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Der Einsatz von computergestützten Analysen hat sich zu einem festen Bestandteil der biowissenschaftlichen Forschung etabliert. Im Rahmen dieser vorliegenden Arbeit wurden systembiologische Untersuchungen auf verschiedene biologische Themengebiete und Organismen angewendet. In diesem Zusammenhang liefert die Arbeit einen innovativen und interdisziplinären methodischen Ansatz. Die grundlegende Frage lautet: Wie verstehe und beschreibe ich Signalwege und wie kann ich sie beeinflussen? Der Ansatz verknüpft verschiedene biologische Datensätze und Datenebenen miteinander, beginnend vom Genom und Interaktionskontext über semiquantitative Simulationen hin zu neuen Interventionen und Experimenten, welche therapeutisch und biotechnologisch genutzt werden können. Die Analysen können auf diese Weise
- zu einem besseren Verständnis experimenteller Daten und biologischer Fragestellungen beitragen und ermöglichen ein systematisches Verständnis der zugrunde liegenden Signalwege und Netzwerkeffekte (z.B. in Pflanzen).
- Darüber hinaus ermöglichen sie die Identifizierung wichtiger funktioneller Hubproteine und die Entwicklung neuer therapeutischer Strategien für weitere experimentelle Testungen (z.B. Tumormodelle),
- stellen zudem einen hilfreichen Schritt auf dem Weg zur personalisierten Medizin (z.B. lncRNAs und Tumormodelle) und Medikamentenentwicklung (z.B. Datenbank DrumPID) dar.
(i) Als Grundlage wurde hierzu eine integrierte systembiologische Methode entwickelt, welche experimentelle Daten (z.B. Transkriptomdaten) hinsichtlich ihrer biologischen Funktionen untersucht und die Identifizierung relevanter funktioneller Cluster und Hubproteine ermöglicht. In einem ersten Teil wurden Analysen zum pflanzlichen Immunsystem durchgeführt. Mithilfe der entwickelten Methode wurden Genexpressionsdatensätze von A. thaliana, die mit dem Pathogen Pst DC3000 infiziert wurden, untersucht, um den Einfluss verschiedener Virulenzfaktoren auf das Interaktom der Wirtspflanze zu untersuchen und neue Modulatoren einer CK-vermittelten Immunabwehr zu finden. In diesem Zusammenhang konnte gezeigt werden, dass die von Pst DC3000 sekretierten Abwehrstoffe wichtige pflanzliche Hormonsignalwege für die Immunabwehr in A. thaliana beeinflussen. Die Ergebnisse zeigen zudem, dass sich der Einfluss auf das Netzwerkverhalten der Effektorproteine und COR-Phytotoxine von dem der PAMPs unterscheidet, sich jedoch auch eine Regulierung gemeinsamer Signalwege und eine Überlappung der beiden Phasen der Immunantwort (PTI und ETI) in A. thaliana finden lassen. Die komplexe Immunantwort auf eine Infektion spiegelt sich zudem in einer höheren Anzahl an funktionellen Clustern und Hubproteinen in Pst DC3000 gegenüber den beiden untersuchten Mutanten wider, wobei sich für Pst DC3000 insbesondere ein stark vernetztes immunrelevantes Cluster um den JA-Signalweg zeigt. Weiterhin wurden anhand der entwickelten Methode wichtige Hubproteine für die Immunabwehr identifiziert. Als bedeutende Vertreter sind AHK2 und AAR14 zu nennen, welche Teil des Zweikomponentensystems der Signalübertragung von CK sind und hierbei wichtige Modulatoren für eine CK-vermittelte Immunabwehr darstellen.
(ii) Im zweiten Teil der Arbeit schließen sich Untersuchungen an einem in vitro-Experiment einer 2D- und 3D-Zellkultur einer HSP90-Behandlung in einem Lungentumormodell an. In diesem Zusammenhang wurden mithilfe der entwickelten Methode Unterschiede zwischen den beiden Zellkultursystemen gefunden, die das unterschiedliche Behandlungsansprechen erklären, und für die beiden KRAS-mutierten Zelllinien A549 und H441 des 3D-Testsystems neue prognostische und therapeutische Kandidaten identifiziert. Hierbei haben die durchgeführten Analysen zwei funktionelle Cluster von Protein-Interaktionen um p53 und die STAT-Familie gefunden, welche eine Verbindung zu HSP90 haben und die entsprechenden Behandlungsunterschiede nach einer HSP90-Inhibierung zwischen den beiden Zellkultursystemen erklären können. Unter Berücksichtigung des zelllinien-spezifischen Mutationshintergrunds wurde eine prognostische Markersignatur und daraus abgeleitet HIF1A für die H441-Zelllinie und AMPK für die A549-Zelllinie als neue therapeutische Targets gefunden, wobei die anschließend durchgeführten in silico-Simulationen einen potentiellen therapeutischen Effekt aufzeigen konnten. Weiterhin wurden wichtige experimentelle Readout-Parameter in ein in silico-Lungentumormodell integriert, wobei unter Einbeziehung des Mutationshintergrunds für die verwendeten Zelllinien die HSP90-Behandlung des 3D-Testsystems computergestützt abgebildet werden konnte. Im weiteren Verlauf wurden im in silico-Lungentumormodell Resistenzmechanismen nach einer Gefitinib-Behandlung mit bekanntem Mutationsstatus für die Zelllinien HCC827 und A549 untersucht und daraus folgend neue Therapieansätze abgeleitet, die von potentieller klinischer Bedeutung sein können. Die durchgeführten in silico-Simulationen für HCC827 konnten hierbei zeigen, dass eine EGFR- und c-MET-Koaktivierung zu einer Gefitinib-Resistenz führen kann, wohingegen bei den A549 eine Komutation von KRAS und IGF-1R zu einem geringen Behandlungsansprechen beiträgt. Die Simulationen lassen zudem erkennen, dass eine direkte Inhibierung der an der Resistenzentwicklung beteiligten Rezeptoren c-MET und IGF-1R in beiden Fällen nicht die bestmögliche Therapiestrategie darstellt. In beiden Zelllinien konnte gezeigt werden, dass eine kombinierte Inhibierung von PI3K und MEK den bestmöglichen therapeutischen Effekt liefert, was demnach einen vielversprechenden Therapieansatz bei Gefitinib-resistenten Lungentumorpatienten darstellt. In einem weiteren Schritt wurde das therapeutische Potential der miRNA-21 im in silico-Modell für die HCC827-Zelllinie untersucht. Die durchgeführten Simulationen zeigen, dass eine miRNA-21-Überexpression zu einer Resistenzentwickung nach Gefitinib-Behandlung beitragen kann, wobei eine Inhibierung der miRNA-21 diesen Effekt umkehren kann. Die Ergebnisse lassen zudem erkennen, dass eine PTEN-Aktivierung als potentieller Marker einer erfolgreichen therapeutischen Inhibierung der miRNA-21 fungieren kann, wohingegen eine reduzierte miRNA-21-Expression als möglicher Marker für eine erfolgreiche Gefitinib-Behandlung dienen kann.
(iii) Im dritten Teil der Arbeit wurden systematisch RNA- und Protein-Interaktionen untersucht. Hierzu wurden integrierte systembiologische Analysen an neu identifizierten und funktionell bislang unbekannten lncRNAs durchgeführt. Die Analysen für die infolge einer Herzhypertrophie hochregulierte lncRNA Chast haben umfassend gezeigt, dass diese Proteine und Transkriptionsfaktoren regulieren und binden kann, welche die Signalübertragung und Genexpression regulieren, aber auch eine Verbindung zum kardiovaskulären System und stressinduzierter Herzhypertrophie besitzt. Anhand der Ergebnisse lässt sich schlussfolgern, dass Chast direkt und indirekt (a) Proteine binden und die Translation beeinflussen kann, zudem eine Chromatin-modifizierende Funktion besitzt und so die Transkription, z.B. für herz- und stress-assoziierte Gene, reguliert, und/oder (b) in einem negativen Feedbackloop seine eigene Transkription reguliert. Obwohl lncRNAs meist eine geringe Konservierung aufweisen, konnten die durchgeführten Analysen für Chast eine Sequenz-Struktur-Konservierung in Säugetieren aufzeigen. Weiterhin haben die Untersuchungen an zwei hypoxie-induzierten lncRNAs in Endothelzellen gezeigt, dass die lncRNA MIR503HG eine hohe Sequenz-Struktur-Konservierung in Säugetieren besitzt, wohingegen die LINC00323-003 eine geringe Konservierung aufzeigt. Dies untermauert die Tatsache, dass lncRNAs häufig eine geringe Konservierung aufweisen, was Untersuchungen in Modellorganismen hinsichtlich einer therapeutischen Nutzung schwierig machen.
Da sich zahlreiche Untersuchungen auf Interaktionen und Signalwege konzentriert haben, wurde abschließend eine Datenbank entwickelt, welche Analysen von Protein-Interaktionen und Signalwegen nachhaltig voranbringt. Die entwickelte DrumPID-Datenbank stellt insbesondere die Interaktion zwischen einem Medikament und seinem Target in den Fokus und ermöglicht Analysen einzelner Interaktionen und beteiligter Signalwege, bietet zusätzlich aber auch verschiedene Links zu anderen Datenbanken für individuelle weiterführende Analysen. DrumPID ermöglicht ein geeignetes Medikament u. a. für ein vorgegebenes Zielprotein zu finden und dessen Wirkmechanismus und Interaktionskontext zu untersuchen, was zu einem besseren experimentellen Verständnis beitragen kann. Zudem erlaubt DrumPID eine potentielle chemische Leitstruktur für ein Zielprotein zu entwickeln, was z.B. spezifisch ein parasitisches Protein inhibiert, ohne dabei einen toxischen Effekt im Menschen zu haben. Zahlreiche weitere Pharmakabeispiele belegen, dass DrumPID für den täglichen wissenschaftlichen Gebrauch auf dem Gebiet der Analyse von Protein-Pharmaka-Interaktionen und der Medikamentenentwicklung geeignet ist.
Die beschriebenen Ergebnisse der Promotionsarbeit wurden in fünf Originalarbeiten, zwei Übersichtsartikeln und einem Buchteil, u. a. in Science Translational Medicine, veröffentlicht, sechs dieser Publikationen erfolgten im Rahmen von Erstautorschaften.
Pancreatic cancer (PC) remains one of the most challenging solid tumors to treat with a high unmet medical need as patients poorly respond to standard-of-care-therapies. Prominent desmoplastic reaction involving cancer-associated fibroblasts (CAFs) and the immune cells in the tumor microenvironment (TME) and their cross-talk play a significant role in tumor immune escape and progression. To identify the key cellular mechanisms induce an immunosuppressive tumor microenvironment, we established 3D co-culture model with pancreatic cancer cells, CAFs, monocyte as well as T cells.
Using this model, we analysed the influence of tumor cells and fibroblasts on monocytes and their immune suppressive phenotype. Phenotypic characterization of the monocytes after 3D co-culture with tumor/fibroblast spheroids was performed by analysing the expression of defined cell surface markers and soluble factors. Functionality of these monocytes and their ability to influence T cell phenotype and proliferation was investigated.
3D co-culture of monocytes with pancreatic cancer cells and fibroblasts induced the production of immunosuppressive cytokines which are known to promote polarization of M2 like macrophages and myeloid derived suppressive cells (MDSCs). These co-culture spheroid polarized monocyte derived macrophages (MDMs) were poorly differentiated and had an M2 phenotype. The immunosuppressive function of these co-culture spheroids polarized MDMs was demonstrated by their ability to inhibit autologous CD4+ and CD8+ T cell activation and proliferation in vitro, which we could partially reverse by 3D co-culture spheroid treatment with therapeutic molecules that are able to re-activate spheroid polarized MDMs or block immune suppressive factors such as Arginase-I.
In conclusion, we generated a physiologically relevant 3D co-culture model, which can be used as a promising tool to study complex cell-cell interactions between different cell types within the tumor microenvironment and to support drug screening and development. In future, research focused on better understanding of resistance mechanisms to existing cancer immunotherapies will help to develop new therapeutic strategies in order to combat cancer.
The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer
(2017)
The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far.
In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters.
Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis.
This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.
Am Rebstock werden in der Natur von Agrobacterium vitis, dem Auslöser Wurzelhalsgallenerkrankung, charakteristische Wurzelhalsgallentumore induziert. Virulente Vertreter der Gattung der Agrobacteria schleusen bakterielle DNA in das pflanzliche Genom ein, wodurch die Pflanze Tumore produziert. Die Wurzelhalsgallenerkrankung wird seit einem Jahrhundert als ein Beispiel der Pflanzen-Pathogen-Interaktion untersucht. Die Rolle der bakteriellen Flora im Zusammenhang mit der Wurzelhalsgallenerkrankung beim Rebstock wurde bisher kaum betrachtet. Um dieser Frage nachzugehen, habe ich die endophytische mikrobielle Zusammensetzung von Rebstöcken mit und ohne Wurzelhalsgalle analysiert. Es werden Proben von drei Zeitpunkten einer Wachstumsperiode (Frühling, Sommer und Herbst) und von den Organen der Rebstöcke (Wurzeln, Pfropfstelle und einjährige Triebe) sowie dem Boden in einer Weinanlage bei Himmelstadt in Unterfranken genommen. Die Bakterienflora dieser Umweltproben wird mit kultivierungsabhängigen (Isolierung von Bakterien) und kultivierungsunabhängigen (Hochdurchsatzsequenzierungen) Methoden untersucht. Zudem werden i) die Virulenz der verschiedenen Agrobacterium-Isolate in Tumorassays bestimmt, ii) synthetische Bakteriengemeinschaften von in vitro kultivierten Weinpflänzchen mit Wurzelhalsgallen analysiert, iii) die Genome von einem virulenten und einem nicht-virulenten Agrobacteria-Isolat aus der Wurzelhalsgalle verglichen, iv) erste Interaktionsstudien auf festen Nährmedien durchgeführt und v) virulente Agrobacteria mittels bildgebender Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) in Wurzelhalsgallen lokalisiert.
Die Rebstöcke dieser Studie haben eine organspezifische Bakterienflora, die innerhalb einer Wachstumsperiode variiert. Nur die Bakterienflora der Pfropfstelle (mit oder ohne Wurzelhalsgalle) aber nicht die des Bodens, der Wurzeln, und der einjährigen Triebe unterscheidet sich strukturell zwischen gesunden und erkrankten Rebstöcken. Mikroskopisch konnten virulente Agrobacteria punktuell in Interzellularen, sklerenchymatischen Geweben und assoziiert mit Leitgefäßen nachgewiesen werden. Dadurch ist ausreichend Lebensraum vorhanden, der zusätzlich von tumorspezifischen Bakterien besiedelt werden kann. Im Gegensatz zur gesunden Pfropfstelle ist in der Wurzelhalsgalle eine saisonal stabile Kernmikroflora, bestehend aus Vertreter von A. vitis, Pseudomonas, Enterobacteriaceae, Agrobacterium tumefaciens, Gammaproteobacteria und Burkholderiales, vorhanden. Diese Bakterien werden überwiegend aus dem Boden rekrutiert und profitieren von der Nährstoffsituation in der Wurzelhalsgalle. Wurzelhalsgallen enthalten Opine, die nur von der transformierten Pflanzenzelle produziert werden. Interessanterweise hat in dieser Arbeit ein Agrobacterium-Isolat Gene, die zum Opinkatabolismus beitragen und ein Pseudomonas-Isolat kann Opine als einzige Kohlenstoffquelle nutzen. Trotzdem sind beide Isolate weder virulent noch verdrängen sie die virulenten A. vitis, die ebenso Opine nutzen, aus der Wurzelhalsgalle. In synthetischen Bakteriengemeinschaften an in vitro kultivierten Weinpflänzchen konnte gezeigt werden, dass diese und weitere tumorspezifischen Bakterien, neben A. vitis, nicht essentiell zur Entstehung der Wurzelhalsgalle nötig sind aber unterschiedliche Funktionen in der Wurzelhalsgalle übernehmen. Ein Serratia-Isolat hemmt das Wachstum von A. vitis auf festen Nährmedium, andere fördern oder hemmen das Wachstum der Wurzelhalsgalle. Nach Studien in der Literatur erhöhen weitere Bakterien die Resistenz des Rebstocks gegenüber biotischem und abiotischem Stress.
Zusammengefasst identifizierten und isolierte ich in dieser Studie unter 150 unterschiedlichen Bakterien in der Wurzelhalsgalle jene Bakterien, die neben A. vitis von der neuen ökologischen Nische profitieren und somit wahrscheinlich Opportunisten mit unterschiedlichen Funktionen sind. In Folge von multiplen Interaktionen in der Wurzelhalsgalle entsteht ein ökologisches Gleichgewicht zwischen den opportunistischen Bakterien, der Wurzelhalsgalle und dem Rebstock, das den Fortbestand des Rebstocks mit Wurzelhalsgalle ermöglicht.
Single-molecule fluorescence microscopy in live \(Trypanosoma\) \(brucei\) and model membranes
(2018)
The eukaryotic parasite Trypanosoma brucei has evolved sophisticated strategies to escape
the host immune response and maintain a persistent infection inside a host. One central
feature of the parasite’s defense mechanism relies on the shielding function of their surface
protein coat. This coat is composed of a dense arrangement of one type of glycosylphosphatidylinositol
(GPI)-anchored variant surface glycoproteins (VSGs) which impair the
identification of epitopes of invariant surface proteins by the immune system. In addition
to the importance of understanding the function of the VSG coat and use it as a potential
target to efficiently fight the parasite, it is also crucial to study its biophysical properties as it is not yet understood sufficiently. This is due to the fact that microscopic investigations
on living trypanosomes are limited to a great extent by the intrinsic motility of the parasite.
In the present study, state-of-the-art single-molecule fluorescence microscopy (SMFM)
is introduced as a tool for biophysical investigations in the field of trypanosome research.
The work encompasses studies of VSG dynamics under the defined conditions of an
artificial supported lipid bilayer (SLB). First, the impact of the lateral protein density on
VSG diffusion was systematically studied in SLBs. Ensemble fluorescence after photobleaching
(FRAP) and complementary single-particle tracking experiments revealed that a
molecular crowding threshold (MCT) exists, above which a density dependent decrease
of the diffusion coefficient is measured. A relative quantification of reconstituted VSGs
illustrated that the VSG coat of living trypanosomes operates very close to its MCT and
is optimized for high density while maintaining fluidity. Second, the impact of VSG
N-glycosylation on VSG diffusion was quantitatively investigated. N-glycosylation was
shown to contribute to preserving protein mobility at high protein concentrations. Third,
a detailed analysis of VSG trajectories revealed that two distinct populations of freely
diffusing VSGs were present in a SLB, which is in agreement with the recent finding, that
VSGs are able to adopt two main structurally distinct conformations. The results from
SLBs were further complemented by single-particle tracking experiments of surface VSGs
on living trypanosomes. A high mobility and free diffusion were measured on the cell
surface, illustrating the overall dynamic nature of the VSG coat. It was concluded that
the VSG coat on living trypanosomes is a protective structure that combines density and
mobility, which is supported by the conformational flexibility of VSGs. These features are
elementary for the persistence of a stable infection in the host.
Different hydrogel embedding methods are presented, that facilitated SMFM in immobilized,
living trypanosomes. The hydrogels were found to be highly cytocompatible for one
hour after cross-linking. They exhibited low autofluorescence properties in the spectral
range of the investigations, making them suitable for super-resolution microscopy (SRM).
Exemplary SRM on living trypanosomes illustrated that the hydrogels efficiently immobilized
the cells on the nanometer lever. Furthermore, the plasma membrane organization was studied in living trypanosomes. A statistical analysis of a tracer molecule inside the
inner leaflet of the plasma membrane revealed that specific membrane domains exist, in
which the tracer appeared accumulated or diluted. It was suggested that this distribution
was caused by the interaction with proteins of the underlying cytoskeleton.
In conclusion, SMFM has been successfully introduced as a tool in the field of trypanosome
research. Measurements in model membranes facilitated systematic studies of VSG dynamics
on the single-molecule level. The implementation of hydrogel immobilization
allowed for the study of static structures and dynamic processes with high spatial and
temporal resolution in living, embedded trypanosomes for the first time.
Die autosomal-dominant vererbte facioscapulohumerale Muskeldystrophie (FSHD) ist mit einer Prävalenz von etwa 1:20.000 die dritthäufigste Form der hereditären Myopathien. Erste Beschwerden werden meist in der zweiten Lebensdekade beobachtet. Betroffen sind vor allem die Muskulatur von Gesicht, Schultern, Oberarmen, die Fußhebermuskulatur und die Muskeln des Hüftgürtels.
