TY - JOUR A1 - Brunet, Frédéric G. A1 - Volff, Jean-Nicolas A1 - Schartl, Manfred T1 - Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates JF - Genome Biology Evolution N2 - The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling. KW - receptor tyrosine kinase KW - vertebrates KW - deuterostomes KW - whole genome duplications Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146988 VL - 8 IS - 15 ER - TY - THES A1 - Bruder, Jessica T1 - Antigenerkennung bei autoaggressiven Lymphozyten T1 - Antigen recognition of autoaggressive lymphocytes N2 - Millionen Menschen weltweit leiden an den verschiedensten Autoimmunerkrankungen. Diese Krankheiten entstehen, wenn das Immunsystem gesundes körpereigenes Gewebe angreift und zerstört. An der Pathogenese sind sowohl Komponenten des angeborenen Immunsystems als auch Bestandteile des adaptiven Immunsystems, wie Lymphozyten und Antikörper, beteiligt. Da die Ursachen und molekularen Mechanismen der Pathogenese dieser Erkrankungen bis heute weitgehend unbekannt sind, wurden in dieser Arbeit autoaggressive Lymphozyten bei den humanen Autoimmunerkrankungen Polymyositis und Multiple Sklerose näher untersucht. Die Polymyositis ist eine chronisch entzündliche Erkrankung der Skelettmuskulatur. Die Muskelfasern werden dabei von zytotoxischen CD8+ gd-T-Lymphozyten infiltriert, attackiert und schließlich zerstört. In einem seltenen Fall der Polymyositis wurden die Muskelzellen hingegen in ähnlicher Weise von CD8- gd-T-Lymphozyten angegriffen. Die gd-T-Lymphozyten waren monoklonal expandiert und ihr Rezeptor, im Folgenden als M88 bezeichnet, wurde als Vg1.3+Vd2+ identifiziert. Frühere Untersuchungen der Antigenspezifität dieser Zellen zeigten, dass M88 mehrere funktionell und strukturell verschiedene Proteine aus unterschiedlichen Spezies erkennt. Die Bindung erfolgt spezifisch durch die Antigenerkennungsregionen beider Rezeptorketten von M88. In dieser Arbeit wurden verschiedene bakterielle und humane Proteine des Translationsapparates als Antigene von M88 identifiziert. Weitere ausführliche Untersuchungen eines paradigmatischen bakteriellen Antigens, dem Translationsinitiationsfaktor EcIF1, zeigten, dass M88 an Oberflächen-exponierte Konformationsepitope von Proteinen bindet. Interessanterweise erkennt M88 mehrere humane Aminoacyl-tRNA-Synthetasen, Antigene, die in anderen Formen der Myositis von Autoantikörpern angegriffen werden. Diese Beobachtung ergibt eine bemerkenswerte Verbindung zwischen T-Zell- und Antikörper-vermittelten B-Zell-Antworten bei der autoimmunen Myositis. Bei der Multiplen Sklerose ist das zentrale Nervensystem betroffen. Autoaggressive Lymphozyten greifen die Myelinschicht der Nervenzellen im Gehirn und Rückenmark an und zerstören sie. Im Liquor cerebrospinalis von Patienten lassen sich klonal expandierte und affinitätsgereifte B-Zellen sowie „oligoklonale Banden“ (OKB) Antikörper nachweisen. Obwohl diese Merkmale auf eine Antigen-induzierte Immunantwort hindeuten, sind die zugrundeliegenden Antigene und die Rolle der OKB bei der Pathogenese bis heute unbekannt. In dieser Arbeit wurde die Antigenspezifität von fünf IgG OKB-Antikörpern aus drei Patienten untersucht. Durch verschiedene proteinbiochemische Methoden konnten intrazelluläre Kandidatenantigene identifiziert werden. Interessanterweise sind darunter mehrere nukleäre Proteine, die an der Transkriptionsregulation oder der RNA-Prozessierung beteiligt sind. Reaktivitäten gegen intrazelluläre Antigene treten auch bei anderen Autoimmunerkrankungen, wie beispielsweise dem systemischen Lupus erythematodes, auf. Diese Ergebnisse könnten auf einen allgemeinen Mechanismus der Entstehung und Funktion von Autoantikörpern bei diesen humanen Autoimmunerkrankungen hindeuten. N2 - Millions of people worldwide suffer from various autoimmune diseases. These disorders occur if the immune system reacts to and destroys healthy body tissue. Pathogenesis is mediated by components of the innate immune system as well as by constituents of the adaptive immune system, like lymphocytes and antibodies. However, the origin and molecular mechanisms of these diseases remain still largely unknown. Therefore we investigated the role of autoaggressive lymphocytes in the human autoimmune diseases polymyositis and multiple sclerosis. Polymyositis is a chronically inflammatory disease of the skeletal muscles. Cytotoxic CD8+ gd-T lymphocytes invade, attack and finally destroy muscle fibers. However, in a rare variant of polymyositis, muscle fibers are similarly attacked by CD8- gd-T lymphocytes. These gd-T lymphocytes were monoclonally expanded and their receptor, termed M88, was identified as Vg1.3+Vd2+. Previous investigations of the antigen specificity of these cells revealed that M88 recognizes several structurally and functionally different proteins from various species. This recognition is specifically mediated by the antigen recognition sites of both receptor chains of M88. In this work we have identified different bacterial and human proteins of the translation apparatus as antigens of M88. Further detailed investigations of one paradigmatic bacterial antigen, the translation initiation factor EcIF1, have shown that M88 binds to surface-exposed conformational protein epitopes. Interestingly, M88 recognizes several human aminoacyl-tRNA-synthetases. These antigens have been described to be also targeted by autoantibodies in other forms of myositis. This observation reveals a remarkable link between T cell and antibody-mediated B cell responses in autoimmune myositis. In multiple sclerosis the central nervous system is affected. Autoaggressive lymphocytes attack and destroy the myelin sheet of nerve cells of the brain and spinal cord. In the cerebrospinal fluid of these patients clonally expanded and affinity-maturated B cells as well as „oligoclonal band“ (OCB) antibodies can be detected. Although these features indicate an antigen-induced immune response, the underlying antigens and the role of the OCB antibodies in the pathogenesis are still unknown. In this work we describe the antigen specificity of five IgG OCB antibodies from three patients. Through various biochemical methods we have identified intracellular candidate antigens. Interestingly, these include several nuclear proteins involved in transcription regulation and RNA processing. Reactivity against intracellular antigens also occurs in other autoimmune diseases such as systemic lupus erythematosus. These findings suggest a general mechanism for the generation and function of autoantibodies in these human autoimmune diseases. KW - Multiple Sklerose KW - gamma-delta T-Lymphozyten KW - Polymyositis KW - B-Lymphozyten KW - Antikörper KW - Multiple Sklerose KW - gamma-delta T-lymphocytes KW - polymyositis KW - B-lymphocytes KW - antibodies KW - Multiple Sclerosis KW - Polymyositis KW - Lymphozyt KW - Antikörper Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-73342 ER - TY - JOUR A1 - Broster Reix, Christine E. A1 - Florimond, Célia A1 - Cayrel, Anne A1 - Mailhé, Amélie A1 - Agnero-Rigot, Corentin A1 - Landrein, Nicolas A1 - Dacheux, Denis A1 - Havlicek, Katharina A1 - Bonhivers, Mélanie A1 - Morriswood, Brooke A1 - Robinson, Derrick R. T1 - Bhalin, an essential cytoskeleton-associated protein of Trypanosoma brucei linking TbBILBO1 of the flagellar pocket collar with the hook complex JF - Microorganisms N2 - Background: In most trypanosomes, endo and exocytosis only occur at a unique organelle called the flagellar pocket (FP) and the flagellum exits the cell via the FP. Investigations of essential cytoskeleton-associated structures located at this site have revealed a number of essential proteins. The protein TbBILBO1 is located at the neck of the FP in a structure called the flagellar pocket collar (FPC) and is essential for biogenesis of the FPC and parasite survival. TbMORN1 is a protein that is present on a closely linked structure called the hook complex (HC) and is located anterior to and overlapping the collar. TbMORN1 is essential in the bloodstream form of T. brucei. We now describe the location and function of BHALIN, an essential, new FPC-HC protein. Methodology/Principal Findings: Here, we show that a newly characterised protein, BHALIN (BILBO1 Hook Associated LINker protein), is localised to both the FPC and HC and has a TbBILBO1 binding domain, which was confirmed in vitro. Knockdown of BHALIN by RNAi in the bloodstream form parasites led to cell death, indicating an essential role in cell viability. Conclusions/Significance: Our results demonstrate the essential role of a newly characterised hook complex protein, BHALIN, that influences flagellar pocket organisation and function in bloodstream form T. brucei parasites. KW - trypanosoma KW - flagellar pocket KW - hook complex KW - endocytosis KW - cytoskeleton KW - protozoan KW - flagellar pocket collar Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250301 SN - 2076-2607 VL - 9 IS - 11 ER - TY - JOUR A1 - Brosch, Philippa K. A1 - Korsa, Tessa A1 - Taban, Danush A1 - Eiring, Patrick A1 - Hildebrand, Sascha A1 - Neubauer, Julia A1 - Zimmermann, Heiko A1 - Sauer, Markus A1 - Shirakashi, Ryo A1 - Djuzenova, Cholpon S. A1 - Sisario, Dmitri A1 - Sukhorukov, Vladimir L. T1 - Glucose and inositol transporters, SLC5A1 and SLC5A3, in glioblastoma cell migration JF - Cancers N2 - (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment. KW - volume regulation KW - transportome KW - phlorizin Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297498 SN - 2072-6694 VL - 14 IS - 23 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 - JOUR A1 - Brocher, Jan A1 - Vogel, Benjamin A1 - Hock, Robert T1 - HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation N2 - Background: High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results: We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable overexpression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions: Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours. KW - HMG-Proteine KW - High mobility group Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67914 ER - TY - THES A1 - Brocher, Jan T1 - Einfluss von HMGA1-Proteinen auf die Myogenese und Heterochromatinorganisation während der Differenzierung T1 - Influence of HMGA1 proteins on myogenesis and heterochromatin organization during differentiation N2 - HMG-Proteine sind nach den Histonen die zweithäufigste Superfamilie nukleärer Proteine. Sie binden an DNA und Nukleosomen und induzieren strukturelle Veränderungen im Chromatin. Sie spielen eine wichtige Rolle in der Dynamik des Chromatins und beeinflussen dadurch DNA-abhängige Prozesse, wie Transkription und Replikation. Proteine der HMGA-Familie sind charakterisiert durch konservierte DNA-Bindungsmotive, den AT-Hooks, welche eine Bindung an AT-reiche DNA-Sequenzen vermitteln und durch einen sauren C-Terminus. HMGA-Proteine sind verstärkt im Heterochromatin konzentriert und stehen in Verbindung mit der Expressionsregulation spezifischer Gene aufgrund der Stabilisierung von Nukleoproteinkomplexen, so genannten Enhanceosomen. HMGA-Proteine spielen des Weiteren eine entscheidende Rolle in verschiedenen Entwicklungsprozessen und bei der Tumorprogression . Um den Einfluss von HMGA1 auf die zelluläre Differenzierung und die Chromatinmodulation zu untersuchen, wurden C2C12 Maus-Myoblastenzellen verwendet. Die Induktion der Myogenese in diesen Zellen geht mit der Herunterregulierung von HMGA1 einher. Durch die Etablierung einer C2C12-Zelllinie, welche ein EGFP-markiertes HMGA1a stabil exprimierte, konnte gezeigt werden, dass eine anhaltende HMGA1-Expression spezifisch die Myogeneseprozess inhibierte, während die Osteogenese davon unbeeinflusst zu bleiben schien. Dieser hemmende Effekt kann durch die HMGA1-abhängige Fehlexpression verschiedener Gene, welche für eine einwandfreie Muskeldifferenzierung nötig sind und in die Zellzyklusregulation eingreifen, erklärt werden. Unter der Verwendung von RNAi konnte gezeigt werden, dass die Herunterregulierung von HMGA1-Proteinen für eine korrekte Genexpression und den Muskeldifferenzierungsprozess notwendig ist. Während der terminalen Differenzierung wird die Umorganisation des Chromatins durch die Fusion der Chromozentren offensichtlich. Fotobleichtechniken, wie „fluorescence recovery after photobleaching“ (FRAP) zeigten, dass HMGA1-Proteine mit dem Methyl-CpG-bindenden Protein 2 (MeCP2), welches eine wichtige Rolle in der Chromozentrenfusion spielt, um DNA-Bindungsstellen konkurriert und dieses vom Chromatin verdrängt. Diese dynamische Konkurrenz zwischen einem anhaltend exprimierten HMGA1 und MeCP2 trägt somit zur Inhibition der differenzierungsabhängigen Modulation des Chromatins während der späten Myogenese bei. Die Untersuchungen in C2A1a-Zellen lieferten weitere Hinweise dafür, dass der wesentlichste Umbau des Chromatins in einem Zeitfenster um den dritten Tag nach Induktion der Myogenese stattfindet, an welchem HMGA1 natürlicherweise nahezu vollständig herunterreguliert sind. In diesem Zeitraum kommt es zur Dissoziation der Chromozentren, zu veränderten Expressionsmustern in bestimmten Genen, zu Modulationen in Histonmodifikationen (H3K4me2, H3K4me3, H3K27me3), zur Replikations-unabhängigen Akkumulation von Histon H3 in den Chromozentren über ungefähr einen Zellzyklus hinweg und zu eine signifikanten Erhöhung der HP1-Dynamik. Durch den Einsatz von Bimolekularer Fluoreszenzkomplementierung (BiFC), die es erlaubt Protein-Protein-Interaktionen in vivo zu visualisieren, konnte gezeigt werden, dass der saure C-Terminus des HMGA mit der Chromodomäne (CD) des HP1 interagiert. Zusätzlich ist für diese Interaktion die korrekte DNA-Bindung des HMGA nötig. FRAP-Messungen mit HP1-EGFP-Fusionsproteinen in Zellen die wildtypisches oder ein mutiertes HMGA koexprimierten, bestätigten diese Daten und wiesen darauf hin, dass die HP1-Verweildauer im Heterochromatin maßgeblich von der Gegenwart eines funktionellen HMGA1 abhängig ist. Des Weiteren zeigten C2C12-Myoblasten, die HMGA1 natürlicherweise exprimieren, eine hohe HP1-Verweildauer, die nach HMGA1-knock down drastisch verringert ist. Umgekehrt ist die HP1-Verweildauer nach einer Herunterregulierung von HMGA1 an Tag 3 der Myogenese gering und steigt durch die Koexpression von HMGA1 auf das in Myoblasten gemessene Niveau an. Zusammengenommen zeigen diese Daten, dass die differenzielle Expression von HMGA1 und ihre Fähigkeit mit HP1 zu interagieren, sowie ihre Konkurrenz mit MeCP2 um DNA-Bindungsstellen einen entscheidende Rolle in der Regulation der Aufrechterhaltung und Plastizität des Heterochromatins während der Differenzierung spielen. Daher ist eine zeitlich festgelegte Herunterregulierung von HMGA1 notwendig, um die Modulation des Chromatins und dadurch den Differenzierungsprozess zu ermöglichen N2 - HMG proteins are an abundant superfamily of nuclear proteins that bind to DNA and nucleosomes and induce structural changes in the chromatin fiber. These proteins play an important role in chromatin dynamics and thereby impact DNA-related processes like transcription and replication. Proteins of the HMGA family are characterized by conserved DNA-binding domains, the AT hooks, which mediate binding to AT-rich DNA, and an acidic c-terminal domain. HMGA proteins concentrate in heterochromatin and are linked to specific gene regulation by stabilizing nucleoprotein complexes called enhanceosomes. Furthermore, HMGA proteins play an important role in several developmental processes and in tumor progression. C2C12 mouse myoblast cells were used to explore the impact of HMGA1 proteins on differentiation and chromatin modulation. After induction of myogenesis HMGA1 proteins revealed a downregulation. By establishing a C2C12 cell line stably expressing an EGFP tagged HMGA1a (C2A1a) it could be shown that sustained HMGA expression inhibited specifically the myogenic process while osteogenesis seemed to be unaffected. This inhibition can be explained by an HMGA1-dependent misexpression of several genes that are required for proper myogenic differentiation and genes involved in cell cycle regulation. Using RNAi techniques it could be demonstrated that downregulation of HMGA1 proteins is required to restore proper gene expression and to enable the myogenic program. During terminal differentiation chromatin remodeling is apparent by fusion of chromocenters. Photobleaching experiments like “fluorescence recovery after photobleaching” (FRAP) revealed that HMGA1 proteins compete with the methyl-CpG-binding protein 2 (MeCP2), which plays an important role during the fusion of chromocenters, for DNA-binding sites. Thereby MeCP2 is displaced from chromatin. This dynamic competition between constitutively expressed HMGA1 and MeCP2 thereby leads to an inhibition of the differentiation dependent modulation of the chromatin during late myogenesis. Studies in C2A1a cells revealed a set of evidences indicating that further major chromatin remodeling occurs around day three after induction when HMGA1 proteins are downregulated. At this time-frame chromocenters dissociate, expression patterns of genes are switching, histone modifications are modulated (H3K4me2, H3K4me3, H3K27me3), histone H3 accumulates in a replication independent mode in chromocenters for approximately one cell cycle, and dynamics of HP1 proteins are significantly increased. Applying bimolecular fluorescence complementation (BiFC) that allows visualization of protein-protein interactions in living cells I could show that the acidic domain of HMGA interacts with the chromodomain (CD) of HP1. In Addition, the proper DNA-binding of HMGA1 is necessary to accomplish a functional interaction between HP1 and HMGA. FRAP measurements of HP1-EGFP in cells coexpressing wild type or mutated HMGAs corroborated theses findings and indicated that the HP1 residence time in heterochromatin strongly depends on the presence of functional HMGA proteins. Furthermore, HP1 residence time is high in C2C12 myoblasts which express HMGA1 but low after HMGA1 knock down. Vice versa, it is low in C2C12 cells at day 3 of differentiation when HMGA proteins are downregulated, but high when HMGA1 proteins are coexpressed. Together, these data indicate that the differential expression of HMGAs and their capacity to interact with HP1 proteins and compete with MeCP2 plays an important role in the regulation of heterochromatin maintenance and plasticity during differentiation. Therefore, the downregulation of HMGA1 proteins is required to allow chromatin remodeling and to enable the differentiation program. KW - HMG-Proteine KW - Differenzierung KW - Muskelentwicklung KW - Heterochromatin KW - C2C12-Zellen KW - HMG proteins KW - myogenesis KW - heterochromatin KW - differentiation KW - C2C12 cells Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-24456 ER - TY - JOUR A1 - Britz, Sebastian A1 - Markert, Sebastian Matthias A1 - Witvliet, Daniel A1 - Steyer, Anna Maria A1 - Tröger, Sarah A1 - Mulcahy, Ben A1 - Kollmannsberger, Philip A1 - Schwab, Yannick A1 - Zhen, Mei A1 - Stigloher, Christian T1 - Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy JF - Frontiers in Neuroanatomy N2 - At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments. KW - FIB-SEM KW - 3D reconstruction KW - neuroanatomy KW - IL2 branching KW - amphids KW - Caenorhabditis elegans (C. elegans) KW - dauer Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249622 SN - 1662-5129 VL - 15 ER - TY - THES A1 - Brink, Andreas T1 - The biological significance of chemically-induced DNA adducts in relation to background DNA damage T1 - Die biologische Bedeutung von chemisch induzierten DNA-Addukten in Relation zum Hintergrund-DNA-Schaden N2 - No abstract available KW - DNS-Schädigung KW - DNS-Strangbruch KW - HPLC-MS KW - API-Massenspektrometrie KW - LC-MS KW - Gentoxikologie KW - Mutagenitätstest KW - Dosis-Wirkungs-Beziehung KW - DNA-Addukte KW - Dosis-Wirkungs-Beziehung KW - Hintergrund-DNA-Schaden KW - Comet assay KW - DNA adducts KW - Dose response relationships KW - Background DNA damage Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-23850 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 - JOUR A1 - Breyer, Maximilian A1 - Grüner, Julia A1 - Klein, Alexandra A1 - Finke, Laura A1 - Klug, Katharina A1 - Sauer, Markus A1 - Üçeyler, Nurcan T1 - \(In\) \(vitro\) characterization of cells derived from a patient with the GLA variant c.376A>G (p.S126G) highlights a non-pathogenic role in Fabry disease JF - Molecular Genetics and Metabolism Reports N2 - Highlights • The GLA variant S126G is not associated with Fabry symptoms in the presented case • S126G has no effect on α-GAL A activity or Gb3 levels in this patient • S126G sensory neurons show no electrophysiological abnormalities Abstract Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice. KW - Fabry disease KW - variants of unknown significance KW - C.376A>G (p.S126G) KW - globotriaosylceramide KW - induced pluripotent stem cells KW - sensory neurons KW - disease model KW - α-Galactosidase A Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350295 SN - 22144269 VL - 38 ER - TY - JOUR A1 - Brenzinger, Kristof A1 - Maihoff, Fabienne A1 - Peters, Marcell K. A1 - Schimmer, Leonie A1 - Bischler, Thorsten