TY - THES A1 - Heiby, Julia T1 - Insight into molecular mechanisms of folding and self-association of spider silk protein domains T1 - Einblicke in molekulare Mechanismen der Faltung und Selbstassoziation von Spinnenseidenproteindomänen N2 - Spider silk is a biomaterial of extraordinary toughness paired with elasticity. The assembly of silk proteins, so-called spidroins (from “spider” and “fibroin”), generates the silk threads we typically see in our garden or the corners of our houses. Although spider webs from different species vary considerably in geometry and size, many sections of spidroin sequences are conserved. Highly conserved regions, found in all spidroins, relate to the terminal domains of the protein, i.e., the N-terminal (NTD) and C-terminal domains (CTD). Both have an essential function in the silk fibre association and polymerisation. The NTD is a 14 kDa five-helix bundle, which self-associates via a pH-driven mechanism. This process is critical for starting the polymerisation of the fibre. However, detailed insights into how conserved this mechanism is in different species and the quantitative thermodynamic comparison between homologous NTDs was missing. For this reason, four homologous NTDs of the major ampullate gland (MaSp) from spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus were investigated. I analysed and quantified equilibrium thermodynamics, kinetics of folding, and self-association. Methods involved dynamic light scattering (MALS), stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. The results showed conserved, cooperative two-state folding on a sub-millisecond time scale. All homologous NTDs showed a similarly fast association in the order of 10^9 M^−1 s^−1, while the resulting equilibrium dissociation constants were in the low nanomolar range. Electrostatic forces were found to be of great importance for protein association. Monomeric protein stability increased with salt concentration while enhancing its folding speed. However, due to Debye-Hückel effects, we found intermolecular electrostatics to be shielded, which reduced the NTDs association capacity significantly at high ionic strength. Altogether, the energetics and kinetics of the NTD dimerisation was conserved for all analysed homologs. Comparable to the NTD, the spider silks CTD is also a α-helix bundle, which covalently links two spidroins. The orientation of the domains predetermines the future fibre geometry. Here again, the detailed quantitative characterisation of the folding and dimerisation was missing. Therefore, the CTD from the E. australis was analysed in-depth. The protein folded via a three-state mechanism and was placed in the family of knotted proteins. By analysing the amino acid composition of the NTD of the MaSp1 of the Euprosthenops australis, we found an unusually high content of methionine residues (Met). To elucidate why this protein exhibits so many Met residues, I mutated all core Mets simultaneously to leucine (Leu). Results revealed a dramatically stabilised NTD, which now folded 50 times faster. After solving the tertiary structure of the mutant by NMR (nuclear magnetic resonance) spectroscopy, the structure of the monomeric mutant was found to be identical with the wild-type protein. However, when probing the dimerisation of the NTD, I could show that the association capacity was substantially impaired for the mutant. Our findings lead to the conclusion that Met provides the NTD with enhanced conformational dynamics and thus mobilises the protein, which results in tightly associated dimers. In additional experiments, I first re-introduced new Met residues into the Met-depleted protein at sequence positions containing native Leu. Hence, the mutated NTD protein was provided with the same number of Leu, which were previously removed by mutation. However, the protein did not regain wild-type characteristics. The functionality was not restored, but its stability was decreased as expected. To probe our hypothesis gained from the MaSp NTD, I transferred the experiment to another protein, namely the Hsp90 chaperone. Therefore, I incorporated methionine residues in the protein, which resulted in a slight improvement of its function. Finally, trial experiments were performed aiming at the synthesis of shortened spidroin constructs containing less repetitive middle-segments than the wild-type protein. The objective was to study the findings of the terminal domains in the context of an intact spidroin. The synthesis of these engineered spidroins was challenging. Nevertheless, preliminary results encourage the assumption that the characteristics observed in the isolated domains hold true in the context of a full-length spidroin. N2 - Spinnenseide ist ein Biomaterial mit außergewöhnlicher Widerstandsfähigkeit welche gepaart ist mit Elastizität. Das Zusammenfügen von Seidenproteinen aus so ge-nannten Spidroinen (ein Kunstwort aus „Spinne“ und „Fibroin“) erzeugt die Seiden-fäden, die wir typischerweise in unseren Gärten oder in den Ecken unserer Häuser finden. Obwohl Spinnennetze von verschiedenen Spinnenarten in Geometrie und Größe stark variieren, sind große Teile der Spidroin-Sequenzen konserviert. Stark konservierte Bereiche, die in allen Spidroinen vorkommen, sind die endständigen Bereiche des Proteins, die N-terminale (NTD) und C-terminale Domäne (CTD) ge-nannt werden. Beide haben wichtige Funktionen in der Assoziation der Proteine im Spinnkanal und deren Polymerisation zur Ausbildung des Seidenfadens. Die NTD ist ein kleines 14 kDa Protein, bestehend aus einem Bündel aus fünf Helices, dessen Selbstorganisation pH-abhängig ist. Dieser Prozess leitet die Poly-merisation der Faser ein. Allerdings fehlten bis heute Informationen darüber, ob dieser Mechanismus bei homologen Domänen aus verschiedenen Spinnenarten konser¬viert ist, da kaum quantitative biophysikalische Daten vorhanden sind. Aus diesem Grund wurden vier homologe NTDs der Spinnenarten Euprosthenops australis, Nephila clavipes, Latrodectus hesperus und Latrodectus geometricus vergleichend untersucht und deren Gleichgewichts-Thermodynamik, die Kinetik der Faltung und die Selbstassoziation quantitativ analysiert. Dazu wurden Methoden wie dynamische Mehrwinkel-Lichtstreuung (MALS), Stopped-Flow Fluoreszenz-spektroskopie und Zirkulardichroismus in Kombination mit thermischen und chemischen Denaturierungs¬experimenten angewandt. Die Ergebnisse lieferten die Erkenntnis einer kooperativen Zwei-Zustands-Faltung, die auf einer Zeitskala von weniger als einer Millisekunde stattfand. Alle homologen NTDs zeigten eine schnelle Assoziationsratenkonstante in der Größenordnung von 10^9 M^-1 s^-1, während die Gleichgewichts-Dissoziationskonstante für alle Homologe im nied¬rigen nano-molaren Bereich lag. Die Proteinassoziation wurde durch elektrostatische Kräfte gesteuert, wobei hohe Salzkonzentrationen die Stabilität des monomeren Proteins und dessen Faltungsgeschwindigkeit erhöhten. Die Assoziation zweier Domänen wurde jedoch durch Abschirmung intermolekularer elektrostatischer Kräfte, dem Debye-Hückel-Gesetz zufolge, reduziert. Die Energetik und Kinetik der NTD-Dimerisierung aller untersuchten Homologen erwies sich konserviert. Ebenso wie die NTD, ist auch die CTD der Spinnenseide ein α-helikales Bündel, welche jedoch zwei Spidroine kovalent miteinander verbindet. Die Orientierung der verknüpften Domäne bestimmt bereits die zukünftige Faserstruktur. Ähnlich wie bei der NTD, waren Faltung und Dimerisierung der CTD bisher nicht im Detail be-schrieben. Durch eine detaillierte Analyse der CTD der E. australis konnte gezeigt werden, dass das Protein sich in einem dreistufigen Mechanismus faltet und außerdem der Familie der geknoteten Proteine angehört. Bei genauerer Betrachtung der Aminosäurezusammensetzung der E. australis NTD konnte ein ungewöhnlich hoher Anteil der Aminosäure Methionin (Met) festge¬stellt werden. Um diesen überraschenden Sachverhalt zu verstehen, habe ich alle im Kern liegenden Met zu Leucin (Leu) mutiert. Die Ergebnisse zeigten eine extrem stabilisierte NTD, welche sich nun 50-fach schneller faltete. Die Protein¬struktur der Mutante wurde in Lösung mittels NMR Spektroskopie ermittelt. Das Ergebnis lieferte deckungsgleiche Strukturen von Mutante und Wildtyp im monomeren Zustand. Allerdings zeigten NTD Dimerisierungs-Versuche, dass die Assoziations-fähigkeit der Mutante erheblich beeinträchtigt war. Untersuchungen der nativen Dynamik mittels NMR und Fluoreszenzkorrelationsspektroskopie zeigten, dass Met diese entscheidend verstärkt, was zu einem eng assoziierten Dimer führte. Im Versuch die Dynamik wieder künstlich herzustellen, habe ich neue Met in die Mutante eingeführt, auf Sequenzpositionen welche natürlicherweise Leu aufweisen. Somit wurde die ursprüngliche Anzahl an Met in der NTD wiederher¬gestellt, jedoch an anderen Positionen. Obwohl das Protein wie erwartet an Stabilität verlor, konnte dessen Funktionalität nicht wiederhergestellt werden. Um unsere Erkenntnisse auf andere Proteine zu übertragen, wurden Met Reste künstlich in ein Hsp90 Protein eingeführt. Es konnte eine leicht verbesserte Funktionalität des Proteins beobachtet werden. Schließlich wurde versucht, die für die CTD und NTD gewonnen Erkenntnisse auf intakte, jedoch verkürzte Spidroine zu übertragen. Dazu wurden Spidroine mit weniger repetitiven Mittelsegmenten mittels rekombinanten Methoden hergestellt. Die Synthese dieser Spidroine erwies sich als Herausforderung. Allerdings zeigten die vorläufigen Ergebnisse, dass eine Verallgemeinerung der Erkenntnisse der isolierten Domänen auf das Volllängen-Spidroin möglich ist. KW - Spinnenseide KW - Fluoreszenzspektroskopie KW - Terminale Domaine KW - Spider Silk KW - Fluorescence spectroscopy KW - Terminal domains Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193455 ER - TY - JOUR A1 - Grimm, Jonathan B. A1 - Klein, Teresa A1 - Kopek, Benjamin G. A1 - Shtengel, Gleb A1 - Hess, Harald F. A1 - Sauer, Markus A1 - Lavis, Luke D. T1 - Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy JF - Angewandte Chemie International Edition N2 - The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules. KW - fluorophore KW - microscopy KW - photoactivation KW - Si-rhodamine KW - super-resolution imaging Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191069 VL - 55 IS - 5 ER - TY - THES A1 - Hofrichter, Michaela Angelika Hedwig T1 - Charakterisierung von angeborenen Hörstörungen mit Hilfe von Hochdurchsatz-Sequenziermethoden T1 - Characterization of inherited hearing loss by high throughput sequencing methods N2 - Fast 500 Millionen Menschen weltweit sind von einer Hörstörung betroffen. Es wird sogar angenommen, dass diese Anzahl laut der Weltgesundheitsorganisation (WHO) noch steigen und 2050 jeder zehnte Mensch eine Hörstörung aufweisen wird. Mindestens in 50% aller Fälle ist die Hörstörung genetisch bedingt. Durch die jüngsten Fortschritte der Sequenzierungstechnologien hat die genetische Analyse von Hörstörungen an Bedeutung gewonnen, vor allem hinsichtlich Familienplanung, geeigneter Therapien und zukünftiger möglichen Therapieansätzen, um das Hörvermögen wiederherzustellen. Die folgende Arbeit stellt 155 familiäre Fälle vor, die genetisch untersucht wurden. Diese Fälle konnten in zwei Kohorten unterteilt werden. Eine Kohorte (n = 74) umfasste Patienten mit kaukasischem Hintergrund, während die andere Kohorte (n = 81) Patienten beinhaltete, die aus dem Iran rekrutiert wurden. Für die Untersuchung wurde zum einen eine Panel-Analyse mit dem TruSight One Panel (Illumina, San Diego, USA) und zum anderen eine Exom-Sequenzierung durchgeführt. Anschließend wurden die Daten mit Analyse-Programmen wie GensearchNGS (PhenoSystems, Wallonia, Belgien) ausgewertet. Insgesamt konnte für 55% aller Fälle eine pathogene oder wahrscheinlich pathogene Variante durch Next Generation Sequencing diagnostiziert werden. Die meisten der gelösten Fälle (ca. 73%) stammten aus der iranischen Kohorte, was durch elterliche Blutsverwandtschaft und erhöhte Inzidenz von Hörstörungen im Iran zu erklären ist. 27% der gelösten Fälle gehörten der zweiten Kohorte an. Mutationen in den Genen MYO15A, LHFPL5, TECTA und SLC26A4 konnten überwiegend bei iranischen Patienten identifiziert werden. Varianten im Gen TECTA als auch im Gen SLC26A4 wurden ebenfalls in der kaukasischen Kohorte identifiziert. Beide Ethnien wiesen jeweils ein eigenes Mutationsspektrum auf. Jedoch wurden in beiden Gruppen Überschneidungen im klinischen Bild durch pathogene Varianten in einer Vielzahl von Hörstörungsgenen, sowie unterschiedliche klinische Phänotypen, deren Ursache pathogene Varianten im gleichen Hörstörungsgen zugrunde liegen, und familiäre Locus-Heterogenität beobachtet.. In dieser Arbeit konnte eine De Novo Mutation im CEACAM16-Gen (DFNA4B) bestätigt und der Effekt von einer wiederholt betroffenen Aminosäure im S1PR2-Gen (DFNB68) beschrieben werden. Darüber hinaus wurden mehrere Patienten mit X-chromosomalem Hörverlust aufgrund von Defekten im POU3F4-Gen (DFNX2) und Deletionen im SMPX-Gen (DFNX4) diagnostiziert. Zusätzlich konnte mit Hilfe einer Exom-basierten Copy Number Variation-Analyse eine Deletion im OTOA-Gen (DFNB22) gefunden werden, welche sich bis in die Tandempseudogenregion erstreckte. Diese Untersuchung zeigt die enormen Möglichkeiten zur Detektion von Mutationen bei heterogenen Erkrankungen durch Anwendung von Next Generation Sequencing. Weiterhin konnte eine intragenische Deletion im Gen COL9A1 identifiziert werden, die im Zusammenhang mit einer scheinbar isolierten Hörstörung steht und durch den komplexen Umlagerungsmechanismus FoSTeS/MMBIR (Fork Stalling und Template Switching/Microhomology-mediated Break-induced Replication) entstand, der so bei Hörstörungen noch nicht beschrieben wurde. Auf der Suche nach Genen, die bisher noch nicht mit Hörstörungen assoziiert werden konnten, wurden acht Familien in eine Kandidatengenuntersuchung miteinbezogen und eine Exom-weite Analyse durchgeführt. Bei fünf Familien konnte noch keine ursächliche Variante identifiziert werden. Jedoch wurde bei drei Familien mit einer autosomal dominanten Schwerhörigkeit eine genetische Ursache identifiziert und TECTB, ATP11A und THBS2 konnten als Kandidatengene ermittelt werden. Diese Arbeit zeigt, wie wichtig es ist, die kausale Variante bei Hörstörungspatienten zu detektieren. Eine genetische Diagnostik ermöglicht eine endgültige Diagnose eines Syndroms, ist für die Klassifizierung der Hörstörung notwendig und trägt zu einer zukünftigen Therapie der Patienten bei. N2 - Nearly 500 million people are affected by hearing impairment. According to the World Health Organization (WHO), the prevalence of hearing loss will increase to one in ten people in 2050. It is expected that at least half of all cases have a genetic etiology. Due to recent advancements in sequencing technologies the genetic analysis of hearing loss gain in importance, especially in regard to family planning, directing appropriate therapies and engaging in future therapeutic approaches for hearing restoration. The following thesis describes the genetic causes of 155 familial cases with hearing loss. These cases were divided into two cohorts. One cohort (n = 74) included patients with a Caucasian background, while the other cohort (n = 81) comprised patients who were recruited from the Iran. A panel analysis using the TruSight One Panel (Illumina, San Diego, USA) as well as an exome sequencing approach were applied. The data were subsequently analyzed using bioinformatics programs such as GensearchNGS (PhenoSystems, Wallonia, Belgium). Overall, 55% of all cases disclosed a pathogenic or likely pathogenic genetic variant by utilizing next generation sequencing methods. Most of the resolved cases (ca. 73%) were detected