FSHD wird durch einen Gendefekt ausgelöst, der den langen Arm des Chromosoms vier (4q35) betrifft, wobei es zur teilweisen Deletion des polymorphen Abschnitts D4Z4, der für das Protein DUX4 codiert, kommt. Dabei treten unter anderem Störungen in der DUX4-Expression, Veränderungen der myogenen Genexpression, eine Unterdrückung der Muskelzelldifferenzierung und eine Inhibition der Muskelbildung auf.
FSHD und eine andere Form der Muskeldystrophie, die Emery-Dreifuss-Muskeldystrophie (EDMD), zeigen trotz unterschiedlicher genetischer Ursachen phänotypisch Ähnlichkeiten in der Ausprägung der Erkrankungen. In früheren Studien zeigte die Kernhülle von EDMD-Myoblasten morphologische Auffälligkeiten. In anderen Untersuchungen waren morphologische Veränderungen der Mitochondrien von FSHD-Patienten festzustellen.
Daher wurden elektronenmikroskopische Untersuchungen der Kernhülle und der Mitochondrien von FSHD-Myoblasten durchgeführt und mit der entsprechenden Kontrolle verglichen.
Hierfür wurden drei verschiedene Zelllinien-Paare in unterschiedlichen Passagen, das heißt unterschiedlicher Anzahl an Subkultivierungen, eingesetzt, wobei in den höheren Passagen vermehrt morphologische Atypien beobachtet werden konnten.
Die eingesetzten Zelllinien differenzieren sich durch verschiedene Parameter wie beispielsweise Alter und Geschlecht der Patienten. Dabei zeigten sich sowohl zwischen den Kontrollzellen als auch zwischen den FSHD-Myoblasten Unterschiede.
Im Rahmen der Probenvorbereitung für die Elektronenmikroskopie kamen zwei verschiedene Fixierungsmethoden zum Einsatz: die konventionelle chemische Fixierung, Entwässerung und Flacheinbettung von Kulturzellen und die Hochdruckgefrierung mit anschließender Gefriersubstitution. In Bezug auf die Qualität des Strukturerhalts, die beim Hochdruckgefrieren erreicht wird, wird dieser Art der Fixierung eine Überlegenheit gegenüber allen anderen Verfahren zugeschrieben. Diese allgemeine Aussage kann nicht vollständig auf die Untersuchungen an den Myoblasten übertragen werden.
Für die Untersuchung der Kernmembranen sind beide Methoden geeignet, wobei der Abstand zwischen innerer und äußerer Kernmembran nach der HPF-Fixierung schärfer abgebildet wurde. Bei der Darstellung der Mitochondrien zeigten die elektronenmikroskopischen Aufnahmen nach dem Hochdruckgefrieren bessere und schärfere Ergebnisse. Die Kernporen waren bei beiden Fixierungsmethoden gut erkennbar.
Beim Vergleich der gesunden und erkrankten Myoblasten wiesen die Kontrollzellen deutlich weniger Auffälligkeiten auf als die Myoblasten von FSHD-Patienten.
Innere und äußere Kernmembran verliefen bei den Kontrollzellen meist parallel und die Mitochondrien zeigten in den meisten Fällen eine typische wurmartige, längliche Form mit Cristae. Dies traf sowohl für die konventionelle Fixierung als auch für das Hochdruckgefrieren zu.
Die erkrankten Myoblasten wiesen im Vergleich zur Kontrolle bei beiden Fixierungsmethoden deutliche Auffälligkeiten in der Mitochondrien-Morphologie auf. Neben einer oft großen Variationsbreite hinsichtlich Form und Länge war auch das teilweise Fehlen der Cristae festzustellen.
Bei Betrachtung der Kernhülle fielen jedoch deutliche Unterschiede zwischen konventioneller und HPF-Fixierung auf. Die äußere Kernmembran der konventionell fixierten FSHD-Myoblasten verlief unregelmäßig und gewellt. Im Gegensatz dazu wies die Kernhülle der HPF-fixierten erkrankten Myoblasten einen erstaunlich parallelen Verlauf auf.
Da bei EDMD in vorangegangenen Untersuchungen auch fluoreszenzmikroskopisch Veränderungen der erkrankten Zellen auffällig waren, wurde neben den Methoden der Elektronenmikroskopie das Vorliegen und die Verteilung verschiedener Proteine in FSHD-Myoblasten mittels indirekter Immunfluoreszenz untersucht und mit den Kontrollzellen verglichen.
Zur Beurteilung der Kernhülle wurden Antikörper gegen Lamin A/C und Nukleoporine eingesetzt. Die Mitochondrien wurden mithilfe des Antikörpers ANT1/2, der an den Adenin-Nukleotid-Translokator der inneren Mitochondrienmembran bindet, untersucht.
Im Gegensatz zu den Untersuchungen an EDMD-Myoblasten waren die Lamine A und C sowie die Kernporen sowohl bei den Myoblasten der FSHD-Patienten als auch bei den Kontrollzellen nachweisbar und gleichmäßig verteilt.
Bei der indirekten Immunfluoreszenz mit ANT1/2 zeigten sich Unterschiede zwischen den untersuchten Myoblasten-Paaren.
Durch die vorliegenden Ergebnisse ist darauf zu schließen, dass die Myoblasten von FSHD-Patienten Veränderungen Mitochondrien aufweisen. Die Untersuchungen der Kernhülle liefern abhängig von der Fixierungsmethode unterschiedliche Ergebnisse.
Neuropeptides and peptide hormones carrying neural or physiological information are intercellular signalling substances. They control most if not all biological processes in vertebrates and invertebrates by acting on specific receptors on the target cell. In mammals, many different neuropeptides and peptide hormones are involved in the regulation of feeding and sleep. In \textit{Drosophila}, allatostatin A (AstA) and myoinhibitory peptides (MIPs) are brain-gut peptides. The AstA receptors are homologues of the mammalian galanin receptors and the amino acid sequences of MIPs are similar to a part of galanin, which has an orexigenic effect and is implicated in the control of sleep behaviour in mammals. I am interested in dissecting pleiotropic functions of AstA and MIPs in the regulation of food intake and sleep in \textit{Drosophila}. \par
In the first part of the dissertation the roles of brain-gut peptide allatostatin A are analysed. Due to the genetic and molecular tools available, the fruit fly \textit{Drosophila melanogaster} is chosen to investigate functions of AstA. The aims in this part are to identify pleiotropic functions of AstA and assign specific effects to the activity of certain subsets of AstA expressing cells in \textit{Drosophila} adults. A new and restricted \textit{AstA\textsuperscript{34}-Gal4} line was generated. The confocal imaging result showed that AstA neurons are located in the posterior lateral protocerebrum (PLP), the gnathal ganglia (GNG), the medullae, and thoracic-abdominal ganglion (TAG). AstA producing DLAa neurons in the TAG innervate hindgut and the poterior part of midgut. In addition, AstA are detected in the enteroendocrine cells (EECs).\par
Thermogenetic activation and neurogenetic silencing tools with the aid of the \textit{UAS/Gal4} system were employed to manipulate the activity of all or individual subsets of AstA cells and investigate the effects on food intake, locomotor activity and sleep. Our experimental results showed that thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs reduced food intake, which can be traced to AstA signalling by using \textit{AstA} mutants. In the locomotor activity, thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs resulted in strongly inhibited locomotor activity and promoted sleep without sexual difference, which was most apparent during the morning and evening activity peaks. The experimental and control flies were not impaired in climbing ability. In contrast, conditional silencing of the PLP neurons and/or AstA expressing EECs reduced sleep specifically in the siesta. The arousal experiment was employed to test for the sleep intensity. Thermogenetically activated flies walked significantly slower and a shorter distance than controls for all arousal stimulus intensities. Furthermore, PDF receptor was detected in the PLP neurons and the PLP neurons reacted with an intracellular increase of cAMP upon PDF, only when PDF receptor was present. Constitutive activation of AstA cells by tethered PDF increased sleep and thermogenetic activation of the PDF producing sLNvs promoted sleep specifically in the morning and evening. \par
The study shows that the PLP neurons and/or EECs vis AstA signalling subserve an anorexigenic and sleep-regulating function in \textit{Drosophila}. The PLP neurons arborise in the posterior superior protocerebrum, where the sleep relevant dopaminergic neurons are located, and EECs extend themselves to reach the gut lumen. Thus, the PLP neurons are well positioned to regulate sleep and EECs potentially modulate feeding and possibly locomotor activity and sleep during sending the nutritional information from the gut to the brain. The results of imaging, activation of the PDF signalling pathway by tethered PDF and thermoactivation of PDF expressing sLNvs suggest that the PLP neurons are modulated by PDF from sLNv clock neurons and AstA in PLP neurons is the downstream target of the central clock to modulate locomotor activity and sleep. AstA receptors are homologues of galanin receptors and both of them are involved in the regulation of feeding and sleep, which appears to be conserved in evolutionary aspect.\par
In the second part of the dissertation, I analysed the role of myoinhibitory peptides. MIPs are brain-gut peptides in insects and polychaeta. Also in \textit{Drosophila}, MIPs are expressed in the CNS and EECs in the gut. Previous studies have demonstrated the functions of MIPs in the regulation of food intake, gut motility and ecdysis in moths and crickets. Yet, the functions of MIPs in the fruit fly are little known. To dissect effects of MIPs regarding feeding, locomotor activity and sleep in \textit{Drosophila melanogater}, I manipulated the activity of MIP\textsuperscript{WÜ} cells by using newly generated \textit{Mip\textsuperscript{WÜ}-Gal4} lines. Thermogenetical activation or genetical silencing of MIP\textsuperscript{WÜ} celles did not affect feeding behaviour and resulted in changes in the sleep status. \par
My results are in contradiction to a recent research of Min Soohong and colleagues who demonstrated a role of MIPs in the regulation of food intake and body weight in \textit{Drosophila}. They showed that constitutive silencing of MIP\textsuperscript{KR} cells increased food intake and body weight, whereas thermogenetic activation of MIP\textsuperscript{KR} cells decreased food intake and body weight by using \textit{Mip\textsuperscript{KR}-Gal4} driver. Then I repeated the experiments with the \textit{Mip\textsuperscript{KR}-Gal4} driver, but could not reproduce the results. Interestingly, I just observed the opposite phenotype. When MIP\textsuperscript{KR} cells were silenced by expressing UAS-tetanus toxin (\textit{UAS-TNT}), the \textit{Mip\textsuperscript{KR}$>$TNT} flies showed reduced food intake. The thermogenetic activation of MIP\textsuperscript{KR} cells did not affect food intake. Furthermore, I observed that the thermogenetic activation of MIP\textsuperscript{KR} cells strongly reduced the sleep duration.\par
In the third part of the dissertation, I adapted and improved a method for metabolic labelling for \textit{Drosophila} peptides to quantify the relative amount of peptides and the released peptides by mass spectrometry under different physiological and behavioural conditions. qRT-PCR is a practical technique to measure the transcription and the corresponding mRNA level of a given peptide. However, this is not the only way to measure the translation and production of peptides. Although the amount of peptides can be quantified by mass spectrometry, it is not possible to distinguish between peptides stored in vesicles and released peptides in CNS extracts. I construct an approach to assess the released peptides, which can be calculated by comparing the relative amount of peptides between two timepoints in combination with the mRNA levels which can be used as semiquantitative proxy reflecting the production of peptides during this period. \par
After optimizing the protocol for metabolic labelling, I carried out a quantitative analysis of peptides before and after eclosion as a test. I was able to show that the EH- and SIFa-related peptides were strongly reduced after eclosion. This is in line with the known function and release of EH during eclosion. Since this test was positive, I next used the metabolic labelling in \textit{Drosophila} adult, which were either fed \textit{ad libitum} or starved for 24 hrs, and analysed the effects on the amount of AstA and MIPs. In the mRNA level, my results showed that in the brain \textit{AstA} mRNA level in the 24 hrs starved flies was increased compared to in the \textit{ad libitum} fed flies, whereas in the gut the \textit{AstA} mRNA level was decreased. Starvation induced the reduction of \textit{Mip} mRNA level in the brain and gut. Unfortunately, due to technical problems I was unable to analyse the metabolic labelled peptides during the course of this thesis.\par
Plant-associated fungi can affect the plants‘ interaction with herbivores and
other microorganisms. For example, many common forage grasses are infected
with Epichloë endophytes. The endophytes systemically colonize the aerial
parts of the plants. They produce bioprotective alkaloids that can negatively
affect insects and livestock feeding on the grasses, and interact with other
fungal species which living from the plants‘ nutrients. Environmental conditions
strongly influence Epichloë endophytes. Endophyte-mediated effects
on herbivores are more pronounced under increased temperatures and the
endophytes may benefit from land use in managed grasslands. Under the
framework of the large-scale German project “Biodiversity Exploratories”, I
investigated whether infection rates and alkaloid concentrations of Epichloë
festucae var. lolii in Lolium perenne (Chapter I) and Epichloë endophytes (E.
uncinata, E. siegelii) in Festuca pratensis (Chapter II) depend on land use and
season. Further I analysed, whether foliar fungal assemblages of L. perenne
are affected by the presence of Epichloë endophytes (Chapter IV).
Die Evolution der Primaten zeigt eine Verbindung zwischen der zunehmenden Komplexität des sozialen Verhaltens und der Vergrößerung des humanen Gehirns, insbesondere des präfrontalen Cortex. Deshalb stellt der präfrontale Cortex bezüglich der Evolution des Menschen eine der interessantesten Strukturen im humanen Gehirn dar. Es wird angenommen, dass nicht allein die Größe, sondern auch die Funktion, vor allem das Zusammenspiel von Neuronen und nicht-neuronalen Zellen, wie z.B. Gliazellen, zur Differenzierung des menschlichen Gehirns von dem rezenter Primaten geführt hat. Daraus lässt sich schließen, dass die Gehirnfunktionen über eine ausgeglichene und gut aufeinander abgestimmte transkriptionelle Landschaft kontrolliert werden, die durch ein zugrundeliegendes genetisches und epigentisches Rückgrat organisiert ist. In dieser Studie wurden das Methylierungsprofil neuronaler und nicht-neuronaler Zellen des präfrontalen Cortex (Brodmann-Areal 10) von drei Menschen und drei Schimpansen miteinander verglichen. Die intra- und interspezifischen differenziell methylierten Regionen (DMRs) waren in bestimmten genomischen Regionen angereichert. Intraspezifische Methylierungsunterschiede zwischen neuronalen und nicht-neuronalen Zellen konnten dreimal häufiger beobachtet werden als interspezifische Unterschiede in den einzelnen Zelltypen. Rund 90% der humanen intraspezifischen DMRs wiesen eine Hypomethylierung in den neuronalen Zellen im Vergleich zu den nicht-neuronalen Zellen auf. In den intraspezifischen DMRs (Mensch und Schimpanse) waren Gene angereichert, die mit verschiedenen neuropsychiatrischen Erkrankungen assoziiert sind. Der Vergleich zwischen Menschen und Schimpanse in den neuronalen und nicht-neuronalen Zelltypen zeigte eine Anreicherung von Genen mit human-spezifischer Histonsignatur. In den nicht-neuronalen Zellen konnten mehr interspezifische DMRs (n=666) detektiert werden als in den neuronalen Zellen (n=96). Ungefähr 95% der nicht-neuronalen interspezifischen DMRs waren im Menschen, im Vergleich zum Schimpansen, hypermethyliert. Daraus ergibt sich der Eindruck, dass mehrere hundert der nicht-neuronalen Gene während der humanen Gehirnevolution einer Methylierungswelle unterlagen. Dies führt zu der Annahme, dass der Einfluss dieser Veränderungen in den nicht-neuronalen Zellen auf die Vergößerung des menschlichen Gehirns bisher stark unterschätzt wurde.
Die bekannteste genetische Ursache für erblichen Brust- und Eierstockkrebs sind Mutationen in den Tumorsuppressorgenen (TSG) BRCA1 und BRCA2. Dennoch können nur rund 20-25% der familiären Brustkrebserkrankungen über Keimbahnmutationen in BRCA1/BRCA2 erklärt werden, besonders bei Frauen, deren Erkrankung vor dem vierzigsten Lebensjahr auftritt. Epigenetische Veränderungen, die zu einer aberranten Genexpression führen, spielen ebenfalls eine wichtige Rolle bei der Karzinogenese und der Entwicklung einer Brustkrebserkrankung. Es ist bekannt, dass TSG nicht nur durch den Verlust der Heterozygotie (engl. loss of heterozygosity, LOH) oder homozygote Deletionen, sondern auch durch transkriptionelle Stilllegung via DNA-Methylierung inaktiviert werden können. Im Rahmen dieser Arbeit wurde überprüft, welchen Einfluss aberrante Methylierungsmuster im Promotorbereich von TSG auf die Brustkrebskarzinogenese und die Expression der Gene haben. Für die Quantifizierung der Epimutationen wurden die Promotorbereiche von acht TSG (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) und des estrogene receptor (ESR1) Gens, welches eine Rolle in der Tumorprogression spielt, mittels Deep Bisulfite Amplicon Sequencing (DBAS) analysiert. Es wurden Blutproben von zwei unabhängigen BRCA1/BRCA2-mutationsnegativen Brustkrebs (BC)-Patientenkohorten, sowie von zwei unabhängigen alters-gematchten, gesunden Kontrollkohorten untersucht. BC-Kohorte 1 beinhaltet early-onset (EO) BC-Patientinnen. Kohorte 2 enthält BC-Patientinnen mit einem Risiko von >95% eine heterozygote Mutation in BRCA1/BRCA2 (high-risk, HR) zu tragen. Allele mit >50% methylierten CpGs werden als funktionell relevante Epimutationen erachtet, da bekannt ist, dass TSG über eine Methylierung im Promotorbereich transkriptionell stillgelegt werden. Im Vergleich zu ESR1 (Ø Methylierung, 3%), welches die Methylierungslevel eines durchschnittlichen Promotors wiederspiegelt, zeigten die TSG sehr geringe durchschnittliche Methylierungswerte von weniger als 1%. Zudem waren die durchschnittlichen Epimutationsraten (EMR; <0,0001-0,1%) der TSG sehr gering. Mit der Ausnahme von BRCA1, welches eine erhöhte EMR in der BC-Kohorte verglichen zu den Kontrollen (0,31% gegen 0,06%) zeigte, gab es keine signifikanten Gruppenunterschiede zwischen BC-Patientinnen und Kontrollen. Eine von 36 HR BC-Patientinnen zeigte im Vergleich zu den restlichen Proben eine stark erhöhte EMR von 14,7% in BRCA1. Rund ein Drittel (15/44) der EO BC-Patientinnen wiesen eine erhöhte Rate an Einzel-CpG Fehlern in mehreren TSG auf. Die nachfolgenden Expressionsanalysen ergaben eine erniedrigte Expression vieler TSG je analysierter Patientin. Diese Ergebnisse führen zu der Annahme, dass epigenetische Veränderungen in normalen Körperzellen als ein möglicher Indikator für einen gestörten Mechanismus, der für die Aufrechterhaltung des unmethylierten Status und der daraus resultierenden normalen Genexpression zuständig ist, angesehen werden können. Dies kann mit einem erhöhten BC-Risiko assoziiert werden.
The interaction of synaptic proteins orchestrate the function of one of the most complex organs, the brain. The multitude of molecular elements influencing neurological correlations makes imaging processes complicated since conventional fluorescence microscopy methods are unable to resolve structures beyond the diffraction-limit.
The implementation of super-resolution fluorescence microscopy into the field of neuroscience allows the visualisation of the fine details of neural connectivity. The key element of my thesis is the super-resolution technique dSTORM (direct Stochastic Optical Reconstruction Microscopy) and its optimisation as a multi-colour approach. Capturing more than one target, I aim to unravel the distribution of synaptic proteins with nanometer precision and set them into a structural and quantitative context with one another. Therefore dSTORM specific protocols are optimized to serve the peculiarities of particular neural samples.
In one project the brain derived neurotrophic factor (BDNF) is investigated in primary, hippocampal neurons. With a precision beyond 15 nm, preand post-synaptic sites can be identified by staining the active zone proteins bassoon and homer. As a result, hallmarks of mature synapses can be exhibited. The single molecule sensitivity of dSTORM enables the measurement of endogenous BDNF and locates BDNF granules aligned with glutamatergic pre-synapses. This data proofs that hippocampal neurons are capable of enriching BDNF within the mature glutamatergic pre-synapse, possibly influencing synaptic plasticity.