A1 - Classen, Alice T1 - Temperature and livestock grazing trigger transcriptome responses in bumblebees along an elevational gradient JF - iScience N2 - Climate and land-use changes cause increasing stress to pollinators but the molecular pathways underlying stress responses are poorly understood. Here, we analyzed the transcriptomic response of Bombus lucorum workers to temperature and livestock grazing. Bumblebees sampled along an elevational gradient, and from differently managed grassland sites (livestock grazing vs unmanaged) in the German Alps did not differ in the expression of genes known for thermal stress responses. Instead, metabolic energy production pathways were upregulated in bumblebees sampled in mid- or high elevations or during cool temperatures. Extensive grazing pressure led to an upregulation of genetic pathways involved in immunoregulation and DNA-repair. We conclude that widespread bumblebees are tolerant toward temperature fluctuations in temperate mountain environments. Moderate temperature increases may even release bumblebees from metabolic stress. However, transcriptome responses to even moderate management regimes highlight the completely underestimated complexity of human influence on natural pollinators. KW - bumblebees KW - stress KW - transcriptomic response KW - climate changes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301276 VL - 25 IS - 10 ER - TY - JOUR A1 - Brenzinger, Kristof A1 - Costa, Ohana Y. A. A1 - Ho, Adrian A1 - Koorneef, Guusje A1 - Robroek, Bjorn A1 - Molenaar, Douwe A1 - Korthals, Gerard A1 - Bodelier, Paul L. E. T1 - Steering microbiomes by organic amendments towards climate-smart agricultural soils JF - Biology and Fertility of Soils N2 - We steered the soil microbiome via applications of organic residues (mix of cover crop residues, sewage sludge + compost, and digestate + compost) to enhance multiple ecosystem services in line with climate-smart agriculture. Our result highlights the potential to reduce greenhouse gases (GHG) emissions from agricultural soils by the application of specific organic amendments (especially digestate + compost). Unexpectedly, also the addition of mineral fertilizer in our mesocosms led to similar combined GHG emissions than one of the specific organic amendments. However, the application of organic amendments has the potential to increase soil C, which is not the case when using mineral fertilizer. While GHG emissions from cover crop residues were significantly higher compared to mineral fertilizer and the other organic amendments, crop growth was promoted. Furthermore, all organic amendments induced a shift in the diversity and abundances of key microbial groups. We show that organic amendments have the potential to not only lower GHG emissions by modifying the microbial community abundance and composition, but also favour crop growth-promoting microorganisms. This modulation of the microbial community by organic amendments bears the potential to turn soils into more climate-smart soils in comparison to the more conventional use of mineral fertilizers. KW - greenhouse gases KW - agricultural soils KW - organic amendment KW - flux measurements KW - microbial community abundance and compositions KW - plant growth Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-326930 VL - 57 IS - 8 ER - TY - JOUR A1 - Brenner, Daniela A1 - Geiger, Nina A1 - Schlegel, Jan A1 - Diesendorf, Viktoria A1 - Kersting, Louise A1 - Fink, Julian A1 - Stelz, Linda A1 - Schneider-Schaulies, Sibylle A1 - Sauer, Markus A1 - Bodem, Jochen A1 - Seibel, Jürgen T1 - Azido-ceramides, a tool to analyse SARS-CoV-2 replication and inhibition — SARS-CoV-2 is inhibited by ceramides JF - International Journal of Molecular Sciences N2 - Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication. KW - ceramides KW - SARS-CoV-2 KW - azido-ceramides KW - sphingolipids Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313581 SN - 1422-0067 VL - 24 IS - 8 ER - TY - THES A1 - Brembs, Björn T1 - An Analysis of Associative Learning in Drosophila at the Flight Simulator T1 - Eine Ananlyse des assoziativen Lernens von Drosophila im Flugsimulator N2 - Most natural learning situations are of a complex nature and consist of a tight conjunction of the animal's behavior (B) with the perceived stimuli. According to the behavior of the animal in response to these stimuli, they are classified as being either biologically neutral (conditioned stimuli, CS) or important (unconditioned stimuli, US or reinforcer). A typical learning situation is thus identified by a three term contingency of B, CS and US. A functional characterization of the single associations during conditioning in such a three term contingency has so far hardly been possible. Therefore, the operational distinction between classical conditioning as a behavior-independent learning process (CS-US associations) and operant conditioning as essentially behavior-dependent learning (B-US associations) has proven very valuable. However, most learning experiments described so far have not been successful in fully separating operant from classical conditioning into single-association tasks. The Drosophila flight simulator in which the relevant behavior is a single motor variable (yaw torque), allows for the first time to completely separate the operant (B-US, B-CS) and the classical (CS-US) components of a complex learning situation and to examine their interactions. In this thesis the contributions of the single associations (CS-US, B-US and B-CS) to memory formation are studied. Moreover, for the first time a particularly prominent single association (CS-US) is characterized extensively in a three term contingency. A yoked control shows that classical (CS-US) pattern learning requires more training than operant pattern learning. Additionally, it can be demonstrated that an operantly trained stimulus can be successfully transferred from the behavior used during training to a new behavior in a subsequent test phase. This result shows unambiguously that during operant conditioning classical (CS-US) associations can be formed. In an extension to this insight, it emerges that such a classical association blocks the formation of an operant association, which would have been formed without the operant control of the learned stimuli. Instead the operant component seems to develop less markedly and is probably merged into a complex three-way association. This three-way association could either be implemented as a sequential B-CS-US or as a hierarchical (B-CS)-US association. The comparison of a simple classical (CS-US) with a composite operant (B, CS and US) learning situation and of a simple operant (B-US) with another composite operant (B, CS and US) learning situation, suggests a hierarchy of predictors of reinforcement. Operant behavior occurring during composite operant conditioning is hardly conditioned at all. The associability of classical stimuli that bear no relation to the behavior of the animal is of an intermediate value, as is operant behavior alone. Stimuli that are controlled by operant behavior accrue associative strength most easily. If several stimuli are available as potential predictors, again the question arises which CS-US associations are formed? A number of different studies in vertebrates yielded amazingly congruent results. These results inspired to examine and compare the properties of the CS-US association in a complex learning situation at the flight simulator with these vertebrate results. It is shown for the first time that Drosophila can learn compound stimuli and recall the individual components independently and in similar proportions. The attempt to obtain second-order conditioning with these stimuli, yielded a relatively small effect. In comparison with vertebrate data, blocking and sensory preconditioning experiments produced conforming as well as dissenting results. While no blocking could be found, a sound sensory preconditioning effect was obtained. Possible reasons for the failure to find blocking are discussed and further experiments are suggested. The sensory preconditioning effect found in this study is revealed using simultaneous stimulus presentation and depends on the amount of preconditioning. It is argued that this effect is a case of 'incidental learning', where two stimuli are associated without the need of reinforcement. Finally, the implications of the results obtained in this study for the general understanding of memory formation in complex learning situations are discussed. N2 - Die meisten Lernsituationen sind von komplexer Natur und bestehen aus einer engen Verknüpfung des Verhaltens eines Tieres (B) mit den wahrgenommenen Stimuli. Entsprechend der Reaktion des Tieres auf diese Stimuli werden diese als entweder biologisch neutral (konditionierte Stimuli, CS) oder signifikant (unkonditionierte Stimuli, US oder Verstärker) klassifiziert. Eine typische Lernsituation ist also durch eine Dreiwegebeziehung zwischen B, CS und US gekennzeichnet. Eine funktionelle Charakterisierung der Einzelassoziationen während des Lernens in einer solchen Dreiwegebeziehung war experimentell bisher kaum zugänglich. Operationell wird daher zwischen klassischer Konditionierung als verhaltensunabhängigem Lernvorgang (CS-US Assoziationen) und operanter Konditionierung als essentiell verhaltensabhängigem Lernen (B-US Assoziationen) unterschieden. In den meisten bisher beschriebenen Lernexperimenten ist noch nicht einmal diese Trennung in Einzelassoziationen vollständig durchzuführen gewesen. Im Drosophila Flugsimulator, in dem das relevante Verhalten eine einzelne Bewegungsvariable (das Gierungsdrehmoment) ist, können zum ersten Mal die operanten (B-US, B-CS) und die klassischen (CS-US) Bestandteile einer komplexen Lernsituation völlig getrennt und auf ihre Interaktionen hin untersucht werden. In der vorliegenden Arbeit wurden sowohl die Beiträge der Einzelassoziationen (CS-US, B-US und B-CS) bei der Akquisition der Gedächtnismatrize in komplexen Lernsituationen untersucht, als auch die Eigenschaften einer besonders prominenten Einzelassoziation (CS-US) während einer komplexen Lernsituation zum ersten Mal weitgehend charakterisiert. Mit einer gejochten (yoked) Kontrolle kann gezeigt werden, dass das klassische (CS-US) Musterlernen umfangreicheres Training als das operante Musterlernen erfordert. Außerdem kann die Fliege einen operant gelernter Stimulus von dem Verhalten mit dem er gelernt wurde, auf ein anderes Verhalten im Test übertragen. Dieses Resultat zeigt eindeutig, dass während der operanten Konditionierung klassische (CS-US) Assoziationen gebildet werden können. In einer Erweiterung dieses Ergebnisses zeigt sich, dass solch eine klassische Assoziation, wenn sie gebildet wird, die Bildung einer operanten Assoziation blockiert, die ohne operante Kontrolle der klassisch assoziierten Stimuli gebildet würde. Stattdessen scheint sich der operante Bestandteil weniger ausgeprägt zu entwickeln und ist eventuell in einer komplexen Dreiwege-Assoziation eingebunden. Die Dreiwege-Assoziation könnte entweder als sequentielle B-CS-US oder als hierarchische (B-CS)-US Assoziation implementiert sein. Der Vergleich einer einfachen klassischen (CS-US) mit einer komplexen operanten (B, CS und US) Lernsituation und einer einfachen operanten (B-US) mit einer anderen komplexen operanten (B, CS und US) Lernsituation, ermöglicht das Postulat einer Hierarchie der Prädiktoren für Verstärker. Operantes Verhalten während einer komplexen operanten Lernsituation wird wenig oder überhaupt nicht konditioniert. Die Assoziierbarkeit der klassischen Stimuli ohne Relation zum Verhalten des Tieres (CS-US) sind - wie operantes Verhalten alleine (B-US) auch - von mittlerer Assoziierbarkeit. Stimuli die von operantem Verhalten kon-trolliert werden, erhöhen am schnellsten ihre assoziative Stärke. Sind mehrere Stimuli während des Lernvorgangs zugänglich, stellt sich erneut die Frage, welche von den CS-US Assoziationen gebildet werden. Eine Vielzahl verschiedenster Studien in Vertebraten wiesen erstaunlich übereinstimmende Ergebnisse auf. Diese Ergebnisse inspirierten dazu, die Eigenschaften der CS-US Assoziationen in der komplexen Lernsituation am Flugsimulator zu untersuchen und mit Ergebnissen in Vertebraten zu vergleichen. Es wird erstmals gezeigt, dass Drosophila zusammengesetzte Stimuli lernen und die Einzelkomponenten unabhängig voneinander und in etwa ähnlichen Proportionen wiedererkennen kann. Der Versuch "Lernen zweiter Ordnung" mit diesen Stimuli zu erzielen, liefert einen relativ kleinen Effekt. Die Gegenüberstellung mit Daten aus Vertebraten liefert sowohl Abweichungen als auch Übereinstimmungen hinsichtlich der Lernregeln, die beim klassischen Konditionieren von Vertebraten gefunden wurden. Während es ein deutliches "sensorisches Präkonditionieren" gibt, konnte kein "Blocken" gefunden werden. Das sensorische Präkonditionieren in dieser Studie zeigt sich bei gleichzeitiger Stimuluspräsentation und ist vom Mass der Präkonditionierung abhängig. Es wird argumentiert, dass dieser Effekt ein Fall "beiläufigen Lernens" ist, bei dem zwei Stimuli ohne die Notwendigkeit der Verstärkung assoziiert werden. Für das nicht gefundene Blocken werden mögliche Gründe diskutiert und weiterführende Experimente vor-geschlagen. Abschließend wird über die Implikationen der Resultate dieser Arbeit für das allgemeine Verständnis der Gedächtnisbildung in komplexen Lernsituationen nachgedacht. KW - Taufliege KW - Lernen KW - Flugsimulator KW - Drosophila KW - Lernen KW - Gedächtnis KW - Assoziation KW - assoziativ KW - Flugsimulator KW - Lernregeln KW - operantes Konditionieren KW - klassisches Konditionieren KW - Drosophila KW - learning KW - memory KW - association KW - associative KW - flight simulator KW - learning rules KW - operant conditioning KW - classical conditioning Y1 - 2000 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-1039 ER - TY - JOUR A1 - Breitenbach, Tim A1 - Lorenz, Kristina A1 - Dandekar, Thomas T1 - How to steer and control ERK and the ERK signaling cascade exemplified by looking at cardiac insufficiency JF - International Journal of Molecular Sciences N2 - Mathematical optimization framework allows the identification of certain nodes within a signaling network. In this work, we analyzed the complex extracellular-signal-regulated kinase 1 and 2 (ERK1/2) cascade in cardiomyocytes using the framework to find efficient adjustment screws for this cascade that is important for cardiomyocyte survival and maladaptive heart muscle growth. We modeled optimal pharmacological intervention points that are beneficial for the heart, but avoid the occurrence of a maladaptive ERK1/2 modification, the autophosphorylation of ERK at threonine 188 (ERK\(^{Thr188}\) phosphorylation), which causes cardiac hypertrophy. For this purpose, a network of a cardiomyocyte that was fitted to experimental data was equipped with external stimuli that model the pharmacological intervention points. Specifically, two situations were considered. In the first one, the cardiomyocyte was driven to a desired expression level with different treatment strategies. These strategies were quantified with respect to beneficial effects and maleficent side effects and then which one is the best treatment strategy was evaluated. In the second situation, it was shown how to model constitutively activated pathways and how to identify drug targets to obtain a desired activity level that is associated with a healthy state and in contrast to the maleficent expression pattern caused by the constitutively activated pathway. An implementation of the algorithms used for the calculations is also presented in this paper, which simplifies the application of the presented framework for drug targeting, optimal drug combinations and the systematic and automatic search for pharmacological intervention points. The codes were designed such that they can be combined with any mathematical model given by ordinary differential equations. KW - optimal pharmacological modulation KW - efficient intervention points KW - ERK signaling KW - optimal treatment strategies KW - optimal drug targeting KW - optimal drug combination Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285164 SN - 1422-0067 VL - 20 IS - 9 ER - TY - JOUR A1 - Breitenbach, Tim A1 - Helfrich-Förster, Charlotte A1 - Dandekar, Thomas T1 - An effective model of endogenous clocks and external stimuli determining circadian rhythms JF - Scientific Reports N2 - Circadian endogenous clocks of eukaryotic organisms are an established and rapidly developing research field. To investigate and simulate in an effective model the effect of external stimuli on such clocks and their components we developed a software framework for download and simulation. The application is useful to understand the different involved effects in a mathematical simple and effective model. This concerns the effects of Zeitgebers, feedback loops and further modifying components. We start from a known mathematical oscillator model, which is based on experimental molecular findings. This is extended with an effective framework that includes the impact of external stimuli on the circadian oscillations including high dose pharmacological treatment. In particular, the external stimuli framework defines a systematic procedure by input-output-interfaces to couple different oscillators. The framework is validated by providing phase response curves and ranges of entrainment. Furthermore, Aschoffs rule is computationally investigated. It is shown how the external stimuli framework can be used to study biological effects like points of singularity or oscillators integrating different signals at once. The mathematical framework and formalism is generic and allows to study in general the effect of external stimuli on oscillators and other biological processes. For an easy replication of each numerical experiment presented in this work and an easy implementation of the framework the corresponding Mathematica files are fully made available. They can be downloaded at the following link: https://www.biozentrum.uni-wuerzburg.de/bioinfo/computing/circadian/. KW - computational biology and bioinformatics KW - systems biology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261655 VL - 11 IS - 1 ER - TY - THES A1 - Breitenbach, Tim T1 - A mathematical optimal control based approach to pharmacological modulation with regulatory networks and external stimuli T1 - Ein auf mathematischer Optimalkontrolle basierender Ansatz für pharmakologische Modulation mit regulatorischen Netzwerken und externen Stimuli N2 - In this work models for molecular networks consisting of ordinary differential equations are extended by terms that include the interaction of the corresponding molecular network with the environment that the molecular network is embedded in. These terms model the effects of the external stimuli on the molecular network. The usability of this extension is demonstrated with a model of a circadian clock that is extended with certain terms and reproduces data from several experiments at the same time. Once the model including external stimuli is set up, a framework is developed in order to calculate external stimuli that have a predefined desired effect on the molecular network. For this purpose the task of finding appropriate external stimuli is formulated as a mathematical optimal control problem for which in order to solve it a lot of mathematical methods are available. Several methods are discussed and worked out in order to calculate a solution for the corresponding optimal control problem. The application of the framework to find pharmacological intervention points or effective drug combinations is pointed out and discussed. Furthermore the framework is related to existing network analysis tools and their combination for network analysis in order to find dedicated external stimuli is discussed. The total framework is verified with biological examples by comparing the calculated results with data from literature. For this purpose platelet aggregation is investigated based on a corresponding gene regulatory network and associated receptors are detected. Furthermore a transition from one to another type of T-helper cell is analyzed in a tumor setting where missing agents are calculated to induce the corresponding switch in vitro. Next a gene regulatory network of a myocardiocyte is investigated where it is shown how the presented framework can be used to compare different treatment strategies with respect to their beneficial effects and side effects quantitatively. Moreover a constitutively activated signaling pathway, which thus causes maleficent effects, is modeled and intervention points with corresponding treatment strategies are determined that steer the gene regulatory network from a pathological expression pattern to physiological one again. N2 - In dieser Arbeit werden Modelle für molekulare Netzwerke bestehend aus gewöhnlichen Differentialgleichungen durch Terme erweitert, die die Wechselwirkung zwischen dem entsprechenden molekularen Netzwerk und der Umgebung berücksichtigen, in die das molekulare Netzwerk eingebettet ist. Diese Terme