in the Iranian cohort, a fact which is traced back to parental consanguinity and increased incidence of overall hearing impairment in the Iran. 27% of resolved cases were revealed in the second cohort. Variants in the genes MYO15A (DFNB3), LHFPL5 (DFNB67), TECTA (DFNB21), and SLC26A4 (DFNB4) were especially prevalent in Iranian patients. Variants in the genes TECTA and SLC26A4 were also identified in the Caucasian cohort. The two ethnic groups each exhibited a distinctly unique mutational landscape. Additionally, the overlapping clinical outcomes caused by pathogenic variants in a multitude of hearing impairment genes as well as the phenotypical different characters of variants in the same gene generating hearing loss and familial locus heterogeneity were observed. This work also described a de novo mutation in the CEACAM16 (DFNA4B) gene and described the effect of a recurrently substituted amino acid residue in the S1PR2 (DFNB68) gene. In addition, several X-linked hearing loss patients were diagnosed due to defects in the POU3F4 (DFNX2) gene and deletions in the SMPX (DFNX4) gene. Furthermore, exome-based copy number variation analysis identified a deletion in the OTOA (DFNB22) gene extending into the tandem pseudogene region. This study demonstrates the enormous potential for the detection of mutations in a genetically heterogeneous disorder applying next generation sequencing. Furthermore, an intragenic deletion in the gene COL9A1 was identified, which is related to an apparent isolated hearing impairment and was likely caused by the complex rearrangement of FoSTeS/MMBIR mechanism (fork stalling and template switching/microhomology-mediated break-induced replication), which has not been previously described in hearing disorders. In order to reveal new genes associated with hearing loss, eight families were investigated with an exome-wide analysis in a candidate gene study. In five families, no causal variant could be identified. However, a genetic cause was identified in three families with autosomal dominant hearing loss and TECTB, ATP11A, and THBS2 were identified as candidate genes. This work shows the importance of the identification of the causal variant. Herein, genetic diagnostic could be necessary for the final diagnosis of a syndrome, is important for classification of the hearing loss and contributes to a future therapy. KW - Hörstörungen KW - High throughput screening Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-185331 ER - TY - JOUR A1 - Hassouna, I. A1 - Ott, C. A1 - Wüstefeld, L. A1 - Offen, N. A1 - Neher, R. A. A1 - Mitkovski, M. A1 - Winkler, D. A1 - Sperling, S. A1 - Fries, L. A1 - Goebbels, S. A1 - Vreja, I. C. A1 - Hagemeyer, N. A1 - Dittrich, M. A1 - Rossetti, M. F. A1 - Kröhnert, K. A1 - Hannke, K. A1 - Boretius, S. A1 - Zeug, A. A1 - Höschen, C. A1 - Dandekar, T. A1 - Dere, E. A1 - Neher, E. A1 - Rizzoli, S. O. A1 - Nave, K.-A. A1 - Sirén, A.-L. A1 - Ehrenreich, H. T1 - Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus JF - Molecular Psychiatry N2 - Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of similar to 20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a \(^{15}\)N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated \(^{15}\)N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration. KW - neural stem-cells KW - recombinat-human-erythropoietin KW - cognitive functions KW - pyramidal neurons KW - nervous-sytem KW - brain-injury KW - mouse-brain KW - progenitors KW - mice KW - memory Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186669 VL - 21 IS - 12 ER - TY - JOUR A1 - Schneider, Eberhard A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - El Hajj, Nady A1 - Haaf, Thomas T1 - CpG sites with continuously increasing or decreasing methylation from early to late human fetal brain development JF - Gene N2 - Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767 m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny. KW - Autism spectrum disorders KW - DNA methylation KW - Genome KW - Autism KW - Frontal cortex KW - Human prefrontal cortex KW - Gene-expression KW - Schizophrenia KW - Patterns KW - Transcription KW - Epigenetics KW - Environment KW - Fetal brain development KW - DNA methylation dynamics KW - Methylome Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186936 VL - 592 IS - 1 ER - TY - JOUR A1 - Markert, Sebastian Matthias A1 - Britz, Sebastian A1 - Proppert, Sven A1 - Lang, Marietta A1 - Witvliet, Daniel A1 - Mulcahy, Ben A1 - Sauer, Markus A1 - Zhen, Mei A1 - Bessereau, Jean-Louis A1 - Stigloher, Christian T1 - Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome JF - Neurophotonics N2 - Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses. KW - caenorhabditis elegans KW - localization micoscopy KW - fluorescent-probes KW - junction proteins KW - resolution limit KW - direct stochasticoptical reconstruction microscopy KW - structured illumination microscopy KW - correlative light and electron microscopy KW - gap junction KW - neural circuits KW - nervous-system KW - image data KW - reconstruction KW - innexins KW - super-resolution microscopy Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187292 VL - 3 IS - 4 ER - TY - THES A1 - Markert, Sebastian Matthias T1 - Enriching the understanding of synaptic architecture from single synapses to networks with advanced imaging techniques T1 - Vertiefung des Verständnisses synaptischer Architektur von der einzelnen Synapse bis zum Netzwerk mit modernsten bildgebenden Verfahren N2 - Because of its complexity and intricacy, studying the nervous system is often challenging. Fortunately, the small nematode roundworm Caenorhabditis elegans is well established as a model system for basic neurobiological research. The C. elegans model is also the only organism with a supposedly complete connectome, an organism-wide map of synaptic connectivity resolved by electron microscopy, which provides some understanding of how the nervous system works as a whole. However, the number of available data-sets is small and the connectome contains errors and gaps. One example of this concerns electrical synapses. Electrical synapses are formed by gap junctions and difficult to map due to their often ambiguous morphology in electron micrographs, leading to misclassification or omission. On the other hand, chemical synapses are more easily mapped, but many aspects of their mode of operation remain elusive and their role in the C. elegans connectome is oversimplified. A comprehensive understanding of signal transduction of neurons between each other and other cells will be indispensable for a comprehensive understanding of the nervous system. In this thesis, I approach these challenges with a combination of advanced light and electron microscopy techniques. First, this thesis describes a strategy to increase synaptic specificity in connectomics. Specifically, I classify gap junctions with a high degree of confidence. To achieve this, I utilized array tomography (AT). In this thesis, AT is adapted for high-pressure freezing to optimize for structure preservation and for super-resolution light microscopy; in this manner, I aim to bridge the gap between light and electron microscopy resolutions. I call this adaptation super-resolution array tomography (srAT). The srAT approach made it possible to clearly identify and map gap junctions with high precision and accuracy. The results from this study showcased the feasibility of incorporating electrical synapses into connectomes in a systematic manner, and subsequent studies have used srAT for other models and questions. As mentioned above, the C. elegans connectomic model suffers from a shortage of datasets. For most larval stages, including the special dauer larval stage, connectome data is completely missing up to now. To obtain the first partial connectome data-set of the C. elegans dauer larva, we used