TY - JOUR A1 - Schartl, Manfred A1 - Shen, Yingjia A1 - Maurus, Katja A1 - Walter, Ron A1 - Tomlinson, Chad A1 - Wilson, Richard K. A1 - Postlethwait, John A1 - Warren, Wesley C. T1 - Whole body melanoma transcriptome response in medaka JF - PLoS ONE N2 - The incidence of malignant melanoma continues to increase each year with poor prognosis for survival in many relapse cases. To reverse this trend, whole body response measures are needed to discover collaborative paths to primary and secondary malignancy. Several species of fish provide excellent melanoma models because fish and human melanocytes both appear in the epidermis, and fish and human pigment cell tumors share conserved gene expression signatures. For the first time, we have examined the whole body transcriptome response to invasive melanoma as a prelude to using transcriptome profiling to screen for drugs in a medaka (Oryzias latipes) model. We generated RNA-seq data from whole body RNA isolates for controls and melanoma fish. After testing for differential expression, 396 genes had significantly different expression (adjusted p-value <0.02) in the whole body transcriptome between melanoma and control fish; 379 of these genes were matched to human orthologs with 233 having annotated human gene symbols and 14 matched genes that contain putative deleterious variants in human melanoma at varying levels of recurrence. A detailed canonical pathway evaluation for significant enrichment showed the top scoring pathway to be antigen presentation but also included the expected melanocyte development and pigmentation signaling pathway. Results revealed a profound down-regulation of genes involved in the immune response, especially the innate immune system. We hypothesize that the developing melanoma actively suppresses the immune system responses of the body in reacting to the invasive malignancy, and that this mal-adaptive response contributes to disease progression, a result that suggests our whole-body transcriptomic approach merits further use. In these findings, we also observed novel genes not yet identified in human melanoma expression studies and uncovered known and new candidate drug targets for further testing in this malignant melanoma medaka model. KW - metastatic melanoma KW - expression KW - fish KW - cancer KW - stage III KW - melanogenesis KW - genome cells KW - gene KW - contributes Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144714 VL - 10 IS - 12 ER - TY - THES A1 - Gnamlin, Prisca T1 - Use of Tumor Vasculature for Successful Treatment of Carcinomas by Oncolytic Vaccinia Virus T1 - Die Tumorvasulatur in der erfolgreichen Therapie von Carcinomen durch onkolytische Vaccinia Viren N2 - Tumor-induced angiogenesis is of major interest for oncology research. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor characterized so far. VEGF blockade was shown to be sufficient for angiogenesis inhibition and subsequent tumor regression in several preclinical tumor models. Bevacizumab was the first treatment targeting specifically tumor-induced angiogenesis through VEGF blockade to be approved by the Food and Drugs Administration (FDA) for cancer treatment. However, after very promising results in preclinical evaluations, VEGF blockade did not show the expected success in patients. Some tumors became resistant to VEGF blockade. Several factors have been accounted responsible, the over-expression of other angiogenic factors, the noxious influence of VEFG blockade on normal tissues, the selection of hypoxia resistant neoplastic cells, the recruitment of hematopoietic progenitor cells and finally the transient nature of angiogenesis inhibition by VEGF blockade. The development of blocking agents against other angiogenic factors like placental growth factor (PlGF) and Angiopoietin-2 (Ang-2) allows the development of an anti-angiogenesis strategy adapted to the profile of the tumor. Oncolytic virotherapy uses the natural propensity of viruses to colonize tumors to treat cancer. The recombinant vaccinia virus GLV-1h68 was shown to infect, colonize and lyse several tumor types. Its descendant GLV-1h108, expressing an anti-VEGF antibody, was proved in previous studies to inhibit efficiently tumor induced angiogenesis. Additional VACVs expressing single chain antibodies (scAb) antibodies against PlGF and Ang-2 alone or in combination with anti VEGF scAb were designed. In this study, VACV-mediated anti-angiogenesis treatments have been evaluated in several preclinical tumor models. The efficiency of PlGF blockade, alone or in combination with VEGF, mediated by VACV has been established and confirmed. PlGF inhibition alone or with VEGF reduced tumor burden 5- and 2-folds more efficiently than the control virus, respectively. Ang-2 blockade efficiency for cancer treatment gave controversial results when tested in different laboratories. Here we demonstrated that unlike VEGF, the success of Ang-2 blockade is not only correlated to the strength of the blockade. A particular balance between Ang-2, VEGF and Ang-1 needs to be induced by the treatment to see a regression of the tumor and an improved survival. We saw that Ang-2 inhibition delayed tumor growth up to 3-folds compared to the control virus. These same viruses induced statistically significant tumor growth delays. This study unveiled the need to establish an angiogenic profile of the tumor to be treated as well as the necessity to better understand the synergic effects of VEGF and Ang-2. In addition angiogenesis inhibition by VACV-mediated PlGF and Ang-2 blockade was able to reduce the number of metastases and migrating tumor cells (even more efficiently than VEGF blockade). VACV colonization of tumor cells, in vitro, was limited by VEGF, when the use of the anti-VEGF VACV GLV-1h108 drastically improved the colonization efficiency up to 2-fold, 72 hours post-infection. These in vitro data were confirmed by in vivo analysis of tumors. Fourteen days post-treatment, the anti-VEGF virus GLV-1h108 was colonizing 78.8% of the tumors when GLV-1h68 colonization rate was 