modellieren die Effekte von externen Stimuli auf das molekulare Netzwerk. Die Nutzbarkeit dieser Erweiterung wird mit einem Modell der circadianen Uhr demonstriert, das mit gewissen Termen erweitert wird und Daten von mehreren verschiedenen Experimenten zugleich reproduziert. Sobald das Modell einschließlich der externen Stimuli aufgestellt ist, wird eine Grundstruktur entwickelt um externe Stimuli zu berechnen, die einen gewünschten vordefinierte Effekt auf das molekulare Netzwerk haben. Zu diesem Zweck wird die Aufgabe, geeignete externe Stimuli zu finden, als ein mathematisches optimales Steuerungsproblem formuliert, für welches, um es zu lösen, viele mathematische Methoden zur Verfügung stehen. Verschiedene Methoden werden diskutiert und ausgearbeitet um eine Lösung für das entsprechende optimale Steuerungsproblem zu berechnen. Auf die Anwendung dieser Grundstruktur pharmakologische Interventionspunkte oder effektive Wirkstoffkombinationen zu finden, wird hingewiesen und diese diskutiert. Weiterhin wird diese Grundstruktur in Bezug zu existierenden Netzwerkanalysewerkzeugen gesetzt und ihre Kombination für die Netzwerkanalyse diskutiert um zweckbestimmte externe Stimuli zu finden. Die gesamte Grundstruktur wird mit biologischen Beispielen verifiziert, indem man die berechneten Ergebnisse mit Daten aus der Literatur vergleicht. Zu diesem Zweck wird die Blutplättchenaggregation untersucht basierend auf einem entsprechenden genregulatorischen Netzwerk und damit assoziierte Rezeptoren werden detektiert. Weiterhin wird ein Wechsel von einem T-Helfer Zelltyp in einen anderen in einer Tumorumgebung analysiert, wobei fehlende Agenzien berechnet werden um den entsprechenden Wechsel in vitro zu induzieren. Als nächstes wird ein genregulatorisches Netzwerk eines Myokardiozyten untersucht, wobei gezeigt wird wie die präsentierte Grundstruktur genutzt werden kann um verschiedene Behandlungsstrategien in Bezug auf ihre nutzbringenden Wirkungen und Nebenwirkungen quantitativ zu vergleichen. Darüber hinaus wird ein konstitutiv aktivierter Signalweg, der deshalb unerwünschte Effekte verursacht, modelliert und Interventionspunkte mit entsprechenden Behandlungsstrategien werden bestimmt, die das genregulatorische Netzwerk wieder von einem pathologischen Expressionsmuster zu einem physiologischen steuern. KW - Bioinformatik KW - systematic drug targeting KW - optimal drug combination KW - disease modelling KW - external stimuli KW - intervention point analyzing KW - Molekülsystem KW - Reiz Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-174368 ER - TY - JOUR A1 - Brehm, Klaus A1 - Koziol, Uriel A1 - Rauschendorfer, Theresa A1 - Rodríguez, Luis Zanon A1 - Krohne, Georg T1 - The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis N2 - Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae. KW - Cestoda KW - Echinococcus KW - Neoblast KW - Germinative cell KW - Stem cell KW - Nanos KW - Argonaute KW - Mucin KW - Alkaline phosphatase Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110315 ER - TY - JOUR A1 - Brehm, Klaus A1 - Koziol, Uriel A1 - Krohne, Georg T1 - Anatomy and development of the larval nervous system in Echinococcus multilocularis JF - Frontiers in Zoology N2 - Background The metacestode larva of Echinococcus multilocularis (Cestoda: Taeniidae) develops in the liver of intermediate hosts (typically rodents, or accidentally in humans) as a labyrinth of interconnected cysts that infiltrate the host tissue, causing the disease alveolar echinococcosis. Within the cysts, protoscoleces (the infective stage for the definitive canid host) arise by asexual multiplication. These consist of a scolex similar to that of the adult, invaginated within a small posterior body. Despite the importance of alveolar echinococcosis for human health, relatively little is known about the basic biology, anatomy and development of E. multilocularis larvae, particularly with regard to their nervous system. Results We describe the existence of a subtegumental nerve net in the metacestode cysts, which is immunoreactive for acetylated tubulin-α and contains small populations of nerve cells that are labeled by antibodies raised against several invertebrate neuropeptides. However, no evidence was found for the existence of cholinergic or serotoninergic elements in the cyst wall. Muscle fibers occur without any specific arrangement in the subtegumental layer, and accumulate during the invaginations of the cyst wall that form brood capsules, where protoscoleces develop. The nervous system of the protoscolex develops independently of that of the metacestode cyst, with an antero-posterior developmental gradient. The combination of antibodies against several nervous system markers resulted in a detailed description of the protoscolex nervous system, which is remarkably complex and already similar to that of the adult worm. Conclusions We provide evidence for the first time of the existence of a nervous system in the metacestode cyst wall, which is remarkable given the lack of motility of this larval stage, and the lack of serotoninergic and cholinergic elements. We propose that it could function as a neuroendocrine system, derived from the nervous system present in the bladder tissue of other taeniids. The detailed description of the development and anatomy of the protoscolex neuromuscular system is a necessary first step toward the understanding of the developmental mechanisms operating in these peculiar larval stages. KW - Echinococcus KW - Metacestode KW - Protoscolex KW - Nervous system KW - Neuropeptide KW - Serotonin KW - Acetylated tubulin Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96504 UR - http://www.frontiersinzoology.com/content/10/1/24 ER - TY - JOUR A1 - Brehm, Klaus A1 - Hemer, Sarah A1 - Konrad, Christian A1 - Spiliotis, Markus A1 - Koziol, Uriel A1 - Schaack, Dominik A1 - Förster, Sabine A1 - Gelmedin, Verena A1 - Stadelmann, Britta A1 - Dandekar, Thomas A1 - Hemphill, Andrew T1 - Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development N2 - Background The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. Results Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. Conclusions Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs. KW - Cestode KW - Tapeworm KW - Echinococcus KW - Echinococcosis KW - Insulin KW - Receptor kinase KW - Kinase inhibitor KW - Host-parasite interaction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110357 ER - TY - JOUR A1 - Brehm, Klaus A1 - Haas, Albert A1 - Goebel, Werner A1 - Kreft, Jürgen T1 - A gene encoding a superoxide dismutase of the facultative intracellular bacterium Listeria monocytogenes N2 - A gene (Imsod) encoding superoxide dismutase (SOD; EC 1.15.1.1) of the facultative intracellular pathogen, Listeria monocytogenes, was cloned by functional complementation of an SOD-deficient Escherichia coli mutant. The nucleotide sequence was determined and the deduced amino acid (aa) sequence (202 aa) showed close similarity to manganese-containing SOD's from other organisms. Subunits of the recombinant L. monocytogenes SOD (re-SOD) and of both E. coli SODs formed enzymatically active hybrid enzymes in vivo. DNA/DNA-hybridization experiments showed that this type of recombinant re-sod gene is conserved within the genus Listeria. KW - Biologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60515 ER - TY - THES A1 - Breher, Stephanie T1 - Die kardiale Funktion von Popdc1 in der Maus: Vom Gen zum Phän T1 - The cardiac function of Popdc1 in mouse: From gene to phene N2 - Die Popeye domain containing (Popdc)-Gene bilden eine evolutionär stark konservierte Genfamilie mit präferenzieller Expression im Herzen und in der Skelettmuskulatur. In dieser Arbeit konnte gezeigt werden, dass Popdc1 in kardialen Myozyten in Glanzstreifen, lateralen Membranen und im T-Tubuli-System exprimiert wird und mit Ionenkanälen und anderen myozytären Membranproteinen wie Cav1.2, Caveolin 3 und NCX1 kolokalisiert ist. Im ventrikulären Reizleitungssystem ist die Expression von Popdc1 gegenüber dem ventrikulären Arbeitsmyokard erhöht, während Atrium und Sinusknoten nahezu äquivalente Expressionsdomänen aufweisen. Mithilfe von elektrophysiologischen Untersuchungen konnte bei den Popdc1-Nullmutanten eine stressinduzierte Sinusbradykardie festgestellt werden, die altersabhängig auftritt und auf Sinuspausen zurückzuführen ist. Histologische Untersuchungen, unter Zuhilfenahme des Sinusknotenmarkers HCN4, zeigten einen Zellverlust im inferioren Teil des Sinusknotens. Popdc1 ist ein Transmembranprotein, das eine 150 Aminosäure umfassende, stark konservierte Popeye-Domäne aufweist. Für diese Domäne konnte auf struktureller Ebene eine Homologie zu zyklischen Nukleotid-Bindungsdomänen vorhergesagt und eine Bindung an cAMP und cGMP experimentell demonstriert werden. Es handelt sich bei den Popdc-Proteinen um einen neuen Zweig der Bindungsproteine für zyklische Nukleotidmonophosphate (cNMP). Die Bindungssequenz weist signifikante Unterschiede zu anderen bereits identifizierten cNMP-Bindungsproteinen auf. Weiterhin wurde die Interaktion von Popdc1 mit TREK1, einem Mitglied der Tandemporenkanäle untersucht. Es zeigte sich, dass Popdc1 nach Koexpression in Froschoozyten, den TREK1-Strom erhöht und dass die β-adrenerge Inhibition des TREK1 Kanals durch Popdc1 verstärkt wird. Im Arbeitsmyokard, im kardialen Reizleitungssystem und in kotransfizierten Cos7-Zellen werden beide Proteine überlappend exprimiert. Diese Daten zeigen, dass Popdc1 eine wichtige Funktion bei der Regulation der Schrittmacheraktivität, der Aufrechterhaltung der Sinusknotenmorphologie und der Modulation von Ionenkanälen aufweist. Interessanterweise wurden von unserer Arbeitsgruppe bereits die gleichen Phänotypen für die Popdc2 Maus beschrieben, sodass die Popdc Genfamilie überlappende und redundante Funktionen aufweist. N2 - The Popeye domain containing (Popdc) family is a highly evolutionary conserved gene family, which shows no homology to other genes. This family shows a preferential expression in the heart and skeletal muscle. In the present study it is shown that Popdc1 protein in the heart was predominantly localized to the intercalated disc, lateral membranes and T-tubularsystem, where it was co-localized with other cardiac membrane proteins such as Cav1.2, Caveolin 3 and NCX1. The expression of Popdc1-LacZ transgene as well as Popdc1 protein was elevated in the ventricular conduction system compared to the ventricular working myocardium. In contrast, expression in atrial tissue was equivalent to the expression in the sinus node. Electrophysiological measurements in Popdc1 null mutants revealed a stressinduced and age-dependent sinus bradycardia, which was due to an increase in sinus pauses and independent of the nature of stress. Histological examinations with the help of the sinus node marker HCN4 revealed structural alterations in the inferior part of the sinus node in 8 months old Popdc1-mice. Biochemical examinations of Popdc1 showed that Popdc1 is a transmembrane protein. The N-terminus is extracellular and glycosylated, while the Cterminus is intracellular and harbours a highly conserved 150 amino acid-long Popeye domain. For this domain, a predicted homology to cyclic nucleotide binding domains was observed. Binding of cAMP and cGMP was experimentally demonstrated and thus, the Popdc proteins constitute a novel branch of the cyclic nucleotide binding protein family. Furthermore interaction of Popdc1 with the tandem pore channel TREK1 was examined. After co-injection of Popdc1 the TREK1 current was increased in Xenopus oocytes. Furthermore, β-adrenergic inhibition of TREK1 current was enhanced in the presence of Popdc1. In working myocardium, conduction tissue as well as in co-transfected Cos7 cells the two proteins showed a similar distribution. In conclusion, Popdc1 is involved in cardiac pacemaker activity, maintaining sinus node morphology and modulating ion channels that contribute to the setting of the membrane potential in cardiac myocytes. Interestingly, a highly similar phenotype was observed for the Popdc2 mouse mutant and therefore the Popdc gene family displays overlapping and redundant functions. KW - Sinusknoten KW - Genexpression KW - Elektrophysiologie KW - Popdc1 KW - Transmembranprotein KW - Sinusknotenbradykardie KW - cAMP-Bindung KW - Popdc1 KW - transmembrane protein KW - sinus bradycardia KW - cAMP binding Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-37283 ER - TY - JOUR A1 - Breeze, Tom D. A1 - Vaissiere, Bernhard E. A1 - Bommarco, Riccardo A1 - Petanidou, Theodora A1 - Seraphides, Nicos A1 - Kozak, Lajos A1 - Scheper, Jeroen A1 - Biesmeijer, Jacobus C. A1 - Kleijn, David A1 - Gyldenkærne, Steen A1 - Moretti, Marco A1 - Holzschuh, Andrea A1 - Steffan-Dewenter, Ingolf A1 - Stout, Jane C. A1 - Pärtel, Meelis A1 - Zobel, Martin A1 - Potts, Simon G. T1 - Agricultural Policies Exacerbate Honeybee Pollination Service Supply-Demand Mismatches Across Europe JF - PLOS ONE N2 - Declines in insect pollinators across Europe have raised concerns about the supply of pollination services to agriculture. Simultaneously, EU agricultural and biofuel policies have encouraged substantial growth in the cultivated area of insect pollinated crops across the continent. Using data from 41 European countries, this study demonstrates that the recommended number of honeybees required to provide crop pollination across Europe has risen 4.9 times as fast as honeybee stocks between 2005 and 2010. Consequently, honeybee stocks were insufficient to supply >90% of demands in 22 countries studied. These findings raise concerns about the capacity of many countries to cope with major losses of wild pollinators and highlight numerous critical gaps in current understanding of pollination service supplies and demands, pointing to a pressing need for further research into this issue. KW - economy services KW - fruit set KW - sequential introduction KW - enhance KW - biodiversity KW - abundance KW - declines KW - crops KW - colonies KW - density Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117692 SN - 1932-6203 VL - 9 IS - 1 ER - TY - JOUR A1 - Brandstätter, Andreas A1 - Rössler, W. A1 - Kleineidam, C. J. T1 - Friends and foes from an ant brain's point of view - neuronal correlates of colony odors in a social insect N2 - Background: Successful cooperation depends on reliable identification of friends and foes. Social insects discriminate colony members (nestmates/friends) from foreign workers (non-nestmates/foes) by colony-specific, multi-component colony odors. Traditionally, complex processing in the brain has been regarded as crucial for colony recognition. Odor information is represented as spatial patterns of activity and processed in the primary olfactory neuropile, the antennal lobe (AL) of insects, which is analogous to the vertebrate olfactory bulb. Correlative evidence indicates that the spatial activity patterns reflect odor-quality, i.e., how an odor is perceived. For colony odors, alternatively, a sensory filter in the peripheral nervous system was suggested, causing specific anosmia to nestmate colony odors. Here, we investigate neuronal correlates of colony odors in the brain of a social insect to directly test whether they are anosmic to nestmate colony odors and whether spatial activity patterns in the AL can predict how odor qualities like ‘‘friend’’ and ‘‘foe’’ are attributed to colony odors. Methodology/Principal Findings: Using ant dummies that mimic natural conditions, we presented colony odors and investigated their neuronal representation in the ant Camponotus floridanus. Nestmate and non-nestmate colony odors elicited neuronal activity: In the periphery, we recorded sensory responses of olfactory receptor neurons (electroantennography), and in the brain, we measured colony odor specific spatial activity patterns in the AL (calcium imaging). Surprisingly, upon repeated stimulation with the same colony odor, spatial activity patterns were variable, and as variable as activity patterns elicited by different colony odors. Conclusions: Ants are not anosmic to nestmate colony odors. However, spatial activity patterns in the AL alone do not provide sufficient information for colony odor discrimination and this finding challenges the current notion of how odor quality is coded. Our result illustrates the enormous challenge for the nervous system to classify multi-component odors and indicates that other neuronal parameters, e.g., precise timing of neuronal activity, are likely necessary for attribution of odor quality to multi-component odors. KW - Ameisen KW - Geruch Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69046 ER - TY - THES A1 - Brandstaetter, Andreas Simon T1 - Neuronal correlates of nestmate recognition in the carpenter ant, Camponotus floridanus T1 - Neuronale Korrelate der Nestgenossen-Erkennung bei der Rossameise, Camponotus floridanus N2 - Cooperation is beneficial for social groups and is exemplified in its most sophisticated form in social insects. In particular, eusocial Hymenoptera, like ants and honey bees, exhibit a level of cooperation only rarely matched by other animals. To assure effective defense of group members, foes need to be recognized reliably. Ants use low-volatile, colony-specific profiles of cuticular hydrocarbons (colony odor) to discriminate colony members (nestmates) from foreign workers (non-nestmates). For colony recognition, it is assumed that multi-component colony odors are compared to a neuronal template, located in a so far unidentified part of the nervous system, where a mismatch results in aggression. Alternatively, a sensory filter in the periphery of the nervous system has been suggested to act as a template, causing specific anosmia to nestmate colony odor due to sensory adaptation and effectively blocking perception of nestmates. Colony odors are not stable, but change over time due to environmental influences. To adjust for this, the recognition system has to be constantly updated (template reformation). In this thesis, I provide evidence that template reformation can be induced artificially, by modifying the sensory experience of carpenter ants (Camponotus floridanus; Chapter 1). The results of the experiments showed that template reformation is a relatively slow process taking several hours and this contradicts the adaptation-based sensory filter hypothesis. This finding is supported by first in-vivo measurements describing the neuronal processes underlying template reformation (Chapter 5). Neurophysiological measurements were impeded at the beginning of this study by the lack of adequate technical means to present colony odors. In a behavioral assay, I showed that tactile interaction is not necessary for colony recognition, although colony odors are of very low volatility (Chapter 2). I developed a novel stimulation technique (dummy-delivered stimulation) and tested its suitability for neurophysiological experiments (Chapter 3). My experiments showed that dummy-delivered stimulation is especially advantageous for presentation of low-volatile odors. Colony odor concentration in headspace was further increased by moderately heating the dummies, and this allowed me to measure neuronal correlates of colony odors in the peripheral and the central nervous system using electroantennography and calcium imaging, respectively (Chapter 4). Nestmate and non-nestmate colony odor elicited strong neuronal responses in olfactory receptor neurons of the antenna and in the functional units of the first olfactory neuropile of the ant brain, the glomeruli of the antennal lobe (AL). My results show that ants are not anosmic to nestmate colony odor and this clearly invalidates the previously suggested sensory filter hypothesis. Advanced two-photon microscopy allowed me to investigate the neuronal representation of colony odors in different neuroanatomical compartments of the AL (Chapter 5). Although neuronal activity was distributed inhomogeneously, I did not find exclusive representation restricted to a single AL compartment. This result indicates that information about colony odors is processed in parallel, using the computational power of the whole AL network. In the AL, the patterns of glomerular activity (spatial activity patterns) were variable, even in response to repeated stimulation with the same colony odor (Chapter 4&5). This finding is surprising, as earlier studies indicated that spatial activity patterns in the AL reflect how an odor is perceived by an animal (odor quality). Under natural conditions, multi-component odors constitute varying and fluctuating stimuli, and most probably animals are generally faced with the problem that these elicit variable neuronal responses. Two-photon microscopy revealed that variability was higher in response to nestmate than to non-nestmate colony odor (Chapter 5), possibly reflecting plasticity of the AL network, which allows template reformation. Due to their high variability, spatial activity patterns in response to different colony odors were not sufficiently distinct to allow attribution of odor qualities like ‘friend’ or ‘foe’. This finding challenges our current notion of how odor quality of