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On the existence of arrested transcriptional machinery in late stages of avian erythropoiesis
(1976)
No abstract available
Computer Science approaches (software, database, management systems) are powerful tools to boost research. Here they are applied to metabolic modelling in infections as well as health care management. Starting from a comparative analysis this thesis shows own steps and examples towards improvement in metabolic modelling software and health data management. In section 2, new experimental data on metabolites and enzymes induce high interest in metabolic modelling including metabolic flux calculations. Data analysis of metabolites, calculation of metabolic fluxes, pathways and their condition-specific strengths is now possible by an advantageous combination of specific software. How can available software for metabolic modelling be improved from a computational point of view? A number of available and well established software solutions are first discussed individually. This includes information on software origin, capabilities, development and used methodology. Performance information is obtained for the compared software using provided example data sets. A feature based comparison shows limitations and advantages of the compared software for specific tasks in metabolic modeling. Often found limitations include third party software dependence, no comprehensive database management and no standard format for data input and output. Graphical visualization can be improved for complex data visualization and at the web based graphical interface. Other areas for development are platform independency, product line architecture, data standardization, open source movement and new methodologies. The comparison shows clearly space for further software application development including steps towards an optimal user friendly graphical user interface, platform independence, database management system and third party independence especially in the case of desktop applications. The found limitations are not limited to the software compared and are of course also actively tackled in some of the most recent developments. Other improvements should aim at generality and standard data input formats, improved visualization of not only the input data set but also analyzed results. We hope, with the implementation of these suggestions, metabolic software applications will become more professional, cheap, reliable and attractive for the user. Nevertheless, keeping these inherent limitations in mind, we are confident that the tools compared can be recommended for metabolic modeling for instance to model metabolic fluxes in bacteria or metabolic data analysis and studies in infection biology. ...
This article is about a measurement analysis based approach to help software practitioners in managing the additional level complexities and variabilities in software product line applications. The architecture of the proposed approach i.e. ZAC is designed and implemented to perform preprocessesed source code analysis, calculate traditional and product line metrics and visualize results in two and three dimensional diagrams. Experiments using real time data sets are performed which concluded with the results that the ZAC can be very helpful for the software practitioners in understanding the overall structure and complexity of product line applications. Moreover the obtained results prove strong positive correlation between calculated traditional and product line measures.
Dynamic interactions and their changes are at the forefront of current research in bioinformatics and systems biology. This thesis focusses on two particular dynamic aspects of cellular adaptation: miRNA and metabolites.
miRNAs have an established role in hematopoiesis and megakaryocytopoiesis, and platelet miRNAs have potential as tools for understanding basic mechanisms of platelet function. The thesis highlights the possible role of miRNAs in regulating protein translation in platelet lifespan with relevance to platelet apoptosis and identifying involved pathways and potential key regulatory molecules. Furthermore, corresponding miRNA/target mRNAs in murine platelets are identified. Moreover, key miRNAs involved in aortic aneurysm are predicted by similar techniques. The clinical relevance of miRNAs as biomarkers, targets, resulting later translational therapeutics, and tissue specific restrictors of genes expression in cardiovascular diseases is also discussed.
In a second part of thesis we highlight the importance of scientific software solution development in metabolic modelling and how it can be helpful in bioinformatics tool development along with software feature analysis such as performed on metabolic flux analysis applications. We proposed the “Butterfly” approach to implement efficiently scientific software programming. Using this approach, software applications were developed for quantitative Metabolic Flux Analysis and efficient Mass Isotopomer Distribution Analysis (MIDA) in metabolic modelling as well as for data management. “LS-MIDA” allows easy and efficient MIDA analysis and, with a more powerful algorithm and database, the software “Isotopo” allows efficient analysis of metabolic flows, for instance in pathogenic bacteria (Salmonella, Listeria). All three approaches have been published (see Appendices).
Embryonale Stammzellen (ESCs) sind durch zwei charakteristische Eigenschaften definiert. Neben einer kontinuierlichen Selbsterneuerungskapazität weisen ESCs die Fähigkeit auf, in alle Zelltypen der drei Keimblätter differenzieren zu können. Diese Eigenschaften werden unter anderem durch ein Netzwerk wichtiger Pluripotenzfaktoren als auch durch epigenetische Mechanismen reguliert, welche die Transkription von Pluripotenz- und Differenzierungsgenen kontrollieren.
In murinen ESCs sind an der Repression von Differenzierungsgenen auch Polycomb group (PcG) Proteine beteiligt. Diese Proteine bauen zwei Chromatin-modifizierende Komplexe auf, die als Polycomb repressive complex 1 bzw. 2 (PRC1 bzw. PRC2) bezeichnet werden. Nach dem klassischen Modell der Polycombfunktion, katalysieren PRC1 und PRC2 gemeinsam zwei charakteristische Histonmodifikationen, die zur Repression PRC-spezifischer Zielgene beitragen. Zahlreiche Studien in den letzten Jahren belegen, dass der Proteinaufbau der PRC1 Komplexe stark variieren kann, wobei die Familie der Polycomb group RING finger (Pcgf) Proteine eine wichtige Rolle spielt. In diesem Zusammenhang definieren einzelne Pcgf Paraloge (Pcgf1 – 6) verschiedene PRC1 Varianten (PRC1.1 – 1.6), die Komplex-spezifische Bindestellen im Genom aufweisen. Diese Erkenntnisse lassen auf unterschiedliche Mechanismen der PRC1 Varianten und Pcgf Paralog-spezifische Funktionen schließen, die zum jetzigen Zeitpunkt nur wenig erforscht sind.
Für manche Pcgf Paraloge sind wichtige Rollen in verschiedenen Stammzelltypen und während der iPS Reprogrammierung bekannt. Pcgf1 (Nspc1), Pcgf2 (Mel18) und Pcgf4 (Bmi1) zeigen eine Funktion in verschiedenen adulten Stammzellen. Pcgf4 spielt darüber hinaus eine wichtige Rolle in der murinen iPS Reprogrammierung. Für Pcgf6 (Mblr) wird eine Pluripotenz-assoziierte Funktion angenommen, denn Pcgf6 ist das einzige Pcgf Paralog, das eine erhöhte Expression in murinen ESCs aufweist, die jedoch im Verlauf der ESC-Differenzierung absinkt. Außerdem zeigen murine Pcgf6 KD ESCs eine verminderte Expression der Pluripotenzgene Oct4, Sox2 und Nanog, eine De-Repression mesodermaler und Testes-spezifischer Gene als auch eine erhöhte Tendenz zur hämatopoetischen Differenzierung. Wie genau Pcgf6 an der Regulation dieser Prozesse in murinen ESCs beteiligt ist, ist nicht bekannt.
In der hier vorliegenden Dissertation wurde die Funktion von Pcgf6 in der murinen iPS Reprogrammierung untersucht. Da bereits für Pcgf4 eine Rolle in der Reprogrammierung somatischer Zellen gezeigt wurde und Pcgf6 eine erhöhte Expression in ESCs aufweist, wurde auch für Pcgf6 eine Funktion in der iPS Reprogrammierung angenommen. Zunächst konnte in dieser Arbeit gezeigt werden, dass Pcgf6 während der iPS Reprogrammierung verstärkt exprimiert wird und in iPS Zellen eine ESC-ähnliche Expression aufweist. Darüber hinaus konnte Pcgf6 in Kombination mit Oct4, Klf4 und c-Myc spezifisch den Transkriptionsfaktor Sox2 in der iPS Reprogrammierung ersetzen. Zudem wurden für OPKM-induzierte iPS Zellen charakteristische Eigenschaften pluripotenter Zellen nachgewiesen. Außerdem konnte eine Rolle von Pcgf6 als Enhancer-Faktor für die iPS Reprogrammierung ausgeschlossen werden, da die Überexpression von Pcgf6 zusammen mit den OSKM Faktoren keine additiven Effekte auf die Reprogrammierungseffizienz erzielte. Im Gegensatz dazu führte der Knockdown (KD) von Pcgf6 in embryonalen Mausfibroblasten (MEFs) zu verminderten Effizienzen nach OSKM Reprogrammierung. Darüber hinaus handelte es sich bei der Mehrheit der AP+ Kolonien, die unter Pcgf6 KD Konditionen entstanden, um partiell-reprogrammierte iPS Zellen.
Zusammengefasst zeigen die Ergebnisse der hier vorliegenden Arbeit, dass Pcgf6 ein neuer und essentieller Faktor der iPS Reprogrammierung ist, der in Kombination mit Oct4, Klf4 und c-Myc spezifisch den Transkriptionsfaktor Sox2 ersetzen kann.
Die molekularen Mechanismen der Wirt-Parasit-Interaktion bei der durch den Zestoden Echinococcus multilocularis ausgelösten Erkrankung der alveolären Echinokokkose sind bislang ungeklärt. Zudem liegen keine Daten über Entwicklungs- und Differenzierungsmechanismen dieses Parasiten vor, die für die Entwicklung neuer Antiparasitika genutzt werden könnten. Ein bei der Evolution der Metazoen bereits frühzeitig entstandener Signaltransduktionsmechanismus zur Steuerung von Entwicklungsvorgängen ist das TGFβ/BMP-System, das aus strukturell verwandten Zytokinen der TGFβ (transforming growth factor β) bzw. BMP (bone morphogenetic protein)-Familie, oberflächenständigen Rezeptoren der TGFβ-Rezeptorfamilie (Typ I und Typ II) und intrazellulären Signaltransduktoren der Smad-Familie besteht. Außer an Entwicklungsvorgängen tierischer Organismen könnte diesem System eine wichtige Rolle bei der Wirt-Helminth-Kommunikation während Infektionsprozessen zukommen, wie in vorherigen Studien am Nematoden Brugia malayi und am Trematoden Schistosoma mansoni gezeigt werden konnte. Erste, wichtige Schritte zur Charakterisierung von TGFβ und BMP-Signalsystemen in Zestoden wurden in der vorliegenden Arbeit getan. Aufbauend auf einem vorherigen Bericht zu einem Transmembranrezeptor (EmRSK1) und einem Smad-Homologen (EmSmadA) aus Echinococcus multilocularis wurde die Liste der TGFβ/BMP Signaltransduktionsfaktoren in E. multilocularis in dieser Arbeit deutlich erweitert und erstmals umfangreiche funktionelle Studien durchgeführt. Die hier charakterisierten Faktoren umfassen zwei weitere Serin/Threonin-Kinasen der TGFβ/BMP-Rezeptorfamilie (EmRSK2, EmRSK3) sowie intrazelluläre Transduktoren der R-Smad-Subfamilie (EmSmadB, EmSmadC) und ein Homologes zur MAP-kinase-kinase-kinase TAK1 (TGFβ activated kinase 1), genannt EmTAK1. Zudem konnte erstmals für einen parasitären Helminthen ein Zytokin der BMP-Subfamilie, EmBMP, auf molekularer Ebene charakterisiert werden. Strukturelle und funktionelle Untersuchungen legen nahe, dass E. multilocularis sowohl ein TGFβ wie auch ein BMP-Signalsystem exprimiert. Ersteres wird sehr wahrscheinlich durch die Kinase EmRSK2 und den Smad-Faktor EmSmadC gebildet, letzteres durch EmRSK1 und EmSmadB. EmSmadA nimmt eine Sonderstellung ein, da es sowohl durch TGFβ- wie auch durch BMP-Rezeptoren aktiviert werden kann. Die genaue Rolle von EmRSK1 und EmTAK1 wäre durch weitere Untersuchungen zu klären. Signifikante funktionelle Homologien zwischen den TGFβ/BMP-Signalsystemen des Parasiten und Säugern konnten nachgewiesen werden, die sich u.a. darin äußern, dass die Echinococcus Smad-Proteine durch entsprechende Rezeptoren des Menschen aktiviert werden können. Darüber hinaus konnten jedoch auch einige deutliche Unterschiede zwischen den Systemen aus Parasit und Wirt nachgewiesen werden, die sich als Angriffspunkte zur Entwicklung von Chemotherapeutika eignen könnten. So fehlt den Smad-Faktoren EmSmadA und EmSmadC eine MH1-Domäne, die sonst unter allen R-Smads hoch konserviert ist. Zudem sind einige bislang noch nie beschriebene, strukturelle Besonderheiten der Echinococcus TGFβ/BMP-Rezeptoren zu verzeichnen. Auch die Regulation dieser Faktoren und die Kreuz-Interaktion mit weiteren intrazellulären Signalwegen (z.B. der MAP Kinase Kaskade) scheint in E. multilocularis anders zu verlaufen als bislang für Vertebraten, Insekten oder Nematoden beschrieben. Schließlich konnte, als sehr wichtiger Befund, auch nachgewiesen werden dass mindestens ein Rezeptor des Parasiten, EmRSK1, mit einem Zytokin des Wirts (BMP2) in vitro funktionell interagiert. Da BMP2 in Zellkultursystemen, die das Wachstum des Parasiten am befallenen Wirtsorgan nachstellen, einen deutlichen Effekt auf E. multilocularis ausübt, könnte die hier beschriebene EmRSK1/BMP2 – Interaktion von entscheidender Bedeutung für die Wirt-Parasit-Interaktion bei der alveolären Echinokokkose sein.
During infection, bacteria need to adapt to a changing environment and have to endure various stress conditions. Small non-coding RNAs are considered as important regulators of bacterial gene expression and so allow quick adaptations by altering expression of specific target genes. Regulation of gene expression in the human-restricted pathogen Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhoea, is only poorly understood. The present study aims a better understanding of gene regulation in N. gonorrhoeae by studying small non-coding RNAs.
The discovery of antisense RNAs for all opa genes led to the hypothesis of asRNA-mediated degradation of out-of-frame opa transcripts. Analysis of asRNA expression revealed a very low abundance of the transcripts and inclusion of another phase-variable gene in the study indicates that the asRNAs are not involved in degradation of out-of-frame transcripts.
This doctoral thesis focuses on the analysis of trans-acting sRNAs. The sibling sRNAs NgncR_162 and NgncR_163 were discovered as post-transcriptional regulators altering expression of genes involved in metabolic processes, amino acid uptake and transcriptional regulation. A more detailed analysis by in silico and transcriptomic approaches showed that the sRNAs regulate a broad variety of genes coding for proteins of central metabolism, amino acid biosynthesis and degradation and several transport processes. Expression levels of the sibling sRNAs depend on the growth phase of the bacteria and on the growth medium. This indicates that NgncR_162 and NgncR_163 are involved in the adaptation of the gonococcal metabolism to specific growth conditions.
This work further initiates characterisation of the sRNA NgncR_237. An in silico analysis showed details on sequence conservation and a possible secondary structure. A combination of in silico target prediction and differential RNA sequencing resulted in the identification of several target genes involved in type IV pilus biogenesis and DNA recombination. However, it was not successful to find induction conditions for sRNA expression. Interestingly, a possible sibling sRNA could be identified that shares the target interaction sequence with NgncR_237 and could therefore target the same mRNAs.
In conclusion, this thesis provides further insights in gene regulation by non-coding RNAs in N. gonorrhoeae by analysing two pairs of sibling sRNAs modulating bacterial metabolism or possibly type IV pilus biogenesis.
Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.
Eye structure, activity rhythms, and visually-driven behavior are tuned to visual niche in ants
(2014)
Insects have evolved physiological adaptations and behavioral strategies that allow them to cope with a broad spectrum of environmental challenges and contribute to their evolutionary success. Visual performance plays a key role in this success. Correlates between life style and eye organization have been reported in various insect species. Yet, if and how visual ecology translates effectively into different visual discrimination and learning capabilities has been less explored. Here we report results from optical and behavioral analyses performed in two sympatric ant species, Formica cunicularia and Camponotus aethiops. We show that the former are diurnal while the latter are cathemeral. Accordingly, F. cunicularia workers present compound eyes with higher resolution, while C. aethiops workers exhibit eyes with lower resolution but higher sensitivity. The discrimination and learning of visual stimuli differs significantly between these species in controlled dual-choice experiments: discrimination learning of small-field visual stimuli is achieved by F. cunicularia but not by C. aethiops, while both species master the discrimination of large-field visual stimuli. Our work thus provides a paradigmatic example about how timing of foraging activities and visual environment match the organization of compound eyes and visually-driven behavior. This correspondence underlines the relevance of an ecological/evolutionary framework for analyses in behavioral neuroscience.
Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte\(^®\)S3.
Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte\(^®\)S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.
Past experience contributes to behavioural organization mainly via learning: Animals learn otherwise ordinary cues as predictors for biologically significant events. This thesis studies such predictive, associative learning, using the fruit fly Drosophila melanogaster. I ask two main questions, which complement each other: One deals with the processing of those cues that are to be learned as predictors for an important event; the other one deals with the processing of the important event itself, which is to be predicted. Do fruit flies learn about combinations of olfactory and visual cues? I probe larval as well as adult fruit flies for the learning about combinations of olfactory and visual cues, using a so called ‘biconditional discrimination’ task: During training, one odour is paired with reinforcement only in light, but not in darkness; the other odour in turn is reinforced only in darkness, but not in light. Thus, neither the odours nor the visual conditions alone predict reinforcement, only combinations of both do. I find no evidence that either larval or adult fruit flies were to solve such task, speaking against a cross-talk between olfactory and visual modalities. Previous studies however suggest such cross-talk. To reconcile these results, I suggest classifying different kinds of interaction between sensory modalities, according to their site along the sensory-motor continuum: I consider an interaction ‘truly’ cross-modal, if it is between the specific features of the stimuli. I consider an interaction ’amodal’ if it instead engages the behavioural tendencies or ‘values’ elicited by each stimulus. Such reasoning brings me to conclude that different behavioural tasks require different kinds of interaction between sensory modalities; whether a given kind of interaction will be found depends on the neuronal infrastructure, which is a function of the species and the developmental stage. Predictive learning of pain-relief in fruit flies Fruit flies build two opposing kinds of memory, based on an experience with electric shock: Those odours that precede shock during training are learned as predictors for punishment and are subsequently avoided; those odours that follow shock during training on the other hand are learned as signals for relief and are subsequently approached. I focus on such relief learning. I start with a detailed parametric analysis of relief learning, testing for reproducibility as well as effects of gender, repetition of training, odour identity, odour concentration and shock intensity. I also characterize how relief memories, once formed, decay. In addition, concerning the psychological mechanisms of relief learning, first, I show that relief learning establishes genuinely associative conditioned approach behaviour and second, I report that it is most likely not mediated by context associations. These results enable the following neurobiological analysis of relief learning; further, they will form in the future the basis for a mathematical model; finally, they will guide the researchers aiming at uncovering relief learning in other experimental systems. Next, I embark upon neurogenetic analysis of relief learning. First, I report that fruit flies mutant for the so called white gene build overall more ‘negative’ memories about an experience with electric shock. That is, in the white mutants, learning about the painful onset of shock is enhanced, whereas learning about the relieving offset of shock is diminished. As they are coherently affected, these two kinds of learning should be in a balance. The molecular mechanism of the effect of white on this balance remains unresolved. Finally, as a first step towards a neuronal circuit analysis of relief learning, I compare it to reward learning and punishment learning. I find that relief learning is distinct from both in terms of the requirement for biogenic amine signaling: Reward and punishment are respectively signalled by octopamine and dopamine, for relief learning, either of these seem dispensible. Further, I find no evidence for roles for two other biogenic amines, tyramine and serotonin in relief learning. Based on these findings I give directions for further research.
