TY - JOUR A1 - Adolfi, Mateus C. A1 - Carreira, Ana C. O. A1 - Jesus, Lázaro W. O. A1 - Bogerd, Jan A1 - Funes, Rejane M. A1 - Schartl, Manfred A1 - Sogayar, Mari C. A1 - Borella, Maria I. T1 - Molecular cloning and expression analysis of dmrt1 and sox9 during gonad development and male reproductive cycle in the lambari fish, Astyanax altiparanae JF - Reproductive Biology and Endocrinology N2 - Background The dmrt1 and sox9 genes have a well conserved function related to testis formation in vertebrates, and the group of fish presents a great diversity of species and reproductive mechanisms. The lambari fish (Astyanax altiparanae) is an important Neotropical species, where studies on molecular level of sex determination and gonad maturation are scarce. Methods Here, we employed molecular cloning techniques to analyze the cDNA sequences of the dmrt1 and sox9 genes, and describe the expression pattern of those genes during development and the male reproductive cycle by qRT-PCR, and related to histology of the gonad. Results Phylogenetic analyses of predicted amino acid sequences of dmrt1 and sox9 clustered A. altiparanae in the Ostariophysi group, which is consistent with the morphological phylogeny of this species. Studies of the gonad development revealed that ovary formation occurred at 58 days after hatching (dah), 2 weeks earlier than testis formation. Expression studies of sox9 and dmrt1 in different tissues of adult males and females and during development revealed specific expression in the testis, indicating that both genes also have a male-specific role in the adult. During the period of gonad sex differentiation, dmrt1 seems to have a more significant role than sox9. During the male reproductive cycle dmrt1 and sox9 are down-regulated after spermiation, indicating a role of these genes in spermatogenesis. Conclusions For the first time the dmrt1 and sox9 were cloned in a Characiformes species. We show that both genes have a conserved structure and expression, evidencing their role in sex determination, sex differentiation and the male reproductive cycle in A. altiparanae. These findings contribute to a better understanding of the molecular mechanisms of sex determination and differentiation in fish. KW - spermatogenesis KW - SOX9 KW - DMRT1 KW - sex differentiation KW - teleostei Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126486 VL - 13 IS - 2 ER - TY - JOUR A1 - Alizadehrad, Davod A1 - Krüger, Timothy A1 - Engstler, Markus A1 - Stark, Holger T1 - Simulating the complex cell design of Trypanosoma brucei and its motility JF - PLOS Computational Biology N2 - The flagellate Trypanosoma brucei, which causes the sleeping sickness when infecting a mammalian host, goes through an intricate life cycle. It has a rather complex propulsion mechanism and swims in diverse microenvironments. These continuously exert selective pressure, to which the trypanosome adjusts with its architecture and behavior. As a result, the trypanosome assumes a diversity of complex morphotypes during its life cycle. However, although cell biology has detailed form and function of most of them, experimental data on the dynamic behavior and development of most morphotypes is lacking. Here we show that simulation science can predict intermediate cell designs by conducting specific and controlled modifications of an accurate, nature-inspired cell model, which we developed using information from live cell analyses. The cell models account for several important characteristics of the real trypanosomal morphotypes, such as the geometry and elastic properties of the cell body, and their swimming mechanism using an eukaryotic flagellum. We introduce an elastic network model for the cell body, including bending rigidity and simulate swimming in a fluid environment, using the mesoscale simulation technique called multi-particle collision dynamics. The in silico trypanosome of the bloodstream form displays the characteristic in vivo rotational and translational motility pattern that is crucial for survival and virulence in the vertebrate host. Moreover, our model accurately simulates the trypanosome's tumbling