The distribution of the metabotropic glutamate receptor mGlu4 is investigated in physiological brain slices enabling the analysis of the receptor in its natural environment. With dual-colour dSTORM, the spatial arrangement of the mGlu4 receptor in the pre-synaptic sites of parallel fibres in the molecular layer of the mouse cerebellum is visualized, as well as a four to six-fold increase in the density of the receptor in the active zone compared to the nearby environment. Prior functional measurements show that metabotropic glutamate receptors influence voltage-gated calcium channels and proteins that are involved in synaptic vesicle priming. Corresponding dSTORM data indeed suggests that a subset of the mGlu4 receptor is correlated with the voltage-gated calcium channel Cav2.1 on distances around 60 nm.
These results are based on the improvement of the direct analysis of localisation data. Tools like coordinated based correlation analysis and nearest neighbour analysis of clusters centroids are used complementary to map protein connections of the synapse. Limits and possible improvements of these tools are discussed to foster the quantitative analysis of single molecule localisation microscopy data.
Performing super-resolution microscopy on complex samples like brain slices benefits from a maximised field of view in combination with the visualisation of more than two targets to set the protein of interest in a cellular context. This challenge served as a motivation to establish a workflow for correlated structured illumination microscopy (SIM) and dSTORM. The development of the visualisation software coSIdSTORM promotes the combination of these powerful super-resolution techniques even on separated setups. As an example, synapses in the cerebellum that are affiliated to the parallel fibres and the dendrites of the Purkinje cells are identified by SIM and the protein bassoon of those pre-synapses is visualised threedimensionally with nanoscopic precision by dSTORM.
In this work I placed emphasis on the improvement of multi-colour super-resolution imaging and its analysing tools to enable the investigation of synaptic proteins. The unravelling of the structural arrangement of investigated proteins supports the building of a synapse model and therefore helps to understand the relation between structure and function in neural transmission processes.
The present work investigates the influence of environmental stimuli on the building behavior of workers of the leaf-cutting ant Atta vollenweideri. It focuses on cues related to the airflow-driven ventilation of their giant underground nests, i.e., air movements and their direction, carbon dioxide concentrations and humidity levels of the nest air. First, it is shown that workers are able to use airflow and its direction as learned orientation cue by performing learning experiments with individual foragers using a classical conditioning paradigm. This ability is expected to allow workers to also navigate inside the nest tunnels using the prevailing airflow directions for orientation, for example during tasks related to nest construction and climate control.
Furthermore, the influence of carbon dioxide on the digging behavior of workers is investigated. While elevated CO2 levels hardly affect the digging rate of the ants, workers prefer to excavate at locations with lower concentrations and avoid higher CO2 levels when given a choice. Under natural conditions, shifting their digging activity to soil layers containing lower carbon dioxide levels might help colonies to excavate new or to broaden existing nest openings, if the CO2 concentration in the underground rises.
It is also shown that workers preferably transport excavated soil along tunnels containing high CO2 concentrations, when carbon dioxide levels in the underground are elevated as well. In addition, workers prefer to carry soil pellets along outflow tunnels instead of inflow tunnels, at least for high humidity levels of the air. The material transported along tunnels providing outflow of CO2-rich air might be used by workers for the construction of ventilation turrets on top of the nest mound, which is expected to promote the wind-induced ventilation and the removal of carbon dioxide from the underground.
The climatic conditions inside the nest tunnels also influence the structural features of the turrets constructed by workers on top the nest. While airflow and humidity have no effect on turret structure, outflow of CO2-rich air from the nest causes workers to construct turrets with additional openings and increased aperture, potentially enhancing the airflow-driven gas exchanges within the nest.
Finally, the effect of airflow and ventilation turrets on the gas exchanges in Atta vollenweideri nests is tested experimentally on a physical model of a small nest consisting of a single chamber and two nest tunnels. The carbon dioxide clearance rate from the underground was measured depending on both the presence of airflow in the nest and the structural features of the built turrets. Carbon dioxide is removed faster from the physical nest model when air moves through the nest, confirming the contribution of wind-induced flow inside the nest tunnels to the ventilation of Atta vollenweideri nests. In addition, turrets placed on top of one of the tunnel openings of the nest further enhance the CO2 clearance rate and the effect is positively correlated with turret aperture.
Taken together, climatic variables like airflow, carbon dioxide and humidity levels strongly affect the building responses of Atta vollenweideri leaf-cutting ants. Workers use these environmental stimuli as orientation cue in the nest during tasks related to excavation, soil transport and turret construction. Although the effects of these building responses on the microclimatic conditions inside the nest remain elusive so far, the described behaviors are expected to allow ant colonies to restore and maintain a proper nest climate in the underground.
Solitary bees in seasonal environments have to align their life-cycles with favorable environmental conditions and resources. Therefore, a proper timing of their seasonal activity is highly fitness relevant. Most species in temperate environments use temperature as a trigger for the timing of their seasonal activity. Hence, global warming can disrupt mutualistic interactions between solitary bees and plants if increasing temperatures differently change the timing of interaction partners. The objective of this dissertation was to investigate the mechanisms of timing in spring-emerging solitary bees as well as the resulting fitness consequences if temporal mismatches with their host plants should occur. In my experiments, I focused on spring-emerging solitary bees of the genus Osmia and thereby mainly on O. cornuta and O. bicornis (in one study which is presented in Chapter IV, I additionally investigated a third species: O. brevicornis).
Chapter II presents a study in which I investigated different triggers solitary bees are using to time their emergence in spring. In a climate chamber experiment I investigated the relationship between overwintering temperature, body size, body weight and emergence date. In addition, I developed a simple mechanistic model that allowed me to unite my different observations in a consistent framework. In combination with the empirical data, the model strongly suggests that solitary bees follow a strategic approach and emerge at a date that is most profitable for their individual fitness expectations. I have shown that this date is on the one hand temperature dependent as warmer overwintering temperatures increase the weight loss of bees during hibernation, which then advances their optimal emergence date to an earlier time point (due to an earlier benefit from the emergence event). On the other hand I have also shown that the optimal emergence date depends on the individual body size (or body weight) as bees adjust their emergence date accordingly. My data show that it is not enough to solely investigate temperature effects on the timing of bee emergence, but that we should also consider individual body conditions of solitary bees to understand the timing of bee emergence.
In Chapter III, I present a study in which I investigated how exactly temperature determines the emergence date of solitary bees. Therefore, I tested several variants degree-day models to relate temperature time series to emergence data. The basic functioning of such degree-day models is that bees are said to finally emerge when a critical amount of degree-days is accumulated. I showed that bees accumulate degree-days only above a critical temperature value (~4°C in O. cornuta and ~7°C in O. bicornis) and only after the exceedance of a critical calendar date (~10th of March in O. cornuta and ~28th of March in O. bicornis). Such a critical calendar date, before which degree-days are not accumulated irrespective of the actual temperature, is in general less commonly used and, so far, it has only been included twice in a phenology model predicting bee emergence. Furthermore, I used this model to retrospectively predict the emergence dates of bees by applying the model to long-term temperature data which have been recorded by the regional climate station in Würzburg. By doing so, the model estimated that over the last 63 years, bees emerged approximately 4 days earlier.
In Chapter IV, I present a study in which I investigated how temporal mismatches in bee-plant interactions affect the fitness of solitary bees. Therefore, I performed an experiment with large flight cages serving as mesocosms. Inside these mesocosms, I manipulated the supply of blossoms to synchronize or desynchronize bee-plant interactions. In sum, I showed that even short temporal mismatches of three and six days in bee-plant interactions (with solitary bee emergence before flower occurrence) can cause severe fitness losses in solitary bees. Nonetheless, I detected different strategies by solitary bees to counteract impacts on their fitness after temporal mismatches. However, since these strategies may result in secondary fitness costs by a changed sex ratio or increased parasitism, I concluded that compensation strategies do not fully mitigate fitness losses of bees after short temporal mismatches with their food plants. In the event of further climate warming, fitness losses after temporal mismatches may not only exacerbate bee declines but may also reduce pollination services for later-flowering species and affect populations of animal-pollinated plants.
In conclusion, I showed that spring-emerging solitary bees are susceptible to climate change as in response to warmer temperatures bees advance their phenology and show a decreased fitness state. As spring-emerging solitary bees not only consider overwintering temperature but also their individual body condition for adjusting emergence dates, this may explain differing responses to climate warming within and among bee populations which may also have consequences for bee-plant interactions and the persistence of bee populations under further climate warming. If in response to climate warming plants do not shift their phenologies according to the bees, bees may experience temporal mismatches with their host plants. As bees failed to show a single compensation strategy that was entirely successful in mitigating fitness consequences after temporal mismatches with their food plants, the resulting fitness consequences for spring-emerging solitary bees would be severe. Furthermore, I showed that spring-emerging solitary bees use a critical calendar date before which they generally do not commence the summation of degree-days irrespective of the actual temperature. I therefore suggest that further studies should also include the parameter of a critical calendar date into degree-day model predictions to increase the accuracy of model predictions for emergence dates in solitary bees. Although our retrospective prediction about the advance in bee emergence corresponds to the results of several studies on phenological trends of different plant species, we suggest that more research has to be done to assess the impacts of climate warming on the synchronization in bee-plant interactions more accurately.
Altersassoziierte und strahleninduzierte Veränderungen des genomweiten DNA-Methylierungs-Profils
(2018)
Der Prozess des Alterns ist ein komplexer multifaktorieller Vorgang, der durch eine sukzessive Verschlechterung der physiologischen Funktionen charakterisiert ist. Ein hohes Alter ist der Hauptrisikofaktor für die meisten Krankheiten, einschließlich Krebs und Herz-Kreislauf-Erkrankungen. Das Verständnis der epigenetischen Mechanismen, die in den Prozess des Alterns involviert sind, könnte zur Entwicklung pharmakologischer Interventionen beitragen, die nicht nur die Lebenserwartung erhöhen, sondern auch den Beginn des altersassoziierten funktionellen Abbaus verzögern könnten. Durch die Langzeit-Kultivierung primärer humaner Fibroblasten wurde ein in vitro Modell für das Altern etabliert, das die Identifizierung altersassoziierter DNA-Methylierungs-Veränderungen ermöglichte. Die in vitro Alterung konnte mit einer globalen Hypomethylierung und einer erhöhten DNA-Methylierung der ribosomalen DNA assoziiert werden. Darüber hinaus konnten DNA-Methylierungs-Veränderungen in Genen und Signalwegen, die für das Altern relevant sind, und ein erhöhtes epigenetisches Alter nachgewiesen werden.
Das in vitro Modell für das Altern wurde verwendet, um neben den direkten Effekten ionisierender Strahlung auf die DNA-Methylierung auch deren Langzeit-Effekte zu untersuchen. Die Strahlentherapie ist ein entscheidendes Element der Krebstherapie, hat aber auch negative Auswirkungen und kann unter anderem das Risiko für die Entwicklung eines Zweittumors erhöhen. Bei externer Bestrahlung wird neben dem Tumor auch gesundes Gewebe ionisierender Strahlung ausgesetzt. Daher ist es wichtig zu untersuchen, wie Zellen mit intakten DNA-Reparatur-Mechanismen und funktionierenden Zellzyklus-Checkpoints durch diese beeinflusst werden. In der frühen Phase der DNA-Schadensantwort auf Bestrahlung wurden in normalen Zellen keine wesentlichen DNA-Methylierungs-Veränderungen beobachtet. Mehrere Populations-Verdoppelungen nach Strahlenexposition konnten dagegen eine globale Hypomethylierung, eine erhöhte DNA-Methylierung der ribosomalen DNA und ein erhöhtes epigenetisches Alter detektiert werden. Des Weiteren zeigten Gene und Signalwege, die mit Krebs in Verbindung gebracht wurden, Veränderungen in der DNA-Methylierung. Als Langzeit-Effekte ionisierender Strahlung traten somit die mit der in vitro Alterung assoziierten DNA-Methylierungs-Veränderungen verstärkt auf und ein epigenetisches Muster, das stark an das DNA-Methylierungs-Profil von Tumorzellen erinnert, entstand. Man geht davon aus, dass Veränderungen der DNA-Methylierung eine aktive Rolle in der Entwicklung eines Tumors spielen. Die durch ionisierende Strahlung induzierten DNA-Methylierungs-Veränderungen in normalen Zellen könnten demnach in die Krebsentstehung nach Strahlenexposition involviert sein und zu dem sekundären Krebsrisiko nach Strahlentherapie beitragen. Es ist bekannt, dass Patienten unterschiedlich auf therapeutische Bestrahlung reagieren. Die Ergebnisse dieser Arbeit weisen darauf hin, dass die individuelle Sensitivität gegenüber ionisierender Strahlung auch auf epigenetischer Ebene beobachtet werden kann.
In einem zweiten Projekt wurden Gesamtblutproben von Patienten mit Werner-Syndrom, einer segmental progeroiden Erkrankung, und gesunden Kontrollen analysiert, um mit dem vorzeitigen Altern in Verbindung stehende DNA-Methylierungs-Veränderungen zu identifizieren. Werner-Syndrom konnte nicht mit einer globalen Hypomethylierung, jedoch mit einer erhöhten DNA-Methylierung der ribosomalen DNA und einem erhöhten epigenetischen Alter assoziiert werden. Das vorzeitige Altern geht demzufolge mit spezifischen epigenetischen Veränderungen einher, die eine Beschleunigung der mit dem normalen Altern auftretenden DNA-Methylierungs-Veränderungen darstellen.
Im Rahmen dieser Arbeit konnte die Bedeutung epigenetischer Mechanismen im Prozess des Alterns hervorgehoben werden und gezeigt werden, dass sowohl exogene Faktoren, wie ionisierende Strahlung, als auch endogene Faktoren, wie das in Werner-Syndrom-Patienten mutiert vorliegende WRN-Gen, altersassoziierte DNA-Methylierungs-Veränderungen beeinflussen können.
Trypanosoma brucei is an obligate parasite and causative agent of severe diseases affecting humans and livestock. The protist lives extracellularly in the bloodstream of the mammalian host, where it is prone to attacks by the host immune system. As a sophisticated means of defence against the immune response, the parasite’s surface is coated in a dense layer of the variant surface glycoprotein (VSG), that reduces identification of invariant epitopes on the cell surface by the immune system to levels that prevent host immunity. The VSG has to form a coat that is both dense and mobile, to shield invariant surface proteins from detection and to allow quick recycling of the protective coat during immune evasion. This coat effectively protects the parasite from the harsh environment that is the mammalian bloodstream and leads to a persistent parasitemia if the infection remains untreated. The available treatment against African Trypanosomiasis involves the use of drugs that are themselves severely toxic and that can lead to the death of the patient. Most of the drugs used as treatment were developed in the early-to-mid 20th century, and while developments continue, they still represent the best medical means to fight the parasite. The discovery of a fluorescent VSG gave rise to speculations about a potential interaction between the VSG coat and components of the surrounding medium, that could also lead to a new approach in the treatment of African Trypanosomiasis that involves the VSG coat. The initially observed fluorescence signal was specific for a combination of a VSG called VSG’Y’ and the triphenylmethane (TPM) dye phenol red. Exchanging this TPM to a bromo-derivative led to the observation of another fluorescence effect termed trypanicidal effect which killed the parasite independent of the expressed VSG and suggests a structurally conserved feature between VSGs that could function as a specific drug target against T. b. brucei. The work of this thesis aims to identify the mechanisms that govern the unique VSG’Y’ fluorescence and the trypanocidal effect. Fluorescence experiments and protein mutagenesis of VSG’Y’ as well as crystallographic trials with a range of different VSGs were utilized in the endeavour to identify the binding mechanisms between TPM compounds and VSGs, to find potentially conserved structural features between VSGs and to identify the working mechanisms of VSG fluorescence and the trypanocidal effect. These trials have the potential to lead to the formulation of highly specific drugs that
target the parasites VSG coat.
During the crystallographic trials of this thesis, the complete structure of a VSG was solved experimentally for the first time. This complete structure is a key component in furthering the understanding of the mechanisms governing VSG coat formation. X-ray scattering techniques, involving x-ray crystallography and small angle x-ray scattering were applied to elucidate the first complete VSG structures, which reveal high flexibility of the protein and supplies insight into the importance of this flexibility in the formation of a densely packed but highly mobile surface coat.
Staphylococcus aureus asymptomatically colonises one third of the healthy human population, finding its niche in the nose and on skin. Apart from being a commensal, it is also an important opportunistic human pathogen capable of destructing tissue, invading host cells and killing them from within. This eventually contributes to severe hospital- and community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA), resistant to commonly used antibiotics are protected when residing within the host cell.
This doctoral thesis is focused on the investigation of staphylococcal factors governing intracellular virulence and subsequent host cell death. To initiate an unbiased approach to conduct this study, complex S. aureus mutant pools were generated using transposon insertional mutagenesis. Genome-wide infection screens were performed using these S. aureus transposon mutant pools in vitro and in vivo, followed by analysis using Transposon insertion site deep sequencing (Tn-seq) technology.
Amongst several other factors, this study identified a novel regulatory system in S. aureus that controls pathogen-induced host cytotoxicity and intra-host survival. The primary components of this system are an AraC-family transcription regulator called Repressor of surface proteins (Rsp) and a virulence associated non-coding RNA, SSR42. Mutants within rsp exhibit enhanced intra-host survival in human epithelial cells and delayed host cytotoxicity. Global gene-expression profiling by RNA-seq demonstrated that Rsp controls the expression of SSR42, several cytotoxins and other bacterial factors directed against the host immune system. Rsp enhances S. aureus toxin response when triggered by hydrogen peroxide, an antimicrobial substance employed by neutrophils to destroy pathogens. Absence of rsp reduces S. aureus-induced neutrophil damage and early lethality during mouse pneumonia, but still permits blood stream infection. Intriguingly, S. aureus lacking rsp exhibited enhanced survival in human macrophages, which hints towards a Trojan horse-like phenomenon and could facilitate dissemination within the host.
Hence, Rsp emerged as a global regulator of bacterial virulence, which has an impact on disease progression with prolonged intra-cellular survival, delayed-lethality but allows disseminated manifestation of disease. Moreover, this study exemplifies the use of genome-wide approaches as useful resources for identifying bacterial factors and deduction of its pathogenesis.
The rotation of the earth leads to a cyclic change of night and day. Numerous strategies evolved to cope with diurnal change, as it is generally advantageous to be synchronous to the cyclic change in abiotic conditions. Diurnal rhythms are regulated by the circadian clock, a molecular feedback loop of RNA and protein levels with a period of circa 24 hours. Despite its importance for individuals as well as for species interactions, our knowledge of circadian clocks is mostly confined to few model organisms.
While the structuring of activity is generally adaptive, a rigid temporal organization also has its drawbacks. For example, the specialization to a diurnal pattern limits the breadth of the temporal niche. Organisms that are adapted to a diurnal life style are often poor predators or foragers during night time, constraining the time budget to only diurnal parts of the day/night cycle.
Climate change causes shifts in phenology (seasonal timing) and northward range expansions, and changes in season or in latitude are associated with novel day length – temperature correlations. Thus, seasonal organisms will have some life history stages exposed to novel day lengths, and I hypothesized that the diurnal niche determines whether the day length changes are beneficial or harmful for the organism. I thus studied the effects of day length on life-history traits in a multi-trophic system consisting of the pea aphid Acyrthosiphon pisum and predatory larvae of Chrysoperla carnea (common green lacewing) and Episyrphus balteatus (marmalade hoverfly). In order to identify the mechanisms for phenological constraints I then focused on diurnal rhythms and the circadian clock of the pea aphid.
Aphids reacted to shorter days with a reduced fecundity and shorter reproductive period. Short days did however not impact population growth, because the fitness constraints only became apparent late in the individual’s life. In contrast, E. balteatus grew 13% faster in the shorter day treatment and preyed on significantly more aphids, whereas C. carnea grew 13% faster under longer days and the elevation of predation rates was marginally significant. These results show that day length affects vital life-history traits, but that the direction and effect size depends on species.
I hypothesized that the constraints or fitness benefits are caused by a constricted or expanded time budget, and hence depend on the temporal niche. E. balteatus is indeed night-active and C. carnea appears to be crepuscular, but very little data exists for A. pisum. Hence, I reared the pea aphid on an artificial diet and recorded survival, moulting and honeydew excretion. The activity patterns were clearly rhythmic and molting and honeydew excretion were elevated during day-time. Thus, the diurnal niche could explain the observed, but weak, day length constraints of aphids.
