome was generated and assembled. The present findings indicate that lungfish, not coelacanths, are the closest relatives to land-adapted vertebrates. Whereas protein-coding genes evolve at a very slow rate, possibly reflecting a “living fossil” status, transposable elements appear to be active and show high diversity, suggesting a role for them in the remarkable expansion of the lungfish genome. Analyses of single genes and gene families documented changes connected to the water to land transition and demonstrated the value of the lungfish reference transcriptome for comparative studies of vertebrate evolution. KW - lungfish KW - transcriptome KW - genome KW - sarcopterygian fish Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167753 VL - 6 IS - 21571 ER - TY - THES A1 - Eck, Saskia T1 - The impact of thermogenetic depolarizations of specific clock neurons on Drosophila melanogaster's circadian clock T1 - Der Einfluss thermogenetischer Depolarisationen spezifischer Uhrneurone auf Drosophila melanogasters circadiane Uhr N2 - The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms’ behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly’s circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock’s interneuronal communication. Drosophila melanogaster’s circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly’s locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature. Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs). The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs & l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day. To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner. However, the molecular link between clock neuron depolarization and shifts in the molecular clock’s cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock. Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways. N2 - Die Rotation der Erde um ihre eigene Achse hat periodisch verändernde Umweltbedingungen, wie beispielsweise Veränderungen in den Lichtverhältnissen und der Temperatur, zur Folge. Um das Verhalten, die Physiologie und den Metabolismus eines Organismus an stets wiederkehrende Veränderungen anzupassen, haben sich endogene/circadiane Uhren entwickelt, die es dem Organismus erlauben diese Umweltbedingungen zu antizipieren. In der Chronobiologie, einem wissenschaftlichen Fachbereich, der sich mit der Untersuchung der zugrunde liegenden Mechanismen der Inneren Uhr befasst, dient die Taufliege Drosophila melanogaster als nützlicher Modellorganismus. Die Innere Uhr der Taufliege ist anatomisch eher einfach organisiert, weist trotz alledem jedoch Homologien zum Säugersystem auf. Auch im Rahmen dieser Doktorarbeit diente die Taufliege daher dazu grundlegende Netzwerkeigenschaften der circadianen Uhr zu untersuchen. Die Innere Uhr von Drosophila melanogaster besteht aus ungefähr 150 Uhrneuronen, die sich im zentralen Nervensystem der Fliege befinden. Diese Uhrneurone können, bezüglich ihrer anatomischen Position im Gehirn in die Gruppe der dorsalen Neurone (DN1, DN2, DN3), sowie in die der lateralen Neurone untergliedert werden (LPN, LNd, s-LNv, l-LNv). Funktionell werden diese Uhrneuronengruppen als Morgen- und Abendoszillatoren (M- und E-Oszillatoren) klassifiziert, da sie für unterschiedliche Verhaltensanteile in der Laufaktivität der Fliege unter Licht-Dunkel-Verhältnissen (LD) verantwortlich sind. Die s-LNv stellen dabei die Morgenoszillatoren (M-Oszillatoren) dar und werden als Hauptschrittmacher betrachtet, da sie die Geschwindigkeit der Uhr unter konstanten Bedingungen (Dauerdunkel; DD) bestimmen. Die Gruppe der Abendoszillatoren (EOszillatoren) besteht aus den LNd, einigen DN1 und der fünften s-LNv (5th s-LNv) und ist für die richtige Terminierung der Abendaktivität in LD zuständig. All diese Uhrneurone sind funktionell nicht unabhängig voneinander, sondern bilden komplexe neuronale Verschaltungen untereinander aus, die durch einen hohen Grad an Plastizität bezüglich ihrer Reaktion auf unterschiedliche Umweltparameter (Zeitgeber), wie Licht oder Temperatur, gekennzeichnet sind. Obwohl bereits vieles hinsichtlich der Funktion und der Bedeutung einiger Gruppen von Uhrneuronen bekannt ist, ist das genaue Zusammenspiel unter ihnen immer noch recht unklar. Um die Mechanismen, die in den Kommunikationsprozessen zwischen verschiedenen Uhrneuronen involviert sind, zu untersuchen, machten wir Gebrauch von dTrpA1, einem thermosensitiven Kationenkanal, der es durch die Applizierung von Temperaturpulsen (TP) über 29°C ermöglicht, Neuronen in der intakten und sich frei bewegenden Fliege zeitlich begrenzt und zellspezifisch zu depolarisieren. Mithilfe verschiedener Uhr-spezifischer GAL4-Treiberlinien und der Verabreichung von TP zu verschiedenen Zeitpunkten des circadianen Zyklus in DD, war es uns möglich Rückschlüsse auf die Eigenschaften der Inneren Uhr anhand von Phasen-Verschiebungsexperimenten zu ziehen. Die hervorgerufenen Phasenverschiebungen im Laufverhalten, die durch die Aktivierung spezieller Uhrneuronen hervorgerufen wurden, wurden dabei als Phasen Responz Kurve (engl. phase response curve; PRC) dargestellt. Die Depolarisierung aller Uhrneurone verschob die Phase der Aktivität am stärksten, insbesondere in der Phasen-Verzögerungszone der PRC. Wurden ausschließlich die M-Oszillatoren zusammen mit den l-LNv (PDF+ Neurone: s-LNv & l-LNv) depolarisiert, wurden ebenso Phasenverschiebungen nach vorne, wie auch nach hinten hervorgerufen, jedoch reichten die Verschiebungen nach vorne deutlich in den subjektiven Tag hinein. Daraus schlussfolgerten wir, dass Licht inhibitorischen Einfluss in diesem Bereich der PRC haben muss, da typische Licht-PRCs nicht derart ausgeprägte Vorverschiebungen aufweisen. Aufgrund des vollständigen Lichtausschlusses in den PRC-Versuchen dieser Doktorarbeit fehlt jedoch dieser Licht-vermittelte inhibitorische Einfluss zu den PDF+ Neuronen und führt daher zur zeitlich stark ausgeprägten Phasen-Vorverschiebungszone. Diese Ergebnisse lassen daher vermuten, dass ein inhibitorisch wirkender Licht-vermittelter Eingang zu den PDF+ Neuronen von den photorezeptiven Organen (Hofbauer-Buchner Äuglein, Photorezeptoren der Komplexaugen, Ocellen) oder von anderen Uhrneuronen existieren muss, der die Phasen-Vorverschiebungen während des subjektiven Tages unterdrückt. Um Kenntnis über den molekularen Status der Uhr in der Verzögerungs- und Phasen-Vorverschiebungszone zu erlangen, wurden Färbungen gegen das Protein Period (PER), eines der zentralen Bestandteile der Inneren Uhr und gegen das Neuropeptid Pigment Dispersing Factor (PDF) angefertigt. Der zeitliche Verlauf im Auf- und Abbau des PER Proteins spiegelte die Phasenverschiebungen im Verhalten der Experimentalfliegen wider, wohingegen die Kontrollen weitestgehend unauffällig blieben. Zudem waren nur diejenigen Neurone von einer unmittelbaren Verschiebung der PER Protein Oszillation betroffen, die depolarisiert wurden, was auf einen schnellen Zell-autonomen Prozess schließen lässt. Die molekulare Verknüpfung, die zwischen der Depolarisation der Uhrneuronen und der Verschiebung der molekularen Uhr-Oszillation fungiert, ist immer noch unbekannt. Diesem Thema wurde nachgegangen, indem CREB (engl. cAMP responsive element binding protein) in den großen ventrolateralen Neuronen (l-LNv) quantifiziert wurde, da diese Neuronen unerwarteterweise und am wirksamsten auf die artifizielle Depolarisation mit einer starken PER-Akkumulation reagiert haben. In vorherigen Arbeiten wurde bereits angenommen, dass CREB in die circadiane Rhythmik involviert sei, indem es an Regulationssequenzen des period Gens bindet (Belvin et al., 1999) und somit dessen Transkription aktiviert. Wir konnten zeigen, dass die Menge an CREB Protein in den l-LNv circadian reguliert wird, da diese am Ende der subjektiven Nacht im Vergleich zum Ende des subjektiven Tages deutlich erhöht ist. Dieser Effekt konnte durch die artifizielle Depolarisation, aber unabhängig von deren Zeitpunkt, weiter verstärkt werden. Zudem deuten die Ergebnisse darauf hin, dass die Akkumulation des CREB Proteins mit dem Zeitpunkt des Anstiegs des PER Proteins in den l-LNv koinzidiert. Das lässt die Vermutung zu, dass CREB als molekulare Verbindung zwischen dem elektrischen neuronalen Status und der molekularen Uhr dienen kann. Zusammenfassend lässt sich sagen, dass die zeitlich begrenzte Depolarisation mithilfe von dTrpA1 signifikante Phasenverschiebungen im Verhalten wie auch auf der Proteinebene hervorrufen kann. Eine artifizielle Depolarisation zu Beginn der subjektiven Nacht verursacht Phasenverschiebungen nach hinten, wohingegen eine Depolarisation zum Ende der subjektiven Nacht Phasenverschiebungen nach vorne zur Folge hat. Die Aktivierung aller Uhrneurone brachte eine PRC hervor, die weitestgehend einer Licht-PRC gleicht. Die Depolarisierung der PDF+ Zellen hingegen ergab eine PRC, die sich insbesondere bezüglich der ausgeprägten Phasen-Vorverschiebungszone von einer Licht-vermittelten PRC unterscheidet. Die Innere Uhr scheint somit die Fähigkeit zu besitzen, exzitatorische und inhibitorische Eingänge in komplexer Art und Weise zu verarbeiten. Obwohl der in dieser Doktorarbeit gewählte experimentelle Ansatz hochgradig artifiziell ist, war es uns gerade durch den Ausschluss von Licht möglich, neue Schlussfolgerungen bezüglich der Kommunikation innerhalb des Netzwerks und dessen Lichtinformations-Eingänge zu ziehen. KW - Chronobiologie KW - Circadian clock KW - Tagesrhythmus KW - Taufliege Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137118 ER - TY - THES A1 - Kupper, Maria T1 - The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus T1 - Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus N2 - The evolutionary success of insects is believed to be at least partially facilitated by symbioses between insects and prokaryotes. Bacterial endosymbionts confer various fitness advantages to their hosts, for example by providing nutrients lacking from the insects’ diet thereby enabling the inhabitation of new ecological niches. The Florida carpenter ant Camponotus floridanus harbours endosymbiotic bacteria of the genus Blochmannia. These primary endosymbionts mainly reside in the cytoplasm of bacteriocytes, specialised cells interspersed into the midgut tissue, but they were also found in oocytes which allows their vertical transmission. The social lifestyle of C. floridanus may facilitate the rapid spread of infections amongst genetically closely related animals living in huge colonies. Therefore, the ants require an immune system to efficiently combat infections while maintaining a “chronic” infection with their endosymbionts. In order to investigate the immune repertoire of the ants, the Illumina sequencing method was used. The previously published genome sequence of C. floridanus was functionally re-annotated and 0.53% of C. floridanus proteins were assigned to the gene ontology (GO) term subcategory “immune system process”. Based on homology analyses, genes encoding 510 proteins with possible immune function were identified. These genes are involved in microbial recognition and immune signalling pathways but also in cellular defence mechanisms, such as phagocytosis and melanisation. The components of the major signalling pathways appear to be highly conserved and the analysis revealed an overall broad immune repertoire of the ants though the number of identified genes encoding pattern recognition receptors (PRRs) and antimicrobial peptides (AMPs) is comparatively low. Besides three genes coding for homologs of thioester-containing proteins (TEPs), which have been shown to act as opsonins promoting phagocytosis in other insects, six genes encoding the AMPs defesin-1 and defensin-2, hymenoptaecin, two tachystatin-like peptides and one crustin-like peptide are present in the ant genome. Although the low number of known AMPs in comparison to 13 AMPs in the honey bee Apis mellifera and 46 AMPs in the wasp Nasonia vitripennis may indicate a less potent immune system, measures summarised as external or social immunity may enhance the immune repertoire of C. floridanus, as it was discussed for other social insects. Also, the hymenoptaecin multipeptide precursor protein may be processed to yield seven possibly bioactive peptides. In this work, two hymenoptaecin derived peptides were heterologously expressed and purified. The preliminary antimicrobial activity assays indicate varying bacteriostatic effects of different hymenoptaecin derived peptides against Escherichia coli D31 and Staphylococcus aureus which suggests a functional amplification of the immune response further increasing the antimicrobial potency of the ants. Furthermore, 257 genes were differentially expressed upon immune challenge of C. floridanus and most of the immune genes showing differential expression are involved in recognition of microbes or encode immune effectors rather than signalling components. Additionally, genes coding for proteins involved in storage and metabolism were downregulated upon immune challenge suggesting a trade-off between two energy-intensive processes in order to enhance effectiveness of the immune response. The analysis of gene expression via qRT-PCR was used for validation of the transcriptome data and revealed stage-specific immune gene regulation. Though the same tendencies of regulation were observed in larvae and adults, expression of several immune-related genes was generally more strongly induced in larvae. Immune gene expression levels depending on the developmental stage of C. floridanus are in agreement with observations in other insects and might suggest that animals from different stages revert to individual combinations of external and internal immunity upon infection. The haemolymph proteome of immune-challenged ants further established the immune-relevance of several proteins involved in classical immune signalling pathways, e.g. PRRs, extracellularly active proteases of the Toll signalling pathway and effector molecules such as AMPs, lysozymes and TEPs. Additionally, non-canonical proteins with putative immune function were enriched in immune-challenged haemolymph, e.g. Vitellogenins, NPC2-like proteins and Hemocytin. As known from previous studies, septic wounding also leads to the upregulation of genes involved in stress responses. In the haemolymph, proteins implicated in protein stabilisation and in the protection against oxidative stress and insecticides were enriched upon immune challenge. In order to identify additional putative immune effectors, haemolymph peptide samples from immune-challenged larvae and adults were analysed. The analysis in this work focussed on the identification of putative peptides produced via the secretory pathway as previously described for neuropeptides of C. floridanus. 