focused ion-beam scanning electron microscopy (FIB-SEM). This technique offers an excellent axial resolution and is useful for acquiring large volumes for connectomics. Together with our collaborators, I acquired several data-sets which enable the analysis of dauer stage-specific “re-wiring” of the nervous system and thus offer valuable insights into connectome plasticity/variability. While chemical synapses are easy to map relative to electrical synapses, signal transduction via chemical transmitters requires a large number of different proteins and molecular processes acting in conjunction in a highly constricted space. Because of the small spatial scale of the synapse, investigating protein function requires very high resolution, which electron tomography provides. I analyzed electron tomograms of a worm-line with a mutant synaptic protein, the serine/threonine kinase SAD-1, and found remarkable alterations in several architectural features. My results confirm and re-contextualize previous findings and provide new insight into the functions of this protein at the chemical synapse. Finally, I investigated the effectiveness of our methods on “malfunctioning,” synapses, using an amyotrophic lateral sclerosis (ALS) model. In the putative synaptopathy ALS, the mechanisms of motor neuron death are mostly unknown. However, mutations in the gene FUS (Fused in Sarcoma) are one known cause of the disease. The expression of the mutated human FUS in C. elegans was recently shown to produce an ALS-like phenotype in the worms, rendering C. elegans an attractive disease model for ALS. Together with our collaboration partners, I applied both srAT and electron tomography methods to “ALS worms” and found effects on vesicle docking. These findings help to explain electrophysiological recordings that revealed a decrease in frequency of mini excitatory synaptic currents, but not amplitudes, in ALS worms compared to controls. In addition, synaptic endosomes appeared larger and contained electron-dense filaments in our tomograms. These results substantiate the idea that mutated FUS impairs vesicle docking and also offer new insights into further molecular mechanisms of disease development in FUS-dependent ALS. Furthermore, we demonstrated the broader applicability of our methods by successfully using them on cultured mouse motor neurons. Overall, using the C. elegans model and a combination of light and electron microscopy methods, this thesis helps to elucidate the structure and function of neuronal synapses, towards the aim of obtaining a comprehensive model of the nervous system. N2 - Das Nervensystem ist ein definierendes Merkmal aller Tiere, unter anderem verantwortlich für Sinneswahrnehmung, Bewegung und „höhere“ Hirnfunktionen. Wegen dessen Komplexität und Feingliedrigkeit stellt das Erforschen des Nervensystems oft eine Herausforderung dar. Jedoch ist der kleine Fadenwurm Caenorhabditis elegans als Modellsystem für neurobiologische Grundlagenforschung gut etabliert. Erbesitzt eines der kleinsten und unveränderlichsten bekannten Nervensysteme. C.elegans ist auch das einzige Modell, für das ein annähernd vollständiges Konnektom vorliegt, eine durch Elektronenmikroskopie erstellte Karte der synaptischen Verbindungen eines gesamten Organismus, die Einblicke in die Funktionsweise des Nervensystems als Ganzes erlaubt. Allerdings ist die Anzahl der verfügbaren Datensätze gering und das Konnektom enthält Fehler und Lücken. Davon sind beispielsweise elektrische Synapsen betroffen. Elektrische Synapsen werden von Gap Junctions gebildet und sind auf Grund ihrer oft uneindeutigen Morphologie in elektronenmikroskopischen Aufnahmen schwierig zu kartieren, was dazu führt, dass einige falsch klassifiziert oder übersehen werden. Chemische Synapsen sind dagegen einfacher zu kartieren, aber viele Aspekte ihrer Funktionsweise sind schwer zu erfassen und ihre Rolle im Konnektom von C.elegans ist daher zu vereinfacht dargestellt. Ein umfassendes Verständnis der Signaltransduktion von Neuronen untereinander und zu anderen Zellen wird Voraussetzung für ein vollständiges Erfassen des Nervensystems sein. In der vorliegenden Arbeit gehe ich diese Herausforderungen mithilfe einer Kombination aus modernsten licht- und elektronenmikroskopischen Verfahren an. Zunächst beschreibt diese Arbeit eine Strategie, um die synaptische Spezifität in der Konnektomik zu erhöhen, indem ich Gap Junctions mit einem hohen Maß an Genauigkeit klassifiziere. Um dies zu erreichen, nutzte ich array tomography (AT), eine Technik, die Licht- und Elektronenmikrokopie miteinander korreliert. In dieser Arbeit wird AT adaptiert für Hochdruckgefrierung, um die Strukturerhaltung zu optimieren, sowie für ultrahochauflösende Lichtmikroskopie; so wird die Kluft zwischen den Auflösungsbereichen von Licht- und Elektronenmikroskopie überbrückt. Diese Adaption nenne ich super-resolution array tomography (srAT). Der srATAnsatz machte es möglich, Gap Junctions mit hoher Präzision und Genauigkeit klar zu identifizieren. Für diese Arbeit konzentrierte ich mich dabei auf Gap Junctions des retrovesikulären Ganglions von C.elegans. Die Ergebnisse dieser Studie veranschaulichen, wie es möglich wäre, elektrische Synapsen systematisch in Konnektome aufzunehmen. Nachfolgende Studien haben srAT auch auf andere Modelle und Fragestellungen angewandt ... KW - Caenorhabditis elegans KW - Synapse KW - Elektronenmikroskopie KW - Myatrophische Lateralsklerose KW - connectomics KW - focused ion-beam scanning electron microscopy KW - super-resolution array tomography Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189935 ER - TY - JOUR A1 - Kilinc, Mehmet Okyay A1 - Ehrig, Klaas A1 - Pessian, Maysam A1 - Minev, Boris R. A1 - Szalay, Aladar A. T1 - Colonization of xenograft tumors by oncolytic vaccinia virus (VACV) results in enhanced tumor killing due to the involvement of myeloid cells JF - Journal of Translational Medicine N2 - Background The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment. Methods Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS\(^{+}\) myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student’s t test was used for comparison between groups in all of the experiments. Results Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b\(^{+}\) ly6G\(^{+}\) myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS\(^{+}\) MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7–10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments. Conclusions We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production. KW - MDSCs KW - VACV KW - iNOS KW - oncolytic virus therapy KW - NO KW - innate immune system KW - antitumor immune response KW - antiviral immunity Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168914 VL - 14 IS - 340 ER - TY - JOUR A1 - Vogtmann, Emily A1 - Hua, Xing A1 - Zeller, Georg A1 - Sunagawa, Shinichi A1 - Voigt, Anita Y. A1 - Hercog, Rajna A1 - Goedert, James J. A1 - Shi, Jianxin A1 - Bork, Peer A1 - Sinha, Rashmi T1 - Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing JF - PLoS ONE N2 - Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing. KW - colorectal cancer KW - gut microbiota KW - whole-genome shotgun sequencing Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166904 VL - 11 IS - 5 ER - TY - JOUR A1 - Hölldobler, Bert T1 - Queen Specific Exocrine Glands in Legionary Ants and Their Possible Function in Sexual Selection JF - PLoS ONE N2 - The colonies of army ants and some other legionary ant species have single, permanently wingless queens with massive post petioles and large gasters. Such highly modified queens are called dichthadiigynes. This paper presents the unusually rich exocrine gland endowment of dichthadiigynes, which is not found in queens of other ant species. It has been suggested these kinds of glands produce secretions that attract and maintain worker retinues around