49.6%. These data confirmed the synergistic effect of VEGF blockade and VACV replication for tumor regression. Three of the tumor cell lines used to assess VACV-mediated angiogenesis inhibition were found, in certain conditions, to mimic either endothelial cell or pericyte functions, and participate directly to the vascular structure. The expression by these tumor cells of e-selectin, p-selectin, ICAM-1 and VCAM-1, normally expressed on activated endothelial cells, corroborates our findings. These proteins play an important role in immune cell recruitment, and there amount vary in presence of VEGF, PlGF and Ang-2, confirming the involvement of angiogenic factors in the immuno-modulatory abilities of tumors. In this study VACV-mediated angiogenesis blockade proved its potential as a therapeutic agent able to treat different tumor types and prevent resistance observed during bevacizumab treatment by acting on different factors. First, the expression of several antibodies by VACV would prevent another angiogenic factor to take over VEGF and stimulate angiogenesis. Then, the ability of VACV to infect tumor cells would prevent them to form blood vessel-like structures to sustain tumor growth, and the localized delivery of the antibody would decrease the risk of adverse effects. Next, the blockade of angiogenic factors would improve VACV replication and decrease the immune-modulatory effect of tumors. Finally the fact that angiogenesis blockade lasts until total regression of the tumor would prevent the recovery of the tumor-associated vasculature and the relapse of patients. N2 - Ein Hauptinteresse der onkologischen Forschung liegt auf dem Verständnis der Tumor-induzierten Angiogenese. Es wurde bereits festgestellt, dass die meisten Tumortypen eine abnorme Expression angiogener Faktoren zeigen. Der vascular endothelial growth factor (VEGF) wurde als der effektivste angiogene Faktor beschrieben. Es wurde gezeigt, dass die Hemmung des VEGF zur Inhibition der Angiogenese führt, das wiederum zu Tumorregression in vorklinischen Tumormodellen führte. Bevacizumab ist das erste FDA zugelassene Krebs-Therapeutikum, welches spezifisch auf die Tumor-induzierte Angiogenese durch VEGF-Inhibition abzielt. Der erwartete Erfolg durch VEGF-Hemmung konnte im Patienten allerdings nicht erzielt werden. Die Entwicklung von neuen Angiogenese hemmenden Stoffen wie gegen den placental growth factor (PIGF) oder Angiopoietin-2 (Ang-2), ermöglichen eine an das Tumor-Profil angepasste anti-angiogene Strategie. Die onkolytische Virustherapie die natürliche Eigenschaft der Viren Tumore zu kolonisieren. Das Vaccinia-Virus (VACV) gehört zur Familie der Poxviridae und wurde bereits lange Zeit als Vakzin zur Immunisierung gegen Pocken eingesetzt. Es konnte gezeigt werden, dass das rekombinante VACV GLV-1h68 effizient verschiedene Tumortypen infiziert, kolonisiert und lysiert. Das VACV GLV-1h108, welches auf der Basis des GLV-1h68 generiert wurde, kodiert einen anti-VEGF Antikörper. Dieses Virus ist in der Lage ist die Tumor-induzierte Angiogenese effizient zu inhibieren. Zusätzlich zu diesem VACV wurden weitere Konstrukte kloniert, welche für Antikörper gegen PIGF und Ang-2 kodieren. Zusätzlich wurden Virusstämme konstruiert, die gleichzeitig zwei Angiogenesefaktoren anzielen. Es wurde verschiedene VACV-vermittelte anti-Angiogenese Therapien in vorklinischen Tumormodellen wie Lungenadenokarzinome, KolonKarzinom, Melanom und Lungenadenokarzinome evaluiert. Die Effizienz der VACV-vermittelten Hemmung von PIGF und Ang-2, singulär oder in Kombination mit VEGF, wurde mit Tumor-Xenotransplantaten ermittelt. Die Inhibition von PlGF alleine oder in Kombination mit VEGF reduzierten die Tumorbelastung bis zu fünf, beziehungsweise zwei mal effizienter als GLV-1h68. Weiterhin wurde gezeigt, dass anders als VEGF, der Erfolg der Ang-2 Hemmung nicht nur mit der Stärke der Hemmung korreliert. Um Tumorregression sowie eine verbesserte Überlebensrate zu verursachen muss eine Balance zwischen Ang-2, VEGF und Ang-1 induziert werden. GLV-1h68 behandelte Tumore waren drei mal gröβer als Tumore, die mit den anti-Ang2 exprimierenden Viren behandelt wurden. Dieselben Virusstämme verursachten eine erhebliche Verspätung des Wachstums der Tumoren. Ausserdem hat diese Arbeit die Notwendigkeit enthüllt, ein angiogenes Profil des zu behandelnden Tumors zu etablieren sowie den Bedarf die synergistischen Effekte von VEGF und Ang-2 besser zu verstehen. Durch die Inhibition der Angiogenese durch VACV-verursachte PIGF und Ang-2 Hemmung wurde die Anzahl der Metastasen und der migrierenden Tumorzellen reduziert. Es wurde gezeigt, dass VEGF die VACV-Kolonisierung von Tumorzellen limitiert, da der Einsatz eines anti-VEGF VACV zu einer Verbesserung der Kolonisierung führt. In vivo Analysen bestätigten diese in vitro Daten. Nach vierzehn Tagen kolonisierte das anti-VEGF Virus 78,85% der Tumoren während die Kolonizationsquote des Kontrollviruses 49,64 % war. Dies resultierte in Tumorregression. Drei der getesteten Tumorzelllinien, in welchen die VACV-vermittelte Angiogenese-Inhibition untersucht wurde, waren in der Lage als Teil der Vaskulatur zu fungieren. Die Expression von Adhäsionsproteinen in diesen Tumorzellen untermauert die Ergebnisse. Weiterhin konnte ein unterschiedliches Expressionsmuster in Anwesenheit von VEGF, PIGF und Ang-2 festgestellt werden, wodurch die Beteiligung angiogener Faktoren bei den immunmodulatorischen Eigenschaften von Tumoren gezeigt werden konnte. In dieser Arbeit konnte gezeigt werden, dass eine VACV-vermittelte anti-angiogene Behandlung für verschiedene Tumorvarianten erfolgsversprechend ist. Die Möglichkeit verschiedene Antikörper gegen unterschiedliche angiogene Faktoren zu exprimieren würde verhindern, dass diese die Angiogenese stimulierende Wirkung des VEGF übernehmen. Die Eigenschaft von VACV Tumorzellen zu infizieren verhindert, dass diese Blutgefäß-ähnliche Strukturen bilden, welche das Tumorwachstum gewährleisten würde. Weiterhin würde die lokal begrenzte Antikörper-Freisetzung das Risiko von Nebenwirkungen