and backward motion. We show that the distinctive course of the attached flagellum around the cell body is one important aspect to produce the observed swimming behavior in a viscous fluid, and also required to reach the maximal swimming velocity. Changing details of the flagellar attachment generates less efficient swimmers. We also simulate different morphotypes that occur during the parasite's development in the tsetse fly, and predict a flagellar course we have not been able to measure in experiments so far. KW - multiparticle collision dynamics KW - human african trypanosomiasis KW - biology KW - cytoskeleton KW - flow KW - flagellar motility KW - tsetse fly KW - propulsion KW - cytokinesis KW - parasites Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144610 VL - 11 IS - 1 ER - TY - JOUR A1 - Andronic, Joseph A1 - Shirakashi, Ryo A1 - Pickel, Simone U. A1 - Westerling, Katherine M. A1 - Klein, Teresa A1 - Holm, Thorge A1 - Sauer, Markus A1 - Sukhorukov, Vladimir L. T1 - Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy JF - PLoS One N2 - Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells. KW - electrolytes KW - isotonic KW - membrane proteins KW - cell membranes KW - hypotonic KW - hypotonic solutions KW - tonicity KW - permeability Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126408 VL - 10 IS - 3 ER - TY - JOUR A1 - Appel, Mirjam A1 - Scholz, Claus-Jürgen A1 - Müller, Tobias A1 - Dittrich, Marcus A1 - König, Christian A1 - Bockstaller, Marie A1 - Oguz, Tuba A1 - Khalili, Afshin A1 - Antwi-Adjei, Emmanuel A1 - Schauer, Tamas A1 - Margulies, Carla A1 - Tanimoto, Hiromu A1 - Yarali, Ayse T1 - Genome-Wide Association Analyses Point to Candidate Genes for Electric Shock Avoidance in Drosophila melanogaster JF - PLoS ONE N2 - Electric shock is a common stimulus for nociception-research and the most widely used reinforcement in aversive associative learning experiments. Yet, nothing is known about the mechanisms it recruits at the periphery. To help fill this gap, we undertook a genome-wide association analysis using 38 inbred Drosophila melanogaster strains, which avoided shock to varying extents. We identified 514 genes whose expression levels and/or sequences covaried with shock avoidance scores. We independently scrutinized 14 of these genes using mutants, validating the effect of 7 of them on shock avoidance. This emphasizes the value of our candidate gene list as a guide for follow-up research. In addition, by integrating our association results with external protein-protein interaction data we obtained a shock avoidance- associated network of 38 genes. Both this network and the original candidate list contained a substantial number of genes that affect mechanosensory bristles, which are hairlike organs distributed across the fly's body. These results may point to a potential role for mechanosensory bristles in shock sensation. Thus, we not only provide a first list of candidate genes for shock avoidance, but also point to an interesting new hypothesis on nociceptive mechanisms. KW - functional analysis KW - disruption project KW - natural variation KW - complex traits KW - networks KW - behavior KW - flies KW - temperature KW - genetics KW - painful Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-152006 VL - 10 IS - 5 ER - TY - JOUR A1 - Basset, Yves A1 - Cizek, Lukas A1 - Cuénoud, Philippe A1 - Didham, Raphael K. A1 - Novotny, Vojtech A1 - Ødegaard, Frode A1 - Roslin, Tomas A1 - Tishechkin, Alexey K. A1 - Schmidl, Jürgen A1 - Winchester, Neville N. A1 - Roubik, David W. A1 - Aberlenc, Henri-Pierre A1 - Bail, Johannes A1 - Barrios, Hector A1 - Bridle, Jonathan R. A1 - Castaño-Meneses, Gabriela A1 - Corbara, Bruno A1 - Curletti, Gianfranco A1 - da Rocha, Wesley Duarte A1 - De Bakker, Domir A1 - Delabie, Jacques H. C. A1 - Dejean, Alain A1 - Fagan, Laura L. A1 - Floren, Andreas A1 - Kitching, Roger L. A1 - Medianero, Enrique A1 - de Oliveira, Evandro Gama A1 - Orivel, Jerome A1 - Pollet, Marc A1 - Rapp, Mathieu A1 - Ribeiro, Servio P. A1 - Roisin, Yves A1 - Schmidt, Jesper B. A1 - Sørensen, Line A1 - Lewinsohn, Thomas M. A1 - Leponce, Maurice T1 - Arthropod Distribution in a Tropical Rainforest: Tackling a Four Dimensional Puzzle JF - PLoS ONE N2 - Quantifying the spatio-temporal distribution of arthropods in tropical rainforests represents a first step towards scrutinizing the global distribution of biodiversity on Earth. To date most studies have focused on narrow taxonomic groups or lack a design that allows partitioning of the components of diversity. Here, we consider an exceptionally large dataset (113,952 individuals representing 5,858 species), obtained from the San Lorenzo forest in Panama, where the phylogenetic breadth of arthropod taxa was surveyed using 14 protocols targeting the soil, litter, understory, lower and upper canopy habitats, replicated across seasons in 2003 and 2004. This dataset is used to explore the relative influence of horizontal, vertical and seasonal drivers of arthropod distribution in this forest. We considered arthropod abundance, observed and estimated species richness, additive decomposition of species richness, multiplicative partitioning of species diversity, variation in species composition, species turnover and guild structure as components of diversity. At the scale of our study (2km of distance, 40m in height and 400 days), the effects related to the vertical and seasonal dimensions were most important. Most adult arthropods were collected from the soil/litter or the upper canopy and species richness was highest in the canopy. We compared the distribution of arthropods and trees within our study system. Effects related to the seasonal dimension were stronger for arthropods than for trees. We conclude that: (1) models of beta diversity developed for tropical trees are unlikely to be applicable to tropical arthropods; (2) it is imperative that estimates of global biodiversity derived from mass collecting of arthropods in tropical rainforests embrace the strong vertical and seasonal partitioning observed here; and (3) given the high species turnover observed between seasons, global climate change may have severe consequences for rainforest arthropods. KW - trees KW - species richness KW - beta-diveristy KW - strategy KW - turnover KW - similarity KW - biodiversity KW - specialization KW - herbivorous insects KW - assemblages Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136393 VL - 10 IS - 12 ER - TY - JOUR A1 - Brill, Martin F. A1 - Meyer, Anneke A1 - Roessler, Wolfgang T1 - It takes two—coincidence coding within the dual olfactory pathway of the honeybee JF - Frontiers in Physiology N2 - To rapidly process biologically relevant stimuli, sensory systems have developed a broad variety of coding mechanisms like parallel processing and coincidence detection. Parallel processing (e.g., in the visual system), increases both computational capacity and processing speed by simultaneously coding different aspects of the same stimulus. Coincidence detection is an efficient way to integrate information from different sources. Coincidence has been shown to promote associative learning and memory or stimulus feature detection (e.g., in auditory delay lines). Within the dual olfactory pathway of the honeybee both of these mechanisms might be implemented by uniglomerular projection neurons (PNs) that transfer information from the primary olfactory centers, the antennal lobe (AL), to a multimodal integration center, the mushroom body (MB). PNs from anatomically distinct tracts respond to the same stimulus space, but have different physiological properties, characteristics that are prerequisites for parallel processing of different stimulus aspects. However, the PN pathways also display mirror-imaged like anatomical trajectories that resemble neuronal coincidence detectors as known from auditory delay lines. To investigate temporal processing of olfactory information, we recorded PN odor responses simultaneously from both tracts and measured coincident activity of PNs within and between tracts. Our results show that coincidence levels are different within each of the two tracts. Coincidence also occurs between tracts, but to a minor extent compared to coincidence within tracts. Taken