TY - THES A1 - Zhu, Ana Cheng T1 - Metagenomic analysis of genetic variation in human gut microbial species T1 - Metagenomische Analysen der genetischen Variationen in menschlichen Darmbakterien N2 - Microbial species (bacteria and archaea) in the gut are important for human health in various ways. Not only does the species composition vary considerably within the human population, but each individual also appears to have its own strains of a given species. While it is known from studies of bacterial pan-genomes, that genetic variation between strains can differ considerably, such as in Escherichia coli, the extent of genetic variation of strains for abundant gut species has not been surveyed in a natural habitat. This is mainly due to the fact that most of these species cannot be cultured in the laboratory. Genetic variation can range from microscale genomic rearrangements such as small nucleotide polymorphism (SNP) to macroscale large genomic rearrangements like structural variations. Metagenomics offers an alternative solution to study genetic variation in prokaryotes, as it involves DNA sequencing of the whole community directly from the environment. However, most metagenomic studies to date only focus on variation in gene abundance and hence are not able to characterize genetic variation (in terms of presence or absence of SNPs and genes) of gut microbial strains of individuals. The aim of my doctorate studies was therefore to study the extent of genetic variation in the genomic sequence of gut prokaryotic species and its phenotypic effects based on: (1) the impact of SNP variation in gut bacterial species, by focusing on genes under selective pressure and (2) the gene content variation (as a proxy for structural variation) and their effect on microbial species and the phenotypic traits of their human host. In the first part of my doctorate studies, I was involved in a project in which we created a catalogue of 10.3 million SNPs in gut prokaryotic species, based on metagenomes. I used this to perform the first SNP-based comparative study of prokaryotic species evolution in a natural habitat. Here, I found that strains of gut microbial species in different individuals evolve at more similar rates than the strains within an individual. In addition, I found that gene evolution can be uncoupled from the evolution of its originating species, and that this could be related to selective pressure such as diet, exemplified by galactokinase gene (galK). Despite the individuality (i.e. uniqueness of each individual within the studied metagenomic dataset) in the SNP profile of the gut microbiota that we found, for most cases it is not possible to link SNPs with phenotypic differences. For this reason I also used gene content as a proxy to study structural variation in metagenomes. In the second part of my doctorate studies, I developed a methodology to characterize the variability of gene content in gut bacterial species, using metagenomes. My approach is based on gene deletions, and was applied to abundant species (demonstrated using a set of 11 species). The method is sufficiently robust as it captures a similar range of gene content variability as has been detected in completely sequenced genomes. Using this procedure I found individuals differ by an average of 13% in their gene content of gut bacterial strains within the same species. Interestingly no two individuals shared the same gene content across bacterial species. However, this variation corresponds to a lower limit, as it is only accounts for gene deletion and not insertions. This large variation in the gene content of gut strain was found to affect important functions, such as polysaccharide utilization loci (PULs) and capsular polysaccharide synthesis (CPS), which are related with digestion of dietary fibers. In summary, I have shown that metagenomics based approaches can be robust in characterizing genetic variation in gut bacterial species. I also illustrated, using examples both for SNPs and gene content (galK, PULs and CPS), that this genetic variation can be used to predict the phenotypic characteristics of the microbial species, as well as predicting the phenotype of their