The diurnal niche of some organisms is remarkably flexible, and a flexible diurnal niche may explain why the day length constrains were relatively low in A. pisum. I thus studied its circadian clock, the mechanism that regulates diurnal rhythms. First, I improved an artificial diet for A. pisum, and added the food colorant Brilliant Blue FCF. This food colorant stained gut and honeydew in low concentration without causing mortalities, and thus made honeydew excretion visible under dim red light. I then used the blue diet to raise individual aphids in 16:08 LD and constant darkness (DD), and recorded honeydew excretion and molting under red light every three hours. In addition, we used a novel monitoring setup to track locomotor activity continuously in LD and DD. Both the locomotor rhythm and honeydew excretion of A. pisum appeared to be bimodal, peaking in early morning and in the afternoon in LD. Both metabolic and locomotor rhythm persisted also for some time under constant darkness, indicating that the rhythms are driven by a functional circadian clock. However, the metabolic rhythm damped within three to four days, whereas locomotor rhythmicity persisted with a complex distribution of several free-running periods. These results fit to a damped circadian clock that is driven by multiple oscillator populations, a model that has been proposed to link circadian clocks and photoperiodism, but never empirically tested.
Overall, my studies integrate constraints in phenological adaptation with a mechanistic explanation. I showed that a shorter day length can constrain some species of a trophic network while being beneficial for others, and linked the differences to the diurnal niche of the species. I further demonstrated that a flexible circadian clock may alleviate the constraints, potentially by increasing the plasticity of the diurnal niche.
The rotation of the earth around its axis causes recurring and predictable changes in the environment. To anticipate those changes and adapt their physiology and behavior accordingly, most organisms possess an endogenous clock. The presence of such a clock has been demonstrated for several ant species including Camponotus ants, but its involvement in the scheduling of daily activities within and outside the ant nest is fairly unknown. Timing of individual behaviors and synchronization among individuals is needed to generate a coordinated collective response and to maintain colony function. The aim of this thesis was to investigate the presence of a circadian clock in different worker castes, and to determine the daily timing of their behavioral tasks within the colonies of two nectar-collecting Camponotus species.
In chapter I, I describe the general temporal organization of work throughout the worker life in the species Camponotus rufipes. Continuous tracking of behavioral activity of individually- marked workers for up to 11 weeks in subcolonies revealed an age-dependent division of labor between interior and exterior workers. After eclosion, the fairly immobile young ants were frequently nurtured by older nurses, yet they started nursing the brood themselves within the first 48 hours of their life. Only 60% of workers switched to foraging at an age range of one to two weeks, likely because of the reduced needs within the small scale of the subcolonies. Not only the transition rates varied between subcolonies, but also the time courses of the task sequences between workers did, emphasizing the timed allocation of workers to different tasks in response to colony needs.
Most of the observed foragers were present outside the nest only during the night, indicating a distinct timing of this behavioral activity on a daily level as well. As food availability, humidity and temperature levels were kept constant throughout the day, the preference for nocturnal activity seems to be endogenous and characteristic for C. rufipes. The subsequent monitoring of locomotor activity of workers taken from the subcolonies revealed the presence of a functional endogenous clock already in one-day old ants. As some nurses displayed activity rhythms in phase with the foraging rhythm, a synchronization of these in-nest workers by social interactions with exterior workers can be hypothesized.
Do both castes use their endogenous clock to schedule their daily activities within the colony? In chapter II, I analyzed behavioral activity of C. rufipes foragers and nurses within the social context continuously for 24 hours. As time-restricted access to food sources may be one factor affecting daily activities of ants under natural conditions, I confronted subcolonies with either daily pulses of food availability or ad libitum feeding. Under nighttime and ad libitum feeding, behavioral activity of foragers outside the nest was predominantly nocturnal, confirming the results from the simple counting of exterior workers done in chapter I. Foragers switched to diurnality during daytime feeding, demonstrating the flexible and adaptive timing of a daily behavior. Because they synchronized their activity with the short times of food availability, these workers showed high levels of inactivity. Nurses, in contrast, were active all around the clock independent of the feeding regime, spending their active time largely with feeding and licking the brood. After the feeding pulses, however, a short bout of activity was observed in nurses. During this time period, both castes increasingly interacted via trophallaxis within the nest. With this form of social zeitgeber, exterior workers were able to entrain in-nest workers, a phenomenon observed already in chapter I. Under the subsequent monitoring of locomotor activity under LD conditions the rhythmic workers of both castes were uniformly nocturnal independent of the feeding regime. This endogenous activity pattern displayed by both worker castes in isolation was modified in the social context in adaption to task demands.
Chapter III focuses on the potential factors causing the observed plasticity of daily rhythms in the social context in the ant C. rufipes. As presence of brood and conspecifics are likely indicators of the social context, I tested the effect of these factors on the endogenous rhythms of otherwise isolated individuals. Even in foragers, the contact to brood triggered an arrhythmic activity pattern resembling the arrhythmic behavioral activity pattern seen in nurses within the social context. As indicated in chapter I and II, social interaction could be one crucial factor for the synchronization of in nest activities. When separate groups were entrained to phase-shifted light-dark-cycles and monitored afterwards under constant conditions in pairwise contact through a mesh partitioning, both individuals shifted parts of their activity towards the activity period of the conspecific. Both social cues modulated the endogenous rhythms of workers and contribute to the context dependent plasticity in ant colonies.
Although most nursing activities are executed arrhythmically throughout the day (chapter II), previous studies reported rhythmic translocation events of the brood in Camponotus nurses. As this behavior favors brood development, the timing of the translocations within the dark nest seems to be crucial. In chapter IV, I tracked translocation activity of all nurses within subcolonies of C. mus. Under the confirmed synchronized conditions of a LD-cycle, the daily pattern of brood relocation was based on the rhythmic, alternating activity of subpopulations with preferred translocation direction either to the warm or to the cold part of the temperature gradient at certain times of the day. Although the social interaction after pulse feeding had noticeable effects on the in-nest activity in C. rufipes (chapter I and II), it was not sufficient to synchronize the brood translocation rhythm of C. mus under constant darkness (e.g. when other zeitgebers were absent). The free-running translocation activity in some nurses demonstrated nevertheless the involvement of an endogenous clock in this behavior, which could be entrained under natural conditions by other potential non-photic zeitgebers like temperature and humidity cycles.
Daily cycling of temperature and humidity could not only be relevant for in-nest activities, but also for the foraging activity outside the nest. Chapter V focuses on the monitoring of field foraging rhythms in the sympatric species C. mus and C. rufipes in relation to abiotic factors. Although both species had comparable critical thermal limits in the laboratory, foragers in C. mus were strictly diurnal and therefore foraged under higher temperatures than the predominant nocturnal foragers in C. rufipes. Marking experiments in C. rufipes colonies with higher levels of diurnal activity revealed the presence of temporally specialized forager subpopulations. These results suggest the presence of temporal niches not only between the two Camponotus species, but as well between workers within colonies of the same species.
In conclusion, the temporal organization in colonies of Camponotus ants involves not only the scheduling of tasks performed throughout the worker life, but also the precise timing of daily activities. The necessary endogenous clock is already functioning in all workers after eclosion. Whereas the light-dark cycle and food availability seem to be the prominent zeitgebers for foragers, nurses may rely more on non-photic zeitgeber like social interaction, temperature and humidity cycles.
The molecular architecture of the meiotic chromosome axis as revealed by super-resolution microscopy
(2018)
During meiosis proteins of the chromosome axis are important for monitoring chromatin structure and condensation, for pairing and segregation of chromosomes, as well as for accurate recombination. They include HORMA-domain proteins, proteins of the DNA repair system, synaptonemal complex (SC) proteins, condensins and cohesins. To understand more about their function in shaping the meiotic chromosome it is crucial to establish a defined model of their molecular architecture. Up to now their molecular organization was analysed using conventional methods, like confocal scanning microscopy (CLSM) and transmission electron microscopy (TEM). Unfortunately, these techniques are limited either by their resolution power or their localization accuracy. In conclusion, a lot of data on the molecular organization of chromosome axis proteins stays elusive. For this thesis the molecular structure of the murine synaptonemal complex (SC) and the localization of its proteins as well as of three cohesins was analysed with isotropic resolution, providing new insights into their architecture and topography on a nanoscale level. This was done using immunofluorescence labelling in combination with super-resolution microscopy, line profiles and average position determination. The results show that the murine SC has a width of 221.6 nm ± 6.1 nm including a central region (CR) of 148.2 nm ± 2.6 nm. In the CR a multi-layered organization of the central element (CE) proteins was verified by measuring their strand diameters and strand distances and additionally by imaging potential anchoring sites of SYCP1 (synaptonemal complex protein 1) to the lateral elements (LEs). We were able to show that the two LEs proteins SYCP2 and SYCP3 do co-localize alongside their axis and that there is no significant preferential localization towards the inner LE axis of SYCP2.
The presented results also predict an orderly organization of murine cohesin complexes (CCs) alongside the chromosome axis in germ cells and support the hypothesis that cohesins in the CR of the SC function independent of CCs.
In the end new information on the molecular organization of two main components of the murine chromosome axis were retrieved with nanometer precision and previously unknown details of their molecular architecture and topography were unravelled.
N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated.
To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif.
Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin.
The sequencing of several ant genomes within the last six years open new research avenues for understanding not only the genetic basis of social species but also the complex systems such as immune responses in general. Similar to other social insects, ants live in cooperative colonies, often in high densities and with genetically identical or closely related individuals. The contact behaviours and crowd living conditions allow the disease to spread rapidly through colonies. Nevertheless, ants can efficiently combat infections by using diverse and effective immune mechanisms. However, the components of the immune system of carpenter ant Camponotus floridanus and also the factors in bacteria that facilitate infection are not well understood.
To form a better view of the immune repository and study the C. floridanus immune responses against the bacteria, experimental data from Illumina sequencing and mass-spectrometry (MS) data of haemolymph in normal and infectious conditions were analysed and integrated with the several bioinformatics approaches. Briefly, the tasks were accomplished in three levels. First, the C. floridanus genome was re-annotated for the improvement of the existing annotation using the computational methods and transcriptomics data. Using the homology based methods, the extensive survey of literature, and mRNA expression profiles, the immune repository of C. floridanus were established. Second, large-scale protein-protein interactions (PPIs) and signalling network of C. floridanus were reconstructed and analysed and further the infection induced functional modules in the networks were detected by mapping of the expression data over the networks. In addition, the interactions of the immune components with the bacteria were identified by reconstructing inter-species PPIs networks and the interactions were validated by literature. Third, the stage-specific MS data of larvae and worker ants were analysed and the differences in the immune response were reported.
Concisely, all the three omics levels resulted to multiple findings, for instance, re-annotation and transcriptome profiling resulted in the overall improvement of structural and functional annotation and detection of alternative splicing events, network analysis revealed the differentially expressed topologically important proteins and the active functional modules, MS data analysis revealed the stage specific differences in C. floridanus immune responses against bacterial pathogens.
Taken together, starting from re-annotation of C. floridanus genome, this thesis provides a transcriptome and proteome level characterization of ant C. floridanus, particularly focusing on the immune system responses to pathogenic bacteria from a biological and a bioinformatics point of view. This work can serve as a model for the integration of omics data focusing on the immuno-transcriptome of insects.
Methoden der Fluoreszenz-Lokalisationsmikroskopie (engl. single-molecule localization microscopy, SMLM) ermöglichen es Moleküle zu quantifizieren und deren Verteilung zu analysieren. Im Rahmen dieser Arbeit wurden verschiedene Membranmoleküle auf unterschiedlichen eukaryotischen Zellen, aber auch auf Prokaryoten mit dSTORM (engl. direct stochastic optical reconstruction microscopy) oder PALM (engl.: photoactivated localization microscopy) aufgenommen und quantifiziert. Bevor jedoch diese hochauflösende fluoreszenzbasierte Technik für biologische Fragestellungen angewendet werden konnten, mussten zunächst potentielle Artefakt-auslösende Quellen identifiziert und Strategien gefunden werden, um diese zu eliminieren.
Eine mögliche Artefakt-Quelle ist eine zu niedrige Photonenzahl, die von Fluorophoren emittiert wird. Werden zu wenige Photonen detektiert, kann die Lokalisation eines Fluorophors weniger präzise bestimmt werden. Dies kann zu einer falschen Abbildung von Strukturen führen oder zu falschen Rückschlüssen über die Verteilung von Molekülen. Eine Möglichkeit die Anzahl der emittierten Photonen zu erhöhen, ist chemische Additive als Triplettlöscher einzusetzen. Sie bewirken, dass die Fluorophore wieder in den Grundzustand relaxieren und somit wieder angeregt werden können. Es wurden verschiedene Additive, die in der Literatur als Triplettlöscher beschrieben sind, getestet. Dazu wurden zunächst ihre Auswirkungen auf den Triplettzustand verschiedener Fluorophore (Alexa Fluor (Al) 488, 532 und 647 und Atto655) mit Hilfe von Fluoreszenzkorrelationsspektroskopie (FCS) untersucht. Cyclooctatetraen (COT) bewirkte dabei eine Abnahme der Triplettausbeute von Al488, Al532 und Al647 um ~ 40-60%, bei Atto655 veränderte sie sich nicht. Obwohl die Ergebnisse der FCS-Messungen darauf hindeuten, dass COT in einer erhöhten Anzahl an emittierten Photonen resultiert, konnte dies bei dSTORM-Messungen nicht bestätigt werden. Hier hatte COT nur einen größeren positiven Effekt auf das Fluorophor Al647 (Zunahme um ~ 60%). Eine Erklärung für diese Widersprüchlichkeit zu den Ergebnissen aus den FCS-Messungen, könnte das Vorhandensein des Schaltpuffers bei dSTORM-Messungen sein. Dieser bewirkt den Übergang der Fluorophore in den Aus-Zustand bzw. entzieht dem Puffer Sauerstoff.
Bei der Zugabe von 5 mM Kaliumiodid (KI) nahm die Triplettamplitude bei FCS-Messungen nur bei Al488 ab (um ~ 80%). Eine geringe Steigerung (um ~ 10%) der Intensität von Al488 mit KI konnte bei dSTORM-Messungen mit niedrigen Konzentrationen (~ 0,5 mM) erzielt werden. Bei einer Konzentration von 5 mM sank die Intensität jedoch wieder um 40%.
Deuteriumoxid (D2O) soll, anders als die Triplettlöscher, eine Verbesserung der Photonenausbeute dadurch bewirken, dass strahlungslose Relaxationsprozesse minimiert werden. Mit dSTORM-Messungen konnte gezeigt werden, dass Atto655 und Al647 in D2O zwar pro An-Zustand mehr Photonen emittieren als in Schaltpuffer ohne D2O, da die Fluorophore hier jedoch schneller bleichen, letztendlich die gleiche Anzahl an Photonen detektiert werden.
Um die Anzahl an emittierten Photonen zu erhöhen, eignet sich also nur COT bei dSTORM-Messungen mit AL647 und KI in sehr geringen Konzentrationen bei Al488. D2O kann eingesetzt werden, wenn eine Probe schnell vermessen werden muss, wie zum Beispiel bei Lebendzellmessungen.
Nicht nur eine zu niedrige Photonenzahl, auch eine zu geringe Photoschaltrate kann Artefakte bei dSTORM-Messungen erzeugen. Dies wurde anhand von verschiedenen biologischen Strukturen, die mit unterschiedlichen Anregungsintensitäten aufgenommen wurden, deutlich gemacht. Besonders die Aufnahmen von Plasmamembranen sind anfällig für die Generierung von Artefakten. Sie weisen viele inhomogene und lokal dichte Regionen auf. Wenn nun mehr als ein Emitter pro µm² gleichzeitig an ist, erzeugt das Auswertungsprogramm große artifizielle Cluster. Die hier durchgeführten Messungen machen deutlich, wie wichtig es ist, dSTORM-Bilder immer auf mögliche Artefakte hin zu untersuchen, besonders wenn Moleküle quantifiziert werden sollen. Dafür müssen die unbearbeiteten Rohdaten sorgfältig gesichtet werden und notfalls die Messungen mit einer höheren Laserleistung wiederholt werden. Da dSTORM mittlerweile immer mehr zur Quantifizierung eingesetzt wird und Clusteranalysen durchgeführt werden, wäre es sinnvoll bei Veröffentlichungen die Rohdaten von entscheidenden Aufnahmen der Öffentlichkeit zur Verfügung zu stellen.
Die Färbemethode ist ein weiterer Punkt, durch den Artefakte bei der Abbildung von Molekülen mittels SMLM entstehen können. Häufig werden Antikörper zum Markieren verwendet. Dabei sollte darauf geachtet werden, dass möglichst kleine Antikörper oder Antikörperfragmente verwendet werden, besonders wenn Clusteranalysen durchgeführt werden sollen. Anderenfalls leidet die Auflösung darunter, bzw. erhöht sich die Gefahr der Kreuzvernetzung von Molekülen.
Im zweiten Teil der vorliegenden Arbeit, wurden Plasmamembran-Ceramide untersucht. Ceramide gehören zu den Sphingolipiden und regulieren diverse zelluläre Prozesse. Verschiedene Stimuli bewirken eine Aktivierung von Sphingomyelinasen (SMasen), die Ceramide in der Plasmamembran synthetisieren. Steigt die Konzentration von Ceramiden in der Plasmamembran an, kondensieren diese zu Ceramid-reichen Plattformen (CRPs). Bisher ist noch wenig über die Verteilung der Ceramide und die Größe der CRPs bekannt. Sie wurden hier über IgG-Antikörper in der Plasmamembran von Jurkat-, U2OS-, HBME- und primären T-Zellen angefärbt und erstmals mit dSTORM hochaufgelöst, um sie dann zu quantifizieren. Unabhängig von der Zelllinie befanden sich 50% aller Ceramidmoleküle in ~ 75 nm großen CRPs. Im Mittel bestanden die CRPs aus ~ 20 Ceramiden. Mit Hilfe einer Titrationsreihe konnte ausgeschlossen werden, dass diese Cluster nur durch die Antikörper-Färbung artifiziell erzeugt wurden. Bei Inkubation der Zellen mit Bacillus cereus Sphingomyelinase (bSMase) stieg die Gesamtkonzentration der Ceramide in der Plasmamembran an, ebenso wie die Ceramidanzahl innerhalb der CRPs, außerdem die Anzahl und Größe der CRPs. Dies könnte zu einer Veränderung der Löslichkeit von Membrankomponenten führen, was wiederum eine Akkumulation bestimmter Rezeptoren oder eine Kompartimentierung bestimmter Proteine erleichtern könnte. Die Anhäufung der Ceramide in den CRPs könnte ebenfalls die lokale Interaktion mit anderen Membranmolekülen erleichtern und dadurch möglicherweise die Reaktivität von Rezeptoren verändern.
Mittels Azid-modifizierten Ceramidanaloga und kupferfreier Click-Chemie wurden Plasmamembran-Ceramide auch in lebenden Jurkat-Zellen mit Hilfe konfokaler Laser-Raster-Mikroskopie (CLSM, engl. confocal laser scanning microscopy) und Strukturierter Beleuchtungsmikroskopie (SIM, engl. structured illumination microscopy) untersucht. Dabei konnte gezeigt werden, dass die Fettsäure-Kettenlänge und die Position des Azids bei den Ceramidanaloga eine entscheidende Rolle spielt, wie hoch das detektierte Signal in der Plasmamembran letztendlich ist. Die Versuche machen auch deutlich, dass die klickbaren Ceramidanaloga lebendzellkompatibel sind, sodass sie eine hervorragende Möglichkeit darstellen, zelluläre Reaktionen zu verfolgen.
Es wurden hier nicht nur Ceramide in eukaryotischen Zellen analysiert, sondern auch in Bakterien. Neisseria meningitidis (N. meningitidis) sind gramnegative Bakterien, die im Menschen eine Sepsis oder eine Meningitis auslösen können. Es wurde mittels immunhistochemischen Färbungen mit dem anti-Ceramid IgG-Antikörper, aber auch mit den klickbaren Ceramidanaloga, ein Signal in der Membran erhalten, was mit dSTORM hochaufgelöst wurde. In anderen Bakterien wurden ebenfalls schon Sphingolipide nachgewiesen. Studien zu Ceramiden in N. meningitidis wurden bisher jedoch noch nicht veröffentlicht. Im Rahmen dieser Arbeit konnten erstmals Ergebnisse erhalten werden, die darauf hinweisen, dass N. meningitidis ebenfalls Ceramide besitzen könnten.