567 regulated peptides derived from 39 proteins were identified in the larval haemolymph, whereas 342 regulated peptides derived from 13 proteins were found in the adult haemolymph. Most of the peptides are derived from hymenoptaecin or from putative uncharacterised proteins. One haemolymph peptide of immune-challenged larvae comprises the complete amino acid sequence of a predicted peptide derived from a Vitellogenin. Though the identified peptide lacks similarities to any known immune-related peptide, it is a suitable candidate for further functional analysis. To establish a stable infection with the endosymbionts, the bacteria have to be transmitted to the next generation of the ants. The vertical transmission of B. floridanus is guaranteed by bacterial infestation of oocytes. This work presents the first comprehensive and detailed description of the localisation of the bacterial endosymbionts in C. floridanus ovaries during oogenesis. Whereas the most apical part of the germarium, which contains the germ-line stem cells, is not infected by the bacteria, small somatic cells in the outer layers of each ovariole were found to be infected in the lower germarium. Only with the beginning of cystocyte differentiation, endosymbionts are exclusively transported from follicle cells into the growing oocytes, while nurse cells were never infected with B. floridanus. This infestation of the oocytes by bacteria very likely involves exocytosis-endocytosis processes between follicle cells and the oocytes. A previous study suggested a down-modulation of the immune response in the midgut tissue which may promote endosymbiont tolerance. Therefore, the expression of several potentially relevant immune genes was analysed in the ovarial tissue by qRT-PCR. The relatively low expression of genes involved in Toll and IMD signalling, and the high expression of genes encoding negative immune regulators, such as PGRP-LB, PGRP-SC2, and tollip, strongly suggest that a down-modulation of the immune response may also facilitate endosymbiont tolerance in the ovaries and thereby contribute to their vertical transmission. Overall, the present thesis improves the knowledge about the immune repertoire of C. floridanus and provides new candidates for further functional analyses. Moreover, the involvement of the host immune system in maintaining a “chronic” infection with symbiotic bacteria was confirmed and extended to the ovaries. N2 - Der evolutionäre Erfolg von Insekten wird zumindest teilweise Symbiosen zwischen Insekten und Prokaryonten zugeschrieben. Dabei übertragen bakterielle Symbionten verschiedenste Fitnessvorteile an ihre Wirte. Beispielsweise ermöglicht die Bereitstellung von Nährstoffen, welche in der Nahrung des Insekts fehlen, die Erschließung neuer ökologischer Nischen. Die Florida Rossameise Camponotus floridanus trägt endosymbiontische Bakterien der Gattung Blochmannia. Diese primären Endosymbionten kommen hauptsächlich im Zytoplasma von spezialisierten Zellen des Mitteldarms, den sogenannten Bakteriozyten, vor. Blochmannien wurden aber auch in Oozyten und Eiern gefunden, was ihre vertikale Übertragung an Individuen der nächsten Generation ermöglicht. Als soziale Insekten leben C. floridanus in großen Kolonien von nah verwandten Individuen. Ihre Lebensweise begünstigt möglicherweise die schnelle Ausbreitung von Infektionen, weshalb erwartet werden müsste, dass die Ameisen ein effizientes Immunsystem besitzen. Gleichzeitig muss jedoch die „chronische“ Infektion mit den bakteriellen Symbionten aufrechterhalten werden. In der vorliegenden Arbeit wurde das Immunrepertoire der Ameisen mittels Illumina Sequenzierung charakterisiert. Zunächst wurde das vor kurzem publizierte Genom von C. floridanus funktionell re-annotiert. Dabei wurden 0.53% der annotierten Proteine der GO-Unterkategorie “Prozesse des Immunsystems” zugeordnet. Basierend auf Homologieanalysen wurden Gene identifiziert, die für 510 Immunproteine kodieren. Die Genprodukte spielen eine Rolle bei der Erkennung von Mikroben und in den Signalwegen des Immunsystems, sind jedoch auch an Prozessen der zellulären Immunantwort, wie beispielsweise Phagozytose und Melanisierung, beteiligt. Dabei sind Komponenten der Hauptsignalwege hoch konserviert. Obwohl die Anzahl der identifizierten Proteine, die Fremdorganismen erkennen (PRRs), und die Anzahl an antimikrobiellen Peptiden (AMPs) vergleichsweise gering ist, verfügt C. floridanus insgesamt über ein umfangreiches Immunrepertoire. Neben drei Genen, die für Thioester-enthaltende Proteine (TEPs) kodieren und wie in anderen Insekten möglicherweise eine Rolle als Opsonine bei der Phagozytose spielen, wurden sechs AMP-Gene identifiziert. Diese kodieren für Defesin-1 und Defensin-2, Hymenoptaecin, zwei Tachystatin-ähnliche und ein Crustin-ähnliches Peptid. Die geringe Anzahl an bekannten AMPs im Vergleich zur Honigbiene Apis mellifera (13 AMPs) und Wespe Nasonia vitripennis (46 AMPs) könnte ein möglicherweise geringeres Potential des Immunsystems anzeigen. Allerdings könnten zusätzliche Maßnahmen, die unter dem Begriff „Soziale Immunität“ zusammengefasst werden, das Immunrepertoire von C. floridanus ergänzen, wie es schon für andere Insekten diskutiert wurde. Zudem könnten durch proteolytische Prozessierung des Hymenoptaecin Multipeptid Präkursormoleküls sieben mögliche antimikrobielle Peptide freigesetzt werden. Für die vorliegende Arbeit wurden zwei verschiedene dieser Hymenoptaecin Peptide heterolog exprimiert und aufgereinigt. Die vorläufige funktionelle Charakterisierung der Peptide zeigt, dass diese Peptide möglicherweise bakteriostatische Wirkung mit einem unterschiedlichen Wirkspektrum gegen Escherichia coli D31 und Staphylococcus aureus entfalten. Dies erlaubt die Annahme, dass die Expression des Hymenoptaecins zu einer funktionellen Amplifikation der Immunantwort führt und das Immunrepertoire der Ameisen erweitert. Nach Injektion von bakteriellem Material in die Ameisen wurde die Expression von 257 Genen reguliert. Viele dieser Gene kodieren für Proteine zur Erkennung von Pathogenen oder kodieren für Effektoren des Immunsystems. Komponenten der Signalwege zeigten dagegen kaum Veränderungen in ihrer Expression auf. Außerdem zeigten Gene, die für Speicherproteine oder Proteine des Metabolismus kodieren, generell eine geringere Expression nach Stimulierung des Immunsystems auf. Dies lässt einen Ausgleich zwischen zwei energieintensiven Prozessen vermuten, um eine effektive Immunantwort zu ermöglichen. Darüber hinaus zeigt die Validierung der Expressionsdaten mittels qRT-PCR eine Abhängigkeit der Expression mehrerer Gene vom Entwicklungsstadium der Ameisen auf. Generell wurden die gleichen Tendenzen in der Regulation der Expression dieser Gene nach Immunstimulierung beobachtet. Allerdings wurde die Expression mehrerer immunrelevanter Gene in Larven weit stärker induziert als in Adulten. Wie es auch schon für andere Insekten gezeigt wurde, scheinen C. floridanus Larven und Arbeiterinnen auf individuelle Kombinationen externer und interner Immunfaktoren zurückzugreifen. Die vorher beschriebenen Transkriptomdaten wurden durch die Charakterisierung des Hämolymph-Proteoms von C. floridanus nach Immunstimulation ergänzt, wodurch die Immunrelevanz vieler Faktoren auch auf Proteinebene bestätigt werden konnte. Beispielsweise wurden zahlreiche PRRs und extrazellulär aktive Proteasen des Toll-Signalwegs, aber auch Immuneffektoren wie AMPs, Lysozyme und TEPs in der Hämolymphe identifiziert. Zusätzlich führte die Immunstimulation in Larven und Adulten zur Anreicherung nicht-kanonischer Proteine mit möglicher Immunfunktion, beispielsweise Vitellogenine, NPC2-ähnliche Proteine und Hemocytin. Aus einer früheren Arbeit ist bekannt, dass septische Verwundungen zusätzlich die transkriptionelle Aktivierung von Genen der Stressantwort hervorrufen können. So wurden auch in der Hämolymphe Proteine entdeckt, die eine Rolle bei der Stabilisierung von Proteinen, und dem Schutz gegen oxidativen Stress und Insektizide spielen. Zur Identifizierung weiterer möglicher Peptideffektoren wurden Hämolymphpeptid-Proben von immunstimulierten Larven und Adulten analysiert. Der Fokus der Analyse lag dabei auf der Identifizierung von Peptiden, die auf dem sekretorischen Weg gebildet werden, wie es zuvor für Neuropeptide von C. floridanus beschrieben worden war. 567 differentiell regulierte Peptide, die von 39 Proteinen abstammen, wurden in Larvenhämolymphe identifiziert, wohingegen in der Hämolymphe von Adulttieren 342 derartige Peptide, die 13 Proteinen zugeordnet werden können, gefunden wurden. Die meisten dieser Peptide können Hymenoptaecin oder bisher noch nicht charakterisierte Proteinen zugeordnet werden. Jedoch wurde ein Peptid in larvaler Hämolymphe identifiziert, dessen Aminosäuresequenz vollständig mit der Sequenz eines vorhergesagten, von Vitellogenin stammenden Peptids übereinstimmt. Weil dieses Peptid keine Ähnlichkeiten zu anderen bereits charakterisierten antimikrobiellen Peptiden aufweist, stellt es einen geeigneten Kandidaten für weitere funktionelle Analysen dar. Die bakterielle Infektion von Oozyten ermöglicht die transovariale Übertragung von B. floridanus und ermöglicht damit die Etablierung einer stabilen Infektion in der nächsten Wirtsgeneration. Die vorliegende Arbeit beinhaltet die erste umfassende und detaillierte Beschreibung der Lokalisation bakterieller Endosymbionten in Ovarien von C. floridanus. Im apikalen Germarium, in welchem sich die Keimbahn-Stammzellen befinden, liegt noch keine bakterielle Infektion des Gewebes vor. In späteren Segmenten des Germariums jedoch können Blochmannien das erste Mal in kleinen somatischen Zellen der äußeren Schicht jeder Ovariole detektiert werden. Mit beginnender Zystozytendifferenzierung werden die Endosymbionten von Follikelzellen ausschließlich in die heranwachsenden Oozyten transportiert, wobei sehr wahrscheinlich Exozytose-Endozytose-Prozesse involviert sind. Nährzellen zeigen zu keinem Zeitpunkt während der Oogenese eine bakterielle Infektion auf. Da in einer früheren Studie vorgeschlagen wurde, dass eine signifikant reduzierte Anregung der Immunantwort im Mitteldarmgewebe zur Toleranz der Endosymbionten beitragen könnte, wurde auch die Expression ausgewählter Immungene in den Ovarien durch qRT-PCR untersucht. Die relativ geringe Expression von Genen des Toll- und des IMD-Signalwegs und die zusätzlich vergleichsweise starke Genexpression negativer Regulatoren des Immunsystems, wie PGRP-LB, PGRP-SC2 und tollip, sind Indikatoren einer reduzierten Immunantwort in den Ovarien von C. floridanus. Wie schon für den Mitteldarm der Tiere vorgeschlagen, könnte dies möglicherweise sowohl zur Toleranz von Blochmannia als auch zur vertikalen Übertragung der Endosymbionten beitragen. Die vorliegende Doktorarbeit erweitert das Wissen über das Immunrepertoire von C. floridanus und es konnten vielversprechende Kandidaten für weitere funktionelle Analysen von möglichen Immunfaktoren identifiziert werden. Darüber hinaus konnten weitere Hinweise auf die Bedeutung von Immunfaktoren der Ameisen bei der Toleranz gegenüber den symbiontischen Bakterien gefunden und auf die Ovarien der Tiere ausgeweitet werden. KW - Camponotus floridanus KW - Oogenese KW - Symbiose KW - endosymbiosis KW - Blochmannia floridanus KW - ant oogenesis KW - immune genes KW - antimicrobial peptides KW - Endosymbiosen KW - Ameisenoogenese KW - Immungene KW - Antimikrobielle Peptide Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142534 ER - TY - JOUR A1 - Künstner, Axel A1 - Hoffmann, Margarete A1 - Fraser, Bonnie A. A1 - Kottler, Verena A. A1 - Sharma, Eshita A1 - Weigel, Detlef A1 - Dreyer, Christine T1 - The Genome of the Trinidadian Guppy, Poecilia reticulata, and Variation in the Guanapo Population JF - PLoS ONE N2 - For over a century, the live bearing guppy, Poecilia reticulata, has been used to study sexual selection as well as local adaptation. Natural guppy populations differ in many traits that are of intuitively adaptive significance such as ornamentation, age at maturity, brood size and body shape. Water depth, light supply, food resources and predation regime shape these traits, and barrier waterfalls often separate contrasting environments in the same river. We have assembled and annotated the genome of an inbred single female from a high-predation site in the Guanapo drainage. The final assembly comprises 731.6 Mb with a scaffold N50 of 5.3 MB. Scaffolds were mapped to linkage groups, placing 95% of the genome assembly on the 22 autosomes and the X-chromosome. To investigate genetic variation in the population used for the genome assembly, we sequenced 10 wild caught male individuals. The identified 5 million SNPs correspond to an average nucleotide diversity (π) of 0.0025. The genome assembly and SNP map provide a rich resource for investigating adaptation to different predation regimes. In addition, comparisons with the genomes of other Poeciliid species, which differ greatly in mechanisms of sex determination and maternal resource allocation, as well as comparisons to other teleost genera can begin to reveal how live bearing evolved in teleost fish. KW - Trinidadian guppy KW - Poecilia reticulata KW - genetics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166755 VL - 11 IS - 12 ER - TY - JOUR A1 - Scharaw, Sandra A1 - Iskar, Murat A1 - Ori, Alessandro A1 - Boncompain, Gaelle A1 - Laketa, Vibor A1 - Poser, Ina A1 - Lundberg, Emma A1 - Perez, Franck A1 - Beck, Martin A1 - Bork, Peer A1 - Pepperkok, Rainer T1 - The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR JF - Journal of Cell Biology N2 - Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COP II) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COP II components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane. KW - Epidermal growth-factor KW - finger protein 11 KW - receptor tyrosine kinases KW - early secretory pathway KW - breast-cancer KW - brefeldin-a KW - E3 ligase KW - trafficking KW - export KW - endoplasmic-reticulum Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186731 VL - 215 IS - 4 ER - TY - JOUR A1 - Kunz, Meik A1 - Liang, Chunguang A1 - Nilla, Santosh A1 - Cecil, Alexander A1 - Dandekar, Thomas T1 - The drug-minded protein interaction database (DrumPID) for efficient target analysis and drug development JF - Database N2 - The drug-minded protein interaction database (DrumPID) has been designed to provide fast, tailored information on drugs and their protein networks including indications, protein targets and side-targets. Starting queries include compound, target and protein interactions and organism-specific protein families. Furthermore, drug name, chemical structures and their SMILES notation, affected proteins (potential drug targets), organisms as well as diseases can be queried including various combinations and refinement of searches. Drugs and protein interactions are analyzed in detail with reference to protein structures and catalytic domains, related compound structures as well as potential targets in other organisms. DrumPID considers drug functionality, compound similarity, target structure, interactome analysis and organismic range for a compound, useful for drug development, predicting drug side-effects and structure–activity relationships. KW - drug-minded protein KW - database Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147369 VL - 2016 ER - TY - THES A1 - Ruf, Franziska T1 - The circadian regulation of eclosion in \(Drosophila\) \(melanogaster\) T1 - Die zeitliche Steuerung des Adultschlupfes in \(Drosophila\) \(melanogaster\) N2 - Eclosion is the emergence of an adult insect from the pupal case at the end of development. In the fruit fly Drosophila melanogaster, eclosion is a circadian clock-gated event and is regulated by various peptides. When studied on the population level, eclosion reveals a clear rhythmicity with a peak at the beginning of the light-phase that persists also under constant conditions. It is a long standing hypothesis that eclosion gating to the morning hours with more humid conditions is an adaption to reduce water loss and increase the survival. Eclosion behavior, including the motor pattern required for the fly to hatch out of the puparium, is orchestrated by a well-characterized cascade of peptides. The main components are ecdysis-triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP). The molt is initiated by a peak level and pupal ecdysis by a subsequent