human host (for example, their capacity to digest different food components). Overall, the results of my thesis highlight the importance of characterizing the strains in the gut microbiome analogous to the emerging variability and importance of human genomics. N2 - Mikrobielle Arten (Bakterien und Archaeen) im menschlichen Darm sind wichtige Begleiter für unsere Gesundheit. Jedoch gibt es nicht nur starke Unterschiede zwischen individuellen Wirten in der Artenzusammensetzung des Darmmikrobioms, sondern es scheint sogar Individuen-spezifische Bakterienstämme zu geben. Analysen von Bakterien wie z.B. Escherichia coli haben schon früh gezeigt, dass die Genome von Bakterienstämmen derselben Art große Unterschiede aufzeigen können; jedoch wurden diese Unterschiede bisher noch nicht in einer natürlichen Umgebung gezeigt. Genetische Variation kann viele Ausprägungen haben und reicht von kleinen Veränderungen wie „small nucleotide polymorphism“ (SNP) zu makroskopischen Veränderung, wie z.B. chromosomalen Restrukturierungen. All diese genetischen Variationen wurden bis jetzt nicht in der natürlichen Umgebung der Bakterien studiert, vorallem bedingt durch fehlende Methoden um die meisten dieser Bakterien um Labor zu kultivieren. Metagenomische Studien können hier helfen, da sie unabhängig von Kultivierungen jegliche DNS aus einer natürlichen Bakteriengemeinschaft untersuchen. Jedoch wurde dies in den meisten bisher veröffentlichten metagenomischen Studien nicht ausgenutzt da diese hauptsächlich auf die Anzahl der gefunden Gene ausgerichtet waren. Das Ziel meiner Doktorarbeit war es, die genetische Variation in Darmbakterien zu beschreiben und phenotypische Veränderungen zu untersuchen. Dies habe ich umgesetzt durch die Erforschung (1) der SNP-Varianz in Darmbakterien, mit besonderem Augenmerk auf Gene, die unter einem selektivem Druck stehen und (2) der Variationen in der Genzusammensetzung eines Genomes (als eine Annäherung an strukturelle Variationen) und welchen Effekt dies auf Mikrobenarten und Wirtsphenotypen hat. Im ersten Kapitel meiner Doktorarbeit beschreibe ich meine Arbeit in einem Projekt unserer Gruppe, in dem wir basierend auf metagenomischen Daten 10 Millionen SNPs in menschlichen Darmbakterien beschrieben haben. Diesen Datensatz habe ich verwendet um die erste SNP-basierte, vergleichende Studie der Bakterienevolution in einem natürlichen Habitat zu realisieren. Ich entdeckte, dass Bakterienstämme unabhängig vom Wirt ähnliche evolutionäre Raten haben. Genauer gesagt, die evolutionäre Rate für eine Art ist stabiler zwischen Wirten, als die von verschiedenen Spezies innerhalb eines Wirtes. Ausserdem fand ich heraus, dass die Evolution von einzelnen Genen unabhängig vom restlichen Genom einer Spezies ist. Dies könnte durch einen Selektionsdruck wie z.B. die Ernährung des Wirtes ausgelöst werden, was ich am Beispiel des Galactokinasegenes (galK) gezeigt habe. Obwohl wir zeigen konnten, dass das SNP-Profil der Darmbakterien spezifisch für den jeweiligen Wirt ist, konnten wir keine Assoziation zwischen SNPs und Wirtsphänotypen finden. Auch aus diesem Grund habe ich mich in meiner weiteren Arbeit verstärkt auf makroskopische Genomvariationen konzentriert. Im zweiten Teil meiner Doktoarbeit entwickelte ich eine neue Methode, um Variationen in der genomische Zusammensetzung von einzelnen Bakterienarten zu beschreiben, wieder basierend auf metagenomischen Daten. Hierbei fokussiere ich mich insbesondere auf Gene, die in unseren metagenomischen Daten im Verglich zum Referengenom fehlen und wende dies auf die 11 dominantesten Bakterienspezies an. Diese neue Methode ist robust, da die gefundene Genomvarianz in unseren metagenomischen Daten übereinstimmt mit Daten aus komplett sequenzierten Genomen. So konnte ich herausfinden, dass im Durchschnitt 13% der Gene einer Bakterienart zwischen einzelen Wirten varieren. Besonders interessant ist hier, dass wir keine zwei Wirte gefunden haben, die für eine Bakterienart genau diesselben Gene haben. Jedoch ist die erwarte Varianz aller Wahrscheinlichkeit nach noch größer, da ich mit dieser Methode nur fehlende Gene beschreiben kann, aber nicht neu hinzugekommende. Diese Varianz kann auch wichtige bakterielle