TY - JOUR A1 - Bensaad, Karim A1 - Favaro, Elena A1 - Lewis, Caroline A. A1 - Peck, Barrie A1 - Lord, Simon A1 - Collins, Jennifer M. A1 - Pinnick, Katherine E. A1 - Wigfield, Simon A1 - Buffa, Francesca M. A1 - Li, Ji-Liang A1 - Zhang, Qifeng A1 - Wakelam, Michael J. O. A1 - Karpe, Fredrik A1 - Schulze, Almut A1 - Harris, Adrian L. T1 - Fatty Acid Uptake and Lipid Storage Induced by HIF-1 alpha Contribute to Cell Growth and Survival after Hypoxia-Reoxygenation JF - Cell Reports N2 - An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1 alpha (HIF-1 alpha)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O-2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via beta-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo. KW - inducible factor-I KW - binding protein KW - triglyceride accumulation KW - cancer cell KW - complex-III KW - beta-oxidation KW - metabolism KW - lipogenesis KW - proliferation KW - resistance Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115162 SN - 2211-1247 VL - 9 IS - 1 ER - TY - JOUR A1 - Akhoon, Bashir A. A1 - Singh, Krishna P. A1 - Varshney, Megha A1 - Gupta, Shishir K. A1 - Shukla, Yogeshwar A1 - Gupta, Shailendra K. T1 - Understanding the Mechanism of Atovaquone Drug Resistance in Plasmodium falciparum Cytochrome b Mutation Y268S Using Computational Methods JF - PLOS ONE N2 - The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance. KW - molecular-dynamics simulations KW - HIV-1 protease KW - structure prediction KW - saccharomyces cerevisiae KW - I-tasser KW - inhibitors KW - binding KW - malaria KW - complex KW - protein-protein interactions Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114882 VL - 9 IS - 10 ER - TY - JOUR A1 - Senecal, Jean-Luc A1 - Isabelle, Catherine A1 - Fritzler, Marvin J. A1 - Targoff, Ira N. A1 - Goldstein, Rose A1 - Gagne, Michel A1 - Raynauld, Jean-Pierre A1 - Joyal, France A1 - Troyanov, Yves A1 - Dabauvalle, Marie-Christine T1 - An Autoimmune Myositis-Overlap Syndrome Associated With Autoantibodies to Nuclear Pore Complexes Description and Long-Term Follow-up of the Anti-Nup Syndrome JF - Medicine N2 - Autoimmune myositis encompasses various myositis-overlap syndromes, each being identified by the presence of serum marker autoantibodies. We describe a novel myositis-overlap syndrome in 4 patients characterized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes. The clinical phenotype was characterized by prominent myositis in association with erosive, anti-CCP, and rheumatoid factor-positive arthritis, trigeminal neuralgia, mild interstitial lung disease, Raynaud phenomenon, and weight loss. The myositis was typically chronic, relapsing, and refractory to corticosteroids alone, but remitted with the addition of a second immuno-modulating drug. There was no clinical or laboratory evidence for liver disease. The prognosis was good with 100% long-term survival (mean follow-up 19.5 yr). By indirect immunofluorescence on HEp-2 cells, sera from all 4 patients displayed a high titer of antinuclear autoantibodies (ANA) with a distinct punctate peripheral (rim) fluorescent pattern of the nuclear envelope characteristic of nuclear pore complexes. Reactivity with nuclear pore complexes was confirmed by immunoelectron microscopy. In a cohort of 100 French Canadian patients with autoimmune myositis, the nuclear pore complex fluorescent ANA pattern was restricted to these 4 patients (4%). It was not observed in sera from 393 adult patients with systemic sclerosis (n = 112), mixed connective tissue disease (n = 35), systemic lupus (n = 94), rheumatoid arthritis (n = 45), or other rheumatic diseases (n = 107), nor was it observed in 62 normal adults. Autoantibodies to nuclear pore complexes were predominantly of IgG isotype. No other IgG autoantibody markers for defined connective tissue diseases or overlap syndromes were present, indicating a selective and highly focused immune response. In 3 patients, anti-nuclear pore complex autoantibody titers varied in parallel with myositis activity, suggesting a pathogenic link to pathophysiology. The nuclear pore complex proteins, that is, nucleoporins (nup), recognized by these sera were heterogeneous and included Nup358/RanBP2 (n = 2 patients), Nup90 (n = 1), Nup62 (n = 1), and gp210 (n = 1). Taken together the data suggest that nup autoantigens themselves drive the anti-nup autoimmune response. Immunogenetically, the 4 patients shared the DQA1*0501 allele associated with an increased risk for autoimmune myositis. In conclusion, we report an apparent novel subset of autoimmune myositis in our population of French Canadian patients with connective tissue diseases. This syndrome is recognized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes that react with nups, consistent with an "anti-nupsyndrome.'' KW - idiopathic inflammatory myopathies KW - primary biliary-cirrhosis KW - transfer RNA-synthetases KW - major histocompatibility complex KW - systemic sclerosis KW - French-Canadian patients KW - protein KW - predictive factors KW - envelope KW - antibodies Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114829 SN - 0025-7974 VL - 93 IS - 24 ER - TY - JOUR A1 - Hudson, Lawrence N. A1 - Newbold, Tim A1 - Contu, Sara A1 - Hill, Samantha L. L. A1 - Lysenko, Igor A1 - De Palma, Adriana A1 - Phillips, Helen R. P. A1 - Senior, Rebecca A. A1 - Bennett, Dominic J. A1 - Booth, Hollie A1 - Choimes, Argyrios A1 - Correia, David L. P. A1 - Day, Julie A1 - Echeverria-Londono, Susy A1 - Garon, Morgan A1 - Harrison, Michelle L. K. A1 - Ingram, Daniel J. A1 - Jung, Martin A1 - Kemp, Victoria A1 - Kirkpatrick, Lucinda A1 - Martin, Callum D. A1 - Pan, Yuan A1 - White, Hannah J. A1 - Aben, Job A1 - Abrahamczyk, Stefan A1 - Adum, Gilbert B. A1 - Aguilar-Barquero, Virginia A1 - Aizen, Marcelo A1 - Ancrenaz, Marc A1 - Arbelaez-Cortes, Enrique A1 - Armbrecht, Inge A1 - Azhar, Badrul A1 - Azpiroz, Adrian B. A1 - Baeten, Lander A1 - Báldi, András A1 - Banks, John E. A1 - Barlow, Jos A1 - Batáry, Péter A1 - Bates, Adam J. A1 - Bayne, Erin M. A1 - Beja, Pedro A1 - Berg, Ake A1 - Berry, Nicholas J. A1 - Bicknell, Jake E. A1 - Bihn, Jochen H. A1 - Böhning-Gaese, Katrin A1 - Boekhout, Teun A1 - Boutin, Celine A1 - Bouyer, Jeremy A1 - Brearley, Francis Q. A1 - Brito, Isabel A1 - Brunet, Jörg A1 - Buczkowski, Grzegorz A1 - Buscardo, Erika A1 - Cabra-Garcia, Jimmy A1 - Calvino-Cancela, Maria A1 - Cameron, Sydney A. A1 - Cancello, Eliana M. A1 - Carrijo, Tiago F. A1 - Carvalho, Anelena L. A1 - Castro, Helena A1 - Castro-Luna, Alejandro A. A1 - Cerda, Rolando A1 - Cerezo, Alexis A1 - Chauvat, Matthieu A1 - Clarke, Frank M. A1 - Cleary, Daniel F. R. A1 - Connop, Stuart P. A1 - D'Aniello, Biagio A1 - da Silva, Pedro Giovani A1 - Darvill, Ben A1 - Dauber, Jens A1 - Dejean, Alain A1 - Diekötter, Tim A1 - Dominguez-Haydar, Yamileth A1 - Dormann, Carsten F. A1 - Dumont, Bertrand A1 - Dures, Simon G. A1 - Dynesius, Mats A1 - Edenius, Lars A1 - Elek, Zoltán A1 - Entling, Martin H. A1 - Farwig, Nina A1 - Fayle, Tom M. A1 - Felicioli, Antonio A1 - Felton, Annika M. A1 - Ficetola, Gentile F. A1 - Filgueiras, Bruno K. C. A1 - Fonte, Steve J. A1 - Fraser, Lauchlan H. A1 - Fukuda, Daisuke A1 - Furlani, Dario A1 - Ganzhorn, Jörg U. A1 - Garden, Jenni G. A1 - Gheler-Costa, Carla A1 - Giordani, Paolo A1 - Giordano, Simonetta A1 - Gottschalk, Marco S. A1 - Goulson, Dave A1 - Gove, Aaron D. A1 - Grogan, James A1 - Hanley, Mick E. A1 - Hanson, Thor A1 - Hashim, Nor R. A1 - Hawes, Joseph E. A1 - Hébert, Christian A1 - Helden, Alvin J. A1 - Henden, John-André A1 - Hernández, Lionel A1 - Herzog, Felix A1 - Higuera-Diaz, Diego A1 - Hilje, Branko A1 - Horgan, Finbarr G. A1 - Horváth, Roland A1 - Hylander, Kristoffer A1 - Horváth, Roland A1 - Isaacs-Cubides, Paola A1 - Ishitani, Mashiro A1 - Jacobs, Carmen T. A1 - Jaramillo, Victor J. A1 - Jauker, Birgit A1 - Jonsell, Matts A1 - Jung, Thomas S. A1 - Kapoor, Vena A1 - Kati, Vassiliki A1 - Katovai, Eric A1 - Kessler, Michael A1 - Knop, Eva A1 - Kolb, Annette A1 - Körösi, Àdám A1 - Lachat, Thibault A1 - Lantschner, Victoria A1 - Le Féon, Violette A1 - LeBuhn, Gretchen A1 - Légaré, Jean-Philippe A1 - Letcher, Susan G. A1 - Littlewood, Nick A. A1 - López-Quintero, Carlos A. A1 - Louhaichi, Mounir A1 - Lövei, Gabor