complex, multi-component odors is coded. Additional neuronal parameters, e.g. precise timing of neuronal activity, are most likely necessary to allow discrimination. The lower variability of activity patterns elicited by non-nestmate compared to nestmate colony odor might facilitate recognition of non-nestmates at the next level of the olfactory pathway. My research efforts made the colony recognition system accessible for direct neurophysiological investigations. My results show that ants can perceive their own nestmates. The neuronal representation of colony odors is distributed across AL compartments, indicating parallel processing. Surprisingly, the spatial activity patterns in response to colony are highly variable, raising the question how odor quality is coded in this system. The experimental advance presented in this thesis will be useful to gain further insights into how social insects discriminate friends and foes. Furthermore, my work will be beneficial for the research field of insect olfaction as colony recognition in social insects is an excellent model system to study the coding of odor quality and long-term memory mechanisms underlying recognition of complex, multi-component odors. N2 - Kooperation innerhalb sozialer Gruppen ist vorteilhaft und zeigt sich bei sozialen Insekten in seiner am höchsten entwickelten Form. Besonders eusoziale Hymenopteren, wie Ameisen und Honigbienen, zeigen ein Maß an Kooperation, das nur selten von anderen Tierarten erreicht wird. Um eine effektive Verteidigung der Gruppenmitglieder sicher zu stellen, ist die zuverlässige Erkennung von Feinden unerlässlich. Ameisen verwenden schwerflüchtige, koloniespezifische Profile kutikulärer Kohlenwasserstoffe (Kolonieduft) zur Unterscheidung zwischen Gruppenmitgliedern (Nestgenossen) und fremden Arbeiterinnen (Nestfremdlinge). Man geht davon aus, dass die aus einer Vielzahl von Komponenten bestehenden Koloniedüfte zum Zweck der Kolonieerkennung mit einer neuronalen Schablone, welche sich an bisher unbestimmter Stelle im Nerven-system befindet, abgeglichen werden. Dabei führt eine Diskrepanz zwischen Schablone und Kolonieduft zu Aggression. Eine alternative Hypothese besagt, dass ein sensorischer Filter in der Peripherie des Nervensystems die Aufgabe einer neuronalen Schablone übernimmt. Dies würde mittels sensorischer Adaptation zu spezifischer Anosmie gegenüber Nestgenossen-Kolonieduft führen, so dass die Wahrnehmung von Nestgenossen effektiv verhindert wäre. Allerdings sind Koloniedüfte nicht stabil, sondern verändern sich im Lauf der Zeit aufgrund von Umwelteinflüssen. Um dies zu kompensieren, muss das Erkennungssystem fortwährend aktualisiert werden (Schablonenerneuerung). In dieser Arbeit erbringe ich den Nachweis, dass bei Rossameisen (Camponotus floridanus) die Schablonenerneuerung artifiziell durch Modifizierung der sensorischen Erfahrung induziert werden kann (Kapitel 1). Die Ergebnisse der in Kapitel 1 beschriebenen Experimente zeigen, dass die Schablonenerneuerung ein relativ langsamer Prozess ist, der mehrere Stunden in Anspruch nimmt. Dies widerspricht der Hypothese eines sensorischen Filters, welcher auf sensorischer Adaptation beruht. Dieser Befund konnte mittels erster in-vivo Messungen bestätigt werden, mit Hilfe derer die der Schablonenerneuerung zugrunde liegenden neuronalen Prozesse beschrieben wurden (Kapitel 5). Die neurophysiologischen Messungen wurden zu Beginn dieser Studie durch das Fehlen eines adäquaten Mittels zur Präsentation von Koloniedüften erschwert. In einem Verhaltensversuch konnte ich zeigen, dass taktile Interaktionen für die Kolonieerkennung nicht notwendig sind (Kapitel 2). Ich entwickelte eine neuartige Stimulierungsmethode (Dummy-vermittelte Stimulierung) und testete deren Eignung für neurophysiologische Experimente (Kapitel 3). Meine Experimente zeigten, dass die Dummy-vermittelte Stimulierung besonders für die Präsentation von schwerflüchtigen Düften geeignet ist. Die Konzentration von Koloniedüften im Gasraum konnte durch moderates Aufheizen der Dummys weiter gesteigert werden. Dies erlaubte mir, die neuronalen Korrelate von Koloniedüften im peripheren und im zentralen Nervensystem mittels Elektroantennographie bzw. funktionaler Bildgebung (Calcium Imaging) zu messen (Kapitel 4). Nestgenossen- und Nestfremdlings-Koloniedüfte riefen starke neuronale Antworten in den olfaktorischen Rezeptorneuronen der Antenne und in den funktionalen Einheiten des ersten olfaktorischen Neuropils des Ameisengehirns, den Glomeruli des Antennallobus (AL), hervor. Meine Ergebnisse zeigen, dass Ameisen nicht anosmisch gegenüber Nestgenossen-Koloniedüften sind, womit die vorgeschlagene Hypothese eines sensorischen Filters eindeutig für ungültig erklärt werden kann. Mittels fortschrittlicher Zwei-Photonen-Mikroskopie konnte ich die neuronale Repräsentation von Koloniedüften in verschiedenen neuroanatomischen Kompartimenten des AL messen (Kapitel 5). Obgleich die neuronale Aktivität inhomogen verteilt war, konnte ich keine exklusive Repräsentation finden, die auf ein einzelnes AL-Kompartiment beschränkt gewesen wäre. Dieses Ergebnis weist darauf hin, dass Informationen über Koloniedüfte parallel verarbeitet werden und dies erlaubt die Nutzung der Rechenleistung des kompletten AL-Netzwerkes. Im AL waren die Muster glomerulärer Aktivität (räumliche Aktivitätsmuster) variabel, selbst wenn sie durch wiederholte Stimulierung mit dem gleichen Kolonieduft hervorgerufen wurden (Kapitel 4&5). Dieser Befund ist insofern überraschend, als frühere Studien darauf hinwiesen, dass die räumlichen Aktivitätsmuster im AL widerspiegeln, wie ein Duft von einem Tier wahrge¬nommen wird (Duftqualität). Unter natürlichen Bedingungen stellen Düfte, die aus einer Vielzahl von Komponenten bestehen, variable und fluktuierende Stimuli dar. Höchstwahrscheinlich sind Tiere generell mit dem Problem konfrontiert, dass solche Düfte variable neuronale Antworten hervorrufen. Mittels Zwei-Photonen-Mikroskopie konnte ich zeigen, dass die Variabilität in Antwort auf Nestgenossen-Kolonieduft höher war als in Antwort auf Nestfremdlings-Kolonieduft (Kapitel 5). Möglicherweise spiegelt dies jene Plastizität im AL-Netzwerk wider, welche die Schablonenerneuerung ermöglicht. Aufgrund ihrer hohen Variabilität waren die von verschiedenen Koloniedüften hervorgerufenen räumlichen Aktivierungsmuster nicht hinreichend unterschiedlich, um eine Zuordnung von Duft-qualitäten wie ‚Freund‘ oder ‚Feind‘ zu erlauben. Dieser Befund stellt unsere momentane Auffassung in Frage, wie die Duftqualität komplexer, aus vielen Komponenten bestehender Düfte kodiert wird. Höchstwahrscheinlich sind zusätzliche neuronale Parameter, wie z.B. die präzise, zeitliche Koordinierung neuronaler Aktivität, zur Diskriminierung notwendig. Die geringere Variabilität der von Nestfremdlings-Kolonieduft hervorgerufenen Aktivitätsmuster könnte die Erkennung von Nestfremdlingen auf der nächsten Ebene der olfaktorischen Bahn begünstigen. Meine Forschungsarbeit hat das Kolonieerkennungssystem für direkte neurophysiologische Untersuchungen zugänglich gemacht. Meine Ergebnisse zeigen, dass Ameisen ihre eigenen Nest-genossen wahrnehmen können. Die neuronale Repräsentation von Koloniedüften ist über die AL-Kompartimente verteilt, was auf eine parallele Verarbeitung hinweist. Desweiteren könnte die geringere Variabilität der von Nestfremdlings-Kolonieduft hervorgerufenen Aktivitätsmuster die Erkennung von Nestfremdlingen auf der nächsten Ebene der olfaktorischen Bahn begünstigen. Erstaunlicherweise sind die räumlichen Aktivitätsmuster in Antwort auf Koloniedüfte hochvariabel. Die wirft die Frage auf, wie in diesem System die Duftqualität kodiert wird. Der experimentelle Fortschritt, den ich in dieser Doktorarbeit vorstelle, wird nützlich sein, um weitere Erkenntnisse zu gewinnen, wie soziale Insekten Freunde von Feinden unterscheiden. Desweiteren wird meine Arbeit dem Forschungsbereich Insektenolfaktion zuträglich sein, da die Kolonieerkennung bei sozialen Insekten ein hervorragendes Modelsystem darstellt, um die Kodierung von Duftqualität zu erforschen, sowie Langzeitmechanismen, die der Erkennung komplexer, aus vielen Komponenten bestehender Düfte zugrunde liegen. KW - Neuroethologie KW - Camponotus floridanus KW - Ameisenstaat KW - Kutikula KW - Kohlenwasserstoffe KW - Kolonieerkennung KW - kutikuläre Kohlenwasserstoffe KW - funktionale Bildgebung KW - Verhalten KW - Neurophysiologie KW - Soziobiologie KW - Erkennung KW - Geruch KW - neuroethology KW - colony recognition KW - cuticular hydrocarbons KW - social insects KW - aggressive behavior Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-55963 ER - TY - THES A1 - Brandes, Nicolas T1 - Oxidative Thiol Modifications in Pro- and Eukaryotic Organisms T1 - Oxidative Thiol Modifikationen in Pro- und Eukaryotischen Organismen N2 - Cystein spielt eine wichtige Rolle in der Biochemie vieler Proteine. Aufgrund der Redox-Eigenschaften und der hohen Reaktivität der freien Thiol-Gruppe sowie dessen Fähigkeit Metallionen zu koordinieren, ist Cystein oft Bestandteil von katalytischen Zentren vieler Enzyme. Zudem lassen sich Cysteine durch reaktive Sauerstoff- und Stickstoffspezies leicht reversibel oxidativ modifizieren. In den letzten Jahren wurde gezeigt, dass Proteine redox-bedingte Thiol-Modifikationen nutzen, um Veränderungen ihrer Aktivität zu steuern. Diese redox-regulierten Proteine spielen eine zentrale Rolle in vielen physiologischen Prozessen. Das erste Ziel meiner Arbeit war die Identifizierung von Stickstoffmonoxid (NO)-sensitiven Proteinen in E. coli. Die redox-bedingten Funktionsänderungen solcher Proteine erklären möglicherweise die veränderte Physiologie von E. coli Zellen, die unter NO-Stress leiden. Um E. coli Proteine zu identifizieren, die unter Einwirkung von NO-Stress reversibel Thiol-modifiziert werden, wandte ich eine Kombination aus differentiellem Thiol-Trapping und 2D Gel-Elektrophorese an. Es wurden zehn Proteinen identifiziert, welche NO-sensitive Thiol-Gruppen enthalten. Genetische Studien ergaben, dass Modifikationen an AceF & IlvC mitverantwortlich sind für die NO-induzierte Wachstumshemmung. Bemerkenswert ist es, dass die Mehrheit der identifizierten Proteine speziell nur gegen reaktive Stickstoffspezies empfindlich ist, welches an einem der identifizierten Stickstoffmonoxid-sensitiven Proteinen, der kleinen Untereinheit von Glutamate synthase, getestet wurde. In vivo und in vitro Aktivitätsstudien zeigten, dass es zu einer schnellen Inaktivierung von Glutamate synthase nach NO-Behandlung kommt, das Protein aber resistent gegenüber anderen Oxidationsmittel ist. Diese Resultate implizieren, dass reaktive Sauerstoff- und Stickstoffspezies unterschiedliche physiologische Vorgänge in Bakterien beeinflussen. Das zweite Ziel meiner Arbeit war es, redox-sensitive Proteine in S. cerevisiae zu identifizieren und deren Redox-Zustand als in vivo Read-Out zu verwenden, um die Rolle von oxidativen Stress während des Alterungsprozess eukaryotischer Zellen zu analysieren. Zunächst bestimmte ich in Hefezellen mit Hilfe von OxICAT, einer hochsensiblen quantitativen Methode, die Thiol-Trapping mit Massenspektrometrie verbindet, den exakten in vivo Thiol-Status von fast 300 Proteinen. Diese Proteine lassen sich in vier Gruppen einteilen: 1) Proteine, deren Cysteinreste resistent gegen Oxidation sind; 2) Proteine, in denen Cysteinmodifikationen strukturelle Aufgaben übernehmen; 3) Proteine mit oxidationsempfindlichen Cysteinen, die bereits eine gewisse Oxidation in exponentiell wachsenden Hefezellen aufweisen; 4) Proteine, die reduziert sind, aber redox-sensitive Cysteinreste enthalten, die die Funktion der Proteine bei Vorhandensein von oxidativen Stress beeinflussen. Die Sensitivität dieser Proteine gegenüber oxidativen Stress wurde durch Exposition subletaler Konzentrationen von H2O2 oder Superoxid auf Hefezellen nachgewiesen. Es wurde gezeigt, dass die wichtigsten zellulären Angriffspunkte von H2O2- und Superoxid-bedingtem Stress Proteine sind, die an Vorgängen der Translation, Glykolyse, des Citratzyklus und der Aminosäure-Biosynthese beteiligt sind. Diese Zielproteine zeigen, dass Zellen für die Bekämpfung von oxidativen Stress Metabolite schnell in Richtung des Pentosephosphatweges umleiten, um die Produktion des Reduktionsmittels NADPH sicherzustellen. Die hier präsentierten Ergebnisse belegen, dass die quantitative Bestimmung des Oxidationsstatus von Proteinen eine wertvolle Methode ist, um redox-sensitive Cysteinreste zu identifizieren. Die OxICAT Technologie wurde dann verwendet, um das genaue Ausmaß und die Entstehung von oxidativen Stress in chronologisch alternden S. cerevisiae Zellen zu bestimmen. Für diese Bestimmung wurde der Oxidationsstatus von Proteinen in alternden Hefezellen als physiologischer Read-Out verwendet. Ich zeigte, dass die zelluläre Redox-Homöostase in chronologisch alternden Hefezellen global zusammenbricht, wobei es sich dabei um einen Prozess handelt, der dem Zelltod vorausgeht. Der Beginn dieses Zusammenbruchs scheint mit der Lebensdauer der Hefezellen zu korrelieren, da Kalorienrestriktion die Lebensdauer der Hefezellen erhöht und den Zusammenbruch des Redox-Gleichgewichts verzögert. Die Oxidation einer kleinen Anzahl an Proteinen (z.B. Thioredoxin reductase) geht dem Redox-Zusammenbruch deutlich voraus, was maßgeblich zum Verlust der Redox-Homöostase beitragen könnte. Diese Studien an alternden Hefezellen erweitern unser Verständnis, wie sich Veränderungen in der Redox-Homöostase auf die Lebensdauer von Hefezellen auswirken. Zudem bestätigen die hier präsentierten Ergebnisse die Bedeutung von oxidativen Thiol-Modifikationen als eine der wichtigsten posttranslationalen Proteinmodifikationen in pro-und eukaryotischen Organismen N2 - Cysteines play important roles in the biochemistry of many proteins. The high reactivity, redox properties, and ability of the free thiol group to coordinate metal ions designate cysteines as the amino acids of choice to form key catalytic components of many enzymes. Also, cysteines readily react with reactive oxygen and nitrogen species to form reversible oxidative thiol modifications. Over the last few years, an increasing number of proteins have been identified that use redox-mediated thiol modifications to modulate their function, activity, or localization. These redox-regulated proteins are central players in numerous important cellular processes. First aim of this study was to discover nitric oxide (NO) sensitive proteins in E. coli, whose redox-mediated functional changes might explain the physiological alterations observed in E. coli cells suffering from NO-stress. To identify E. coli proteins that undergo reversible thiol modifications upon NO-treatment in vivo, I applied a differential thiol trapping technique combined with two-dimensional gel analysis. 10 proteins were found to contain thiol groups sensitive to NO-treatment. Subsequent genetic studies revealed that the oxidative modifications of AceF & IlvC are, in part, responsible for the observed NO-induced growth inhibition. Noteworthy, the majority of identified protein targets turned out to be specifically sensitive towards reactive nitrogen species. This oxidant specificity was tested on one NO-sensitive protein, the small subunit of glutamate synthase. In vivo and in vitro activity studies demonstrated that glutamate synthase rapidly inactivates upon nitric oxide treatment but is resistant towards other oxidative stressors. These results imply that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. The second aim of my study was to identify redox-sensitive proteins in S. cerevisiae and to use their redox state as in vivo read-out to assess the role of oxidative stress during the eukaryotic aging process. I first determined the precise in vivo thiol status of almost 300 yeast proteins located in the cytosol and sub-cellular compartments of yeast cells using a highly quantitative mass spectrometry based thiol trapping technique, called OxICAT. The identified proteins can be clustered in four groups: 1) proteins, whose cysteine residues are oxidation resistant; 2) proteins with structurally or functionally important cysteine modifications 3) proteins with highly oxidation-sensitive active site cysteines, which are partially oxidized in exponentially growing yeast cells due to their exquisite sensitivity towards low amounts of ROS; 4) proteins that are reduced in exponentially growing cells but harbor redox-sensitive cysteine(s) that affect the catalytic function of the protein during oxidative stress. These oxidative stress sensitive proteins were identified by exposure of yeast cells to sublethal concentrations of H2O2 or superoxide. It was shown that the major targets of peroxide- and superoxide-mediated stress in the cell are proteins involved in translation, glycolysis, TCA cycle and amino acid biosynthesis. These targets indicate that cells rapidly redirect the metabolic flux and energy towards the pentose phosphate pathway in an attempt to ensure the production of the reducing equivalent NADPH to counterattack oxidative stress. These results reveal that the quantitative assessment of a protein’s oxidation state is a valuable tool to identify catalytically active and redox-sensitive cysteine residues. The OxICAT technology was then used to precisely determine extent and onset of oxidative stress in chronologically aging S. cerevisiae cells by utilizing the redox status of proteins as physiological read-out. I found that chronological aging yeast cells undergo a global collapse of the cellular redox homeostasis, which precedes cell death. The onset of this collapse appears to correlate with the yeast life span, as caloric restriction increases the life span and delays the redox collapse. These results suggest that maintenance of the redox balance might contribute to the life expanding benefits of regulating the caloric intake of yeast. Clustering analysis of all oxidatively modified proteins in chronological aging yeast revealed a subset of proteins whose oxidative thiol modifications significantly precede the general redox collapse. Oxidation of these early target proteins, which most likely results in a loss of their activity, might contribute to or even cause the observed loss of redox homeostasis (i.e., thioredoxin reductase) in chronologically aging yeast. These studies in aging yeast expand our understanding how changes in redox homeostasis affect the life span of yeast cells and confirm the importance of oxidative thiol modifications as key posttranslational modifications in pro- and eukaryotic organisms. KW - Oxidativer Stress KW - Cystein KW - Saccharomyces cerevisiae KW - Escherichia coli KW - Wasserstoffperoxid KW - Hyperoxide KW - Sauerstoffradikal KW - Thiolgruppe KW - Altern KW - Oxidation KW - Biologische Oxidation KW - Oxidative Thiol Modifikationen KW - Reaktive Sauerstoffspezies KW - Chronologisches Altern KW - Reversibel KW - Posttranslational KW - oxidative thiol modification KW - chronological aging KW - reactive oxygen species KW - Saccharomyces cerevisiae KW - thioredoxin reductase Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46542 ER - TY - THES A1 - Brambrink, Tobias T1 - Entwicklung und Evaluierung eines Verfahrens zur Genexpressionsanalyse bei individuellen präimplantatorischen Säugerembryonen über die cDNA-Array-Technologie T1 - Development and evaluation of a methodology for cDNA-array gene expression profiling in individual mammalian preimplantation embryos N2 - Untersuchungen der Transkriptionsebene individueller präimplantatorischer Embryonalstadien können wertvolle Informationen über den physiologischen Status der betrachteten Embryonen, die z.B. zur Verbesserung der Systeme zur In vitro-Produktion von Embryonen genutzt werden können, liefern. Bisher fehlte es jedoch an einer geeigneten Technologie, um eine große Anzahl von Transkripten in einzelnen Embryonen zu erfassen. Zielsetzung der vorliegenden Arbeit war es, ein Verfahren zur globalen Amplifikation embryonaler mRNA-Präparationen zu entwickeln, das die Analyse der Transkriptionsebene einzelner präimplantatorischer Embryonalstadien über die cDNA-Array-Technologie ermöglicht. Dazu wurde die Strategie gewählt, zwei bereits etablierte Amplifikationsverfahren, Polymerasekettenreaktion und In vitro-Transkription, zu kombinieren, um so synergistische Effekte beider Verfahren zu nutzen. Die Evaluierung des entwickelten Verfahrens zeigte eine hohe Reproduzierbarkeit der erhaltenen Genexpressionsdaten und belegte, dass die relativen Mengenverhältnisse einzelner mRNA-Spezies zueinander während der globalen mRNA-Amplifikation nur unwesentlich verändert wurden. Die entwickelte Methodik ist somit geeignet, komplexe Genexpressionsprofile einzelner Blastozysten zu erstellen und Unterschiede in der Expressionsstärke einzelner Transkripte zu detektieren. Es konnte weiterhin gezeigt werden, dass es möglich ist, über heterologe Hybridisierung Genexpressionsprofile boviner Blastozysten mit cDNA-Arrays, die murine Probensequenzen enthalten, reproduzierbar darzustellen. Neben der Detektion individueller Unterschiede in den Genexpressionsprofilen diverser muriner Embryonalstadien und boviner Blastozysten lag ein Schwerpunkt