decline of the ecdysteroid ecdysone. Ecdysteroids are produced by the prothoracic gland (PG), an endocrine tissue that contains a peripheral clock and degenerates shortly after eclosion. Production and release of ecdysteroids are regulated by the prothoracicotropic hormone (PTTH). Although many aspects of the circadian clock and the peptidergic control of the eclosion behavior are known, it still remains unclear how both systems are interconnected. The aim of this dissertation research was to dissect this connection and evaluate the importance of different Zeitgebers on eclosion rhythmicity under natural conditions. Potential interactions between the central clock and the peptides regulating ecdysis motor behavior were evaluated by analyzing the influence of CCAP on eclosion rhythmicity. Ablation and silencing of CCAP neurons, as well as CCAP null-mutation did not affect eclosion rhythmicity under either light or temperature entrainment nor under natural conditions. To dissect the connection between the central and the peripheral clock, PTTH neurons were ablated. Monitoring eclosion under light and temperature entrainment revealed that eclosion became arrhythmic under constant conditions. However, qPCR expression analysis revealed no evidence for cycling of Ptth mRNA in pharate flies. To test for a connection with pigment-dispersing factor (PDF)-expressing neurons, the PDF receptor (PDFR) and short neuropeptide F receptor (sNPFR) were knocked down in the PTTH neurons. Knockdown of sNPFR, but not PDFR, resulted in arrhythmic eclosion under constant darkness conditions. PCR analysis of the PTTH receptor, Torso, revealed its expression in the PG and the gonads, but not in the brain or eyes, of pharate flies. Knockdown of torso in the PG lead to arrhythmicity under constant conditions, which provides strong evidence for the specific effect of PTTH on the PG. These results suggest connections from the PDF positive lateral neurons to the PTTH neurons via sNPF signaling, and to the PG via PTTH and Torso. This interaction presumably couples the period of the peripheral clock in the PG to that of the central clock in the brain. To identify a starting signal for eclosion and possible further candidates in the regulation of eclosion behavior, chemically defined peptidergic and aminergic neurons were optogenetically activated in pharate pupae via ChR2-XXL. This screen approach revealed two candidates for the regulation of eclosion behavior: Dromyosuppressin (DMS) and myo-inhibitory peptides (MIP). However, ablation of DMS neurons did not affect eclosion rhythmicity or success and the exact function of MIP must be evaluated in future studies. To assess the importance of the clock and of possible Zeitgebers in nature, eclosion of the wildtype Canton S and the clock mutant per01 and the PDF signaling mutants pdf01 and han5304 was monitored under natural conditions. For this purpose, the Würzburg eclosion monitor (WEclMon) was developed, which is a new open monitoring system that allows direct exposure of pupae to the environment. A general decline of rhythmicity under natural conditions compared to laboratory conditions was observed in all tested strains. While the wildtype and the pdf01 and han5304 mutants stayed weakly rhythmic, the per01 mutant flies eclosed mostly arrhythmic. PDF and its receptor (PDFR encoded by han) are required for the synchronization of the clock network and functional loss can obviously be compensated by a persisting synchronization to external Zeitgebers. The loss of the central clock protein PER, however, lead to a non-functional clock and revealed the absolute importance of the clock for eclosion rhythmicity. To quantitatively analyze the effect of the clock and abiotic factors on eclosion rhythmicity, a statistical model was developed in cooperation with Oliver Mitesser and Thomas Hovestadt. The modelling results confirmed the clock as the most important factor for eclosion rhythmicity. Moreover, temperature was found to have the strongest effect on the actual shape of the daily emergence pattern, while light has only minor effects. Relative humidity could be excluded as Zeitgeber for eclosion and therefore was not further analyzed. Taken together, the present dissertation identified the so far unknown connection between the central and peripheral clock regulating eclosion. Furthermore, a new method for the analysis of eclosion rhythms under natural conditions was established and the necessity of a functional clock for rhythmic eclosion even in the presence of multiple Zeitgebers was shown. N2 - Der Schlupf adulter Fliegen aus dem Puparium wird in der Taufliege Drosophila melanogaster zum einen von der inneren Uhr und zum anderen von Peptiden gesteuert. Beobachtet man den Schlupf auf der Populationsebene, lässt sich erkennen, dass die meisten Fliegen zu Beginn der Lichtphase schlüpfen. Diese Rhythmizität im Schlupfverhalten von Fliegenpopulationen hält auch unter konstanten Bedingungen an. Seit langer Zeit wird angenommen, dass der Schlupf am Morgen eine Anpassung an feuchte Bedingungen ist, wodurch der Wasserverlust verringert und die Überlebenswahrscheinlichkeit erhöht werden könnte. Das stereotype motorische Schlupfverhalten, mit dem sich die Fliege aus der Puppenhülle befreit, wird durch das gut untersuchte Zusammenspiel zahlreicher Peptide gesteuert. Die wichtigsten Peptide sind hierbei das ecdysis-triggering hormone (ETH), das Schlupfhormon (EH) und das crustacean cardioactive peptide (CCAP). Wie bei jedem Schlupf wird die Häutung durch eine stark erhöhte Produktion des Ecdysteroids Ecdyson ausgelöst. Der anschließende Abfall der Ecdyson-Titer löst dann den Adultschlupf aus. Ecdysteroide werden in der Prothorakaldrüse (PD) gebildet, die eine periphere Uhr besitzt und kurz nach dem Adultschlupf zurückgebildet wird. Das prothorakotrope Hormon (PTTH) reguliert sowohl die Produktion als auch die Freisetzung der Ecdysteroide aus der PD. Obwohl bereits viel über den Aufbau und die Funktionsweise der inneren Uhr und der Kontrolle des Adultschlupfes durch Peptide bekannt ist, weiß man bisher nicht, wie beide Systeme miteinander interagieren. Das Hauptziel der vorliegenden Arbeit war es, einerseits diese Verbindung zu untersuchen und andererseits die Gewichtung verschiedener Zeitgeber für den Adultschlupf unter natürlichen Bedingungen zu bewerten. Um eine mögliche Verbindung zwischen der zentralen Uhr und den Peptiden, die das motorische Verhalten während des Schlupfes steuern, zu untersuchen, wurde der Einfluss von CCAP auf die Schlupfrhythmik betrachtet. Hierzu wurden die CCAP-exprimierenden Neurone genetisch ablatiert oder elektrisch stillgelegt, sowie zusätzlich eine CCAP-defiziente Mutante getestet. Weder unter künstlichen Licht- oder Temperaturzyklen, noch unter natürlichen Bedingungen wurden Effekte auf den Schlupfrhythmus bei veränderter CCAP Verfügbarkeit beobachtet. Die Verbindung zwischen der zentralen und der peripheren Uhr der PD wurde untersucht, indem die PTTH-exprimierenden Neurone in Fliegen ablatiert wurden. Dies führte sowohl unter konstanten Licht- als auch Temperaturbedingungen zu arrhythmischem Schlupf der Populationen. Die Analyse der Expression von Ptth mRNA mittels qPCR lieferte keine Hinweise auf eine zyklische Regulation des Ptth Transkripts in pharaten Tieren. Um eine Verbindung zu pigment-dispersing factor (PDF)-exprimierenden Uhrneuronen nachzuweisen, wurden die Rezeptoren von PDF (PDFR) und dem short Neuropeptide F (sNPFR) in den PTTH- Neuronen herunterreguliert. Nur der Verlust von sNPFR führte unter konstanten Bedingungen zu arrhythmischem Schlupf. RT-PCR-Analyse der mRNA Expression des Rezeptors von PTTH, Torso, ergab, dass torso mRNA in pharaten Fliegen nur in der PD und in den Gonaden exprimiert wird, nicht jedoch im Gehirn. Das Herrunterregulieren der torso mRNA in der PD führte unter konstanten Bedingungen zu arrhythmischem Schlupf und lieferte deutliche Hinweise zur spezifischen Funktion von PTTH in der PD. Diese Ergebnisse zeigen eine sNPF-vermittelte Verbindung zwischen den PDF-positiven lateralen Neuronen und den PTTH-Neuronen, welche über PTTH und Torso weiter bis in die PD reicht. Durch diese Verbindung wird vermutlich die Periode der peripheren Uhr in der PD an die Periode der zentralen Uhr im Gehirn angepasst. Um ein Startsignal für den Adultschlupf und weitere mögliche Kandidaten, die eine Rolle in der Steuerung des Schlupfes spielen, zu identifizieren, wurden chemisch definierte kleine Gruppen peptiderger und aminerger Neurone optogenetisch durch das Kanalrhodopsin ChR2-XXL aktiviert. In dieser Testreihe wurden Dromyosuppressin (DMS) und myoinhibitorisches Peptid (MIP) als mögliche Kandidaten ermittelt. Eine Ablation der DMS-Neurone hatte jedoch keine Auswirkungen auf Schlupfrhythmik und -erfolg. Die genaue Funktion von MIP sollte in zukünftigen Experimenten untersucht werden. Um die Gewichtung der Uhr und möglicher Zeitgeber für das natürliche Verhalten zu bestimmen, wurde der Schlupf des Wildtyps Canton S, der Uhrmutante per01 sowie der PDF-Signalwegsmutanten pdf01 und han5304 (han codiert für den PDFR) unter natürlichen Bedingungen beobachtet. Hierfür wurde ein neues und offenes Aufzeichnungssystem entwickelt: der Würzburger Schlupfmonitor (WEclMon), der einen direkten Kontakt der Puppen mit den sie umgebenden abiotischen Bedingungen ermöglicht. Im Vergleich zu Laborbedingungen war die Rhythmizität des Schlupfes unter natürlichen Bedingungen in allen getesteten Fliegenlinien weniger ausgeprägt. Während der Wildtyp sowie die pdf01 und han5304 Mutanten weiterhin schwach rhythmisch schlüpften, schlüpfte die per01 Mutante hauptsächlich arrhythmisch. Das Zusammenspiel zwischen PDF und seinem Rezeptor synchronisiert das Uhrnetzwerk, und der Verlust dieser Interaktion kann durch tägliches neues Ausrichten an den Zeitgebern ausgeglichen werden. Der Verlust des Uhrproteins PER unterbindet jedoch die komplette Funktionsfähigkeit der Uhr. Dadurch wird die Notwendigkeit der Uhr für einen rhythmischen Schlupf unterstrichen. Um den Einfluss der Uhr und abiotischer Faktoren auf den Schlupfrhythmus zu untersuchen, wurde im Rahmen einer Kooperation mit Oliver Mitesser und Thomas Hovestadt ein statistisches Modell entwickelt. Die Ergebnisse der Modellierung unterstützen die Hypothese, dass die Uhr der wichtigste Faktor für einen rhythmischen Schlupf auch unter Zeitgeber-Bedingungen ist. Die Umgebungstemperatur übt hingegen den stärksten Einfluss auf die Form des täglichen Schlupfmusters aus, während Licht hier nur einen schwachen Einfluss hat. Es konnte gezeigt werden, dass sich relative Luftfeuchtigkeit nicht als Zeitgeber für den Schlupf eignet, weshalb sie in weiteren Untersuchungen nicht berücksichtigt wurde. Zusammenfassend lässt sich sagen, dass mit der vorliegenden Arbeit die Verbindung zwischen der zentralen und peripheren Uhr in der Steuerung des Schlupfes identifiziert werden konnten, die bisher nicht bekannt war. Außerdem wurde eine neue Methode der Untersuchung des Adultschlupfes unter natürlichen Bedingungen etabliert und die Notwendigkeit einer intakten Uhr für einen rhythmischen Adultschlupf selbst in Anwesenheit mehrerer Zeitgeber konnte herausgestellt werden. KW - Taufliege KW - Tagesrhythmus KW - Adultschlupfes Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146265 ER - TY - JOUR A1 - Ankenbrand, Markus J. A1 - Weber, Lorenz A1 - Becker, Dirk A1 - Förster, Frank A1 - Bemm, Felix T1 - TBro: visualization and management of de novo transcriptomes JF - Database N2 - RNA sequencing (RNA-seq) has become a powerful tool to understand molecular mechanisms and/or developmental programs. It provides a fast, reliable and cost-effective method to access sets of expressed elements in a qualitative and quantitative manner. Especially for non-model organisms and in absence of a reference genome, RNA-seq data is used to reconstruct and quantify transcriptomes at the same time. Even SNPs, InDels, and alternative splicing events are predicted directly from the data without having a reference genome at hand. A key challenge, especially for non-computational personnal, is the management of the resulting datasets, consisting of different data types and formats. Here, we present TBro, a flexible de novo transcriptome browser, tackling this challenge. TBro aggregates sequences, their annotation, expression levels as well as differential testing results. It provides an easy-to-use interface to mine the aggregated data and generate publication-ready visualizations. Additionally, it supports users with an intuitive cart system, that helps collecting and analysing biological meaningful sets of transcripts. TBro’s modular architecture allows easy extension of its functionalities in the future. Especially, the integration of new data types such as proteomic quantifications or array-based gene expression data is straightforward. Thus, TBro is a fully featured yet flexible transcriptome browser that supports approaching complex biological questions and enhances collaboration of numerous researchers. KW - database Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147954 VL - 2016 ER - TY - THES A1 - Jung, Lisa Anna T1 - Targeting MYC Function as a Strategy for Tumor Therapy T1 - Hemmung der MYC-Funktion als Strategie für die zielgerichtete Tumortherapie N2 - A large fraction of human tumors exhibits aberrant expression of the oncoprotein MYC. As a transcription factor regulating various cellular processes, MYC is also crucially involved in normal development. Direct targeting of MYC has been a major challenge for molecular cancer drug discovery. The proof of principle that its inhibition is nevertheless feasible came from in vivo studies using a dominant-negative allele of MYC termed OmoMYC. Systemic expression of OmoMYC triggered long-term tumor regression with mild and fully reversible side effects on normal tissues. In this study, OmoMYC’s mode of action was investigated combining methods of structural biology and functional genomics to elucidate how it is able to preferentially affect oncogenic functions of MYC. The crystal structure of the OmoMYC homodimer, both in the free and the E-box-bound state, was determined, which revealed that OmoMYC forms a stable homodimer, and as such, recognizes DNA via the same base-specific DNA contacts as the MYC/MAX heterodimer. OmoMYC binds DNA with an equally high affinity as MYC/MAX complexes. RNA-sequencing showed that OmoMYC blunts both MYC-dependent transcriptional activation and repression. Genome-wide DNA-binding studies using chromatin immunoprecipitation followed by high-throughput sequencing revealed that OmoMYC competes with MYC/MAX complexes on chromatin, thereby reducing their occupancy at consensus DNA binding sites. The most prominent decrease in MYC binding was seen at low-affinity promoters, which were invaded by MYC at oncogenic levels. Strikingly, gene set enrichment analyses using OmoMYC-regulated genes enabled the identification of tumor subgroups with high MYC levels in multiple tumor entities. Together with a targeted shRNA screen, this identified novel targets for the eradication of MYC-driven tumors, such as ATAD3A, BOP1, and ADRM1. In summary, the findings suggest that OmoMYC specifically inhibits tumor cell growth by attenuating the expression of rate-limiting proteins in cellular processes that respond to elevated levels of MYC protein using a DNA-competitive mechanism. This opens up novel strategies to target oncogenic MYC functions for tumor therapy. N2 - Eine Vielzahl humaner Tumore entsteht durch die aberrante Expression des Onkoproteins MYC. Da MYC als