Funktionen betreffen, z.B. Gene für „polysaccharide utilization loci“ (PULs) und „capsular polysaccharide synthesis“ (CPS), welche wichtig sind um Ballaststoffe in der Nahrung zu verwerten. Zusammenfassend konnte ich in dieser Arbeit zeigen, dass metagenomische Methoden robust genug sind um die genetische Varianz von Darmbakterien zu beschreiben. Ausserdem konnte ich zeigen, dass die beschriebene Varianz benutzt werden kann, um phenotypische Veränderungen von Bakterien vorherzusagen (demonstriert für die galK, PULs and CPS-Gene). Dies wiederrum könnte benutzt werden um Vorhersagen für den Wirt über z.B. seine Ernährung zu machen. Meine Doktorarbeit zeigt wie wichtig es ist, einzelne Bakterienstämme zu charakterisieren, ganz analog zu der Bedeutsamkeit der genetischen Varianz des menschlichen Genomes. KW - metagenomic KW - Darmflora KW - Metagenom Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113890 ER - TY - JOUR A1 - Wäldchen, Sina A1 - Lehmann, Julian A1 - Klein, Teresa A1 - van de Linde, Sebastian A1 - Sauer, Markus T1 - Light-induced cell damage in live-cell super-resolution microscopy JF - Scientific Reports N2 - Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of similar to 1 kW cm\(^{-2}\) at 640 nm for several minutes, the maximum dose at 405 nm is only similar to 50 J cm\(^{-2}\), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. KW - optical reconstruction microscopy KW - tag fusion proteins KW - localization microscopy KW - photodynamic therapy KW - diffraction limit KW - illumination microscopy KW - structured illumination KW - fluorescent probes KW - in vitro KW - dynamics Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145207 VL - 5 IS - 15348 ER - TY - JOUR A1 - Wolf, Beat A1 - Kuonen, Pierre A1 - Dandekar, Thomas A1 - Atlan, David T1 - DNAseq workflow in a diagnostic context and an example of a user friendly implementation JF - BioMed Research International N2 - Over recent years next generation sequencing (NGS) technologies evolved from costly tools used by very few, to a much more accessible and economically viable technology. Through this recently gained popularity, its use-cases expanded from research environments into clinical settings. But the technical know-how and infrastructure required to analyze the data remain an obstacle for a wider adoption of this technology, especially in smaller laboratories. We present GensearchNGS, a commercial DNAseq software suite distributed by Phenosystems SA. The focus of GensearchNGS is the optimal usage of already existing infrastructure, while keeping its use simple. This is achieved through the integration of existing tools in a comprehensive software environment, as well as custom algorithms developed with the restrictions of limited infrastructures in mind. This includes the possibility to connect multiple computers to speed up computing intensive parts of the analysis such as sequence alignments. We present a typical DNAseq workflow for NGS data analysis and the approach GensearchNGS takes to implement it. The presented workflow goes from raw data quality control to the final variant report. This includes features such as gene panels and the integration of online databases, like Ensembl for annotations or Cafe Variome for variant sharing. KW - next generation sequencing KW - genome browser KW - mutation KW - algorithm KW - database KW - format KW - discovery KW - exome KW - variants KW - alignment Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144527 IS - 403497 ER - TY - THES A1 - Winkler, Ann-Cathrin Nicole T1 - Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection T1 - Identifizierung von humanen Wirtszellfaktoren die eine Rolle bei der \(Staphylococcus\) \(aureus\) Infektion spielen N2 - Staphylococcus aureus is both a human commensal and a pathogen. 