dieser Arbeit in der Untersuchung der Auswirkungen verschiedener in vitro-Produktionssysteme auf die embryonale Genexpression. Die erhaltenen cDNA-Array Expressionsdaten muriner Oozyten, Zweizeller und Blastozysten befanden sich dabei in Übereinstimmung mit Daten früherer Publikationen anderer Arbeitsgruppen. Genexpressionsprofile in vitro fertilisierter boviner Blastozysten ließen eine Beurteilung der Auswirkungen unterschiedlicher Proteinsupplemente des Kulturmediums auf die embryonale Genexpression zu. Im Rahmen dieser Arbeit wurden zum ersten Mal Genexpressionsprofile einzelner präimplantatorischer Säugerembryonen über cDNA-Array-Analyse erstellt. Die entwickelte Technologie ermöglicht es -bei Verwendung entsprechender cDNA-Array-Systeme-, eine theoretisch unbegrenzte Zahl von Transkripten in individuellen Säugerembryonen semiquantitativ zu erfassen. Dies ist ein wichtiger Schritt hin zu einem besseren Verständnis komplexer Regulationsabläufe während der frühen Embryonalentwicklung und einer besseren Beurteilung der Lebensfähigkeit und Entwicklungskompetenz in vitro produzierter Embryonen, was für die Verbesserung von In vitro-Produktionssystemen für Embryonen sowohl bei Tieren als auch beim Menschen unerlässlich ist. N2 - Transcript expression profiling in single mammalian embryos can provide valuable information about their physiological status and developmental competence that can be exploited to improve systems for embryo in vitro production. Conventional methodologies such as RT-PCR limit the number of transcripts that can be quantitatively screened in a single embryo to only a few. The purpose of this study was to develop and evaluate a methodology for the global amplification of mRNA that permits cDNA-array analysis of individual preimplantation embryos. For this purpose, two conventional amplification procedures – polymerase chain reaction and in vitro transcription – were combined to a global amplification procedure. Evaluation of methodology developed revealed that data produced were high reproducible and that the relative transcript levels found in the original (non-amplified) sample were maintained throughout the amplification process. Thus, this method is suitable to generate complex gene expression profiles and to detect differentially expressed transcripts in individual mammalian embryos. Furthermore, this study demonstrates that expression profiles can reproducibly be produced from bovine embryos using arrays consisting of murine cDNA-probes by heterologous hybridization. The focus of this study was to establish a methodology to detect differentially expressed genes in different murine developmental stages and in bovine embryos derived from different in vitro production systems. The data obtained from murine oocyte, 2-cell stage and blastocyst expression profiles were in agreement with data previously published by other groups. Expression profiles from bovine in vitro fertilized embryos cultured in different media revealed effects of different media protein supplementation on embryonic gene expression. In this study, for the first time, gene expression profiles were generated from single mammalian preimplantation embryos via model cDNA-arrays. Using state-of-the-art cDNA-arrays this technology features the quantitative screening of a virtually unlimited number of transcripts in individual blastocysts and cleavage stages. Complex expression profiles of preimplantation embryos will contribute to the understanding of the molecular mechanisms essential for embryogenesis. This is crucial for the improvement of systems for in vitro production of mammalian embryos. KW - Embryo KW - Säugetiere KW - Array-Technologie KW - Messenger-RNS KW - Genexpression KW - Embryo KW - Genexpression KW - cDNA-Arrays KW - mRNA-Amplifikation KW - embryo KW - gene expression KW - cDNA-arrays KW - mRNA-amplification Y1 - 2002 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-1787 ER - TY - THES A1 - Braasch, Ingo T1 - Evolution by genome duplication: insights from vertebrate neural crest signaling and pigmentation pathways in teleost fishes T1 - Evolution durch Genomverdoppelung: Erkenntnisse aus Analysen der Signalwege in der Neuralleiste der Vertebraten und in den Pigmentzellen im Fisch N2 - Gene and genome duplications are major mechanisms of eukaryotic genome evolution. Three rounds of genome duplication have occurred in the vertebrate lineage, two rounds (1R, 2R) during early vertebrate evolution and a third round, the fish-specific genome duplication (FSGD), in ray-finned fishes at the base of the teleost lineage. Whole genome duplications (WGDs) are considered to facilitate speciation processes and to provide the genetic raw material for major evolutionary transitions and increases in morphological complexity. In the present study, I have used comparative genomic approaches combining molecular phylogenetic reconstructions, synteny analyses as well as gene function studies (expression analyses and knockdown experiments) to investigate the evolutionary consequences and significance of the three vertebrate WGDs. First, the evolutionary history of the endothelin signaling system consisting of endothelin ligands and receptors was reconstructed. The endothelin system is a key component for the development of a major vertebrate innovation, the neural crest. This analysis shows that the endothelin system emerged in an ancestor of the vertebrate lineage and that its members in extant vertebrate genomes are derived from the vertebrate WGDs. Each round of WGD was followed by co-evolution of the expanding endothelin ligand and receptor repertoires. This supports the importance of genome duplications for the origin and diversification of the neural crest, but also underlines a major role for the co-option of new genes into the neural crest regulatory network. Next, I have studied the impact of the FSGD on the evolution of teleost pigment cell development and differentiation. The investigation of 128 genes showed that pigmentation genes have been preferentially retained in duplicate after the FSGD so that extant teleost genomes contain around 30% more putative pigmentation genes than tetrapods. Large parts of pigment cell regulatory pathways are present in duplicate being potentially involved in teleost pigmentary innovations. There are also important differences in the retention of duplicated pigmentation genes among divergent teleost lineages. Functional studies of pigment synthesis enzymes in zebrafish and medaka, particularly of the tyrosinase family, revealed lineage-specific functional evolution of duplicated pigmentation genes in teleosts, but also pointed to anciently conserved gene functions in vertebrates. These results suggest that the FSGD has facilitated the evolution of the teleost pigmentary system, which is the most complex and diverse among vertebrates. In conclusion, the present study supports a major role of WGDs for phenotypic evolution and biodiversity in vertebrates, particularly in fish. N2 - Gen- und Genomverdopplungen sind wichtige Mechanismen der Genomevolution in Eukaryonten. Im Verlauf der Evolution der Wirbeltiere gab es drei wichtige Genomduplikationen. Zwei Genomverdopplungen (1R, 2R) fanden während der sehr frühen Vertebratenevolution statt. In der Linie der Fische kam es an der Basis der Teleostier zu einer weiteren, fischspezifischen Genomduplikation (FSGD). Man nimmt an, dass Genomduplizierungen Artbildungsprozesse begünstigen und dass sie zusätzliches genetisches Material für wichtige evolutionäre Übergänge und für die Steigerung morphologischer Komplexität erzeugen. In der vorliegenden Arbeit wurden Methoden der vergleichenden und funktionellen Genomik gewählt, um die Auswirkungen und die Bedeutung der drei Genomverdopplungen bei Vertebraten zu untersuchen. Dazu wurden molekularphylogenetische Stammbaumanalysen und Synteniedaten mit Genexpressionsstudien und Knockdown-Experimenten kombiniert. Zunächst wurde die Evolution des Endothelin-Signalsystems rekonstruiert. Dieses besteht aus Endothelin-Liganden und -Rezeptoren und hat eine Schlüsselrolle in die Entwicklung der Neuralleiste. Die Neuralleiste und die von ihr abgeleiteten Zelltypen sind wirbeltierspezifische Innovationen. Die Analyse zeigt, dass das Endothelin-System in einem gemeinsamen Vorfahren der Vertebraten entstanden ist. Die in den Genomen rezenter Vertebraten vorkommenden Komponenten des Endothelin-Systems sind durch die drei Genomverdoppelungen entstanden. Nach jeder der Duplizierungen kam es zur Ko-Evolution der Liganden- und Rezeptorenfamilien. Die Evolution des Endothelin-System unterstreicht daher die Bedeutung der Genomduplizierungen für den Ursprung und die Diversifizierung der Neuralleiste. Sie weist aber auch auf eine wichtige Rolle für die Integrierung neuer Gene in das regulatorische Netzwerk der Neuralleiste hin. Im Weiteren wurde der Einfluss der FSGD auf die Evolution der Pigmentzellentwicklung und differenzierung in Teleostiern untersucht. Die evolutionäre Analyse von 128 Genen zeigte, dass Pigmentierungsgene nach der FSGD bevorzugt in zwei Kopien erhalten geblieben sind. Daher besitzen rezente Teleostier im Vergleich zu Landwirbeltieren zusätzlich ca. 30% mehr Gene mit potentiellen Funktionen für die Pigmentierung. Große Teile der regulatorischen Signalwege in den Pigmentzellen liegen daher als zwei Kopien vor. Diese waren möglicherweise an der Evolution von Innovationen in der Körperfärbung von Teleostiern beteiligt. In der vorliegenden Arbeit wurden auch wichtige Unterschiede zwischen verschiedenen Fischgruppen im Erhalt duplizierter Pigmentierungsgene gefunden. Funktionelle Studien bei Zebrafish und bei Medaka an Enzymen der Pigmentsynthese, insbesondere der Tyrosinase-Familie, gaben Hinweise darauf, dass die funktionelle Evolution duplizierter Pigmentierungsgene in Fischen linienspezifisch verlaufen kann. Die Studien ergaben außerdem, dass bestimmte Funktionen der Pigmentsyntheseenzyme innerhalb der Vertebraten konserviert sind. Die Evolution des Pigmentierungssystems der Fische, welches das vielfältigste und komplexeste innerhalb der Wirbeltiere ist, wurde somit maßgeblich durch die FSGD beeinflusst. Zusammenfassend weisen die Ergebnisse der vorliegenden Arbeit darauf hin, dass die Verdopplung ganzer Genome ein wichtiger Mechanismus der phänotypische Evolution bei Vertebraten ist und damit in besonderem Maße zur ihrer Biodiversität beiträgt. KW - Molekulare Evolution KW - Fische KW - Entwicklungsbiologie KW - Evolutionsbiologie KW - Genanalyse KW - Pigmentierung KW - Melanin KW - Vertebrat KW - Neuralleiste KW - Gen-/Genomverdoppelung KW - gene/genome duplication KW - fish KW - vertebrate KW - neural crest KW - pigmentation Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-35702 ER - TY - THES A1 - Boyanova, Desislava Veselinova T1 - Systems biological analysis of the platelet proteome and applications of functional module search in proteome networks T1 - Systembiologische Analyse des Blutplättchenproteoms und funktionelle Modulsuche in Proteinnetzwerken N2 - Recent development of proteomic approaches and generation of large-scale proteomic datasets calls for new methods for biological interpretation of the obtained results. Systems biological approaches such as integrated network analysis and functional module search have become an essential part of proteomic investigation. Proteomics is especially applied in anucleate cells such as platelets. The underlying molecular mechanisms of platelet activation and their pharmacological modulation are of immense importance for clinical research. Advances in platelet proteomics have provided a large amount of proteomic data, which has not yet been comprehensively investigated in a systems biological perspective. To this end, I assembled platelet specific data from proteomic and transcriptomic studies by detailed manual curation and worked on the generation of a comprehensive human platelet repository for systems biological analysis of platelets in the functional context of integrated networks (PlateletWeb) (http:/PlateletWeb.bioapps.biozentrum.uni-wuerzburg.de). I also added platelet-specific experimentally validated phosphorylation data and generated kinase predictions for 80% of the newly identified platelet phosphosites. The combination of drug, disease and pathway information with phosphorylation and interaction data makes this database the first integrative platelet platform available for platelet research. PlateletWeb contains more than 5000 platelet proteins, which can also be analyzed and visualized in a network context, allowing identification of all major signaling modules involved in platelet activation and inhibition. Using the wealth of integrated data I performed a series of platelet-specific analyses regarding the platelet proteome, pathways, drug targets and novel platelet phosphorylation events involved in crucial signaling events. I analyzed the statistical enrichment of known pathways for platelet proteins and identified endocytosis as a highly represented pathway in platelets. Further results revealed that highly connected platelet proteins are more often targeted by drugs. Using integrated network analysis offered by PlateletWeb, I analyzed the crucial activation signaling pathway of adenosine diphosphate (ADP), visualizing how the signal flow from receptors to effectors is maintained. My work on integrin inside-out signaling was also based on the integrated network approach and examined new platelet-specific phosphorylation sites and their regulation using kinase predictions. I generated hypothesis on integrin signaling, by investigating the regulation of Ser269 phosphorylation site on the docking protein 1 (DOK1). This phosphorylation site may influence the inhibiting effect of DOK1 on integrin a2bb3. Extending the integrated network approach to further cell lines, I used the assembled human interactome information for the analysis of functional modules in cellular networks. The investigation was performed with a previously developed module detection algorithm, which finds maximum-scoring subgraphs in transcriptomic datasets by using assigned values to the network nodes. We extended the algorithm to qualitative proteomic datasets and enhanced the module search by adding functional information to the network edges to concentrate the solution onto modules with high functional similarity. I performed a series of analyses to validate its performance in small-sized (virus-infected gastric cells) and medium-sized networks (human lymphocytes). In both cases the algorithm extracted characteristic modules of sample proteins with high functional similarity. The functional module search is especially useful in site-specific phosphoproteomic datasets, where kinase regulation of the detected sites is often sparse or lacking. Therefore, I used the module detection algorithm in quantitative phosphoproteomic datasets. In a platelet phosphorylation dataset, I presented a pipeline for network analysis of detected phosphorylation sites. In a second approach, the functional module detecting algorithm was used on a phosphoproteome network of human embryonic stem cells, in which nodes represented the maximally changing phosphorylation sites in the experiment. Additional kinases from the human phosphoproteome in PlateletWeb were included to the network to investigate the regulation of the signal flow. Results indicated important phosphorylation sites and their upstream kinases and explained changes observed in embryonic stem cells during differentiation. This work presents novel approaches for integrated network analysis in cells and introduces for the first time a systematic biological investigation of the human platelet proteome based on the platelet-specific knowledge base PlateletWeb. The extended methods for optimized functional module detection offer an invaluable tool for exploring proteomic datasets and covering gaps in complex large-scale data analysis. By combining exact module detection approaches with functional information data between interacting proteins, characteristic functional modules with high functional resemblance can be extracted from complex datasets, thereby focusing on important changes in the observed networks. N2 - Jüngste Entwicklungen der Proteomik und die damit einhergehende Erzeugung großer Datensätze erfordern neue Methoden zur biologischen Interpretation der gewonnenen Ergebnisse. Systembiologische Ansätze wie die integrierte Netzwerkanalyse sowie die funktionelle Modulsuche sind zu einem wesentlichen Bestandteil bei der Untersuchung von Proteinen geworden. Die Proteomik wird vor allem in kernlosen Zellen wie den Blutplättchen angewandt. Die zu Grunde liegenden molekularen Mechanismen bei der Aktivierung von Thrombozyten und deren pharmakologische Modulation sind von immenser Bedeutung für die klinische Forschung. Aktuelle Studien in der Proteomforschung haben insbesondere bei Thrombozyten große Mengen an Daten erzeugt, die bisher noch nicht umfassend systembiologisch untersucht wurden. Zu diesem Zweck stellte ich manuell thrombozyten-spezifische Daten aus Proteom- und Transkriptomstudien zusammen und arbeitete an der Entwicklung einer umfassenden menschlichen Thrombozytendatenbank für die systembiologische Analyse der Funktion von Blutplättchen mittels integrierter Netzwerkanalyse (PlateletWeb) (http:/PlateletWeb.bioapps.biozentrum.uni-wuerzburg.de). Zusätzlich habe ich plättchen-spezifische, experimentell validierte Phosphorylierungsinformationen hinzugefügt und generierte Kinasenvorhersagen für 80% der neu identifizierten Phosphorylierungsstellen. Die Kombination aus Medikamenten, assoziierten Krankheiten und Signalweginformation zusammen mit Phosphorylierungs- und Interaktionsdaten macht diese Datenbank zu einer ersten und umfassenden Anlaufstelle für Thrombozytenforschung. PlateletWeb enthält mehr als 5000 Plättchenproteine, die in einem Netzwerk analysiert und dargestellt werden können. Dabei ist die Identifizierung aller wichtigen Signalmodule zur Plättchenaktivierung und -inhibierung möglich. Mit der Fülle an verfügbaren Daten führte ich eine Reihe thrombozyten-spezifischer Analysen am Plättchenproteom, an Signalwegen, pharmakologischen Wirkstoffzielen und Phosphorylierungsreaktionen in grundlegenden Signalprozessen durch. Ich analysierte die statistische Anreicherung bekannter Signalwege für Plättchenproteine und identifizierte Endozytose als einen sehr repräsentativen Signalweg in Thrombozyten. Weitere Ergebnisse zeigten, dass stark vernetzte Plättchenproteine häufiger Ziel von Medikamenten sind. Mittels der Netzwerkanalyse von PlateletWeb untersuchte ich den grundlegenden Signalaktivierungspfad von Adenosindiphosphat (ADP), und veranschaulichte den Signalfluss von Rezeptor zu Effektor. Meine Arbeit an der Integrin-Inside-Out-Signalisierung beinhaltete zudem die Untersuchung neuer thrombozyten-spezifischer Phosphorylierungsstellen und ihre Regulation durch Kinasenvorhersagen mit Hilfe des integrierten Netzwerkanalyseansatzes. Durch die Untersuchung der Regulation bei der Phosphorylierungsstelle Ser269 im Docking-Protein (DOK1) stellte ich eine neue Hypothese zur Integrinsignalisierung auf. Diese Phosphorylierungsstelle könnte den inhibitorischen Effekt von DOK1 auf integrin a2bb3 beeinflussen. Ich erweiterte den integrierten Netzwerkanalyseansatz für andere Zelllinien, indem ich die gesammelten Informationen aus dem menschlichen Interaktom für die Analyse von funktionellen Modulen in zellulären Netzen nutzte. Die Untersuchung wurde mit einem zuvor entwickelten Algorithmus zur Modulerkennung durchgeführt, der maximal bewertete Teilgraphen in Transkriptomdatensätzen anhand zugewiesener Werte für Netzwerkknoten findet. Wir erweiterten den Algorithmus zur Anwendung auf qualitative Proteomdatensätze und optimierten die Modulsuche durch Integration funktioneller Informationen in die Netzwerkkanten. Dies fokussierte die Optimierung auf Proteinmodule mit hoher funktioneller Ähnlichkeit. Ich führte eine Reihe von Analysen durch, um die Effizienz des Algorithmus in kleinen (durch Viren infizierte Magenzellen) und mittelgroßen Netzwerken (menschliche Lymphozyten) zu überprüfen. In beiden Fällen extrahierte der Algorithmus charakteristische Module der untersuchten Proteine mit hohen funktionellen Ähnlichkeiten. Die funktionelle Modulsuche ist besonders bei positionsspezifischen Phosphoproteomikdatensätzen nützlich, in denen die Kinasenregulation der detektierten Phosphorylierungsstellen nur spärlich oder gar nicht vorhanden ist. Daher habe ich den Algorithmus der Moduldetektion auf quantitative Phosphoproteomikdatensätze angewandt. Anhand eines Datensatzes bestehend aus phosphorylierten Plättchenproteinen habe ich eine Vorgehensweise zur Netzwerkanalyse von Phosphorylierungsstellen entwickelt. In einer zweiten Studie wurde der Algorithmus der Moduldetektion auf ein phosphoproteomisches Netzwerk menschlich embryonaler Stammzellen angewandt, in dem Phosphorylierungsstellen mit maximaler Veränderung durch Netzwerkknoten repräsentiert wurden. Um die Regulation des Signalflusses zu untersuchen wurden weitere Kinasen aus dem menschlichen Phosphoproteom beziehungsweise PlateletWeb integriert. Ergebnisse wiesen