together our findings support the relevance of spike timing in coding of olfactory information (temporal code). KW - olfaction KW - mushroom body KW - insect KW - coincidence KW - multi-electrode-recording KW - antennal lobe Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126179 VL - 6 IS - 208 ER - TY - THES A1 - Brockmann, Markus T1 - Inhibition von Aurora-A als neue Therapiestrategie gegen MYCN-amplifizierte Neuroblastome T1 - Inhibition of Aurora-A as a novel therapeutic strategy against MYCN-amplified Neuroblastoma N2 - Im Neuroblastom ist die Amplifikation des MYCN-Gens, das für den Transkriptionsfaktor N-Myc kodiert, der klinisch bedeutendste Faktor für eine schlechte Prognose. Als Mitglied der onkogenen Myc-Familie induziert N-Myc die Expression von Genen, die in vielen biologischen Prozessen wie Metabolismus, Zellzyklusprogression, Zellwachstum und Apoptose eine wichtige Rolle spielen. Die Deregulation der MYCN-Expression führt zu einem charakteristischen Genexpressionsprofil und einem aggressiven Phenotyp in den Tumorzellen. In normalen neuronalen Vorläuferzellen wird N-Myc gewöhnlich sehr schnell proteasomal abgebaut. Während der Mitose wird N-Myc an Serin 62 phosphoryliert. Diese Phosphorylierung dient als Erkennungssignal für die Kinase GSK3β, die die Phosphorylierung an Threonin 58 katalysiert. Das Phosphodegron wird von Fbxw7, einer Komponente des E3-Ubiquitinligase-Komplex SCFFbxw7, erkannt. Die anschließende Ubiquitinierung induziert den proteasomalen Abbau des Proteins. Die Reduktion der N-Myc–Proteinlevel ermöglicht den neuronalen Vorläuferzellen den Austritt aus dem Zellzyklus und führt zu einer terminalen Differenzierung. In einem shRNA Screen konnte AURKA als essentielles Gen für die Proliferation MYCN-amplifizierter Neuroblastomzellen identifiziert werden. Eine Aurora-A–Depletion hatte jedoch keinen Einfluss auf das Wachstum nicht-amplifizierter Zellen. Während dieser Doktorarbeit konnte gezeigt werden, dass Aurora-A speziell den Fbxw7-vermittelten Abbau verhindert und dadurch N-Myc stabilisiert. Für die Stabilisierung ist zwar die Interaktion der beiden Proteine von entscheidender Bedeutung, überraschenderweise spielt die Kinaseaktivität von Aurora-A jedoch keine Rolle. Zwei spezifische Aurora-A–Inhibitoren, MLN8054 und MLN8237, sind allerdings in der Lage, nicht nur die Kinaseaktivität zu hemmen, sondern auch die N-Myc-Proteinlevel zu reduzieren. Beide Moleküle induzieren eine Konformationsänderung in der Kinasedomäne von Aurora-A. Diese ungewöhnliche strukturelle Veränderung hat zur Folge, dass der N-Myc/Aurora-A–Komplex dissoziiert und N-Myc mit Hilfe von Fbxw7 proteasomal abgebaut werden kann. In MYCN-amplifizierten Zellen führt diese Reduktion an N-Myc zu einem Zellzyklusarrest in der G1-Phase. Die in vitro Daten konnten in einem transgenen Maus-Modell für das MYCN-amplifizierte Neuroblastom bestätigt werden. Die Behandlung mit MLN8054 und MLN8237 führte in den Tumoren ebenfalls zu einer N-Myc-Reduktion. Darüber hinaus konnte ein prozentualer Anstieg an differenzierten Zellen, die vollständige Tumorregression in der Mehrzahl der Neuroblastome und eine gesteigerte Lebenserwartung beobachtet werden. Insgesamt zeigen die in vitro und in vivo Daten, dass die spezifischen Aurora-A–Inhibitoren ein hohes therapeutisches Potential gegen das MYCN-amplifizierte Neuroblastom besitzen. N2 - Amplification of MYCN, encoding the transcription factor N-Myc, is one of the strongest clinical predictors of poor prognosis in neuroblastoma. As a member of the oncogenic Myc family, N-Myc activates genes that are involved in several biological processes like metabolism, cell cycle progression, cell growth and apoptosis. Deregulation of MYCN expression leads to a distinct gene expression profile and an aggressive phenotype in neuroblastoma cells. In normal neuronal progenitor cells, N-Myc is rapidly degraded by the ubiquitin-proteasome system. N-Myc degradation is controlled by two phospho-sites. During mitosis, Cyclin B/Cdk1 phosphorylates N-Myc at serine 62, which primes the protein for a second phosphorylation at threonine 58 via GSK3β. This phospho-degron provides a