20%-30% of all individuals are permanently or occasionally carriers of S. aureus without any symptoms. In contrast to this, S. aureus can cause life-threatening diseases e.g. endocarditis, osteomyelitis or sepsis. Here, the increase in antibiotic resistances makes it more and more difficult to treat these infections and hence the number of fatalities rises constantly. Since the pharmaceutical industry has no fundamentally new antibiotics in their pipeline, it is essential to better understand the interplay between S. aureus and the human host cell in order to find new, innovative treatment options. In this study, a RNA interference based whole genome pool screen was performed to identify human proteins, which play a role during S. aureus infections. Since 1,600 invasion and 2,271 cell death linked factors were enriched at least 2 fold, the big challenge was to filter out the important ones. Here, a STRING pathway analysis proved to be the best option. Subsequently, the identified hits were validated with the help of inhibitors and a second, individualised small interfering RNA-based screen. In the course of this work two important steps were identified, that are critical for host cell death: the first is bacterial invasion, the second phagosomal escape. The second step is obligatory for intracellular bacterial replication and subsequent host cell death. Invasion in turn is determining for all following events. Accordingly, the effect of the identified factors towards these two crucial steps was determined. Under screening conditions, escape was indirectly measured via intracellular replication. Three inhibitors (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) could be identified for the invasion process. In addition, siRNAs targeted against 16 different genes (including CAPN2, CAPN4 and PIK3CG), could significantly reduce bacterial invasion. Seven siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) were able to inhibit intracellular replication significantly. Further studies showed that the IP3 receptor inhibitor 2-APB, the calpain inhibitor calpeptin and the proteasome inhibitor MG-132 are able to prevent phagosomal escape and as a consequence intracellular replication and host cell death. In this context the role of calpains, calcium, the proteasome and the mitochondrial membrane potential was further investigated in cell culture. Here, an antagonistic behaviour of calpain 1 and 2 during bacterial invasion was observed. Intracellular calcium signalling plays a major role, since its inhibition protects host cells from death. Beside this, the loss of mitochondrial membrane potential is characteristic for S. aureus infection but not responsible for host cell death. The reduction of membrane potential can be significantly diminished by the inhibition of the mitochondrial Na+/Ca2+ exchanger. All together, this work shows that human host cells massively contribute to different steps in S. aureus infection rather than being simply killed by bacterial pore-forming toxins. Various individual host cell factors were identified, which contribute either to invasion or to phagosomal escape and therefore to S. aureus induced cytotoxicity. Finally, several inhibitors of S. aureus infection were identified. One of them, 2-APB, was already tested in a sepsis mouse model and reduced bacterial load of kidneys. Thus, this study shows valuable evidence for novel treatment options against S. aureus infections, based on the manipulation of host cell signalling cascades. N2 - Staphylococcus aureus kann sowohl ein Bestandteil der natürlichen Hautflora als auch ein Krankheitserreger sein. 20%-30% aller Menschen werden, permanent oder zeitweise, von S. aureus besiedelt, ohne Krankheitssymptome aufzuweisen. Im Gegensatz dazu kann S. aureus lebensbedrohliche Krankheiten wie Endokarditis, Osteomyelitis oder Sepsis verursachen. Diese Infektionen können immer schlechter behandelt werden, da immer mehr Stämme Resistenzen gegen die vorhandenen Antibiotika aufweisen. Dies führt zu einer steigenden Anzahl an Todesfällen, die auf Staphylokokkeninfektionen zurückzuführen sind. Da die Pharmaindustrie keine grundlegend neuen Antibiotika kurz vor der Marktreife hat, ist ein besseres Verständnis für das Wechselspiel zwischen Staphylokokken und ihren menschlichen Wirtszellen unbedingt notwendig, um neue, innovative Behandlungsmöglichkeiten finden zu können. Dafür wurde in dieser Arbeit ein genomweiter RNA-interferenz basierter Screen durchgeführt. Es sollten so die Proteine identifiziert werden, die eine Rolle bei der Staphylokokkeninfektion spielen. Da 1.600 