auf wichtige Phosphorylierungsstellen und ihre Upstream-Kinasen hin und verdeutlichten Vorgänge, die während der Differenzierung in den embryonalen Stammzellen stattgefunden haben. Diese Arbeit bietet neue Vorgehensweisen der integrierten Netzwerkanalyse in Zellen und präsentiert zum ersten Mal eine systembiologische Untersuchung des menschlichen Proteoms mit Hilfe der Trombozytendatenbank PlateletWeb. Die erweiterten Methoden zur verbesserten Erkennung funktioneller Module bieten ein wertvolles Werkzeug für die Erforschung proteomischer Datensätze und vervollständigen die komplexe und umfangreiche Datenanalyse. Charakteristische Module, die große Ähnlichkeit auf funktioneller Ebene aufweisen, können durch die Kombination von exakten Modulerkennungsansätzen mit funktionellen Daten extrahiert werden. Dabei werden wichtige Änderungen besonders bei der Analyse komplexer Netzwerke hervorgehoben. KW - Netzwerkanalyse KW - Thrombozyt KW - Proteomanalyse KW - Systembiologie KW - Funktionelle Modulsuche KW - Plättchenphosphoproteom KW - Netzwerkalgorithmen KW - Systems Biology KW - Integrated network analysis KW - Plättchennetzwerk KW - Proteome KW - Phosphoproteomic analysis KW - Functional module search KW - Functional interaction Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72165 ER - TY - JOUR A1 - Bowler, Diana E. A1 - Bjorkman, Anne D. A1 - Dornelas, Maria A1 - Myers‐Smith, Isla H. A1 - Navarro, Laetitia M. A1 - Niamir, Aidin A1 - Supp, Sarah R. A1 - Waldock, Conor A1 - Winter, Marten A1 - Vellend, Mark A1 - Blowes, Shane A. A1 - Böhning‐Gaese, Katrin A1 - Bruelheide, Helge A1 - Elahi, Robin A1 - Antão, Laura H. A1 - Hines, Jes A1 - Isbell, Forest A1 - Jones, Holly P. A1 - Magurran, Anne E. A1 - Cabral, Juliano Sarmento A1 - Bates, Amanda E. T1 - Mapping human pressures on biodiversity across the planet uncovers anthropogenic threat complexes JF - People and Nature N2 - Climate change and other anthropogenic drivers of biodiversity change are unequally distributed across the world. Overlap in the distributions of different drivers have important implications for biodiversity change attribution and the potential for interactive effects. However, the spatial relationships among different drivers and whether they differ between the terrestrial and marine realm has yet to be examined. We compiled global gridded datasets on climate change, land‐use, resource exploitation, pollution, alien species potential and human population density. We used multivariate statistics to examine the spatial relationships among the drivers and to characterize the typical combinations of drivers experienced by different regions of the world. We found stronger positive correlations among drivers in the terrestrial than in the marine realm, leading to areas with high intensities of multiple drivers on land. Climate change tended to be negatively correlated with other drivers in the terrestrial realm (e.g. in the tundra and boreal forest with high climate change but low human use and pollution), whereas the opposite was true in the marine realm (e.g. in the Indo‐Pacific with high climate change and high fishing). We show that different regions of the world can be defined by Anthropogenic Threat Complexes (ATCs), distinguished by different sets of drivers with varying intensities. We identify 11 ATCs that can be used to test hypotheses about patterns of biodiversity and ecosystem change, especially about the joint effects of multiple drivers. Our global analysis highlights the broad conservation priorities needed to mitigate the impacts of anthropogenic change, with different priorities emerging on land and in the ocean, and in different parts of the world. KW - Anthropocene KW - biodiversity threats KW - direct drivers KW - global change Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213634 VL - 2 IS - 2 SP - 380 EP - 394 ER - TY - JOUR A1 - Boschert, Verena A1 - Klenk, Nicola A1 - Abt, Alexander A1 - Raman, Sudha Janaki A1 - Fischer, Markus A1 - Brands, Roman C. A1 - Seher, Axel A1 - Linz, Christian A1 - Müller-Richter, Urs D. A. A1 - Bischler, Thorsten A1 - Hartmann, Stefan T1 - The influence of Met receptor level on HGF-induced glycolytic reprogramming in head and neck squamous cell carcinoma JF - International Journal of Molecular Sciences N2 - Head and neck squamous cell carcinoma (HNSCC) is known to overexpress a variety of receptor tyrosine kinases, such as the HGF receptor Met. Like other malignancies, HNSCC involves a mutual interaction between the tumor cells and surrounding tissues and cells. We hypothesized that activation of HGF/Met signaling in HNSCC influences glucose metabolism and therefore substantially changes the tumor microenvironment. To determine the effect of HGF, we submitted three established HNSCC cell lines to mRNA sequencing. Dynamic changes in glucose metabolism were measured in real time by an extracellular flux analyzer. As expected, the cell lines exhibited different levels of Met and responded differently to HGF stimulation. As confirmed by mRNA sequencing, the level of Met expression was associated with the number of upregulated HGF-dependent genes. Overall, Met stimulation by HGF leads to increased glycolysis, presumably mediated by higher expression of three key enzymes of glycolysis. These effects appear to be stronger in Met\(^{high}\)-expressing HNSCC cells. Collectively, our data support the hypothesized role of HGF/Met signaling in metabolic reprogramming of HNSCC. KW - HNSCC KW - head and neck cancer KW - HGF KW - Met KW - cancer metabolism Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235995 SN - 1422-0067 VL - 21 IS - 2 ER - TY - THES A1 - Borst, Andreas T1 - Apoptosis & senescence: cell fate determination in inhibitor-treated melanoma cells T1 - Apoptose & Seneszenz: Bestimmung der Zell-spezifischen Reaktion von Melanomzellen auf Inhibitoren N2 - Neoplasms of the skin represent the most frequent tumors worldwide; fortunately, most of them are benign or semi-malignant and well treatable. However, the two most aggressive and deadly forms of malignant skin-neoplasms are melanoma and Merkel cell carcinoma (MCC), being responsible for more than 90% of skin-cancer related deaths. The last decade has yielded enormous progress in melanoma therapy with the advent of targeted therapies, like BRAF or MEK inhibitors, and immune-stimulating therapies, using checkpoint antibodies targeting CTLA- 4, PD-1 or PD-L1. Very recent studies suggest that also MCC patients benefit from a treatment with checkpoint antibodies. Nevertheless, in an advanced metastatic stage, a cure for both of these aggressive malignancies is still hard to achieve: while only a subset of patients experience durable benefit from the immune-based therapies, the widely applicable targeted therapies struggle with development of resistances that inevitably occur in most patients, and finally lead to their death. The four articles included in this thesis addressed current questions concerning therapy and carcinogenesis of melanoma and MCC. Moreover, they are discussed in the light of the up-to-date research regarding targeted and immune-based therapies. In article I we demonstrated that besides apoptosis, MAPK pathway inhibition in BRAF-mutated melanoma cells also induces senescence, a permanent cell cycle arrest. These cells may provide a source for relapse, as even permanently arrested cancer cells can contribute to a pro-tumorigenic milieu. To identify molecular factors determining the differential response, we established M14 melanoma cell line derived single cell clones that either undergo cell death or arrest when treated with BRAF/MEK inhibitors. Using these single cell clones, we demonstrated in article IV that downregulation of the pro-apoptotic BH3-only protein BIK via epigenetic silencing is involved in apoptosis deficiency, which can be overcome by HDAC inhibitors. These observations provide a possible explanation for the lack of a complete and durable response to MAPK inhibitor treatment in melanoma patients, and suggest the application of HDAC inhibitors as a complimentary therapy to MAPK pathway inhibition. Concerning MCC, we scrutinized the interactions between the Merkel cell polyomavirus’ (MCV) T antigens (TA) and the tumor suppressors p53 and Rb in article II and III, respectively. In article III, we demonstrated that the cell cycle master regulator Rb is the crucial target of MCV large T (LT), while it - in contrast to other polyomavirus LTs - exhibits much lower affinity to the related proteins p107 and p130. Knockdown of MCV LT led to proliferation arrest in MCC cells, which can be rescued by knockdown of Rb, but not by knockdown of p107 and p130. Contrary to Rb, restriction of p53 in MCC seems to be independent of the MCV TAs, as we demonstrated in article II. In conclusion, the presented thesis has revealed new molecular details, regarding the response of melanoma cells towards an important treatment modality and the mechanisms of viral carcinogenesis in MCC. N2 - Die häufigsten Tumore weltweit sind Neoplasien der Haut; glücklicherweise sind die meisten dieser benigne oder semi-maligne und gut behandelbar. Die beiden aggressivsten und tödlichsten Formen bösartiger Hauttumoren sind das Melanom und das Merkelzell-Karzinom (MCC), welche verantwortlich für über 90% aller durch Hauttumore verursachten Todesfälle sind. Im letzten Jahrzehnt gab es jedoch erstaunliche Fortschritte in der Therapie des malignen Melanoms, was vor allem durch das Aufkommen der zielgerichteten Therapien wie den BRAF oder MEK Inhibitoren und den immunstimulierenden Therapien, welche Checkpoint-Antikörper gegen CTLA-4, PD-1 oder PD-L1 verwenden, bedingt ist. Neueste Studien legen nahe, dass auch MCC Patienten von diesen Checkpoint-Antikörpern profitieren können. In fortgeschrittenen, metastasierten Stadien ist jedoch für beide Malignitäten eine Heilung immer noch sehr schwer erreichbar: nur eine kleine Gruppe der Patienten erreichen einen dauerhaften Nutzen durch die Immuntherapien, während die breit anwendbaren zielgerichteten Therapien mit der Entwicklung von Resistenzen zu kämpfen haben, welche unausweichlich in den meisten Patienten entstehen und letztendlich zu deren Tod führen. Die vier dieser Dissertation beigefügten Publikationen adressierten aktuelle Fragestellungen bezüglich Therapie und Karzinogenese des Melanoms und des MCCs. Des Weiteren werden diese im Licht des heutigen Forschungsstandes diskutiert, im Besonderen mit Blick auf die zielgerichteten und immunbasierten Therapien. In Publikation I zeigten wir, dass Inhibition des MAPK Signalwegs in BRAF-mutierten Melanom-Zellen neben Apoptose auch zu Seneszenz, einem permanenten Zellzyklusarrest, führen kann. Diese Zellen können der Ursprung der Resistenzbildung sein, da auch permanent arretierte Krebszellen zu einem Tumor-fördernden Milieu beitragen können. Um molekulare Faktoren zu identifizieren, die für diese unterschiedliche Behandlungsreaktion ursächlich sind, haben wir Einzelzellklone aus der M14 Melanom-Zelllinie etabliert, welche entweder mit Zelltod oder Arrest auf die BRAF/MEK Inhibitor Behandlung reagieren. Mit Hilfe dieser Klone zeigten wir in Publikation IV, dass die Herunterregulierung des pro-apoptotischen BH3-only Proteins BIK durch einen epigenetischen Mechanismus zur Apoptose-Resistenz dieser Zellen führt, was durch den Einsatz von HDAC-Inhibitoren umgangen werden kann. Diese Beobachtungen bieten eine mögliche Erklärung für das Ausbleiben eines vollständigen und dauerhaften Ansprechens auf die MAPK-Inhibitor Behandlung der Melanom-Patienten, und legen den Einsatz von HDAC-Inhibitoren als komplementäre Therapieoption nahe. Beim MCC haben wir jeweils die Interaktion zwischen den Merkelzell-Polyomavirus (MCV) T Antigenen (TA) und den Tumor-Suppressoren p53 und Rb in Publikation II und III näher betrachtet. In Publikation III haben wir gezeigt, dass das zentrale, Zellzyklus-regulierende Protein Rb das vorrangige Ziel des MCV large T Antigens (LT) ist, während es - im Gegensatz zu anderen Polyomavirus-LTs - viel weniger Affinität zu den verwandten Proteinen p107 und p 130 aufweist. Der Knockdown des MCV LT führte zu Proliferationsarrest in MCC Zellen, welcher durch Knockdown von Rb aufgehoben werden konnte, nicht jedoch durch Knockdown von p107 und p130. Die Restriktion von p53 scheint im Gegensatz zu Rb im MCC unabhängig von den MCV TAs zu sein, wie wir in Publikation II gezeigt haben. Zusammenfassend gibt diese Dissertation Aufschluss über neue molekulare Zusammenhänge bezüglich der Reaktion von Melanom-Zellen gegenüber einer wichtigen Behandlungsmöglichkeit und den Mechanismen der viralen Karzinogenese des MCC. KW - Melanom KW - Apoptosis KW - MAP-Kinase KW - Senescence KW - BRAF inhibition Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-155085 ER - TY - JOUR A1 - Borges, Alyssa R. A1 - Link, Fabian A1 - Engstler, Markus A1 - Jones, Nicola G. T1 - The Glycosylphosphatidylinositol Anchor: A Linchpin for Cell Surface Versatility of Trypanosomatids JF - Frontiers in Cell and Developmental Biology N2 - The use of glycosylphosphatidylinositol (GPI) to anchor proteins to the cell surface is widespread among eukaryotes. The GPI-anchor is covalently attached to the C-terminus of a protein and mediates the protein’s attachment to the outer leaflet of the lipid bilayer. GPI-anchored proteins have a wide range of functions, including acting as receptors, transporters, and adhesion molecules. In unicellular eukaryotic parasites, abundantly expressed GPI-anchored proteins are major virulence factors, which support infection and survival within distinct host environments. While, for example, the variant surface glycoprotein (VSG) is the major component of the cell surface of the bloodstream form of African trypanosomes, procyclin is the most abundant protein of the procyclic form which is found in the invertebrate host, the tsetse fly vector. Trypanosoma cruzi, on the other hand, expresses a variety of GPI-anchored molecules on their cell surface, such as mucins, that interact with their hosts. The latter is also true for Leishmania, which use GPI anchors to display, amongst others, lipophosphoglycans on their surface. Clearly, GPI-anchoring is a common feature in trypanosomatids and the fact that it has been maintained throughout eukaryote evolution indicates its adaptive value. Here, we explore and discuss GPI anchors as universal evolutionary building blocks that support the great variety of surface molecules of trypanosomatids. KW - cell surface proteome KW - evolution KW - GPI-anchor KW - Kinetoplastea Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249253 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Bonte, Dries A1 - Travis, Justin M. J. A1 - De Clercq, Nele A1 - Zwertvaegher, Ingrid A1 - Lens, Luc T1 - Thermal conditions during juvenile development affect adult dispersal in a spider N2 - Abstract: Understanding the causes and consequences of dispersal is a prerequisite for the effective management of natural populations. Rather than treating dispersal as a fixed trait, it should be considered a plastic process that responds to both genetic and environmental conditions. Here, we consider how the ambient temperature experienced by juvenile Erigone atra, a spider inhabiting crop habitat, influences adult dispersal. This species exhibits 2 distinct forms of dispersal, ballooning (long distance) and rappelling (short distance). Using a half-sib design we raised individuals under 4 different temperature regimes and quantified the spiders' propensity to balloon and to rappel. Additionally, as an indicator of investment in settlement, we determined the size of the webs build by the spiders following dispersal. The optimal temperature regimes for reproduction and overall dispersal investment were 20 °C and 25 °C. Propensity to perform short-distance movements was lowest at 15 °C, whereas for long-distance dispersal it was lowest at 30 °C. Plasticity in dispersal was in the direction predicted on the basis of the risks associated with seasonal changes in habitat availability; long-distance ballooning occurred more frequently under cooler, spring-like conditions and short-distance rappelling under warmer, summer-like conditions. Based on these findings, we conclude that thermal conditions during development provide juvenile spiders with information about the environmental conditions they are likely to encounter as adults and that this information influences the spider's dispersal strategy. Climate change may result in suboptimal adult dispersal behavior, with potentially deleterious population level consequences. KW - Erigone atra KW - emigration KW - dispersal distance KW - immigration KW - behavior KW - plasticity KW - silk KW - body condition KW - seasonality Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48691 ER - TY - JOUR A1 - Bonte, Dries A1 - Maes, Dirk T1 - Trampling affects the distribution of specialised coastal dune arthropods N2 - Abstract: From a conservation point of view, species- tolerances towards disturbance are often generalised and lack reference to spatial scales and underlying processes. In order to investigate how average typical species react to habitat fragmentation and disturbance, we adopted a multi-species approach to address occupancy patterns of five specialised dune arthropods (butterflies Hipparchia semele, Issoria lathonia; grasshopper Oedipoda caerulescens; spiders Alopecosa fabrilis, Xysticus sabulosus) in recently fragmented coastal dune habitats which are subjected to varying levels and modes of local disturbance, i.e. trampling by cattle or people. Occupancy patterns were assessed during two successive years in 133 grey dune fragments of the Flemish coastal dunes (Belgium, France). By treating species as a random factor in our models, emphasis was placed on generalisations rather than documenting species-specific patterns. Our study demonstrates that deteriorating effects of local disturbance on arthropod incidence cannot be interpreted independent of its landscape context, and appear to be more severe when patch area and connectivity decrease. When controlled for patch area and trampling intensity, the probability of species occupancy in poorly connected patches is higher under cattle trampling than under recreation. Incidences additionally decrease with increasing intensity of cattle trampling, but increases with trampling by tourists. This study provides evidence of mode- and landscape-dependent effects of local disturbance on species occupancy patterns. Most importantly, it demonstrates that trampling of sensitive dune fragments will lead to local and metapopulation extinction in landscapes where trampling occurs in a spatially autocorrelated way, but that the outcome (spatial patterns) varies in relation to disturbance mode, indicating that effects of disturbance cannot be generalised. KW - Araneae KW - grazing KW - grey dunes KW - Lepidoptera KW - multispecies metapopulation KW - Orthoptera KW - recreation KW - trampling Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48274 ER - TY - JOUR A1 - Bonte, Dries A1 - Lanckacker, Kjell A1 - Wiersma, Elisabeth A1 - Lens, Luc T1 - Web building flexibility of an orb-web spider in a heterogeneous agricultural landscape N2 - Abstract: Intensification of land-use in agricultural landscapes is responsible for a decline of biodiversity which provide important ecosystem services like pest-control. Changes in landscape composition may also induce behavioural changes of predators in response to variation in the biotic or abiotic environment. By controlling for environmentally confounding factors, we here demonstrate that the orb web spider Araneus diadematus alters its web building behaviour in response to changes in the composition of agricultural landscapes. Thereby, the species increases its foraging efficiency (i.e. investments in silk and web asymmetry) with an increase of agricultural land-use at intermediate spatial scales. This intensification is also related to a decrease in the abundance of larger prey. A negative effect of landscape properties at similar spatial scales on spider fitness was recorded when controlling for relative investments in capture thread length. This study consequently documents the web building flexibility in response to changes in landscape composition, possibly due to changes in prey availability. KW - Araneus diadematus KW - Araneidae KW - behavioural flexibility KW - orb web geometry KW - landscape KW - model selection KW - semi-natural habitats Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48262 ER - TY - JOUR A1 - Bonte, Dries A1 - Hovestadt, Thomas A1 - Poethke, Hans-Joachim T1 - Male-killing endosymbionts: influence of environmental conditions on persistance of host metapopulation N2 - Background: Male killing endosymbionts manipulate their arthropod host reproduction by only allowing female embryos to develop into infected females and killing all male offspring. Because of the reproductive manipulation, we expect them to have an effect on the evolution of host dispersal rates. In addition, male killing endosymbionts are expected to approach fixation when fitness of infected individuals is larger than that