In einem dritten Projekt wurde die Interaktion zwischen NK-Zellen und Aspergillus fumigatus untersucht. Der Schimmelpilz kann eine Invasive Aspergillose in immunsupprimierten Menschen auslösen, was zum Tod führen kann. Verschiedene Studien konnten schon zeigen, dass NK-Zellen eine wichtige Rolle bei der Bekämpfung des Pilzes spielen. Der genaue Mechanismus ist jedoch noch unbekannt. Im Rahmen dieser Arbeit konnte nachgewiesen werden, dass der NK-Zell-Marker CD56 entscheidend für die Pilzerkennung ist. Mit immunhistochemischen Färbungen und LSM-, aber auch dSTORM-Messungen, konnte gezeigt werden, dass die normalerweise homogen verteilten CD56-Rezeptoren auf der Plasmamembran von NK-Zellen aktiv an die Interaktionsstelle zu A. fumigatus transportiert werden. Mit der Zeit akkumulieren hier immer mehr CD56-Proteine, während das Signal in der restlichen Membran immer weiter abnimmt. Es konnte erstmals CD56 als wichtiger Erkennungsrezeptor für A. fumigatus identifiziert werden.
In dem letzten bearbeiteten Projekt, wurde die Bindung von Anti-N-Methyl-D-Aspartat (NMDA)-Rezeptor Enzephalitis Autoantikörper an Neuronen untersucht. Bei einer Anti-NMDA-Rezeptor Enzephalitis bilden die Patienten Autoantikörper gegen die NR1-Untereinheit ihrer eigenen postsynaptischen NMDA-Rezeptoren. Da die Krankheit oft sehr spät erkannt wird und die Behandlungsmöglichkeiten noch sehr eingeschränkt sind, führt sie noch oft zum Tod. Sie wurde erst vor wenigen Jahren beschrieben, sodass der genaue Mechanismus noch unbekannt ist. Im Rahmen dieser Arbeit, konnten erste Färbungen mit aufgereinigten Antikörper aus Anti-NMDA-Rezeptor Enzephalitis Patienten an NMDA-Rezeptor-transfizierte HEK-Zellen und hippocampalen Maus-Neuronen durchgeführt und mit dSTORM hochaufgelöst werden. Mit den Messungen der HEK-Zellen konnte bestätigt werden, dass die Autoantikörper an die NR1-Untereinheit der Rezeptoren binden. Es konnten erstmals auch die Bindung der Antikörper an Neuronen hochaufgelöst werden. Dabei wurde sichtbar, dass die Antikörper zum einen dicht gepackt in den Synapsen vorliegen, aber auch dünner verteilt in den extrasynaptischen Regionen. Basierend auf der Ripley’s H-Funktion konnten in den Synapsen große Cluster von ~ 90 nm Durchmesser und im Mittel ~ 500 Lokalisationen und extrasynaptisch kleinere Cluster mit einem durchschnittlichen Durchmesser von ~ 70 nm und ~ 100 Lokalisationen ausgemacht werden. Diese ersten Ergebnisse legen den Grundstein für weitere Messungen, mit denen der Mechanismus der Krankheit untersucht werden kann.
Chronic Obstructive Pulmonary Disease (COPD) exacerbations are a considerable reason for increased morbidity and mortality in patients. Infections with influenza virus (H1N1), respiratory syncytial virus (RSV) or nontypeable Haemophilus influenzae (NTHi) are important triggers of exacerbations. To date, no treatments are available which can stop the progression of COPD. Novel approaches are urgently needed. Pre-clinical models of the disease are crucial for the development of novel therapeutic options.
In order to establish pre-clinical models which mimic aspects of human COPD exacerbations, mice were exposed to cigarette smoke (CS) and additionally infected with H1N1, RSV and/or NTHi. Clinically relevant treatments such as the corticosteroids Fluticasone propionate and Dexamethasone, the phosphodiesterase-4 (PDE-4) inhibitor Roflumilast and the long-acting muscarinic receptor antagonist Tiotropium were tested in the established models. Furthermore, a novel treatment approach using antibodies (Abs) directed against IL-1α, IL-1β or IL-1R1 was examined in the established CS/H1N1 model. Levels of IFN-γ, IL-1β, IL-2, IL-6, KC, TNF-α, RANTES, IL-17, MCP-1, MIP 1α and MIP-1β were measured in lung homogenate. Numbers of total cells, neutrophils and macrophages were assessed in bronchoalveolar lavage (BAL) fluid. Hematoxylin- and eosin- (H&E-) stained lung slices were analyzed to detect pathological changes. Quantitative polymerase-chain-reaction (qPCR) was used to investigate gene expression of ICAM-1 and MUC5 A/C. The viral/bacterial load was investigated in lung homogenate or BAL fluid. In addition to the in vivo studies, the effects of the above mentioned treatments were investigated in vitro in H1N1, RSV or NTHi-infected (primary) human bronchial epithelial cells using submerged or air-liquid-interface (ALI) cell culture systems.
Four pre-clinical models (CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi) were established depicting clinically relevant aspects of COPD exacerbations such as increased inflammatory cells and cytokines in the airways and impaired lung function.
In the CS/H1N1 model, Tiotropium improved lung function and was superior in reducing inflammation in comparison to Fluticasone or Roflumilast. Moreover, Fluticasone increased the loss of body-weight, levels of IL-6, KC and TNF-α and worsened lung function. In CS/RSV-exposed mice Tiotropium but not Fluticasone or Roflumilast treatment reduced neutrophil numbers and IL-6 and TNF α levels in the lung. The viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after Fluticasone and Dexamethasone treatment. The results from these studies demonstrate that Tiotropium has anti-inflammatory effects on CS/virus-induced inflammation and might help to explain the observed reduction of exacerbation rates in Tiotropium-treated COPD patients. Furthermore, the findings from this work indicate that treatment with Fluticasone or Dexamethasone might not be beneficial to reduce inflammation in the airways of COPD patients and supports clinical studies that link treatment with corticosteroids to an increased risk for pneumonia.
Testing of anti-IL-1α, anti-IL-1β or anti-IL-1R1 Abs in the CS/H1N1 model suggests that, in line with clinical data, antagonization of IL-1β is not sufficient to reduce pulmonary inflammation and indicates a predominant role of IL-1α in CS/virus-induced airway inflammation. In line with the in vivo findings, anti-IL-1α but not anti-IL-1β Abs reduced levels of TNF-α and IL-6 in H1N1-infected primary human bronchial epithelial ALI cell culture. Blocking the IL-1R1 provided significant inhibitory effects on inflammatory cells in vivo but was inferior compared to inhibiting both its soluble ligands IL-1α and IL-1β. Concomitant usage of Abs against IL-1α/IL-1β revealed strong effects and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1β were significantly reduced and ICAM-1 mRNA expression was attenuated. These results suggest that combined inhibition of IL-1α/IL-1β might be beneficial to reduce inflammation and exacerbations in COPD patients. Moreover, combined targeting of both IL-1α/IL-1β might be more efficient compared to inhibition of the IL-1R1.
As in the CS/virus models, corticosteroid treatment failed to reduce inflammatory cells in the CS/NTHi and CS/H1N1/NTHi models, increased the loss of body-weight and the bacterial load. Furthermore, Roflumilast administration had no significant effects on cell counts or cytokines. However, it improved compliance in the CS/NTHi model. Treatment with Azithromycin reduced the bacterial load in the CS/NTHi model and reduced numbers of total cells, neutrophils, macrophages and levels of KC and TNF-α in the CS/H1N1/NTHi model.
In conclusion, the established CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi models depict clinically relevant aspects of human COPD exacerbations in mice and provide the opportunity to investigate underlying disease mechanisms and to test novel therapies.
Due to the earth´s rotation around itself and the sun, rhythmic daily and seasonal changes in illumination, temperature and many other environmental factors occur. Adaptation to these environmental rhythms presents a considerable advantage to survival. Thus, almost all living beings have developed a mechanism to time their behavior in accordance. This mechanism is the endogenous clock. If it fulfills the criteria of (1) entraining to zeitgebers (2) free-running behavior with a period of ~ 24 hours (3) temperature compensation, it is also referred to as “circadian clock”. Well-timed behavior is crucial for eusocial insects, which divide their tasks among different behavioral castes and need to respond to changes in the environment quickly and in an orchestrated fashion. Circadian rhythms have thus been studied and observed in many eusocial species, from ants to bees. The underlying mechanism of this clock is a molecular feedback loop that generates rhythmic changes in gene expression and protein levels with a phase length of approximately 24 hours. The properties of this feedback loop are well characterized in many insects, from the fruit fly Drosophila melanogaster, to the honeybee Apis mellifera. Though the basic principles and components of this loop are seem similar at first glance, there are important differences between the Drosophila feedback loop and that of hymenopteran insects, whose loop resembles the mammalian clock loop. The protein PERIOD (PER) is thought to be a part of the negative limb of the hymenopteran clock, partnering with CRYPTOCHROME (CRY). The anatomical location of the clock-related neurons and the PDF-network (a putative in- and output mediator of the clock) is also well characterized in Drosophila, the eusocial honeybee as well as the nocturnal cockroach Leucophea maderae. The circadian behavior, anatomy of the clock and its molecular underpinnings were studied in the carpenter ant Camponotus floridanus, a eusocial insect Locomotor activity recordings in social isolation proved that the majority of ants could entrain to different LD cycles, free-ran in constant darkness and had a temperature-compensated clock with a period slightly shorter than 24 hours. Most individuals proved to be nocturnal, but different types of activity like diurnality, crepuscularity, rhythmic activity during both phases of the LD, or arrhythmicity were also observed. The LD cycle had a slight influence on the distribution of these activities among individuals, with more diurnal ants at shorter light phases. The PDF-network of C. floridanus was revealed with the anti-PDH antibody, and partly resembled that of other eusocial or nocturnal insects. A comparison of minor and major worker brains, only revealed slight differences in the number of somata and fibers crossing the posterior midline. All in all, most PDF-structures that are conserved in other insects where found, with numerous fibers in the optic lobes, a putative accessory medulla, somata located near the proximal medulla and many fibers in the protocerebrum. A putative connection between the mushroom bodies, the optic lobes and the antennal lobes was found, indicating an influence of the clock on olfactory learning. Lastly, the location and intensity of PER-positive cell bodies at different times of a 24 hour day was established with an antibody raised against Apis mellifera PER. Four distinct clusters, which resemble those found in A. mellifera, were detected. The clusters could be grouped in dorsal and lateral neurons, and the PER-levels cycled in all examined clusters with peaks around lights on and lowest levels after lights off.
In summary, first data on circadian behavior and the anatomy and workings of the clock of C. floridanus was obtained. Firstly, it´s behavior fulfills all criteria for the presence of a circadian clock. Secondly, the PDF-network is very similar to those of other insects. Lastly, the location of the PER cell bodies seems conserved among hymenoptera. Cycling of PER levels within 24 hours confirms the suspicion of its role in the circadian feedback loop.
Simkania negevensis (Sn) repliziert als Chlamydia-ähnliches obligat intrazelluläres Bakterium auch in einer membranumschlossenen Vakuole, die als SCV (engl. Simkania-containing vacuole) bezeichnet wird. Die SCV ist ein bis jetzt einzigartiges Kompartiment, das stark mit ER-Membranen assoziiert ist, wobei ER-Stress von den Bakterien blockiert wird. Die Morphologie der SCV scheint dabei in Epithelzellen (HeLa229, A549, HEp-2) als auch in Makrophagen (THP1) gleich zu sein. Die SCV stellt die erste intrazelluläre Berührungsfläche dar, an der Interaktionen zwischen Wirt und Pathogen erfolgen, und dient gleichzeitig als Replikationsnische. Mithilfe der Identifizierung von humanen und bakteriellen Faktoren, die mit der ER-SCV-Membran assoziiert sind, sollten die Zusammensetzung der SCV sowie mögliche Interaktionen mit dem ER oder zellulären Transportwegen ermittelt werden. Vergleichsstudien von SCV-assoziierten Proteinen sollten Ähnlichkeiten zur chlamydialen Inklusion aufzeigen, für die bereits einige interagierende Wirtszellfaktoren beschrieben wurden. In dieser Arbeit wurde ein Aufreinigungsprotokoll etabliert, das sowohl für ER-SCVMembranen aus HeLa229 als auch THP1 geeignet war und für spätere Proteom- oder Lipidomanalysen diente. Über markierungsfreie massenspektrometrische Messungen konnten 302 bakterielle und 1178 humane Proteine in ER-(SCV-) Membranen und 885 bakterielle Proteine in aufgereinigten Sn identifiziert werden. In ER-(SCV-) Membranen von nicht und Sn-infizierten HeLa229 Zellen befanden sich 51 unterschiedlich verteilte und 57 transportassoziierte humane Proteine, die auf infektionsinduzierte Unterschiede beim intrazellulären Proteintransport hindeuteten. Eine entgegengesetzte Regulation von retro- und anterograd transportierten Proteinen konnte mithilfe von RNA-Interferenz und dem Einsatz geeigneter Inhibitoren bestätigt werden, wobei sich zeigte, dass Clathrin-assoziierte und COPI-Vesikel eine zentrale Rolle spielen. Retro-Inhibitoren, die den retrograden Transport zwischen Endosomen zum Golgi oder zwischen frühen und späten Endosomen inhibieren, sowie Bafilomycin A1 (retrograd, späte Endosomen und Lysosomen) und Brefeldin A (anterograd, ER und Golgi), beeinflussten massiv die SCV-Ausbildung, SCV-Morphologie und den intrazellulären Lipidtransport. Über markierungsfreie massenspektrometrische und dünnschichtchromatographische Analysen konnten erste Unterschiede im Lipidvorkommen in Sn-infizierten Zellen, an ER-SCVMembranen und in aufgereinigten Sn im Vergleich zu nicht infizierten Zellen ermittelt werden. Dabei konnten neben An- und Abreicherungen in Gesamtzellextrakten sowie ER-SCVMembranen zwei infektionsspezifische Lipide, Cholesterol-ß-D-Glykosid und PE 30:0, identifiziert werden. Weiterführende umfangreiche RNA-Interferenzstudien weisen darauf hin, dass die Sn-Infektion vom Endosomen-zum-Golgi-Transport und vom Clathrin-assoziierten Vesikeltransport abhängig ist. Zusammenfassend konnten erste mögliche SCV-assoziierte Proteine und Lipide identifiziert werden, die mit der bakteriellen Infektion verbunden waren. Des Weiteren hängen eine stabile SCV-Ausbildung und Sn-Infektivität von verschiedenen retrograden Transportwegen und damit von dem Erwerb von Nährstoffen wie bspw. Lipiden ab.
Characterization of motility and erythrocyte adherence as virulence factors in African trypanosomes
(2018)
Pathogens causing African animal trypanosomiasis (AAT), the major livestock disease in sub-Saharan Africa, belong to the salivarian group of the African trypanosomes, which are transmitted by the bite of the tsetse fly (Glossina spec.). T. vivax, T. congolense and T. brucei brucei are major pathogens of cattle in particular, causing nagana, with dramatic socio-economic consequences for the affected regions. The parasites additionally have a huge reservoir of other livestock and wild animal hosts. T. brucei, the species which also includes the subspecies pathogenic to humans causing sleeping sickness, has been extensively studied as the cultivatable model trypanosome. But less is known about the other salivarian species, which are not routinely held in culture, if at all possible. A hallmark of trypanosomal lifestyle is the protozoan flagellates incessant motility, which enables them to populate an enormous range of habitats in very diverse hosts. We were now able to characterize, for the first time with high spatiotemporal resolution microscopy, the swimming behaviour and mechanism of the most relevant salivarian species isolated directly from blood. We show the influence of viscosity on the motility of bloodstream form (BSF) cells and simulate their movement between erythrocytes, giving a clear picture of how all analyzed species move under varying environmental conditions. We show that although the basic mechanism of flagellar motility applies to all analyzed species, there are clear morphological differences that produce different reactions to the physical environment. We could define specific conditions for highly increased swimming persistence and speed for compared to the behaviour in standard culture. These results have important implications for the parasites survival strategies in the host, e.g. regarding the capacity for antibody clearance. Although we show all species to effectively remove antibodies from the cell surface, T. congolense differed markedly in its motility behaviour, which gives rise to interesting questions about this species behaviour in the bloodstream. Most of the T. congolense parasites (and to a lesser extent T. vivax) adhere to sheep erythrocytes. Further in vitro studies showed that T. congolense and T. vivax adhered to rabbit, goat, pig and cattle erythrocytes- but binding behaviour was absent in murine blood. Notably, both T. brucei and T. evansi lacked adherence to all studied host erythrocytes. Generally, attachment to blood cells caused reduction of swimming velocities. Judging from its cell architecture, as well as the motility studies in higher media viscosity and in micropillar arrays, T. congolense is not adapted to swim at high speeds in the mammalian bloodstream. Low swimming speeds could allow these purely intravascular parasites to remain bound to the host erythrocytes.
African trypanosomes are the causative agents of fatal diseases in humans and livestock. Trypanosomes show a complex lifecycle and shuttle between the transmitting vector, the tsetse (Glossina spec.), and the mammalian host. As a result of this the parasite undergoes tremendous changes in morphology and metabolism to adapt to the different living environments.
The two best-studied lifecycle stages are the procyclic forms (PCF) that live in the tsetse fly and the proliferative bloodstream form (BSF) that resides in the mammalian blood. The most conspicuous weapon that trypanosomes use to evade the host immune attack is a dense layer of a single protein type, the variant surface glycoprotein (VSG), which shields the entire cell surface. Immune evasion required high rates of surface membrane turnover and surface coat recycling.
Trypanosomes show highly polarised cell architecture with all major eukaryotic organelles (endoplasmic reticulum, Golgi apparatus, endosomal apparatus, lysosome, mitochondrion and peroxisome-like glycosomes) generally present in single copy. Furthermore, trypanosomes possess a single flagellum, which is important not only for cellular motility but also for cell division.
How the duplication of all these cellular components is coordinated in order to progresss through the cell division cycle is poorly understood.
We used trypanosomes as a model organism due to the relative simplicity and the polarised nature of their cell architecture and determined the duplication of all their compartments. This was only possible due to a new synchronisation approach developed during this project.
In the first part of the thesis a precise temporal map of the cell division cycle of the BSF T. brucei cell division cycle was generated. By the use of well-described morphological markers (K/N status, new flagellum outgrowth and DNA synthesis) the position of individual cells was determined with high temporal resolution; this allowed us for the first time to synchronise a cell population in silico without affecting the naturally asynchronous growth.
In the second part of the thesis we used this tool to follow duplication events of the Major organelles during progression through the cell division cycle. We precisely determined the time points of organelle duplication and found that it is ordered in trypanosomes. Furthermore we found that BSF T. brucei cells do not grow continuously, cell size start to increase rapidly, during a short period of time, late in the cell division cycle. We speculate that the initiation of cell volume increase is temporally separated from the formation of all secretory organelles in order to ensure maintenance of the protective coat, which must remain intact at all times in order for BSF trypanosomes to be able to evade the host immune response.
The Dual Olfactory Pathway in the Honeybee Brain: Sensory Supply and Electrophysiological Properties
(2018)
The olfactory sense is of utmost importance for honeybees, Apis mellifera. Honeybees use olfaction for communication within the hive, for the identification of nest mates and non-nest mates, the localization of food sources, and in case of drones (males), for the detection of the queen and mating. Honeybees, therefore, can serve as excellent model systems for an integrative analysis of an elaborated olfactory system.
To efficiently filter odorants out of the air with their antennae, honeybees possess a multitude of sensilla that contain the olfactory sensory neurons (OSN). Three types of olfactory sensilla are known from honeybee worker antennae: Sensilla trichoidea, Sensilla basiconica and Sensilla placodea. In the sensilla, odorant receptors that are located in the dendritic arborizations of the OSNs transduce the odorant information into electrical information. Approximately 60.000 OSN axons project in two parallel bundles along the antenna into the brain. Before they enter the primary olfactory brain center, the antennal lobe (AL), they diverge into four distinct tracts (T1-T4). OSNs relay onto ~3.000-4.000 local interneurons (LN) and ~900 projection neurons (PN), the output neurons of the AL. The axons of the OSNs together with neurites from LNs and PNs form spheroidal neuropil units, the so-called glomeruli. OSN axons from the four AL input tracts (T1-T4) project into four glomerular clusters. LNs interconnect the AL glomeruli, whereas PNs relay the information to the next brain centers, the mushroom body (MB) - associated with sensory integration, learning and memory - and the lateral horn (LH). In honeybees, PNs project to the MBs and the LH via two separate tracts, the medial and the lateral antennal-lobe tract (m/lALT) which run in parallel in opposing directions. The mALT runs first to the MB and then to the LH, the lALT runs first to the LH and then to the MB. This dual olfactory pathway represents a feature unique to Hymenoptera. Interestingly, both tracts were shown to process information about similar sets of odorants by extracting different features. Individual mALT PNs are more odor specific than lALT PNs. On the other hand, lALT PNs have higher spontaneous and higher odor response action potential (AP) frequencies than mALT PNs. In the MBs, PNs form synapses with ~184.000 Kenyon cells (KC), which are the MB intrinsic neurons. KCs, in contrast to PNs, show almost no spontaneous activity and employ a spatially and temporally sparse code for odor coding.