Transkriptionsfaktor viele zelluläre Prozesse reguliert, ist er auch maßgeblich an der Entwicklung von normalem Gewebe beteiligt. Die direkte Hemmung von MYC stellt eine große Herausforderung für die Wirkstoffentwicklung dar. Studien mit dem dominant-negativen MYC-Allel namens OmoMYC belegten, dass MYC ein potenzieller Angriffspunkt für die zielgerichtete Tumortherapie ist. Die systemische Expression dieser MYC-Mutante löste eine dauerhafte Tumorregression aus und zeigte milde sowie vollständig reversible Nebenwirkungen. In der vorliegenden Arbeit wurde der molekulare Wirkmechanismus von OmoMYC untersucht, wobei sowohl Methoden der Strukturbiologie als auch der funktionalen Genomik angewendet wurden. Die Kristallstruktur des OmoMYC Proteins wurde im freien und E-Box-gebundenen Zustand bestimmt. Dadurch konnte gezeigt werden, dass OmoMYC ein stabiles Homodimer bildet. Als solches erkennt es DNA mittels derselben basenspezifischen Interaktionen wie der MYC/MAX-Komplex. Dabei bindet OmoMYC DNA mit einer ähnlichen Affinität wie das MYC/MAX-Heterodimer. Die genomweite Expressionsanalyse mittels RNA-Sequenzierung identifiziert eine Reduktion sowohl der MYC-abhängigen Transkriptionsaktiverung als auch der Transkriptionsrepression durch OmoMYC. Mittels Chromatin-Immunpräzipitation gefolgt von einer Hochdurchsatz-Sequenzierung wird gezeigt, dass OmoMYC mit MYC/MAXKomplexen auf Chromatin konkurriert und so deren Besetzung global an Konsensus-Bindestellen verringert. Die stärkste Reduktion zeigt sich an Promoterregionen mit schwacher Affinität für die MYC-Bindung, welche durch onkogene MYC-Proteinmengen aufgefüllt werden. Gene set enrichment-Analysen unter Berücksichtigung von OmoMYC-regulierten Genen erlaubten die Identifizierung von Tumor-Subgruppen mit hohen MYC-Proteinmengen in zahlreichen Tumorentitäten. Zusammen mit einem fokussierten shRNA-Screen können so neue Zielproteine für die Bekämpfung von MYC-getriebenen Tumoren, wie zum Beispiel ATAD3A, BOP1 und ADRM1, identifiziert werden. Zusammenfassend weisen die Ergebnisse darauf hin, dass OmoMYC spezifisch das Tumorzellwachstum inhibiert, indem es die Expression von zentralen Proteinen limitiert, welche durch erhöhte MYC-Proteinmengen reguliert werden. Somit können neue Strategien zur Tumortherapie identifiziert werden, die auf onkogene Funktionen von MYC zielen. KW - Myc KW - Kristallstruktur KW - Transkription KW - Bauchspeicheldrüsenkrebs KW - DNS-Bindung KW - OmoMYC KW - promoter invasion Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146993 ER - TY - JOUR A1 - Kaltdorf, Martin A1 - Srivastava, Mugdha A1 - Gupta, Shishir K. A1 - Liang, Chunguang A1 - Binder, Jasmin A1 - Dietl, Anna-Maria A1 - Meir, Zohar A1 - Haas, Hubertus A1 - Osherov, Nir A1 - Krappmann, Sven A1 - Dandekar, Thomas T1 - Systematic Identification of Anti-Fungal Drug Targets by a Metabolic Network Approach JF - Frontiers in Molecular Bioscience N2 - New antimycotic drugs are challenging to find, as potential target proteins may have close human orthologs. We here focus on identifying metabolic targets that are critical for fungal growth and have minimal similarity to targets among human proteins. We compare and combine here: (I) direct metabolic network modeling using elementary mode analysis and flux estimates approximations using expression data, (II) targeting metabolic genes by transcriptome analysis of condition-specific highly expressed enzymes, and (III) analysis of enzyme structure, enzyme interconnectedness (“hubs”), and identification of pathogen-specific enzymes using orthology relations. We have identified 64 targets including metabolic enzymes involved in vitamin synthesis, lipid, and amino acid biosynthesis including 18 targets validated from the literature, two validated and five currently examined in own genetic experiments, and 38 further promising novel target proteins which are non-orthologous to human proteins, involved in metabolism and are highly ranked drug targets from these pipelines. KW - metabolism KW - targets KW - antimycotics KW - modeling KW - structure KW - interaction KW - fungicide Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147396 VL - 3 ER - TY - JOUR A1 - Gatto, Francesco A1 - Schulze, Almut A1 - Nielsen, Jens T1 - Systematic Analysis Reveals that Cancer Mutations Converge on Deregulated Metabolism of Arachidonate and Xenobiotics JF - Cell Reports N2 - Mutations are the basis of the clonal evolution of most cancers. Nevertheless, a systematic analysis of whether mutations are selected in cancer because they lead to the deregulation of specific biological processes independent of the type of cancer is still lacking. In this study, we correlated the genome and transcriptome of 1,082 tumors. We found that nine commonly mutated genes correlated with substantial changes in gene expression, which primarily converged on metabolism. Further network analyses circumscribed the convergence to a network of reactions, termed AraX, that involves the glutathione- and oxygen-mediated metabolism of arachidonic acid and xenobiotics. In an independent cohort of 4,462 samples, all nine mutated genes were consistently correlated with the deregulation of AraX. Among all of the metabolic pathways, AraX deregulation represented the strongest predictor of patient survival. These findings suggest that oncogenic mutations drive a selection process that converges on the deregulation of the AraX network. KW - Cancer genetics KW - Genetics research Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164814 VL - 16 IS - 3 ER - TY - JOUR A1 - Grimm, Jonathan B. A1 - Klein, Teresa A1 - Kopek, Benjamin G. A1 - Shtengel, Gleb A1 - Hess, Harald F. A1 - Sauer, Markus A1 - Lavis, Luke D. T1 - Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy JF - Angewandte Chemie International Edition N2 - The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules. KW - fluorophore KW - microscopy KW - photoactivation KW - Si-rhodamine KW - super-resolution imaging Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191069 VL - 55 IS - 5 ER - TY - THES A1 - Costea, Paul Igor T1 - Stratification and variation of the human gut microbiota T1 - Stratifikation und Variation des menschlichen Darmmikrobioms N2 - The microbial communities that live inside the human gastrointestinal tract -the human gut microbiome- are important for host health and wellbeing. Characterizing this new “organ”, made up of as many cells as the human body itself, has recently become possible through technological advances. Metagenomics, the high-throughput sequencing of DNA directly from microbial communities, enables us to take genomic snapshots of thousands of microbes living together in this complex ecosystem, without the need for isolating and growing them. Quantifying the composition of the human gut microbiome allows us to investigate its properties and connect it to host physiology and disease. The wealth of such connections was unexpected and is probably still underestimated. Due to the fact that most of our dietary as well as medicinal intake affects the microbiome and that the microbiome itself interacts with our immune system through a multitude of pathways, many mechanisms have been proposed to explain the observed correlations, though most have yet to be understood in depth. An obvious prerequisite to characterizing the microbiome and its interactions with the host is the accurate quantification of its composition, i.e. determining which microbes are present and in what numbers they occur. Historically, standard practices have existed for sample handling, DNA extraction and data analysis for many years. However, these were generally developed for single microbe cultures and it is not always feasible to implement them in large scale metagenomic studies. Partly because of this and partly because of the excitement that new technology brings about, the first metagenomic studies each took the liberty to define their own approach and protocols. From early meta-analysis of these studies it became clear that the differences in sample handling, as well as differences in computational approaches, made comparisons across studies very difficult. This restricts our ability to cross-validate findings of individual studies and to pool samples from larger cohorts. To address the pressing need for standardization, we undertook an extensive comparison of 21 different DNA extraction methods as well as a series of other sample manipulations that affect quantification. We developed a number of criteria for determining the measurement quality in the absence of a mock community and used these to propose best practices for sampling, DNA extraction and library preparation. If these were to be accepted as standards in the field, it would greatly improve comparability across studies, which would dramatically increase the power of our inferences and our ability to draw general conclusions about the microbiome. Most metagenomics studies involve comparisons between microbial communities, for example between fecal samples from cases and controls. A multitude of approaches have been proposed to calculate community dissimilarities (beta diversity) and they are often combined with various preprocessing techniques. Direct metagenomics quantification usually counts sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to technology-inherent differences in sampling depth, normalizing counts is necessary, for instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by subsampling. To derive a single value for community (dis-)similarity, multiple distance measures have been proposed. Although it is theoretically difficult to benchmark these approaches, we developed a biologically motivated framework in which distance measures can be evaluated. This highlights the importance of data transformations and their impact on the measured distances. Building on our experience with accurate abundance estimation and data preprocessing techniques, we can now try and understand some of the basic properties of microbial communities. In 2011, it was proposed that the space of genus level variation of the human gut microbial community is structured into three basic types, termed enterotypes. These were described in a multi-country cohort, so as to be independent of geography, age and other host properties. Operationally defined through a clustering approach, they are “densely populated areas in a multidimensional space of community composition”(source) and were proposed as a general stratifier for the human population. Later studies that applied this concept to other datasets raised concerns about the optimum number of clusters and robustness of the clustering approach. This heralded a long standing debate about the existence of structure and the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in the context of the vastly increased amounts of available data. We propose a refined framework in which the different types should be thought of as weak attractors in compositional space and we try to implement an approach to determining which attractor a sample is closest to. To this end, we train a classifier on a reference dataset to assign membership to new samples. This way, enterotypes assignment is no longer dataset dependent and effects due to biased sampling are minimized. Using a model in which we assume the existence of three enterotypes characterized by the same driver genera, as originally postulated, we show the relevance of this stratification and propose it to be used in a clinical setting as a potential marker for disease development. Moreover, we believe that these attractors underline different rules of community assembly and we recommend they be accounted for when analyzing gut microbiome samples. While enterotypes describe structure in the community at genus level, metagenomic sequencing can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis methodology for this level of resolution has only recently been developed and little exploration has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the landscape of genomic variation seems highly structured even beyond species resolution, indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several cases, these subspecies exhibit geo-stratification, with some subspecies only found in the Chinese population. Generally however, they present only minor dispersion limitations and are seen across most of our study populations. Within one individual, one subspecies is commonly found to dominate and only rarely are several subspecies observed to co-occur in the same ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable over periods of more than three years. When interrogating their functional properties we find many differences, with specific ones appearing relevant to the host. For example, we identify a subspecies of E. rectale that is lacking the flagellum operon and find its presence to be significantly associated with lower body mass index and lower insulin resistance of their hosts; it also correlates with higher microbial community diversity. These associations could not be seen at the species level (where multiple subspecies are convoluted), which illustrates the importance of this increased resolution for a more comprehensive understanding of microbial interactions within the microbiome and with the host. Taken together, our results provide a rigorous basis for performing comparative metagenomics of the human gut, encompassing recommendations for both experimental sample processing and computational analysis. We furthermore refine the concept of community stratification into enterotypes, develop a reference-based approach for enterotype assignment and provide compelling evidence for their relevance. Lastly, by harnessing the full resolution of metagenomics, we discover a highly structured genomic variation landscape below the microbial species level and identify common subspecies of the human gut microbiome. By developing these high-precision metagenomics analysis tools, we thus hope to contribute to a greatly improved understanding of the properties and dynamics of the human gut microbiome. N2 - Die mikrobiellen Gemeinschaften innerhalb des menschlichen Darmtrakts – das menschliche Darm-Mikrobiom - sind wichtig für das Wohlbefinden und die Gesundheit des Wirts. Die Charakterisierung dieses neuen “Organs”, welches aus ähnlich vielen Zellen besteht wie der menschliche Körper, ist in jüngster Zeit durch technologische Fortschritte möglich geworden. Die Metagenomik, die direkte Hochdurchsatz-Sequenzierung mikrobieller DNA, ermöglicht die Aufnahme “genomischer Schnappschüsse” tausender verschiedener, in einem komplexen Ökosystem zusammenlebender Bakterien, ohne dafür auf deren Isolierung und Wachstum angewiesen zu sein. Die Quantifizierung des menschlichen Mikrobioms erlaubt es uns, seine Eigenschaften zu untersuchen und Verbindungen zu Wirtsphysiologie und -krankheiten zu knüpfen. Der Reichtum dieser Informationen ist unerwartet hoch und wahrscheinlich noch immer unterbewertet. Aufgrund der Tatsache, dass der Großteil unserer Ernährung und unseres Medikamentenkonsums unser Mikrobiom, welches wiederum selbst über verschiedene Arten mit unserem Immunsystem interagiert, beeinflusst, wurden viele Mechanismen vorgeschlagen, um die beobachteten Korrelationen zu erklären. Die meisten davon sind jedoch noch nicht vollständig verstanden. Eine offensichtliche Komponente zur Charakterisierung des Mikrobioms und dessen Interaktionen mit dem Wirt ist eine akkurate Quantifizierung seiner genauen Zusammensetzung, womit sowohl die Anwesenheit von bestimmten Bakterien als auch deren