of uninfected ones and when transmission from mother to offspring is nearly perfect. They then vanish as the host population crashes. High observed infection rates and among-population variation in natural systems can consequently not be explained if defense mechanisms are absent and when transmission efficiency is perfect. Results: By simulating the host-endosymbiont dynamics in an individual-based metapopulation model we show that male killing endosymbionts increase host dispersal rates. No fitness compensations were built into the model for male killing endosymbionts, but they spread as a group beneficial trait. Host and parasite populations face extinction under panmictic conditions, i.e. conditions that favor the evolution of high dispersal in hosts. On the other hand, deterministic 'curing' (only parasite goes extinct) can occur under conditions of low dispersal, e.g. under low environmental stochasticity and high dispersal mortality. However, high and stable infection rates can be maintained in metapopulations over a considerable spectrum of conditions favoring intermediate levels of dispersal in the host. Conclusion: Male killing endosymbionts without explicit fitness compensation spread as a group selected trait into a metapopulation. Emergent feedbacks through increased evolutionary stable dispersal rates provide an alternative explanation for both, the high male-killing endosymbiont infection rates and the high among-population variation in local infection rates reported for some natural systems. KW - Metapopulation KW - Parasit KW - Wirt KW - Endosymbiont KW - Theoretische Ökologie KW - Host-parasite interactions KW - individual-based model Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-45344 ER - TY - JOUR A1 - Bonte, Dries A1 - Hovestadt, Thomas A1 - Poethke, Hans-Joachim T1 - Evolution of dispersal polymorphism and local adaptation of dispersal distance in spatially structured landscapes N2 - Many organisms show polymorphism in dispersal distance strategies. This variation is particularly ecological relevant if it encompasses a functional separation of short- (SDD) and long-distance dispersal (LDD). It remains, however, an open question whether both parts of the dispersal kernel are similarly affected by landscape related selection pressures. We implemented an individual-based model to analyze the evolution of dispersal traits in fractal landscapes that vary in the proportion of habitat and its spatial configuration. Individuals are parthenogenetic with dispersal distance determined by two alleles on each individual‘s genome: one allele coding for the probability of global dispersal and one allele coding for the variance of a Gaussian local dispersal with mean value zero. Simulations show that mean distances of local dispersal and the probability of global dispersal, increase with increasing habitat availability, but that changes in the habitat's spatial autocorrelation impose opposing selective pressure: local dispersal distances decrease and global dispersal probabilities increase with decreasing spatial autocorrelation of the available habitat. Local adaptation of local dispersal distance emerges in landscapes with less than 70% of clumped habitat. These results demonstrate that long and short distance dispersal evolve separately according to different properties of the landscape. The landscape structure may consequently largely affect the evolution of dispersal distance strategies and the level of dispersal polymorphism. Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47856 ER - TY - JOUR A1 - Bonte, Dries A1 - Hovestadt, Thomas A1 - Poethke, Hans Joachim T1 - Sex-specific dispersal and evolutionary rescue in metapopulations infected by male killing endosymbionts N2 - Background: Male killing endosymbionts manipulate their arthropod host reproduction by only allowing female embryos to develop into infected females and killing all male offspring. Because the resulting change in sex ratio is expected to affect the evolution of sex-specific dispersal, we investigated under which environmental conditions strong sex-biased dispersal would emerge, and how this would affect host and endosymbiont metapopulation persistence. Results: We simulated host-endosymbiont metapopulation dynamics in an individual-based model, in which dispersal rates are allowed to evolve independently for the two sexes. Prominent male-biased dispersal emerges under conditions of low environmental stochasticity and high dispersal mortality. By applying a reshuffling algorithm, we show that kin-competition is a major driver of this evolutionary pattern because of the high within-population relatedness of males compared to those of females. Moreover, the evolution of sex-specific dispersal rescues metapopulations from extinction by (i) reducing endosymbiont fixation rates and (ii) by enhancing the extinction of endosymbionts within metapopulations that are characterized by low environmental stochasticity. Conclusion: Male killing endosymbionts induce the evolution of sex-specific dispersal, with prominent male-biased dispersal under conditions of low environmental stochasticity and high dispersal mortality. This male-biased dispersal emerges from stronger kin-competition in males compared to females and induces an evolutionary rescue mechanism. KW - Metapopulation KW - Theoretische Ökologie KW - Endosymbiont KW - Wirt KW - Parasit KW - Host-parasite interactions KW - individual-based model Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-45351 ER - TY - JOUR A1 - Bonte, Dries A1 - Clercq, Nele De A1 - Zwertvaegher, Ingrid A1 - Lens, Luc T1 - Repeatability of dispersal behaviour in a common dwarf spider: evidence for different mechanisms behind short- and long-distance dispersal N2 - Abstract: 1. The response of dispersal towards evolution largely depends on its heritability for which upper limits are determined by the trait's repeatability. 2. In the Linyphiid spider E. atra, we were able to separate long- and short-distance dispersal behaviours (respectively ballooning and rappelling) under laboratory conditions. By performing repeated behavioural trials for females, we show that average dispersal trait values decrease with increasing testing days. By comparing mated and unmated individuals during two periods (before and after mating for the mated group, and the same two periods for the unmated group), we show that mating has no effect on the mean displayed dispersal behaviour or its within-individual variation. Repeatabilities were high and consistent for ballooning motivation, but not for rappelling. 3. Ballooning motivation can be regarded as highly individual-specific behaviour, while general pre-dispersal and rappelling behaviours showed more individual variation. Such difference in repeatability between long-and short-distance dispersal suggests that short-and long-distance dispersal events are triggered by different ecological and evolutionary mechanisms. Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48242 ER - TY - JOUR A1 - Bona, Marion A1 - Scheer, Ulrich A1 - Bautz, Ekkehard K. F. T1 - Antibodies to RNA polymerase II (B) inhibit transcription in lampbrush chromosomes after microinjection into living amphibian oocytes N2 - Antibodies directed against RNA polymerase II (B) from Drosophila melanogaster were obtained from rabbit sera and, as monoclonal immunoglobulins, from mouse hybridomas and shown to cross-react with the amphibian enzyme protein. Localization by indirect immunofluorescence microscopy revealed the association of this enzyme with chromatin of interphase nuclei of amphibian cells and its absence in nucleoli. Purified immunoglobulins were microinjected in to nuclei ofliving vitellogenic oocytes of Ple1lrodeles waltlii and X enopus laevis and their effects on transcriptional processes were monitored by biochemical and light and electron microscopic stud ies. RNA polymerase II antibodies from rabbit sera caused a rapid and almost complete release of nascent transcripts from the chromatin axis of the loops of lampbrush chromosomes, followed by collapse of the loops and their retraction on the main chromosome axis. Monoclonal murine antibodies to the Iarge RNA polymerase II subunits also inhibited transcription in chromosome Ioops but appeared to inhibit initiation rather than elongation events. Activities of class land III RNA polymerases were not significantly affected by injection of antibodies to polymerase II, indicating immunological differences between the three RNA polymerases. The potential value of the in vitro test system described , as a very sensitive assay for detecting proteins involved in transcription in living cells, is discussed. 1 Y1 - 1981 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33128 ER - TY - THES A1 - Bollmann, Stefan T1 - Structural Dynamics of Oligopeptides determined by Fluorescence Quenching of Organic Dyes T1 - Bestimmung struktureller Dynamiken von Oligopeptiden mittels Fluoreszenzlöschung von organischen Fluorophoren N2 - For determination of structures and structural dynamics of proteins organic fluorophores are a standard instrument. Intra- and intermolecular contact of biomolecular structures are determined in time-resolved and stationary fluorescence microscopy experiments by quenching of organic fluorophores due to Photoinduced Electron Transfer (PET) and dimerization interactions. Using PET we show in this work that end-to-end contact dynamics of serine-glycine peptides are slowed down by glycosylation. This slow down is due to a change in reaction enthalpy for end-to-end contact and is partly compensated by entropic effects. In a second step we test how dimerization of MR121 fluorophore pairs reports on end-to-end contact dynamics. We show that in aqueous solutions containing strong denaturants MR121 dimerization reports advantageously on contact dynamics for glycine-serine oligopeptides compared to the previously used MR121/tryptophane PET reporters. Then we analyze dimer interactions and quenching properties of different commercially available fluorophores being standards in Förster Resonance Energy Transfer (FRET) measurements. Distances in biomolecules are determinable using FRET, but for very flexible biomolecules the analysis of masurement data can be distorted if contact of the two FRET fluorophores is likely. We quantify how strong the quenching of fluorophore pairs with two different or two identical fluorophores is. Dimer spectra and association constants are quantified to estimate if fluophores are applicable in various applications, e.g. in FRET measurements with unstructured peptides and proteins. N2 - Zur Charakterisierung von Proteinen werden in der fluoreszenzbasierten Mikroskopie organische Farbstoffe benutzt, um strukturelle Informationen bzw. Informationen über dynamische Prozesse zu gewinnen. In der zeitaufgelösten und stationären Fluoreszenzmikroskopie können hiermit Kontaktprozesse durch photoinduzierten Elektronentransfer und auch Dimerisierung der Fluorophore analysiert werden. In dieser Arbeit wird mittels photoinduziertem Elektronentransfer PET gezeigt, dass Glykosylierung End-zu-End Kontaktkinetiken verändert. Sehr flexible Serin-Glycin Peptide zeigen glykosyliert langsamere Kinetiken durch Veränderung der Reaktionsenthalpie der Kontaktreaktion beider Peptidenden verglichen zu unglykosylierten. Diese enthalpischen Beiträge werden zum Teil von entropischen Beiträgen kompensiert. Außerdem wird gezeigt, dass Glycin-Serin Peptiddynamiken auch mittels Farbstoffpaaren gemessen werden können, die auf Löschwechselwirkungen durch Dimerisierung beruhen. Die Stärke dieser Löschwechselwirkungen hängt vom Farbstoffpaar ab. In Lösungen mit Denaturierungsmitteln können Farbstoffpaare des Fluoreszenzfarbstoffes MR121 vorteilhaft für Messungen von Dynamiken von Glycin-Serin Peptiden genutzt werden. Die Dimerwechselwirkungen können bei sehr flexiblen Biomolekülen und möglichem Kontakt von Fluorophoren die konventionelle Analyse von Förster Resonanz Energie Transfer (FRET) Messungen erschweren. Wir untersuchen an Glycin-Serin Oligopeptiden das Dimerisierungsverhalten kommerziell erhältlicher Fluorophore, die in FRET Messungen verwendet werden. Für gleiche und verschiedene Fluorophore wird die Löschung durch Dimerwechselwirkungen quantifiziert. Dabei werden Dimerspektren und Assoziationskonstanten für Dimerisierungsreaktionen bestimmt. Letztere helfen bei der Abschätzung, ob Fluorophorpaare für verschiedene Anwendungen geeignet sind, zum Beispiel in FRET-Messungen in unstrukturierten Peptiden und Proteinen. KW - Fluorophore KW - Fluoreszenzlöschung KW - h-dimerization KW - Lumineszenzlöschung KW - Fluoreszenzkorrelationsspektroskopie KW - Glykosylierung KW - Dimerisierung Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-92191 ER - TY - THES A1 - Bollazzi Sosa, Leonardo Martin T1 - Building behaviour and the control of nest climate in Acromyrmex leaf-cutting ants T1 - Bauverhalten und die Kontrolle des Nestklimas in der Blattschneiderameisen Acromyrmex N2 - This work was aimed at experimentally studying whether climatic variables act as environmental cues for workers’ building behaviour in leaf-cutting ants of the genus Acromyrmex, and to what extent building responses account for the maintenance of nest climate in a proper range for the inhabiting colony. Specifically, this work presents independent analysis in different Acromyrmex species with disparate ecology and nesting habits, aimed at understanding to what extent: i) temperature and humidity act as cues for workers’ building behaviour, ii) inter- and intraspecific differences in the nesting habits observed in South American Acromyrmex are based on distinct building behaviours and on the variation in regional climate across continent, iii) differences in nest architecture account for the maintenance of nest climate in a proper range for colony members and, iv) climatic variables trigger building responses aimed at controlling short-term changes in nest climate. It is first experimentally shown that soil temperature acts as a cue for workers’ digging behaviour. Acromyrmex lundi workers were observed to respond to both soil temperature as well as its changes, and to decide accordingly where to start or whether to stop digging. The soil temperature range preferred by workers to dig, between 20°C and maximally 30.6°C, matches the range at which colony growth is expected to be maximized. Temperature-sensitive digging might therefore lead to the establishment of the fungus chambers in soil layers with a proper range of temperatures for colony growth. Based on that, it was hypothesized that nest depth in Acromyrmex largely depends on the depth at which this temperature range is located across the soil profile, i.e., the higher the temperature in the superficial soil layers, the deeper the nest location, since soil temperature decreases with increasing depth. A bibliographic survey on nesting habits of 21 South American Acromyrmex species confirmed that the warmer the soil temperature at 50 cm depth throughout the South American continent, the higher the number of species presenting subterranean nests, compared with those inhabiting superficial nests. Temperature-sensitive digging in Acromyrmex would therefore explain the geographical distribution of nesting habits observed for this genus in the South American continent, i.e., subterranean in the northern tropical regions, and superficial in the southern temperate ones. In addition, results showed that Acromyrmex colonies from temperate regions indeed achieve thermoregulatory benefits through the determination of nest depth based on thermoregulatory needs. In sympatrically-occurring colonies of the grass-cutting ant A. heyeri, temperature inside superficial thatched nests was higher, and more suitable for colony growth, than that inside subterranean nests. This temperature surplus was even higher in spring, at the time of production of sexual brood, than in winter or summer. It was demonstrated that such temperature surplus was brought about by the low thermal diffusivity of the nest thatch, which prevents diurnal nest overheating by the incoming solar radiation, and avoids losses of the accumulated daily heat into the cold air during night, thus leading to high average nest temperatures. Although highly advantageous for colonies in terms of nest temperature, the determination of nest depth based on thermoregulatory needs may differentially affect nest ventilation and humidity depending on how nest exposition influences the exchange of nest air with the outside air. For instance, colonies with a superficial nesting habit might benefit from improved nest ventilation, but be at risk of desiccation due to their exposition and the consequent humidity losses into the dry outside air. Results demonstrated that in two Acromyrmex species, short-term regulatory building responses triggered and spatially organized by climatic variables occur, and may counteract undesired changes in internal nest humidity. Workers of the thatching grass-cutting ant A. heyeri, for instance, closed a number of nest-thatch openings as a response to desiccation of the outside air, even at a nest temperature that otherwise triggered the response of opening them so as to reduce nest temperature. In the leaf-cutting ant A. ambiguus, the direction of the airflow inside nest tunnels was shown to act as a cue for spatially guiding the building behaviour of plugging nest entrances. However, workers only responded if the humidity content of the circulating air was low, trading therefore nest ventilation for humidity maintenance. N2 - Die vorliegende Arbeit untersucht, inwiefern das Bauverhalten von Blattschneiderameisen der Gattung Acromyrmex durch klimatische Variablen beeinflusst wird und dem Erhalt für die Ameisen geeigneter klimatischer Bedingungen dient. Betrachtet werden verschiedene Acromyrmex-Arten, die sich in ihrer Ökologie und ihren Nistgewohnheiten unterscheiden. Ziel ist es zu verstehen, in wie fern: i) Temperatur und Feuchtigkeit als Reize das Bauverhalten der Arbeiterinnen beeinflussen, ii) Unterschiede im Bauverhalten und die regionale Variation des Klimas über den südamerikanischen Kontinent die beobachteten, inter- und intraspezifischen Unterschiede zwischen den Nesttypen südamerikanischer Acromyrmex-Arten erklären, iii) unterschiedliche Nestarchitekturen für die Aufrechterhaltung für die Ameisen geeigneter klimatischer Bedingungen im Nest sorgen, iv) klimatische Variablen Verhaltensweisen auslösen, die der Kontrolle kurzfristiger Änderungen des Nestklimas dienen. Zunächst wird experimentell gezeigt, dass die Bodentemperatur ein Reiz ist, der das Bauverhalten von Ameisen beeinflusst. Es wurde beobachtet, dass Acromyrmex lundi-Arbeiterinnen sowohl auf Temperaturen als auch auf Temperaturänderungen reagieren, und, abhängig von diesen Variablen, über die Aufnahme oder den Abbruch des Grabeverhaltens entscheiden. Der Temperaturbereich im Boden, in dem die Arbeiterinnen zu Graben bevorzugen, also zwischen 20°C und maximal 30.6°C, entspricht dem Temperaturbereich, bei dem ein maximales Koloniewachstum erwartet werden sollte. Zudem legen die Ergebnisse nahe, dass die Orientierung des kollektiven Grabenverhaltens an der Bodentemperatur den Ameisen ermöglicht, Nestkammern in Bodenschichten zu etablieren die geeignete Temperaturbedingungen bieten. Es wird angenommen, dass die Nesttiefe bei Acromyrmex stark davon abhängt, wie tief im Boden geeignete Temperaturbedingungen anzutreffen sind. Je höher die Temperatur in den obersten Bodenschichten, desto tiefer das Nest, denn die Bodentemperatur sinkt mit zunehmender Tiefe. Literaturdaten zu den Nistgewohnheiten von 21 südamerikanischen Acromyrmex-Arten wurden verglichen. Hierbei bestätigte sich, dass über den südamerikanischen Kontinent mit zunehmender, mittlerer Bodentemperatur in einer Tiefe von 50 cm auch der Anteil der Arten zunimmt, die ausschließlich unterirdische Nester bauen im Verhältnis zu den Arten mit Oberflächennestern zunimmt. Temperaturabhängiges Graben würde die geographische Verteilung der Nistgewohnheiten von Acromyrmex in Südamerika erklären: Unterirdische Nester überwiegen in den nördlichen, tropischen Regionen und Oberflächennester in den gemäßigten Regionen im Süden. Zudem konnte gezeigt werden, dass Acromyrmex-Kolonien der gemäßigten Regionen tatsächlich ihre Nesttemperatur durch Anpassung der Nesttiefe an klimatische Bedingungen regulieren. Bei der Grassschneiderameise A. heyeri, bei der Kolonien mit unterirdischen Nestern und solche mit oberflächlichen Hügelnestern sympatrisch vorkommen, war die Temperatur in den Oberflächennestern höher, und für das Koloniewachstum günstiger, als in unterirdischen Nestern. Dieser Temperaturvorteil war im Frühling, der Zeit, in der die Geschlechtstierbrut herangezogen wird, größer als in Winter oder Sommer. Es wurde gezeigt, dass dieser Vorteil durch die niedrige Wärmeleitfähigkeit der Nesthügels bedingt ist. Tagsüber verhindert der Nesthügel zunächst die Überhitzung durch Sonneneinstrahlung, und minimiert dann während der Nacht den Wärmeverlust an die kalte Umgebungsluft. Dies führt zu hohen Durchschnittstemperaturen innerhalb solcher