invasionsrelevante und 2.271 zelltodrelevante Faktoren mindestens 2-fach angereichert waren, musste ein Weg gefunden werden die wichtigen Faktoren herauszufiltern. Eine STRING-Pathwayanalyse stellte sich als die beste Methode hierfür heraus. In einem zweiten Schritt wurden die so identifizierten Faktoren mit Inhibitoren oder einzelnen siRNAs ein weiteres Mal herunterreguliert, um ihre tatsächlichen Auswirkungen auf den Infektionsverlauf zu untersuchen. Im Verlauf dieser Arbeit konnte gezeigt werden, dass dem S. aureus induzierten Wirtszelltod mindestens zwei wichtige Schritte vorausgehen müssen. Erstens die Invasion der Wirtszelle und zweitens der Ausbruch aus dem Phagosom. Nur so können sich im dritten Schritt die Bakterien intrazellulär vermehren und die Zelle töten. Daher wurde der Einfluss der identifizierten Faktoren auf diese beiden entscheidenden Prozesse untersucht. Der Ausbruch wurde unter Screenkonditionen indirekt über die intrazelluläre Vermehrung bestimmt. Es konnten drei Inhibitoren (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) identifiziert werden, die die bakterielle Invasion vermindern. Darüber hinaus wurden 16 Proteine (unter anderem CAPN2, CAPN4 und PIK3CG) gefunden, deren Herunterregulation durch siRNAs, eine signifikant reduzierte Invasion zur Folge hatten. Sieben siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) waren in der Lage die intrazelluläre Vermehrung signifikant zu verringern. In nachfolgenden Versuchen konnte gezeigt werden, dass der IP3-Rezeptorinhibitor 2-APB, der Calpaininhibitor Calpeptin und der Proteasominhibitor MG-132 den Ausbruch aus dem Phagosom, sowie die darauffolgenden Ereignisse (intrazelluläre Vermehrung und Wirtszelltod) inhibieren können. In diesem Zusammenhang wurden die Einflüsse von Calpainen, Calcium, dem Proteasom sowie dem mitochondrialen Membranpotentialverlust im Zellkulturmodell im Detail weiter untersucht. So konnte eine gegensätzliche Rolle von Calpain 1 und 2 bei der S. aureus Invasion festgestellt werden. Die intrazelluläre calciumabhängige Signalweiterleitung spielt eine bedeutende Rolle bei der S. aureus Infektion, da ihre Inhibition eine normale Infektion verhindert. Das mitochondriale Membranpotential (MMP) sinkt während einer S. aureus infektion, ist aber nicht für den Zelltod verantwortlich. Das Sinken des MMPs kann mit einem Inhibitor, der den mitochondrialen Na+/Ca2+ Austausch verhindert, signifikant reduziert werden. Zusammenfassend zeigt diese Arbeit, dass die menschliche Wirtszelle selbst relevant zu den verschiedenen Schritten der Staphylokokkeninfektion beiträgt, und nicht einfach, wie häufig angenommen, von porenbildenden bakteriellen Toxinen zerstört wird. Entsprechend konnten einzelne Wirtszellproteine identifiziert werden, die entweder zur bakteriellen Invasion oder zum phagosomalen Ausbruch und somit zum induzierten Wirtszelltod beitragen. Überdies konnte gezeigt werden, dass Inhibitoren, die diese Wirtszellproteine hemmen, die Wirtszellen zu unterschiedlichen Zeitpunkten vor einer S. aureus Infektion schützen können. Folglich liefert diese Arbeit wertvolle Hinweise für neue Behandlungsmöglichkeiten von S. aureus Infektionen, die auf der Manipulation von Wirtszellsignalkaskaden beruhen. KW - Staphylococcus aureus KW - Wirtszelle KW - RNS-Interferenz KW - Host cell death KW - RNAi KW - 2-APB KW - intracellular replication KW - calpain KW - Human Host KW - Inhibitor Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114300 ER - TY - JOUR A1 - Williams, Richard D. A1 - Chagtai, Tasnim A1 - Alcaide-German, Marisa A1 - Apps, John A1 - Wegert, Jenny A1 - Popov, Sergey A1 - Vujanic, Gordan A1 - Van Tinteren, Harm A1 - Van den Heuvel-Eibrink, Marry M A1 - Kool, Marcel A1 - De Kraker, Jan A1 - Gisselsson, David A1 - Graf, Norbert A1 - Gessler, Manfred A1 - Pritchard-Jones, Kathy T1 - Multiple mechanisms of MYCN dysregulation in Wilms tumour JF - Oncotarget N2 - Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation. KW - integrative genomics viewer KW - oncogene amplification KW - sequencing data KW - gene KW - gain KW - copy number KW - somatic mutations KW - beta-catenin KW - histology KW - reveals KW - Wilms tumour KW - MYCN KW - DNA methylation