senken. Die Inhibition angiogener Faktoren würde die VACV Replikationsrate steigern und den immunmodulatorischen Effekt der Tumore abschwächen. Letztlich würde die Hemmung der Angiogenese bis zur völligen Regression des Tumors aufrechterhalten, die Neubildung Tumor-assoziierter Vaskulatur verhindern und somit den Rückfall des Patienten. KW - Vaccinia-Virus KW - cancer KW - vaccinia virus KW - virotherapy KW - tumor vascularization KW - oncolytic virotherapy KW - Onkolyse KW - Angiogenese Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119019 ER - TY - JOUR A1 - Kuhn, Joachim A1 - Gripp, Tatjana A1 - Flieder, Tobias A1 - Dittrich, Marcus A1 - Hendig, Doris A1 - Busse, Jessica A1 - Knabbe, Cornelius A1 - Birschmann, Ingvild T1 - UPLC-MRM Mass Spectrometry Method for Measurement of the Coagulation Inhibitors Dabigatran and Rivaroxaban in Human Plasma and Its Comparison with Functional Assays JF - PLOS ONE N2 - Introduction The fast, precise, and accurate measurement of the new generation of oral anticoagulants such as dabigatran and rivaroxaban in patients' plasma my provide important information in different clinical circumstances such as in the case of suspicion of overdose, when patients switch from existing oral anticoagulant, in patients with hepatic or renal impairment, by concomitant use of interaction drugs, or to assess anticoagulant concentration in patients' blood before major surgery. Methods Here, we describe a quick and precise method to measure the coagulation inhibitors dabigatran and rivaroxaban using ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry in multiple reactions monitoring (MRM) mode (UPLC-MRM MS). Internal standards (ISs) were added to the sample and after protein precipitation; the sample was separated on a reverse phase column. After ionization of the analytes the ions were detected using electrospray ionization-tandem mass spectrometry. Run time was 2.5 minutes per injection. Ion suppression was characterized by means of post-column infusion. Results The calibration curves of dabigatran and rivaroxaban were linear over the working range between 0.8 and 800 mu g/L (r > 0.99). Limits of detection (LOD) in the plasma matrix were 0.21 mu g/L for dabigatran and 0.34 mu g/L for rivaroxaban, and lower limits of quantification (LLOQ) in the plasma matrix were 0.46 mu g/L for dabigatran and 0.54 mu g/L for rivaroxaban. The intraassay coefficients of variation (CVs) for dabigatran and rivaroxaban were < 4% and 6%; respectively, the interassay CVs were < 6% for dabigatran and < 9% for rivaroxaban. Inaccuracy was < 5% for both substances. The mean recovery was 104.5% (range 83.8-113.0%) for dabigatran and 87.0%(range 73.6-105.4%) for rivaroxaban. No significant ion suppressions were detected at the elution times of dabigatran or rivaroxaban. Both coagulation inhibitors were stable in citrate plasma at -20 degrees C, 4 degrees C and even at RT for at least one week. A method comparison between our UPLC-MRM MS method, the commercially available automated Direct Thrombin Inhibitor assay (DTI assay) for dabigatran measurement from CoaChrom Diagnostica, as well as the automated anti-Xa assay for rivaroxaban measurement from Chromogenix both performed by ACL-TOP showed a high degree of correlation. However, UPLC-MRM MS measurement of dabigatran and rivaroxaban has a much better selectivity than classical functional assays measuring activities of various coagulation factors which are susceptible to interference by other coagulant drugs. Conclusions Overall, we developed and validated a sensitive and specific UPLC-MRM MS assay for the quick and specific measurement of dabigatran and rivaroxaban in human plasma. KW - LC-MS/MS KW - validation KW - serum KW - quantification KW - apixaban KW - diagnostic accuracy KW - performance liquid chromatography KW - factor XA inhibitor KW - direct oral anticoagulants KW - direct thrombin inhibitor Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136023 VL - 10 IS - 12 ER - TY - JOUR A1 - Kang, Ji Hyoun A1 - Manousaki, Tereza A1 - Franchini, Paolo A1 - Kneitz, Susanne A1 - Schartl, Manfred A1 - Meyer, Axel T1 - Transcriptomics of two evolutionary novelties: how to make a sperm-transfer organ out of an anal fin and a sexually selected "sword" out of a caudal fin JF - Ecology and Evolution N2 - Swords are exaggerated male ornaments of swordtail fishes that have been of great interest to evolutionary biologists ever since Darwin described them in the Descent of Man (1871). They are a novel sexually selected trait derived from modified ventral caudal fin rays and are only found in the genus Xiphophorus. Another phylogenetically more widespread and older male trait is the gonopodium, an intromittent organ found in all poeciliid fishes, that is derived from a modified anal fin. Despite many evolutionary and behavioral studies on both traits, little is known so far about the molecular mechanisms underlying their development. By investigating transcriptomic changes (utilizing a RNA-Seq approach) in response to testosterone treatment in the swordtail fish, Xiphophorus hellerii, we aimed to better understand the architecture of the gene regulatory networks underpinning the development of these two evolutionary novelties. Large numbers of genes with tissue-specific expression patterns were identified. Among the sword genes those involved in embryonic organ development, sexual character development and coloration were highly expressed, while in the gonopodium rather more morphogenesis-related genes were found. Interestingly, many genes and genetic pathways are shared between both developing novel traits derived from median fins: the sword and the gonopodium. Our analyses show that a larger set of gene networks was co-opted during the development and evolution of the older gonopodium than in the younger, and morphologically less complex trait, the sword. We provide a catalog of candidate genes for future efforts to dissect the development of those sexually selected exaggerated male traits in swordtails. KW - mouse testis differentiation KW - fishes Xiphophorus KW - beetle horns KW - gonopodium KW - RNA-Seq KW - swordtails KW - Xiphophorus KW - key innovation KW - male-specific traits KW - Co-option KW - genus Xiphophorus KW - hybrid origin KW - Drosophila melanogaster KW - expression analysis KW - cell proliferation KW - preexisting bias KW - sex combs Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144139 VL - 5 IS - 4 ER - TY - THES A1 - Pusch, Tobias T1 - The transcription factor NFATc1 mediates cytotoxic T cell function in vitro and in vivo T1 - Der Transkriptionsfaktor NFATc1 vermittelt die Funktion von zytotoxischen T Zellen in vitro und in vivo N2 - While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment. Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells. Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade. We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation. Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse. N2 - Viele Experimente zur Rolle von NFAT wurden bereits anhand von CD4+ T Zellen durchgeführt und zeigten eine veränderte Zellphysiologie. Hingegen wurden CD8+ T Zellen diesbezüglich noch nicht intensiv studiert. Deshalb untersuchten wir den Einfluss von NFATc1 und NFATc2 auf die Funktion von CD8+ T Zellen in vitro und in vivo anhand des murinen Infektionsmodells mit dem Bakterium Listeria monocytogenes. Für die Versuche benutzen wir Mäuse, in denen das Protein NFATc1 spezifisch im T Zellkompartiment entfernt wurde. Erste Ergebnisse zeigten eine typische Translokation von NFATc1 und NFATc2 in den Zellkern. Eine Veränderung in der mRNA Expression nach Aktivierung, sowohl in CD4+ T Zellen als auch in CD8+ T Zellen, fand ebenfalls statt. NFATc defiziente CD4+ und CD8+ T Zellen wiesen eine verminderte Expression von Th1/Tc1 Zytokinen wie z.B. Interleukin-2 und Interferon γ auf, welche für die Bekämpfung intrazellulärer Pathogene wichtig sind. In unserem in vitro Modell fanden wir eine verminderte Abtötungsfähigkeit und eine Reduktion in der Freisetzung lytischer Granula in NFATc1-/- CD8+ T Zellen wohingegen eine NFATc2 Defizienz keine Auswirkungen auf die Zytotoxizität - verglichen mit wildtypischen Zellen - aufweist. Interessanterweise fanden wir eine Anhäufung von lytischen Granula und eine verminderte intrazelluläre Migration von Mitochondrien nach Ausbildung einer immunologischen Synapse in NFATc1-/- CD8+ T Zellen. Zusammen mit den Ergebnissen unserer CsA-Inhibierungsversuche nehmen wir an, dass einige allgemeine zelluläre Prozesse von NFATc1 beeinflusst werden, die nicht direkt von der T Zellrezeptor-induzierten Signalkaskade abhängen. Anhand eines in vivo Mausmodells zeigten wir auch die wichtige Rolle von NFATc1 in T Zellen während der Infektion mit Listeria monocytogenes. Fünf Tage nach Infektion konnten aus Nfatc1fl/fl x Cd4 cre Mäusen mehr Bakterienpartikel extrahiert werden als aus wt Mäusen. Wie in den in vitro Versuchen konnte auch hier eine geringere Zytokinproduktion der CD8+ T Zellen festgestellt werden allerdings wiesen die Mäuse auch eine geringere Bildung von Gedächniszellen auf. Unsere Ergebnisse zeigen, dass NFATc1 in CD8+ T Zellen eine wichtige Rolle spielt und auch Auswirkungen auf grundlegendere zelluläre Funktionen, wie die Ausbildung einer immunologischen Synapse, hat. KW - Transkriptionsfaktor KW - Killerzelle KW - Antigen CD8 KW - Cytotoxizität KW - NFAT KW - CTL function KW - CD8 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123690 ER - TY - JOUR A1 - Frank, Daniel O. A1 - Dengjel, Jörn A1 - Wilfling, Florian A1 - Kozjak-Pavlovic, Vera A1 - Häcker, Georg A1 - Weber, Arnim T1 - The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM) JF - PLoS ONE N2 - The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knockdowns of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated. KW - bax KW - preproteins KW - phosphorylation KW - proteomics KW - degradation KW - cells KW - family KW - import KW - BH3 domains KW - Bcl-2 proteins Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143301 VL - 10 IS - 4 ER - TY - JOUR A1 - Pasch, Elisabeth A1 - Link, Jana A1 - Beck, Carolin A1 - Scheuerle, Stefanie A1 - Alsheimer, Manfred T1 - The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility JF - Biology Open N2 - LINC complexes are evolutionarily conserved nuclear envelope bridges, physically connecting the nucleus to the peripheral cytoskeleton. They are pivotal for dynamic cellular and developmental processes, like nuclear migration, anchoring and positioning, meiotic chromosome movements and maintenance of cell polarity and nuclear shape. Active nuclear reshaping is a hallmark of mammalian sperm development and, by transducing cytoskeletal forces to the nuclear envelope, LINC complexes could be vital for sperm head formation as well. We here analyzed in detail the behavior and function of Sun4, a bona fide testis-specific LINC component. We demonstrate that Sun4 is solely expressed in spermatids and there localizes to the posterior nuclear envelope, likely interacting with Sun3/Nesprin1 LINC components. Our study revealed that Sun4 deficiency severely impacts the nucleocytoplasmic junction, leads to mislocalization of other LINC components and interferes with the formation of the microtubule manchette, which finally culminates in a globozoospermia-like phenotype. Together, our study provides direct evidence for a critical role of LINC complexes in mammalian sperm head formation and male fertility. KW - SUN domain proteins KW - sperm head formation KW - nuclear envelope KW - LINC complex KW - spermiogenesis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125212 VL - 4 ER - TY - JOUR A1 - García-Martínez, Jorge A1 - Brunk, Michael A1 - Avalos, Javier A1 - Terpitz, Ulrich T1 - The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination JF - Scientific Reports N2 - Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO\(^{-}\) mutant and carO\(^{+}\) control strains showed a faster development of light-exposed carO-germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin. KW - microbial rhodopsins KW - intracellular pH