queens, especially during migration. However, large worker retinues also occur in non-legionary species whose queens do not have such an exuberance of exocrine glands. We argue and present evidence in support of our previously proposed hypothesis that the enormous outfit of exocrine glands found in dichthadiigynes is due to sexual selection mediated by workers as the main selecting agents KW - exocrine glands KW - dichthadiigynes KW - legionary ants KW - queens KW - sexual selection KW - army ants Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167057 VL - 11 IS - 3 ER - TY - JOUR A1 - Joschinski, Jens A1 - Beer, Katharina A1 - Helfrich-Förster, Charlotte A1 - Krauss, Jochen T1 - Pea Aphids (Hemiptera: Aphididae) Have Diurnal Rhythms When Raised Independently of a Host Plant JF - Journal of Insect Science N2 - Seasonal timing is assumed to involve the circadian clock, an endogenous mechanism to track time and measure day length. Some debate persists, however, and aphids were among the first organisms for which circadian clock involvement was questioned. Inferences about links to phenology are problematic, as the clock itself is little investigated in aphids. For instance, it is unknown whether aphids possess diurnal rhythms at all. Possibly, the close interaction with host plants prevents independent measurements of rhythmicity. We reared the pea aphid Acyrthosiphon pisum (Harris) on an artificial diet, and recorded survival, moulting, and honeydew excretion. Despite their plant-dependent life style, aphids were independently rhythmic under light–dark conditions. This first demonstration of diurnal aphid rhythms shows that aphids do not simply track the host plant’s rhythmicity. KW - artificial diet KW - circadian clock KW - hourglass clock KW - Acyrthosiphon pisum KW - photoperiodism Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168783 VL - 16 IS - 1 ER - TY - THES A1 - Blättner, Sebastian T1 - The role of the non-ribosomal peptide synthetase AusAB and its product phevalin in intracellular virulence of Staphylococcus aureus T1 - Die Rolle der nicht-ribosomalen Peptidsynthetase AusAB und ihres Produktes Phevalin in der intrazellulären Virulenz von Staphylococcus aureus N2 - Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis. In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70% of the CA infections. The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur. S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient. In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae. The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria. N2 - Staphylococcus aureus ist ein weit verbreitetes kommensales Bakterium, welches zugleich einer der häufigsten Verursacher von Krankenhausinfektionen ist, und eine Reihe verschiedener Krankheiten, angefangen bei simplen Abszessen, bis hin zu schweren Erkrankungen wie Lungenentzündung, Osteomylitis und Sepsis verursachen kann. Das Risiko durch nosokomiale sowie epidemische S. aureus Infektionen ist in den vergangenen Jahren weiter gestiegen. Dazu beigetragen hat das Auftreten multiresistenter und hoch cytotoxischer Stämme, vor allem in den USA. Als Konsequenz hat die WHO S. aureus inzwischen als „Superbug“ tituliert und als globales Gesundheitsrisiko eingestuft. Bei CA-Infektionen dominieren die Isolate der Klassifizierung USA300 und USA400, wobei den Erstgenannten bis zu 70% aller in den USA registrierten CA-MRSA Infektionen der letzten Jahre zugesprochen werden. Lange Zeit wurde angenommen, dass S. aureus strikt extrazellulär im Infektionsbereich vorliegt und die cytotoxische Wirkung von z.B. α-Toxin für Wirtszelltod und Gewebeschädigungen verantwortlich ist. Erst vor kurzem wurde festgestellt, dass S. aureus auch durch fakultativ phagozytotische Zellen, wie Epithel- oder Endothelzellen, mittels zahlreicher Adhäsine aufgenommen wird. Die Aufnahme in die Zelle erfolgt zunächst in ein Phagoendosom, in dem die Pathogene durch antimikrobielle Mechanismen abgebaut würden. Um dies zu verhindern, verfügt S. aureus über Virulenzfaktoren, welche die endosomale Membran schädigen. Die Bakterien gelangen so in das Zellzytoplasma, wo sie sich vervielfältigen können, bevor die Wirtszelle schließlich getötet wird. Eine wichtige Funktion in diesem Vorgang konnte bereits in mehreren Studien den Phenol löslichen Modulinen (PSM) zugesprochen werden, Arbeiten unserer Gruppe deuten jedoch darauf hin, dass diese nicht alleine für den phagosomalen Ausbruch von S. aureus verantwortlich sind. In dieser Arbeit verwendete ich eine Transposon Mutantenbibliothek des S. aureus Stammes JE2 (USA300) in Verbindung mit automatisierter Fluoreszenzmikroskopie, um Gene zu identifizieren, die den phagosomalen Ausbruch von S. aureus beeinflussen. Unter den Mutanten, welche eine Minderung der Ausbruchsraten zeigten, fanden sich auch Mutanten in beiden Genen eines Operons, welches für die nicht-ribosomale Peptidsynthetase AusA/B codiert, die die beiden Dipeptide Phevalin und Tyrvalin produziert. Verminderte Ausbruchsraten konnten sowohl durch genetische Komplementation als auch mittels des Zusatzes synthetischen Phevalins wiederhergestellt werden. In Leukozyten verhindert Phevalin effizienten Calcium-Flux und die Aktivierung von Neutrophilen. Zudem fördert Phevalin die Cytotoxizität intrazellulärer Bakterien sowohl in Makrophagen, als auch Neutrophilen. Darüber hinaus konnten wir zeigen, dass die NRPS AusAB und ihre Produkte eine Rolle beim Überleben der Bakterien während einer Infektion im Tiermodell einnehmen. Die hier präsentierten Daten hinsichtlich des Einflusses von Phevalin auf Virulenz und der Interaktion zwischen Wirt und Pathogen lassen den Schluss zu, dass Phevalin direkt auf einen Wirtszellfaktor wirkt, um die Cytotoxicität intrazellulärer Bakterien zu stärken. KW - Staphylococcus aureus KW - MRSA KW - Virulenz KW - Intracellular virulence KW - Non-ribosomal peptide synthetase KW - USA300 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146662 ER - TY - JOUR A1 - Horn, Hannes A1 - Keller, Alexander A1 - Hildebrandt, Ulrich A1 - Kämpfer, Peter A1 - Riederer, Markus A1 - Hentschel, Ute T1 - Draft genome of the \(Arabidopsis\) \(thaliana\) phyllosphere bacterium, \(Williamsia\) sp. ARP1 JF - Standards in Genomic Sciences N2 - The Gram-positive actinomycete \(Williamsia\) sp. ARP1 was originally isolated from the \(Arabidopsis\) \(thaliana\) phyllosphere. Here we describe the general physiological features of this microorganism together with the draft genome sequence and annotation. The 4,745,080 bp long genome contains 4434 protein-coding genes and 70 RNA genes. To our knowledge, this is only the second reported genome from the genus \(Williamsia\) and the first sequenced strain from the phyllosphere. The presented genomic information is interpreted in the context of an adaptation to the phyllosphere habitat. KW - arabidopsis thaliana KW - whole genome sequencing KW - adaption KW - Williamsia sp. ARP1 KW - phyllosphere KW - draft genome KW - next generation sequencing KW - assembly KW - annotation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146008 VL - 11 IS - 8 ER - TY - THES A1 - Mekala, SubbaRao T1 - Generation of cardiomyocytes from vessel wall-resident stem cells T1 - Erzeugung von Kardiomyozyten aus Gefäßwand-residenten Stammzellen N2 - Myocardial infarction (MI) is a major cause of health problems and is among the leading deadly ending diseases. Accordingly, regenerating functional myocardial tissue and/or cardiac repair by stem cells is one of the most desired aims worldwide. Indeed, the human heart serves as an ideal target for regenerative intervention, because the capacity of the adult myocardium to restore itself after injury or infarct is limited. Thus, identifying new sources of tissue resident adult stem or progenitor cells with cardiovascular potential would help to establish more sophisticated therapies in