L. A1 - Lucas-Borja, Manuel Esteban A1 - Luja, Victor H. A1 - Maeto, Kaoru A1 - Magura, Tibor A1 - Mallari, Neil Aldrin A1 - Marin-Spiotta, Erika A1 - Marhall, E. J. P. A1 - Martínez, Eliana A1 - Mayfield, Margaret M. A1 - Mikusinski, Gregorz A1 - Milder, Jeffery C. A1 - Miller, James R. A1 - Morales, Carolina L. A1 - Muchane, Mary N. A1 - Muchane, Muchai A1 - Naidoo, Robin A1 - Nakamura, Akihiro A1 - Naoe, Shoji A1 - Nates-Parra, Guiomar A1 - Navarerete Gutierrez, Dario A. A1 - Neuschulz, Eike L. A1 - Noreika, Norbertas A1 - Norfolk, Olivia A1 - Noriega, Jorge Ari A1 - Nöske, Nicole M. A1 - O'Dea, Niall A1 - Oduro, William A1 - Ofori-Boateng, Caleb A1 - Oke, Chris O. A1 - Osgathorpe, Lynne M. A1 - Paritsis, Juan A1 - Parrah, Alejandro A1 - Pelegrin, Nicolás A1 - Peres, Carlos A. A1 - Persson, Anna S. A1 - Petanidou, Theodora A1 - Phalan, Ben A1 - Philips, T. Keith A1 - Poveda, Katja A1 - Power, Eileen F. A1 - Presley, Steven J. A1 - Proença, Vânia A1 - Quaranta, Marino A1 - Quintero, Carolina A1 - Redpath-Downing, Nicola A. A1 - Reid, J. Leighton A1 - Reis, Yana T. A1 - Ribeiro, Danilo B. A1 - Richardson, Barbara A. A1 - Richardson, Michael J. A1 - Robles, Carolina A. A1 - Römbke, Jörg A1 - Romero-Duque, Luz Piedad A1 - Rosselli, Loreta A1 - Rossiter, Stephen J. A1 - Roulston, T'ai H. A1 - Rousseau, Laurent A1 - Sadler, Jonathan P. A1 - Sáfián, Szbolcs A1 - Saldaña-Vásquez, Romeo A. A1 - Samnegård, Ulrika A1 - Schüepp, Christof A1 - Schweiger, Oliver A1 - Sedlock, Jodi L. A1 - Shahabuddin, Ghazala A1 - Sheil, Douglas A1 - Silva, Fernando A. B. A1 - Slade, Eleanor A1 - Smith-Pardo, Allan H. A1 - Sodhi, Navjot S. A1 - Somarriba, Eduardo J. A1 - Sosa, Ramón A. A1 - Stout, Jane C. A1 - Struebig, Matthew J. A1 - Sung, Yik-Hei A1 - Threlfall, Caragh G. A1 - Tonietto, Rebecca A1 - Tóthmérész, Béla A1 - Tscharntke, Teja A1 - Turner, Edgar C. A1 - Tylianakis, Jason M. A1 - Vanbergen, Adam J. A1 - Vassilev, Kiril A1 - Verboven, Hans A. F. A1 - Vergara, Carlos H. A1 - Vergara, Pablo M. A1 - Verhulst, Jort A1 - Walker, Tony R. A1 - Wang, Yanping A1 - Watling, James I. A1 - Wells, Konstans A1 - Williams, Christopher D. A1 - Willig, Michael R. A1 - Woinarski, John C. Z. A1 - Wolf, Jan H. D. A1 - Woodcock, Ben A. A1 - Yu, Douglas W. A1 - Zailsev, Andreys A1 - Collen, Ben A1 - Ewers, Rob M. A1 - Mace, Georgina M. A1 - Purves, Drew W. A1 - Scharlemann, Jörn P. W. A1 - Pervis, Andy T1 - The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts JF - Ecology and Evolution N2 - Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species' threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project - and avert - future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups - including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems - ). We make site-level summary data available alongside this article. The full database will be publicly available in 2015. KW - urban-rural gradient KW - instensively managed farmland KW - Mexican coffee plantations KW - Bombus Spp. Hymenoptera KW - bumblebee nest density KW - data sharing KW - land use KW - habitat destruction KW - global change KW - land-use change KW - plant community composition KW - Northeastern Costa Rica KW - dung beetle coleoptera KW - bird species richness Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114425 VL - 4 IS - 24 ER - TY - JOUR A1 - Rozycka, Miroslawa A1 - Wojtas, Magdalena A1 - Jakob, Michal A1 - Stigloher, Christian A1 - Grzeszkowiak, Mikolaj A1 - Mazur, Maciej A1 - Ozyhar, Andrzej T1 - Intrinsically Disordered and Pliable Starmaker-Like Protein from Medaka (Oryzias latipes) Controls the Formation of Calcium Carbonate Crystals JF - PLOS ONE N2 - Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed. KW - circular-dichroism KW - unstructured proteins KW - olyelectrolyte domains KW - modulating KW - biominarlization proteins KW - nacreous layer formation KW - alpha-helical structure KW - dye stains-all KW - polyelectrolyte domains KW - phosphorylation sites KW - procambarus-clarkii KW - secondary structure Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114251 SN - 1932-6203 VL - 9 IS - 12 ER - TY - THES A1 - Sibilski, Claudia T1 - Identification and characterization of the novel mKSR1 phosphorylation site Tyr728 and its role in MAPK signaling T1 - Identifizierung und Charakterisierung der neuartigen mKSR1-Phosphorylierungsstelle Tyr728 und deren Rolle in der MAPK-Signalkaskade N2 - In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work. Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases. Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity. Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1. Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses. N2 - KSR1 fungiert bei Säugetieren als zentrales Gerüstprotein, welches die Anordnung von RAF/MEK/ERK-Komplexen koordiniert und die intrazelluläre Signalweiterleitung nach extrazellulärer Stimulation reguliert. Eine abweichende Aktivierung des entsprechenden MAPK-Signalwegs wurde mit vielen humanen Krebsformen und einigen Entwicklungsstörungen in Verbindung gebracht. Der Mechanismus der KSR1-Regulierung ist hochgradig komplex und involviert mehrfach Schritte der Phosphorylierung/Dephosphorylierung. In der vorliegenden Studie wurden etliche neue in-vivo-Phosphorylierungsstellen in mKSR1 mittels massenspektrometrischer Analyse entdeckt. Neben anderen wurde Tyr728 als besonderer regulatorischer Rest identifiziert, welcher durch LCK, einem Mitglied der Src-Kinase-Familie, phosphoryliert wird. Um zu verstehen wie die Phosphorylierung von Tyr728 die Funktion von KSR1 innerhalb der Signalweiterleitung und zellulärer Prozesse regulieren könnte, wurden strukturelle Modellierungen und biochemische Untersuchungen in diese Arbeit integriert. Die Computermodellierung der mKSR1(KD)-Proteinstruktur zeigte starke Wasserstoff- brückenbindungen zwischen Phospho-Tyr728 und den Resten in der Umgebung von Arg649 auf. Dieses Muster war auffällig verändert, wenn Tyr728 nicht phosphoryliert oder substituiert war. Wie anhand biochemischer Analyse untermauert wurde, könnte Arg649 für phospho-Tyr728 als Hauptankerpunkt dienen, um interne Strukturen in KSR1 zu stabilisieren. In Übereinstimmung mit den Ergebnissen der Proteinmodellierung enthüllten die Mutationsstudien, dass die Substitution von Tyr728 mit Phenylalanin zu einer weniger kompakten Interaktion zwischen KSR1 und MEK, einer erleichterten KSR1/B-RAF-Bindung und einer ansteigenden Phosphorylierung von MEK im Komplex mit KSR1 führt. Anhand dieser Erkenntnisse kann man rückschließen, dass Phospho-Tyr728 in die Verstärkung der Interaktionen innerhalb der KSR1/MEK-Grenzfläche und in die Regulierung der Phosphorylierung von KSR1-gebundenem MEK durch entweder RAF- oder KSR1-Kinasen involviert ist. Neben Tyr728 wurde Ser722 als eine neuartige regulatorische Phosphorylierungsstelle identifiziert. Aminosäureaustausche an der betreffenden Position demonstrierten, dass Ser722 die Phosphorylierung von KSR1-gebundenem MEK reguliert ohne die KSR1/MEK-Bindung selbst zu beeinträchtigen. Bedingt durch seine Lokalisierung könnte Ser722 folglich die katalytische Aktivität von KSR1 kontrollieren, indem es den Zugang des Substrates (möglicherweise MEK) zur aktiven Seite der KSR1-Kinase behindert. Zusammen mit Ser722 könnte phosphoryliertes Tyr728 ferner die Kinaseaktivität von KSR1 positiv beeinflussen, infolge von dessen Nähe zur Aktivierungs- und katalytischen Schleife in der KSR1(KD). Wie mittels Strukturmodellierung offengelegt wurde, bildet Phospho-Tyr728 eine Wasserstoffbrücke mit dem hochgradig konservierten Lys685 aus. Folglich hat Phospho-Tyr728 einen stabilisierenden Effekt auf interne Strukturen, welche in die katalytische Reaktion involviert sind, und erleichtert möglicherweise den Phosphattransfer innerhalb der katalytischen Spalte in KSR1. In Anbetracht dieser Fakten scheint es sehr wahrscheinlich, dass die LCK-abhängige Phosphorylierung von Tyr728 eine äußerst wichtige Rolle in der Regulierung der katalytischen Aktivität von KSR1 spielt. Die Ergebnisse der Fraktionierungs- und Morphologieanalysen enthüllten, dass KSR1 für die Phosphorylierung an Tyr728 LCK zum Zytoskelett rekrutiert, was darauf hindeutet, dass dieser Rest die Dynamik des Zytoskeletts und