recognition site for Fbxw7, an F-box protein of the E3-Ligase complex SCFFbxw7, leading to destabilisation of the N-Myc protein. Mitotic degradation of the N-Myc protein allows progenitor cells to exit the cell cycle and enables terminal differentiation. Our group had previously identified AURKA as a gene that is required for cell growth in MYCN-amplified neuroblastoma cells but not essential for cells lacking MYCN. Here, we show that Aurora-A counteracts the Fbxw7-mediated degradation, and that the interaction between Aurora-A and N-Myc is cruical for N-Myc stabilization. Surprisingly, Aurora-A stabilizes the transcription factor in a kinase-independent manner. Interestingly, two Aurora-A-Inhibitors, MLN8054 and MLN8237, inhibit the kinase activity but also destabilize the N-Myc protein. These inhibitors induce an unusual conformation of the kinase domain and resulting in the dissociation of the N-Myc/Aurora-A complex. We demonstrate that the disruption of the complex promotes Fbxw7-mediated degradation of N-Myc. Inhibitor treatment in neuroblastoma cells leads to a cell cycle arrest in G1-phase. In a transgenic mouse model of MYCN-driven neuroblastoma, treatment causes downregulation of N-Myc protein levels, differentiation of the tumor cells and complete tumor regression in the majority of tumors. Consistent with the tumor reduction, treatment with MLN8054 and MLN8234 results in an extension of survival of the transgenic mice. Collectively, these results demonstrate that the Aurora-A–Inhibitors MLN8054 and MLN8237 are potential therapeutics against MYCN-amplified Neuroblastoma. KW - N-Myc KW - Neuroblastoma KW - Therapie KW - Neuroblastom KW - MYCN-amplified KW - Therapy KW - Aurora-A Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135951 ER - TY - THES A1 - Bruttel, Valentin Stefan T1 - Soluble HLA-G binds to dendritic cells which likely suppresses anti-tumour immune responses in regional lymph nodes in ovarian carcinoma T1 - Lösliches HLA-G wird von dendritischen Zellen gebunden, was beim Ovarialkarzinom zur Hemmung von Immunraktionen in regionalen Lymphknoten führen kann N2 - Zusammenfassung Einleitung HLA-G, ein nicht-klassisches HLA bzw. MHC Klasse Ib Molekül, kann sowohl als membrangebundenes als auch als lösliches Molekül verschiedenste Immunzellpopulationen effektiv inhibieren. Unter physiologischen Bedingungen wird HLA-G vor allem in der Plazenta exprimiert, wo es dazu beiträgt den semiallogenen Embryo vor einer Abstoßung durch das mütterliche Immunsystem zu beschützen. Außerdem wird HLA-G in einer Vielzahl von Tumoren wie zum Beispiel in Ovarialkarzinomen überexprimiert. Ziel dieser Arbeit war es besonders die Rolle von löslichem HLA-G im Ovarialkarzinom und die Expression von HLA-G in verschiedenen Subtypen des Ovarialkarzinoms genauer zu untersuchen. Ergebnisse Anhand eines Tissue Microarrays wurde bestätigt dass HLA-G unter physiologischen Bedingungen nur in sehr wenigen Geweben wie Plazenta oder Testes exprimiert wird. Außerdem wurden erstmals auch im Nebennierenmark hohe Expressionslevel detektiert. Im Gegensatz zur physiologischen Expression wurde HLA-G in serösen, muzinösen, endometrioiden und Klarzellkarzinomen und somit in Tumoren aller untersuchten Subtypen des Ovarialkarzinoms detektiert. Am häufigsten war HLA-G in hochgradigen serösen Karzinomen überexprimiert. Hier konnte gezeigt werden dass auf Genexpressionslevel in Ovarialkarzinomen die Expression des immunsuppressiven HLA-G mit der Expression von klassischen MHC Molekülen wie HLA-A, -B oder -C hochsignifikant korreliert. Außerdem konnte in Aszitesproben von Patientinnen mit Ovarialkarzinomen hohe Konzentrationen von löslichem HLA-G nachgewiesen werden. Auch auf metastasierten Tumorzellen in regionalen Lymphknoten war HLA-G nachweisbar. Überraschenderweise wurde aber besonders viel HLA-G auf Dendritischen Zellen in Lymphknoten detektiert. Da in Monozyten und Dendritischen Zellen von gesunden Spendern durch IL-4 oder IL-10 im Gegensatz zu Literatur keine Expression von HLA-G induzierbar war, untersuchten wir ob Dendritische Zellen lösliches HLA-G binden. Es konnte gezeigt werden, dass besonders Dendritische