The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development.
Folliculin Controls the Intracellular Survival and Trans-Epithelial Passage of Neisseria gonorrhoeae
(2020)
Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases. Emerging multi-antibiotic resistant gonococci and increasing numbers of infections complicate the treatment of infected patients. Here, we used an shRNA library screen and next-generation sequencing to identify factors involved in epithelial cell infection. Folliculin (FLCN), a 64 kDa protein with a tumor repressor function was identified as a novel host factor important for N. gonorrhoeae survival after uptake. We further determined that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for its survival in the cells by modulating autophagy. In addition, FLCN was also required to maintain cell to cell contacts in the epithelial layer. In an infection model with polarized cells, FLCN inhibited the polarized localization of E-cadherin and the transcytosis of gonococci across polarized epithelial cells. In conclusion, we demonstrate here the connection between FLCN and bacterial infection and in particular the role of FLCN in the intracellular survival and transcytosis of gonococci across polarized epithelial cell layers.
Metabolic adaptation to the host cell is important for obligate intracellular pathogens such as Chlamydia trachomatis (Ct). Here we infer the flux differences for Ct from proteome and qRT-PCR data by comprehensive pathway modeling. We compare the comparatively inert infectious elementary body (EB) and the active replicative reticulate body (RB) systematically using a genome-scale metabolic model with 321 metabolites and 277 reactions. This did yield 84 extreme pathways based on a published proteomics dataset at three different time points of infection. Validation of predictions was done by quantitative RT-PCR of enzyme mRNA expression at three time points. Ct’s major active pathways are glycolysis, gluconeogenesis, glycerol-phospholipid (GPL) biosynthesis (support from host acetyl-CoA) and pentose phosphate pathway (PPP), while its incomplete TCA and fatty acid biosynthesis are less active. The modeled metabolic pathways are much more active in RB than in EB. Our in silico model suggests that EB and RB utilize folate to generate NAD(P)H using independent pathways. The only low metabolic flux inferred for EB involves mainly carbohydrate metabolism. RB utilizes energy -rich compounds to generate ATP in nucleic acid metabolism. Validation data for the modeling include proteomics experiments (model basis) as well as qRT-PCR confirmation of selected metabolic enzyme mRNA expression differences. The metabolic modeling is made fully available here. Its detailed insights and models on Ct metabolic adaptations during infection are a useful modeling basis for future studies.
Background: Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes).
Methodology and Principal Findings: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1 similar to MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by similar to 12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo.
Conclusions: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.
Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling
(2016)
In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.
Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. Here, we dissect the function of the key regulator of serotonin signaling—the serotonin transporter–in innate olfactory attraction to ethanol in Drosophila melanogaster. We generated a mutated version of the serotonin transporter that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. We identified four serotonergic neurons that inhibit the olfactory attraction to ethanol and two additional neurons that counteract this inhibition by strengthening olfactory information. Our results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior.
In contrast to c-Myc, a deregulated expression of the MYCN gene is restricted to human neuroendocrine tumours. In most cases, the excessive activity of N-Myc results from a MYCN amplification. In neuroblastoma, amplification of MYCN is a predictor of poor prognosis and resistance to therapy. The inability to target the N-Myc protein directly necessitates the search for alternative targets. This project aimed at identifying genes specifically required for growth and survival of cells that express high levels of N-Myc using high-throughput shRNA screening combined with next generation sequencing. The identification and analysis of these genes will shed light on functional interaction partners of N-Myc.
We screened a shRNA library containing 18,327 shRNAs and identified 148 shRNAs, which were selectively depleted in the presence of active N-Myc. In addition, shRNAs targeting genes that are involved in p53 and ARF turnover and apoptosis were depleted in the cell population during the screen. These processes are known to affect N-Myc-mediated apoptosis. Consequently, these results biologically validated the screen. The 148 shRNAs that showed a significant synthetic lethal interaction with high levels of N-Myc expression were further analysed using the bioinformatics program DAVID. We found an enrichment of shRNAs that target genes involved in specific biological processes. For example, we validated synthetic lethal interactions for genes such as, THOC1, NUP153 and LARP7, which play an important role in the process of RNA polymerase II-mediated transcription elongation. We also validated genes that are involved in the neddylation pathway.
In the screen we identified Cullin 3, which is a component of the BTB-CUL3-Rbx1 ubiquitin ligase that is involved in the turnover of Cyclin E. Depletion of cullin 3 and activation of N-Myc was found to synergistically increase Cyclin E expression to supraphysiological levels, inducing S-phase arrest and a strong DNA damage response.
Together with results from a proteomics analysis of N-Myc associated proteins, our results lead us to the following hypothesis: In a neuroblastoma cell, the high levels of N-Myc result in a conflict between RNA polymerase II and the replication machinery during S-phase. The newly identified interaction partners of N- Myc are required to solve this conflict. Consequently, loss of the interaction leads to a massive DNA damage and the induction of apoptosis. In addition, inhibition or depletion of the essential components of the neddylation pathway also results in an unresolvable problem during S-phase.
Neisseria gonorrhoeae is a human-specific pathogen that causes gonorrhea. It is defined as a super bacterium by the WHO due to the emergence of gonococci that are resistant to a variety of antibiotics and a rapidly increasing infection incidence. Genome-wide investigation of neisserial gene essentiality and novel virulence factors is urgently required in order to identify new targets for anti-neisserial therapeutics. To identify essential genes and new virulence factors, a high-density mutant library in N. gonorrhoeae MS11 was generated by in vitro transposon mutagenesis. The transposon library harbors more than 100,000 individual mutants, a density that is unprecedented in gonococcal research. Essential genes in N. gonorrhoeae were determined by enumerating frequencies of transposon insertion sites (TIS) with Illumina deep sequencing (Tn-seq). Tn-seq indicated an average distance between adjacent TIS of 25 bp. Statistical analysis unequivocally demonstrated 781 genes that were significantly depleted in TIS and thus are essential for Neisseria survival. A subset of the genes was experimentally verified to comprise essential genes and thus support the outcome of the study. The hereby identified candidate essential genes thus may constitute excellent targets for the development of new antibiotics or vaccines.
In a second study, the transposon mutant library was applied in a genome-scale “negative-selection strategy” to identify genes that are involved in low phosphate-dependent invasion (LPDI). LPDI is dependent on the Neisseria porin subtype PorBIA which acts as an epithelial cell invasin in absence of phosphate and is associated with severe pathogenicity in disseminated gonococcal infections (DGI). Tn-seq demonstrated 98 genes, which were involved in adherence to host cells and 43 genes involved in host cell invasion. E.g. the hypothetical protein NGFG_00506, an ABC transporter ATP-binding protein NGFG_01643, as well as NGFG_04218 encoding a homolog of mafI in N. gonorrhoeae FA1090 were experimentally verified as new invasive factors in LPDI. NGFG_01605, a predicted protease, was identified to be a common factor involved in PorBIA, Opa50 and Opa57-mediated neisserial engulfment by the epithelial cells. Thus, this first systematic Tn-seq application in N. gonorrhoeae identified a set of previously unknown N. gonorrhoeae invasive factors which demonstrate molecular mechanisms of DGI.
In der vorliegenden Arbeit wurden die Mechanismen der Hochdosis-GC-Pulstherapie im Zusammenhang mit akuten Schüben von MS-Patienten anhand des Tiermodells der MS, der Experimentellen Autoimmunen Enzephalomyelitis (EAE), untersucht. Die EAE wurde in C57Bl/6 Mäusen und diversen GR-defizienten Mäusen durch Immunisierung mit Myelinoligodendrozytenglykoprotein (MOG35-55) induziert. Es konnte gezeigt werden, dass die Gabe von Dexamethason (Dex) den Krankheitsverlauf dosisabhängig verbessert. Die Untersuchung heterozygoter GR Knock-out Mäuse und hämatopoetischer Stammzellchimären verdeutlichte, dass der zytosolische GR (cGR) für die Vermittlung therapeutischer GC-Effekte von sehr großer Bedeutung ist. Der Einsatz zelltyp-spezifischer GR-defizienter Mäuse zeigte auf zellulärer Ebene, dass für die Vermittlung von GC-Wirkungen die Expression des GR vor allem in T-Zellen unabdingbar ist, wohingegen die GR-Expression in myeloiden Zellen in diesem Kontext keine Bedeutung hat. Durch die Analyse des molekularen Mechanismus konnte festgestellt werden, dass diese Effekte durch Apoptoseinduktion und Herunterregulieren von Adhäsionsmolekülen in peripheren, aber nicht ZNS-residenten T-Zellen erzielt wurden. Überdies wurde ersichtlich, dass Dex die T-Zellmigration in das ZNS verhinderte. Diese Beobachtung unterstützt die Hypothese, dass Dex durch Apoptoseinduktion und Immunmodulation hauptsächlich auf periphere T-Zellen wirkt und somit den ständigen Influx neuer Immunzellen in das ZNS verhindert. Ferner konnte in dieser Arbeit gezeigt werden, dass die therapeutische Gabe hochdosierten Methylprednisolons (MP) in diesem EAE-Modell ebenfalls zu einer dosisabhängigen Verbesserung der EAE führte. Diese beruhte auf einer reduzierten Lymphozyteninfiltration in das ZNS, war allerdings im Vergleich zur Dex-Therapie aufgrund geringerer Wirkpotenz weniger stark ausgeprägt. Im Gegensatz dazu führte die präventive MP-Applikation zu einem verstärkten EAE-Verlauf, der nach der Beeinflussung peripherer, hämatopoetischer Immunzellen auf eine verstärkte Proliferation autoreaktiver T-Zellen zurückzuführen ist. Im weiteren Verlauf der vorliegenden Arbeit wurde als möglicher Ersatz für die Hochdosis-GC-Pulstherapie eine nicht-steroidale, antiinflammatorische Substanz im chronischen EAE-Modell der C57Bl/6 Maus etabliert. Erste tierexperimentelle Untersuchungen mit Compound A (CpdA) offenbarten eine lediglich geringe therapeutische Breite dieser Substanz, wobei innerhalb pharmakologischer Dosierungen dennoch therapeutische Wirkungen vermittelt werden konnten. Anhand von in vitro Experimenten konnte eindeutig nachgewiesen werden, dass CpdA GR-unabhängig Apoptose induzierte, wobei Immunzellen und neuronale Zellen gegenüber CpdA besonders empfindlich reagierten. Der Einsatz T-Zell-spezifischer GR-defizienter Mäuse konnte zeigen, dass CpdA für die Vermittlung therapeutischer Wirkungen den cGR benötigt. Ferner wurde offensichtlich, dass CpdA in Abwesenheit des cGR in T-Zellen eine signifikante Verschlechterung der EAE verursachte. Durch die Anwendung physikochemischer Analysenmethoden, wie der Massenspektrometrie und 1H-NMR-Spektroskopie, konnte festgestellt werden, dass CpdA in vitro in gepufferten Medien in eine zyklische, chemisch sehr reaktive Verbindung (Aziridin) metabolisiert wird. Diese kann sehr wahrscheinlich für die Apoptose-Induktion in Zellen und die in Mäusen beobachteten neurotoxischen Ausfallerscheinungen verantwortlich gemacht werden. Durch chemische Analysen konnte in vitro in wässriger CpdA-Lösung ein weiterer Metabolit, das sympathomimetisch wirksame Synephrin, identifiziert werden. Um die Wirksamkeit adrenerger Substanzen in vivo zu testen, wurde das ß1/2-Sympathomimetikum Isoproterenol appliziert. Dieses verbesserte die EAE-Symptomatik, was sehr wahrscheinlich auf eine reduzierte Antigenpräsentation und einer damit verbundenen verminderten T-Zellinfiltration in das ZNS zurückzuführen ist.
Die zellulären Rho GTPasen kontrollieren und regulieren zentrale elementare Zellvorgänge wie Phagozytose, Migration und epitheliale Integrität. Aufgrund ihrer zentralen Stellung, interagiert eine Vielzahl von bakteriellen Cytotoxinen und Modulinen mit den Rho GTPasen und wirken so als Pathogenitätsfaktoren. Die zur W-xxx-E Familie gehörenden Effektoren IpgB1 und IpgB2 von Shigella und Map von E. coli (Pathotypen EHEC und EPEC) werden über ein Typ 3 Sekretionssystem (T3SS) in Wirtszellen injiziert und wirken als Rac1, RhoA bzw. Cdc42 GEF Mimetikum. In der vorliegenden Arbeit wurden die Effektor Funktionen von IpgB1 IpgB2 und Map mit Hilfe des Yersinia (Ysc)-T3SS untersucht, was zur Etablierung der „Yersinia-Toolbox“ führte. Damit können heterologe Effektoren isoliert im physiologischen Kontext der Erreger-Zell-Interaktion zellbiologisch untersucht werden unter Vermeidung von simultaner Injektion redundanter oder unbekannter Effektoren. Zur Etablierung der Yersinia-Toolbox wurden zunächst die Gene für die Rho GTPasen modulierenden Shigella Effektoren IpgB1 und IpgB2 sowie der E. coli (EHEC)-Effektor Map mit unterschiedlich langen Gensequenzen der N-terminalen Bereiche des Yersinia-Effektorproteins YopE fusioniert (Hybridproteine: YopEi-X:i = 18, 53 bzw. 138 Aminosäurereste, X = IpgB1, IpgB2 bzw. Map). In der vorliegenden Arbeit wird gezeigt, dass die Hybridproteine YopE53-X und YopE138-X (X=IpgB1, IpgB2, Map) in den Kulturüberstand sezerniert bzw. in Zielzellen injiziert wurden. In einem weiteren Schritt konnte die zellbiologische Aktivität der heterologen Proteine fluoreszenzmikroskopisch durch Aktinzytoskelettumlagerungen gezeigt werden. So wurden „Membrane Ruffles“ (Rac1-Aktivierung) durch YopE138-IpgB1, Stressfasern (RhoA-Aktivierung) durch E138-IpgB2 und „Mikrospikes“ (Cdc42-Aktivierung) durch YopE138-Map nachgewiesen. Invasionstudien zeigten, dass YopEi-IpgB1 (i = 53, 138) die Yersinia-Invasion induzierte, wohingegen YopEi-IpgB2 die Invasionsrate der Stämme WA (pT3SS, pEi-IpgB2) (i=53, 138) verglichen mit dem Stamm WA (pT3SS) reduziert war. Durch Kombination verschiedener Yersinia-Toolbox-Stämme konnte im Co-Infektionsmodell mit HeLa-Zellen gezeigt werden, dass (1) die YopE138-IpgB1 vermittelte Invasion durch YopE138-IpgB2 signifikant inhibiert werden kann, was auf eine antagonistische Wirkung zwischen IpgB1 und IpgB2 schließen lässt, dass (2) YopT ebenfalls die IpgB1 vermittelte Invasionsrate reduziert (inhibitorische Wirkung auf Rac1), und dass (3) YopE als GAP für RhoG/Rac1 (bevorzugt RhoG) praktisch nicht die IpgB1-vermittelte Invasion hemmt. Durch Klonierung der YopE138-IpgB1 und YopE138-IpgB2 kodierenden Fusionsgene in zwei kompatible Plasmidvektoren konnten die Hybridproteine simultan transloziert werden und die Co-Infektionsergebnisse bestätigt werden. In der Literatur ist beschrieben, dass die Ysc-Translokationspore YopB/YopD Rho-abhängig Membranporen-bedingte Zellschädigungen verursacht (LDH-Freisetzung, PI-Kernfärbung). Mit der Yersinia-Toolbox konnte mit dem Stamm WA (pT3SS) Zytoplasmamembranschädigung / Zytotoxizität nachgewiesen werden, nicht aber mit den Stämmen WA (pE138-X) X = IpgB1, IpgB2 oder Map. Co-Infektionen jedoch zeigen, dass vermehrt LDH bei der Infektion mit WA (pT3SS) + WA (pT3SS, pE138-IpgB1) detektiert wurde, wohingegen dieser Effekt von YopE138-IpgB2 in einer Co-Infektion von WA (pT3SS) + WA (pT3SS, pE138-IpgB2) inhibiert wurde. Auch hier wurde der Antagonismus zwischen IpgB1 und IpgB2 erneut sichtbar. Diese Befunde widersprechen publizierten Daten, die eine RhoA-Aktivierung/Aktinpolymerisierung mit verstärkter Porenbildung in einen Zusammenhang bringen. Rho GTPasen sind beteiligt an der Erhaltung der polarisierten Eipthelzellschichtintegrität über Adhäsionskomplexbildung. Mittels Infektion von polarisierten MDCK-Zellschichten mit verschiedenen Yersinia-Stämmen und Messung des transepithelialen elektrischen Widerstandes/Resistenz (TER) konnte gezeigt werden, dass die Ysc-T3SS vermittelte Injektion von YopE138-IpgB1 (Rac1-Aktivierung) oder YopE138-Map (Cdc42-Aktivierung) zur Abnahme der TER und damit Schädigung der Zellschichtintegrität führt, wogegen bei YopE138-IpgB2-Injektion der TER-Wert unverändert blieb. Um bakterielle Rho GTPasen-modulierende Effektorproteine detailliert untersuchen zu können und um die Rolle von Rho GTPasen im Mausinfektionsmodell mit Yersinia enterocolitica und Salmonellen zu bestimmen, wurden Mäuse mit deletierten Genen für RhoA, Rac1 bzw. Cdc42 in Makrophagen hergestellt.