KW - membrane proteins KW - mutants KW - virulence KW - channelrhodopsin-2 KW - growth KW - gene KW - expression KW - bacteriorhodopsin Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149049 VL - 5 IS - 7798 ER - TY - JOUR A1 - Scholz, Nicole A1 - Gehring, Jennifer A1 - Guan, Chonglin A1 - Ljaschenko, Dmitrij A1 - Fischer, Robin A1 - Lakshmanan, Vetrivel A1 - Kittel, Robert J. A1 - Langenhan, Tobias T1 - The adhesion GPCR Latrophilin/CIRL shapes mechanosensation JF - Cell Reports N2 - G-protein-coupled receptors (GPCRs) are typically regarded as chemosensors that control cellular states in response to soluble extracellular cues. However, the modality of stimuli recognized through adhesion GPCR (aGPCR), the second largest class of the GPCR superfamily, is unresolved. Our study characterizes the Drosophila aGPCR Latrophilin/dCirl, a prototype member of this enigmatic receptor class. We show that dCirl shapes the perception of tactile, proprioceptive, and auditory stimuli through chordotonal neurons, the principal mechanosensors of Drosophila. dCirl sensitizes these neurons for the detection of mechanical stimulation by amplifying their input-output function. Our results indicate that aGPCR may generally process and modulate the perception of mechanical signals, linking these important stimuli to the sensory canon of the GPCR superfamily. KW - \(\alpha\)-latrotoxin KW - chordotonal organs KW - Johnstons organ KW - ligand CD55 KW - hearing KW - binding KW - shear stress KW - protein-coupled receptors KW - drosophila larvae KW - domain Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148626 VL - 11 ER - TY - JOUR A1 - Ehmann, Nadine A1 - Sauer, Markus A1 - Kittel, Robert J. T1 - Super-resolution microscopy of the synaptic active zone JF - Frontiers in Cellular Neuroscience N2 - Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins KW - excitation-secretion coupling KW - Ca\(^{2+}\) channels KW - structure-function relationships KW - super-resolution microscopy KW - active zone KW - presynaptic calcium KW - neurotransmitter release Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148997 VL - 9 IS - 7 ER - TY - JOUR A1 - Laine, Romain F. A1 - Albecka, Anna A1 - van de Linde, Sebastian A1 - Rees, Eric J. A1 - Crump, Colin M. A1 - Kaminski, Clemens F. T1 - Structural analysis of herpes simplex virus by optical super-resolution imaging JF - Nature Communications N2 - Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument. KW - tegument protein pUL36 KW - fluorescence microscopy KW - monoclonal antibodies KW - 3-dimensional structure KW - type-1 KW - nuclear pore complex KW - reconstruction microscopy KW - localization microscopy KW - resolution KW - envelopment Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144623 VL - 6 IS - 5980 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 - JOUR A1 - Herter, Eva K. A1 - Stauch, Maria A1 - Gallant, Maria A1 - Wolf, Elmar A1 - Raabe, Thomas A1 - Gallant, Peter T1 - snoRNAs are a novel class of biologically relevant Myc targets JF - BMC Biology N2 - Background Myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. They function as transcription factors that (in vertebrates) control many growth- and proliferation-associated genes, and in some contexts contribute to global gene regulation. Results We combine chromatin immunoprecipitation-sequencing (ChIPseq) and RNAseq approaches in Drosophila tissue culture cells to identify a core set of less than 500 Myc target genes, whose salient function resides in the control of ribosome biogenesis. Among these genes we find the non-coding snoRNA genes as a large novel class of Myc targets. All assayed snoRNAs are affected by Myc, and many of them are subject to direct transcriptional activation by Myc, both in Drosophila and in vertebrates. The loss of snoRNAs impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors. Conclusions This work shows that Myc acts as a master regulator of snoRNP biogenesis. In addition, in combination with recent observations of snoRNA involvement in human cancer, it raises the possibility that Myc’s transforming effects are partially mediated by this class of non-coding transcripts. KW - Drosophila KW - ribosome KW - snoRNA KW - Myc Transcription KW - growth Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124956 VL - 13 IS - 25 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 - Tuchscherr, Lorena A1 - Bischoff, Markus A1 - Lattar, Santiago M. A1 - Noto Llana, Mariangeles A1 - Pförtner, Henrike A1 - Niemann, Silke A1 - Geraci, Jennifer A1 - Van de Vyver, Hélène A1 - Fraunholz, Martin J. A1 - Cheung, Ambrose L. A1 - Herrmann, Mathias A1 - Völker, Uwe A1 - Sordelli, Daniel O. A1 - Peters, Georg A1 - Loeffler, Bettina T1 - Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, \(\Delta\)sigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections. KW - gene regulator agr KW - endothelial cells KW - modulates virulence KW - death pathway sar locus KW - factor B KW - small-colony variants KW - alpha-toxin KW - epithelial cells KW - in vitro Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143419 VL - 11 IS - 4 ER - TY - THES A1 - Wiese, Katrin Evelyn T1 - Sensing supraphysiological levels of MYC : mechanisms of MIZ1-dependent MYC-induced Apoptosis in Mammary Epithelial Cells T1 - Mechanismen der MIZ1-abhängigen MYC-induzierten Apoptose in Brustepithelzellen N2 - Deregulated MYC expression contributes to cellular transformation as well as progression and maintenance of human tumours. Interestingly, in the absence of additional genetic alterations, potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced apoptosis has long been recognised as a major barrier against cancer development. However, it is largely unknown how cells discriminate physiological from supraphysiological levels of MYC in order to execute an appropriate biological response. The experiments described in this thesis demonstrate that induction of apoptosis in mammary epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required to invade low-affinity binding sites and repress target genes of the serum response factor SRF. These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1- dependent gene repression and increases AKT phosphorylation and downstream function. Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC’s transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive role for the MYC/MIZ1 complex in epithelial cells. N2 - Eine Deregulation der MYC Expression trägt entscheidend zur malignen Transformation und Progression humaner Tumoren bei. In Abwesenheit von zusätzlichen genetischen Läsionen machen potentiell onkogene MYC Proteinmengen Zellen jedoch anfällig für eine Reihe Apoptoseauslösender Reize. Daher kann MYC-induzierte Apoptose als bedeutende tumorsuppressive Maßnahme und wichtige Barriere gegen die Entstehung von Krebs betrachtet werden. Mechanistisch unklar ist allerdings wie genau Zellen physiologische von supraphysiologischen MYC-Mengen unterscheiden um adäquat darauf reagieren zu können. Die Experimente in dieser Dissertation zeigen, dass die repressive Eigenschaft von MYC/MIZ1 Komplexen für die Induktion von Apoptose in Brustepithelzellen essentiell ist. Die Analyse von Genexpressions- und ChIP-Sequenzier-Experimenten verdeutlicht, dass hohe Level an MYC benötigt werden um niedrig-affine Bindestellen im Genom zu besetzen und Zielgene des SRF (serum response factor ) Transkriptionsfaktors zu reprimieren. Diese Gene haben eine wichtige Funktion in Prozessen wie Zytoskelettdynamik und Zelladhäsion und sind vermutlich daran beteiligt notwendige Überlebenssignale an die Kinase AKT weiterzuleiten. Eine Wiederherstellung der SRF Aktivität revertiert die MIZ1-abhängige Repression der Zielgene und führt zu einer vermehrten AKT Phosphorylierung und Funktion. Insgesamt deuten diese Resultate auf eine tumorsuppressive Rolle des MYC/MIZ1 Komplexes in epithelialen Zellen hin, da eine Veränderung der genregulatorischen Aktivität als Folge der Assoziation mit MIZ1 dazu beiträgen könnte onkogene Mengen an MYC zu erkennen. KW - Myc KW - Apoptosis KW - Myc KW - Miz1 KW - Apoptose KW - Repression KW - ChIP-sequencing KW - Repression Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132532 ER - TY - JOUR A1 - Gupta, Shishir K. A1 - Kupper, Maria A1 - Ratzka, Carolin A1 - Feldhaar, Heike A1 - Vilcinskas, Andreas A1 - Gross, Roy A1 - Dandekar, Thomas A1 - Förster, Frank T1 - Scrutinizing the immune defence inventory of Camponotus floridanus applying total transcriptome sequencing JF - BMC Genomics N2 - Background Defence mechanisms of organisms are shaped by their lifestyle, environment and pathogen pressure. Carpenter ants are social insects which live in huge colonies comprising genetically closely related individuals in high densities within nests. This lifestyle potentially facilitates the rapid spread of pathogens between individuals. In concert with their innate immune system, social insects may apply external immune defences to manipulate the microbial community among individuals and within nests. Additionally, carpenter ants carry a mutualistic intracellular and obligate endosymbiotic bacterium, possibly maintained and regulated by the innate immune system. Thus, different selective forces could shape internal immune defences of Camponotus floridanus. Results The immune gene repertoire of C. floridanus was investigated by re-evaluating its genome sequence combined with a full transcriptome analysis of immune challenged and control animals using Illumina sequencing. The genome was re-annotated by mapping transcriptome reads and masking repeats. A total of 978 protein sequences were characterised further by annotating functional domains, leading to a change in their original annotation regarding function and domain composition in about 8 % of all proteins. Based on homology analysis with key components of major immune pathways of insects, the C. floridanus immune-related genes were compared to those of Drosophila melanogaster, Apis mellifera, and other hymenoptera. This analysis revealed that overall the immune system of carpenter ants comprises many components found in these insects. In addition, several C. floridanus specific genes of yet unknown functions but which are strongly induced after immune challenge were discovered. In contrast to solitary insects like Drosophila or the hymenopteran Nasonia vitripennis, the number of genes encoding pattern recognition receptors specific for bacterial peptidoglycan (PGN) and a variety of known antimicrobial peptide (AMP) genes is lower in C. floridanus. The comparative analysis of gene expression post immune-challenge in different developmental stages of C. floridanus suggests a stronger induction of immune gene expression in larvae in comparison to adults. Conclusions The comparison of the immune system of C. floridanus with that of other insects revealed the presence of a broad immune repertoire. However, the relatively low number of PGN recognition proteins and AMPs, the identification of Camponotus specific putative immune genes, and stage specific differences in immune gene regulation reflects Camponotus specific evolution including adaptations to its lifestyle. KW - immune system KW - transcriptome KW - carpenter ant KW - camponotus floridanus KW - re-annotation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125279 VL - 16 IS - 540 ER - TY - JOUR A1 - Dandekar, Thomas A1 - Fieselmann, Astrid A1 - Fischer, Eva A1 - Popp, Jasmin A1 - Hensel, Michael A1 - Noster, Janina T1 - Salmonella - how a metabolic generalist adopts an intracellular lifestyle during infection JF - Frontiers in Cellular and Infection Microbiology N2 - The human-pathogenic bacterium Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, "-omics" data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology. KW - enterica serovar Typhimurium KW - bacterial invasion KW - mouse model KW - defenses KW - regulation KW - "-omics" KW - virulence KW - Salmonella-containing vacuole (SCV) KW - metabolism KW - nitric oxide Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149029 VL - 4 IS - 191 ER - TY - THES A1 - Schmitt, Alexandra T1 - Role of Peroxiredoxin 6 in human melanoma T1 - Die Funktion von Peroxiredoxin 6 im humanen Melanom N2 - Peroxiredoxin 6 (PRDX6) is a bifunctional enzyme comprising a peroxidase and a Ca2+-independent phospholipase (iPLA2) activity. This renders the enzyme capable of detoxifying reactive oxygen species (ROS) and of catalyzing the liberation of arachidonic acid (AA) from cellular membranes. Released AA can be further metabolized to bioactive lipids including eicosanoids, which are involved in inflammation, cell growth, differentiation, invasion and proliferation. Human melanoma cells are often characterized by imbalances in both ROS and lipid levels, which can be generated by oncogenic signaling, altered metabolism or UV irradiation. In previous studies, a comparative proteome analysis of the Xiphophorus fish melanoma model revealed a strong upregulation of Prdx6 in benign and malignant lesions compared to healthy skin. As the Xiphophorus melanoma model displays in many respects molecular characteristics that are similar to human melanoma, I investigated the functional role of PRDX6 in human melanoma cells. The first part of the study deals with the regulation of PRDX6 in melanocytes and human melanoma cells. I could demonstrate that the protein level of PRDX6 was strongly enhanced by the induction of the EGFR orthologue Xmrk from the Xiphophorus fish as well as the human EGFR. The upregulation of PRDX6 was further shown to be mediated in a PI3K-dependent and ROS-independent manner. The main part of the thesis comprises the investigation of the functional role of PRDX6 in human melanoma cells as well as the analysis of the underlying mechanism. I could show that knockdown of PRDX6 enhanced the oxidative stress response and led to decreased proliferation of melanoma cells. This cell growth effect was mainly mediated by the iPLA2 activity of PRDX6. Under conditions of strongly enhanced oxidative stress, the peroxidase activity became also important for cellular proliferation. Furthermore, the anti-proliferative effect in cells with lowered PRDX6 levels was the result of reduced cellular AA content and the decrease in the activation of SRC family proteins. Similarly, supplementation with AA led to regeneration of SRC family kinase activity and to an improvement in the reduced proliferation after knockdown of PRDX6. Since AA can be further processed into the prostaglandin PGE2, which has a pro-tumorigenic function in some cancer types, I further examined whether this eicosanoid is involved in the proliferative function of PRDX6. In contrast to AA, PGE2 was not consistently required for melanoma proliferation. In summary, I could demonstrate that PRDX6 plays a major role in AA-dependent lipid signaling in melanoma cells and thereby regulates proliferation. Interestingly, the proliferation relevant iPLA2 activity can be pharmacologically targeted, and melanoma cell growth was clearly blocked by the inhibitor BEL. Thus, I could identify the phospholipase activity of PRDX6 as a new therapeutically interesting target for melanoma treatment. N2 - Peroxiredoxin 6 (PRDX6) ist ein bifunktionales Enzym, welches neben seiner Peroxidase-Aktivität auch eine Ca2+-unabhängige Phospholipase-Aktivität besitzt. Aufgrund dieser beiden Aktivitäten ist das Enzym in der Lage, sowohl oxidativen Stress zu bekämpfen als auch die Freisetzung von Arachidonsäure aus zellulären Membranen zu katalysieren. Freie Arachidonsäure (AA) dient der Generierung von bioaktiven Lipiden wie zum Beispiel Eicosanoiden, welche an Entzündungsreaktionen, Zellwachstum, Differenzierung, Invasion und Proliferation beteiligt sind. Humane Melanomzellen zeichnen sich oft durch ein gestörtes Gleichgewicht reaktiver Sauerstoffspezies und zellulärer Lipide aus. Dieses Ungleichgewicht kann durch onkogene Signalgebung, einen veränderten Metabolismus oder UV-Bestrahlung hervorgerufen werden. Eine vorangegangene Proteomanalyse des Xiphophorus-Fisch-Melanommodells zeigte, dass im Vergleich zur gesunden Haut die Menge an PRDX6 in benignen und malignen Läsionen stark erhöht ist. Da das Xiphophorus-Melanommodell in vielerlei Hinsicht die molekulare Situation des humanen Melanoms wiederspiegelt, habe ich die funktionale Rolle von PRDX6 in humanen Melanomzellen untersucht. Der erste Teil der Studie beschäftigt sich mit der Regulierung von PRDX6 in Melanozyten und humanen Melanomzellen. Ich konnte nachweisen, dass die Menge an PRDX6 Protein durch die Induktion des EGFR Orthologs Xmrk aus Xiphophorus Fischen, sowie des humanen EGFR stark erhöht wurde. Auch konnte ich zeigen, dass die Heraufregulierung von PRDX6 von der Signalgebung der PI3 Kinase, aber nicht von reaktiven Sauerstoffspezies abhängig war. Der Hauptteil der vorliegenden Forschungsarbeit befasst sich mit der Ermittlung der funktionalen Rolle von PRDX6 in humanen Melanomzellen und der Analyse des zugrundeliegenden Mechanismus. Ich konnte nachweisen, dass ein Knockdown von PRDX6 die oxidative Stress-Antwort verstärkte und die Proliferation von Melanomzellen reduzierte. Der Effekt auf das zelluläre Wachstum wurde hierbei hauptsächlich durch die iPLA2-Aktivität von PRDX6 verursacht. Bei stark erhöhtem oxidativem Stress konnte auch eine Relevanz der Peroxidase-Aktivität für die zelluläre Proliferation nachgewiesen werden. Auch ging der anti-proliferative Effekt mit einer Abnahme zellulärer AA und der Reduktion aktiver Kinasen der SRC-Familie einher. Die Zugabe von AA zu Zellen mit PRDX6-Knockdown führte zur Regeneration der SRC-Kinase-Aktivität und konnte die Proliferation wieder verbessern. Da AA zum Prostaglandin PGE2 prozessiert werden kann, welches in einigen Krebsarten pro-tumorigene Funktionen erfüllt, untersuchte ich, ob dieses Eicosanoid auch für die proliferative Funktion von PRDX6 relevant ist. Im Gegensatz zu AA wies PGE2 jedoch keine kontinuierliche pro-proliferative Funktion auf. Zusammenfassend konnte ich zeigen, dass PRDX6 eine entscheidende Rolle im AA- Stoffwechsel von Melanomzellen spielt und hierdurch die Proliferation reguliert. Interessanterweise