Anzahl gemeint ist. Obwohl etablierte Standardprozeduren zur Probenbehandlung, DNA- Extrahierung und Datenanalyse existieren, sind sie nicht immer für metagenomische Studien anwendbar, da sie für isolierte Bakterienkulturen entwickelt worden. Deswegen und auch wegen der Begeisterung, die neuartige Technologien mit sich bringen, nahmen sich die ersten metagenomischen Studien jeweils die Freiheit, ihre eigenen Protokolle und Herangehensweisen zu definieren. Die Metaanalyse dieser Studien zeigte, dass Unterschiede sowohl in der Probenbehandlung als auch in der statistischen Auswertung den Vergleich zwischen Studien sehr schwierig machen. Das wiederum beschneidet unsere Fähigkeit, Entdeckungen zu bestätigen und Daten über Studien hinweg zu kombinieren. Um die zwingend notwendige Standardisierung voranzutreiben haben wir einen umfassenden Vergleich von 21 verschiedenen DNA-Extraktionsmethoden sowie verschiedener weiterer Probenbehandlungen, welche Quantifizierungen beeinflussen, vorgenommen. Wir haben eine Reihe von Kriterien entwickelt, um die Messqualität in Abwesenheit von Mock-Kontrollen zu bestimmen und schlagen anhand dieser Methoden für Probenbeschaffung, DNA-Extraktion und Library- Generierung optimale Verfahren vor. Wenn diese als Standard akzeptiert werden, würde das eine stark verbesserte Vergleichbarkeit zwischen Studien ermöglichen und damit sowohl einen extremen Zuwachs an statistischer Power als auch unserer Fähigkeit, generelle Schlüsse über das Mikrobiom zu ziehen, zur Folge haben. Die meisten metagenomischen Studien teilen ihre Datensätze auf um Vergleiche anzustellen, z.B. zwischen Stuhlproben gesunder und erkrankter Menschen. Eine Vielzahl verschiedener Ansätze, welche wiederum oft mit verschiedenen Datenvorbehandlungen kombiniert werden, wurden vorgeschlagen, um Dissimilarität zwischen Gemeinschaften (Beta-Diversität) zu berechnen. Um metagenomische Daten auf Spezies-, Genus- und höheren Ebenen zu quantifizieren werden üblicherweise reads auf Referenzgenome bestimmter taxonomischer Einheiten aligniert und gezählt. Aufgrund technologieabhängiger Unterschiede in Sequenziertiefe müssen reads normalisiert werden, z.B. indem man alle counts durch die Gesamtanzahl der counts einer Sequenzierung teilt (total sum scaling), oder durch subsampling. Für die Messung der Gemeinschafts(dis)similarität wurden viele Distanzmaße vorgeschlagen. Da es schwierig ist diese Ansätze theoretisch zu vergleichen, haben wir ein biologisch motiviertes Konzept entwickelt, mit dem man Distanzmaße evaluieren kann. Dies unterstreicht die Wichtigkeit der Datentransformation und dessen Einwirkung auf Distanzmaße. Aufbauend auf unserer Erfahrung mit Häufigkeitsabschätzungen und Techniken zur Datenvorbehandlung können wir nun versuchen, grundlegende Eigenschaften mikrobieller Gemeinschaften zu verstehen. 2011 wurde vorgeschlagen, dass sich die Variation auf Genusebene im menschlichen Darm auf drei grundlegende Typen beschränkt, welche Enterotypen getauft wurden. Diese wurden in Datensätzen verschiedener Länder als unabhängig von Herkunft, Alter und anderer Wirtseigenschaften beschrieben. Die Enterotypen sind durch einen Cluster-Ansatz als „dicht besiedelte Bereiche in einem multidimensionalen Raum der Gemeinschaftszusammensetzung“ definiert und wurden als grundlegende Stratifikatoren für die menschlichen Population vorgeschlagen. Spätere Studien, welche dieses Konzept auf andere Datensätze anwandten, erhoben Zweifel bezüglich der optimalen Anzahl an Clustern und an der generellen Robustheit des Ansatzes. Dies leitete erneut eine langanhaltende Debate über die Existenz von Strukturen und die besten Wege, diese zu bestimmen und einzufangen, ein. Hier überdenken wir, in Anbetracht der stark gestiegenen Anzahl an verfügbaren Daten, das Enterotypen-Konzept. Wir schlagen ein überarbeitetes Konzept vor, in welchem die verschiedenen Enterotypen als schwache Attraktoren im multidimensionalen Raum verstanden werden und implementieren einen Ansatz zur Berechnung des Attraktors, der dem Datensatz am ähnlichsten ist. Dafür trainieren wir einen Klassifizierer auf einen Referenz- Datensatz, um neue Datensätze zuzuordnen. Damit ist Enterotypisierung nicht mehr datensatzabhängig und der Effekt von sampling bias ist minimiert. Indem wir ein Modell nutzen für das wir die Existenz dreier Enterotypen (definiert durch die selben Genera wie ursprünglich postuliert) annehmen, zeigen wir die Relevanz dieser Stratifikation und schlagen es in einem klinischen Zusammenhang als potentiellen Marker für Krankheitsfortschritt vor. Außerdem glauben wir, dass diese Attraktoren verschiedene Regeln mikrobieller Zusammensetzung widerspiegeln und schlagen vor, sie bei der Analyse von mikrobiellen Daten zu berücksichtigen. Während Enterotypen Struktur in der Gemeinschaft auf Genusebene beschreiben, kann metagenomische Sequenzierung prinzipiell Auflösung auf Nukleotidebene erreichen, womit single nucleotide polymorphisms (SNPs) und andere genomische Variationen im Darm- Mikrobiom identifiziert werden können. Analysemethoden für dieses Auflösungsniveau wurden erst kürzlich entwickelt und bis heute wurden diese erst wenig erforscht. Wir zeigen, dass die Landschaft an genomischer Variation von SNPs in einer großen, multinationalen Kohorte sogar über die Speziesebene hinaus geht und hochgradig strukturiert ist, was das Vorkommen klar abgrenzbarer Subspezies unter Darmmikroben suggeriert. In mehreren Fällen zeigen diese Subspezies geographische Stratifikation, wobei einige Subspezies nur in chinesischen Populationen vorkommen. Im Allgemein zeigen Sie jedoch nur eine geringfügige Beschränkung der Dispersion und sind in der Mehrzahl der Populationen vorhanden. Innerhalb eines Individuums dominiert häufig eine bestimmte Subspezies, nur selten dominieren verschieden gemeinsam im gleichen Ökosystem. Eine Analyse von Zeitreihenexperimenten deutet darauf hin, dass die dominante Subspezies über Zeiträume von mehr als drei Jahren stabil bleibt. Wenn man ihre funktionalen Eigenschaften untersucht findet man viele Unterschiede, von denen bestimmte relevant für den Wirt erscheinen. Zum Beispiel identifizieren wir eine Subspezies von E. rectale, welcher das Flagellum-Operon fehlt, die signifikant assoziiert ist mit geringerem BMI und geringerer Insulinresistenz ihres Wirts; sie korreliert zudem mit höherer mikrobieller Diversität. Diese Assoziationen konnten auf Speziesebene nicht gesehen werden (auf der mehrere Subspezies überlagert sind), was die Wichtigkeit dieser erhöhten Auflösung für ein umfassenderes Verständnis mikrobieller Interaktionen innerhalb des Mikrobioms und mit dem Wirt illustriert. Zusammenfassend bieten unsere Ergebnisse eine präzise Grundlage für vergleichende Metagenomik des menschlichen Darms, einschließlich Empfehlungen über experimentelles Sampling und statistische Analysen. Weiterhin verfeinern wir das Konzept der Enterotypen- Stratifikation in Gemeinschaften, entwickeln referenzbasierte Ansätze für Enterotypen- Zuordnung und bieten überzeugende Beweise für ihre Relevanz. Indem wir die volle Auflösung metagenomischer Sequenzierungen nutzen entdecken wir eine Landschaft hochgradig strukturierter genomischer Variation unterhalb der Speziesebene und identifizieren gemeinsame Subspezies des menschlichen Darm-Mikrobioms. Durch die Entwicklung dieser hochpräzisen metagenomischen Untersuchungsansätze tragen wir zu einem verbesserten KW - metagenomics KW - microbiology KW - Mensch KW - Darmflora KW - Metagenom Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139649 ER - TY - JOUR A1 - Mena, Wilson A1 - Diegelmann, Sören A1 - Wegener, Christian A1 - Ewer, John T1 - Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators JF - eLife N2 - Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors. KW - neuropeptides Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165003 VL - 5 ER - TY - JOUR A1 - Blättner, Sebastian A1 - Das, Sudip A1 - Paprotka, Kerstin A1 - Eilers, Ursula A1 - Krischke, Markus A1 - Kretschmer, Dorothee A1 - Remmele, Christian W. A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Schuelein-Voelk, Christina A1 - Hertlein, Tobias A1 - Mueller, Martin J. A1 - Huettel, Bruno A1 - Reinhardt, Richard A1 - Ohlsen, Knut A1 - Rudel, Thomas A1 - Fraunholz, Martin J. T1 - Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes JF - PLoS Pathogens N2 - Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. KW - cell death KW - cytotoxicity KW - Staphylococcus aureus KW - host cells KW - neutrophils KW - macrophages KW - transposable elements KW - epithelial cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180380 VL - 12 IS - 9 ER - TY - JOUR A1 - Bargul, Joel L. A1 - Jung, Jamin A1 - McOdimba, Francis A. A1 - Omogo, Collins O. A1 - Adung'a, Vincent O. A1 - Krüger, Timothy A1 - Masiga, Daniel K. A1 - Engstler, Markus T1 - Species-Specific Adaptations of Trypanosome Morphology and Motility to the Mammalian Host JF - PLoS Pathogens N2 - African trypanosomes thrive in the bloodstream and tissue spaces of a wide range of mammalian hosts. Infections of cattle cause an enormous socio-economic burden in sub-Saharan Africa. A hallmark of the trypanosome lifestyle is the flagellate’s incessant motion. This work details the cell motility behavior of the four livestock-parasites Trypanosoma vivax, T. brucei, T. evansi and T. congolense. The trypanosomes feature distinct swimming patterns, speeds and flagellar wave frequencies, although the basic mechanism of flagellar propulsion is conserved, as is shown by extended single flagellar beat analyses. Three-dimensional analyses of the trypanosomes expose a high degree of dynamic pleomorphism, typified by the ‘cellular waveform’. This is a product of the flagellar oscillation, the chirality of the flagellum attachment and the stiffness of the trypanosome cell body. The waveforms are characteristic for each trypanosome species and are influenced by changes of the microenvironment, such as differences in viscosity and the presence of confining obstacles. The distinct cellular waveforms may be reflective of the actual anatomical niches the parasites populate within their mammalian host. T. vivax displays waveforms optimally aligned to the topology of the bloodstream, while the two subspecies T. brucei and T. evansi feature distinct cellular waveforms, both additionally adapted to motion in more confined environments such as tissue spaces. T. congolense reveals a small and stiff waveform, which makes these parasites weak swimmers and destined for cell adherence in low flow areas of the circulation. Thus, our experiments show that the differential dissemination and annidation of trypanosomes in their mammalian hosts may depend on the distinct swimming capabilities of the parasites. KW - swimming KW - viscosity KW - flagella KW - host-pathogen interactions KW - cell motility KW - blood KW - parasitic diseases KW - trypanosoma brucei gambiense Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146513 VL - 12 IS - 2 ER - TY - THES A1 - König, Sebastian T1 - Spatially selective visual attention in Drosophila melanogaster T1 - Räumlich selektive visuelle Aufmerksamkeit in Drosophila melanogaster N2 - Finding the right behavior at the right time is one of the major tasks of brains. In a natural scenery there is often an abundance of stimuli present and the brain has to separate the relevant from the irrelevant ones. Selective visual attention (SVA) is a property of higher visual systems that achieves this separation, as it allows to ‘[…] focus on one source of sensory input to the exclusion of others’ (Luck and Mangun, 1996). There are probably several forms of SVA depending upon the criteria used for the separation, such as salience, color, location in space, novelty, or motion. Many studies have investigated SVA in humans and non-human primates. However, complex functions like attention were initially not expected to be already implemented in the brains of simple organisms like Drosophila. After a first demonstration of selective attention in the fly (Wolf and Heisenberg, 1980), it took some time until other studies included attentional mechanisms in their argumentation to explain certain behaviors of Drosophila. However, their definition and characterization of attention differed and often was ambiguous. Here, one particular form, spatially selective visual attention in the fly Drosophila is investigated. It has been shown earlier that the fly spontaneously may restrict its behavioral responses in stationary flight to the visual stimuli on one side of the visual field. On the basis of experiments of Sareen et al., (2011) it has been conjectured that the fly has a focus of attention (FoA) and that the fly responds to the visual stimuli within this area of the visual field. Whether the FoA is the adequate concept for this spatial property of SVA in the fly needs to be further discussed and is a subject also of the present study. At this stage, the concept will be used in the description of the new results expanding the characterization of SVA. This study continued the investigation of SVA during tethered flight with variable but controlled visual input and an automated primary data evaluation. This standardized paradigm allowed for analysis of wild-type behavior as well as for a comparison of several mutant and pharmacologically manipulated strains to the wild-type. Some properties of human SVA like the occurrence of externally as well as internally caused shifts of attention were found in Drosophila and it could be shown, that SVA in the fly can be externally guided and has an attention span. Additionally, a neurotransmitter and proteins, which play a significant role in SVA were discovered. Based on this, the genetic tools available for Drosophila provided the means to a first examination of cells and circuits involved in SVA. Finally, the free walk behavior of flies that had been shown to have compromised SVA was characterized. The results suggested that the observed phenotypes of SVA were not behavior specific. Covert shifts of the FoA were investigated. The FoA can be externally guided by visual cues to one or the other side of the visual field and even after the cue has disappeared it remains there for <4s. An intriguing finding of this study is the fact, that the quality of the cue determines whether it is attractive or repellent. For example a cue can be changed from being repellent (negative) to being attractive (positive) by changing its oscillation amplitude from 4° to 2°. Testing the effectiveness of cues in the upper and lower visual field separately, revealed that the perception of a cue by the fly is not exclusively based on a sum of its specifications. Because positive cueing did not have an after-effect in each of the two half-fields alone, but did so if the cue was shown in both, the fly seems to evaluate the cue for each combination of parameters specifically. Whether this evaluation of the cue changed on a trial-to-trial basis or if the cue in some cases failed to shift the FoA can at this point not be determined. Looking at the responses of the fly to the displacement of a black vertical stripe showed that they can be categorized as no responses, syn-directional responses (following the direction of motion of the stripe) and anti-directional responses (in the opposite direction of the motion of the stripe). The yaw-torque patterns of the latter bared similarities with spontaneous body saccades and they most likely represented escape attempts of the fly. Syn-directional responses, however, were genuine object responses, distinguishable by a longer latency until they