folglich Zellmotilität regulieren könnte. Darüber hinaus ist die Phosphorylierung von Tyr728 in die Regulierung der Zellproliferation involviert, wie anhand einer bedeutend reduzierten Populationsverdopplungszeit von KSR1-Y728F-Zellen im Vergleich zu Zellen, welche wildtypisches KSR1 exprimieren, gezeigt wurde. Zusammenfassend lässt sich sagen, dass die Tyrosin-Phosphorylierung in KSR1 eine neue Verknüpfung zwischen Kinasen der Src-Familie und der MAPK-Signalwirkung enthüllt. Tyr728, die neuartige regulatorische Phosphorylierungsstelle in Maus-KSR1, könnte den Übergang zwischen der Gerüst- und der katalytischen Funktion von KSR1 koordinieren und damit als Kontrollpunkt dienen, um zelluläre Reaktionen fein abzustimmen. KW - MAP-Kinase KW - Signaltransduktion KW - Regulation KW - tyrosine phosphorylation KW - KSR1 KW - LCK KW - MAPK KW - phosphorylation KW - signaling Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114672 ER - TY - THES A1 - Wenzel, Jens T1 - Regulation of TLR-induced macrophage responses by cytoskeleton-associated phosphoproteins T1 - Regulation der Antwort von Makrophagen auf TLR-Stimulation durch Zytoskelett-assoziierte Phosphoproteine N2 - Toll-like receptors (TLR) are pattern recognition receptors (PRR) by which macrophages (MØ) sense pathogen-associated molecular patterns (PAMPs). The recognition of lipopolysaccharide (LPS), the PAMP of gram negative bacteria, by TLR4 triggers signaling cascades and leads to the pro-inflammatory activation of the cells. A recent quantitative and kinetic analysis of the phosphoproteome of LPS-activated primary macrophages highlighted the cytoskeleton as a cell compartment with an enriched protein phosphorylation. In total 44 cytoskeleton-associated proteins were regulated by this post-translational modification and thus might be involved in the control and regulation of key macrophage functions like spreading, motility and phagocytosis. To investigate the control of cytoskeleton-associated cell functions by TLR4 activation, we first developed a method to quantitatively measure the spreading response of bone marrow MØ after stimulation with LPS. Fluorescence microscopy was used for cell imaging and visualisation of the MØ contact area. In collaboration with the Fraunhofer Institute Erlangen, we developed and validated a software tool for the semi-automated segmentation and quantitation of MØ fluorescence microscopy data, which allowed fast, robust and objective image analysis. Using this method, we observed that LPS caused time-dependent spreading, which was detectable after 1-2 h and maximal after 24 h. Next, the impact of genetic or pharmacological inhibition of known TLR signaling components was investigated. Deficiency in the adapter protein MYD88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of ERK1/2 signaling, indicating that ERK1/2 mediates MYD88-dependent MØ spreading. In contrast, MØ lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation. The genetic deletion of the MAPK phosphatases DUSP1 and DUSP16 resulted in impaired late spreading, corroborating the essential role for functional MAPK signaling in TLR4-driven MØ spreading. To identify the contribution of other cytoskeletal phosphoproteins to MØ spreading, siRNA knockdown of selected candidate genes in primary murine MØ was employed and combined with automated quantitative image analysis. These experiments revealed a functional role for the Myosins MYO1e and MYO1f in MØ spreading. These motor proteins are strongly phosphorylated in LPS-activated MØ. Because of their ability to simultaneously bind to actin filaments and cell membrane or other proteins, we investigated their role in phagocytosis, cytokine production and antigen presentation. Phagocytosis and killing of bacteria were not affected in Myo1e-/- macrophages. However, MYO1e plays a role in chemokine secretion and antigen presentation processes. MCP1 (CCL2) release was selectively increased in Myo1e-deficient MØ and dendritic cells (DC), while cytokine secretion was unaffected. Furthermore, macrophages and DCs lacking MYO1e showed lower levels of MHC-II on the cell surface. However, mRNA levels of CCL2 and of MHC-II were unaltered. These data suggest a role for MYO1e in the transport of selected chemokines and of MHC-II molecules to the cell surface. MHC-II-restricted antigen presentation assays revealed an impaired capacity of macrophages and DC lacking MYO1e to stimulate antigen-specific T cells, suggesting that the reduced MHC-II expression is functionally relevant. Taken together, in this study first a quantitative image analysis method was developed which allows the unbiased, robust and efficient investigation of the macrophage spreading response. Combination of this method with siRNA knockdown of selected cytoskeleton-associated phosphoproteins led to the identification of MYO1e and MYO1f as regulators of macrophage spreading. Furthermore, we identified MYO1e in MØ and DC to be essential for the intracellular transport of CCL2 and MHC-II to the cell surface and for optimal stimulation of antigen-specific CD4 T cells. N2 - Toll-like Rezeptoren (TLR) sind Mustererkennungsrezeptoren (PRR) durch die Makrophagen (MØ) pathogen-assoziierte molekulare Muster (PAMPs) erkennen. Die Erkennung von Lipopolysacchariden (LPS), dem PAMP gramnegativer Bakterien, durch TLR4 löst Signalkaskaden aus, die zu einer pro-inflammatorischen Aktivierung der Zellen führen. Eine quantitative und kinetische Analyse des Phosphoproteoms LPS-aktivierter primärer Makrophagen identifizierte das Zytoskelett als ein Zellkompartiment mit gesteigerter Proteinphosphorylierung. Insgesamt wurden 44 Zytoskelett-assoziierte Proteine identifiziert, die durch diese post-translationale Modifikation reguliert wurden und demzufolge an der Regulation wichtiger Zellfunktionen von Makrophagen wie Spreading, Motilität und Phagozytose beteiligt sein könnten. Um die Kontrolle Zytoskelett-vermittelter Zellfunktionen nach TLR4 Aktivierung zu untersuchen, entwickelten wir zunächst eine Methode zur quantitativen Messung der Spreadingantwort von Knochenmarksmakrophagen nach LPS Stimulation. Die Visualisierung der Zellen sowie ihrer Kontaktfläche erfolgte hierbei mittels Fluoreszenzmikroskopie. Für eine schnelle, robuste und objektive Analyse der Fluoreszenzaufnahmen entwickelten und validierten wir in Kollaboration mit dem Fraunhofer Institut in Erlangen eine Software zur halbautomatischen Segmentierung und Quantifizierung der Kontaktfläche. Unter Verwendung dieser Methode konnte eine zeitabhängige LPS-induzierte Zunahme der Zellkontaktfläche beobachtet werden, die nach 1-2 Stunden detektierbar war und ein Maximum nach 24 Stunden erreichte. Durch den Einsatz pharmakologischer Inhibitoren sowie genetisch veränderter Zellen wurde anschließend der Einfluss bekannter TLR4-Signalwegkomponenten untersucht. Die genetische Defizienz des Adapterproteins MYD88 führte hierbei zu einer stark reduzierten Spreadingaktivität der Zellen während der späten LPS Stimulationsphase, wohingegen das initiale Spreading nicht beeinflusst wurde. Ein vergleichbarer Effekt konnte unter Verwendung eines pharmakologischen Inhibitors zur Hemmung des ERK1/2 Signalweges identifiziert werden. Diese Beobachtungen deuten darauf hin, dass ERK1/2 für die Weiterleitung des MYD88 vermittelten Spreading notwendig ist. Im Gegensatz dazu wurde in p38-defizienten Makrophagen ein beeinträchtigtes initiales Spreading beobachtet, wohingegen das späte Spreading nach 8 – 24 Stunden nicht beeinflusst war. Die genetische Deletion der MAPK Phosphatasen DUSP1 und DUSP16 resultierte ebenfalls in einer Minderung des späten Spreadings, ebenfalls ein Hinweis auf die essentielle Rolle funktioneller MAPK Signalwege. Um die Beteiligung weiter Zytoskelett-Phosphoproteine am Zellspreading zu identifizieren, wurde die Expression ausgewählter Kandidatengene in primären Makrophagen mittels spezifischer siRNA unterdrückt und das Zellspreading mit Hilfe der entwickelten Software quantifiziert. Diese Versuche zeigten eine funktionelle Rolle der Myosine MYO1e und MYO1f. Diese Motorproteine weisen ebenfalls eine starke Phosphorylierung nach LPS Stimulation auf. Aufgrund ihrer Eigenschaft simultan mit Aktinfilamenten und Zellmembranen sowie anderen Proteinen