Zellen die in Gegenwart von IL-4, IL-10 und GM-CSF aus Monozyten generiert wurden (DC-10) effektiv lösliches HLA-G über ILT Rezeptoren binden. In Abhängigkeit von ihrer Beladung mit HLA-G hemmen auch fixierte DC-10 Zellen noch die Proliferation von zytotoxischen CD8+ T Zellen. Zudem wurden regulatorische T Zellen induziert. Schlussfolgerungen Besonders in den am häufigsten diagnostizierten hochgradigen serösen Ovarialkarzinomen ist HLA-G in den meisten Fällen überexprimiert. Durch die Expression immunsuppressiver MHC Klasse Ib Moleküle wie HLA-G können wahrscheinlich auch Tumore wachsen, die noch klassische MHC Moleküle exprimieren und aufgrund ihrer Mutationslast eigentlich vom Immunsystem erkannt und eliminiert werden müssten. Lösliches HLA-G könnte zudem lokal Immunantworten gegen Tumorantigene unterdrücken indem es an Dendritische Zellen in regionalen Lymphknoten bindet. Diese Zellen präsentieren nomalerweise zytotoxischen T Zellen Tumorantigene und spielen daher eine entscheidende Rolle in der Entstehung von protektiven Immunantworten. Mit löslichem HLA-G beladene Dendritische Zellen hemmen jedoch die Proliferation von CD8+ T Zellen und induzieren regulatorische T Zellen. Dadurch könnten Ovarialkarzinome “aus der Ferne” auch in metastasenfreien Lymphknoten die Entstehung von gegen den Tumor gerichteten Immunantworten unterdrücken. Dieser erstmals beschriebene Mechanismus könnte auch in anderen malignen Erkrankungen eine Rolle spielen, da lösliches HLA-G in einer Vielzahl von Tumorindikationen nachgewiesen wurde. N2 - Abstract Background HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases. Results As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde. Conclusion The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G. KW - HLA-G KW - HLA-G KW - Dendritische Zelle KW - Eierstocktumor KW - ovarian cancer KW - ovarian carcinoma KW - transfer KW - dendritic cells KW - immune escape Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127252 ER - TY - THES A1 - Czakai, Kristin Bernadette T1 - Interaktionen des humanpathogenen Pilzes Aspergillus fumigatus mit dem angeborenen Immunsystem und Thrombozyten T1 - Interaction of the human pathogenic mold Aspergillus fumigatus with the innate immune system and platelets N2 - Pilze sind in unserer Umwelt allgegenwärtig und besiedeln im Fall von Candida albicans (C. albicans) sogar bei über 50% der Menschen die Schleimhäute, während Sporen von Aspergillus fumigatus (A. fumigatus) täglich über die Atmung in die Lunge des Menschen gelangen. Dennoch sind Erkrankungen, die durch diese zwei Pilze ausgelöst werden, bei gesunden Menschen selten. Ist jedoch das Immunsystem beeinträchtigt, können diese Pilze zu systemischen und damit lebensbedrohlichen Erkrankungen wie der invasiven Aspergillose und der systemischen Candidiasis führen. Für eine Verbesserung der Behandlung solcher Infektionen ist das genaue Verständnis der Immunabwehrmechanismen entscheidend. Da A. fumigatus über die Lunge in den Körper gelangt, wurden in dieser Arbeit die häufigsten Immunzellen der Lunge, die Makrophagen, und deren Immunantwort auf A. fumigatus untersucht. Parallel hierzu wurden dendritische Zellen (DCs) verwendet, die als Brücke zwischen dem angeborenen und adaptiven Immunsystem wirken. Ein besonderes Augenmerk wurde hierbei auf A. fumigatus induzierte Genexpressionsänderungen und deren Regulationsmechanismen gelegt. Dabei wurden kurze, regulatorische RNAs, die sogenannten miRNAs, untersucht, die eine wichtige Rolle in der post-transkriptionalen Genregulation spielen. Bislang ist nur wenig über die miRNA-abhängigen Genregulationen in DCs, die auf eine Infektion mit A. fumigatus oder C. albicans reagieren, bekannt. Um alle durch A. fumigatus und C. albicans regulierten miRNAs zu identifizieren, wurden DCs mit A. fumigatus und C. albicans ko-kultiviert und anschließend eine Komplettsequenzierung der kurzen RNAs durchgeführt. Die Pilz-spezifische Induktion der miRNA-Regulation wurde zudem mit der miRNA-Regulation durch den bakteriellen Zellwandbestandteil Lipopolysaccharid verglichen. Durch die Stimulation mit Keimschläuchen von A. fumigatus wurden