Quantitative community-wide moth surveys frequently employ flight-interception traps equipped with UV-light emitting sources as attractants. It has long been known that moth species differ in their responsiveness to light traps. We studied how the settling behaviour of moths at a light trap may further contribute to sampling bias. We observed the behaviour of 1426 moths at a light tower. Moths were classified as either, settling and remaining still after arrival, or continually moving on the gauze for extended periods of time. Moths that did not move after settling may not end up in the sampling container of the light trap and therefore are under-represented in automated trap samples relative to their true proportions in the community. Our analyses revealed highly significant behavioural differences between moths that differed in body size. Small moths were more likely to remain stationary after settling. As a corollary, representatives of three taxa, which in Europe are predominantly small species (Nolidae, Geometridae: Eupitheciini, Erebidae: Lithosiini), usually settled down immediately, whereas most other moths remained active on or flying around the trap for some time. Moth behaviour was also modulated by ambient temperature. At high temperatures, they were less likely to settle down immediately, but this behavioural difference was most strongly apparent among medium-sized moths. These results indicate the likely extent of the sampling bias when analysing and interpreting automated light-trap samples. Furthermore, to control for temperature modulated sampling bias temperature should always be recorded when sampling moths using flight-interception traps.
Plant diversity is known to affect success of host location by pest insects, but its effect on olfactory orientation of non-pest insect species has hardly been addressed. First, we tested in laboratory experiments the hypothesis that non-host plants, which increase odour complexity in habitats, affect the host location ability of herbivores and parasitoids. Furthermore, we recorded field data of plant diversity in addition to herbivore and parasitoid abundance at 77 grassland sites in three different regions in Germany in order to elucidate whether our laboratory results reflect the field situation. As a model system we used the herb Plantago lanceolata, the herbivorous weevil Mecinus pascuorum, and its larval parasitoid Mesopolobus incultus. The laboratory bioassays revealed that both the herbivorous weevil and its larval parasitoid can locate their host plant and host via olfactory cues even in the presence of non-host odour. In a newly established two-circle olfactometer, the weevils capability to detect host plant odour was not affected by odours from non-host plants. However, addition of non-host plant odours to host plant odour enhanced the weevils foraging activity. The parasitoid was attracted by a combination of host plant and host volatiles in both the absence and presence of non-host plant volatiles in a Y-tube olfactometer. In dual choice tests the parasitoid preferred the blend of host plant and host volatiles over its combination with non-host plant volatiles. In the field, no indication was found that high plant diversity disturbs host (plant) location by the weevil and its parasitoid. In contrast, plant diversity was positively correlated with weevil abundance, whereas parasitoid abundance was independent of plant diversity. Therefore, we conclude that weevils and parasitoids showed the sensory capacity to successfully cope with complex vegetation odours when searching for hosts.
Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of similar to 1 kW cm\(^{-2}\) at 640 nm for several minutes, the maximum dose at 405 nm is only similar to 50 J cm\(^{-2}\), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
Die Entwicklung hochauflösender Fluoreszenzmikroskopiemethoden hat die Lichtmikroskopie revolutioniert. Einerseits ermöglicht die höhere erzielte räumliche Auflösung die Abbildung von Strukturen, die deutlich unterhalb der beugungsbedingten Auflösungsgrenze liegen. Andererseits erhält man durch Einzelmoleküllokalisationsmikroskopiemethoden
wie dSTORM (Direct Stochastic Optical Reconstruction Microscopy) Informationen, welche man für quantitative Analysen heranziehen kann. Aufgrund der sich dadurch bietenden neuen Möglichkeiten, hat sich die hochauflösende Fluoreszenzmikroskopie rasant entwickelt und kommt mittlerweile zur Untersuchung einer Vielzahl biologischer und medizinischer Fragestellungen zum Einsatz. Trotz dieses Erfolgs ist jedoch nicht zu verleugnen, dass auch diese neuen Methoden ihre Nachteile haben. Dazu zählt die Notwendigkeit relativ hoher Laserleistungen, welche Voraussetzung für hohe Auflösung ist und bei lebenden Proben zur Photoschädigung führen kann.
Diese Arbeit widmet sich sowohl dem Thema der Photoschädigung durch Einzelmoleküllokalisationsmikroskopie,
als auch der Anwendung von dSTORM und SIM (Structured Illumination Microscopy) zur Untersuchung neurobiologischer Fragestellungen auf Proteinebene.
Zur Ermittlung der Photoschädigung wurden lebende Zellen unter typischen Bedingungen bestrahlt und anschließend für 20−24 h beobachtet. Als quantitatives Maß für den Grad der Photoschädigung wurde der Anteil sterbender Zellen bestimmt. Neben der zu erwartenden Intensitäts- und Wellenlängenabhängigkeit, zeigte sich, dass die Schwere der Photoschädigung auch von vielen weiteren Faktoren abhängt und dass sich Einzelmoleküllokalisationsmikroskopie bei Berücksichtigung der gewonnenen Erkenntnisse durchaus mit Lebendzellexperimenten vereinbaren lässt.
Ein weiteres Projekt diente der Untersuchung der A- und B-Typ-Glutamatrezeptoren an der neuromuskulären Synapse von Drosophila melanogaster mittels dSTORM. Dabei konnte eine veränderte Anordnung beider Rezeptortypen infolge synaptischer Plastizität beobachtet, sowie eine absolute Quantifizierung des A-Typ-Rezeptors durchgeführt werden.
Im Mittelpunkt eines dritten Projekts standen Cadherin-13 (CDH13) sowie der Glucosetransporter Typ 3 (GluT3), welche beide mit der Aufmerksamkeitsdefizit-Hyperaktivitätsstörung in Verbindung gebracht werden. CDH13 konnte mittels SIM in serotonergen Neuronen, sowie radiären Gliazellen der dorsalen Raphekerne des embryonalen Mausgehirns nachgewiesen werden. Die Rolle von GluT3 wurde in aus induzierten pluripotenten Stammzellen differenzierten Neuronen analysiert, welche verschiedene Kopienzahlvariation des für GluT3-codierenden SLC2A3-Gens aufwiesen. Die Proteine GluT3, Bassoon und Homer wurden mittels dSTORM relativ quantifiziert. Während die Deletion des Gens zu einer erwartenden Verminderung von GluT3 auf Proteinebene führte, hatte die Duplikation keinen Effekt auf die GluT3-Menge. Für Bassoon und Homer zeigte sich weder durch die Deletion noch die Duplikation eine signifikante Veränderung.
Pocket-Proteine und E2F-Transkriptionsfaktoren regulieren die Expression von Zellzyklus-assoziierten Genen und spielen eine zentrale Rolle bei der Koordination der Zellteilung, Differenzierung und Apoptose. Störungen dieser Signalwege tragen zur Entstehung zahlreicher Tumorentitäten beim Menschen bei. Trotz der intensiven Untersuchung der Zellzyklusregulation sind viele Details noch unverstanden.
Der LIN-Komplex (LINC / DREAM) ist ein kürzlich entdeckter humaner Multiprotein-komplex, welcher dynamisch mit Pocket-Proteinen und E2F-Transkriptionsfaktoren interagiert. Eine essentielle Komponente des LIN-Komplexes ist das LIN9-Protein. Um die Funktion dieses Proteins bei der Zellzyklusregulation und Tumorentstehung genauer untersuchen zu können, wurde in unserer Arbeitsgruppe ein konditionelles Lin9-Knockout-Mausmodell etabliert.
Primäres Ziel der Arbeit war es, den Phänotyp embryonaler Fibroblasten (MEFs) aus diesen Mäusen zu charakterisieren. Bereits kurz nach Inaktivierung von Lin9 konnte ein stark verlangsamtes Zellwachstums beobachtet werden. In Lin9-depletierten MEFs wurden multiple mitotische Defekte detektiert, die u. a. strukturelle Auffälligkeiten des Spindelapparates, aberrante Zellkerne, Störungen der Chromosomensegregation sowie zytokinetische Defekte umfassen und in einer dramatischen Zunahme polyploider und aneuploider Zellen resultieren. Im Langzeitverlauf führen diese erheblichen Aberrationen zu einer vorzeitigen zellulären Seneszenz. Wird diese durch das Large T-Protoonkogen durchbrochen, können sich MEFs an den Verlust von Lin9 adaptieren, zeigen dann jedoch eine hochgradige genomische Instabilität und Substrat-unabhängiges Wachstum im Weichagar als Zeichen onkogener Transformation.
Im zweiten Abschnitt der vorliegenden Arbeit wurde die Genexpression in Lin9-defizienten MEFs mittels quantitativer Real Time-PCR untersucht um zu klären, ob die beschriebenen Defekte auf Veränderungen der transkriptionellen Aktivität zurück-zuführen sind. Dabei wurde eine erhebliche Reduktion der Expressionslevel mitotischer Gene nach Verlust von Lin9 beobachtet. Des Weiteren wurden zur Klärung der zu Grunde liegenden molekularen Mechanismen Chromatin-Immunpräzipitations-Experimente (ChIP) durchgeführt. Im Vergleich zu Kontrollzellen wurden dabei in Lin9-defizienten Zellen signifikante epigenetische Veränderungen bezüglich aktivierender Histon-Modifikationen an den Promotoren mitotischer Lin9-Zielgene festgestellt.
Im letzten Abschnitt der Arbeit sollten die Auswirkungen des heterozygoten Verlustes von Lin9 analysiert werden. Dabei zeigte sich, dass Lin9-haploinsuffiziente Zellen normal proliferieren, obwohl die Expression verschiedener G2/M-Gene leicht vermindert war. Es wurde jedoch eine Schwächung des mitotischen Spindelkontrollpunktes und in der Folge über mehrere Zellgenerationen eine Zunahme polyploider Zellen beobachtet. Mit Weichagar-Assays konnte gezeigt werden, dass bereits der heterozygote Verlust des Lin9-Gens zur onkogenen Transformation beiträgt.
Zusammengenommen dokumentieren diese Studien, dass LIN9 eine entscheidende Bedeutung bei der Regulation von Zellzyklus-assoziierten Genen spielt und sowohl einen essentiellen Faktor für die Zellproliferation darstellt als auch durch die Gewährleistung genomischer Stabilität tumorsuppressive Eigenschaften aufweist.
Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC- and UHPLC-MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2).
Ca2+-empfindliche K+-Kanäle mittlerer Leitfähigkeit (IK1-Kanäle) übernehmen wichtige Funktionen bei vielen physiologischen Prozessen wie z.B. bei der Zell-Proliferation, der epithelialen Salz- und Wasser-Sekretion und der Zellmigration. Die Kanäle werden durch die intrazeluläre Ca2+-Konzentration reguliert, wobei ihre Ca2+-Sensitivität durch Phosphorylierungsreaktionen moduliert werden kann. Ziel dieser Arbeit war die funktionelle Charakterisierung des aus transformierten Nierenepithelzellen (MDCK-F-Zellen) klonierten Ca2+-sensitiven K+-Kanals mittlerer Leitfähigkeit (cIK1) und die Untersuchung seiner Regulierung durch die Proteinkinase C (PKC). Dazu wurde der Kanal heterolog in CHO- und HEK293-Zellen exprimiert. Seine biophysikalischen und pharmakologischen Eigenschaften sowie der Einfluß der Proteinkinase C auf die Kanalaktivität wurden mit Hilfe der Patch-Clamp-Technik untersucht. Die cIK1-Ströme sind schwach einwärtsrektifizierend, zeigen keine Aktivierungs- oder Inaktivierungskinetik und weisen im physiologischen Bereich keine Spannungsabhängigkeit auf. Der cIK1 ist K+-selektiv und wird durch einen Anstieg der intrazellulären Ca2+-Konzentration aktiviert. Der Kanal wird durch Barium, Charybdotoxin und Clotrimazol blockiert und durch 1-Ethyl-2-Benzimidazolon aktiviert. Die funktionellen und pharmakologischen Eigenschaften des klonierten cIK1 entsprechen damit denen des nativen Kanals aus MDCK-F-Zellen und stimmen mit denen anderer Mitglieder der IK1-Kanalfamilie überein. Neben der Regulierung durch die intrazelluläre Ca2+-Konzentration wird der cIK1 auch durch eine PKC-abhängige Phosphorylierung reguliert. Sowohl ATP als auch ATP?S stimulieren die Kanalaktivität. Die ATP-abhängige Aktivierung wird durch Inhibitoren der Proteinkinase C (Bisindolylmaleimid, Calphostin C) gehemmt, während die mit ATP?S induzierte Kanalaktivität weitgehend resistent gegen diese PKC-Inhibitoren ist. Eine Stimulierung der Proteinkinase C mit Phorbol 12-Myristat 13-Acetat (PMA) führt zu einer sofortigen Aktivierung des cIK1. Im Gegensatz dazu sind die cIK1-Kanäle nach fast vollständigem Abbau der Proteinkinase C durch eine langfristige Inkubierung der Zellen mit PMA nicht mehr aktiv. Um zu untersuchen, ob diese Regulierung eine direkte Interaktion der Proteinkinase C mit dem Kanalprotein erfordert, wurden die drei putativen PKC-Konsensussequenzen des cIK1 mittels zielgerichteter Mutagenese so verändert, daß eine Phosphorylierung an diesen Stellen nicht mehr möglich ist. Weder die einzelne Mutation der PKC-Konsensussequenzen (T101, S178, T329) noch die gleichzeitige Mutation aller drei Phosporylierungsstellen zu Alanin beeinflußt die akute Regulierung des cIK1 durch die Proteinkinase C. Die cIK1-Mutante T329A und die Dreifachmutante reagieren jedoch nach einem Abbau der Proteinkinase C mit einem extremen Anstieg der Kanalaktivität und demaskieren damit einen zweiten Weg der Kanalregulierung. Die Ergebnisse zeigen, daß der cIK1 durch zwei voneinander unabhängige Mechanismen reguliert wird. Eine PKC-abhängige Phosphorylierung erhöht die Aktivität der Kanäle, findet jedoch nicht an den bekannten PKC-Konsensusesquenzen des Kanalproteins statt. Dagegen werden die cIK1-Kanäle über einen zweiten ATP-abhängigen Mechanismus, der wahrschenlich eine direkte Interaktion mit dem Kanalprotein erfordert, gehemmt.
Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identifed the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efciently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.
Aim of this thesis was to study the contribution of the hosts immune system during tumor regression. A wild-type rejection model was studied in which tumor regression is mediated through an adaptive, T cell host response (Research article 1). Additionally, the relationship between VACV infection and cancer rejection was assessed by applying organism-specific microarray platforms to infected and non-infected xenografts. It could be shown that tumor rejection in this nude mouse model was orchestrated solely by the hosts innate immune system without help of the adaptive immunity. In a third study the inflammatory baseline status of 75 human cancer cell lines was tested in vitro which was correlated with the susceptibility to VACV and Adenovirus 5 (Ad5) replication of the respective cell line (Manuscript for Research article 3). Although xenografts by themselves lack the ability to signal danger and do not provide sufficient proinflammatory signals to induce acute inflammation, the presence of viral replication in the oncolytic xenograft model provides the "tissue-specific trigger" that activates the immune response and in concordance with the hypothesis, the ICR is activated when chronic inflammation is switched into an acute one. Thus, in conditions in which a switch from a chronic to an acute inflammatory process can be induced by other factors like the immune-stimulation induced by the presence of a virus in the target tissue, adaptive immune responses may not be necessary and immune-mediated rejection can occur without the assistance of T or B cells. However, in the regression study using neu expressing MMC in absence of a stimulus such as a virus and infected cancer cells thereafter, adaptive immunity is needed to provoke the switch into an acute inflammation and initiate tissue rejection. Taken together, this work is supportive of the hypothesis that the mechanisms prompting TSD differ among immune pathologies but the effect phase converges and central molecules can be detected over and over every time TSD occurs. It could be shown that in presence of a trigger such as infection with VACV and functional danger signaling pathways of the infected tumor cells, innate immunity is sufficient to orchestrate rejection of manifested tumors.
How insects promote crop pollination remains poorly understood in terms of the contribution of functional trait differences between species. We used meta-analyses to test for correlations between community abundance, species richness and functional trait metrics with oilseed rape yield, a globally important crop. While overall abundance is consistently important in predicting yield, functional divergence between species traits also showed a positive correlation. This result supports the complementarity hypothesis that pollination function is maintained by non-overlapping trait distributions. In artificially constructed communities (mesocosms), species richness is positively correlated with yield, although this effect is not seen under field conditions. As traits of the dominant species do not predict yield above that attributed to the effect of abundance alone, we find no evidence in support of the mass ratio hypothesis. Management practices increasing not just pollinator abundance, but also functional divergence, could benefit oilseed rape agriculture.