zu interagieren, untersuchten wir ihre Rolle während der Phagozytose, Zytokinfreisetzung und Antigenpräsentation. Obwohl Myo1e defiziente Makrophagen keine Beeinträchtigung der Phagozytose oder Abtötung von Bakterien aufwiesen, spielte das Motorprotein eine wichtige Rolle in der Chemokinfreisetzung und Antigenpräsentation. Interessanterweise war die Sekretion des Chemokins MCP1 (CCL2) in Myo1e-defizienten Makrophagen und dendritischen Zellen (DC) selektiv erhöht, während die Zytokinfreisetzung unbeeinträchtigt war. Des Weiteren wiesen Myo1e KO Makrophagen und DC eine reduzierte MHC-II Oberflächen-Expression auf, obwohl die MHC-II als auch die CCL2 Transkription auf mRNA Ebene nicht beeinflusst war. Diese Daten legen nahe, dass MYO1e während des Transports bestimmter Chemokine, sowie von MHC-II zur Zelloberfläche eine wichtige Rolle spielt. Zudem zeigten Myo1e KO Makrophagen und DC in einem MHC-II-abhängigen Antigenpräsentationsassay eine abgeschwächte Fähigkeit zur Antigen-spezifischen T-Zell Aktivierung, was die funktionelle Relevanz der reduzierten Expression von MHC-II nahelegt. Zusammenfassend wurde in dieser Studie zunächst eine Methode zur quantitativen Bildanalyse entwickelt, welche eine unvoreingenommene, robuste und effiziente Untersuchung des Spreadings von Makrophagen erlaubte. Die Kombination dieser Methode mit dem spezifischen siRNA Knockdown ausgewählter Zytoskelett-assoziierter Phosphoproteine führte zur Identifizierung von MYO1e und MYO1f als wichtige Regulatoren dieser Zellfunktion. Darüber hinaus konnte in Makrophagen und DC eine essentielle Rolle für MYO1e im intrazellulären Transport von CCL2 und MHC-II an die Zelloberfläche identifiziert werden, sowie dessen Notwendigkeit für eine vollständige Aktivierung antigen-spezifischer CD4 T Zellen. KW - Toll-like-Rezeptoren KW - Makrophage KW - Phosphoproteine KW - Zellskelett KW - macrophage KW - cytoskeleton KW - phosphorylation KW - TLR4 Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-98843 ER - TY - JOUR A1 - Remmele, Christian W. A1 - Xian, Yibo A1 - Albrecht, Marco A1 - Faulstich, Michaela A1 - Fraunholz, Martin A1 - Heinrichs, Elisabeth A1 - Dittrich, Marcus T. A1 - Müller, Tobias A1 - Reinhardt, Richard A1 - Rudel, Thomas T1 - Transcriptional landscape and essential genes of Neisseria gonorrhoeae N2 - The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes. KW - Neisseria gonorrhoeae Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113676 ER - TY - JOUR A1 - Linder, Bastian A1 - Hirmer, Anja A1 - Gal, Andreas A1 - Rüther, Klaus A1 - Bolz, Hanno Jörn A1 - Winkler, Christoph A1 - Laggerbauer, Bernhard A1 - Fischer, Utz T1 - Identification of a PRPF4 Loss-of-Function Variant That Abrogates U4/U6.U5 Tri-snRNP Integration and Is Associated with Retinitis Pigmentosa N2 - Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina. KW - zebrafish KW - embryos KW - immunoprecipitation KW - arginine KW - messenger RNA KW - spliceosomes KW - mutation KW - RNA splicing Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113663 ER - TY - JOUR A1 - Vergho, Daniel Claudius A1 - Kneitz, Susanne A1 - Kalogirou, Charis A1 - Burger, Maximilian A1 - Krebs, Markus A1 - Rosenwald, Andreas A1 - Spahn, Martin A1 - Löser, Andreas A1 - Kocot, Arkadius A1 - Riedmiller, Hubertus A1 - Kneitz, Burkhard T1 - Impact of miR-21, miR-126 and miR-221 as Prognostic Factors of Clear Cell Renal Cell Carcinoma with Tumor Thrombus of the Inferior Vena Cava N2 - Clear cell renal cell carcinoma (ccRCC) characterized by a tumor thrombus (TT) extending into the inferior vena cava (IVC) generally indicates poor prognosis. Nevertheless, the risk for tumor recurrence after nephrectomy and thrombectomy varies. An applicable and accurate prediction system to select ccRCC patients with TT of the IVC (ccRCC/TT) at high risk after nephrectomy is urgently needed, but has not been established up to now. To our knowledge, a possible role of microRNAs (miRs) for the development of ccRCC/TT or their impact as prognostic markers in ccRCC/TT has not been explored yet. Therefore, we analyzed the expression of the previously described onco-miRs miR-200c, miR-210, miR-126, miR-221, let-7b, miR-21, miR-143 and miR-141 in a study collective of 74 ccRCC patients. Using the expression profiles of these eight miRs we developed classification systems that accurately differentiate ccRCC from non-cancerous renal tissue and ccRCC/TT from tumors without TT. In the subgroup of 37 ccRCC/TT cases we found that miR-21, miR-126, and miR-221 predicted cancer related death (CRD) accurately and independently from other clinico-pathological features. Furthermore, a combined risk score based on the expression of miR-21, miR-126 and miR-221 was developed and showed high sensitivity and specificity to predict cancer specific survival (CSS) in ccRCC/TT. Using the combined risk score we were able to classify ccRCC/TT patients correctly into high and low risk cases. The risk stratification by the combined risk score (CRS) will benefit from further cohort validation and might have potential for clinical application as a molecular prediction system to identify high- risk ccRCC/TT patients. KW - forecasting KW - metastasis KW - renal cancer KW - renal cell carcinoma KW - kidneys KW - surgical oncology KW - surgical and invasive medical procedures KW - regression analysis Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113633 ER - TY - THES A1 - Gjorgjevikj, Maja T1 - IL-4 analogues with site-specific chemical modification as screening tools for foldamers T1 - IL-4-Muteine mit ortsspezifische chemische Modifikation als Screening-Tools für Foldamere N2 - The cytokine Interleukin-4 (IL-4) plays a crucial role in the pathophysiology and progression of asthma and other atopic diseases. Its activities are signaled into the cells upon binding to and signaling through a shared receptor complex composed of the subunits IL-4Rα and common γc. Another cytokine, Interleukin-13 shares many functions with IL-4. This can be explained by the fact that both, IL-4 and IL-13, can signal via a shared receptor complex comprising the IL-4R and the IL-13R1 subunit. Therefore, the IL-4Rα receptor subunit has become a highly promising drug target, since it mediates IL-4 and IL-13 responses and blocking IL-4Rα will abrogate IL-4 as well as IL-13 effector functions. Currently, an IL-4 based mutein (Pitrakinra), acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. This work describes the generation and production of biologically active IL-4 muteins, which contain a single additional engineered cysteine. The introduction of a free thiol group allows site-specific chemical modification. The muteins were expressed in E. coli in insoluble form, refolded and purified. The thiol group of the mutein was protected as mixed disulfide with the tripeptide glutathione. A first attempt to chemically reduce the engineered cysteine residue failed, because the three native disulfide bonds of IL-4 exhibit a similar reactivity and chemical reduction of the native disulfide resulted in full deactivation and precipitation of the IL-4 protein. Therefore, an enzymatic approach was developed which specifically reduces the mixed disulfide bonds with an attached glutathion moiety and thus leaves the native structurally essential disulfide bonds unaltered. For optimization, four different IL-4 cysteine muteins with four cysteine residues introduced at positions close to the IL-4Rα binding site were tested and their reduction rates by glutaredoxin was determined. The enzymatic reduction occured at different rates for all four muteins indicating that accessibility is an important influence and must be determined individually for each mutant protein. After optimization of the pH value and particularly the reaction time, all muteins could be prepared with the engineered thiol group being released in reasonable yield. The proteins exhibiting the free thiol group were then modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effects of these modifications at different positions on binding to IL-4R were measured employing SPR biosensor technology. In the second project of this study, foldamers, which represent a new class of stable, compactly folded biomolecules and can specifically