order to either prevent cardiac failure or to achieve a functional repair. Ongoing research worldwide in this field is focusing on a) induced pluripotent stem (iPS) cells, b) embryonic stem (ES) cells and c) adult stem cells (e. g. mesenchymal stem cells) as well as cardiac fibroblasts or myofibroblasts. However, thus far, these efforts did not result in therapeutic strategies that were transferable into the clinical management of MI and heart failure. Hence, identifying endogenous and more cardiac-related sources of stem cells capable of differentiating into mature cardiomyocytes would open promising new therapeutic opportunities. The working hypothesis of this thesis is that the vascular wall serves as a niche for cardiogenic stem cells. In recent years, various groups have identified different types of progenitors or mesenchymal stem cell-like cells in the adventitia and sub-endothelial zone of the adult vessel wall, the so called vessel wall-resident stem cells (VW-SCs). Considering the fact that heart muscle tissue contains blood vessels in very high density, the physiological relevance of VW-SCs for the myocardium can as yet only be assumed. The aim of the present work is to study whether a subset of VW-SCs might have the capacity to differentiate into cardiomyocyte-like cells. This assumption was challenged using adult mouse aorta-derived cells cultivated in different media and treated with selected factors. The presented results reveal the generation of spontaneously beating cardiomyocyte-like cells using specific media conditions without any genetic manipulation. The cells reproducibly started beating at culture days 8-10. Further analyses revealed that in contrast to several publications reporting the Sca-1+ cells as cardiac progenitors the Sca-1- fraction of aortic wall-derived VW-SCs reproducibly delivered beating cells in culture. Similar to mature cardiomyocytes the beating cells developed sarcomeric structures indicated by the typical cross striated staining pattern upon immunofluorescence analysis detecting α-sarcomeric actinin (α-SRA) and electron microscopic analysis. These analyses also showed the formation of sarcoplasmic reticulum which serves as calcium store. Correspondingly, the aortic wall-derived beating cardiomyocyte-like cells (Ao-bCMs) exhibited calcium oscillations. This differentiation seems to be dependent on an inflammatory microenvironment since depletion of VW-SC-derived macrophages by treatment with clodronate liposomes in vitro stopped the generation of Ao bCMs. These locally generated F4/80+ macrophages exhibit high levels of VEGF (vascular endothelial growth factor). To a great majority, VW-SCs were found to be positive for VEGFR-2 and blocking this receptor also stopped the generation VW-SC-derived beating cells in vitro. Furthermore, the treatment of aortic wall-derived cells with the ß-receptor agonist isoproterenol or the antagonist propranolol resulted in a significant increase or decrease of beating frequency. Finally, fluorescently labeled aortic wall-derived cells were implanted into the developing chick embryo heart field where they became positive for α-SRA two days after implantation. The current data strongly suggest that VW-SCs resident in the vascular adventitia deliver both progenitors for an inflammatory microenvironment and beating cells. The present study identifies that the Sca-1- rather than Sca-1+ fraction of mouse aortic wall-derived cells harbors VW-SCs differentiating into cardiomyocyte-like cells and reveals an essential role of VW-SCs-derived inflammatory macrophages and VEGF-signaling in this process. Furthermore, this study demonstrates the cardiogenic capacity of aortic VW-SCs in vivo using a chimeric chick embryonic model. N2 - Der Myokardinfarkt (MI) ist einer der Hauptgründe für gesundheitliche Probleme und zählt zu einer der am häufigsten tödlich verlaufenden Krankheiten weltweit. Daher ist es nicht verwunderlich, dass die Regeneration von funktionellem Myokardgewebe und/oder die kardiale Reparatur durch Stammzellen eines der weltweit am meisten angestrebten Ziele darstellt. Das adulte menschliche Herz stellt aufgrund seiner äußerst eingeschränkten endogenen Regenerationskapazität, die bei weitem nicht ausreicht, das geschädigte Gewebe zu erneuern, ein ideales Zielorgan für regenerative Therapieverfahren dar. Folglich könnte die Identifizierung neuer Quellen adulter Stamm- oder Vorläuferzellen mit kardiovaskulärem Differenzierungspotential dabei helfen, verfeinerte Therapien zu entwickeln, um entweder kardiale Fehlfunktionen zu verhindern oder eine deutlich verbesserte myokardiale Reparatur zu erreichen. Die aktuelle weltweite Forschung auf diesem Gebiet fokussiert sich auf: a) induzierte pluripotente Stammzellen (iPS), b) embryonale Stammzellen (ES) und c) adulte Stammzellen, wie z. B. mesenchymale Stammzellen, kardiale Fibroblasten und Mesangioblasten sowie Myofibroblasten. Bisher haben jedoch alle Bemühungen noch zu keinem Durchbruch geführt, so dass die teilweise vielversprechenden experimentellen Ergebnisse nicht in die klinische Therapie des MI und der kardialen Defekte mittels Stammzellen transferiert werden können. Abgesehen davon, ob und wie stark so ein endogenes herzeigenes Potential wäre, würde die Identifizierung neuer endogener Stammzellen mit kardiogenem Potential, die genaue Charakterisierung ihrer Nischen und der Mechanismen ihrer Differenzierung einen Meilenstein in der kardioregenerativen Stammzelltherapie darstellen. Die Arbeitshypothese der hier vorgelegten Dissertation besagt, dass die Gefäßwand als Nische solcher Zellen dienen könnte. Innerhalb der letzten Jahre konnte die Adventitia und die subendotheliale Zone der adulten Gefäßwand als Nische für unterschiedliche Typen von Vorläuferzellen und multipotenten Stammzellen, die sogenannten Gefäßwand-residenten Stammzellen (VW-SCs) identifiziert werden. In Anbetracht der Tatsache, dass die Blutgefäße aufgrund ihrer hohen Dichte im Herzen eine essentielle stromale Komponente des Herzgewebes darstellen, kann die mögliche klinische Relevanz von VW-SCs für das Myokardium im Moment nur erahnt werden. Ausgehend von der Annahme, dass eine Subpopulation dieser VW-SCs die Fähigkeit besitzt, sich in Kardiomyozyten-ähnliche Zellen zu differenzieren, sollte im Rahmen dieser Dissertationsarbeit das myokardiale Potential der Gefäßwand-residenten Stammzellen aus der Aorta adulter Mäuse studiert werden, indem die Zellen unter unterschiedlichen definierten Bedingungen kultiviert und dann sowohl morphologisch als auch funktionell charakterisiert werden. Erstaunlicherweise zeigten die ersten Ergebnisse die Generierung spontan schlagender Kardiomyozyten-ähnlicher Zellen, nur durch Verwendung eines speziellen Nährmediums und ohne jegliche genetische Manipulation. Die im Rahmen dieser Arbeit durchgeführten Analysen belegen zudem, dass die Kardiomyozyten-ähnlichen Zellen reproduzierbar nach ca. 9-11 Tagen in der Kultur anfangen, spontan zu schlagen. In immunzytochemischen Analysen zeigten die schlagenden Zellen ein quergestreiftes Färbemuster für α sarkomeres Actinin. Passend dazu wiesen diese spontan schlagenden Zellen, wie reife Kardiomyozyten, Sarkomerstrukturen mit Komponenten des sarkoplasmatischen Retikulums in elektronenmikroskopischen Analysen auf. Sie zeigten dementsprechend eine mit dem spontanen Schlag assoziierte Kalzium-Oszillation. Erstaunlicherweise zeigten die hier vorgelegten Befunde erstmalig, dass es nicht die Sca-1+ (stem cell antigen-1) Zellen waren, denen seit Jahren eine kardiomyozytäre Kapazität zugeschrieben wird, sondern es waren die Sca-1- Zellen der Mausaorta, die sich zu den spontan schlagenden Zellen differenzierten. Des Weiteren scheint diese Differenzierung von einer endogen generierten inflammatorischen Mikroumgebung abhängig zu sein. Die hier vorgelegten Ergebnisse legen daher den Schluss nahe, dass die