ist die proliferationsrelevante iPLA2-Aktivität pharmakologisch hemmbar, und auch das Wachstum der Melanomzellen wurde durch den Inhibitor BEL deutlich inhibiert. Mit der Phospholipase-Aktivität von PRDX6 konnte ich somit einen neuen therapeutisch nutzbaren Angriffspunkt für das Melanom identifizieren. KW - Melanom KW - Peroxiredoxin 6 KW - peroxiredoxin 6 KW - Melanom KW - melanoma KW - Arachidonsäure KW - arachidonic acid KW - Prostaglandin E2 KW - prostaglandin E2 KW - Melanomzellen KW - melanoma cells KW - Peroxiredoxin KW - Arachidonsäure KW - Prostaglandin E2 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111465 ER - TY - THES A1 - Jänicke, Laura Annika T1 - Regulation of MYC Activity by the Ubiquitin-Proteasome System T1 - Regulation der MYC Aktivität durch das Ubiquitin-Proteasom-System N2 - The oncogenic MYC protein is a transcriptional regulator of multiple cellular processes and is aberrantly activated in a wide range of human cancers. MYC is an unstable protein rapidly degraded by the ubiquitin-proteasome system. Ubiquitination can both positively and negatively affect MYC function, but its direct contribution to MYC-mediated transactivation remained unresolved. To investigate how ubiquitination regulates MYC activity, a non-ubiquitinatable MYC mutant was characterized, in which all lysines are replaced by arginines (K-less MYC). The absence of ubiquitin-acceptor sites in K-less MYC resulted in a more stable protein, but did not affect cellular localization, chromatin-association or the ability to interact with known MYC interaction partners. Unlike the wild type protein, K-less MYC was unable to promote proliferation in immortalized mammary epithelial cells. RNA- and ChIP-Sequencing analyses revealed that, although K-less MYC was present at MYC-regulated promoters, it was a weaker transcriptional regulator. The use of K-less MYC, a proteasomal inhibitor and reconstitution of individual lysine residues showed that proteasomal turnover of MYC is required for MYC target gene induction. ChIP-Sequencing of RNA polymerase II (RNAPII) revealed that MYC ubiquitination is dispensable for RNAPII recruitment and transcriptional initiation but is specifically required to promote transcriptional elongation. Turnover of MYC is required to stimulate histone acetylation at MYC-regulated promoters, which depends on a highly conserved region in MYC (MYC box II), thereby enabling the recruitment of BRD4 and P-TEFb and the release of elongating RNAPII from target promoters. Inhibition of MYC turnover enabled the identification of an intermediate in MYC-mediated transactivation, the association of MYC with the PAF complex, a positive elongation factor, suggesting that MYC acts as an assembly factor transferring elongation factors onto RNAPII. The interaction between MYC and the PAF complex occurs via a second highly conserved region in MYC’s amino terminus, MYC box I. Collectively, the data of this work show that turnover of MYC coordinates histone acetylation with recruitment and transfer of elongation factors on RNAPII involving the cooperation of MYC box I and MYC box II. N2 - Der Transkriptionsfaktor MYC ist an der Regulation einer Vielzahl biologischer Prozesse beteiligt ist und spielt bei der Tumorentstehung und des Tumorwachstum eine entscheidende Rolle. MYC ist ein kurzlebiges Protein, das durch das Ubiquitin-Proteasom-System abgebaut wird. Die Ubiquitinierung von MYC hat auch einen stimulierenden Einfluss auf dessen transkriptionelle Aktivität. Dabei blieb jedoch der Mechanismus, der dieser Beobachtung zugrunde liegt, bislang ungeklärt. Um den direkten Einfluss von Ubiquitinierung auf die Aktivität von MYC zu untersuchen, wurde in der vorliegenden Arbeit eine MYC Mutante analysiert, in der alle Lysine zu Argininen mutiert wurden (K-less MYC). Die Mutation der Ubiquitin-Verknüpfungsstellen resultierte in einem stabileren Protein, hatte jedoch keinen Einfluss auf die zelluläre Lokalisation oder Assoziation mit bekannten Interaktionspartnern. Im Vergleich zu Wildtyp (WT) MYC war K-less MYC jedoch in der Vermittlung MYC-induzierter biologischer Phänotypen stark beeinträchtigt. Mittels RNA- und ChIP-Sequenzierungen konnte gezeigt werden, dass K-less MYC zwar an MYC-regulierte Promotoren bindet, in der transkriptionellen Aktivität aber stark beeinträchtigt ist und diese Zielgene nicht aktivieren kann. Dabei war K-less MYC noch in der Lage, RNA Polymerase II (RNAPII) zu den Zielpromotoren zu rekrutieren und die Transkription dort zu initiieren, jedoch war der Übergang zur Elongation blockiert. Die Verwendung eines Proteasom-Inhibitors sowie die Rekonstitution einzelner Lysine in K-less MYC zeigten, dass der proteasomale Abbau von MYC für die Aktivierung von Zielgenen benötigt wird. Der proteasomale Abbau ist für die Histon-Acetylierung von Bedeutung, die von einer hoch konservieren Region in MYC, der MYC Box II, abhängt. Durch die WT MYC-vermittelte Induktion der Histon-Acetylierung können folglich die Proteine BRD4 und P-TEFb an die Promotoren rekrutiert werden. Diese Proteine spielen bei dem Übergang der initiierenden RNAPII zur elongierenden RNAPII eine essentielle Rolle. Darüber hinaus ermöglichte die Inhibition des MYC Abbaus die Identifizierung eines Zwischenschritts der MYC-abhängigen Transaktivierung: die Assoziation von MYC mit dem positiven Elongationskomplex, dem PAF-Komplex. Dieser wird über eine zweite hochkonservierte Region in MYC, der MYC Box I, rekrutiert. Somit kann angenommen werden, dass MYC als eine Verbindungsstelle fungiert, die positive Elongationsfaktoren auf die RNAPII transferiert. Zusammenfassend resultieren die Daten dieser Arbeit in einem Model, nach dem der proteasomale Abbau von MYC die Histon-Acetylierung mit der Rekrutierung und dem Transfer von Elongationsfaktoren auf die RNAPII koordiniert, was der Kooperation von MYC Box I und MYC Box II bedarf. KW - Myc KW - Ubiquitinierung KW - Transkription KW - RNS-Polymerase II KW - Elongation (Transkription) KW - Proteasomaler Abbau Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123339 ER -