interact with proteins and nucleic acids, were examined to identify their potential as new drugs to interfere with IL-4 activities. Fragment-based drug discovery offers great promise for providing new starting points for drug discovery and facilitates the lead optimization. As foldamers equipped with a thiol-group for tethering could not to be produced; only the effect of foldamers present in a synthesized foldamer library on the binding to IL-4R could be tested. Two libraries containing different foldamers based on aromatic amide were synthesized by Michael Grotz and Dr. Michael Deligny and tested in our lab for their capability to disrupt the ligand-receptor interaction of IL-4 and its receptor IL-4Rα [ECD] using surface plasmon resonance technology. None of the studied foldamers could specifically inhibit the IL-4/IL-4Rα interaction. Some foldamers showed non-specific binding. The study presented here shows the design and production of a potentially new type of IL-4 antagonists, which employ site-specific chemical modification to exert their antagonistic function. N2 - Das Zytokin Interleukin-4 (IL-4) spielt eine entscheidende Rolle in der Entstehung und Pathophysiologie von Asthma und anderen atopischen Krankheiten. Seine Aktivitäten können in die Zelle durch die Bindung an einen Rezeptorkomplex übertragen werden, welcher aus den Untereinheiten IL-4Rα und γc besteht. Interleukin-13 (IL-13), ein verwandtes Zytokin, und IL-4 besitzen viele gemeinsame Funktionen. Das kann dadurch erklärt werden, dass IL-4 wie auch IL-13 ihre Signale über einen gemeinsamen Rezeptorkomplex übertragen können, der aus der IL-4R und der IL-13R1 Untereinheit besteht. Die IL-4R Untereinheit ist ein vielversprechendes Zielmolekül für die Entwicklung von Pharmaka, da sie IL-4 und IL-13 Reaktionen vermittelt. Durch Blockieren von IL-4R werden die Aktivitäten von IL-4 sowie IL-13 unterdrückt. Ein IL-4 basiertes Doppelmutein (Pitrakinra), welches als Gegenspieler zu IL-4 und IL-13 Rezeptoren fungiert, befindet sich derzeit in der klinischen Entwicklung. In dieser Arbeit wird die Bildung und Produktion von biologisch aktiven IL-4 Muteinen mit einem einzelnen zusätzlich eingefügten Cysteinrest beschrieben. Die Einführung einer freien Thiol-Gruppe ermöglicht ortsspezifische chemische Modifizierungen. Ein „Tethering“ Ansatz sollte dann auch eine sehr schwach Bindung von thiol-reaktiven Verbindungen an IL-4 messbar machen. Die Muteine wurden in unlöslicher Form in E. coli exprimiert, zurückgefaltet und auf gereinigt. Dabei wurde die Thiolgruppe des Muteins als Disulfid mit dem Tripeptid Glutathion geschützt. Erste Versuche gezielt den eingeführten Cysteinrest selektiv chemisch zu reduzieren schlugen fehl, da die drei proteineigenen Disulfidbrücken von IL-4 eine ähnliche Reaktivität zeigten, und die Reduktion zur vollständigen Desaktivierung und Fällung des IL-4 Proteins führte. Daher wurde ein enzymatischer Ansatz entwickelt, der gezielt die Disulfidbrücke zum Glutathionrest reduziert und die proteineigenen strukturell essentiellen Disulfidbrücken unverändert lässt. Zur Optimierung wurden vier verschiedene IL-4 Cystein-Muteine mit Cysteinresten an verschiedenen Positionen nahe der IL-4Rα Bindungsstelle getestet und die Reduktionsgeschwindigkeit in Gegenwart von Glutaredoxin bestimmt. Die enzymatische Reduktion verlief für alle vier Muteine mit verschiedenen Geschwindigkeiten. Dies deutet darauf hin, dass die Zugänglichkeit der Disulfidgruppe einen wichtigen Einfluss besitzt. Die Reduktionsbedingungen mussten daher für jedes Mutein neu bestimmt werden. Nach Optimierung des pH Wertes und insbesondere der Reaktionszeit konnten alle Muteine mit einer freien Thiolgruppe in angemessener Ausbeute erhalten werden. Die Proteine mit jeweils einer freien Thiolgruppe wurden daraufhin mit N-Ethylmaleinimid (NEM) oder Maleimido-PEG modifiziert. Die Effekte der Modifizierung an verschiedenen Positionen des IL-4 auf die Bindung an IL-4R wurden mit Hilfe der SPR-Spektroskopie (Oberflächen Plasmon Resonanz Spektroskopie) gemessen. Im zweiten Teil dieser Arbeit wurde die Interaktion von Foldameren mit der IL-4Ra Rezeptorkette untersucht. Foldamere stellen eine neue Klasse von stabilen, kompakt gefalteten Biomolekülen dar, die möglicherweise spezifisch mit Proteinen und Nukleinsäuren wechselwirken können. Es sollten Vorversuche durchgeführt werden um zu sondieren, ob aus Foldameren Hemmstoffe für IL-4 und IL-13 entwickelt werden können. Da Foldamere mit einer Thiolgruppe zur Anbindung (Tethering) an IL-4 nicht hergestellt werden konnten, wurden zunächst nur nichtreaktive Foldamare aus einer synthetisierten Foldamer-Bibliothek getestet. Zwei Bibliotheken mit verschiedenen auf aromatischen Amiden basierenden Foldameren wurden von Michael Grotz und Dr. Michael Deligny synthetisiert und von mir mit Hilfe der SPR Spektroskopie auf ihre Fähigkeit getestet, die Ligand-Rezeptor Wechselwirkung von IL-4 und derIL-4Rα Rezeptoruntereinheit zu unterbinden. Keines der untersuchten Foldamere konnte die IL-4/IL-4Rα Wechselwirkung spezifisch hemmen. Einige Foldamere zeigten eine unspezifische Bindung. Die hier dargestellten Studien zeigen das Design und die Herstellung eines potentiell neuen Typs von Gegenspieler zu IL-4, welcher ortsspezifische chemische Modifikationen ausnutzt um seine antagonistische Funktion zu erfüllen. KW - Il 4 KW - Foldamere KW - Modifizierung KW - Foldamers KW - PEG chemical modification KW - Zutokin KW - chemische Modifizierung KW - SPR-Spektroskopie Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113531 ER - TY - JOUR A1 - Morton, Charles Oliver A1 - Fliesser, Mirjam A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Bauer, Ruth A1 - Kneitz, Susanne A1 - Hope, William A1 - Rogers, Thomas Richard A1 - Einsele, Hermann A1 - Löffler, Jürgen T1 - Gene Expression Profiles of Human Dendritic Cells Interacting with Aspergillus fumigatus in a Bilayer Model of the Alveolar Epithelium/Endothelium Interface N2 - The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA. KW - aspergillus fumigatus KW - gene expression KW - immune receptors KW - immune response KW - denritic cells KW - B cell receptors KW - gene regulation KW - RNA extraction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112893 ER - TY - JOUR A1 - Hopfenmueller, Sebastian A1 - Steffan-Dewenter, Ingolf A1 - Holzschuh, Andrea T1 - Trait-Specific Responses of Wild Bee Communities to Landscape Composition, Configuration and Local Factors N2 - Land-use intensification and loss of semi-natural habitats have induced a severe decline of bee diversity in agricultural landscapes. Semi-natural habitats like calcareous grasslands are among the most important bee habitats in central Europe, but they are threatened by decreasing habitat area and quality, and by homogenization of the surrounding landscape affecting both landscape composition and configuration. In this study we tested the importance of habitat area, quality and connectivity as well as landscape composition and configuration on wild bees in calcareous grasslands. We made detailed trait-specific analyses as bees with different traits might differ in their response to the tested factors. Species richness and abundance of wild bees were surveyed on 23 calcareous grassland patches in Southern Germany with independent gradients in local and landscape factors. Total wild bee richness was positively affected by complex landscape configuration, large habitat area and high habitat quality (i.e. steep slopes). Cuckoo bee richness was positively affected by complex landscape configuration and large habitat area whereas habitat specialists were only affected by the local factors habitat area and habitat quality. Small social generalists were positively influenced by habitat area whereas large social generalists (bumblebees) were positively affected by landscape composition (high percentage of semi-natural habitats). Our results emphasize a strong dependence of habitat specialists on local habitat characteristics, whereas cuckoo bees and bumblebees are more likely affected by the surrounding landscape. We conclude that a combination of large high-quality patches and heterogeneous landscapes maintains high bee species richness and communities with