were elicited and a larger amplitude. These properties as well as the distribution of response polarities were not influenced by the presence or absence of a cue. When two stripes were displaced simultaneously in opposite directions the rate of no responses increased in comparison to the displacement of a single stripe. If one of the stripes was cued, both, the responses towards and away from the side of cue resembled the syn-directional responses. Significant progress was made with the elucidation of the neuronal underpinnings of SVA. Ablation of the mushroom bodies (MB) demonstrated their requirement for SVA. Furthermore, it was shown that dopamine signaling has to be balanced between too much and too little. Either inhibiting the synthesis of dopamine or its re-uptake at the synapse via the dDAT impaired the flies’ susceptibility to cueing. Using the Gal4/UAS system, cell specific expression or knockdown of the dDAT was used to scrutinize the role of MB sub-compartments in SVA. The αβ-lobes turned out to be necessary and sufficient to maintain SVA. The Gal4-line c708a labels only a subset of Kenyon cells (KC) within the αβ-lobes, αβposterior. These cells stand out, because of (A) the mesh-like arrangement of their fibers within the lobes and (B) the fact that unlike the other KCs they bypass the calyx and thereby the main source of olfactory input to the MBs, forming connections only in the posterior accessory calyx (Tanaka et al., 2008). This structure receives no or only marginal olfactory input, suggesting for it a role in tasks other than olfaction. This study shows their requirement in a visual task by demonstrating that they are necessary to uphold SVA. Restoring dDAT function in these approximately only 90 cells was probably insufficient to lower the dopamine concentration at the relevant synapses and hence a rescue failed. Alternatively, the processes mediating SVA at the αβ-lobes might require an interplay between all of their KCs. In conclusion, the results provide an initial point for future research to fully understand the localization of and circuitry required for SVA in the brain. In the experiments described so far, attention has been externally guided. However, flies are also able to internally shift their FoA without any cues from the outside world. In a set of 60 consecutive simultaneous displacements of two stripes, they were more likely to produce a response with the same polarity as the preceding one than a random polarity selection predicted. This suggested a dwelling of the FoA on one side of the visual field. Assuming that each response was influenced by the previous one in a way that the probability to repeat the response polarity was increased by a certain factor (dwelling factor, df), a random selection of response type including a df was computed. Implementation of the df removed the difference between observed probability of polarity repetition and the one suggested by random selection. When the interval between displacements was iteratively increased to 5s, no significant df could be detected anymore for pauses longer than 4s. In conclusion, Drosophila has an attention span of approximately 4s. Flies with a mutation in the radish gene expressed no after-effect of cueing and had a shortened attention span of about 1s. The dDAT inhibitor methylphenidate is able to rescue the first, but does not affect the latter phenotype. Probably, radish is differently involved in the two mechanisms. This study showed, that endogenous (covert) shifts of spatially selective visual attention in the fly Drosophila can be internally and externally guided. The variables determining the quality of a cue turned out to be multifaceted and a more systematic approach is needed for a better understanding of what property or feature of the cue changes the way it is evaluated by the fly. A first step has been made to demonstrate that SVA is a fundamental process and compromising it can influence the characteristics of other behaviors like walking. The existence of an attention span, the dependence of SVA on dopamine as well as the susceptibility to pharmacological manipulations, which in humans are used to treat respective diseases, point towards striking similarities between SVA in humans and Drosophila. N2 - Eine der Hauptaufgaben eines Gehirns ist es, das richtige Verhalten zur richtigen Zeit zu finden. In einer natürlichen Umgebung gibt es eine Vielzahl visueller Reize, die das Gehirn unterteilen muss in solche, die irrelevant und solche, die bedeutsam sind. Selektive visuelle Aufmerksamkeit (SVA) ist eine Eigenschaft hoch entwickelter visueller Systeme, die diese Unterteilung erzielt, indem sie es erlaubt „[…] eine Quelle sensorischen Inputs zu fokussieren und dabei andere auszuschließen“ (Luck and Mangun, 1996). In Abhängigkeit der Kriterien (z.B. Salienz, Farbe, Lage im Raum, Neuartigkeit oder Bewegung), die für die Aufteilung herangezogen werden, existieren wahrscheinlich mehrere Formen von SVA. Viele Studien haben sich mit SVA in Menschen und in Primaten beschäftigt, ohne jedoch zu erwarten, dass eine komplexe Funktion wie Aufmerksamkeit bereits in den Gehirnen von einfachen Organismen wie Drosophila implementiert zu finden. Erst einige Zeit nachdem selektive Aufmerksamkeit ein erstes Mal in der Fliege gezeigt worden war (Wolf, Heisenberg, 1980) begannen auch andere Studien Aufmerksamkeit in ihrer Argumentation als Erklärung für bestimmte Verhaltensweisen von Drosophila heranzuziehen. Definition und Charakterisierung des Begriffes Aufmerksamkeit waren jedoch oft mehrdeutig und unterschieden sich von Studie zu Studie. In dieser Arbeit wird eine ganz bestimmte Form von Aufmerksamkeit – räumlich selektive visuelle Aufmerksamkeit - anhand der Fliege Drosophila untersucht. Es wurde bereits gezeigt, dass die Fliege im stationären Flug ihre Verhaltensantworten spontan auf visuelle Reize einer Seite des visuellen Feldes beschränken kann. Basierend auf Experimenten von Sareen et al. (2011) wurde vermutet, dass die Fliege einen Aufmerksamkeitsfokus (FoA) besitzt und auf Reize, die innerhalb dieses Teils des visuellen Feldes liegen antwortet. Ob der FoA ein angemessenes Konzept für diese räumliche Eigenschaft von SVA in der Fliege ist, steht zur Debatte und ist auch ein Thema dieser Studie. Vorerst soll dieses Konzept jedoch für die Beschreibung der Ergebnisse, die die Charakterisierung von SVA vorantreiben, genutzt werden. Die vorliegende Arbeit führt die Untersuchung von SVA mit variablem aber kontrolliertem visuellem Input im stationären Flug fort und nutzt dazu eine automatisierte Datenerfassung. Dieses standardisierte Paradigma ermöglicht eine Analyse von Verhalten im Wildtyp aber auch einen Vergleich mit verschiedenen mutanten und pharmakologisch manipulierten Fliegenstämmen. Einige im Menschen auftretende Eigenschaften von SVA wurden auch in Drosophila gefunden. Dazu zählt das Auftreten von extern und intern verursachten Aufmerksamkeitsverlagerungen. Es konnte gezeigt werden, dass SVA in der Fliege extern gelenkt werden kann und eine Aufmerksamkeitsspanne aufweist. Zusätzlich wurden ein Neurotransmitter und einige Proteine entdeckt, die eine wichtige Rolle in SVA einnehmen. Darauf basierend ermöglichten es die verfügbaren genetischen Werkzeuge mit einer ersten Untersuchung der an SVA beteiligten Zellen und Netzwerke zu beginnen. Des Weiteren wurde das Laufverhalten von Fliegen, die Einschränkungen in SVA aufwiesen charakterisiert. Die Ergebnisse lassen vermuten, dass die beobachteten Phänotypen von SVA nicht verhaltensspezifisch sind. Als nächstes wurden interne Bewegungen des Aufmerksamkeitskegels (FoA) betrachtet. Der FoA kann durch visuelle Reize von außerhalb zu der einen oder der anderen Seite des visuellen Feldes gelenkt werden. Er verweilt dort für >4s nachdem der lenkende Reiz verschwunden ist. Es ist ein spannender Befund dieser Arbeit, dass dieser Reiz in Abhängigkeit seiner Beschaffenheit abstoßend oder anziehend sein kann. So kann ein abstoßender (negativer) Reiz auf einmal anziehend (positiv) werden, wenn seine Oszillationsamplitude von 4° auf 2° reduziert wird. Eine Überprüfung der Wirksamkeit von Aufmerksamkeitslenkung durch Reize im oberen und unteren Teil des visuellen Feldes ergab, dass die Wahrnehmung eines Reizes durch die Fliege sich nicht ausschließlich aus der Summe seiner Spezifikationen ergibt. Da positive Aufmerksamkeitslenkung in keinem der beiden Halbfelder einen Nacheffekt hatte, ein solcher aber bei der Präsentation von Reizen in beiden Felder gleichzeitig auftrat, kann vermutet werden, dass die Fliege den Reiz für jede Kombination von Parametern spezifisch bewertet. Ob sich diese Bewertung in jedem einzelnen Durchgang änderte oder ob der Reiz in manchen Fällen den FoA nicht auf eine Seite lenkte kann mit dem jetzigen Kenntnisstand nicht bestimmt werden. Betrachtet man die Antworten der Fliege auf eine Versetzung eines schwarzen vertikalen Streifens, so zeigt sich eine mögliche Unterteilung in die Kategorien „keine Antwort“, „syn-direktionale Antwort“ (der Bewegungsrichtung des Streifens folgend) und „anti-direktionale Antwort“ (entgegengesetzt zur Bewegungsrichtung des Streifens). Die Drehmomentmuster der letzteren Kategorie wiesen starke Ähnlichkeit zu spontanen Körpersakkaden auf und es handelte sich bei ihnen sehr wahrscheinlich um Fluchtversuche der Fliege. Syn-direktionale Antworten waren hingegen reine Objekt-Bewegungsantworten, erkennbar an einer längeren Latenz bis zu ihrer Auslösung und einer größeren Amplitude. Diese Eigenschaften und auch die Verteilung der Antworten auf die beiden Kategorien wurden durch die An- oder Abwesenheit eines vorhergehenden Reizes nicht beeinflusst. Wurden zwei Streifen gleichzeitig gegenläufig versetzt, so blieben die Antworten im Vergleich zur Versetzung eines einzelnen Streifens häufiger aus. Wurde der FoA zuvor auf eine Seite gelenkt, so entsprachen die Drehmomentmuster der Antworten auf diese Seite und auch die der Antworten auf die andere Seite denen der syn-direktionalen Antworten. Die Aufklärung der SVA zu Grunde liegenden neuronalen Strukturen konnte bedeutend vorangetrieben werden. Eine Ablation der Pilzkörper (MB) zeigte, dass diese für SVA benötigt werden. Außerdem konnte gezeigt werden, dass die von Dopamin übermittelte Signalstärke weder zu stark, noch zu schwach sein darf. Wurde die Synthese von Dopamin inhibiert oder seine Wiederaufnahme aus dem synaptischen Spalt mittels dDAT blockiert, führte dies dazu, dass die Aufmerksamkeit dieser Fliegen nicht mehr extern gelenkt werden konnte. Mithilfe des Gal4/UAS-Systems und zellspezifischer Expression oder Unterdrückung der Bildung von dDAT wurde die Rolle einzelner Strukturen der Pilzkörper in SVA genauer untersucht. Es zeigte sich, dass die αβ-Loben sowohl ausreichend als auch notwendig sind, um SVA nachhaltig zu lenken. Die Gal4-Linie c708a markiert einen Teil der Kenyonzellen (KC) innerhalb der αβ-Loben, αβposterior. Diese Zellen sind besonders, da (A) ihre Fasern innerhalb der Loben eine netzartige Anordnung aufweisen und (B) da sie anders als die anderen KCs nicht mit der Kalyx, der größten Quelle olfaktorischen Inputs in die MBs, verknüpft sind, sondern nur in der posterioren akzessorischen Kalyx Verbindungen ausbilden (Tanaka et al., 2008). Diese Struktur erhält keinen oder zumindest nur marginalen olfaktorischen Input und es ist anzunehmen, dass sie eher an Aufgaben aus anderen sensorischen Modalitäten beteiligt ist. In dieser Arbeit wird die Beteiligung dieser Zellen an einem visuellen Task gezeigt, genauer ihre Notwendigkeit für einen Nacheffekt der Lenkung von SVA. Eine Wiederherstellung der Funktion von dDAT in diesen ca. 90 Zellen war erfolglos, da die geringe Anzahl möglicherweise nicht ausreichte, um die Konzentration von Dopamin an den relevanten Synapsen zu senken. Es ist jedoch auch möglich, dass die Prozesse, die SVA über die αβ-Loben vermitteln ein Zusammenspiel aller dortigen KCs erfordern. Zusammen bilden die gesammelten Ergebnisse einen Ausgangspunkt für zukünftige Bestrebungen, die für SVA erforderlichen neuronalen Strukturen und deren Verortung komplett zu verstehen. In den bisher beschriebenen Experimenten wurde die Aufmerksamkeit extern gelenkt. Fliegen können ihren FoA aber auch ganz ohne äußerliche Reize intern verlagern. In einer Reihe von 60 aufeinanderfolgenden gleichzeitigen Versetzungen zweier Streifen zeigte sich, dass die Fliegen häufiger Antworten mit der gleichen Polarität wie die vorausgegangene produzierten, als dies eine zufällige Auswahl der Polarität vorhersagte. Dies ließ vermuten, dass der FoA auf einer Seite des visuellen Feldes verweilt. Es wurde angenommen, dass jede Antwort von der vorhergehenden beeinflusst wird, sodass die Wahrscheinlichkeit die Polarität dieser Antwort zu wiederholen um einen gewissen Faktor erhöht wird (dwelling factor, df). Deswegen wurde eine zufällige Verteilung der Antwortpolaritäten unter Berücksichtigung des df berechnet. Dadurch verschwand der Unterschied zwischen der beobachteten Wiederholungswahrscheinlichkeit einer Antwortpolarität und derer einer rein zufälligen Wahl der Antwort. Als das Intervall zwischen den einzelnen Versetzungen schrittweise auf 5s erhöht wurde, konnte bereits bei Pausen über 4s kein signifikanter df mehr festgestellt werden. Als Schlussfolgerung ergibt sich, dass Drosophila eine Aufmerksamkeitsspanne von etwa 4s besitzt. Fliegen mit einer Mutation im radish Gen zeigten keine anhaltende Lenkung von SVA und hatten zudem eine verkürzte Aufmerksamkeitsspanne von ungefähr 1s. Der dDAT-Inhibitor Methylphenidat beseitigte den zuerst erwähnten Phänotyp, verlängerte jedoch nicht die Aufmerksamkeitsspanne. Es ist anzunehmen, dass radish auf unterschiedliche Art und Weise an beiden Mechanismen beteiligt ist. Im Zuge dieser Arbeit wurde gezeigt, dass endogene (covert) Verlagerungen von räumlich selektiver visueller Aufmerksamkeit in der Fliege Drosophila intern und extern gelenkt werden können. Vielfältige Variablen bestimmen die Beschaffenheit eines Reizes. Es bedarf eines systematischeren Ansatzes, um die Eigenschaften eines Reizes genauer zu verstehen, die dessen Wahrnehmung durch die Fliege verändern. Es konnte bereits grundlegend gezeigt werden, dass SVA ein fundamentaler Prozess ist, dessen Fehlfunktion auch die Eigenschaften anderer Verhaltensweisen wie z.B. Laufen beeinflusst. Die Existenz einer Aufmerksamkeitsspanne, die Abhängigkeit von SVA von Dopamin sowie deren Zugänglichkeit für pharmakologische Manipulationen, deren Nutzen für den Menschen in der Behandlung aufmerksamkeitsbezogener Erkrankungen liegt, deuten auf starke Ähnlichkeiten zwischen SVA in Menschen und in Drosophila hin. KW - Taufliege KW - Visueller Reiz KW - visuell KW - Selektive Wahrnehmung KW - Aufmerksamkeit KW - Drosophila Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134452 ER - TY - JOUR A1 - Kramer, Susanne T1 - Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution JF - Nucleic Acids Research N2 - The detection of mRNAs undergoing transcription or decay is challenging, because both processes are fast. However, the relative proportion of an mRNA in synthesis or decay increases with mRNA size and decreases with mRNA half-life. Based on this rationale, I have exploited a 22 200 nucleotide-long, short-lived endogenous mRNA as a reporter for mRNA metabolism in trypanosomes. The extreme 5΄ and 3΄ ends were labeled with red- and green-fluorescent Affymetrix® single mRNA FISH probes, respectively. In the resulting fluorescence images, yellow spots represent intact mRNAs; red spots are mRNAs in transcription or 3΄-5΄ decay, and green spots are mRNAs in 5΄-3΄ degradation. Most red spots were nuclear and insensitive to transcriptional inhibition and thus likely transcription intermediates. Most green spots were cytoplasmic, confirming that the majority of cytoplasmic decay in trypanosomes is 5΄-3΄. The system showed the expected changes at inhibition of transcription or translation and RNAi depletion of the trypanosome homologue to the 5΄-3΄ exoribonuclease Xrn1. The method allows to monitor changes in mRNA metabolism both on cellular and on population/tissue wide levels, but also to study the subcellular localization of mRNA transcription and decay pathways. I show that the system is applicable to mammalian cells. KW - mRNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148002 ER - TY - JOUR A1 - Jahn, Martin T. A1 - Markert, Sebastian M. A1 - Ryu, Taewoo A1 - Ravasi, Timothy A1 - Stigloher, Christian A1 - Hentschel, Ute A1 - Moitinho-Silva, Lucas T1 - Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling JF - Scientific Reports N2 - Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology. KW - high resolution visualisation KW - transcriptional profiling KW - FISH-CLEM KW - cell compartmentation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167513 VL - 6 IS - 35860 ER - TY - JOUR A1 - Singh, Krishna P. A1 - Verma, Neeraj A1 - Akhoon, Bashir A . A1 - Bhatt, Vishal A1 - Gupta, Shishir K. A1 - Gupta, Shailendra K. A1 - Smita, Suchi T1 - Sequence-based approach for rapid identification of cross-clade CD8+ T-cell vaccine candidates from all high-risk HPV strains JF - 3 Biotech N2 - Human papilloma virus (HPV) is the primary etiological agent responsible for cervical cancer in women. Although in total 16 high-risk HPV strains have been identified so far. Currently available commercial vaccines are designed by targeting mainly HPV16 and HPV18 viral strains as these are the most common strains associated with cervical cancer. Because of the high level of antigenic specificity of HPV capsid antigens, the currently available vaccines are not suitable to provide cross-protection from all other high-risk HPV strains. Due to increasing reports of cervical cancer cases from other HPV high-risk strains other than HPV16 and 18, it is crucial to design vaccine that generate reasonable CD8+ T-cell responses for possibly all the high-risk strains. With this aim, we have developed a computational workflow to identify conserved cross-clade CD8+ T-cell HPV vaccine candidates by considering E1, E2, E6 and E7 proteins from all the high-risk HPV strains. We have identified a set of 14 immunogenic conserved peptide fragments that are supposed to provide protection against infection from any of the high-risk HPV strains across globe. KW - HPV KW - Epitope KW - Cytotoxic KW - T lymphocytes KW - Cervical cancer KW - Vaccine Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191056 VL - 6 ER - TY - JOUR A1 - Heurich, Marco A1 - Zeis, Klara A1 - Küchenhoff, Helmut A1 - Müller, Jörg A1 - Belotti, Elisa A1 - Bufka, Luděk A1 - Woelfing, Benno T1 - Selective Predation of a Stalking Predator on Ungulate Prey JF - PLoS ONE N2 - Prey selection is a key factor shaping animal populations and evolutionary dynamics. An optimal forager should target prey that offers the highest benefits in terms of energy content at the lowest costs. Predators are therefore expected to select for prey of optimal size. Stalking predators do not pursue their prey long, which may lead to a more random choice of prey individuals. Due to difficulties in assessing the composition of available prey populations, data on prey selection of stalking carnivores are still scarce. We show how the stalking predator Eurasian lynx (Lynx lynx) selects prey individuals based on species identity, age, sex and individual behaviour. To address the difficulties in assessing prey population structure, we confirm inferred selection patterns by using two independent data sets: (1) data of 387 documented kills of radio-collared lynx were compared to the prey population structure retrieved from systematic camera trapping using Manly’s standardized selection ratio alpha and (2) data on 120 radio-collared roe deer were analysed using a Cox proportional hazards model. Among the larger red deer prey, lynx selected against adult males—the largest and potentially most dangerous prey individuals. In roe deer lynx preyed selectively on males and did not select for a specific age class. Activity during high risk periods reduced the risk of falling victim to a lynx attack. Our results suggest that the stalking predator lynx actively selects for size, while prey behaviour induces selection by encounter and stalking success rates. KW - stalking predators KW - prey selection KW - Lynx lynx Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166827 VL - 11 IS - 8 ER - TY - JOUR A1 - Senthilan, Pingkalai R. A1 - Helfrich-Förster, Charlotte T1 - Rhodopsin 7-The unusual Rhodopsin in Drosophila JF - PeerJ N2 - Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1–Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a “vertebrate-melanopsin-type”–cluster, and Rh3, Rh4 and Rh5 form an “insect-type”-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins. KW - vision KW - Drosophila KW - Opsins KW - Rhodopsins KW - phototransduction Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177998 VL - 4 ER - TY - JOUR A1 - Hassouna, I. A1 - Ott, C. A1 - Wüstefeld, L. A1 - Offen, N. A1 - Neher, R. A. A1 - Mitkovski, M. A1 - Winkler, D. A1 - Sperling, S. A1 - Fries, L. A1 - Goebbels, S. A1 - Vreja, I. C. A1 - Hagemeyer, N. A1 - Dittrich, M. A1 - Rossetti, M. F. A1 - Kröhnert, K. A1 - Hannke, K. A1 - Boretius, S. A1 - Zeug, A. A1 - Höschen, C. A1 - Dandekar, T. A1 - Dere, E. A1 - Neher, E. A1 - Rizzoli, S. O. A1 - Nave, K.-A. A1 - Sirén, A.-L. A1 - Ehrenreich, H. T1 - Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus JF - Molecular Psychiatry N2 - Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of similar to 20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a \(^{15}\)N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated \(^{15}\)N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration. KW - neural stem-cells KW - recombinat-human-erythropoietin KW - cognitive functions KW - pyramidal neurons KW - nervous-sytem KW - brain-injury KW - mouse-brain KW - progenitors KW - mice KW - memory Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186669 VL - 21 IS - 12 ER - TY - JOUR A1 - Adolfi, Mateus C. A1 - Herpin, Amaury A1 - Regensburger, Martina A1 - Sacquegno, Jacopo A1 - Waxman, Joshua S. A1 - Schartl, Manfred T1 - Retinoic acid and meiosis induction in adult versus embryonic gonads of medaka JF - Scientific Reports N2 - In vertebrates, one of the first recognizable sex differences in embryos is the onset of meiosis, known to be regulated by retinoic acid (RA) in mammals. We investigated in medaka a possible meiotic function of RA during the embryonic sex determination (SD) period and in mature gonads. We found RA mediated transcriptional activation in germ cells of both sexes much earlier than the SD stage, however, no such activity during the critical stages of SD. In adults, expression of the RA metabolizing enzymes indicates sexually dimorphic RA levels. In testis, RA acts directly in Sertoli, Leydig and pre-meiotic germ cells. In ovaries, RA transcriptional activity is highest in meiotic oocytes. Our results show that RA plays an important role in meiosis induction and gametogenesis in adult medaka but contrary to common expectations, not for initiating the first meiosis in female germ cells at the SD stage. KW - developmental biology KW - molecular biology Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147843 VL - 6 ER - TY - JOUR A1 - Endres, Marcel A1 - Kneitz, Susanne A1 - Orth, Martin F. A1 - Perera, Ruwan K. A1 - Zernecke, Alma A1 - Butt, Elke T1 - Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1) JF - Oncotarget N2 - The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines. By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines. In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown. Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression. The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies. KW - LASP1 KW - c-Fos KW - extracellular matrix KW - AP-1 KW - matrix metalloproteinases KW - breast cancer Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176920 VL - 7 IS - 39 ER - TY - JOUR A1 - Dreschers, Stephan A1 - Saupp, Peter A1 - Hornef, Mathias A1 - Prehn, Andrea A1 - Platen, Christopher A1 - Morschhäuser, Joachim A1 - Orlikowsky, Thorsten W. T1 - Reduced PICD in Monocytes Mounts Altered Neonate Immune Response to Candida albicans JF - PLoS ONE N2 - Background Invasive fungal infections with Candida albicans (C. albicans) occur frequently in extremely low birthweight (ELBW) infants and are associated with poor outcome. Phagocytosis of C.albicans initializes apoptosis in monocytes (phagocytosis induced cell death, PICD). PICD is reduced in neonatal cord blood monocytes (CBMO). Hypothesis Phagocytosis of C. albicans causes PICD which differs between neonatal monocytes (CBMO) and adult peripheral blood monocytes (PBMO) due to lower stimulation of TLR-mediated immune responses. Methods The ability to phagocytose C. albicans, expression of TLRs, the induction of apoptosis (assessment of sub-G1 and nick-strand breaks) were analyzed by FACS. TLR signalling was induced by agonists such as lipopolysaccharide (LPS), Pam3Cys, FSL-1 and Zymosan and blocked (neutralizing TLR2 antibodies and MYD88 inhibitor). Results Phagocytic indices of PBMO and CBMO were similar. Following stimulation with agonists and C. albicans induced up-regulation of TLR2 and consecutive phosphorylation of MAP kinase P38 and expression of TNF-α, which were stronger on PBMO compared to CBMO (p < 0.005). Downstream, TLR2 signalling initiated caspase-3-dependent PICD which was found reduced in CBMO (p < 0.05 vs PBMO). Conclusion Our data suggest direct involvement of TLR2-signalling in C. albicans-induced PICD in monocytes and an alteration of this pathway in CBMO. KW - Candida albicans KW - monocytes KW - immune response KW - PICD Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166778 VL - 11 IS - 11 ER - TY - JOUR A1 - Konte, Tilen A1 - Terpitz, Ulrich A1 - Plemenitaš, Ana T1 - Reconstruction of the High-Osmolarity Glycerol (HOG) Signaling Pathway from the Halophilic Fungus Wallemia ichthyophaga in Saccharomyces cerevisiae JF - Frontiers in Microbiology N2 - The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like other fungi, W. ichthyophaga detects changes in environmental salinity mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with protein modeling data, our study reveals conserved and non-conserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus. KW - signaling KW - protein-protein interaction KW - protein phosphorylation KW - mitogen activated protein kinase (MAPK) KW - high-osmolarity glycerol (HOG) KW - signaling pathway KW - Saccharomyces cerevisiae KW - halophilic fungus KW - Wallemia ichthyophaga Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165214 ER - TY - JOUR A1 - Hölldobler, Bert T1 - Queen Specific Exocrine Glands in Legionary Ants and Their Possible Function in Sexual Selection JF - PLoS ONE N2 - The colonies of army ants and some other legionary ant species have single, permanently wingless queens with massive post petioles and large gasters. Such highly modified queens are called dichthadiigynes. This paper presents the unusually rich exocrine gland endowment of dichthadiigynes, which is not found in queens of other ant species. It has been suggested these kinds of glands produce secretions that attract and maintain worker retinues around queens, especially during migration. However, large worker retinues also occur in non-legionary species whose queens do not have such an exuberance of exocrine glands. We argue and present evidence in support of our previously proposed hypothesis that the enormous outfit of exocrine glands found in dichthadiigynes is due to sexual selection mediated by workers as the main selecting agents KW - exocrine glands KW - dichthadiigynes KW - legionary ants KW - queens KW - sexual selection KW - army ants Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167057 VL - 11 IS - 3 ER - TY - JOUR A1 - Diao, Wenwen A1 - Mousset, Mathilde A1 - Horsburgh, Gavin J. A1 - Vermeulen, Cornelis J. A1 - Johannes, Frank A1 - van de Zande, Louis A1 - Ritchie, Michael G. A1 - Schmitt, Thomas A1 - Beukeboom, Leo W. T1 - Quantitative Trait Locus Analysis of Mating Behavior and Male Sex Pheromones in Nasonia Wasps JF - G3: Genes Genomes Genetics N2 - A major focus in speciation genetics is to identify the chromosomal regions and genes that reduce hybridization and gene flow. We investigated the genetic architecture of mating behavior in the parasitoid wasp species pair Nasonia giraulti and Nasonia oneida that exhibit strong prezygotic isolation. Behavioral analysis showed that N. oneida females had consistently higher latency times, and broke off the mating sequence more often in the mounting stage when confronted with N. giraulti males compared with males of their own species. N. oneida males produce a lower quantity of the long-range male sex pheromone (4R,5S)-5-hydroxy-4-decanolide (RS-HDL). Crosses between the two species yielded hybrid males with various pheromone quantities, and these males were used in mating trials with females of either species to measure female mate discrimination rates. A quantitative trait locus (QTL) analysis involving 475 recombinant hybrid males (F2), 2148 reciprocally backcrossed females (F3), and a linkage map of 52 equally spaced neutral single nucleotide polymorphism (SNP) markers plus SNPs in 40 candidate mating behavior genes revealed four QTL for male pheromone amount, depending on partner species. Our results demonstrate that the RS-HDL pheromone plays a role in the mating system of N. giraulti and N. oneida, but also that additional communication cues are involved in mate choice. No QTL were found for female mate discrimination, which points at a polygenic architecture of female choice with strong environmental influences. KW - Nasonia courtship KW - female choice KW - sex pheromone KW - QTL analysis KW - speciation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165412 VL - 6 IS - 6 ER - TY - JOUR A1 - Dejung, Mario A1 - Subota, Ines A1 - Bucerius, Ferdinand A1 - Dindar, Gülcin A1 - Freiwald, Anja A1 - Engstler, Markus A1 - Boshart, Michael A1 - Butter, Falk A1 - Janzen, Chistian J. T1 - Quantitative proteomics uncovers novel factors involved in developmental differentiation of Trypanosoma brucei JF - PLoS Pathogens N2 - Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes. KW - cell differentiation KW - cell cycle and cell division KW - parasitic cell cycles KW - proteomes KW - chromatin KW - parasitic life cycles KW - transcriptome analysis KW - host-pathogen interactions Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146362 VL - 12 IS - 2 ER - TY - JOUR A1 - Vendelova, Emilia A1 - de Lima, Jeferson Camargo A1 - Lorenzatto, Karina Rodrigues A1 - Monteiro, Karina Mariante A1 - Mueller, Thomas A1 - Veepaschit, Jyotishman A1 - Grimm, Clemens A1 - Brehm, Klaus A1 - Hrčková, Gabriela A1 - Lutz, Manfred B. A1 - Ferreira, Henrique B. A1 - Nono, Justin Komguep T1 - Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions JF - PLoS Neglected Tropical Diseases N2 - Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts. KW - proteomic analysis KW - excretory-secretory KW - Mesocestoides