Implications of Advanced Glycation Endproducts in Oxidative Stress and Neurodegenerative Disorders
(2001)
The reactions of reducing sugars with primary amino groups are the most common nonenzymatic modifications of proteins. Subsequent rearrangements, oxidations, and dehydrations yield a heterogeneous group of mostly colored and fluorescent compounds, termed "Maillard products" or advanced glycation end products (AGEs). AGE formation has been observed on long-lived proteins such as collagen, eye lens crystalline, and in pathological protein deposits in Alzheimer's (AD) and Parkinson's disease (PD) and dialysis-related amyloidosis. AGE-modified proteins are also involved in the complications of diabetes. AGEs accumulate in the the ß-amyloid plaques and neurofibrillary tangles (NFT) associated with AD and in the Lewy bodies characteristic of PD. Increasing evidence supports a role for oxidative stress in neurodegenerative disorders such as AD and PD. AGEs have been shown to contribute towards oxidative damage and chronic inflammation, whereby activated microglia secrete cytokines and free radicals, including nitric oxide (NO). Roles proposed for NO in the pathophysiology of the central nervous system are increasingly diverse and range from intercellular signaling, through necrosis of cells and invading pathogens, to the involvement of NO in apoptosis. Using in vitro experiments, it was shown that AGE-modified bovine serum albumin (BSA-AGE) and AGE-modified ß-amyloid, but not their unmodified proteins, induce NO production in N-11 murine microglia cells. This was mediated by the receptor for AGEs (RAGE) and upregulation of the inducible nitric oxide synthase (iNOS). AGE-induced enzyme activation and NO production could be blocked by intracellular-acting antioxidants: Ginkgo biloba special extract EGb 761, the estrogen derivative, 17ß-estradiol, R-(+)-thioctic acid, and a nitrone-based free radical trap, N-tert.-butyl-*-phenylnitrone (PBN). Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), common precursors in the Maillard reaction, were also tested for their ability to induce the production of NO in N-11 microglia. However, no significant changes in nitrite levels were detected in the cell culture medium. The significance of these findings was supported by in vivo immunostaining of AD brains. Single and double immunostaining of cryostat sections of normal aged and AD brains was performed with polyclonal antibodies to AGEs and iNOS and monoclonal antibodies to Aß and PHF-1 (marker for NFT) and reactive microglia. In aged normal individuals as well as early stage AD brains (i.e. no pathological findings in isocortical areas), a few astrocytes showed co-localisation of AGE and iNOS in the upper neuronal layers of the temporal (Area 22) and entorhinal (Area 28, 34) cortices compared with no astrocytes detected in young controls. In late AD brains, there was a much denser accumulation of astrocytes co-localised with AGE and iNOS in the deeper and particularly upper neuronal layers. Also, numerous neurons with diffuse AGE but not iNOS reactivity and some AGE and iNOS-positive microglia were demonstrated, compared with only a few AGE-reactive neurons and no microglia in controls. Finally, astrocytes co-localised with AGE and iNOS as well as AGE and ß-amyloid were found surrounding mature but not diffuse ß-amyloid plaques in the AD brain. Parts of NFT were AGE-immunoreactive. Immunohistochemical staining of cryostat sections of normal aged and PD brains was performed with polyclonal antibodies to AGEs. The sections were counterstained with monoclonal antibodies to neurofilament components and a-synuclein. AGEs and a-synuclein were colocalized in very early Lewy bodies in the substantia nigra of cases with incidental Lewy body disease. These results support an AGE-induced oxidative damage due to the action of free radicals, such as NO, occurring in the AD and PD brains. Furthermore, the involvement of astrocytes and microglia in this pathological process was confirmed immunohistochemically in the AD brain. It is suggested that oxidative stress and AGEs participate in the very early steps of Lewy body formation and resulting cell death in PD. Since the iNOS gene can be regulated by redox-sensitive transcription factors, the use of membrane permeable antioxidants could be a promising strategy for the treatment and prevention of chronic inflammation in neurodegenerative disorders.
Localization microscopy is a class of super-resolution fluorescence microscopy techniques. Localization microscopy methods are characterized by stochastic temporal isolation of fluorophore emission, i.e., making the fluorophores blink so rapidly that no two are
likely to be photoactive at the same time close to each other. Well-known localization microscopy methods include dSTORM}, STORM, PALM, FPALM, or GSDIM. The biological community has taken great interest in localization microscopy, since it can enhance the resolution of common fluorescence microscopy by an order of magnitude at little experimental cost.
However, localization microscopy has considerable computational cost since millions of individual stochastic emissions must be located with nanometer precision. The computational cost of this evaluation, and the organizational cost of implementing the complex algorithms, has impeded adoption of super-resolution microscopy for a long time.
In this work, I describe my algorithmic framework for evaluating localization microscopy data.
I demonstrate how my novel open-source software achieves real-time data evaluation, i.e., can evaluate data faster than the common experimental setups can capture them.
I show how this speed is attained on standard consumer-grade CPUs, removing the need for computing on expensive clusters or deploying graphics processing units.
The evaluation is performed with the widely accepted Gaussian PSF model and a Poissonian maximum-likelihood noise model.
I extend the computational model to show how robust, optimal two-color evaluation is realized, allowing correlative microscopy between multiple proteins or structures. By employing cubic B-splines, I show how the evaluation of three-dimensional samples can be made simple and robust, taking an important step towards precise imaging of micrometer-thick samples.
I uncover the behavior and limits of localization algorithms in the face of increasing emission densities.
Finally, I show up algorithms to extend localization microscopy to common biological problems.
I investigate cellular movement and motility by considering the in vitro movement of myosin-actin filaments. I show how SNAP-tag fusion proteins enable imaging with bright and stable organic fluorophores in live cells. By analyzing the internal structure of protein clusters, I show how localization microscopy can provide new quantitative approaches beyond pure imaging.
The MuvB multiprotein complex, together with B-MYB and FOXM1 (MMB-FOXM1), plays an essential role in cell cycle progression by regulating the transcription of genes required for mitosis and cytokinesis. In many tumors, B-MYB and FOXM1 are overexpressed as part of the proliferation signature. However, the transcriptional targets that are important for oncogenesis have not been identified. Given that mitotic kinesins are highly expressed in cancer cells and that selected kinesins have been reported as target genes of MMB-FOXM1, we sought to determine which mitotic kinesins are directly regulated by MMB-FOXM1. We demonstrate that six mitotic kinesins and two microtubule-associated non-motor proteins (MAPs) CEP55 and PRC1 are direct transcriptional targets of MuvB, B-MYB and FOXM1 in breast cancer cells.
Suppression of KIF23 and PRC1 strongly suppressed proliferation of MDA-MB-231 cells. The set of MMB-FOXM1 regulated kinesins genes and 4 additional kinesins which we referred to as the mitotic kinesin signature (MKS) is linked to poor outcome in breast cancer patients. Thus, mitotic kinesins could be used as prognostic biomarker and could be potential therapeutic targets for the treatment of breast cancer.
Compensatory base changes (CBCs) in internal transcribed spacer 2 (ITS2) rDNA secondary structures correlate with Ernst Mayr’s biological species concept. This hypothesis also referred to as the CBC species concept recently was subjected to large-scale testing, indicating two distinct probabilities. (1) If there is a CBC then there are two different species with a probability of ~0.93. (2) If there is no CBC then there is the same species with a probability of ~0.76. In ITS2 research, however, the main problem is the multicopy nature of ITS2 sequences. Most recently, 454 pyrosequencing data have been used to characterize more than 5000 intragenomic variations of ITS2 regions from 178 plant species, demonstrating that mutation of ITS2 is frequent, with a mean of 35 variants per species, respectively per individual organism. In this study, using those 454 data, the CBC criterion is reconsidered in the light of intragenomic variability, a proof of concept, a necessary criterion, expecting no intragenomic CBCs in variant ITS2 copies. In accordance with the CBC species concept, we could demonstrate that the probability that there is no intragenomic CBC is ~0.99.
To ask students what a species is always has something rhetorical about it. Too quickly comes the rote answer, often learned by heart without ever thinking about it: “A species is a reproductive community of populations (reproductively isolated from others), which occupies a specific niche in nature” (Mayr 1982). However, do two people look alike because they are twins or are they twins because they look alike? “Two organisms do not belong to the same species because they mate and reproduce, but they only are able to do so because they belong to the same species” (Mahner and Bunge 1997). Unfortunately, most biology (pre-university) teachers have no opinion on whether species are real or conceptual, simply because they have never been taught the question themselves, but rather one answer they still pass on to their students today, learned by heart without ever thinking about it. Species are either real or conceptual and, in my opinion, it is this “or” that we should teach about. Only then can we discuss those fundamental questions such as who or what is selected, who or what evolves and, finally, what is biodiversity and phylogenetics all about? Individuals related to each other by the tree of life.
The extracellular matrix within connective tissues represents a structural scaffold as well as a barrier for motile cells, such as invading tumor cells or passenger leukocytes. It remains unclear how different cell types utilize matrix-degrading enzymes for proteolytic migration strategies and, on the other hand, non-proteolytic strategies to overcome 3D fibrillar matrix networks. To monitor cell migration, a 3D collagen model in vitro or the mouse dermis in vivo were used, in combination with time-lapse video-, confocal- or intravital multiphoton-microscopy, and computer-assisted cell tracking. Expression of proteases, including several MMPs, ADAMs, serine proteases and cathepsins, was shown by flow cytometry, Western blot, zymography, and RT-PCR. Protease activity by migrating HT-1080 fibrosarcoma cells resulting in collagenolysis in situ and generation of tube-like matrix defects was detected by three newly developed techniques:(i) quantitative FITC-release from FITC-labelled collagen, (ii) structural alteration of the pyhsical matrix structure (macroscopically and microscopically), and (iii) the visualization of focal in situ cleavage of individual collagen fibers. The results show that highly invasive ollagenolytic cells utilized a spindle-shaped "mesenchymal" migration strategy, which involved beta1 integrindependent interaction with fibers, coclustering of beta1 integrins and matrix metalloproteinases (MMPs) at fiber bundling sites, and the proteolytic generation of a tube-like matrix-defect by MMPs and additional proteases. In contrast to tumor cells, activated T cells migrated through the collagen fiber network by flexible "amoeboid" crawling including a roundish, elliptoid shape and morphological adaptation along collagen fibers, which was independent of collagenase function and fiber degradation. Abrogation of collagenolysis in tumor cells was achieved by a cocktail of broad-spectrum protease inhibitors at non-toxic conditions blocking collagenolysis by up to 95%. While in T cells protease inhibition induced neither morphodynamic changes nor reduced migration rates, in tumor cells a time-dependent conversion was obtained from proteolytic mesenchymal to non-proteolytic amoeboid migration in collagen lattices in vitro as well as the mouse dermis in vivo monitored by intravital microscopy. Tumor cells vigorously squeezed through matrix gaps and formed constriction rings in regions of narrow space, while the matrix structure remained intact. MMPs were excluded from fiber binding sites and beta1 integrin distribution was non-clustered linear. Besides for fibrosarcoma cells, this mesenchymal-toameboid transition (MAT) was confirmed for epithelial MDA-MB-231 breast carcinoma cells. In conclusion, cells of different origin exhibit significant diversity as well as plasticity of protease function in migration. In tumor cells, MAT could respresent a functionally important cellular and molecular escape pathway in tumor invasion and migration.
Over recent years next generation sequencing (NGS) technologies evolved from costly tools used by very few, to a much more accessible and economically viable technology. Through this recently gained popularity, its use-cases expanded from research environments into clinical settings. But the technical know-how and infrastructure required to analyze the data remain an obstacle for a wider adoption of this technology, especially in smaller laboratories. We present GensearchNGS, a commercial DNAseq software suite distributed by Phenosystems SA. The focus of GensearchNGS is the optimal usage of already existing infrastructure, while keeping its use simple. This is achieved through the integration of existing tools in a comprehensive software environment, as well as custom algorithms developed with the restrictions of limited infrastructures in mind. This includes the possibility to connect multiple computers to speed up computing intensive parts of the analysis such as sequence alignments. We present a typical DNAseq workflow for NGS data analysis and the approach GensearchNGS takes to implement it. The presented workflow goes from raw data quality control to the final variant report. This includes features such as gene panels and the integration of online databases, like Ensembl for annotations or Cafe Variome for variant sharing.
In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-\(\kappa\) B in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-\(\kappa\) B such as its inhibitor I\(\kappa\)B isoform \(\alpha\) that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-\(\kappa\)B in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-\(\kappa\)B signaling pathways. The system successfully enabled the controlled manipulation of NF-\(\kappa\)B activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e. g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.
Anthropogenic and environmental drivers shape diversity of naturalized plants across the Pacific
(2021)
Aim
The Pacific exhibits an exceptional number of naturalized plant species, but the drivers of this high diversity and the associated compositional patterns remain largely unknown. Here, we aim to (a) improve our understanding of introduction and establishment processes and (b) evaluate whether this information is sufficient to create scientific conservation tools, such as watchlists.
Location
Islands in the Pacific Ocean, excluding larger islands such as New Zealand, Japan, the Philippines and Indonesia.
Methods
We combined information from the most up‐to‐date data sources to quantify naturalized plant species richness and turnover across island groups and investigate the effects of anthropogenic, biogeographic and climate drivers on these patterns. In total, we found 2,672 naturalized plant species across 481 islands and 50 island groups, with a total of 11,074 records.
Results
Most naturalized species were restricted to few island groups, and most island groups have a low number of naturalized species. Island groups with few naturalized species were characterized by a set of widespread naturalized species. Several plant families that contributed many naturalized species globally also did so in the Pacific, particularly Fabaceae and Poaceae. However, many families were significantly over‐ or under‐represented in the Pacific naturalized flora compared to other regions of the world. Naturalized species richness increased primarily with increased human activity and island altitude/area, whereas similarity between island groups in temperature along with richness differences was most important for beta diversity.
Main conclusions
The distribution and richness of naturalized species can be explained by a small set of drivers. The Pacific region contains many naturalized plant species also naturalized in other regions in the world, but our results highlight key differences such as a stronger role of anthropogenic drivers in shaping diversity patterns. Our results establish a basis for predicting and preventing future naturalizations in a threatened biodiversity hotspot.
Der Wilms Tumor (WT), auch Nephroblastom genannt, zählt zu den im Kindesalter am häufigsten auftretenden malignen Tumoren und entsteht meist unilateral (90 – 95 %) und sporadisch (98 – 99 %). Leider sind bis heute die molekularen Ursachen, die zur Entwicklung dieser Tumoren führen nur unzureichend aufgeklärt. So werden bisher nur drei Gene mit dem Auftreten von WT in Verbindung gebracht: WT1, CTNNB1 und WTx. Während WT1 und CTNNB1 jeweils Mutationsraten von etwa 10 – 15 % aufweisen, die zudem häufig gemeinsam vorliegen, werden für WTx Mutationsraten von etwa 30 % beobachtet. Die genetischen Alterationen der anderen Tumoren sind noch immer komplett unbekannt. Ziel dieser Arbeit war aus diesem Grund die Identifikation von relevanten Regionen und Genen, die an der Entstehung bzw. dem klinischen Fortschreiten von Wilms Tumoren beteiligt sind. Zusätzlich sollten weitere Untersuchungen zur Einschätzung ihres prognostischen Potenzials dienen. In einem ersten Ansatz wurden die Chromosomenbereiche 11q und 16q in einer großen Anzahl von Wilms Tumoren auf LOH (=loss of heterozygosity), d.h. den (partiellen) Verlust von genetischem Material, untersucht. In beiden Fällen wurden erhöhte LOH-Raten von etwa 20 % beobachtet, jedoch war keine Eingrenzung der relevanten Regionen möglich, da Allelverluste nicht stets ab einem bestimmten Marker beobachtet wurden. Ein Vergleich mit der Histologie ergab signifikante Assoziationen der Allelverluste mit anaplastischen und Mischtyp-Tumoren (nur für LOH 11q), wohingegen kaum LOHs in epithelialen und stromareichen Tumoren festgestellt wurden. Somit scheinen auf 11q und 16q Gene vorzuliegen, die einerseits die Differenzierung in Epithel und Stroma begünstigen oder andererseits ein blastemreiches und anaplastisches Erscheinungsbild verhindern. Jedoch könnte auch die Assoziation von bestimmten Subtypen mit LOH 11q und 16q auf eine Entstehung aus unterschiedlichen Zellen hindeuten. Weiterhin war das Auftreten von LOH, v.a. wenn jeweils der komplette Chromosomenarm betroffen war, mit einem erhöhten Rezidiv- und Sterberisiko (nur LOH 11q) verbunden. Somit konnte gezeigt werden, dass LOH-Untersuchungen auf 11q und 16q zur Identifikation von Hochrisikopatienten für die Entwicklung von Rezidiven bzw. erhöhter Mortalität eingesetzt werden können, wodurch eine individuelle Anpassung der Therapiemaßnahmen ermöglicht wird. In einem zweiten Ansatz wurden eine Reihe von bereits publizierten potenziellen Markergenen in einer großen Anzahl von Wilms Tumoren mit Hilfe der Realtime RT-PCR auf ihre Relevanz überprüft. Allen diesen Genen wurde zuvor eine Funktion bei der histologischen Klassifikation der Tumoren bzw. bei der Vorhersage bestimmter klinischer Verläufe zugeschrieben. Die univariate Analyse diente der Beurteilung der Relevanz einzelner Gene, wohingegen die multivariate Analyse zur Bestimmung von prognostischen Genkombinationen eingesetzt wurde. Anschließend erfolgte die Validierung mittels eines zweiten und unabhängigen Tumorsatzes. Auch wenn viele der bereits publizierten Marker und in der ersten Analyse erhaltenen Assoziationen in einem weiteren und unabhängigen Tumorsatz nicht verifizierbar waren, konnten dennoch einige frühere Ergebnisse repliziert und die Relevanz der entsprechenden Gene nachgewiesen werden. Neben der Verbindung der Repression von HEY2 und TRIM22 mit Hochrisikotumoren bzw. einer höheren Sterbewahrscheinlichkeit fanden sich schwach signifikante Assoziationen auch für die verminderte Expression von TRIM22 und VEGF mit der Histologie. Ebenso waren erhöhte Level von TERT und die Repression von TRIM22 mit der Entwicklung eines Rezidivs verbunden. Vor allem aber die Korrelation der Repression von HEY2 und VEGF sowie einer Überexpression von CA9 mit Rezidiven, Tumoren hoher Malignität oder primären Metastasen verweisen auf die Notwendigkeit, besonders die Hypoxie- und Angiogenese-Signalkaskaden in Wilms Tumoren zu untersuchen, um deren Einfluss v.a. auf das Fortschreiten und die Ausbreitung der Tumoren zu evaluieren. Auch wenn die multivariate Analyse nicht zu relevanten Genkombinationen führte, konnte hier dennoch eine schwache Assoziation der verminderten Expression von TOP2A und TRIM22 mit primären Metastasen oder einer erhöhten Mortalität, sowie der Überexpression von TERT mit der Rezidivbildung bestätigt werden. Interessanterweise stellte sich die Histologie, die derzeit das Hauptkriterium für die Risikoklassifikation darstellt, weder als geeigneter prognostischer Marker für die Beurteilung des Rezidiv- noch des Sterberisikos heraus. Somit sollten Realtime RT-PCR Analysen in Zukunft als weiterer Faktor zur Beurteilung des Rezidiv- und Sterberisikos eingesetzt werden, um eine individuelle Anpassung der Therapie zu ermöglichen. Basierend auf den Ergebnissen der Realtime RT-PCR Analyse wurde der Einfluss der Expression ausgewählter Gene auf Primärkulturen, die aus nativem Wilms Tumormaterial gewonnen wurden, untersucht. Nach der Überexpression von HEY2, EGR1, MYCN und TRIM22 wurden bei allen Zellen hohe Sterberaten beobachtet, v.a. bei HEY2 und EGR1. Leider konnte weder für HEY2 noch für EGR1 der Grund hierfür aufgeklärt werden, allerdings war bei EGR1 weder die Apoptose noch die Seneszenz beteiligt. Im Gegensatz hierzu wurde die Apoptose als entscheidender Mechanismus bei MYCN und v.a. TRIM22 ermittelt. Außerdem scheint bei MYCN ein großer Anteil an Zellen in die Seneszenz einzutreten. Auch wenn diese ersten Untersuchungen an Primärkulturen von Wilms Tumoren eindeutig die Relevanz dieser Gene für die Entwicklung bzw. das Fortschreiten der Tumoren bestätigten, so sind trotz alledem weitere Experimente v.a. in einer größeren Anzahl genetisch unterschiedlicher Primärkulturen nötig, um das endgültige Potenzial dieser Gene aufzuklären.
Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential Iigand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properlies and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related Iigand-binding domain sequences of the human epidennal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk proteinwas examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transfonning protein equivalent to other tyrosine kinase oncoproteins.
Untersuchungen zum Wasserferntransport wurden mit Hife der Druckmeßsonden- und NMR-Bildgebungstechnik durchgeführt. Dabei wurden Experimente zum Einfluß der Schwerkraft auf den Wasserferntransport an einer Liane bei unterschiedlicher Orientierung der Pflanze durchgeführt. Der zweite Teil der Arbeit beschäftigte sich mit der Korrelation von Flußgeschwindigkeiten und Xylemdruck in den Wasserleitungsbahnen gut gewässerter und trockengestreßter Pflanzen. Der dritte Teil befasste sich mit der Wiederbefüllung von kavitierten oder leeren Xylemgefäßen anhand der Auferstehungspflanze Myrothamnus flabellifolia.
Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(−)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(−) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(−) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(−) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(−) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(−) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm.
Ligand-dependent tumor induction in medakafish embryos by a Xmrk receptor tyrosine kinase transgene
(1994)
Xmrk encodes a subclass 1 receptor tyrosine kinase (RTK) which has been cloned from the melanomainducing locus Tu of the poeciliid fish Xiphophorus. To demonstrate a high oncogenic potential in vivo we transferred the gene into early embryos of the closely related medakafish. Ectopic expression of the Xmrk oncogene under the control of a strong, constitutive promoter (CMVTk) led to the induction of embryonic tumors with high incidence, after short latency periods, and with a specific pattern of affected tissues. We demonstrate ligand-dependent transformation in vivo using a chimeric receptor consisting of the extracellular and transmembrane domains of the human EGF receptor (HER) and the cytoplasmatic domain of Xmrk. Expression of the chimeric receptor alone does not lead to ldnase activation or induction of tumors. Coexpression of the chimera with its corresponding ligand, human transforming growth factor alpha (bTGF(X), however, results in the activation of the chimeric RTK. In injected fish embryos the induction of the neoplastic growth is observed with similar incidence and tissue distribution as in embryos carrying the native Xmrk oncogene suggesting that the ligand as well as factors downstream of tbe RTK are required for tumor formation. In this study we show single-step induction of tumors by ectopic expression of RTKs in vivo substantiating tbe significance of autocrine stimulation in RTK induced tumors in vertebrales.
Species of small fish are becoming useful tools for studies on vertebrate development. Wehave investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporaland spatial expression patterns ofbacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as weil as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.
Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a nurober of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish celllines andin embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoterenhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. From the data obtained, a set of expression vectors for basic research as weH as for aquaculture purposes were established.
Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection
(2015)
Staphylococcus aureus is both a human commensal and a pathogen. 20%-30% of all individuals are permanently or occasionally carriers of S. aureus without any symptoms. In contrast to this, S. aureus can cause life-threatening diseases e.g. endocarditis, osteomyelitis or sepsis. Here, the increase in antibiotic resistances makes it more and more difficult to treat these infections and hence the number of fatalities rises constantly. Since the pharmaceutical industry has no fundamentally new antibiotics in their pipeline, it is essential to better understand the interplay between S. aureus and the human host cell in order to find new, innovative treatment options.
In this study, a RNA interference based whole genome pool screen was performed to identify human proteins, which play a role during S. aureus infections. Since 1,600 invasion and 2,271 cell death linked factors were enriched at least 2 fold, the big challenge was to filter out the important ones. Here, a STRING pathway analysis proved to be the best option. Subsequently, the identified hits were validated with the help of inhibitors and a second, individualised small interfering RNA-based screen.
In the course of this work two important steps were identified, that are critical for host cell death: the first is bacterial invasion, the second phagosomal escape. The second step is obligatory for intracellular bacterial replication and subsequent host cell death. Invasion in turn is determining for all following events. Accordingly, the effect of the identified factors towards these two crucial steps was determined. Under screening conditions, escape was indirectly measured via intracellular replication. Three inhibitors (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) could be identified for the invasion process. In addition, siRNAs targeted against 16 different genes (including CAPN2, CAPN4 and PIK3CG), could significantly reduce bacterial invasion. Seven siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) were able to inhibit intracellular replication significantly. Further studies showed that the IP3 receptor inhibitor 2-APB, the calpain inhibitor calpeptin and the proteasome inhibitor MG-132 are able to prevent phagosomal escape and as a consequence intracellular replication and host cell death.
In this context the role of calpains, calcium, the proteasome and the mitochondrial membrane potential was further investigated in cell culture. Here, an antagonistic behaviour of calpain 1 and 2 during bacterial invasion was observed. Intracellular calcium signalling plays a major role, since its inhibition protects host cells from death. Beside this, the loss of mitochondrial membrane potential is characteristic for S. aureus infection but not responsible for host cell death. The reduction of membrane potential can be significantly diminished by the inhibition of the mitochondrial Na+/Ca2+ exchanger.
All together, this work shows that human host cells massively contribute to different steps in S. aureus infection rather than being simply killed by bacterial pore-forming toxins. Various individual host cell factors were identified, which contribute either to invasion or to phagosomal escape and therefore to S. aureus induced cytotoxicity. Finally, several inhibitors of S. aureus infection were identified. One of them, 2-APB, was already tested in a sepsis mouse model and reduced bacterial load of kidneys.
Thus, this study shows valuable evidence for novel treatment options against S. aureus infections, based on the manipulation of host cell signalling cascades.
Synaptonemal Komplexe (SC) sind evolutionär konservierte, meiosespezifische, proteinöse Strukturen, die maßgeblich an Synapsis, Rekombination und Segregation der homologen Chromosomen beteiligt sind. Sie zeigen eine dreigliedrige strickleiter-artige Organisation, die sich aus i) zwei Lateralelementen (LE), an die das Chromatin der Homologen angelagert ist, ii) zahlreichen Transversalfilamenten (TF), welche die LE in einer reißverschlussartigen Weise miteinander verknüpfen, und iii) einem zentralen Element (CE) zusammensetzt. Die Hauptproteinkomponenten der Säuger-SC sind das Transversalfilamentprotein SYCP1 und die Lateralelementproteine SYCP2 und SYCP3. Wie sich die SC-Struktur zusammenfügt war bisher nur wenig verstanden; es war nicht bekannt wie die TF innerhalb der LE-Strukturen verankert sind und dabei die homologen Chromosomen verknüpfen. Aufgrund dessen wurde die Interaktion zwischen den Proteinen SYCP1 und SYCP2 untersucht. Mit der Hilfe verschiedenster Interaktionssysteme konnte gezeigt werden, dass der C-Terminus von SYCP1 mit SYCP2 interagieren kann. Aufgrund der Bindungsfähigkeit zu beiden Proteinen, SYCP1 und SYCP3, kann angenommen werden, dass SYCP2 als Linker zwischen diesen Proteinen fungiert und somit möglicherweise das fehlende Bindungsglied zwischen den Lateralelementen und Transversalfilamenten darstellt. Obwohl die SC-Struktur in der Evolution hochkonserviert ist, schien dies nicht für seine Protein-Untereinheiten zuzutreffen. Um die Struktur und Funktion des SC besser verstehen zu können, wurde ein Vergleich zwischen den orthologen SYCP1 Proteinen der evolutionär entfernten Spezies Ratte und Medaka erstellt. Abgesehen von den erheblichen Sequenzunterschieden die sich in 450 Millionen Jahren der Evolution angehäuft haben, traten zwei bisher nicht identifizierte Sequenzmotive hervor, CM1 und CM2, die hochgradig konserviert sind. Anhand dieser Motive konnte in Datenbankanalysen erstmals ein Protein in Hydra vulgaris nachgewiesen werden, bei dem es sich um das orthologe Protein von SYCP1 handeln könnte. Im Vergleich mit dem SYCP1 der Ratte zeigten die Proteine aus Medaka und Hydra, neben den hoch konservierten CM1 und CM2, vergleichbare Domänenorganisationen und im heterologen System zudem sehr ähnliche Polymerisationseigenschaften. Diese Ergebnisse sprechen für eine evolutionäre Konservierung von SYCP1.
Für Patienten mit malignem Melanom im Stadium der Fernmetastasierung gibt es bis heute lediglich Therapieoptionen mit sehr eingeschränkten Erfolgsaussichten. Diese Tatsache bestätigt die Notwendigkeit von Biomarkern zur Vorhersage des Erfolgs verschiedener Therapien. Der ATP-basierende ex vivo Chemosensitivitätsassay hat sich als erfolgreiche Methode zur individuellen Vorhersage eines Chemotherapieerfolgs herausgestellt. Tatsächlich zeigte der Assay ein heterogenes Sensitivitätsprofil gegen verschiedene Chemotherapeutika und ließ in getesteten Patienten ein ex vivo wirksames Chemotherapieregime identifizieren, das anschließend auch klinische Therapieerfolge bei Verwendung der Therapie mit dem besten individuellen Chemosensitivitätsindex(BICSI) zeigte. Um diesen sehr aufwendigen Assay zukünftig zu umgehen, sollten in der vorliegenden Arbeit prädiktive molekulare Biomarker der Chemosensitivität identifiziert werden. Hierfür wurden im Voraus durch einen Microarray die Kandidaten Secernin 1 (SCRN1), Lysyl oxidaselike 1 (LOXL1), Thymosin beta 4 X-linked (TMSB4X), Vesicle-associated membrane protein 5 (VAMP5) und Serine protease inhibitor B1 (SERPINB1) als differentiell exprimierte Gene in chemosensitivem gegenüber chemoresistentem Gewebe identifiziert. Die relative Expression dieser Kandidatengene wurde daraufhin in bis zu 128 verschiedenen Melanomgeweben mit dem Chemosensitivitätsindex verschiedener Chemotherapeutika korreliert. Hierbei konnte eine signifikante Korrelation zwischen SerpinB1 mit der Chemosensitivität gegenüber der Therapiekombination mit Paclitaxel und Cisplatin auf Gen- aber nicht auf Proteinebene identifiziert werden. Weiterhin konnte eine differentielle Expression ebenfalls in chemosensitiven und -resistenten Melanomzelllinien nachgewiesen werden, die allerdings im Vergleich mit dem analysierten Gewebe in gegensätzlicher Richtung verlief. Zusammenfassend lässt sich sagen, dass SerpinB1 ein vielversprechender Marker für die Chemosensitivität gegenüber Paclitaxel und Cisplatin ist, dessen funktionelle Bedeutung aber unklar bleibt. Das Merkelzellkarzinom (MCC) ist ein seltener und hoch aggressiver Tumor der mit dem Merkelzellpolyomavirus (MCV) in Zusammenhang steht. Da MCC Zelllinien zur Aufrechterhaltung ihrer Viabilität die MCV T-Antigene benötigen, könnte der Einsatz von Interferonen (IFN) ein möglicher therapeutischer Ansatz zur Behandlung dieser Krebserkrankung sein. In der vorliegenden Arbeit haben wir daher die Effekte von IFNs auf MCC Zelllinien, mit besonderer Berücksichtigung der MCV+ Linien, untersucht. IFNs vom Typ I (hier Multiferon, ein Mix verschiedener IFN α Subtypen, und IFN β) wirkten stark inhibierend auf die zelluläre Viabilität. Die Zellzyklusanalyse zeigte eine Erhöhung des sub-G Anteils der Zellen nach Behandlung mit IFN, was auf Apoptose als ausschlagebenden Grund schließen ließ. Diese Effekte waren für die Behandlung mit IFN β weniger stark ausgeprägt. Der inhibitorische Effekt von Typ I IFNs auf MCV+ MCC Zelllinien war assoziiert mit einer verringerten Expression des viralen großen T-Antigens (LTA) und einer Erhöhung in der Expression von promyelocytic leukemia protein (PML), das dafür bekannt ist, die Funktion des LTA störend zu beeinflussen. Zusätzlich führte die intratumorale Anwendung von Multiferon in vivo zu einer Regression im Wachstum von MCV+, aber nicht MCV- MCC Xenotransplantaten. Die Ergebnisse zeigen das Typ I IFNs einen starken antitumoralen Effekt haben, der zum Teil durch die Regulierung des LTA herbeigeführt wird. Neben diesen direkten Effekten der IFNs auf die Zellproliferation induzieren diese auch die Expression von MHC Klasse I Molekülen in MCC Zelllinien. Die Durchflusszytometrie zeigte eine Induktion der MHC Klasse I Expression in drei MHC I negativen MCC Zelllinien und eine Erhöhung der Expression, die vor der Behandlung eine geringe Menge an MHC I aufwiesen. Diese Effekte konnten auch in den in vivo Xenotransplantaten beobachtet werden. Die Ergebnisse zeigen, dass die Behandlung mit IFN sowohl direkte als auch indirekte Effekte auf das MCC hat und eine breite Anwendung in Patienten mit MCV+ und MCV- Tumoren finden kann. Neben IFNs sind auch Artemisinin und seine Derivate bekannt für ihre antitumoralen und antiviralen Eigenschaften. Daher haben wir den Effekt des Artemisininderivats Artesunate auf MCV+ und MCV- MCC Zelllinien getestet. Tatsächlich konnten wir auch hier einen antiproliferativen Effekt des Stoffes nachweisen, der stärker auf MCV+ als auf MCV- Zelllinien wirkte und bei ersteren wiederum mit einer reduzierten LTA Expression einherging. Im Vergleich dazu blieben Fibroblasten von der Behandlung unbeeinflusst. Das verringerte Tumorwachstum konnte ebenfalls für in vivo Xenotransplantationsmodelle gezeigt werden. Auf Grundlage dieser Erkenntnis sollte eine genauere Untersuchung dieses alten Naturheilstoffes für die Behandlung von MCC Patienten in Betracht gezogen werden.
Das fakultativ intrazelluläre Bakterium Listeria monocytogenes besitzt die Fähigkeit, eukaryotische Wirtszellen zu penetrieren, sich in diesen zu vermehren, fortzubewegen und zwischen den Zellen auszubreiten. Im Zuge des intrazellulären Lebenszyklus gehen Listerien Wechselwirkungen mit ver-schiedenen zellulären Proteinen ein. Als eines dieser Proteine konnte das zelluläre Phosphoprotein Stathmin identifiziert werden. Dieses Protein bindet an Untereinheiten des Tubulins und destabilisiert dadurch Mikrotubuli (MT). Es wird durch Phosphorylierung von vier spezifischen Serinresten in seiner MT-destabilisierenden Aktivität reguliert. Da Stathmin als Antwort auf externe Signale zelluläre Funktionen, z. B. Zell-Proliferation und Differenzierung reguliert, vermutet man seine Funktion in einer Art Relais welches verschiedene Signale aus dem Umfeld der Zelle integriert. In mit L. monocytogenes infizierten Wirtszellen wird Stathmin an die Oberfläche intrazellulärer Bakterien rekrutiert. Inwiefern diese Rekrutierung das Phosphorylierungsmuster von Stathmin und damit dessen Aktivität beeinflusst, konnte im Rahmen dieser Arbeit nicht geklärt werden. Stathmin knock-out Mäuse sollten sich gut eignen, um die Rolle von Stathmin während einer Infektion mit L. monocytogenes EGD in vitro und in vivo zu untersuchen. Es stellte sich heraus, dass in Stathmin(-/-)-Makrophagen der intrazelluläre Lebenszyklus der Listerien nicht signifikant beeinflusst ist. Nach intravenöser Verabreichung von 5x103 L. monocytogenes waren drei Tage nach der Infektion in Leber und Milz der knock-out Mäuse allerdings signifikant mehr Listerien nachzuweisen, als in den Organen wildtypischer Mäuse. Mittels Immunfluoreszenzmikroskopie und einem anti-Stathmin-Antiersum konnte an mit verschiedenen L. monocytogenes-Mutanten infizierten Zellen gezeigt werden, dass Stathmin mit der Oberfläche intrazellulärer Listerien kolokalisiert. Allerdings konnten dabei die Angaben in der Literatur nicht bestätigt werden, wonach für diese Kolokalisation die Expression von ActA notwendig ist. Die Ergebnisse dieser Arbeit sprechen im Gegensatz zu den publizierten Daten dafür, dass Stathmin über einen bisher noch unbekannten Mechanismus ActA-unabhängig an intrazelluläre Listerien rekrutiert wird. Zweikomponentensysteme ermöglichen Bakterien eine rasche Anpassung an sich verändernde Umweltbedingungen, da sie extra- und intrazelluläre Stimuli in zelluläre Signale umwandeln. Um die 16 in der Genomsequenz von L. mono-cytogenes EGDe identifizierten Zweikomponentensysteme charakterisieren zu können, wurden individuelle Mutanten konstruiert, in denen individuelle Response Regulatorgene deletiert sind. Die erhaltenen Mutanten wurden in vitro und in vivo auf ihr Wachstumsverhalten hin untersucht. Es zeigte sich, dass unter den angewandten Kultur- und Versuchsbedingungen keines der Zweikomponentensysteme eine signifikante Rolle bei der Anpassung an Temperatur, sowie an oxidativen oder osmotischen Stress spielt. Die Zugabe von 5 % Ethanol hatte einen stark hemmenden Effekt auf das Wachstum von 4 Mutanten, wohingegen zwei andere Mutanten in Gegenwart des Alkohols deutlich besser wuchsen. Unter anaeroben Bedingungen konnte kein Unterschied im Wachstum beobachtet werden. Die Expression wichtiger Virulenzgene war in keiner der untersuchten Mutanten im Vergleich zum Ausgangsstamm verändert. Die intrazelluläre Replikation sowie intrazelluläre Bewegung und Ausbreitung im Zellrasen waren durch die Deletion der Response Regulatorgene nicht beeinträchtigt. Abgesehen von geringen Unterschieden in der Invasivität einiger Deletionsmutanten für Cos-1 und Caco-2 Zellen zeigte sich keiner der Response Regulatoren für den intrazellulären Lebenszyklus von L. monocytogenes erforderlich. Es zeigte sich, dass der in der vorliegenden Arbeit verwendete L. monocyto-genes-Wildstamm auch bei der für die Flagellenexpression normalerweise nicht-permissiven Temperatur von 37° C noch beweglich ist. Die L. monocytogenes ΔdegU-Mutante war dagegen auf Weichagar temperaturunabhängig unbeweglich. Die elektronenmikroskopische Analyse ergab, dass dieser Stamm im Gegensatz zum Wildtyp auch bei 24° C keine Flagellen ausbildet. Durch vergleichende Proteomanalysen konnte gezeigt werden, dass L. monocytogenes ΔdegU bei 24° C wesentliche Proteine des Flagellenapparates nicht synthetisiert. Mittels Transkriptomanalysen konnten die Ergebnisse der Proteomanalysen bestätigt werden. Es wurden neben Genen, die für Proteine der Flagellenbiosynthese und Chemotaxis codieren, noch weitere Gene identifiziert, die offensichtlich unter der transkriptionellen Kontrolle des Response Regulators DegU stehen. Die Ergebnisse der in vivo Studien zeigten, dass L. monocytogenes ΔdegU deutlich virulenzattenuiert ist. Für die restlichen L. monocytogenes ΔTCS-Mutanten waren im Vergleich zum Wildtyp die Unterschiede in Leber und Milz nur leicht verändert und statistisch nicht signifikant.
Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation.
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.
Deregulated MYC expression contributes to cellular transformation as well as progression and
maintenance of human tumours. Interestingly, in the absence of additional genetic alterations,
potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced
apoptosis has long been recognised as a major barrier against cancer development.
However, it is largely unknown how cells discriminate physiological from supraphysiological levels
of MYC in order to execute an appropriate biological response.
The experiments described in this thesis demonstrate that induction of apoptosis in mammary
epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene
expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required
to invade low-affinity binding sites and repress target genes of the serum response factor SRF.
These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely
needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1-
dependent gene repression and increases AKT phosphorylation and downstream function.
Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC’s
transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive
role for the MYC/MIZ1 complex in epithelial cells.
The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor that stabilises the c-Myc protein. CIP2A is overexpressed in several tumours, and expression levels are an independent marker for long-term outcome. To determine whether CIP2A expression is elevated in colon cancer and whether it might serve as a prognostic marker for survival, we analysed CIP2A mRNA expression by real-time PCR in 104 colon cancer samples. CIP2A mRNA was overexpressed in colon cancer samples and CIP2A expression levels correlated significantly with tumour stage. We found that CIP2A serves as an independent prognostic marker for disease-free and overall survival. Further, we investigated CIP2A-dependent effects on levels of c-Myc, Akt and on cell proliferation in three colon cancer cell lines by silencing CIP2A using small interfering (si) and short hairpin (sh) RNAs. Depletion of CIP2A substantially inhibited growth of colon cell lines and reduced c-Myc levels without affecting expression or function of the upstream regulatory kinase, Akt. Expression of CIP2A was found to be dependent on MAPK activity, linking elevated c-Myc expression to deregulated signal transduction in colon cancer.
Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n = 9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n = 5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC\(_{50}\)< 3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents.
Clinical prognosis of metastasized colorectal carcinoma (CRC) is still not at desired levels and novel drugs are needed. Here, we focused on the multi-tyrosine kinase inhibitor E7080 (Lenvatinib) and assessed its therapeutic efficacy against human CRC cell lines in vitro and human CRC xenografts in vivo. The effect of E7080 on cell viability was examined on 10 humanCRCcell lines and humanendothelial cells (HUVEC). The inhibitory effect of E7080 on VEGF-induced angiogenesis was studied in an ex vivo mouse aortic ring angiogenesis assay. In addition, the efficacy of E7080 against xenografts derived fromCRC cell lines and CRC patient resection specimenswithmutated KRASwas investigated in vivo. Arelatively low cytotoxic effect of E7080 on CRC cell viabilitywas observed in vitro. Endothelial cells (HUVEC)weremore susceptible to the incubation with E7080. This is in line with the observation that E7080 demonstrated an anti-angiogenic effect in a three-dimensional ex vivo mouse aortic ring angiogenesis assay. E7080 effectively disrupted CRC cell-mediated VEGF-stimulated growth of HUVEC in vitro. Daily in vivo treatment with E7080 (5 mg/kg) significantly delayed the growth of KRAS mutated CRC xenografts with decreased density of tumor-associated vessel formations and without tumor regression. This observation is in line with results that E7080 did not significantly reduce the number of Ki67-positive cells in CRC xenografts. The results suggest antiangiogenic activity of E7080 at a dosage thatwas well tolerated by nudemice. E7080 may provide therapeutic benefits in the treatment of CRC with mutated KRAS.
Background
The management of rectal cancer (RC) has substantially changed over the last decades with the implementation of neoadjuvant chemoradiotherapy, adjuvant therapy and improved surgery such as total mesorectal excision (TME). It remains unclear in which way these approaches overall influenced the rate of local recurrence and overall survival.
Methods
Clinical, histological and survival data of 658 out of 662 consecutive patients with RC were analyzed for treatment and prognostic factors from a prospectively expanded single-institutional database. Findings were then stratified according to time of diagnosis in patient groups treated between 1993 and 2001 and 2002 and 2010.
Results
The study population included 658 consecutive patients with rectal cancer between 1993 and 2010. Follow up data was available for 99.6% of all 662 treated patients. During the time period between 2002 and 2010 significantly more patients underwent neoadjuvant chemoradiotherapy (17.6% vs. 60%) and adjuvant chemotherapy (37.9% vs. 58.4%). Also, the rate of reported TME during surgery increased. The rate of local or distant metastasis decreased over time, and tumor related 5-year survival increased significantly with from 60% to 79%.
Conclusion
In our study population, the implementation of treatment changes over the last decade improved the patient’s outcome significantly. Improvements were most evident for UICC stage III rectal cancer.
Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae
(2016)
Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution.
Robotic-assisted colon surgery may contain advantages over the laparoscopic approach, but clear evidence is sparse. This study aimed to analyze postoperative inflammation status, short-term outcome and cost-effectiveness of robotic-assisted versus laparoscopic left hemicolectomy. All consecutive patients who received minimal-invasive left hemicolectomy at the Department of Surgery I at the University Hospital of Wuerzburg in 2021 were prospectively included. Importantly, no patient selection for either procedure was carried out. The robotic-assisted versus laparoscopic approaches were compared head to head for postoperative short-term outcomes as well as cost-effectiveness. A total of 61 patients were included, with 26 patients having received a robotic-assisted approach. Baseline characteristics did not differ among the groups. Patients receiving a robotic-assisted approach had a significantly decreased length of hospital stay as well as lower rates of complications in comparison to patients who received laparoscopic surgery (n = 35). In addition, C-reactive protein as a marker of systemic stress response was significantly reduced postoperatively in patients who were operated on in a robotic-assisted manner. Consequently, robotic-assisted surgery could be performed in a cost-effective manner. Thus, robotic-assisted left hemicolectomy represents a safe and cost-effective procedure and might improve patient outcomes in comparison to laparoscopic surgery.
TNF (Tumor Nekrose Faktor) vermittelt seine biologischen Funktionen durch Interaktionen mit TNFR1 (TNFRezeptor 1) und TNFR2 (TNFRezeptor 2). In früheren Arbeiten konnte gezeigt werden, dass der TNFR2 sowohl durch die Induktion von membrangebundenem TNF als auch durch die proteasomale Degradation von TRAF2 (TNFRezeptor-assozierter Faktor 2) die TNFR1-vermittelte Apoptose verstärken kann. Des Weiteren war bekannt, dass TRAF1 (TNFRezeptor-assozierter Faktor 1), ein anderes Mitglied der TRAF-Familie, mit TRAF2 Heterotrimere bilden kann und zudem nach TNF-induzierter NFkappaB- (nuclear factor kappaB) Aktivierung verstärkt exprimiert wird. In der vorliegenden Arbeit konnte nun erstmals gezeigt werden, dass TRAF1 in beide TNFR-Signalkomplexe rekrutiert wird und darin in einem TRAF2/TRAF1-Heterotrimer TRAF2 funktionell ersetzen kann. Darüber hinaus verhindert TRAF1 die Rekrutierung von TRAF2 in lipid rafts sowie dessen anschließende proteasomale Degradation. Auf diese Weise kann TRAF1 die TNFR2-abhängige Verstärkung der TNFR1-induzierten Apoptose verhindern. Im zweiten Teil der vorliegenden Arbeit wurde die TNF-vermittelte Aktivierung der JNK (c-Jun N-terminale Kinase), dessen Regulation durch ROS (reactive oxygen species), Caspasen (Cysteinyl-Aspartat-spezifische Proteasen) sowie NFkappaB-induzierte Faktoren untersucht. TNF induziert in den meisten Zellen zunächst nach zehn bis 30 Minuten eine transiente JNK-Aktivierung, woraufhin bei NFkB-inhibierten Zellen eine zweite andauernde JNK-Aktivierung folgt. Die meisten in der Literatur beschriebenen Studien gehen dabei von einem ROS-abhängigen, Caspase-unabhängigen Mechanismus der persistierenden JNK-Aktivierung aus. Des Weiteren wurde in den vor allem bei embryonale Mausfibroblasten durchgeführten Untersuchungen davon ausgegangen, dass bestimmte NFkappaB-induzierte Radikalfänger die andauernde Aktivierung der JNK verhindern. In dieser Arbeit konnte gezeigt werden, dass in den humanen Zelllinien KB, Jurkat und HaCaT die andauernde Aktivierung der JNK, im Gegensatz zur transienten JNK-Aktivierung, Caspase-abhängig verläuft. Es ergab sich überdies, dass die inhibierende Wirkung des NFkB-Signalweges auf die persistierende JNK-Aktivierung in diesen Zelllinien in erster Linie auf die indirekte Verhinderung der Apoptose durch die Induktion von antiapoptotischen Proteinen wie Flip-L (FLICE-inhibitory protein long) und IAPs (inhibitor of apoptosis) zurückzuführen ist, als auf die direkte Expression von Radikalfängern. Zudem wurde in den untersuchten Zelllinien die Caspase-vermittelte Spaltung von MEKK-1 (MAP/ERK kinase kinase-1) und p21WAF/Cip 1 nachgewiesen, von denen bekannt ist, dass die Spaltprodukte eine JNK-stimulierende Wirkung haben. Dennoch müssen künftige Studien zeigen, ob die Spaltung von p21WAF/Cip 1 und MEKK-1 in Fragmente mit JNK–stimulierender Aktivität oder andere Caspasesubstrate für die Caspase-vermittelte andauernde Aktivierung der JNK verantwortlich sind.
The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research.
Orthopteran diversity in steep slope vineyards: the role of vineyard type and vegetation management
(2023)
The abandonment of traditional agricultural practices and subsequent succession are major threats to many open-adapted species and species-rich ecosystems. Viticulture on steep slopes has recently suffered from strong declines due to insufficient profitability, thus increasing the area of fallow land considerably. Changing cultivation systems from vertically oriented to modern vineyard terraces offers an opportunity to maintain management economically viable and thus reduces further abandonment. Hillside parallel terraces favor mechanization, and their embankments offer large undisturbed areas that could provide valuable habitats. We investigated the effects of vineyard abandonment, different vineyard management types (vertically oriented vs. terraced), and local parameters on Orthoptera diversity in 45 study sites along the Upper Middle Rhine Valley in Germany. Our results show that woody structures and vineyard abandonment reduced Orthoptera diversity at the local and landscape scale due to decreased habitat quality, especially for open-adapted species. In contrast, open inter-rows of actively managed vineyard types supported heat-adapted Caelifera species. On terrace embankments, extensive management and taller vegetation benefited Ensifera species, while short and mulched vegetation in vertically oriented vineyards favored the dominance of one single Caelifera species. Our results highlight the significance of maintaining viticultural management on steep slopes for the preservation of both open-adapted Orthoptera species and the cultural landscape.
no abstract available
Toll-like receptors (TLR) are pattern recognition receptors (PRR) by which macrophages (MØ) sense pathogen-associated molecular patterns (PAMPs). The recognition of lipopolysaccharide (LPS), the PAMP of gram negative bacteria, by TLR4 triggers signaling cascades and leads to the pro-inflammatory activation of the cells. A recent quantitative and kinetic analysis of the phosphoproteome of LPS-activated primary macrophages highlighted the cytoskeleton as a cell compartment with an enriched protein phosphorylation. In total 44 cytoskeleton-associated proteins were regulated by this post-translational modification and thus might be involved in the control and regulation of key macrophage functions like spreading, motility and phagocytosis.
To investigate the control of cytoskeleton-associated cell functions by TLR4 activation, we first developed a method to quantitatively measure the spreading response of bone marrow MØ after stimulation with LPS. Fluorescence microscopy was used for cell imaging and visualisation of the MØ contact area. In collaboration with the Fraunhofer Institute Erlangen, we developed and validated a software tool for the semi-automated segmentation and quantitation of MØ fluorescence microscopy data, which allowed fast, robust and objective image analysis. Using this method, we observed that LPS caused time-dependent spreading, which was detectable after 1-2 h and maximal after 24 h. Next, the impact of genetic or pharmacological inhibition of known TLR signaling components was investigated. Deficiency in the adapter protein MYD88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of ERK1/2 signaling, indicating that ERK1/2 mediates MYD88-dependent MØ spreading. In contrast, MØ lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation. The genetic deletion of the MAPK phosphatases DUSP1 and DUSP16 resulted in impaired late spreading, corroborating the essential role for functional MAPK signaling in TLR4-driven MØ spreading.
To identify the contribution of other cytoskeletal phosphoproteins to MØ spreading, siRNA knockdown of selected candidate genes in primary murine MØ was employed and combined with automated quantitative image analysis. These experiments revealed a functional role for the Myosins MYO1e and MYO1f in MØ spreading. These motor proteins are strongly phosphorylated in LPS-activated MØ. Because of their ability to simultaneously bind to actin filaments and cell membrane or other proteins, we investigated their role in phagocytosis, cytokine production and antigen presentation. Phagocytosis and killing of bacteria were not affected in Myo1e-/- macrophages. However, MYO1e plays a role in chemokine secretion and antigen presentation processes. MCP1 (CCL2) release was selectively increased in Myo1e-deficient MØ and dendritic cells (DC), while cytokine secretion was unaffected. Furthermore, macrophages and DCs lacking MYO1e showed lower levels of MHC-II on the cell surface. However, mRNA levels of CCL2 and of MHC-II were unaltered. These data suggest a role for MYO1e in the transport of selected chemokines and of MHC-II molecules to the cell surface. MHC-II-restricted antigen presentation assays revealed an impaired capacity of macrophages and DC lacking MYO1e to stimulate antigen-specific T cells, suggesting that the reduced MHC-II expression is functionally relevant.
Taken together, in this study first a quantitative image analysis method was developed which allows the unbiased, robust and efficient investigation of the macrophage spreading response. Combination of this method with siRNA knockdown of selected cytoskeleton-associated phosphoproteins led to the identification of MYO1e and MYO1f as regulators of macrophage spreading. Furthermore, we identified MYO1e in MØ and DC to be essential for the intracellular transport of CCL2 and MHC-II to the cell surface and for optimal stimulation of antigen-specific CD4 T cells.
Bees are subject to permanent threat from predators such as ants. Their nests with large quantities of brood, pollen and honey represent lucrative targets for attacks whereas foragers have to face rivalry at food sources. This thesis focused on the role of stingless bees as third party interactor on ant-aphid-associations as well as on the predatory potential represented by ants and defense mechanisms against this threat. Regular observations of an aphid infested Podocarpus for approaching stingless bees yielded no results. Another aim of this thesis was the observation of foraging habits of four native and one introduced ant species for assessment of their predatory potential to stingless bees. All species turned out to be dietary balanced generalists with one mostly carnivorous species and four species predominantly collecting nectar roughly according to optimal foraging theory. Two of the species monitored, Rhytidoponera metallica and Iridomyrmex rufoniger were considered potential nest robbers. As the name implies, stingless bees lack the powerful weapon of their distant relatives; hence they specialized on other defense strategies. Resin is an important, multipurpose resource for stingless bees that is used as material for nest construction, antibiotic and for defensive means. For the latter purpose highly viscous resin is either directly used to stick down aggressors or its terpenic compounds are included in the bees cuticular surface. In a feeding choice experiment, three ant species were confronted with the choice between two native bee species - Tetragonula carbonaria and Austroplebeia australis - with different cuticular profiles and resin collection habits. Two of the ant species, especially the introduced Tetramorium bicarinatum did not show any preferences. The carnivorous R. metallica predominantly took the less resinous A. australis as prey. The reluctance towards T. carbonaria disappeared when the resinous compounds on its cuticle had been washed off with hexane. To test whether the repulsive reactions were related to the stickiness of the resinous surface or to chemical substances, hexane extracts of bees’ cuticles, propolis and three natural tree resins were prepared. In the following assay responses of ants towards extract treated surfaces were observed. Except for one of the resin extracts, all tested substances had repellent effects to the ants. Efficacy varied with the type of extract and species. Especially to the introduced T. bicarinatum the cuticular extract had no effect. GCMS-analyses showed that some of the resinous compounds were also found in the cuticular profile of T. carbonaria which featured reasonable analogies to the resin of Corymbia torelliana that is highly attractive for stingless bees. The results showed that repellent effects were only partially related to the sticky quality of resin but were rather caused by chemical substances, presumably sesqui- and diterpenes. Despite its efficacy this defense strategy only provides short time repellent effects sufficient for escape and warning of nest mates to initiate further preventive measures.