diverse trait composition. Such diverse communities might stabilize pollination services provided to crops and wild plants on local and landscape scales. KW - habitats KW - bees KW - grasslands KW - species diversity KW - biodiversity KW - pollination KW - flowers KW - foraging Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112872 ER - TY - JOUR A1 - Römer, Daniela A1 - Roces, Flavio T1 - Nest Enlargement in Leaf-Cutting Ants: Relocated Brood and Fungus Trigger the Excavation of New Chambers N2 - During colony growth, leaf-cutting ants enlarge their nests by excavating tunnels and chambers housing their fungus gardens and brood. Workers are expected to excavate new nest chambers at locations across the soil profile that offer suitable environmental conditions for brood and fungus rearing. It is an open question whether new chambers are excavated in advance, or will emerge around brood or fungus initially relocated to a suitable site in a previously-excavated tunnel. In the laboratory, we investigated the mechanisms underlying the excavation of new nest chambers in the leaf-cutting ant Acromyrmex lundi. Specifically, we asked whether workers relocate brood and fungus to suitable nest locations, and to what extent the relocated items trigger the excavation of a nest chamber and influence its shape. When brood and fungus were exposed to unfavorable environmental conditions, either low temperatures or low humidity, both were relocated, but ants clearly preferred to relocate the brood first. Workers relocated fungus to places containing brood, demonstrating that subsequent fungus relocation spatially follows the brood deposition. In addition, more ants aggregated at sites containing brood. When presented with a choice between two otherwise identical digging sites, but one containing brood, ants' excavation activity was higher at this site, and the shape of the excavated cavity was more rounded and chamber-like. The presence of fungus also led to the excavation of rounder shapes, with higher excavation activity at the site that also contained brood. We argue that during colony growth, workers preferentially relocate brood to suitable locations along a tunnel, and that relocated brood spatially guides fungus relocation and leads to increased digging activity around them. We suggest that nest chambers are not excavated in advance, but emerge through a self-organized process resulting from the aggregation of workers and their density-dependent digging behavior around the relocated brood and fungus. KW - fungi KW - ants KW - fungal structure KW - fungal pathogens KW - foraging KW - humidity KW - pupae KW - fungal diseases Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112860 ER - TY - JOUR A1 - Keller, Daniela Barbara A1 - Schultz, Jörg T1 - Word Formation Is Aware of Morpheme Family Size N2 - Words are built from smaller meaning bearing parts, called morphemes. As one word can contain multiple morphemes, one morpheme can be present in different words. The number of distinct words a morpheme can be found in is its family size. Here we used Birth-Death-Innovation Models (BDIMs) to analyze the distribution of morpheme family sizes in English and German vocabulary over the last 200 years. Rather than just fitting to a probability distribution, these mechanistic models allow for the direct interpretation of identified parameters. Despite the complexity of language change, we indeed found that a specific variant of this pure stochastic model, the second order linear balanced BDIM, significantly fitted the observed distributions. In this model, birth and death rates are increased for smaller morpheme families. This finding indicates an influence of morpheme family sizes on vocabulary changes. This could be an effect of word formation, perception or both. On a more general level, we give an example on how mechanistic models can enable the identification of statistical trends in language change usually hidden by cultural influences. KW - linguistic morphology KW - language KW - death rates KW - psycholinguistics KW - chi square tests KW - vocabulary KW - birth rates KW - culture Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112848 ER - TY - JOUR A1 - Leingärtner, Annette A1 - Hoiss, Bernhard A1 - Krauss, Jochen A1 - Steffan-Dewenter, Ingolf T1 - Combined Effects of Extreme Climatic Events and Elevation on Nutritional Quality and Herbivory of Alpine Plants N2 - Climatic extreme events can cause the shift or disruption of plant-insect interactions due to altered plant quality, e.g. leaf carbon to nitrogen ratios, and phenology. However, the response of plant-herbivore interactions to extreme events and climatic gradients has been rarely studied, although climatic extremes will increase in frequency and intensity in the future and insect herbivores represent a highly diverse and functionally important group. We set up a replicated climate change experiment along elevational gradients in the German Alps to study the responses of three plant guilds and their herbivory by insects to extreme events (extreme drought, advanced and delayed snowmelt) versus control plots under different climatic conditions on 15 grassland sites. Our results indicate that elevational shifts in CN (carbon to nitrogen) ratios and herbivory depend on plant guild and season. CN ratios increased with altitude for grasses, but decreased for legumes and other forbs. In contrast to our hypotheses, extreme climatic events did not significantly affect CN ratios and herbivory. Thus, our study indicates that nutritional quality of plants and antagonistic interactions with insect herbivores are robust against seasonal climatic extremes. Across the three functional plant guilds, herbivory increased with nitrogen concentrations. Further, increased CN ratios indicate a reduction in nutritional plant quality with advancing season. Although our results revealed no direct effects of extreme climatic events, the opposing responses of plant guilds along elevation imply that competitive interactions within plant communities might change under future climates, with unknown consequences for plant-herbivore interactions and plant community composition. KW - Plant-herbivore interactions KW - Herbivory KW - Leaves KW - Grasses KW - Legumes KW - Insects KW - Drought KW - Climate Change Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112812 ER - TY - JOUR A1 - Shityakov, Sergey A1 - Förster, Carola A1 - Rethwilm, Axel A1 - Dandekar, Thomas T1 - Evaluation and Prediction of the HIV-1 Central Polypurine Tract Influence on Foamy Viral Vectors to Transduce Dividing and Growth-Arrested Cells N2 - Retroviral vectors are potent tools for gene delivery and various biomedical applications. To accomplish a gene transfer task successfully, retroviral vectors must effectively transduce diverse cell cultures at different phases of a cell cycle. However, very promising retroviral vectors based on the foamy viral (FV) backbone lack the capacity to efficiently transduce quiescent cells. It is hypothesized that this phenomenon might be explained as the inability of foamy viruses to form a pre-integration complex (PIC) with nuclear import activity in growth-arrested cells, which is the characteristic for lentiviruses (HIV-1). In this process, the HIV-1 central polypurine tract (cPPT) serves as a primer for plus-strand synthesis to produce a “flap” element and is believed to be crucial for the subsequent double-stranded cDNA formation of all retroviral RNA genomes. In this study, the effects of the lentiviral cPPT element on the FV transduction potential in dividing and growth-arrested (G1/S phase) adenocarcinomic human alveolar basal epithelial (A549) cells are investigated by experimental and theoretical methods. The results indicated that the HIV-1 cPPT element in a foamy viral vector background will lead to a significant reduction of the FV transduction and viral titre in growth-arrested cells due to the absence of PICs with nuclear import activity. KW - Evaluation KW - Prognose KW - HIV KW - Spumaviren KW - Einfluss Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112763 ER - TY - JOUR A1 - Albert, Štefan A1 - Spaethe, Johannes A1 - Grübel, Kornelia A1 - Rössler, Wolfgang T1 - Royal