In manuscript I of my thesis, I investigated whether the differences in specificity of odor responses between m- and lALT are due to differences in the synaptic input. Therefore, I investigated the axonal projection patterns of OSNs housed in S. basiconica in honeybee workers and compared them with S. trichoidea and S. placodea using selective anterograde labeling with fluorescent tracers and confocal- microscopy analyses of axonal projections in AL glomeruli. Axons of S. basiconica-associated OSNs preferentially projected into the T3 input-tract cluster in the AL, whereas the two other types of sensilla did not show a preference for a specific glomerular cluster. T3- associated glomeruli had previously been shown to be innervated by mALT PNs. Interestingly, S. basiconica as well as a number of T3 glomeruli lack in drones. Therefore I set out to determine whether this was associated with the reduction of glomeruli innervated by mALT PNs. Retrograde tracing of mALT PNs in drones and counting of innervated glomeruli showed that the number of mALT-associated glomeruli was strongly reduced in drones compared to workers. The preferential projections of S. basiconica-associated OSNs into T3 glomeruli in female workers together with the reduction of mALT-associated glomeruli in drones support the presence of a female-specific olfactory subsystem that is partly innervated by OSNs from S. basiconica and is associated with mALT projection neurons. As mALT PNs were shown to be more odor specific, I suppose that already the OSNs in this subsystem are more odor specific than lALT associated OSNs. I conclude that this female-specific subsystem allows the worker honeybees to respond adequately to the enormous variety of odorants they experience during their lifetime.
In manuscript II, I investigated the ion channel composition of mALT and lALT PNs and KCs in situ. This approach represents the first study dealing with the honeybee PN and KC ion channel composition under standard conditions in an intact brain preparation. With these recordings I set out to investigate the potential impact of intrinsic neuronal properties on the differences between m- and lALT PNs and on the sparse odor coding properties of KCs. In PNs, I identified a set of Na+ currents and diverse K+ currents depending on voltage and Na+ or Ca2+ that support relatively high spontaneous and odor response AP frequencies. This set of currents did not significantly differ between mALT and lALT PNs, but targets for potential modulation of currents leading to differences in AP frequencies were found between both types of PNs. In contrast to PNs, KCs have very prominent K+ currents, which are likely to contribute to the sparse response fashion observed in KCs. Furthermore, Ca2+ dependent K+ currents were found, which may be of importance for coincidence detection, learning and memory formation.
Finally, I conclude that the differences in odor specificity between m- and lALT PNs are due to their synaptic input from different sets of OSNs and potential processing by LNs. The differences in spontaneous activity between the two tracts may be caused by different neuronal modulation or, in addition, also by interaction with LNs. The temporally sparse representation of odors in KCs is very likely based on the intrinsic KC properties, whereas general excitability and spatial sparseness are likely to be regulated through GABAergic feedback neurons.
I. Timing is a crucial feature in organisms that live within a variable and changing environment. Complex mechanisms to measure time are wide-spread and were shown to exist in many taxa. These mechanisms are expected to provide fitness benefits by enabling organisms to anticipate environmental changes and adapt accordingly. However, very few studies have addressed the adaptive value of proper timing. The objective of this PhD-project was to investigate mechanisms and fitness consequences of timing decisions concerning colony phenology and foraging activity in the honey bee (Apis mellifera), a social insect species with a high degree of social organization and one of the most important pollinators of wild plants and crops. In chapter II, a study is presented that aimed to identify the consequences of disrupted synchrony between colony phenology and the local environment by manipulating the timing of brood onset after hibernation. In a follow-up experiment, the importance of environmental factors for the timing of brood onset was investigated to assess the potential of climate change to disrupt synchronization of colony phenology (Chapter III). Chapter IV aimed to prove for the first time that honey bees can use interval time-place learning to improve foraging activity in a variable environment. Chapter V investigates the fitness benefits of information exchange between nest mates via waggle dance communication about a resource environment that is heterogeneous in space and time.
II. In the study presented in chapter II, the importance of the timing of brood onset after hibernation as critical point in honey bee colony phenology in temperate zones was investigated. Honey bee colonies were overwintered at two climatically different sites. By translocating colonies from each site to the other in late winter, timing of brood onset was manipulated and consequently colony phenology was desynchronized with the local environment. Delaying colony phenology in respect to the local environment decreased the capability of colonies to exploit the abundant spring bloom. Early brood onset, on the other hand, increased the loads of the brood parasite Varroa destructor later in the season with negative impact on colony worker population size. This indicates a timing related trade-off and illustrates the importance of investigating effects of climate change on complex multi-trophic systems. It can be concluded that timing of brood onset in honey bees is an important fitness relevant step for colony phenology that is highly sensitive to climatic conditions in late winter. Further, phenology shifts and mismatches driven by climate change can have severe fitness consequences.
III. In chapter III, I assess the importance of the environmental factors ambient temperature and photoperiod as well as elapsed time on the timing of brood onset. Twenty-four hibernating honey bee colonies were placed into environmental chambers and allocated to different combinations of two temperature regimes and three different light regimes. Brood onset was identified non-invasively by tracking comb temperature within the winter cluster. The experiment revealed that ambient temperature plays a major role in the timing of brood onset, but the response of honey bee colonies to temperature increases is modified by photoperiod. Further, the data indicate the involvement of an internal clock. I conclude that the timing of brood onset is complex but probably highly susceptible to climate change and especially spells of warm weather in winter.
IV. In chapter IV, it was examined if honey bees are capable of interval time-place learning and if this ability improves foraging efficiency in a dynamic resource environment. In a field experiment with artificial feeders, foragers were able to learn time intervals and use this ability to anticipate time periods during which feeders were active. Further, interval time-place learning enabled foragers to increase nectar uptake rates. It was concluded that interval time-place learning can help honey bee foragers to adapt to the complex and variable temporal patterns of floral resource environments.
V. The study presented in chapter V identified the importance of the honey bee waggle dance communication for the spatiotemporal coordination of honey bee foraging activity in resource environments that can vary from day to day. Consequences of disrupting the instructional component of honey bee dance communication were investigated in eight temperate zone landscapes with different levels of spatiotemporal complexity. While nectar uptake of colonies was not affected, waggle dance communication significantly benefitted pollen harvest irrespective of landscape complexity. I suggest that this is explained by the fact that honey bees prefer to forage pollen in semi-natural habitats, which provide diverse resource species but are sparse and presumably hard to find in intensively managed agricultural landscapes. I conclude that waggle dance communication helps to ensure a sufficient and diverse pollen diet which is crucial for honey bee colony health.
VI. In my PhD-project, I could show that honey bee colonies are able to adapt their activities to a seasonally and daily changing environment, which affects resource uptake, colony development, colony health and ultimately colony fitness. Ongoing global change, however, puts timing in honey bee colonies at risk. Climate change has the potential to cause mismatches with the local resource environment. Intensivation of agricultural management with decreased resource diversity and short resource peaks in spring followed by distinctive gaps increases the probability of mismatches. Even the highly efficient foraging system of honey bees might not ensure a sufficiently diverse and healthy diet in such an environment. The global introduction of the parasitic mite V. destructor and the increased exposure to pesticides in intensively managed landscapes further degrades honey bee colony health. This might lead to reduced cognitive capabilities in workers and impact the communication and social organization in colonies, thereby undermining the ability of honey bee colonies to adapt to their environment.
New experimental methods have drastically accelerated the pace and quantity at which biological data is generated. High-throughput DNA sequencing is one of the pivotal new technologies. It offers a number of novel applications in various fields of biology, including ecology, evolution, and genomics. However, together with those opportunities many new challenges arise. Specialized algorithms and software are required to cope with the amount of data, often requiring substantial training in bioinformatic methods. Another way to make those data accessible to non-bioinformaticians is the development of programs with intuitive user interfaces.
In my thesis I developed analyses and programs to tackle current problems with high-throughput data in biology. In the field of ecology this covers the establishment of the bioinformatic workflow for pollen DNA meta-barcoding. Furthermore, I developed an application that facilitates the analysis of ecological communities in the context of their traits. Information from multiple public databases have been aggregated and can now be mapped automatically to existing community tables for interactive inspection. In evolution the new data are used to reconstruct phylogenetic trees from multiple genes. I developed the tool bcgTree to automate this process for bacteria. Many plant genomes have been sequenced in current years. Sequencing reads of those projects also contain data from the chloroplasts. The tool chloroExtractor supports the targeted extraction and analysis of the chloroplast genome. To compare the structure of multiple genomes specialized software is required for calculation and visualization of the relationships. I developed AliTV to address this. In contrast to existing programs for this task it allows interactive adjustments of produced graphics. Thus, facilitating the discovery of biologically relevant information. Another application I developed helps to analyze transcriptomes even if no reference genome is present. This is achieved by aggregating the different pieces of information, like functional annotation and expression level, for each transcript in a web platform. Scientists can then search, filter, subset, and visualize the transcriptome.
Together the methods and tools expedite insights into biological systems that were not possible before.
The transcription factor MYC is deregulated in over 70% of all human tumors and, in its oncogenic form, plays a major role in the cancer metabolic reprogramming, promoting the uptake of nutrients in order to sustain the biosynthetic needs of cancer cells.
The research presented in this work aimed to understand if MYC itself is regulated by nutrient availability, focusing on the two major fuels of cancer cells: glucose and glutamine.
Initial observations showed that endogenous MYC protein levels strongly depend on the availability of glutamine, but not of glucose. Subsequent analysis highlighted that the mechanism which accounts for the glutamine-mediated regulation of MYC is dependent on the 3´-untranslated region (3´-UTR) of MYC. Enhanced glutamine utilization by tumors has been shown to be directly linked to MYC oncogenic activity and MYC-dependent apoptosis has been observed under glutamine starvation. Such effect has been described in experimental systems which are mainly based on the use of MYC transgenes that do not contain the 3´-UTR. It was observed in the present study that cells are able to survive under glutamine starvation, which leads to cell cycle arrest and not apoptosis, as previously reported. However, enforced expression of a MYC transgene, which lacks the 3´-UTR, strongly increases the percentage of apoptotic cells upon starvation. Evaluation of glutamine-derived metabolites allowed to identify adenosine nucleotides as the specific stimulus responsible for the glutamine-mediated regulation of MYC, in a 3´-UTR-dependent way. Finally, glutamine-dependent MYC-mediated effects on RNA Polymerase II (RNAPII) function were evaluated, since MYC is involved in different steps of global transcriptional regulation. A global loss of RNAPII recruitment at the transcriptional start site results upon glutamine withdrawal. Such effect is overcome by enforced MYC expression under the same condition.
This study shows that the 3´UTR of MYC acts as metabolic sensor and that MYC globally regulates the RNAPII function according to the availability of glutamine. The observations presented in this work underline the importance of considering stress-induced mechanisms impinging on the 3´UTR of MYC.
Genetic foundation of unrivaled survival strategies - Of water bears and carnivorous plants -
(2018)
All living organisms leverage mechanisms and response systems to optimize reproduction, defense, survival, and competitiveness within their natural habitat. Evolutionary theories such as the universal adaptive strategy theory (UAST) developed by John Philip Grime (1979) attempt to describe how these systems are limited by the trade-off between growth, maintenance and regeneration; known as the universal three-way trade-off. Grime introduced three adaptive strategies that enable organisms to coop with either high or low intensities of stress (e.g., nutrient deficiency) and environmental disturbance (e.g., seasons). The competitor is able to outcompete other organisms by efficiently tapping available resources in environments of low intensity stress and disturbance (e.g., rapid growers). A ruderal specism is able to rapidly complete the life cycle especially during high intensity disturbance and low intensity stress (e.g., annual colonizers). The stress tolerator is able to respond to high intensity stress with physiological variability but is limited to low intensity disturbance environments. Carnivorous plants like D. muscipula and tardigrades like M. tardigradum are two extreme examples for such stress tolerators. D. muscipula traps insects in its native habitat (green swamps in North and South Carolina) with specialized leaves and thereby is able to tolerate nutrient deficient soils. M. tardigradum on the other side, is able to escape desiccation of its terrestrial habitat like mosses and lichens which are usually covered by a water film but regularly fall completely dry. The stress tolerance of the two species is the central study object of this thesis. In both cases, high througput sequencing data and methods were used to test for transcriptomic (D. muscipula) or genomic adaptations (M. tardigradum) which underly the stress tolerance. A new hardware resource including computing cluster and high availability storage system was implemented in the first months of the thesis work to effectively analyze the vast amounts of data generated for both projects. Side-by-side, the data management resource TBro [14] was established together with students to intuitively approach complex biological questions and enhance collaboration between researchers of several different disciplines. Thereafter, the unique trapping abilities of D. muscipula were studied using a whole transcriptome approach. Prey-dependent changes of the transcriptional landscape as well as individual tissue-specific aspects of the whole plant were studied. The analysis revealed that non-stimulated traps of D. muscipula exhibit the expected hallmarks of any typical leaf but operates evolutionary conserved stress-related pathways including defense-associated responses when digesting prey. An integrative approach, combining proteome and transcriptome data further enabled the detailed description of the digestive cocktail and the potential nutrient uptake machinery of the plant. The published work [25] as well as a accompanying video material (https://www.eurekalert.org/pub_releases/ 2016-05/cshl-fgr042816.php; Video credit: Sönke Scherzer) gained global press coverage and successfully underlined the advantages of D. muscipula as experimental system to understand the carnivorous syndrome. The analysis of the peculiar stress tolerance of M. tardigradum during cryptobiosis was carried out using a genomic approach. First, the genome size of M. tardigradum was estimated, the genome sequenced, assembled and annotated. The first draft of M. tardigradum and the workflow used to established its genome draft helped scrutinizing the first ever released tardigrade genome (Hypsibius dujardini) and demonstrated how (bacterial) contamination can influence whole genome analysis efforts [27]. Finally, the
M. tardigradum genome was compared to two other tardigrades and all species present in the current release of the Ensembl Metazoa database. The analysis revealed that tardigrade genomes are not that different from those of other Ecdysozoa. The availability of the three genomes allowed the delineation of their phylogenetic position within the Ecdysozoa and placed them as sister taxa to the nematodes. Thereby, the comparative analysis helped to identify evolutionary trends within this metazoan lineage. Surprisingly, the analysis did not reveal general mechanisms (shared by all available tardigrade genomes) behind the arguably most peculiar feature of tardigrades; their enormous stress tolerance. The lack of molecular evidence for individual tardigrade species (e.g., gene expression data for M. tardigradum) and the non-existence of a universal experimental framework which enables hypothesis testing withing the whole phylum Tardigrada, made it nearly impossible to link footprints of genomic adaptations to the unusual physiological capabilities. Nevertheless, the (comparative) genomic framework established during this project will help to understand how evolution tinkered, rewired and modified existing molecular systems to shape the remarkable phenotypic features of tardigrades.
In einem sich entwickelnden multizellulären Organismus ist die räumlich-zeitliche Regulation der Genexpression von entscheidender Bedeutung für die Bildung, Identität und Funktion von Zellen. Der REST (repressor element silencing transcription factor) Komplex spielt bei der neuronalen Differenzierung und bei der Aufrechterhaltung des neuronalen Status eine essentielle Rolle, indem er in nicht neuronalen Zellen und neuralen Vorläufern die Expression neuronaler Gene unterdrückt, in deren Promotorregion eine RE1 (repressor element 1) Erkennungssequenz vorhanden ist. Während der neuronalen Differenzierung wird der REST-Komplex schrittweise inaktiviert, was zur Einleitung eines neuronalen Genexpression-Programms führt. Es wird daher angenommen, dass die Inhibierung des REST-Komplexes ein essentieller Vorgang der Neurogenese ist. Wichtige Bestandteile für die transkriptionell repressive Funktion des REST-Komplexes sind kleine Phosphatasen (CTDSP = C-terminal domain small phosphatases), welche die Polymerase-II-Aktivität an Zielgenen inhibieren. Im Zebrafisch wurde gezeigt, dass ctdsp2 durch die miR-26b negativ reguliert wird. Alle miR-26 Familienmitglieder sind in Vertebraten evolutionär konserviert und in Introns von Ctdsp Genen kodiert. Sie sind in der Lage, die Expression ihres eigenen Wirtsgens mittels einer autoregulatorischen Rückkopplungsschleife zu regulieren.
Im Rahmen dieser Dissertation wurde als Modellsystem für die Neurogenese ein neurales Differenzierungssystem, welches auf murinen, embryonalen Stammzellen (ESCs) aufbaut, eingesetzt. Zur funktionellen Analyse der miR-26 Familie wurden mit Hilfe der CRISPR/Cas9-Methode verschiedene miR-26 Knockout (KO) ESC-Linien hergestellt. Hierbei wurden die Sequenzen der einzelnen Familienmitglieder und der gesamten miR-26 Familie im Genom von Wildtyp (Wt) ESCs deletiert. Diese miR-26-defizienten ESCLinien behielten ihre Pluripotenz und zeigten keinen Phänotyp hinsichtlich Proliferation, Morphologie und Identität der Zellen während der Differenzierung bis zum neuralen Vorläuferzellstadium (NPCs, engl.: neural progenitor cells). Jedoch führte die Deletion sowohl der gesamten miR-26 Familie als auch einzelner Mitglieder bei der terminalen Differenzierung zu einem spezifischen Entwicklungsstillstand im NPC Stadium und infolgedessen zu einer starken Reduktion der Anzahl von Neuronen und Astroglia. Die Transkriptom-Analyse der differenzierten miR-26-KO ESCs mittels RNA-Seq zeigte, dass die Expression von Genen die mit der Neurogenese und der neuronalen Differenzierung, aber auch der Gliogenese assoziert sind, herunterreguliert war. Die Abwesenheit der miR-26 Familie führte außerdem zu einer selektiven Reduzierung bestimmter miRNAs (REST-miRs), die einerseits die Expression von REST-Komplex Komponenten unterdrücken können, und andererseits selbst unter dessen transkriptioneller Kontrolle stehen. Zu diesem REST-miR Netzwerk gehören einige miRNAs (miR-9, miR-124, miR-132 und miR-218), die wichtige Funktionen bei verschiedenen Prozessen der neuronalen Entwicklung haben. Weiterhin führte der miR-26-KO zu einer Derepression der Proteinlevel von REST und CTDSP2 während der terminalen Differenzierung. Funktionelle Analysen mit miRNA mimics zeigten, dass erhöhte miR-26 Level zu einer Hochregulation von REST-miRs führen. Weitere Experimente, die darauf zielten, die Hierarchie des REST-miR Netwerks aufzuklären zeigten, dass die miR-26 Familie stromaufwärts die REST-miR Expression reguliert.
Zusammengefasst weisen die in dieser Arbeit gezeigten Daten darauf hin, dass die miR-26 Familie als Initiator der schrittweisen Inaktivierung des REST-Komplexes eine zentrale Rolle bei der Differenzierung von neuralen Vorläuferzellen zu postmitotischen Neuronen spielt.