corti Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166742 VL - 10 IS - 10 ER - TY - JOUR A1 - Peters, Marcell K. A1 - Hemp, Andreas A1 - Appelhans, Tim A1 - Behler, Christina A1 - Classen, Alice A1 - Detsch, Florian A1 - Ensslin, Andreas A1 - Ferger, Stefan W. A1 - Frederiksen, Sara B. A1 - Gebert, Frederike A1 - Haas, Michael A1 - Helbig-Bonitz, Maria A1 - Hemp, Claudia A1 - Kindeketa, William J. A1 - Mwangomo, Ephraim A1 - Ngereza, Christine A1 - Otte, Insa A1 - Röder, Juliane A1 - Rutten, Gemma A1 - Costa, David Schellenberger A1 - Tardanico, Joseph A1 - Zancolli, Giulia A1 - Deckert, Jürgen A1 - Eardley, Connal D. A1 - Peters, Ralph S. A1 - Rödel, Mark-Oliver A1 - Schleuning, Matthias A1 - Ssymank, Axel A1 - Kakengi, Victor A1 - Zhang, Jie A1 - Böhning-Gaese, Katrin A1 - Brandl, Roland A1 - Kalko, Elisabeth K.V. A1 - Kleyer, Michael A1 - Nauss, Thomas A1 - Tschapka, Marco A1 - Fischer, Markus A1 - Steffan-Dewenter, Ingolf T1 - Predictors of elevational biodiversity gradients change from single taxa to the multi-taxa community level JF - Nature Communications N2 - The factors determining gradients of biodiversity are a fundamental yet unresolved topic in ecology. While diversity gradients have been analysed for numerous single taxa, progress towards general explanatory models has been hampered by limitations in the phylogenetic coverage of past studies. By parallel sampling of 25 major plant and animal taxa along a 3.7 km elevational gradient on Mt. Kilimanjaro, we quantify cross-taxon consensus in diversity gradients and evaluate predictors of diversity from single taxa to a multi-taxa community level. While single taxa show complex distribution patterns and respond to different environmental factors, scaling up diversity to the community level leads to an unambiguous support for temperature as the main predictor of species richness in both plants and animals. Our findings illuminate the influence of taxonomic coverage for models of diversity gradients and point to the importance of temperature for diversification and species coexistence in plant and animal communities. KW - community ecology KW - macroecology KW - tropical ecology KW - biodiversity Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-169374 VL - 7 ER - TY - JOUR A1 - De Palma, Adriana A1 - Abrahamczyk, Stefan A1 - Aizen, Marcelo A. A1 - Albrecht, Matthias A1 - Basset, Yves A1 - Bates, Adam A1 - Blake, Robin J. A1 - Boutin, Céline A1 - Bugter, Rob A1 - Connop, Stuart A1 - Cruz-López, Leopoldo A1 - Cunningham, Saul A. A1 - Darvill, Ben A1 - Diekötter, Tim A1 - Dorn, Silvia A1 - Downing, Nicola A1 - Entling, Martin H. A1 - Farwig, Nina A1 - Felicioli, Antonio A1 - Fonte, Steven J. A1 - Fowler, Robert A1 - Franzen, Markus Franzén A1 - Goulson, Dave A1 - Grass, Ingo A1 - Hanley, Mick E. A1 - Hendrix, Stephen D. A1 - Herrmann, Farina A1 - Herzog, Felix A1 - Holzschuh, Andrea A1 - Jauker, Birgit A1 - Kessler, Michael A1 - Knight, M. E. A1 - Kruess, Andreas A1 - Lavelle, Patrick A1 - Le Féon, Violette A1 - Lentini, Pia A1 - Malone, Louise A. A1 - Marshall, Jon A1 - Martínez Pachón, Eliana A1 - McFrederick, Quinn S. A1 - Morales, Carolina L. A1 - Mudri-Stojnic, Sonja A1 - Nates-Parra, Guiomar A1 - Nilsson, Sven G. A1 - Öckinger, Erik A1 - Osgathorpe, Lynne A1 - Parra-H, Alejandro A1 - Peres, Carlos A. A1 - Persson, Anna S. A1 - Petanidou, Theodora A1 - Poveda, Katja A1 - Power, Eileen F. A1 - Quaranta, Marino A1 - Quintero, Carolina A1 - Rader, Romina A1 - Richards, Miriam H. A1 - Roulston, T’ai A1 - Rousseau, Laurent A1 - Sadler, Jonathan P. A1 - Samnegård, Ulrika A1 - Schellhorn, Nancy A. A1 - Schüepp, Christof A1 - Schweiger, Oliver A1 - Smith-Pardo, Allan H. A1 - Steffan-Dewenter, Ingolf A1 - Stout, Jane C. A1 - Tonietto, Rebecca K. A1 - Tscharntke, Teja A1 - Tylianakis, Jason M. A1 - Verboven, Hans A. F. A1 - Vergara, Carlos H. A1 - Verhulst, Jort A1 - Westphal, Catrin A1 - Yoon, Hyung Joo A1 - Purvis, Andy T1 - Predicting bee community responses to land-use changes: Effects of geographic and taxonomic biases JF - Scientific Reports N2 - Land-use change and intensification threaten bee populations worldwide, imperilling pollination services. Global models are needed to better characterise, project, and mitigate bees' responses to these human impacts. The available data are, however, geographically and taxonomically unrepresentative; most data are from North America and Western Europe, overrepresenting bumblebees and raising concerns that model results may not be generalizable to other regions and taxa. To assess whether the geographic and taxonomic biases of data could undermine effectiveness of models for conservation policy, we have collated from the published literature a global dataset of bee diversity at sites facing land-use change and intensification, and assess whether bee responses to these pressures vary across 11 regions (Western, Northern, Eastern and Southern Europe; North, Central and South America; Australia and New Zealand; South East Asia; Middle and Southern Africa) and between bumblebees and other bees. Our analyses highlight strong regionally-based responses of total abundance, species richness and Simpson's diversity to land use, caused by variation in the sensitivity of species and potentially in the nature of threats. These results suggest that global extrapolation of models based on geographically and taxonomically restricted data may underestimate the true uncertainty, increasing the risk of ecological surprises. KW - bee community KW - land-use change KW - intensification KW - geographic biases KW - taxonomic biases KW - global dataset Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167642 VL - 6 ER - TY - JOUR A1 - Drakulić, Sanja A1 - Feldhaar, Heike A1 - Lisičić, Duje A1 - Mioč, Mia A1 - Cizelj, Ivan A1 - Seiler, Michael A1 - Spatz, Theresa A1 - Rödel, Mark-Oliver T1 - Population-specific effects of developmental temperature on body condition and jumping performance of a widespread European frog JF - Ecology and Evolution N2 - All physiological processes of ectotherms depend on environmental temperature. Thus, adaptation of physiological mechanisms to the thermal environments is important for achieving optimal performance and fitness. The European Common Frog, Rana temporaria, is widely distributed across different thermal habitats. This makes it an exceptional model for studying the adaptations to different thermal conditions. We raised tadpoles from Germany and Croatia at two constant temperature treatments (15°C, 20°C), and under natural temperature fluctuations (in outdoor treatments), and tested how different developmental temperatures affected developmental traits, that is, length of larval development, morphometrics, and body condition, as well as jumping performance of metamorphs. Our results revealed population‐specific differences in developmental time, body condition, and jumping performance. Croatian frogs developed faster in all treatments, were heavier, in better body condition, and had longer hind limbs and better jumping abilities than German metamorphs. The populations further differed in thermal sensitivity of jumping performance. While metamorphs from Croatia increased their jumping performance with higher temperatures, German metamorphs reached their performance maximum at lower temperatures. These population‐specific differences in common environments indicate local genetic adaptation, with southern populations being better adapted to higher temperatures than those from north of the Alps. KW - Amphibians KW - ectotherms KW - physiological traits KW - plasticity KW - thermal adaptation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164960 VL - 6 IS - 10 ER - TY - JOUR A1 - Kramer, Susanne A1 - Piper, Sophie A1 - Estevez, Antonio A1 - Carrington, Mark T1 - Polycistronic trypanosome mRNAs are a target for the exosome JF - Molecular and Biochemical Parasitology N2 - Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNA5 from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5'-3' exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNA5. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. KW - Trypanosoma brucei KW - Exosome KW - NMD KW - Polycistronic mRNA KW - trans-splicing KW - Trypanosomes Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191350 VL - 205 IS - 1-2 ER - TY - JOUR A1 - Mildner, Stephanie A1 - Roces, Flavio T1 - Plasticity of Daily Behavioral Rhythms in Foragers and Nurses of the Ant Camponotus rufipes: Influence of Social Context and Feeding Times JF - PLoS One N2 - Daily activities within an ant colony need precise temporal organization, and an endogenous clock appears to be essential for such timing processes. A clock drives locomotor rhythms in isolated workers in a number of ant species, but its involvement in activities displayed in the social context is unknown. We compared locomotor rhythms in isolated individuals and behavioral rhythms in the social context of workers of the ant Camponotus rufipes. Both forager and nurse workers exhibited circadian rhythms in locomotor activity under constant conditions, indicating the involvement of an endogenous clock. Activity was mostly nocturnal and synchronized with the 12:12h light-dark-cycle. To evaluate whether rhythmicity was maintained in the social context and could be synchronized with non-photic zeitgebers such as feeding times, daily behavioral activities of single workers inside and outside the nest were quantified continuously over 24 hours in 1656 hours of video recordings. Food availability was limited to a short time window either at day or at night, thus mimicking natural conditions of temporally restricted food access. Most foragers showed circadian foraging behavior synchronized with food availability, either at day or nighttime. When isolated thereafter in single locomotor activity monitors, foragers mainly displayed arrhythmicity. Here, high mortality suggested potential stressful effects of the former restriction of food availability. In contrast, nurse workers showed high overall activity levels in the social context and performed their tasks all around the clock with no circadian pattern, likely to meet the needs of the brood. In isolation, the same individuals exhibited in turn strong rhythmic activity and nocturnality. Thus, endogenous activity rhythms were inhibited in the social context, and timing of daily behaviors was flexibly adapted to cope with task demands. As a similar socially-mediated plasticity in circadian rhythms was already shown in honey bees, the temporal organization in C. rufipes and honey bees appear to share similar basic features. KW - honey bees KW - biological locomotion KW - foraging KW - circadian rhythms KW - chronobiology KW - insects KW - nurses KW - ants Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148010 VL - 12 IS - 1 ER - TY - JOUR A1 - Viera, Jonathan Trujillo A1 - El-Merahbi, Rabih A1 - Nieswandt, Bernhard A1 - Stegner, David A1 - Sumara, Grzegorz T1 - Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight JF - PLoS ONE N2 - Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1\(^{-/-}\) and Pld2\(^{-/-}\) mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes. KW - enzyme regulation KW - insulin resistance KW - body weight KW - mouse models KW - bioenergetics KW - insulin KW - hypothalamus KW - adipose tissue Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179729 VL - 11 IS - 6 ER - TY - JOUR A1 - Joschinski, Jens A1 - Beer, Katharina A1 - Helfrich-Förster, Charlotte A1 - Krauss, Jochen T1 - Pea Aphids (Hemiptera: Aphididae) Have Diurnal Rhythms When Raised Independently of a Host Plant JF - Journal of Insect Science N2 - Seasonal timing is assumed to involve the circadian clock, an endogenous mechanism to track time and measure day length. Some debate persists, however, and aphids were among the first organisms for which circadian clock involvement was questioned. Inferences about links to phenology are problematic, as the clock itself is little investigated in aphids. For instance, it is unknown whether aphids possess diurnal rhythms at all. Possibly, the close interaction with host plants prevents independent measurements of rhythmicity. We reared the pea aphid Acyrthosiphon pisum (Harris) on an artificial diet, and recorded survival, moulting, and honeydew excretion. Despite their plant-dependent life style, aphids were independently rhythmic under light–dark conditions. This first demonstration of diurnal aphid rhythms shows that aphids do not simply track the host plant’s rhythmicity. KW - artificial diet KW - circadian clock KW - hourglass clock KW - Acyrthosiphon pisum KW - photoperiodism Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168783 VL - 16 IS - 1 ER - TY - JOUR A1 - Lichthardt, Sven A1 - Kerscher, Alexander A1 - Dietz, Ulrich A. A1 - Jurowich, Christian A1 - Kunzmann, Volker A1 - von Rahden, Burkhard H. A. A1 - Germer, Christoph-Thomas A1 - Wiegering, Armin T1 - Original article: role of adjuvant chemotherapy in a perioperative chemotherapy regimen for gastric cancer JF - BMC Cancer N2 - Background Multimodal treatment strategies – perioperative chemotherapy (CTx) and radical surgery – are currently accepted as treatment standard for locally advanced gastric cancer. However, the role of adjuvant postoperative CTx (postCTx) in addition to neoadjuvant preoperative CTx (preCTx) in this setting remains controversial. Methods Between 4/2006 and 12/2013, 116 patients with locally advanced gastric cancer were treated with preCTx. 72 patients (62 %), in whom complete tumor resection (R0, subtotal/total gastrectomy with D2-lymphadenectomy) was achieved, were divided into two groups, one of which receiving adjuvant therapy (n = 52) and one without (n = 20). These groups were analyzed with regard to survival and exclusion criteria for adjuvant therapy. Results Postoperative complications, as well as their severity grade, did not correlate with fewer postCTx cycles administered (p = n.s.). Long-term survival was shorter in patients receiving postCTx in comparison to patients without postCTx, but did not show statistical significance. In per protocol analysis by excluding two patients with perioperative death, a shorter 3-year survival rate was observed in patients receiving postCTx compared to patients without postCTx (3-year survival: 71.2 % postCTx group vs. 90.0 % non-postCTx group; p = 0.038). Conclusion These results appear contradicting to the anticipated outcome. While speculative, they question the value of post-CTx. Prospectively randomized studies are needed to elucidate the role of postCTx. KW - gastric cancer KW - chemotherapy KW - neoadjuvant KW - multimodal KW - complication KW - adjuvant KW - risk factor KW - survival Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147743 VL - 16 IS - 650 ER - TY - JOUR A1 - Feldbauer, Katrin A1 - Schlegel, Jan A1 - Weissbecker, Juliane A1 - Sauer, Frank A1 - Wood, Phillip G. A1 - Bamberg, Ernst A1 - Terpitz, Ulrich T1 - Optochemokine Tandem for Light-Control of Intracellular Ca\(^{2+}\) JF - PLoS ONE N2 - An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca\(^{2+}\)-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca\(^{2+}\) by tandem endosomes into the cytosol via CatCh was visualized using the Ca\(^{2+}\)-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca\(^{2+}\) in response to light. KW - capacitance KW - endosomes KW - cell membranes KW - membrane proteins KW - intracellular membranes KW - vesicles KW - confocal laser microscopy KW - cytosol Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178921 VL - 11 IS - 10 ER -