die miRNAs miR-132-3p/5p, miR-155-5p, miR129-2-3p, miR-129-5p, miR-212-3p/5p und miR-9-5p in DCs induziert. Diese wurden ebenfalls durch C. albicans induziert, zudem noch die miRNAs miR-147a und miR-147b. Spezifisch für A. fumigatus war die Regulation der miR-129-2-3p. Neben dem miRNA-Profiling wurde auch das mRNA-Transkriptom über Microarrays analysiert und dadurch 18 potentielle Zielgene der Pilz-induzierten miRNAs identifiziert. Neben den Elementen der Translationsregulation wurden auch die Transkriptionsfaktoren untersucht. Als einziger unter den 60 regulierten Transkriptionsfaktoren zeigte KLF4 eine veränderte Expressionsrichtung in DCs, die mit Pilzen oder LPS behandelt waren. Während die Stimulation mit LPS die Expression von KLF4 induzierte, wurde es durch die Pilze A. fumigatus und C. albicans reprimiert. In einer Untersuchung der unterschiedlichen A. fumigatus-Rezeptoren, wurde deren Einfluss auf die KLF4-Regulation gezeigt. Während TLR4-Liganden KLF4 induzierten, führten Liganden, die an die Rezeptoren TLR2/TLR1 und Dectin-1 binden, zu einer Reduktion von KLF4. Nach einem erfolgreich etablierten KLF4-knock-down mittels RNA-Interferenz wurden KLF4-Zielgene untersucht. Während kein bzw. nur ein geringer Effekt auf die Genexpression von CCL2, RANTES, CXCL10 und TNF beobachtet wurde, sorgte der KLF4 knock-down für eine hoch signifikante Reduktion der IL6-Genexpression in LPS-stimulierten DCs. Um die KLF4-Regulation weiter zu untersuchen, wurde zudem eine weitere Zellpopulation des angeborenen Immunsystems, die Makrophagen, verwendet. Auch hier wurde die Immunantwort gegen A. fumigatus analysiert. Zudem wurde die Rolle der Thrombozyten als Immunmediatoren betrachtet. Zuerst wurde ein Zytokinprofil des plättchenreichen Plasmas (PRP), das mit A. fumigatus stimuliert wurde, erstellt. In diesem konnte nur RANTES in hoher Konzentration nachgewiesen werden. Daraufhin wurde der Einfluss von PRP auf die Reifung von DCs, die Phagozytosefähigkeit von Makrophagen und DCs sowie der Einfluss von DCs und Makrophagen auf die metabolische Aktivität von A. fumigatus in An- und Abwesenheit von plättchenreichem Plasma untersucht. Es konnte eine gering verstärkte Reifung der DCs durch PRP gezeigt werden. Isolierte Thrombozyten konnten die Phagozytose von DCs steigern, während Makrophagen durch PRP verstärkt Konidien phagozytierten. In einem genomweiten Transkriptomprofiling wurde die Immunantwort von DCs und Makrophagen verglichen. Zudem wurde untersucht, wie PRP die Immunantwort dieser Immunzellen beeinflusst. Es wurden 2 bzw. 24 Gene identifiziert, die signifikant in A. fumigatus-stimulierten DCs und Makrophagen reguliert waren. Hierbei wurde gezeigt, dass KLF4 durch die Zugabe von PRP herabreguliert wurde. Das zuvor beschriebene Zielgen IL6 wurde durch PRP in A. fumigatus-stimulierten DCs gegenüber stimulierten DCs ohne PRP deutlich reduziert, wodurch sich eine immunmodulatorische Fähigkeit des PRP zeigte. Die Induktion von IL-6, weiteren Zytokinen und der Reifemarker durch A. fumigatus in DCs wurden zudem in einem Booleschen Modell simuliert. Dieses Modell soll in Zukunft Vorhersagen über experimentelle Ergebnisse und dadurch eine optimale Versuchsvorbereitung ermöglichen. N2 - Fungi are ubiquitously distributed and around 50% of human mucosae are colonized by Candida albicans. Spores of Aspergillus fumigatus, called conidia, reach the human lung by inhalation of normal air. Still, fungi normally do not cause severe diseases in healthy individuals. Immunosuppression, however, predisposes to systematic and therefore life-threatening infection like invasive aspergillosis and systemic candidiasis. The treatment of those infections can only be improved by a detailed insight in immune defense mechanisms. The most prominent cell type in the lung, the location where A. fumigatus conidia can germinate and grow into tissue, are macrophages. Furthermore, the immune response of DCs that bridge innate and adaptive immunity was compared to macrophages. The main focus was on A. fumigatus induced changes in gene expression and their regulatory mechanisms. Especially the regulation of small, regulatory