VW-SCs in der vaskulären Adventitia sowohl die inflammatorische Mikroumgebung als auch die spontan schlagenden Kardiomyozyten-ähnlichen Zellen bereitstellten. So entstanden in der Kultur aortaler Zellen unter anderem auch Makrophagen, die hohe Mengen des Gefäßwachstumsfaktors VEGF (Vascular Endothelial Growth Factor) aufweisen. Wurden die Makrophagen in der Zellkultur durch Zugabe von Clodtronat-Liposomen depletiert, so wurde damit auch die Generierung spontan schlagender Zellen aus den aortalen VW-SCs unterbunden. Um zu testen, ob und inwieweit dieser Einfluss der Makrophagen auf die Entstehung spontan schlagender Zellen aus den VW-SCs auf den VEGF zurückzuführen ist, wurden kultivierte Zellen der Mausaorta mit dem VEGF-Rezeptor-2-Blocker (E7080) behandelt. Auch diese Behandlung resultierte wie bei der Depletion von Makrophagen darin, dass keine spontan schlagenden Zellen entstanden. Um die von VW-SCs generierten spontan schlagenden Zellen funktionell zu charakterisieren, wurden die kultivierten Zellen der Mausaorta mit Isoproterenol (ß-Sympathomimetikum) und Propranolol (ß-Blocker) behandelt. Eine signifikante Steigerung der Schlagfrequenz unter Isoproterenol und eine Reduzierung bei Zugabe von Propranolol unterstreichen ebenfalls die Kardiomyozyten-ähnliche Eigenschaft der spontan schlagenden Zellen. Schließlich wurden die aus der Mausaorta isolierten Zellen Fluoreszenz-markiert und dann in das kardiale Feld des sich entwickelnden Hühnerembryos (am fünften Tag der Entwicklung) implantiert. Zwei Tage später wurden die Herzen entnommen. Immunfärbungen zeigten, dass ein Teil der implantierten Zellen auch unter diesen in vivo-Bedingungen für α-sarkomeres Actinin positiv wurde und somit einen kardiomyozytären Phänotyp aufwies. KW - vessel wall resident stem cells KW - cardiomyocytes KW - Herzmuskelzelle KW - Stammzelle Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146046 N1 - My PhD research work has been published in Circ Res. 2018 Aug 31;123(6):686-699. ER - TY - THES A1 - Pischimarov, Jordan Ivanov T1 - Bioinformatische Methoden zur Identifizierung und Klassifizierung somatischer Mutationen in hämatologischen Erkrankungen T1 - Bioinformatics approaches for the detection and classification of somatic mutations in hematological malignancies N2 - Die Sequenzierungstechnologien entwickeln sich stetig weiter, dies ermöglicht eine zuvor nicht erreichte Ausbeute an experimentellen Daten und auch an Neuentwicklungen von zuvor nicht realisierbaren Experimenten. Zugleich werden spezifische Datenbanken, Algorithmen und Softwareprogramme entwickelt, um die neu entstandenen Daten zu analysieren. Während der Untersuchung bioinformatischer Methoden für die Identifizierung und Klassifizierung somatischer Mutationen in hämatologischen Erkrankungen, zeigte sich eine hohe Vielfalt an alternativen Softwaretools die für die jeweiligen Analyseschritte genutzt werden können. Derzeit existiert noch kein Standard zur effizienten Analyse von Mutationen aus Next-Generation-Sequencing (NGS)-Daten. Die unterschiedlichen Methoden und Pipelines generieren Kandidaten, die zum größten Anteil in allen Ansätzen identifiziert werden können, jedoch werden Software spezifische Kandidaten nicht einheitlich detektiert. Um eine einheitliche und effiziente Analyse von NGS-Daten durchzuführen war im Rahmen dieser Arbeit die Entwicklung einer benutzerfreundlichen und einheitlichen Pipeline vorgesehen. Hierfür wurden zunächst die essentiellen Analysen wie die Identifizierung der Basen, die Alignierung und die Identifizierung der Mutationen untersucht. Des Weiteren wurden unter Berücksichtigung von Effizienz und Performance diverse verfügbare Softwaretools getestet, ausgewertet und sowohl mögliche Verbesserungen als auch Erleichterungen der bisherigen Analysen vorgestellt und diskutiert. Durch Mitwirken in Konsortien wie der klinischen Forschergruppe 216 (KFO 216) und International Cancer Genome Consortium (ICGC) oder auch bei Haus-internen Projekten wurden Datensätze zu den Entitäten Multiples Myelom (MM), Burkitt Lymphom (BL) und Follikuläres Lymphom (FL) erstellt und analysiert. Die Selektion geeigneter Softwaretools und die Generierung der Pipeline basieren auf komparativen Analysen dieser Daten, sowie auf geteilte Ergebnisse und Erfahrungen in der Literatur und auch in Foren. Durch die gezielte Entwicklung von Skripten konnten biologische und klinische Fragestellungen bearbeitet werden. Hierzu zählten eine einheitliche Annotation der Gennamen, sowie die Erstellung von Genmutations-Heatmaps mit nicht Variant-Calling-File (VCF)-Syntax konformen Dateien. Des Weiteren konnten nicht abgedeckte Regionen des Genoms in den NGS-Daten identifiziert und analysiert werden. Neue Projekte zur detaillierten Untersuchung der Verteilung von wiederkehrender Mutationen und Funktionsassays zu einzelnen Mutationskandidaten konnten basierend auf den Ergebnissen initiiert werden. Durch eigens erstellte Python-Skripte konnte somit die Funktionalität der Pipeline erweitert werden und zu wichtigen Erkenntnissen bei der biologischen Interpretation der Sequenzierungsdaten führen, wie beispielsweise zu der Detektion von drei neuen molekularen Subgruppen im MM. Die Erweiterungen, der in dieser Arbeit entwickelten Pipeline verbesserte somit die Effizienz der Analyse und die Vergleichbarkeit unserer Daten. Des Weiteren konnte durch die Erstellung eines eigenen Skripts die Analyse von unbeachteten Regionen in den NGS-Daten erfolgen. N2 - The sequencing technologies, while still being under further development, render it possible to develop novel experiments and allow the generation of larger amounts of utilizable data. At the same time novel software tools, databases and algorithms are developed to analyze these larger amounts of data. The analysis of somatic mutations in hematological malignancies showed that a high variety of alternative software tools can be used for different analysis steps. Furthermore there is currently no standardized procedure for the efficient identification and analysis of mutations in NGS data. The different pipeline and methods are, for the most part, able to identify the same mutation candidates, however there are software specific candidates which are not called by all pipelines. The scope of this dissertation was therefore to develop a user-friendly pipeline which is able to call candidate mutations uniformly and efficiently. For this purpose necessary analysis steps including base calling, alignment generation and variant calling were investigated. Furthermore available software tools were tested and evaluated regarding their efficiency and performance. Possible improvements of these software tools and previously performed analysis are explained and discussed in this work. NGS data sets of the different cancer entities multiple myeloma (MM), Burkitt lymphoma (BL) and follicular lymphoma (FL) were generated and analyzed within the framework of cooperate projects like the International Cancer Genome Consortium (ICGC) and the Clinical Research Group 216 (KFO) as well as for internal projects. The development of the pipeline and selection of suitable software tools is based on the comparative analysis of the generated data sets, as well as previously described results and experiences in literature and forums. The selective development of certain python scripts enabled the evaluation of novel biological and clinical questions by standardizing gene names in the annotation step, generating heat- maps of non-standardized VCF-files as well as the identification and analysis of uncovered regions in NGS data sets. This work and the obtained results thereby provide the groundwork for further projects e.g. the analysis of the distribution of recurrent mutations or the functional analysis of specific mutation candidates. This extensions of the developed pipeline with python scripts helped to improve the efficiency and comparability of the NGS data. The interpretation of the NGS data with the extended script for example led to the discovery of three distinct molecular subgroups in MM. Furthermore the generation of the novel python scripts helped to analyze uncovered regions in the NGS data sets.  