Since its first experimental implementation in 2005, single-molecule localization microscopy (SMLM) emerged as a versatile and powerful imaging tool for biological structures with nanometer resolution. By now, SMLM has compiled an extensive track-record of novel insights in sub- and inter- cellular organization.\\
Moreover, since all SMLM techniques rely on the analysis of emission patterns from isolated fluorophores, they inherently allocate molecular information $per$ $definitionem$.\\
Consequently, SMLM transitioned from its origin as pure high-resolution imaging instrument towards quantitative microscopy, where the key information medium is no longer the highly resolved image itself, but the raw localization data set.\\
The work presented in this thesis is part of the ongoing effort to translate those $per$ $se$ molecular information gained by SMLM imaging to insights into the structural organization of the targeted protein or even beyond. Although largely consistent in their objectives, the general distinction between global or segmentation clustering approaches on one side and particle averaging or meta-analyses techniques on the other is usually made.\\
During the course of my thesis, I designed, implemented and employed numerous quantitative approaches with varying degrees of complexity and fields of application.\\ \\
In my first major project, I analyzed the localization distribution of the integral protein gp210 of the nuclear pore complex (NPC) with an iterative \textit{k}-means algorithm. Relating the distinct localization statistics of separated gp210 domains to isolated fluorescent signals led, among others, to the conclusion that the anchoring ring of the NPC consists of 8 homo-dimers of gp210.\\
This is of particular significance, both because it answered a decades long standing question about the nature of the gp210 ring and it showcased the possibility to gain structural information well beyond the resolution capabilities of SMLM by crafty quantification approaches.\\ \\
The second major project reported comprises an extensive study of the synaptonemal complex (SNC) and linked cohesin complexes. Here, I employed a multi-level meta-analysis of the localization sets of various SNC proteins to facilitate the compilation of a novel model of the molecular organization of the major SNC components with so far unmatched extend and detail with isotropic three-dimensional resolution.\\
In a second venture, the two murine cohesin components SMC3 and STAG3 connected to the SNC were analyzed. Applying an adapted algorithm, considering the disperse nature of cohesins, led to the realization that there is an apparent polarization of those cohesin complexes in the SNC, as well as a possible sub-structure of STAG3 beyond the resolution capabilities of SMLM.\\ \\
Other minor projects connected to localization quantification included the study of plasma membrane glycans regarding their overall localization distribution and particular homogeneity as well as the investigation of two flotillin proteins in the membrane of bacteria, forming clusters of distinct shapes and sizes.\\ \\
Finally, a novel approach to three-dimensional SMLM is presented, employing the precise quantification of single molecule emitter intensities. This method, named TRABI, relies on the principles of aperture photometry which were improved for SMLM.\\
With TRABI it was shown, that widely used Gaussian fitting based localization software underestimates photon counts significantly. This mismatch was utilized as a $z$-dependent parameter, enabling the conversion of 2D SMLM data to a virtual 3D space. Furthermore it was demonstrated, that TRABI can be combined beneficially with a multi-plane detection scheme, resulting in superior performance regarding axial localization precision and resolution.\\
Additionally, TRABI has been subsequently employed to photometrically characterize a novel dye for SMLM, revealing superior photo-physical properties at the single-molecule level.\\
Following the conclusion of this thesis, the TRABI method and its applications remains subject of diverse ongoing research.
Viral infections induce a significant impact on various functional categories of biological processes in the host. The understanding of this complex modification of the infected host immune system requires a global and detailed overview on the infection process. Therefore it is essential to apply a powerful approach which identifies the involved components conferring the capacity to recognize and respond to specific pathogens, which in general are defeated in so-called compatible virus-plant infections. Comparative and integrated systems biology of plant-virus interaction progression may open a novel framework for a systemic picture on the modulation of plant immunity during different infections and understanding pathogenesis mechanisms. In this thesis these approaches were applied to study plant-virus infections during two main viral pathogens of cassava: Cassava brown streak virus and African cassava mosaic virus.
Here, the infection process was reconstructed by a combination of omics data-based analyses and metabolic network modelling, to understand the major metabolic pathways and elements underlying viral infection responses in different time series, as well as the flux activity distribution to gain more insights into the metabolic flow and mechanism of regulation; this resulted in simultaneous investigations on a broad spectrum of changes in several levels including the gene expression, primary metabolites, and enzymatic flux associated with the characteristic disease development process induced in Nicotiana benthamiana plants due to infection with CBSV or ACMV.
Firstly, the transcriptome dynamics of the infected plant was analysed by using mRNA-sequencing, in order to investigate the differential expression profile according the symptom developmental stage. The spreading pattern and different levels of biological functions of these genes were analysed associated with the infection stage and virus entity. A next step was the Real-Time expression modification of selected key pathway genes followed by their linear regression model. Subsequently, the functional loss of regulatory genes which trigger R-mediated resistance was observed. Substantial differences were observed between infected mutants/transgenic lines and wild-types and characterized in detail. In addition, we detected a massive localized accumulation of ROS and quantified the scavenging genes expression in the infected wild-type plants relative to mock infected controls.
Moreover, we found coordinated regulated metabolites in response to viral infection measured by using LC-MS/MS and HPLC-UV-MS. This includes the profile of the phytohormones, carbohydrates, amino acids, and phenolics at different time points of infection with the RNA and DNA viruses. This was influenced by differentially regulated enzymatic activities along the salicylate, jasmonate, and chorismate biosynthesis, glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways, as well as photosynthesis, photorespiration, transporting, amino acid and fatty acid biosynthesis. We calculated the flux redistribution considering a gradient of modulation for enzymes along different infection stages, ranging from pre-symptoms towards infection stability.
Collectively, our reverse-engineering study consisting of the generation of experimental data and modelling supports the general insight with comparative and integrated systems biology into a model plant-virus interaction system. We refine the cross talk between transcriptome modification, metabolites modulation and enzymatic flux redistribution during compatible infection progression. The results highlight the global alteration in a susceptible host, correlation between symptoms severity and the alteration level. In addition we identify the detailed corresponding general and specific responses to RNA and DNA viruses at different stages of infection. To sum up, all the findings in this study strengthen the necessity of considering the timing of treatment, which greatly affects plant defence against viral infection, and might result in more efficient or combined targeting of a wider range of plant pathogens.
The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009).
This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo.
In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker.
Die NO-sensitive Guanylyl-Cyclase (NO-GC) ist ein zentrales Enzym der NO/cGMP-Signalkaskade, das über die Aktivierung von NO zur Bildung des second messangers cGMP führt. Die NO-GC setzt sich aus zwei Untereinheiten zusammen, sodass zwei Isoformen des Enzyms gebildet werden können (α1β1 und α2β1). Da die genaue Verteilung der beiden Isoformen im Colon nicht bekannt ist, wurde diese im ersten Teil dieser Arbeit charakterisiert. Immunhistochemie und In-situ-Hybridisierung zeigten die Expression beider Isoformen sowohl in der glatten Muskelschicht als auch in der Submukosa und Lamina propria. Dabei war die α1β1-Isoform ubiquitär, die α2β1-Isoform dagegen hauptsächlich im Bereich des myenterischen Plexus vorzufinden.
In der glatten Muskelschicht des Colons ist die NO-GC in glatten Muskelzellen (SMC), interstitiellen Zellen von Cajal (ICC) sowie Fibroblasten-ähnliche Zellen (FLC) exprimiert und hauptsächlich in die Modulation der gastrointestinalen Motilität involviert. Zur spezifischen Charakterisierung der Funktion der NO-GC in den einzelnen Zelltypen wurden Knockout-Mäuse generiert, denen die NO-GC global (GCKO) oder spezifisch in SMC (SMC-GCKO), ICC (ICC-GCKO) oder beiden Zelltypen (SMC/ICC-GCKO) fehlt. Anhand dieser Mausmodelle sollten im zweiten Teil dieser Arbeit die modulatorischen Effekte der NO-GC auf die spontanen Kontraktionen des Colons bestimmt werden. Zur Charakterisierung der spontanen Kontraktionen der zirkulären Muskelschicht wurden Myographiestudien mit 2,5 mm langen Colonringen durchgeführt. Hierbei konnten drei verschiedene Kontraktionen gemessen werden: Kleine, hochfrequente Ripples, mittlere Kontraktionen und große Kontraktionen. Die detaillierte Analyse der einzelnen Kontraktionen zeigte einerseits eine NO-unabhängige Regulation der Ripples, andererseits eine NO-abhängige Modulation der mittleren und großen Kontraktionen über die NO-GC in SMC und ICC. Die NO-GC in SMC beeinflusst die Kontraktionen vermutlich vor allem über die Regulation des Muskeltonus der zirkulären Muskelschicht. Die NO-GC in ICC dagegen modifiziert die spontanen Kontraktionen möglicherweise über eine Veränderung der Schrittmacheraktivität. Allerdings führt erst ein Funktionsverlust des NO/cGMP-Signalweges in beiden Zelltypen zu einem sichtbar veränderten Kontraktionsmuster, das dem von globalen Knockout-Tieren glich. Dies weist auf eine kompensatorische Wirkung der NO-GC im jeweils anderen Zelltyp hin.
Zur Analyse der propulsiven Kontraktionen entlang des gesamten Colons wurden Videoaufnahmen der Darmbewegungen in Kontraktionsmusterkarten transformiert. Zudem wurde der Darm durchspült und die Ausflusstropfen aufgezeichnet, um die Effektivität der Kontraktionen beurteilen zu können. Hierbei zeigte sich, dass eine Beeinträchtigung des NO/cGMP-Signalweges eine verminderte Effektivität der Kontraktionen zur Folge hat und vermutlich durch eine beeinträchtige Synchronisation der Kontraktionen erklärt werden kann. In diesem Regulationsmechanismus konnte vor allem der NO-GC in SMC eine übergeordnete Rolle zugewiesen werden.
Der dritte Teil der Arbeit thematisierte den Befund, dass SMC-GCKO-Tiere ca. 5 Monate nach Tamoxifen-Behandlung Entartungen der Mukosa entwickelten. Diese Entartung war lediglich in Tamoxifen-induzierten Knockout-Tieren vorzufinden. Histologische Analysen identifizierten die Entartungen als tubulovillöses Adenom. Die Genexpressionsanalyse von Mukosafalten von SMC-GCKO- und heterozygoten Kontrolltieren zeigte eine Vielzahl von Genen, welche spezifisch bei colorectalem Karzinom differenziell exprimiert sind. Einer dieser Faktoren war der BMP-Antagonist Gremlin1. Dieser Faktor erschien von besonderem Interesse, da er in Zellen der Lamina muscularis mucosae und kryptennahen Myofibroblasten exprimiert wird. Immunhistochemische Analysen ließen vermuten, dass diese Zellen sowohl die NO-GC als auch die Cre-Rekombinase unter dem SMMHC-Promotor exprimieren. Diese Arbeit liefert demnach Hinweise darauf, dass die NO-GC einen wichtigen Regulator innerhalb der Stammzellnische bildet. Die Deletion der NO-GC führt vermutlich zu einer verstärkten Bildung bzw. Sekretion von Gremlin1, was die Homöostase der mukosalen Erneuerung stört und somit zur Entwicklung von Adenomen führt.
Staphylococcus aureus asymptomatically colonizes the skin and anterior nares of 20-30% of the healthy human population. As an opportunistic human pathogen it elicits a variety of infections ranging from skin and soft tissue infections to highly severe manifestations such as pneumonia, endocarditis and osteomyelitis. Due to the emergence of multi resistant strains, treatment of staphylococcal infections becomes more and more challenging and the WHO therefore classified S. aureus as a “superbug”. The variety of diseases triggered by S. aureus is the result of a versatile expression of a large set of virulence factors. The most prominent virulence factor is the cytotoxic and haemolytic pore-forming α-toxin whose expression is mediated by a complex regulatory network involving two-component systems such as the agr quorum-sensing system, accessory transcriptional regulators and alternative sigma-factors. However, the intricate regulatory network is not yet understood in its entirety. Recently, a transposon mutation screen identified the AraC-family transcriptional regulator ‘Repressor of surface proteins’ (Rsp) to regulate haemolysis, cytotoxicity and the expression of various virulence associated factors. Deletion of rsp was accompanied by a complete loss of transcription of a 1232 nt long non-coding RNA, SSR42.
This doctoral thesis focuses on the molecular and functional characterization of SSR42. By analysing the transcriptome and proteome of mutants in either SSR42 or both SSR42 and rsp, as well as by complementation of SSR42 in trans, the ncRNA was identified as the main effector of Rsp-mediated virulence. Mutants in SSR42 exhibited strong effects on transcriptional and translational level when compared to wild-type bacteria. These changes resulted in phenotypic alterations such as strongly reduced haemolytic activity and cytotoxicity towards epithelial cells as well as reduced virulence in a murine infection model. Deletion of SSR42 further promoted the formation of small colony variants (SCV) during long term infection of endothelial cells and demonstrated the importance of this molecule for intracellular bacteria. The impact of this ncRNA on staphylococcal haemolysis was revealed to be executed by modulation of sae mRNA stability and by applying mutational studies functional domains within SSR42 were identified.
Moreover, various stressors modulated the transcription of SSR42 and antibiotic challenge resulted in SSR42-dependently increased haemolysis and cytotoxicity. Transcription of SSR42 itself was found under control of various important global regulators including AgrA, SaeS, CodY and σB, thereby illustrating a central position in S. aureus virulence gene regulation.
The present study thus demonstrates SSR42 as a global virulence regulatory RNA which is important for haemolysis, disease progression and adaption of S. aureus to intracellular conditions via formation of SCVs.
Das menschliche Gehirn ist ein Organ, das aufgrund seiner Komplexität und zellulären Diversität noch am wenigsten verstanden ist. Eine der Ursachen dafür sind zahlreiche Herausforderungen in diversen neurobiologischen Bild-gebungsverfahren. Erst seit der Erfindung der hochauflösenden Fluoreszenz-mikroskopie ist es möglich, Strukturen unterhalb der Beugungsgrenze zu visua-lisieren und somit eine maximale Auflösung von bis zu 20 nm zu erreichen. Zusätzlich hängt die Fähigkeit, biologische Strukturen aufzulösen, von der Markierungs-größe und -dichte ab. Derzeit ist die häufigste Methode zur Proteinfärbung die indirekte Antikörperfärbung, bei der ein Fluorophor-markierter Sekundärantikörper an einen Epitop-spezifischen Primärantikörper bindet. Dabei kann der Abstand von Zielstruktur und Fluorophor bis zu 30 nm betragen, was eine Auflösungs-verminderung zur Folge haben kann. Aufgrund dessen wurden in dieser Arbeit alternative Markierungsmethoden getestet, um postsynaptische Proteine sicht-bar zu machen.
Zunächst wurde der postsynaptische N-Methyl-D-Aspartat (NMDA)-Rezeptor mit Hilfe konventioneller indirekter Antikörperfärbung markiert. Hier war die NR1-Untereinheit des NMDA-Rezeptors von besonderem Interesse, da diese in der Autoimmunerkrankung Anti-NMDA-Rezeptor-Enzephalitis invol-viert ist.
Patienten dieser seltenen Krankheit bilden Autoantikörper gegen die NR1-Untereinheit, wodurch ein schneller reversibler Verlust der NMDA-Rezeptoren auf der Postsynapse induziert wird. Wichtige Informationen können nicht mehr ausreichend weitergegeben werden, was psychiatrische und neurologi-sche Störungen zur Folge hat. In dieser Arbeit wurden sowohl kommerzielle NR1-Antikörper, als auch rekombinante monoklonale NR1-Antikörper von Patien-ten mit Anti-NMDA-Rezeptor-Enzephalitis getestet. In konfokalen und in hochaufgelösten SIM- (engl. structured illumination microscopy) und dSTORM- (engl. direct stochastic optical reconstruction microscopy) Messun-gen konnten kommerzielle NR1-Antikörper keine erfolgreichen Färbungen erzielen. Dagegen erwiesen sich die rekombinanten monoklonalen NR1-Patientenantikörper als sehr spezifisch, sowohl in primären Neuronen als auch im Hippocampus von murinen Gehirnschnitten und lieferten gute Kolokalisati-onen mit dem postsynaptischen Markerprotein Homer.
Um die optische Auflösung zu verbessern, wurde eine neue Markierungs-methode mit sog. „Super-Binde-Peptiden“ (SBPs) getestet. SBPs sind modifi-zierte Peptide, die erhöhte Affinitäten und Spezifitäten aufweisen und mit ei-ner Größe von ~ 2,5 nm wesentlich kleiner als Antikörper sind. In dieser Arbeit bestätigte sich ein kleines hochspezifisches SPB, das an den Fluoreszenzfarb-stoff Tetra-
methylrhodamin (TMR) gekoppelt ist, als effektiver Marker für das Ankerpro-tein Gephyrin. Gephyrin ist für die Lokalisation und Verankerung einiger post-synaptischer Rezeptoren zuständig, indem es sie mit dem Cytoskelett der Zelle verbindet. SIM-Messungen in primären Neuronen zeigten eine bessere Clus-terrepräsentation bei der Färbung von Gephyrin mit SBPs, als mit Antikörper-färbung. Zusätzlich wurden Kolokalisationsanalysen von Gephyrin zusammen mit dem inhibito-rischen präsynaptischen vesikulären GABA-Transporter VGAT durchgeführt.
Eine weitere Färbemethode stellte die bioorthogonale Click-Färbung durch die Erweiterung des eukaryotischen genetischen Codes (engl. genetic code ex-pansion, GCE) dar. Dabei wurde eine unnatürliche, nicht-kanonische Amino-säure (engl. non-canonical amino acid, ncAA) ins Zielprotein eingebaut und in Kombination mit der Click-Chemie ortsspezifisch mit organischen Tetrazin-Farbstoff-Konjugaten angefärbt. Organische Fluorophore haben den Vorteil, dass sie mit einer Größe von 0,5 – 2 nm sehr klein sind und damit die natürli-chen Funktionen der Proteine in der Zelle kaum beeinflussen. In dieser Arbeit wurde zum ersten Mal gezeigt, dass der tetramere postsynaptische NMDA-Rezeptor durch die Amber-Supres-sionsmethode bioorthogonal angefärbt werden konnte. Aus sieben verschiede-nen Amber-Mutanten der NR1-Untereinheit stellte sich die Y392TAG-NR1-Mutante als diejenige mit der besten Proteinexpression, Färbeeffizienz und rezeptorfunktionalität heraus. Dies konnte durch Fluoreszenzmikroskopie- und Whole-Cell Patch-Clamp-Experimenten gezeigt werden. Die bioorthogo-nale Click-Färbung durch GCE eignete sich für die Färbung des NMDA-Rezeptors in verschiedenen Zelllinien, mit unterschiedlichen Tetrazin-Farbstoff-Konjugaten und für Lebendzellexperimente. In dSTORM-Messungen erwies sich das Tetrazin-Cy5-Farbstoff-Konjugat als ideal aufgrund seiner Grö-ße, Photostabilität, Helligkeit und seines geeigneten Blinkverhaltens, sodass eine homogene NMDA-Rezeptorverteilung auf der Zellmembran gezeigt wer-den konnte. NR1-Antikörperfärbungen wiesen dagegen starke Clusterbildun-gen auf. Die Ergebnisse konnten belegen, dass kleinere Farbstoffe eine deut-lich bessere Zugänglichkeit zu ihrem Zielprotein haben und somit besser für die hochauflösende Fluoreszenzmikroskopie geeignet sind.
The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels.
In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic.
Charakterisierung der Wechselwirkung zwischen N-Myc und Aurora-A im MYCN-amplifizierten Neuroblastom
(2019)
Im Neuroblastom ist die Amplifikation des MYCN-Gens, eines Mitglieds der MYC-Onkogenfamilie, mit einer ungünstigen Prognose assoziiert. Der von dem Gen kodierte Transkriptionsfaktor N-Myc ist für die Proliferation der MYCN-amplifizierten Neuroblastomzelllinien notwendig und seine Depletion oder Destabilisierung führen zum Proliferationsarrest (Otto et al., 2009). Da N-Myc auf Proteinebene durch die Interaktion mit der mitotischen Kinase Aurora-A stabilisiert wird, bewirkt deren Depletion oder die Hemmung der Interaktion der beiden Proteine mittels spezieller Aurora- A-Inhibitoren (z.B. MLN8054 und MLN8237) ebenso eine Hemmung der Proliferation – in vitro und in vivo (Brockmann et al., 2013). Bisher ist jedoch unklar, über welchen Mechanismus Aurora-A die Stabilisierung von N-Myc erreicht, die Kinaseaktivität spielt hierbei jedoch keine Rolle (Otto et al., 2009). Eine Möglichkeit stellt die Rekrutierung von Usps dar, die das angehängte Ubiquitinsignal so modifizieren, dass die Erkennung und der Abbau des Proteins durch das Proteasom verringert werden. In der vorliegenden Arbeit wurde die Wirkung von Usp7 und Usp11 auf die Stabilität von N-Myc untersucht. Für beide konnte in Immunpräzipitationen die Interaktion mit N-Myc gezeigt werden. Ebenso erhöhten beide Proteasen in Überexpressionsexperimenten die vorhandene Menge an NMyc. Die Depletion von Usp7 mittels shRNAs führte in IMR-32 zu einem Arrest in der G1-Phase und zur Differenzierung der Zellen. Gleichzeitig wurden stark erniedrigte mRNA- und Proteinmengen von N-Myc und Aurora-A nachgewiesen. Es konnte jedoch nicht eindeutig gezeigt werden, ob die beobachteten zellulären Effekte durch eine vermehrte proteasomale Degradation von N-Myc begründet sind oder ob dabei die veränderte Regulation weiterer Zielproteine von Usp7 eine Rolle spielt. Die Depletion von Usp11 mit shRNAs bewirkte eine Abnahme der N-Myc-Mengen auf posttranslationaler Ebene. Somit stellen beide Usps vielversprechende Angriffspunkte einer gezielten Therapie in MYCN-amplifizierten Neuroblastomen dar und sollten deshalb Gegenstand weiterführender Untersuchungen sein. Über welche Proteindomäne in N-Myc die Interaktion mit Aurora-A stattfindet ist nicht bekannt. Eine mögliche Pseudosubstratbindungssequenz in Myc-Box I (Idee Richard Bayliss, University of Leicester) wurde in der vorliegenden Arbeit untersucht. Durch Mutation dieser Sequenz sollte die Bindung von Aurora-A unmöglich gemacht werden. Allerdings wurde die erwartete Abnahme der Stärke der Interaktion von Aurora-A und N-Myc durch die Mutation ebensowenig beobachtet wie eine verringerte Stabilität. Die Regulation der Phosphorylierung von N-Myc im Verlauf des Zellzyklus wurde durch die Mutation beeinträchtigt. Wie diese Veränderung exakt zu begründen ist bedarf weiterer Experimente
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.