Nester. Neben dem Vorteil, den eine geringe Nesttiefe in diesem Fall für die Temperatur in der Nestkammer bietet, spielen auch weitere Aspekte eine Rolle. Kolonien mit oberflächlichen Nestern profitieren zwar von der vergleichsweise guten Nestventilation, setzen sich dabei aber einem Erhöhten Risiko aus, durch den Verlust von Feuchtigkeit an die Außenluft auszutrocknen. Bei zwei Acromyrmex-Arten zeigen die Ergebnisse das Auftreten regulatorischer Bauaktivität, die, ausgelöst und räumlich organisiert durch klimatische Variablen, einem unerwünschten Feuchtigkeitsverlust innerhalb des Nestes entgegenwirkt. Arbeiterinnen der hügelbauenden Grassschneiderameise A. heyeri verschlossen Öffnungen im Nesthügel als Antwort auf die Austrocknung der Aussenluft, und das selbst bei einer Nesttemperatur, auf die unter anderen Umständen mit der Öffnung derselben zur Reduzierung der Nesttemperatur reagiert worden wäre. Bei der Blattschneiderameise A. ambiguus, die unter bestimmten Bedingungen ihre Tunnel mit Pflanzenmaterial verschließt, wurde gezeigt, dass die Richtung der Luftbewegung in den Nestgängen das Verschließen der Eingänge räumlich beeinflusst. Dennoch reagierten Arbeiteinnen nur, wenn der Feuchtigkeitsgehalt der zirkulierenden Luft niedrig war, sie beschränkten somit die Nestventilation um die Feuchtigkeit aufrecht zu erhalten. KW - Verhaltensökologie KW - Bauverhalten KW - Nestklimas KW - Acromyrmex KW - Blattschneiderameisen KW - Building behaviour KW - nest climate KW - Acromyrmex KW - leaf-cutting ants Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-27610 ER - TY - JOUR A1 - Bollazzi, Martin A1 - Roces, Flavio T1 - The thermoregulatory function of thatched nests in the South American grass-cutting ant, Acromyrmex heyeri N2 - The construction of mound-shaped nests by ants is considered as a behavioral adaptation to low environmental temperatures, i.e., colonies achieve higher and more stables temperatures than those of the environment. Besides the well-known nests of boreal Formica wood-ants, several species of South American leaf-cutting ants of the genus Acromyrmex construct thatched nests. Acromyrmex workers import plant fragments as building material, and arrange them so as to form a thatch covering a central chamber, where the fungus garden is located. Thus, the degree of thermoregulation attained by the fungus garden inside the thatched nest largely depends on how the thatch affects the thermal relations between the fungus and the environment. This work was aimed at studying the thermoregulatory function of the thatched nests built by the grass-cutting ant Acromyrmex heyeri Forel (Hymenoptera: Formicidae: Myrmicinae). Nest and environmental temperatures were measured as a function of solar radiation on the long-term. The thermal diffusivity of the nest thatch was measured and compared to that of the surrounding soil, in order to assess the influence of the building material on the nest’s thermoregulatory ability. The results showed that the average core temperature of thatched nests was higher than that of the environment, but remained below values harmful for the fungus. This thermoregulation was brought about by the low thermal diffusivity of the nest thatch built by workers with plant fragments, instead of the readily-available soil particles that have a higher thermal diffusivity. The thatch prevented diurnal nest overheating by the incoming solar radiation, and avoided losses of the accumulated daily heat into the cold air during the night. The adaptive value of thatching behavior in Acromyrmex leaf-cutting ants occurring in the southernmost distribution range is discussed. KW - Acromyrmex heyeri KW - building behaviour KW - thermal biology KW - nest material KW - heat transfer KW - leaf-cutting ants Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68225 ER - TY - JOUR A1 - Bollazzi, Martin A1 - Roces, Flavio T1 - Information Needs at the Beginning of Foraging: Grass-Cutting Ants Trade Off Load Size for a Faster Return to the Nest N2 - Background: Acquisition of information about food sources is essential for animals that forage collectively like social insects. Foragers deliver two commodities to the nest, food and information, and they may favor the delivery of one at the expenses of the other. We predict that information needs should be particularly high at the beginning of foraging: the decision to return faster to the nest will motivate a grass-cutting ant worker to reduce its loading time, and so to leave the source with a partial load. Principal Findings: Field results showed that at the initial foraging phase, most grass-cutting ant foragers (Acromyrmex heyeri) returned unladen to the nest, and experienced head-on encounters with outgoing workers. Ant encounters were not simply collisions in a probabilistic sense: outgoing workers contacted in average 70% of the returning foragers at the initial foraging phase, and only 20% at the established phase. At the initial foraging phase, workers cut fragments that were shorter, narrower, lighter and tenderer than those harvested at the established one. Foragers walked at the initial phase significantly faster than expected for the observed temperatures, yet not at the established phase. Moreover, when controlling for differences in the fragment-size carried, workers still walked faster at the initial phase. Despite the higher speed, their individual transport rate of vegetable tissue was lower than that of similarly-sized workers foraging later at the same patch. Conclusions/Significance: At the initial foraging phase, workers compromised their individual transport rates of material in order to return faster to the colony. We suggest that the observed flexible cutting rules and the selection of partial loads at the beginning of foraging are driven by the need of information transfer, crucial for the establishment and maintenance of a foraging process to monopolize a discovered resource. KW - Blattschneiderameisen Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68940 ER - TY - JOUR A1 - Bohnert, Simone A1 - Wirth, Christoph A1 - Schmitz, Werner A1 - Trella, Stefanie A1 - Monoranu, Camelia-Maria A1 - Ondruschka, Benjamin A1 - Bohnert, Michael T1 - Myelin basic protein and neurofilament H in postmortem cerebrospinal fluid as surrogate markers of fatal traumatic brain injury JF - International Journal of Legal Medicine N2 - The aim of this study was to investigate if the biomarkers myelin basic protein (MBP) and neurofilament-H (NF-H) yielded informative value in forensic diagnostics when examining cadaveric cerebrospinal fluid (CSF) biochemically via an enzyme-linked immunosorbent assay (ELISA) and comparing the corresponding brain tissue in fatal traumatic brain injury (TBI) autopsy cases by immunocytochemistry versus immunohistochemistry. In 21 trauma and 19 control cases, CSF was collected semi-sterile after suboccipital puncture and brain specimens after preparation. The CSF MBP (p = 0.006) and NF-H (p = 0.0002) levels after TBI were significantly higher than those in cardiovascular controls. Immunohistochemical staining against MBP and against NF-H was performed on cortical and subcortical samples from also biochemically investigated cases (5 TBI cases/5 controls). Compared to the controls, the TBI cases showed a visually reduced staining reaction against MBP or repeatedly ruptured neurofilaments against NF-H. Immunocytochemical tests showed MBP-positive phagocytizing macrophages in CSF with a survival time of > 24 h. In addition, numerous TMEM119-positive microglia could be detected with different degrees of staining intensity in the CSF of trauma cases. As a result, we were able to document that elevated levels of MBP and NF-H in the CSF should be considered as useful neuroinjury biomarkers of traumatic brain injury. KW - biofluid KW - CSF KW - cerebrospinal fluid KW - forensic neuropathology KW - forensic neurotraumatology KW - biomarker Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-266929 SN - 1437-1596 VL - 135 IS - 4 ER - TY - JOUR A1 - Bohnert, Simone A1 - Reinert, Christoph A1 - Trella, Stefanie A1 - Schmitz, Werner A1 - Ondruschka, Benjamin A1 - Bohnert, Michael T1 - Metabolomics in postmortem cerebrospinal fluid diagnostics: a state-of-the-art method to interpret central nervous system–related pathological processes JF - International Journal of Legal Medicine N2 - In the last few years, quantitative analysis of metabolites in body fluids using LC/MS has become an established method in laboratory medicine and toxicology. By preparing metabolite profiles in biological specimens, we are able to understand pathophysiological mechanisms at the biochemical and thus the functional level. An innovative investigative method, which has not yet been used widely in the forensic context, is to use the clinical application of metabolomics. In a metabolomic analysis of 41 samples of postmortem cerebrospinal fluid (CSF) samples divided into cohorts of four different causes of death, namely, cardiovascular fatalities, isoIated torso trauma, traumatic brain injury, and multi-organ failure, we were able to identify relevant differences in the metabolite profile between these individual groups. According to this preliminary assessment, we assume that information on biochemical processes is not gained by differences in the concentration of individual metabolites in CSF, but by a combination of differently distributed metabolites forming the perspective of a new generation of biomarkers for diagnosing (fatal) TBI and associated neuropathological changes in the CNS using CSF samples. KW - CSF KW - cerebrospinal fluid KW - forensic neuropathology KW - forensic neurotraumatology KW - biomarker KW - metabolomics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235724 SN - 0937-9827 VL - 135 ER - TY - THES A1 - Bohn, Holger Florian T1 - Biomechanik von Insekten-Pflanzen-Interaktionen bei Nepenthes-Kannenpflanzen T1 - Biomechanics of insect-plant interactions in Nepenthes pitcher plants N2 - Interaktionen zwischen Insekten und Pflanzen können auf chemischen oder mechanischen Faktoren beruhen. Mechanische Faktoren spielen eine besonders wichtige Rolle bei den Fallen karnivorer Pflanzen. Ziel dieser Arbeit war es, die Rolle mechanischer Faktoren in der Interaktion zwischen der Kannenpflanze Nepenthes bicalcarata und der Ameise Camponotus schmitzi aufzuklären, bei der Ameisen Gegenanpassungen zu spezialisierten pflanzlichen Fangstrukturen entwickelt haben. Im Rahmen meiner Arbeit habe ich mich mit den Fragen beschäftigt, 1) welche Kannenstrukturen und welche Mechanismen für den Fang von Arthropoden wichtig sind und 2) welche speziellen Anpassungen C. schmitzi-Ameisen für das Leben auf ihrer karnivoren Wirtspflanze besitzen. Bisher wurde angenommen, dass Nepenthes-Kannen Tiere mit Hilfe von rutschigen Wachskristallschichten fangen. Ich konnte zeigen, dass ein weiterer, bisher unbekannter Fangmechanismus existiert, welcher auf speziellen Oberflächeneigenschaften des Kannenrandes (Peristom) und "Insekten-Aquaplaning" basiert. Das Peristom besitzt eine regelmäßige Mikrostruktur, welche dafür sorgt, dass die Oberfläche vollständig mit Wasser benetzbar ist, so dass sie bei feuchter Witterung von homogenen Flüssigkeitsfilmen überzogen ist. Auf dem trockenen Peristom können Ameisen ohne Schwierigkeiten laufen und Nektar von den am inneren Peristomrand gelegenen Nektarien ernten. Wird die Oberfläche aber beispielsweise durch Regen nass, rutschen die meisten Tiere ab und stürzen in die Kanne. Messungen der Reibungskräfte von Weberameisen (Oecophylla smaragdina) auf dem Peristom von N. bicalcarata zeigten, dass Flüssigkeitsfilme auf der Oberfläche die Anhaftung der Haftorgane (Arolien) verhindern, und dass die Mikrostruktur des Peristoms auch den Einsatz der Krallen unterbindet. Versuche an Nepenthes alata zeigten darüber hinaus, dass dieser Fangmechanismus des Peristoms auch für Nepenthes-Arten mit wachsbereifter Kanneninnenwand essentiell, und die Wachsschicht eher für die Retention gefangener Tiere wichtig ist. Zur Analyse der ökologischen Auswirkungen des "Aquaplaning"-Fangmechanismus habe ich die Peristomfeuchte von Nepenthes rafflesiana var. typica-Kannen zeitgleich mit meteorologischen Daten im Feld kontinuierlich aufgezeichnet und mit Experimenten zur Beurteilung der Fangeffizienz der Kannen kombiniert. Die Ergebnisse dieser Versuche zeigen, dass die Kannen abhängig vom Befeuchtungsgrad des Peristoms zeitweise sehr effiziente Fallen mit Fangraten von 80% sein können, während sie zu anderen Zeiten vollkommen ineffizient sind. Die Variation der Peristomfeuchte wird durch Regen, Kondensation und von den Peristomnektarien sezerniertem Nektar verursacht. Es ist zu vermuten, dass die nur zeitweise und unvorhersehbare Aktivierung der Nepenthes-Kannenfallen durch Nässe der Evolution von Vermeidungsstrategien bei Beutetieren entgegenwirkt. Im Rahmen der Untersuchungen, welche mechanischen Anpassungen C. schmitzi-Ameisen für das Leben auf N. bicalcarata besitzen habe ich mich auf die Fragen konzentriert, wie es den Ameisen gelingt den Peristom-Fangmechanismus zu umgehen und welche Anpassungen sie besitzen um in der Kannenflüssigkeit tauchend und schwimmend nach Nahrung zu suchen. Im Gegensatz zu generalistischen Arten stürzen C. schmitzi-Ameisen auf dem nassen Peristom nicht ab. Durch selektive Manipulation der tarsalen Haftstrukturen konnte ich demonstrieren, dass die Arolien für die Peristomlauffähigkeit der C. schmitzi-Ameisen eine wesentliche Rolle spielen. Für das Furagieren in der Kannenflüssigkeit verfügen C. schmitzi-Ameisen über ein sich wiederholendes, stereotypes Verhaltensmuster, welches aus einer Unterwasserlauf- und einer Oberflächenschwimmphase besteht. Meine Untersuchungen dieses Verhaltensmusters zeigten, dass die Ameisen am Ende der Unterwasserlaufphase mit Hilfe ihres stets vorhandenen Auftriebs zur Flüssigkeitsoberfläche aufsteigen. Dabei taucht ein Teil ihres Hinterleibs aus der Kannenflüssigkeit auf, was den Ameisen die Sauerstoffaufnahme aus der Luft ermöglicht. Nach dem Auftauchen schwimmen C. schmitzi-Ameisen mittels schneller Beinbewegungen an der Oberfläche der Kannenflüssigkeit. Dabei ähnelt die Bewegungskoordination ihrer Beine dem bei Ameisen für die Fortbewegung an Land typischen Dreifußgang. Ein Vergleich der Kinematik von schwimmenden und laufenden C. schmitzi-Ameisen hat gezeigt, dass schwimmende Ameisen ihre Beine in der Schlagphase mit einer höheren Winkelgeschwindigkeit als in der Rückholphase bewegen, während dies bei den laufenden Tieren genau umgekehrt ist. Ferner strecken schwimmende Ameisen ihre Beine während der Schlagphase weiter aus als in der Rückholphase, wohingegen laufende Ameisen in beiden Bewegungsphasen vergleichbare Beinradien aufweisen. Dies lässt den Schluss zu, dass die Schwimmkinematik der C. schmitzi-Ameisen eine abgewandelte Form ihrer Laufkinematik darstellt, welche für die Erzeugung von Vortrieb im Wasser optimiert wurde. N2 - Insect-plant interactions based on either chemical or mechanical factors, play a key role in nature. Mechanical factors are of particular importance for the animal traps of carnivorous plants. The aim of this study is to clarify the role of mechanical factors in the interaction between the pitcher plant Nepenthes bicalcarata and its ant partner, Camponotus schmitzi which has evolved counter adaptations against the specialised capture structures of the plant. This study investigates two questions, firstly, which of the pitchers' structures and which mechanisms are important for the capture of arthropods and secondly, what are the special adaptations that enable the C. schmitzi ants to live on their carnivorous host plant. It has so far been suggested, that Nepenthes pitchers capture prey by means of slippery epicuticular wax crystals. I was however able to show, that another, yet unknown capture mechanism exists. It is based on the special surface properties of the pitcher rim (peristome) and on the phenomenon of insect "aquaplaning". The peristome is characterized by a regular microstructure with radial ridges of smooth overlapping epidermal cells, which form a series of steps toward the pitcher interior. This surface is completely wettable by water, so that under humid weather conditions it is covered by homogenous liquid films. If the peristome is dry, ants can run freely on it and harvest nectar from the nectaries at the inner margin of the peristome. As soon as the peristome surface is wetted, for example by rain, it becomes extremely slippery for insects, so that most of the ant visitors are trapped. By measuring the friction forces of weaver ants (Oecophylla smaragdina) on the peristome of N. bicalcarata, I was able to show that the liquid films on the surface disrupt attachment for the soft adhesive pads (arolia) and that the surface topography impedes the use of claws. Experiments on Nepenthes alata demonstrated that the trapping mechanism of the peristome is also essential in Nepenthes species with waxy inner pitcher walls, indicating that the waxy surfaces are more important for the retention rather than the capture of prey. I investigated the ecological implications of the "aquaplaning" capture mechanism in Nepenthes rafflesiana var. typica by combining meteorological data and continuous field measurements of peristome wetness with experimental assessments of the pitchers’ capture efficiency. My results demonstrate that pitchers can be highly effective traps with capture rates as high as 80% but are completely ineffective at other times. These dramatic changes are due to the wetting conditions of the peristome. Variation of peristome wetness and thus the variation of capture efficiency is caused by rain, condensation, and nectar secreted from the peristome nectaries. I propose that the intermittent and unpredictable activation of Nepenthes pitcher traps prevents the evolution of avoidance strategies in prey animals. In the second part of my study I investigated the mechanical adaptations that the C. schmitzi possess in order to live on N. bicalcarata. I focused on two principal questions, how are the ants able to circumvent the peristome capture mechanism and what adaptations do they need in order to swim and dive in the digestive fluid. In contrast to generalist ants, C. schmitzi ants are capable of running on the wet peristome without difficulties. Through selective manipulation of tarsal attachment structures I was able to demonstrate, that the arolia are essential for the ants’ capability to run on the wet peristome. Whilst foraging in the pitcher fluid C. schmitzi ants show a repetitive stereotyped behaviour pattern, consisting of an underwater running and surface swimming phase. My analysis of this behaviour pattern showed that at the end of the underwater running phase the ants advance to the fluid surface with the aid of buoyancy. When reaching the surface film parts of the ants’ gaster and head emerge. I was able to show that while foraging in the pitcher fluid the emerging of the gaster is crucial for the respiration of the ants. After emersion the ants swim at the surface of the pitcher fluid using fast leg movements. Hence the leg coordination is similar to a tripod gait which is typical for their locomotion on land. A comparison between the kinematics of swimming and running C. schmitzi ants showed that whilst swimming, the angular velocity of their legs is higher in the stroke than in the recovery, whereas the opposite is true whilst running. Furthermore the swimming ants stretch their legs further in the stroke than in the recovery whereas the leg radius of running ants does not vary much throughout a step. It can be concluded that the swimming kinematics of C. schmitzi ants derives from the kinematics of their running and has been optimized for generating thrust in water. KW - Biomechanik KW - Kannenpflanze KW - Rossameise KW - Fleischfressende Pflanzen KW - Aquaplaning KW - Mikrostruktur KW - Symbiose KW - Kinematik KW - Insekten-Pflanzen-Interaktion KW - schwimmende Ameisen KW - insect-plant interactions KW - Camponotus KW - Nepenthes KW - capture mechanism KW - swimming ants Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-26101 ER - TY - JOUR A1 - Bogdan, Sven A1 - Schultz, Jörg A1 - Grosshans, Jörg T1 - Formin’ cellular structures: Physiological roles of Diaphanous (Dia) in actin dynamics JF - Communicative & Integrative Biology N2 - Members of the Diaphanous (Dia) protein family are key regulators of fundamental actin driven cellular processes, which are conserved from yeast to humans. Researchers have uncovered diverse physiological roles in cell morphology, cell motility, cell polarity, and cell division, which are involved in shaping cells into tissues and organs. The identification of numerous binding partners led to substantial progress in our understanding of the differential functions of Dia proteins. Genetic approaches and new microscopy techniques allow important new insights into their localization, activity, and molecular principles of regulation. KW - Drosophila KW - cytoskeleton KW - actin KW - nucleator KW - development KW - formin Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121305 VL - 6 IS - e27634 ER -