KW - Pipeline-Rechner KW - somatische Mutationen KW - Sequenzierung KW - Bioinformatik KW - Identifizierungspipeline KW - Next Generation Sequencing KW - Variantcalling KW - Bioinformatic KW - somatic mutations KW - DNS-Sequenz KW - Somatische Mutation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147773 ER - TY - JOUR A1 - Sommerlandt, Frank M. J. A1 - Spaethe, Johannes A1 - Rössler, Wolfgang A1 - Dyer, Adrian G. T1 - Does Fine Color Discrimination Learning in Free-Flying Honeybees Change Mushroom-Body Calyx Neuroarchitecture? JF - PLoS One N2 - Honeybees learn color information of rewarding flowers and recall these memories in future decisions. For fine color discrimination, bees require differential conditioning with a concurrent presentation of target and distractor stimuli to form a long-term memory. Here we investigated whether the long-term storage of color information shapes the neural network of microglomeruli in the mushroom body calyces and if this depends on the type of conditioning. Free-flying honeybees were individually trained to a pair of perceptually similar colors in either absolute conditioning towards one of the colors or in differential conditioning with both colors. Subsequently, bees of either conditioning groups were tested in non-rewarded discrimination tests with the two colors. Only bees trained with differential conditioning preferred the previously learned color, whereas bees of the absolute conditioning group, and a stimuli-naïve group, chose randomly among color stimuli. All bees were then kept individually for three days in the dark to allow for complete long-term memory formation. Whole-mount immunostaining was subsequently used to quantify variation of microglomeruli number and density in the mushroom-body lip and collar. We found no significant differences among groups in neuropil volumes and total microglomeruli numbers, but learning performance was negatively correlated with microglomeruli density in the absolute conditioning group. Based on these findings we aim to promote future research approaches combining behaviorally relevant color learning tests in honeybees under free-flight conditions with neuroimaging analysis; we also discuss possible limitations of this approach.q KW - bees KW - behavioral conditioning KW - learning KW - color vision KW - vision KW - calyx KW - cognition KW - honey bees Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147932 VL - 11 IS - 10 ER - TY - JOUR A1 - Ankenbrand, Markus J. A1 - Weber, Lorenz A1 - Becker, Dirk A1 - Förster, Frank A1 - Bemm, Felix T1 - TBro: visualization and management of de novo transcriptomes JF - Database N2 - RNA sequencing (RNA-seq) has become a powerful tool to understand molecular mechanisms and/or developmental programs. It provides a fast, reliable and cost-effective method to access sets of expressed elements in a qualitative and quantitative manner. Especially for non-model organisms and in absence of a reference genome, RNA-seq data is used to reconstruct and quantify transcriptomes at the same time. Even SNPs, InDels, and alternative splicing events are predicted directly from the data without having a reference genome at hand. A key challenge, especially for non-computational personnal, is the management of the resulting datasets, consisting of different data types and formats. Here, we present TBro, a flexible de novo transcriptome browser, tackling this challenge. TBro aggregates sequences, their annotation, expression levels as well as differential testing results. It provides an easy-to-use interface to mine the aggregated data and generate publication-ready visualizations. Additionally, it supports users with an intuitive cart system, that helps collecting and analysing biological meaningful sets of transcripts. TBro’s modular architecture allows easy extension of its functionalities in the future. Especially, the integration of new data types such as proteomic quantifications or array-based gene expression data is straightforward. Thus, TBro is a fully featured yet flexible transcriptome browser that supports approaching complex biological questions and enhances collaboration of numerous researchers. KW - database Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147954 VL - 2016 ER - TY - JOUR A1 - Othman, Eman M. A1 - Naseem, Muhammed A1 - Awad, Eman A1 - Dandekar, Thomas A1 - Stopper, Helga T1 - The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells JF - PLoS One N2 - Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing. KW - DNA damage KW - apoptosis KW - oxidative stress KW - fluorescence recovery after photobleaching KW - lymphocytes KW - antioxidants KW - cell staining KW - cytokinins Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147983 VL - 11 IS - 12 ER - TY - JOUR A1 - Kunz, Meik A1 - Wolf, Beat A1 - Schulze, Harald A1 - Atlan, David A1 - Walles, Thorsten A1 - Walles, Heike A1 - Dandekar, Thomas T1 - Non-Coding RNAs in Lung Cancer: Contribution of Bioinformatics Analysis to the Development of Non-Invasive Diagnostic Tools JF - Genes N2 - Lung cancer is currently the leading cause of cancer related mortality due to late diagnosis and limited treatment intervention. Non-coding RNAs are not translated into proteins and have emerged as fundamental regulators of gene expression. Recent studies reported that microRNAs and long non-coding RNAs are involved in lung cancer development and progression. Moreover, they appear as new promising non-invasive biomarkers for early lung cancer diagnosis. Here, we highlight their potential as biomarker in lung cancer and present how bioinformatics can contribute to the development of non-invasive diagnostic tools. For this, we discuss several bioinformatics algorithms and software tools for a comprehensive understanding and functional characterization of microRNAs and long non-coding RNAs. KW - lung cancer KW - non-invasive biomarkers KW - miRNAs KW - lncRNAs KW - bioinformatics KW - early diagnosis KW - algorithm Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147990 VL - 8 IS - 1 ER - TY - JOUR A1 - Kramer, Susanne T1 - Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution JF - Nucleic Acids Research N2 - The detection of mRNAs undergoing transcription or decay is challenging, because both processes are fast. However, the relative proportion of an mRNA in synthesis or decay increases with mRNA size and decreases with mRNA half-life. Based on this rationale, I have exploited a 22 200 nucleotide-long, short-lived endogenous mRNA as a reporter for mRNA metabolism in trypanosomes. The extreme 5΄ and 3΄ ends were labeled with red- and green-fluorescent Affymetrix® single mRNA FISH probes, respectively. In the resulting fluorescence images, yellow spots represent intact mRNAs; red spots are mRNAs in transcription or 3΄-5΄ decay, and green spots are mRNAs in 5΄-3΄ degradation. Most red spots were nuclear and insensitive to transcriptional inhibition and thus likely transcription intermediates. Most green spots were cytoplasmic, confirming that the majority of cytoplasmic decay in trypanosomes is 5΄-3΄. The system showed the expected changes at inhibition of transcription or translation and RNAi depletion of the trypanosome homologue to the 5΄-3΄ exoribonuclease Xrn1. The method allows to monitor changes in mRNA metabolism both on cellular and on population/tissue wide levels, but also to study the subcellular localization of mRNA transcription and decay pathways. I show that the system is applicable to mammalian cells. KW - mRNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148002 ER -