Die Messung der Genexpression ist für viele Bereiche der Biologie und Medizin wichtig geworden und unterstützt Studien über Behandlung, Krankheiten und Entwicklungsstadien. Microarrays können verwendet werden, um die Expression von tausenden mRNA-Molekülen gleichzeitig zu messen und ermöglichen so einen Einblick und einen Vergleich der verschiedenen zellulären Bedingungen. Die Daten, die durch Microarray-Experimente gewonnen werden, sind hochdimensional und verrauscht, eine Interpretation der Daten ist deswegen nicht einfach. Obwohl Programme für die statistische Auswertung von Microarraydaten existieren, fehlt vielen eine Integration der Analyseergebnisse mit einer automatischen Interpretationsmöglichkeit. In dieser Arbeit wurde GEPAT, Genome Expression Pathway Analysis Tool, entwickelt, das eine Analyse der Genexpression unter dem Gesichtspunkten der Genomik, Proteomik und Metabolik ermöglicht. GEPAT integriert statistische Methoden zum Datenimport und -analyse mit biologischer Interpretation für Genmengen oder einzelne Gene, die auf dem Microarray gemessen werden. Verschiedene Typen von Oligonukleotid- und cDNAMicroarrays können importiert werden, unterschiedliche Normalisierungsmethoden können auf diese Daten angewandt werden, anschließend wird eine Datenannotation durchgeführt. Nach dem Import können mit GEPAT verschiedene statische Datenanalysemethoden wie hierarchisches, k-means und PCA-Clustern, ein auf einem linearen Modell basierender t-Test, oder ein Vergleich chromosomaler Profile durchgeführt werden. Die Ergebnisse der Analysen können auf Häufungen biologischer Begriffe und Vorkommen in Stoffwechselwegen oder Interaktionsnetzwerken untersucht werden. Verschiedene biologische Datenbanken wurden integriert, um zu jeder Gensonde auf dem Array Informationen zur Verfügung stellen zu können. GEPAT bietet keinen linearen Arbeitsablauf, sondern erlaubt die Benutzung von beliebigen Teilmengen von Genen oder biologischen Proben als Startpunkt einer neuen Analyse oder Interpretation. Dabei verlässt es sich auf bewährte Datenanalyse-Pakete, bietet einen modularen Ansatz zur einfachen Erweiterung und kann auf einem verteilten Computernetzwerk installiert werden, um eine große Zahl an Benutzern zu unterstützen. Es ist unter der LGPL Open-Source Lizenz frei verfügbar und kann unter http://gepat.sourceforge.net heruntergeladen werden.
Das maligne Melanom ist ein Hauttumor mit steigender Inzidenz und hohen Mortalitätsraten. Da die molekularbiologischen Ereignisse, die der Melanomentwicklung zugrundeliegen, nur unzureichend bekannt sind, gibt es kaum spezifische Therapieansätze. Zur Untersuchung der Melanomentwicklung eignet sich das Xiphophorus-Modell. In diesem System ist die Anwesenheit der RTK Xmrk ausreichend, um durch Aktivierung proliferativer und entdifferenzierender Signalwege und Apoptoseinhibition Melanome zu verursachen. Im Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass Xmrk auch die Migration der Melanozytenzellinie Melan a-Hm induzieren kann. Die Migration der durch Xmrk transformierten Zellen ist amöboid und unabhängig von MAPK- und PI3K-Signalwegen. Eine Funktion bei der Migration haben jedoch die Kinasen FAK und Fyn. Sie bilden möglicherweise einen Proteinkomplex, der für FAK und Src aus zahlreichen anderen Systemen bekannt ist und als Signalplattform für die Zellmigration fungiert. Diese Erkenntnisse können dazu beitragen, das Xiphophorus-Modell weiterzuentwickeln und die Grundlagen der Melanomgenese besser zu verstehen.
Chapter I
The gradual turnover of dead organic material into mineral nutrients is a key ecological function, linking decomposition and primary production, the essential parts of the nutrient-energy cycle. However, disturbances in terms of species or resource losses might impair the equilibrium between production and decomposition. Humanity has converted large proportions of natural landscapes and intensified land-use activity for food production. Globally, only very few areas are totally unaffected by human activity today.
To ensure the maintenance of both essential ecosystem services, knowledge about the interplay of biodiversity and ecosystem functioning as well as effects of intensified management on both is crucial. The vast majority of terrestrial biomass production as well as decomposition take place in forest ecosystems. Though forestry has a long sustainable history in Europe, its intensification during the last century has caused severe impacts on forest features and, consequently, on the associated biota, especially deadwood dependent organisms. Among these, saproxylic beetles are the most diverse group in terms of species numbers and functional diversity, but also most endangered due to habitat loss. These features classify them as ideal research organisms to study effects of intensified forestry on ecosystem services. The BELONGDEAD project located in Germany aimed to investigate deadwood decay and functional consequences of diversity changes in the associated fauna on the decomposition process from the initialisation of deadwood decay to complete degradation.
As part of the BeLongDead project, this dissertation focussed on saproxylic beetle species, thereby evaluating (1) regionally effects of tree species identity of fresh deadwood and (2) forest management of varying intensities on the diversity, abundance and community composition of saproxylic beetles (chapter II); (3) the specialisation degree of different trophic guilds of saproxylic beetles, and thus the stability and robustness of their interaction networks against disturbances (chapter III); (4) the impact of environmental features of local to regional spatial scales on species richness of saproxylic beetles differing in their habitat niche in terms of deadwood decay stages (chapter IV).
Chapter II
The vast majority of European forest ecosystems have been anthropogenically affected, leaving less than 1% of the about 1 milliard hectare as natural forests. A long history of forestry and especially the technological progress during the last century have caused massive habitat fragmentation as well as substantial loss of essential resources in European forest ecosystems. Due to this, the substrate-dependent group of saproxylic beetles has experienced severe species losses. Thus, investigations concerning saproxylic diversity and deadwood volume were badly needed. However, the importance of different deadwood in terms of tree species identity for the colonization by saproxylic beetles under different local and regional management regimes is poorly understood. Therefore, we studied possible regional differences in colonization patterns of saproxylic beetle species in a total of 688 fresh deadwood logs of 13 tree species in 9 sites of managed conifer and beech forests, and unmanaged beech forests, respectively. We found that tree species identity was an important driver in determining saproxylic species composition and abundance within fresh deadwood. However, saproxylic species showed different colonization patterns of deadwood items of the same tree species among the study regions. Regionally consistent, conifer forests were most diverse. We attribute the latter result to the historically adaption of saproxylic beetle species to semi-open forests, which conditions are actually best reflected by conifer forests. To preserve a diverse local species pool of early successional saproxylic beetles, we suggest an equal high degree of deadwood diversity in a tree species context in due consideration of regional differences.
Chapter III
The extinction risk of a particular species corresponds with its species-specific requirements on resources and habitat conditions, in other words with the width of the species` ecological niche. Species with a narrow ecological niche are defined as specialists. Members of this group experience higher extinction risk by resource limitation than generalists, which are able to utilize a variety of resources. For the classification of species as specialists or generalists, thus evaluating possible extinction risks, ecologists use the concept of interaction networks. This method has often been applied for mutualistic or antagonistic plant-animal interactions, but information for networks of detritivores is scarce. Therefore, saproxylic beetle species sampled as described in chapter II were categorised according to their larval diet; additionally their interaction networks (N=108) with 13 dead host tree species were analysed. Specialisation degree was highest for wood-digesting beetles and decreased with increasing trophic level. Also the network indices evaluating robustness and generality indicated a higher susceptibility to species extinctions for xylophagous than for mycetophagous and predatory beetles. The specialisation of xylophagous species on specific tree species might be an adaption to tree species specific ingredients stored for defence against pathogens and pests. However, we conclude that the high specialisation degree of xylophages and thus their higher extinction risk by resource loss harbours certain dangers for ecosystem function and stability as species diversity is positively linked to both.
Chapter IV
Populations depend on individual emigration and immigration events to ensure genetic exchange. For successful migration it is of utmost importance that spatially separated populations are obtainable by specimen. Migratory success depends on the one hand on the species dispersal abilities and on the other on the availability of suitable habitats in the surrounding landscape in which the distinct host populations exist. However, consequences of intensive forest management correspond not only to severe reduction of local deadwood amount, but, among others, also a change in tree species composition and high levels of fragmentation in the surrounding forest area. Saproxylic beetle species differ in their dispersal behaviour according to the temporal availability of their preferred habitat. Generally, early successional saproxylic beetles are able to disperse over large distances, whereas beetles inhabiting advanced decayed wood often remain close to their larval habitat. Due to this, environmental factors might affect saproxylic beetle guilds differently. We classified the saproxylic beetles sampled as described in chapter II according to their calculated habitat niche as early, intermediate or late successional saproxylic beetles. For the different guilds the effects of 14 environmental factors on different spatial scales (stand factors at 0.1 km radius, landscape composition at 2 km radius, and regionally differing abiotic factors in 400 km to 700 km distance) were investigated. Consistently for all guilds, species richness decreased with fragmentation at local and landscape scale, and increased in warmer climate. However, we found contradictory results between the guilds to some extent. We relate this to guild specific habitat requirements of the saproxylic beetles. Therefore, for the development of appropriate conservation practices guild-specific requirements saproxylic beetles have to be considered not only locally but on larger spatial scales.
Chapter V
In conclusion, this dissertation identified main drivers of early successional saproxylic beetle species richness on various spatial scales. Our results emphasize the importance to develop management schemes meeting species-specific and guild-specific habitat requirements of the saproxylic beetle fauna at relevant spatial and temporal scales. Therefore, short-term actions suggested for sustainable forest management should be the focus on a diverse tree species composition consisting of indigenous tree species with respect to regional differences. Moreover, senescent trees, fallen and standing deadwood should remain in the forests, and some tree individuals should be allowed to grow old. Long-term actions should involve the reduction of forest fragmentation and the connection of spatial widely separated forest fragments. Furthermore, to fully understand the effects of forest management long-term research should be conducted to compare habitat requirements of intermediate and late successional beetles with the results presented in this dissertation.
(1) Background: about 10% of Wilms Tumor (WT) patients have a malformation or cancer predisposition syndrome (CPS) with causative germline genetic or epigenetic variants. Knowledge on CPS is essential for genetic counselling. (2) Methods: this retrospective analysis focused on 2927 consecutive patients with WTs registered between 1989 and 2017 in the SIOP/GPOH studies. (3) Results: Genitourinary malformations (GU, N = 66, 2.3%), Beckwith-Wiedemann spectrum (BWS, N = 32, 1.1%), isolated hemihypertrophy (IHH, N = 29, 1.0%), Denys-Drash syndrome (DDS, N = 24, 0.8%) and WAGR syndrome (N = 20, 0.7%) were reported most frequently. Compared to others, these patients were younger at WT diagnosis (median age 24.5 months vs. 39.0 months), had smaller tumors (349.4 mL vs. 487.5 mL), less often metastasis (8.2% vs. 18%), but more often nephroblastomatosis (12.9% vs. 1.9%). WT with IHH was associated with blastemal WT and DDS with stromal subtype. Bilateral WTs were common in WAGR (30%), DDS (29%) and BWS (31%). Chemotherapy induced reduction in tumor volume was poor in DDS (0.4% increase) and favorable in BWS (86.9% reduction). The event-free survival (EFS) of patients with BWS was significantly (p = 0.002) worse than in others. (4) Conclusions: CPS should be considered in WTs with specific clinical features resulting in referral to a geneticist. Their outcome was not always favorable.
We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming.
Spir proteins are the founding members of the novel class of WH2-actin nucleators. A C-terminal modified FYVE zinc finger motif is necessary to target Spir proteins towards intracellular membranes. The function and regulation of the Spir actin organizers at vesicular membranes is almost unknown. Live cell imaging analyses performed in this study show that Spir-2 is localized at tubular vesicles. Cytoplasmic Spir-2-associated vesicles branch and form protrusions, which can make contacts to the microtubule network, where the Spir-2 vesicles stretch and slide along the microtubule filaments. The analysis of living HeLa cells expressing eGFP-tagged Spir-2, Spir-2-ΔKIND and Spir-2-ΔKW (lacking the 4 WH2 domains and the KIND domain) showed Spir-2-associated tubular structures which differ in their length and motility. Throughout the course of that study it could be shown that the tail domain of the actin motor protein myosin Vb, as a force-generating molecule, is colocalizing and co-immunoprecipitating with Spir-2-ΔKW. By using the tail domain of myosin Vb as a dominant negative mutant for myosin Vb-dependent vesicle transport processes it could be shown that Spir-2-ΔKW/MyoVb-cc-tail- associated vesicles exhibit an increased elongation. Moreover, using the microtubule depolymerizing drug nocodazole it could be shown that the elongation and the motility of Spir-2-ΔKW-associated vesicles depends on an intact microtubule cytoskeleton. Motility and morphological dynamics of Spir-2-associated vesicles is therefore dependent on actin, actin motorproteins and microtubule filaments. These results propose a model in which myosin/F-actin forces mediate vesicle branching, allowing the vesicles to move to and in between the microtubule filaments and thereby providing a new degree of freedom in vesicular motility. To determine the exact subcellular localization of Spir-2, colocalization studies were performed. It could be shown that Spir-2 shows a partial colocalization to Rab11a-positive compartments. Furthermore, Spir-2 exhibits an almost identical localization to Arf1 and the Arf1 small G protein but not Rab11a could be immunoprecipitated with Spir-2-ΔKW. This suggests, that Arf1 recruits Spir-2 to Arf1/Rab11a-positive membranes. Another important function of the Spir-2 C-terminus is the membrane targeting by the FYVE domain. By performing a protein-lipid overlay assay, it has been shown that purified GST- and 6xHis-tagged Spir-2-ΔKW bind phosphatidic acid suggesting a mechanism in which Spir-2 is recruited to phosphatidic acid-enriched membranes. To further elucidate the mechanism in which Spir-2 membrane-targeting could be regulated, interaction studies of C-terminal parts of Spir-2 revealed that the Spir-2 proteins interact directly.
Background
The actin cytoskeleton is a hallmark of eukaryotic cells. Its regulation as well as its interaction with other proteins is carefully orchestrated by actin interaction domains. One of the key players is the WH2 motif, which enables binding to actin monomers and filaments and is involved in the regulation of actin nucleation. Contrasting conserved domains, the identification of this motif in protein sequences is challenging, as it is short and poorly conserved.
Findings
To identify divergent members, we combined Hidden-Markov-Model (HMM) to HMM alignments with orthology predictions. Thereby, we identified nearly 500 proteins containing so far not annotated WH2 motifs. This included shootin-1, an actin binding protein involved in neuron polarization. Among others, WH2 motifs of ‘proximal to raf’ (ptr)-orthologs, which are described in the literature, but not annotated in genome databases, were identified.
Conclusion
In summary, we increased the number of WH2 motif containing proteins substantially. This identification of candidate regions for actin interaction could steer their experimental characterization. Furthermore, the approach outlined here can easily be adapted to the identification of divergent members of further domain families.
Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing
(2016)
Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.
A cytochrome b preparation from Neurospora crassa mitochondria is found to consist of three polypeptides (apparent molecular weight 10 000, 11 000 and 32 000), a cytochrome aa3 preparation of six to seven polypeptides (apparent molecular weight 8 000, 11 000, 13 000, 18 000, 28 000 and 36 000). Selective incorporation of radioactive amino acids by eilher mitochondrial protein synthesis when the cytoplasmic one is blocked or by the cytoplasmic protein synthesis, when the mitochondrial one is blocked, indicates that one cytochrome b polypeptide (mw 32 000) and one to three cytochrome aa3 polypeptides (mw 36 000, 28 000 and 18 000) are mitochondrial translation products, the other cytochrome b and cytochrome aa3 polypeptides cytoplasmic translation products. The delayed appearance of labeling in the cytochrome b and cytochrome aa3 polypeptides compared to the average cell protein after a pulse of <~H leueine revealed that these polypeptides are derived from separate pools of precursor polypeptides. The pool sizes range from 2 p. cent to 25 p. cent of the amount of the corresponding polypeptide present in the cytochromes. The 32 000 molecular weight polypeptide of cytochrome band at least the 18 000 molecular weight polypeptide of cytochrome aa\(_3\) are mitochondrial translation products as well in the fungus Neurospora crassa as in the insect Locusta migratoria. So, despite the fact that the size of mitochondrial DNA and mitochondrial ribosomes is reduced in insects, the products have maintained their characteristics.
Radioaetive leueine was ineorporated by N eurospora crassa mitoehondria in vivo in the presence of cyeloheximide. When the membrane protein of these mitochondria was ehromatographieally separated on oleyl polymethaerylie aeid resin, & nurober of fraetions were obtained whieh differ with respeet to their eontents of radioaetivity and eytoehromes. The highest speeifie radioaetivity was found in the fraction eontaining eytoehrome aa3• This fraetion proved to be a pure and enzymatically aetive cytoehrome oxidase. Its ratio of absorbanee at 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodeeylsulfate gel-electrophoresis, this enzymewas separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioaetivity.
A chromatographic procedure 1 is described by means of which cytochrome oxidase has been purified from a variety of organisms including the fungus N eurospora crassa,2,3 the unicellular alga Po/ytoma mirum, 4 the insect Locusta migratoria ,5 the frog Xenopus muel/eri,4 and the mammal Rattus norwegicus. 4 This procedure can be used to equal effect for large-scale preparations, starting from grams of mitochondrial protein, or for small-scale preparations starting from milligrams. The cytochrome oxidase preparations from the different organisms are enzymically active. They show similar subunit compositions.
Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56\(^{bright}\)CD16\(^{dim}\) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
In der vorliegenden Arbeit wurde nach Wegen gesucht, den Import peroxisomaler Matrixproteine, der über ein peroxisomales Targeting Signal Typ 2 gesteuert wird, zu messen. Es war vorgesehen, in erster Linie einen enzymchemischen Nachweis zu etablieren, da diese Methode den Vorteil einer Quantifizierbarkeit der Aktivität der gemessenen Enzyme bietet und somit Rückschlüsse auf den Grad einer Beeinträchtigung des Importes zulassen würden. Von dem Test wurde eine Sensitivität gefordert, die eine Messung auch in Homogenaten kultivierter Zellen, insbesondere von CHO-Zellen, erlaubt. Dieses war deswegen gefordert, weil der Test zur Charakterisierung induzierter CHO-Zell-Mutanten eingesetzt werden sollte, die die Merkmale eines PTS 2-Import-Defektes aufweisen. Dieser Nachweis sollte durch eine Messung der Phytanoyl-CoA-Hydroxylase erfolgen. Dieses Enzym ist eines von drei derzeit bekannten Proteinen, die eine PTS 2 besitzen und über diesen Weg importiert werden. Das Substrat für die Hydroxylase war als Phytansäure mit einer 2,3-3H-Markierung in der Arbeitsgruppe vorrätig und wurde für den Test zum CoA-Thioester chemisch umgesetzt. Nach erfolgter enzymatischer Umsetzung von Phytonoyl-CoA zu a-Hydroxyphytanoyl-CoA durch die Hydroxylase waren dann sowohl Edukt wie auch das Produkt durch eine radioaktive Markierung gekennzeichnet und konnten nach einer dünnschicht-chromatographischen Trennung über Kieselgel durch einem Radiodünnschichtscanner nachgewiesen werden. Zunächst wurde mit Hilfe von Homogenaten aus Rattenlebergewebe ein bereits beschriebenes Verfahren zur Messung der Phytanoyl-CoA-Hydroxylase optimiert. Es stellte sich jedoch heraus, daß die Sensitivität dieses Testes nicht hoch genug ist, um die Hydroxylase-Aktivität in Homogenaten kultivierter CHO-Zellen zu messen. An dieser Stelle wurde die Etablierung eines immunchemischen Nachweises begonnen. Hierzu sollten Antikörper gegen die Hydroxylase des chinesischen Zwerghamsters, des Ursprungsorganismus der CHO-Zellen, generiert werden. Eine Reinigung des Enzyms kam nicht in Betracht, weil die Hamster nicht im Labortierhandel erhältlich waren. Folglich musste die cDNA der Hydroxylase aus einer Hamster-cDNA-Bank kloniert werden, nachdem sie durch ihre bekannten Homologe aus Mensch und Maus identifizierbar war. In den verfügbaren cDNA-Banken fand sich keine vollständige Sequenz, so daß mit einer partiellen Sequenz ohne 5´-Ende weitergearbeitet werden musste. Es bot sich im Institut die Möglichkeit, aus dieser Sequenz Pepetide zu bestimmen, die mit hoher Wahrscheinlichkeit stark immunogen wirken. Solche Peptide wurden synthetisiert und nach Koppelung an Trägerproteine neuseeländischen weißen Kaninchen geimpft. Im Elisa wies das Antiserum zum Zeitpunkt seiner Gewinnung einen Titer von etwa 1:10000 auf, zeigte aber im Westernblot neben einer starken Detektion in Laufweite der Hydroxylase auch eine unspezifische Anfärbung der Proben. In der nun durchgeführten Affinitätsreinigung des Antiserums über einer mit den antigenen Peptiden beladenen Säule tauchte das Problem auf, daß die Antikörper so fest binden, daß sie von ihren Antigenen nicht mehr ohne dentaturierende Bedingungen zu lösen waren. Für die weitere Arbeit sollte sich nun eine affinitätschromatographische Reinigung über Peptide, die den Antikörper mit geringerer Avidität binden, anschließen, so daß nach Trennung der Immunkomplexe native Antikörper isoliert werden könnten. Hierzu wäre ein Epitop-mapping wünschenswert, damit auf dieser Grundlage Peptide mit den geforderten Eigenschaften synthetisiert werden können.
No abstract available
The unicellular pathogen Trypanosoma brucei is the causative agent of African
trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream
form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF.
In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis.
The social organization of insect colonies has long fascinated naturalists. One of the main features of colony organization is division of labor, whereby each member of the colony specializes in a subset of all tasks required for successful group functioning. The most striking aspect of division of labor is its plasticity: workers switch between tasks in response to external challenges and internal perturbations. The mechanisms underlying flexible division of labor are far from being understood. In order to comprehend how the behavior of individuals gives rise to flexible collective behavior, several questions need to be addressed: We need to know how individuals acquire information about their colony's current demand situation; how they then adjust their behavior according; and which mechanisms integrate dozens or thousands of insect into a higher-order unit. With these questions in mind I have examined two examples of collective and flexible behavior in social bees. First, I addressed the question how a honey bee colony controls its pollen collection. Pollen foraging in honey bees is precisely organized and carefully regulated according to the colony's needs. How this is achieved is unclear. I investigated how foragers acquire information about their colony's pollen need and how they then adjust their behavior. A detailed documentation of pollen foragers in the hive under different pollen need conditions revealed that individual foragers modulate their in-hive working tempo according to the actual pollen need of the colony: Pollen foragers slowed down and stayed in the hive longer when pollen need was low and spent less time in the hive between foraging trips when pollen need of their colony was high. The number of cells inspected before foragers unloaded their pollen load did not change and thus presumably did not serve as cue to pollen need. In contrast, the trophallactic experience of pollen foragers changed with pollen need conditions: trophallactic contacts were shorter when pollen need was high and the number and probability of having short trophallactic contacts increased when pollen need increased. Thus, my results have provided support for the hypothesis that trophallactic experience is one of the various information pathways used by pollen foragers to assess their colony's pollen need. The second example of collective behavior I have examined in this thesis is the control of nest climate in bumble bee colonies, a system differing from pollen collection in honey bees in that information about task need (nest climate parameters) is directly available to all workers. I have shown that an increase in CO2 concentration and temperature level elicits a fanning response whereas an increase in relative humidity does not. The fanning response to temperature and CO2 was graded; the number of fanning bees increased with stimulus intensity. Thus, my study has evidenced flexible colony level control of temperature and CO2. Further, I have shown that the proportion of total work force a colony invests into nest ventilation does not change with colony size. However, the dynamic of the colony response changes: larger colonies show a faster response to perturbations of their colony environment than smaller colonies. Thus, my study has revealed a size-dependent change in the flexible colony behavior underlying homeostasis. I have shown that the colony response to perturbations in nest climate is constituted by workers who differ in responsiveness. Following a brief review of current ideas and models of self-organization and response thresholds in insect colonies, I have presented the first detailed investigation of interindividual variability in the responsiveness of all workers involved in a collective behavior. My study has revealed that bumble bee workers evidence consistent responses to certain stimulus levels and differ in their response thresholds. Some consistently respond to low stimulus intensities, others consistently respond to high stimulus intensities. Workers are stimulus specialists rather than task specialists. Further, I have demonstrated that workers of a colony differ in two other parameters of responsiveness: response probability and fanning activity. Response threshold, response probability and fanning activity are independent parameters of individual behavior. Besides demonstrating and quantifying interindividual variability, my study has provided empirical support for the idea of specialization through reinforcement. Response thresholds of fanning bees decreased over successive trials. I have discussed the importance of interindividual variability for specialization and the collective control of nest climate and present a general discussion of self-organization and selection. This study contributes to our understanding of individual behavior and collective structure in social insects. A fascinating picture of social organization is beginning to emerge. In place of centralized systems of communication and information transmission, insect societies frequently employ mechanisms based upon self-organization. Self-organization promises to be an important and unifying principle in physical, chemical and biological systems.
A cDNA clone of about 2500 basepairswas prepared from the human osteosarcoma cellline U-2 OS by hybridizing with a v-sis probe. Sequence analysis showed that this cDNA contains the coding region for the PDGF-B chain. Here we report that the mitogen secreted by these osteosarcoma cells contains the PDGF-B chain and is probably a homodimer of two B-chains.
Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors.
TP53 mutations have been associated with anaplasia in Wilms tumour, which conveys a high risk for relapse and fatal outcome. Nevertheless, TP53 alterations have been reported in no more than 60% of anaplastic tumours, and recent data have suggested their presence in tumours that do not fulfil the criteria for anaplasia, questioning the clinical utility of TP53 analysis. Therefore, we characterized the TP53 status in 84 fatal cases of Wilms tumour, irrespective of histological subtype. We identified TP53 alterations in at least 90% of fatal cases of anaplastic Wilms tumour, and even more when diffuse anaplasia was present, indicating a very strong if not absolute coupling between anaplasia and deregulation of p53 function. Unfortunately, TP53 mutations do not provide additional predictive value in anaplastic tumours since the same mutation rate was found in a cohort of non-fatal anaplastic tumours. When classified according to tumour stage, patients with stage I diffuse anaplastic tumours still had a high chance of survival (87%), but this rate dropped to 26% for stages II–IV. Thus, volume of anaplasia or possible spread may turn out to be critical parameters. Importantly, among non-anaplastic fatal tumours, 26% had TP53 alterations, indicating that TP53 screening may identify additional cases at risk. Several of these non-anaplastic tumours fulfilled some criteria for anaplasia, for example nuclear unrest, suggesting that such partial phenotypes should be under special scrutiny to enhance detection of high-risk tumours via TP53 screening. A major drawback is that these alterations are secondary changes that occur only later in tumour development, leading to striking intratumour heterogeneity that requires multiple biopsies and analysis guided by histological criteria. In conclusion, we found a very close correlation between histological signs of anaplasia and TP53 alterations. The latter may precede development of anaplasia and thereby provide diagnostic value pointing towards aggressive disease.
Retinoic acid pathway activity in Wilms tumors and characterization of biological responses in vitro
(2011)
Background: Wilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear. Results: The association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/ intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however. Conclusions: Altered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.
Der Wilms Tumor (WT), auch Nephroblastom genannt, ist einer der häufigsten bösartigen Tumoren im Kindesalter. Er entsteht aus embryonalem undifferenziertem Nierengewebe und tritt meist als unilateraler und sporadischer Tumor auf. In 10-15% der Wilms Tumoren finden sich WT1- und/oder CTNNB1-Mutationen. Während diese schon länger als genetische Ursachen des Nephroblastoms bekannt sind, wurde erst kürzlich WTX als drittes Gen beschrieben, welches eine Rolle in der Tumorentstehung spielt. Für einen Großteil der WT ist die genetische Ursache jedoch unklar. Da die bisher publizierten WTX-Mutationsraten auf Untersuchungen kleiner Gruppen basieren und sich stark unterscheiden, sollten in dieser Arbeit WTX-, CTNNB1- und WT1-Mutationen in einem großen WT-Set bestimmt werden. Verluste genetischen Materials in der WTX-Region traten in 17% der Fälle auf und waren zwischen den Geschlechtern gleich verteilt. Die Sequenzierung von WT-Proben zeigte, dass nur 2% von WTX-Punktmutationen betroffen sind. In weiteren 11,5% der Proben konnte keine WTX-Expression nachgewiesen werden. Die WTX-Veränderungen traten z. T. gemeinsam mit WT1- und/oder CTNNB1-Mutationen auf. Die unvollständige WTX-Deletion in einigen WT legte die Vermutung nahe, dass innerhalb eines Tumors eine Heterogenität in Bezug auf den WTX-Status möglich ist. Dieser Verdacht konnte durch die detaillierte Untersuchung verschiedener Regionen solcher Tumoren erhärtet werden: Hierzu wurden histologisch unterschiedliche Bereiche auf den Anteil einer WTX-Mutation bzw. eines WTX-LOH hin untersucht. Obwohl alle Regionen des jeweiligen Tumors einen kompletten LOH auf Chromosom 11 aufwiesen, waren die WTX-Veränderungen unterschiedlich stark ausgeprägt. Diese Ergebnisse deuten darauf hin, dass WTX-Veränderungen keine notwendigen und frühen Ereignisse in der Tumorentstehung sind, sondern erst später auftreten und nur einen Teil der Tumorzellen betreffen können. Die Vermutung, dass WTX-Mutationen keinen direkten Einfluss auf die Tumorentwicklung und prognose haben, wird durch das Fehlen eines signifikanten Zusammenhangs zwischen WTX-Deletion bzw. WTX-Expression und den klinischen Eigenschaften der WT gestützt. Um die Rolle von Genen, die potentiell an der Entstehung und Entwicklung des Nephroblastoms beteiligt sind, zu untersuchen oder mögliche neue Therapiestrategien zu überprüfen, sind in vitro-Modelle nötig. Da ein solches für Wilms Tumoren nicht etabliert ist, wurden Primärkulturen aus verschiedenen WT-Proben angelegt. Kulturen aus Tumorgewebe von 12 Patienten mit unterschiedlichen genetischen Veränderungen konnten als echte Tumorzellen validiert werden. Zwei Zelltypen ließen sich morphologisch und immunhistochemisch unterscheiden: Zum einen runde, langsam wachsende Zellen mit Epithelcharakter und zum anderen fibroblastenähnliche Zellen, welche weniger differenziert waren und häufig für viele Passagen kultiviert werden konnten. Somit wurde ein Set verschiedener WT-Primärkulturen etabliert, welches nun für in vitro-Experimente zur Untersuchung grundlegender Mechanismen der WT-Entstehung oder zum Test neuer Therapieansätze eingesetzt werden kann. Frühere Microarray-Analysen deuteten auf eine Deregulation des Retinsäure (RA)-Signalwegs in fortgeschrittenen Wilms Tumoren hin. Diese Ergebnisse sollten in einem großen unabhängigen Proben-Set mittels Realtime-RT-PCR validiert werden. Eine Deregulation des RA-Signalwegs und die Überexpression von NMYC wurden für Tumoren der Hochrisikogruppe im Vergleich zu Tumoren mit geringem/mittlerem Risiko nachgewiesen. So stellte sich die Frage, ob Patienten mit fortgeschrittenem WT von einem Retinsäure-Einsatz in der Therapie profitieren könnten. Um dies zu beantworten, wurde der Effekt von verschiedenen Retinoiden auf WT-Primärkulturen untersucht. Die WT-Zellen wurden mit all-trans RA (ATRA), 9cisRA, dem synthetischen Retinoid Fenretinid (4HPR) und Kombinationen von ATRA bzw. 4HPR und einem HDAC-Inhibitor (SAHA) behandelt. Gene, welche in Hochrisiko-WT differenziell reguliert waren, wurden untersucht und zeigten nach RA-Behandlung eine entgegengesetzte Expression. In sechs der sieben verwendeten Primärkulturen wurde eine RA-vermittelte Proliferationsreduktion nachgewiesen. Für die Kombinationen von Retinoiden mit SAHA wurden keine synergistischen Effekte beobachtet. Während Fenretinid in den meisten Kulturen Apoptose induzierte, verursachten ATRA und 9cisRA morphologische Veränderungen, welche auf Differenzierungsvorgänge hindeuteten. Eine Microarray-Analyse ATRA-behandelter WT-Zellen zeigte die differenzielle Regulation vieler Gene, welche eine Rolle in der Bildung der extrazellulären Matrix oder bei Differenzierungsvorgängen von Knochen-, Knorpel-, Nerven- oder Muskelgewebe spielen. Diese Befunde bieten einen weiteren Hinweis darauf, dass Retinoide für den Einsatz in der Therapie des Nephroblastoms geeignet sein könnten.
Peptidergic neurons are not easily integrated into current connectomics concepts, since their peptide messages can be distributed via non-synaptic paracrine signaling or volume transmission. Moreover, the polarity of peptidergic interneurons in terms of in- and out-put sites can be hard to predict and is very little explored. We describe in detail the morphology and the subcellular distribution of fluorescent vesicle/dendrite markers in CCAP neurons (NCCAP), a well defined set of peptidergic neurons in the Drosophila larva. NCCAP can be divided into five morphologically distinct subsets. In contrast to other subsets, serial homologous interneurons in the ventral ganglion show a mixed localization of in- and output markers along ventral neurites that defy a classification as dendritic or axonal compartments. Ultrastructurally, these neurites contain both pre- and postsynaptic sites preferably at varicosities. A significant portion of the synaptic events are due to reciprocal synapses. Peptides are mostly non-synaptically or parasynaptically released, and dense-core vesicles and synaptic vesicle pools are typically well separated. The responsiveness of the NCCAP to ecdysis-triggering hormone may be at least partly dependent on a tonic synaptic inhibition, and is independent of ecdysteroids. Our results reveal a remarkable variety and complexity of local synaptic circuitry within a chemically defined set of peptidergic neurons. Synaptic transmitter signaling as well as peptidergic paracrine signaling and volume transmission from varicosities can be main signaling modes of peptidergic interneurons depending on the subcellular region. The possibility of region-specific variable signaling modes should be taken into account in connectomic studies that aim to dissect the circuitry underlying insect behavior and physiology, in which peptidergic neurons act as important regulators.
Neuropeptides have gained broad attraction in insect neuroscience and physiology, as new genetic tools are increasingly uncovering their wide-ranging pleiotropic functions with high cellular resolution. Allatostatin A (AstA) peptides constitute one of the best studied insect neuropeptide families. In insects and other panarthropods, AstA peptides qualify as brain-gut peptides and have regained attention with the discovery of their role in regulating feeding, growth, activity/sleep and learning. AstA receptor homologs are found throughout the protostomia and group with vertebrate somatostatin/galanin/kisspeptin receptors. In this review, we summarise the current knowledge on the evolution and the pleiotropic and cell-specific non-allatostatic functions of AstA. We speculate about the core functions of AstA signalling, and derive open questions and challengesfor future research on AstA and invertebrate neuropeptides in general.