KW - prognostic marker Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143471 VL - 6 IS - 9 ER - TY - THES A1 - Wiese, Katrin Evelyn T1 - Sensing supraphysiological levels of MYC : mechanisms of MIZ1-dependent MYC-induced Apoptosis in Mammary Epithelial Cells T1 - Mechanismen der MIZ1-abhängigen MYC-induzierten Apoptose in Brustepithelzellen N2 - Deregulated MYC expression contributes to cellular transformation as well as progression and maintenance of human tumours. Interestingly, in the absence of additional genetic alterations, potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced apoptosis has long been recognised as a major barrier against cancer development. However, it is largely unknown how cells discriminate physiological from supraphysiological levels of MYC in order to execute an appropriate biological response. The experiments described in this thesis demonstrate that induction of apoptosis in mammary epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required to invade low-affinity binding sites and repress target genes of the serum response factor SRF. These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1- dependent gene repression and increases AKT phosphorylation and downstream function. Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC’s transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive role for the MYC/MIZ1 complex in epithelial cells. N2 - Eine Deregulation der MYC Expression trägt entscheidend zur malignen Transformation und Progression humaner Tumoren bei. In Abwesenheit von zusätzlichen genetischen Läsionen machen potentiell onkogene MYC Proteinmengen Zellen jedoch anfällig für eine Reihe Apoptoseauslösender Reize. Daher kann MYC-induzierte Apoptose als bedeutende tumorsuppressive Maßnahme und wichtige Barriere gegen die Entstehung von Krebs betrachtet werden. Mechanistisch unklar ist allerdings wie genau Zellen physiologische von supraphysiologischen MYC-Mengen unterscheiden um adäquat darauf reagieren zu können. Die Experimente in dieser Dissertation zeigen, dass die repressive Eigenschaft von MYC/MIZ1 Komplexen für die Induktion von Apoptose in Brustepithelzellen essentiell ist. Die Analyse von Genexpressions- und ChIP-Sequenzier-Experimenten verdeutlicht, dass hohe Level an MYC benötigt werden um niedrig-affine Bindestellen im Genom zu besetzen und Zielgene des SRF (serum response factor ) Transkriptionsfaktors zu reprimieren. Diese Gene haben eine wichtige Funktion in Prozessen wie Zytoskelettdynamik und Zelladhäsion und sind vermutlich daran beteiligt notwendige Überlebenssignale an die Kinase AKT weiterzuleiten. Eine Wiederherstellung der SRF Aktivität revertiert die MIZ1-abhängige Repression der Zielgene und führt zu einer vermehrten AKT Phosphorylierung und Funktion. Insgesamt deuten diese Resultate auf eine tumorsuppressive Rolle des MYC/MIZ1 Komplexes in epithelialen Zellen hin, da eine Veränderung der genregulatorischen Aktivität als Folge der Assoziation mit MIZ1 dazu beiträgen könnte onkogene Mengen an MYC zu erkennen. KW - Myc KW - Apoptosis KW - Myc KW - Miz1 KW - Apoptose KW - Repression KW - ChIP-sequencing KW - Repression Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132532 ER - TY - JOUR A1 - Weiß, Clemens Leonard A1 - Schultz, Jörg T1 - Identification of divergent WH2 motifs by HMM-HMM alignments JF - BMC Research Notes N2 - Background The actin cytoskeleton is a hallmark of eukaryotic cells. Its regulation as well as its interaction with other proteins is carefully orchestrated by actin interaction domains. One of the key players is the WH2 motif, which enables binding to actin monomers and filaments and is involved in the regulation of actin nucleation. Contrasting conserved domains, the identification of this motif in protein sequences is challenging, as it is short and poorly conserved. Findings To identify divergent members, we combined Hidden-Markov-Model (HMM) to HMM alignments with orthology predictions. Thereby, we identified nearly 500 proteins containing so far not annotated WH2 motifs. This included shootin-1, an actin binding protein involved in neuron polarization. Among others, WH2 motifs of ‘proximal to raf’ (ptr)-orthologs, which are