jelly-like protein localization reveals differences in hypopharyngeal glands buildup and conserved expression pattern in brains of bumblebees and honeybees N2 - Royal jelly proteins (MRJPs) of the honeybee bear several open questions. One of them is their expression in tissues other than the hypopharyngeal glands (HGs), the site of royal jelly production. The sole MRJP-like gene of the bumblebee, Bombus terrestris (BtRJPL), represents a pre-diversification stage of the MRJP gene evolution in bees. Here we investigate the expression of BtRJPL in the HGs and the brain of bumblebees. Comparison of the HGs of bumblebees and honeybees revealed striking differences in their morphology with respect to sex- and caste-specific appearance, number of cells per acinus, and filamentous actin (F-actin) rings. At the cellular level, we found a temporary F-actin-covered meshwork in the secretory cells, which suggests a role for actin in the biogenesis of the end apparatus in HGs. Using immunohistochemical localization, we show that BtRJPL is expressed in the bumblebee brain, predominantly in the Kenyon cells of the mushroom bodies, the site of sensory integration in insects, and in the optic lobes. Our data suggest that a dual glandbrain function preceded the multiplication of MRJPs in the honeybee lineage. In the course of the honeybee evolution, HGs dramatically changed their morphology in order to serve a food-producing function. KW - Hypopharyngeal glands KW - Bumblebee KW - Bombus KW - Brain KW - Labial glands KW - Immunohistochemistry KW - Kenyon cells KW - Mushroom bodies KW - Honeybee Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112733 ER - TY - THES A1 - Vona, Barbara C. T1 - Molecular Characterization of Genes Involved in Hearing Loss T1 - Molekulare Charakterisierung der in Hörstörungen involvierten Genen N2 - The auditory system is an exquisitely complex sensory organ dependent upon the synchronization of numerous processes for proper function. The molecular characterization of hereditary hearing loss is complicated by extreme genetic heterogeneity, wherein hundreds of genes dispersed genome-wide play a central and irreplaceable role in normal hearing function. The present study explores this area on a genome-wide and single gene basis for the detection of genetic mutations playing critical roles in human hearing. This work initiated with a high resolution SNP array study involving 109 individuals. A 6.9 Mb heterozygous deletion on chromosome 4q35.1q35.2 was identified in a syndromic patient that was in agreement with a chromosome 4q deletion syndrome diagnosis. A 99.9 kb heterozygous deletion of exons 58-64 in USH2A was identified in one patient. Two homozygous deletions and five heterozygous deletions in STRC (DFNB16) were also detected. The homozygous deletions alone were enough to resolve the hearing impairment in the two patients. A Sanger sequencing assay was developed to exclude a pseudogene with a high percentage sequence identity to STRC from the analysis, which further solved three of the six heterozygous deletion patients with the hemizygous, in silico predicted pathogenic mutations c.2726A>T (p.H909L), c.4918C>T (p.L1640F), and c.4402C>T (p.R1468X). A single patient who was copy neutral for STRC and without pathogenic copy number variations had compound heterozygous mutations [c. 2303_2313+1del12 (p.G768Vfs*77) and c.5125A>G (p.T1709A)] in STRC. It has been shown that STRC has been previously underestimated as a hearing loss gene. One additional patient is described who does not have pathogenic copy number variation but is the only affected member of his family having hearing loss with a paternally segregating translocation t(10;15)(q26.13;q21.1). Twenty-four patients without chromosomal aberrations and the above described patient with an USH2A heterozygous deletion were subjected to a targeted hearing loss gene next generation sequencing panel consisting of either 80 or 129 hearing-relevant genes. The patient having the USH2A heterozygous deletion also disclosed a second mutation in this gene [c.2276G>T (p.C759F)]. This compound heterozygous mutation is the most likely cause of hearing loss in this patient. Nine mutations in genes conferring autosomal dominant hearing loss [ACTG1 (DFNA20/26); CCDC50 (DFNA44); EYA4 (DFNA10); GRHL2 (DFNA28); MYH14 (DFNA4A); MYO6 (DFNA22); TCF21 and twice in MYO1A (DFNA48)] and four genes causing autosomal recessive hearing loss were detected [GJB2 (DFNB1A); MYO7A (DFNB2); MYO15A (DFNB3), and USH2A]. Nine normal hearing controls were also included. Statistical significance was achieved comparing controls and patients that revealed an excess of mutations in the hearing loss patients compared to the control group. The family with the GRHL2 c.1258-1G>A mutation is only the second family published worldwide with a mutation described in this gene to date, supporting the initial claim of this gene causing DFNA28 hearing loss. Audiogram analysis of five affected family members uncovered the progressive nature of DFNA28 hearing impairment. Regression analysis predicted the annual threshold deterioration in each of the five family members with multiple audiograms available over a number of years. N2 - Das Gehör als komplexes Sinnesorgan ist für eine einwandfreie Funktion abhängig von der Synchronisation zahlreicher Prozesse. Durch die extreme genetische Heterogenität wird die molekulare Charakterisierung einer erblich bedingten Schwerhörigkeit erschwert, da hunderte genomweit verteilter Gene eine zentrale und unersetzliche Rolle beim Hören spielen. Die vorliegende Studie untersucht dieses Forschungsgebiet auf genomweiter Ebene und auf der Basis von Einzelgenen, um genetische Mutationen zu ermitteln, die eine entscheidende Rolle bei der menschlichen auditiven Wahrnehmung besitzen. Diese Arbeit beginnt mit einer Studie an 109 Personen unter Zuhilfenahme von hochauflösenden SNP-Arrays. In dieser Studie wurde eine 6,9 Mb heterozygote Deletion auf Chromosom 4q35.1q35.2 bei einem syndromalen Patienten identifiziert, die eine Übereinstimmung mit einem Chromosom 4q-Deletionssyndrom aufwies. Bei einem weiteren Patienten wurde eine 99,9 kb heterozygote Deletion der Exons 58-64 in USH2A nachgewiesen. Zwei homozygote Deletionen und fünf heterozygote Deletionen in STRC (DFNB16) wurden ebenfalls detektiert. Die homozygoten Deletionen waren ausreichend, um die Schwerhörigkeit bei beiden Patienten zu klären. Ein Sanger-Sequenzierungs-Assay wurde entwickelt, um ein Pseudogen mit einer hohen prozentualen Sequenzidentität zu STRC von der Analyse auszuschließen. Dadurch konnten drei der sechs heterozygoten Deletionspatienten mit hemizygot in silico vorhergesagten pathogenen Mutationen, c.2726A>T (p.H909L), c.4918 C>T (p.L1640F) und c.4402C>T (p.R1468X), aufgeklärt werden. Ein Patient, der eine kopieneutrale STRC Variation und keine pathogenen Kopienzahlvariationen besaß, zeigte eine compound heterozygote Mutation [c.2303_2313+1del12 (p.G768Vfs*77) und c.5125A>G (p.T1709A)] in STRC. Es wurde gezeigt, daß die Beurteilung von STRC als Hörstörungsgen bisher unterschätzt wurde. Zusätzlich wird ein Patient beschrieben, der keine pathogenen Kopienzahlvariationen aufwies, aber das einzige Familienmitglied mit einer Schwerhörigkeit und einer paternalen segregierten Translokation t(10;15)(q26.13;q21.1) war. Vierundzwanzig Patienten ohne Chromosomenstörungen und der oben beschriebene Patient mit einer USH2A heterozygoten Deletion wurden mit einem Next Generation Sequencing Panel bestehend aus entweder 80 oder 129 für das Hören relevanter Gene untersucht. Der Patient mit einer USH2A heterozygoten Deletion zeigte eine zweite Mutation in diesem Gen [c.2276G>T (p.C759F)]. Diese compound heterozygote Mutation ist die wahrscheinlichste Ursache für die Schwerhörigkeit des Patienten. Neun Mutationen in Genen, die zu einem autosomal dominanten Hörverlust führen [ACTG1 (DFNA20/26); CCDC50 (DFNA44); EYA4 (DFNA10); GRHL2 (DFNA28); MYH14 (DFNA4A); MYO6 (DFNA22); TCF21], sowie zwei MYO1A (DFNA48) Mutationen und Mutationen in vier weiteren Genen, verantwortlich für autosomal rezessive Schwerhörigkeit [GJB2 (DFNB1A); MYO7A (DFNB2); MYO15A (DFNB3) und USH2A], konnten identifiziert werden. Neun normal hörende Kontrollen waren ebenfalls