This thesis elucidates patterns and drivers of invertebrate herbivory, herbivore diversity, and community-level biomass along elevational and land use gradients at Mt. Kilimanjaro, Tanzania.
Chapter I provides background information on the response and predictor variables, study system, and the study design. First, I give an overview of the elevational patterns of species diversity/richness and herbivory published in the literature. The overview illuminates existing debates on elevational patterns of species diversity/richness and herbivory. In connection to these patterns, I also introduce several hypotheses and mechanisms put forward to explain macroecological patterns of species richness. Furthermore, I explain the main variables used to test hypotheses. Finally, I describe the study system and the study design used.
Chapter II explores the patterns of invertebrate herbivory and their underlying drivers along extensive elevational and land use gradients on the southern slopes of Mt. Kilimanjaro. I recorded standing leaf herbivory from leaf chewers, leaf miners and gall-inducing insects on 55 study sites located in natural and anthropogenic habitats distributed from 866 to 3060 meters above sea level (m asl) on Mt. Kilimanjaro. Standing leaf herbivory was related to climatic variables [mean annual temperature - (MAT) and mean annual precipitation - (MAP)], net primary productivity (NPP) and plant functional traits (leaf traits) [specific leaf area (SLA), carbon to nitrogen ratio (CN), and nitrogen to phosphorous ratio (NP)]. Results revealed an unimodal pattern of total leaf herbivory along the elevation gradient in natural habitats. Findings also revealed differences in the levels and patterns of herbivory among feeding guilds and between anthropogenic and natural habitats. Changes in NP and CN ratios which were closely linked to NPP were the strongest predictors of leaf herbivory. Our study uncovers the role of leaf nutrient stoichiometry and its linkages to climate in explaining the variation in leaf herbivory along climatic gradients.
Chapter III presents patterns and unravels direct and indirect effects of resource (food) abundance (NPP), resource (food) diversity [Functional Dispersion (FDis)], resource quality (SLA, NP, and CN rations), and climate variables (MAT and MAP) on species diversity of phytophagous beetles. Data were collected from 65 study sites located in natural and anthropogenic habitats distributed from 866 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Sweep net and beating methods were used to collect a total of 3,186 phytophagous beetles representing 21 families and 304 morphospecies. Two groups, weevils (Curculionidae) and leaf beetles (Chrysomelidae) were the largest and most diverse families represented with 898 and 1566 individuals, respectively. Results revealed complex (bimodal) and dissimilar patterns of Chao1-estimated species richness (hereafter referred to as species diversity) along elevation and land use gradients. Results from path analysis showed that temperature and climate-mediated changes in NPP had a significant positive direct and indirect effect on species diversity of phytophagous beetles, respectively. The results also revealed that the effect of NPP (via beetles abundance and diversity of food resources) on species diversity is stronger than that of temperature. Since we found that factors affecting species diversity were intimately linked to climate, I concluded that predicted climatic changes over the coming decades will likely alter the species diversity patterns which we observe today.
Chapter IV presents patterns and unravels the direct and indirect effects of climate, NPP and anthropogenic disturbances on species richness and community-level biomass of wild large mammals which represent endothermic organisms and the most important group of vertebrate herbivores. Data were collected from 66 study sites located in natural and anthropogenic habitats distributed from 870 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Mammals were collected using camera traps and used path analysis to disentangle the direct and indirect effects of climatic variables, NPP, land use, land area, levels of habitat protection and occurrence of domesticated mammals on the patterns of richness and community-level biomass of wild mammals, respectively. Results showed unimodal patterns for species richness and community-level biomass of wild mammals along elevation gradients and that the patterns differed depending on the type of feeding guild. Findings from path analysis showed that net primary productivity and levels of habitat protection had a strong direct effect on species richness and community-level biomass of wild mammals whereas temperature had an insignificant direct effect. Findings show the importance of climate-mediated food resources in determining patterns of species richness of large mammals. While temperature is among key predictors of species richness in several ectotherms, its direct influence in determining species richness of wild mammals was insignificant. Findings show the sensitivity of wild mammals to anthropogenic influences and underscore the importance of protected areas in conserving biodiversity.
In conclusion, despite a multitude of data sets on species diversity and ecosystem functions along broad climatic gradients, there is little mechanistic understanding of the underlying causes. Findings obtained in the three studies illustrate their contribution to the scientific debates on the mechanisms underlying patterns of herbivory and diversity along elevation gradients. Results present strong evidence that plant functional traits play a key role in determining invertebrate herbivory and species diversity along elevation gradients and that, their strong interdependence with climate and anthropogenic activities will shape these patterns in future. Additionally, findings from path analysis demonstrated that herbivore diversity, community-level biomass, and herbivory are strongly influenced by climate (either directly or indirectly). Therefore, the predicted climatic changes are expected to dictate ecological patterns, biotic interactions, and energy and nutrient fluxes in terrestrial ecosystems in the coming decades with stronger impacts probably occurring in natural ecosystems. Furthermore, findings demonstrated the significance of land use effects in shaping ecological patterns. As anthropogenic pressure is advancing towards more pristine higher elevations, I advocate conservation measures which are responsive to and incorporate human dimensions to curb the situation. Although our findings emanate from observational studies which have to take several confounding factors into account, we have managed to demonstrate global change responses in real ecosystems and fully established organisms with a wide range of interactions which are unlikely to be captured in artificial experiments. Nonetheless, I recommend additional experimental studies addressing the effect of top-down control by natural enemies on herbivore diversity and invertebrate herbivory in order to deepen our understanding of the mechanisms driving macroecological patterns along elevation gradients.
The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules.
An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed.
Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and –lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates.
In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60–1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed.
Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models.
Das Multiple Myelom (MM) ist eine maligne B-Zell-Erkrankung, welche von einer großen Heterogenität auf der biologischen und klinischen Ebene sowie in der Therapieantwort geprägt ist. Durch die biologische Interpretation von whole exome sequencing (WES)-Daten der Tumor- und Normalproben von fünf MM-Patienten und sechs MM-Zelllinien (ZL) sowie dem Einbezug von publizierten next generation sequencing (NGS)-Daten von 38 MM-Patienten konnten in dieser Dissertation sowohl somatische tumorrelevante Mutationen identifiziert als auch ein MM-spezifisches Signaltransduktionsnetzwerk definiert werden. Interessanterweise wurde in fast 100 % der MM-Patienten mindestens eine Mutation und in ~50 % der MM-Patienten sogar mehr als eine Mutation innerhalb dieses Netzwerkes beobachtet, was auf eine inter- und intra-individuelle Signalweg-Redundanz hinweist, die für die individuelle Therapieentscheidung möglicherweise von Bedeutung sein könnte. Außerdem konnte bestätigt werden, dass identische, positionsspezifische und genspezifische Mutationen im MM selten wiederholt auftreten. Als häufig mutierte Gene im MM konnten KRAS, NRAS, LRP1B, FAM46C, WHSC1, ALOX12B, DIS3 und PKHD1 identifiziert werden. Interessanterweise wurde die DIS3-Mutation in der MM-ZL OPM2 gemeinsam mit einer copy neutral loss of heterozygosity (CNLOH) im DIS3-Lokus detektiert, und in der MM-ZL AMO1 wurde eine noch nicht näher charakterisierte KRAS-Mutation in Exon 4 in Verbindung mit einem copy number (CN)-Zugewinn und einer erhöhten KRAS-Genexpression gefunden. DIS3 ist ein enzymatisch aktiver Teil des humanen RNA-Exosom-Komplexes und KRAS ein zentrales Protein im RTK-Signalweg, wodurch genetische Aberrationen in diesen Genen möglicherweise in der Entstehung oder Progression des MMs eine zentrale Rolle spielen. Daher wurde die gesamte coding sequence (CDS) der Gene DIS3 und KRAS an Tumorproben eines einheitlich behandelten Patientensets der DSMM-XI-Studie mit einem Amplikon-Tiefen-Sequenzierungsansatz untersucht. Das Patientenset bestand aus 81 MM-Patienten mit verfügbaren zytogenetischen und klinischen Daten. Dies ergab Aufschluss über die Verteilung der Mutationen innerhalb der Gene und dem Vorkommen der Mutationen in Haupt- und Nebenklonen des Tumors. Des Weiteren wurde die Assoziation der Mutationen mit weiteren klassischen zytogenetischen Alterationen (z.B. Deletion von Chr 13q14, t(4;14)-Translokation) untersucht und der Einfluss der Mutationen in Haupt- und Nebenklonen auf den klinischen Verlauf und die Therapieantwort bestimmt. Besonders hervorzuheben war dabei die Entdeckung von sieben neuen Mutationen sowie drei zuvor unbeschriebenen hot spot-Mutationen an den Aminosäure (AS)-Positionen p.D488, p.E665 und p.R780 in DIS3. Es wurde des Weiteren die Assoziation von DIS3-Mutationen mit einer Chr 13q14-Deletion und mit IGH-Translokationen bestätigt. Interessanterweise wurde ein niedrigeres medianes overall survival (OS) für MM-Patienten mit einer DIS3-Mutation sowie auch eine schlechtere Therapieantwort für MM-Patienten mit einer DIS3-Mutation im Nebenklon im Vergleich zum Hauptklon beobachtet. In KRAS konnten die bereits publizierten Mutationen bestätigt und keine Auswirkungen der KRAS-Mutationen in Haupt- oder Nebenklon auf den klinischen Verlauf oder die Therapieantwort erkannt werden. Erste siRNA vermittelte knockdown-Experimente von KRAS und Überexpressionsexperimente von KRAS-Wildtyp (WT) und der KRAS-Mutationen p.G12A, p.A146T und p.A146V mittels lentiviraler Transfektion zeigten eine Abhängigkeit der Phosphorylierung von MEK1/2 und ERK1/2 von dem KRAS-Mutationsstatus.
Zusammenfassend liefert die vorliegende Dissertation einen detaillierten Einblick in die molekularen Strukturen des MMs, vor allem im Hinblick auf die Rolle von DIS3 und KRAS bei der Tumorentwicklung und dem klinischen Verlauf.
Endogenous clocks regulate physiological as well as behavioral rhythms within all organisms. They are well investigated in D. melanogaster on a molecular as well as anatomical level. The neuronal clock network within the brain represents the center for rhythmic activity control. One neuronal clock subgroup, the pigment dispersing factor (PDF) neurons, stands out for its importance in regulating rhythmic behavior. These neurons express the neuropeptide PDF (pigment dispersing factor). A small neuropil at the medulla’s edge, the accessory medulla (AME), is of special interest, as it has been determined as the main center for clock control. It is not only highly innervated by the PDF neurons but also by terminals of all other clock neuron subgroups. Furthermore, terminals of the photoreceptors provide light information to the AME. Many different types of neurons converge within the AME and afterward spread to their next target. Thereby the AME is supplied with information from a variety of brain regions. Among these neurons are the aminergic ones whose receptors’ are expressed in the PDF neurons. The present study sheds light onto putative synaptic partners and anatomical arrangements within the neuronal clock network, especially within the AME, as such knowledge is a prerequisite to understand circadian behavior. The aminergic neurons’ conspicuous vicinity to the PDF neurons suggests synaptic communication among them. Thus, based on former anatomical studies regarding this issue detailed light microscopic studies have been performed. Double immunolabellings, analyses of the spatial relation of pre- and postsynaptic sites of the individual neuron populations with respect to each other and the identification of putative synaptic partners using GRASP reenforce the hypothesis of synaptic interactions within the AME between dopaminergic/ serotonergic neurons and the PDF neurons. To shed light on the synaptic partners I performed first steps in array tomography, as it allows terrific informative analyses of fluorescent signals on an ultrastructural level. Therefore, I tested different ways of sample preparation in order to achieve and optimize fluorescent signals on 100 nm thin tissue sections and I made overlays with electron microscopic images. Furthermore, I made assumptions about synaptic modulations within the neuronal clock network via glial cells. I detected their cell bodies in close vicinity to the AME and PDFcontaining clock neurons. It has already been shown that glial cells modulate the release of PDF from s-LNvs’ terminals within the dorsal brain. On an anatomical level this modulation appears to exist also within the AME, as synaptic contacts that involve PDF-positive dendritic terminals are embedded into glial fibers. Intriguingly, these postsynaptic PDF fibers are often VIIAbstract part of dyadic or even multiple-contact sites in opposite to prolonged presynaptic active zonesimplicating complex neuronal interactions within the AME. To unravel possible mechanisms of such synaptic arrangements, I tried to localize the ABC transporter White. Its presence within glial cells would indicate a recycling mechanism of transmitted amines which allows their fast re-provision. Taken together, synapses accompanied by glial cells appear to be a common arrangement within the AME to regulate circadian behavior. The complexity of mechanisms that contribute in modulation of circadian information is reflected by the complex diversity of synaptic arrangements that involves obviously several types of neuron populations
Cataglyphis ants are famous for their navigational abilities. They live in hostile habitats where they forage as solitary scavengers covering distances of more than hundred thousand times their body lengths. To return to their nest with a prey item – mainly other dead insects that did not survive the heat – Cataglyphis ants constantly keep track of their directions and distances travelled. The navigational strategy is called path integration, and it enables an ant to return to the nest in a straight line using its home vector. Cataglyphis ants mainly rely on celestial compass cues, like the position of the sun or the UV polarization pattern, to determine directions, and they use an idiothetic step counter and optic flow to measure distances. In addition, they acquire information about visual, olfactory and tactile landmarks, and the wind direction to increase their chances of returning to the nest safe and sound. Cataglyphis’ navigational performance becomes even more impressive if one considers their life style. Most time of their lives, the ants stay underground and perform tasks within the colony. When they start their foraging careers outside the nest, they have to calibrate their compass systems and acquire all information necessary for navigation during subsequent foraging. This navigational toolkit is not instantaneously available, but has to be filled with experience. For that reason, Cataglyphis ants perform a striking behavior for up to three days before actually foraging. These so-called learning walks are crucial for the success as foragers later on. In the present thesis, both the ontogeny and the fine-structure of learning walks has been investigated. Here I show with displacement experiments that Cataglyphis ants need enough space and enough time to perform learning walks. Spatially restricted novices, i. e. naïve ants, could not find back to the nest when tested as foragers later on. Furthermore, ants have to perform several learning walks over 1-3 days to gain landmark information for successful homing as foragers. An increasing number of feeder visits also increases the importance of landmark information, whereas in the beginning ants fully rely on their path-integration vector. Learning walks are well-structured. High-speed video analysis revealed that Cataglyphis ants include species-specific rotational elements in their learning walks. Greek Cataglyphis ants (C. noda and C. aenescens) inhabiting a cluttered pine forest perform voltes, small walked circles, and pirouettes, tight turns about the body axis with frequent stopping phases. During the longest stopping phases, the ants gaze back to their nest entrance. The Tunisian Cataglyphis fortis ants inhabiting featureless saltpans only perform voltes without directed gazes. The function of voltes has not yet been revealed. In contrast, the fine structure of pirouettes suggests that the ants take snapshots of the panorama towards their homing direction to memorize the nest’s surroundings. The most likely hypothesis was that Cataglyphis ants align the gaze directions using their path integrator, which gets directional input from celestial cues during foraging. To test this hypothesis, a manipulation experiment was performed changing the celestial cues above the nest entrance (no sun, no natural polarization pattern, no UV light). The accurately directed gazes to the nest entrance offer an easily quantifiable readout suitable to ask the ants where they expect their nest entrance. Unexpectedly, all novices performing learning walks under artificial sky conditions looked back to the nest entrance. This was especially surprising, because neuronal changes in the mushroom bodies and the central complex receiving visual input could only be induced with the natural sky when comparing test animals with interior workers. The behavioral findings indicated that Cataglyphis ants use another directional reference system to align their gaze directions during the longest stopping phases of learning walk pirouettes. One possibility was the earth’s magnetic field. Indeed, already disarraying the geomagnetic field at the nest entrance with an electromagnetic flat coil indicated that the ants use magnetic information to align their looks back to the nest entrance. To investigate this finding further, ants were confronted with a controlled magnetic field using a Helmholtz coil. Elimination of the horizontal field component led to undirected gaze directions like the disarray did. Rotating the magnetic field about 90°, 180° or -90° shifted the ants’ gaze directions in a predictable manner. Therefore, the earth’s magnetic field is a necessary and sufficient reference system for aligning nest-centered gazes during learning-walk pirouettes. Whether it is additionally used for other navigational purposes, e. g. for calibrating the solar ephemeris, remains to be tested. Maybe the voltes performed by all Cataglyphis ant species investigated so far can help to answer this question..
All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism.
The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons.
To further study the functional hierarchy within the clock network and to monitor the “ticking clock“ over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.
A fundamental question in current biology concerns the translational mechanisms leading from genetic variability to phenotypes. Technologies have evolved to the extent that they can efficiently and economically determine an individual’s genomic composition, while at the same time big data on clinical profiles and diagnostics have substantially accumulated. Genome-wide association studies linking genomic loci to certain traits, however, remain limited in their capacity to explain the cellular mechanisms that underlie the given association. For most associations, gene expression has been blamed; yet given that transcript and protein abundance oftentimes do not correlate, that finding does not necessarily decrypt the underlying mechanism. Thus, the integration of further information is crucial to establish a model that could prove more accurate in predicting genotypic effects on the human organism.
In this work we describe the so-called proteotype as a feature of the cell that could provide a substantial link between genotype and phenotype. Rather than looking at the proteome as a set of independent molecules, we demonstrate a consistent modular architecture of the proteome that is driven by molecular cooperativity. Functional modules, especially protein complexes, can be further interrogated for differences between individuals and tackled as imprints of genetic and environmental variability. We also show that subtle stoichiometric changes of protein modules could have broader effects on the cellular system, such as the transport of specific molecular cargos.
The presented work also delineates to what extent temporal events and processes influence the stoichiometry of protein complexes and functional modules. The re-wiring of the glycolytic pathway for example is illustrated as a potential cause for an increased Warburg effect during the ageing of the human bone marrow. On top of analyzing protein abundances we also interrogate proteome dynamics in terms of stability and solubility transitions during the short temporal progression of the cell cycle. One of our main observations in the thesis encompass the delineation of protein complexes into respective sub-complexes according to distinct stability patterns during the cell cycle. This has never been demonstrated before, and is functionally relevant for our understanding of the dis- and assembly of large protein modules.
The insights presented in this work imply that the proteome is more than the sum of its parts, and primarily driven by variability in entire protein ensembles and their cooperative nature. Analyzing protein complexes and functional modules as molecular reflections of genetic and environmental variations could indeed prove to be a stepping stone in closing the gap between genotype and phenotype and customizing clinical treatments in the future.
Die verfügbaren in vitro Genotoxizitätstests weisen hinsichtlich ihrer Spezifität und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschränkungen auf. Um diese Mängel zu überwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Prüfung auf Genotoxizität in der Arzneimittelentwicklung beitragen.
1. Etablierung und Bewertung einer neuen in vitro Genotoxizitätsmethode (MultiFlow Methode)
Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbrüche), phosphorylierten H3 (S10) (mitotische Zellen), nukleären Protein p53 (Genotoxizität) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und ergänzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre Fähigkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen“ gegenüber der „alten“ Methode führte zu einer verbesserten Sensitivität von 95% gegenüber 90%, Spezifität von 90% gegenüber 72% und einer MoA-Klassifizierungsrate von 85% gegenüber 45% (Aneugen vs. Klastogen).
2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen
Die Analyse 67 ausgewählter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen können. Die Kombination der höchstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 ermöglichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivität, Spezifität und Prädiktivität von 86%, 83% und 85%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizität mit TP53I3, Klastogenität mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann.
Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen für BubR1 (S670) und pH3 (S28) mit Aneugenität, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenität, p53 (K373) mit Genotoxizität und Nrf2 (S40) mit oxidativem Stress identifiziert.
Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinveränderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivitäten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden können und daher eine bessere Risikobewertung von Wirkstoffkandidaten ermöglichen.