described in the literature, but not annotated in genome databases, were identified. Conclusion In summary, we increased the number of WH2 motif containing proteins substantially. This identification of candidate regions for actin interaction could steer their experimental characterization. Furthermore, the approach outlined here can easily be adapted to the identification of divergent members of further domain families. KW - WH2 domain KW - spire KW - shootin-1 KW - actin nucleation KW - HHblits Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126413 VL - 8 IS - 18 ER - TY - JOUR A1 - Wallace, Helen Margaret A1 - Leonhardt, Sara Diana T1 - Do Hybrid Trees Inherit Invasive Characteristics? Fruits of Corymbia torelliana X C. citriodora Hybrids and Potential for Seed Dispersal by Bees JF - PLoS One N2 - Tree invasions have substantial impacts on biodiversity and ecosystem functioning, and trees that are dispersed by animals are more likely to become invasive. In addition, hybridisation between plants is well documented as a source of new weeds, as hybrids gain new characteristics that allow them to become invasive. Corymbia torelliana is an invasive tree with an unusual animal dispersal mechanism: seed dispersal by stingless bees, that hybridizes readily with other species. We examined hybrids between C. torelliana and C. citriodora subsp. citriodora to determine whether hybrids have inherited the seed dispersal characteristics of C. torelliana that allow bee dispersal. Some hybrid fruits displayed the characteristic hollowness, resin production and resin chemistry associated with seed dispersal by bees. However, we did not observe bees foraging on any hybrid fruits until they had been damaged. We conclude that C. torelliana and C. citriodora subsp. citriodora hybrids can inherit some fruit characters that are associated with dispersal by bees, but we did not find a hybrid with the complete set of characters that would enable bee dispersal. However, around 20,000 hybrids have been planted in Australia, and ongoing monitoring is necessary to identify any hybrids that may become invasive. KW - resin KW - long-distance dispersal KW - Australian stingless bees KW - plantations KW - hymenoptera KW - populations KW - carbonaria KW - eucalyptus KW - cuticular profiles KW - hybridization Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141777 VL - 10 IS - 9 ER - TY - JOUR A1 - Tuchscherr, Lorena A1 - Bischoff, Markus A1 - Lattar, Santiago M. A1 - Noto Llana, Mariangeles A1 - Pförtner, Henrike A1 - Niemann, Silke A1 - Geraci, Jennifer A1 - Van de Vyver, Hélène A1 - Fraunholz, Martin J. A1 - Cheung, Ambrose L. A1 - Herrmann, Mathias A1 - Völker, Uwe A1 - Sordelli, Daniel O. A1 - Peters, Georg A1 - Loeffler, Bettina T1 - Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, \(\Delta\)sigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections. KW - gene regulator agr KW - endothelial cells KW - modulates virulence KW - death pathway sar locus KW - factor B KW - small-colony variants KW - alpha-toxin KW - epithelial cells KW - in vitro Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143419 VL - 11 IS - 4 ER - TY - JOUR A1 - Tsai, Yu-Chen A1 - Grimm, Stefan A1 - Chao, Ju-Lan A1 - Wang, Shih-Chin A1 - Hofmeyer, Kerstin A1 - Shen, Jie A1 - Eichinger, Fred A1 - Michalopoulou, Theoni A1 - Yao, Chi-Kuang A1 - Chang, Chih-Hsuan A1 - Lin, Shih-Han A1 - Sun, Y. Henry A1 - Pflugfelder, Gert O. T1 - Optomotor-blind negatively regulates Drosophila eye development by blocking Jak/STAT signaling JF - PLoS ONE N2 - Organ formation requires a delicate balance of positive and negative regulators. In Drosophila eye development, wingless (wg) is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb) is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occur predominantly in the ventral margin. Our results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired. KW - morphogenetic furrow progression KW - cell fate KW - compartment boundary KW - reporter gene KW - compound eye KW - gene expression KW - retinal differentiation KW - acts downstream KW - imaginal disk KW - glial cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143577 VL - 10 IS - 3 ER -