in diese Studie einbezogen worden. Durch einen Vergleich der Kontrollen mit den Patienten konnte eine statistische Signifikanz erreicht werden, die einen Überschuss an Mutationen bei der Patientengruppe gegenüber der Kontrollgruppe aufzeigte. Die Familie mit einer GRHL2 c.1258-1G>A Mutation ist die erst zweite Familie weltweit, die mit einer Mutation in diesem Gen publiziert worden ist. Dies unterstützt die initiale Behauptung, dass dieses Gen für eine DFNA28 Schwerhörigkeit verantwortlich ist. Die Audiogrammanalyse von fünf der betroffenen Familienmitglieder lässt eine voranschreitende Natur der DFNA28 Hörschädigung erkennen. Eine jährliche Verschlechterung der Hörschwelle bei jedem der fünf Familienmitglieder konnte eine Regressionsanalyse anhand von Audiogrammen, die über eine Anzahl von Jahren zur Verfügung standen, vorhersagen. KW - Molekularbiologie KW - Hearing loss KW - Hörverlust Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112170 N1 - Dieses Dokument wurde aus Datenschutzgründen - ohne inhaltliche Änderungen - erneut veröffentlicht. Die ursprüngliche Veröffentlichung war am: 09.07.2014 ER - TY - THES A1 - Hovhanyan, Anna T1 - Functional analyses of Mushroom body miniature (Mbm) in growth and proliferation of neural progenitor cells in the central brain of Drosophila melanogaster T1 - Funktionelle Analyse des Mushroom body minature (Mbm) in das Wachstum und die Proliferation von neuronalen Vorläuferzellen im zentralen Gehirn von Drosophila melanogaster N2 - Zellwachstum und Zellteilung stellen zwei miteinander verknüpfte Prozesse dar, die dennoch grundsätzlich voneinander zu unterscheiden sind. Die Wiederaufnahme der Proliferation von neuralen Vorläuferzellen (Neuroblasten) im Zentralhirn von Drosophila nach der spät-embryonalen Ruhephase erfordert zunächst Zellwachstum. Der Erhalt der regulären Zellgröße ist eine wichtige Voraussetzung für die kontinuierliche Proliferation der Neuroblasten über die gesamte larvale Entwicklungsphase. Neben extrinsischen Ernährungssignalen ist für das Zellwachstum eine kontinuierliche Versorgung mit funktionellen Ribosomen notwendig, damit die Proteinsynthese aufrechterhalten werden kann. Mutationen im mushroom body miniature (mbm) Gen wurden über einen genetischen Screen nach strukturellen Gehirnmutanten identifiziert. Der Schwerpunkt dieser Arbeit lag in der funktionellen Charakterisierung des Mbm Proteins als neues nukleoläres Protein und damit seiner möglichen Beteiligung in der Ribosomenbiogenese. Der Vergleich der relativen Expressionslevel von Mbm und anderen nuklearen Proteinen in verschiedenen Zelltypen zeigte eine verstärkte Expression von Mbm in der fibrillären Komponente des Nukleolus von Neuroblasten. Diese Beobachtung legte die Vermutung nahe, dass in Neuroblasten neben generell benötigten Faktoren der Ribosomenbiogenese auch Zelltyp-spezifische Faktoren existieren. Mutationen in mbm verursachen Proliferationsdefekte von Neuroblasten, wirken sich jedoch nicht auf deren Zellpolarität, die Orientierung der mitotischen Spindel oder die Asymmetrie der Zellteilung aus. Stattdessen wurde eine Reduktion der Zellgröße beobachtet, was im Einklang mit einer Beeinträchtigung der Ribosomenbiogenese steht. Insbesondere führt der Verlust der Mbm Funktion zu einer Retention der kleinen ribosomalen Untereinheit im Nukleolus, was eine verminderte Proteinsynthese zur Folge hat. Interessanterweise wurden Störungen der Ribosomenbiogenese nur in den Neuroblasten beobachtet. Zudem ist Mbm offensichtlich nicht erforderlich, um Wachstum oder die Proliferation von Zellen der Flügelimginalscheibe und S2-Zellen zu steuern, was wiederum dafür spricht, dass Mbm eine Neuroblasten-spezifische Funktion erfüllt. Darüber hinaus wurden die transkriptionelle Regulation des mbm-Gens und die funktionelle Bedeutung von posttranslationalen Modifikationen analysiert. Mbm Transkription wird von dMyc reguliert. Ein gemeinsames Merkmal von dMyc Zielgenen ist das Vorhandensein einer konservierten „E-Box“-Sequenz in deren Promotorregionen. In der Umgebung der mbm-Transkriptionsstartstelle befinden sich zwei „E-Box“-Motive. Mit Hilfe von Genreporteranalysen konnte nachgewiesen werden, dass nur eine von ihnen die dMyc-abhängige Transkription vermittelt. Die dMyc-abhängige Expression von Mbm konnte auch in Neuroblasten verifiziert werden. Auf posttranslationaler Ebene wird Mbm durch die Proteinkinase CK2 phosphoryliert. In der C-terminalen Hälfte des Mbm Proteins wurden in zwei Clustern mit einer Abfolge von sauren Aminosäuren sechs Serin- und Threoninreste als CK2- Phosphorylierungsstellen identifiziert. Eine Mutationsanalyse dieser Stellen bestätigte deren Bedeutung für die Mbm Funktion in vivo. Weiterhin ergaben sich Evidenzen, dass die Mbm-Lokalisierung durch die CK2-vermittelte Phosphorylierung gesteuert wird. Obwohl die genaue molekulare Funktion von Mbm in der Ribosomenbiogenese noch im Unklaren ist, unterstreichen die Ergebnisse dieser Studie die besondere Rolle von Mbm in der Ribosomenbiogenese von Neuroblasten um Zellwachstum und Proliferation zu regulieren. N2 - Cell growth and cell division are two interconnected yet distinct processes. Initiation of proliferation of central brain progenitor cells (neuroblasts) after the late embryonic quiescence stage requires cell growth, and maintenance of proper cell size is an important prerequisite for continuous larval neuroblast proliferation. Beside extrinsic nutrition signals, cell growth requires constant supply with functional ribosomes to maintain protein synthesis. Mutations in the mushroom body miniature (mbm) gene were previously identified in a screen for structural brain mutants. This study focused on the function of the Mbm protein as a new nucleolar protein, which is the site of ribosome biogenesis. The comparison of the relative expression levels of Mbm and other nucleolar proteins in different cell types showed a pronounced expression of Mbm in neuroblasts, particularly in the fibrillar component of the nucleolus, suggesting that in addition to nucleolar components generally required for ribosome biogenesis, more neuroblast specific nucleolar factors exist. Mutations in mbm cause neuroblast proliferation defects but do not interfere with cell polarity, spindle orientation or asymmetry of cell division of neuroblasts. Instead a reduction in cell size was observed, which correlates with an impairment of ribosome biogenesis. In particular, loss of Mbm leads to the retention of the small ribosomal subunit in the nucleolus resulting in decreased protein synthesis. Interestingly, the defect in ribosome biogenesis was only observed in neuroblasts. Moreover, Mbm is apparently not required for cell size and proliferation control in wing imaginal disc and S2 cells supporting the idea of a neuroblast-specific function of Mbm. Furthermore, the transcriptional regulation of the mbm gene and the functional relevance of posttranslational modifications were analyzed. Mbm is a transcriptional target of dMyc. A common feature of dMyc target genes is the presence of a conserved E-box sequence in their promoter regions. Two E-box motifs are found in the vicinity of the transcriptional start site of mbm. Gene reporter assays verified that only one of them mediates dMyc-dependent transcription. Complementary studies in flies showed that removal of dMyc function in neuroblasts resulted in reduced Mbm expression levels. At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2. Six serine and threonine residues located in two acidic amino acid rich clusters in the C-terminal half of the Mbm protein were identified as CK2 phosphorylation sites. Mutational analysis of these sites verified their importance for Mbm function in vivo and indicated that Mbm localization is controlled by CK2-mediated phosphorylation. Although the molecular function of Mbm in ribosome biogenesis remains to be determined, the results of this study emphasize the specific role of Mbm in neuroblast ribosome biogenesis to control cell growth and proliferation. KW - Taufliege KW - Mbm KW - Neuroblast KW - cell growth KW - proliferation KW - ribosome biogenesis KW - CK2 KW - Myc KW - Vorläuferzellen KW - Drosophila melanogaster Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-91303 ER -