RNAs, called miRNAs, that are important in the post-transcriptional regulation of gene expression, was examined. So far, little is known about miRNA regulations in DCs, confronted with A. fumigatus or C. albicans. Therefore a complete sequencing of small RNAs was performed to discover all regulated miRNAs. The fungi-induced regulation was compared to DCs, stimulated with the bacterial cell membrane component lipopolysaccharide (LPS). An A. fumigatus and C. albicans dependent regulation of miR-132-3p/5p, miR-155-5p, miR129-2-3p, miR-129-5p, miR-212-3p/5p and miR-9-5p was observed. In C. albicans simulated DCs miR-147a and miR-147b were additionally regulated, whereas miR-129-2-3p was specifically regulated by A. fumigatus. Furthermore a genome wide transcriptome profiling was performed and 18 potential miRNA targets were identified. Beside post-transcriptional regulators of gene expression, an analysis of transcriptional regulators, called transcription factors, was conducted. The transcription factor KLF4 was identified as the only of all 60 differentially regulated transcription factors that was oppositely regulated comparing fungi and LPS. KLF4 was induced by LPS, but strongly down-regulated by A. fumigatus and C. albicans stimulation. Specific Toll-like receptor 4 activation by LPS and ultra-pure LPS induced KLF4. However, ligands to TLR2/TLR1 and Dectin-1 significantly reduced KLF4 mRNA and protein. A KLF4 knock-down by RNA interference was established to analyze KLF4 target genes. Little or no effect was observed on the expression of CCL2, RANTES, CXCL10 and TNF. However, KLF4 knock down induced a significant reduction of IL6 gene expression and IL-6 release by LPS treated DCs. To further examine the KLF4 regulation another cell type of the innate immune system, called macrophages, was used. The A. fumigatus-induced immune response of macrophages was compared to DCs. Furthermore, the role of platelets as immune mediators was examined. At first, a cytokine profile of untreated and A. fumigatus stimulated platelet-rich plasma was performed. RANTES was identified as the only detectable cytokine. Then the influence of PRP on DC maturation, DC and macrophages phagocytosis as well as the metabolic activity of A. fumigatus was determined. Only a weak, but still significant, influence of PRP on DCs maturation induced by A. fumigatus was observed. In the analysis of phagocytosis of A. fumigatus conidia, DCs and macrophages were reacting differently to the addition of PRP and isolated platelets. Isolated platelets were able to enhance the phagocytosis of DCs. On the other hand, PRP and plasma without platelets increased phagocytosis of macrophages. In a genome wide approach the immune response of DCs and macrophages was compared and additionally examined, if PRP alters the DC and macrophage gene expression. PRP induced a significant regulation of 2 and 24 genes of A. fumigatus treated DCs and macrophages. Furthermore, an influence of PRP on the KLF4 expression was monitored. The previously described KLF4 target gene IL6 was significantly down-regulated in PRP and A. fumigatus stimulated DCs and macrophages, compared to A. fumigatus stimulated cells. In conclusion, PRP has only a weak but detectable influence on cell function and gene expression. Furthermore a Boolean model was established to analyze the influence of A. fumigatus stimulation on the cytokine release, especially of IL-6, IL-1B and TNF, and the induction of maturation markers. This model will be used for predictions and optimizations of experimental settings. KW - Aspergillus fumigatus KW - Immunsystem KW - Thrombozyt KW - angeborenes Immunsystem Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117496 ER - TY - JOUR A1 - Dandekar, Thomas A1 - Eisenreich, Wolfgang T1 - Host-adapted metabolism and its regulation in bacterial pathogens JF - Frontiers in Cellular and Infection Microbiology N2 - No abstract available. KW - bacterial pathogens KW - enteric pathogens KW - metabolism KW - host-pathogen adaption KW - isotopolog profiling Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-196876 SN - 2235-2988 VL - 5 IS - 28 ER -