TY - JOUR A1 - Dammert, Marcel A. A1 - Brägelmann, Johannes A1 - Olsen, Rachelle R. A1 - Böhm, Stefanie A1 - Monhasery, Niloufar A1 - Whitney, Christopher P. A1 - Chalishazar, Milind D. A1 - Tumbrink, Hannah L. A1 - Guthrie, Matthew R. A1 - Klein, Sebastian A1 - Ireland, Abbie S. A1 - Ryan, Jeremy A1 - Schmitt, Anna A1 - Marx, Annika A1 - Ozretić, Luka A1 - Castiglione, Roberta A1 - Lorenz, Carina A1 - Jachimowicz, Ron D. A1 - Wolf, Elmar A1 - Thomas, Roman K. A1 - Poirier, John T. A1 - Büttner, Reinhard A1 - Sen, Triparna A1 - Byers, Lauren A. A1 - Reinhardt, H. Christian A1 - Letai, Anthony A1 - Oliver, Trudy G. A1 - Sos, Martin L. T1 - MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer JF - Nature Communications N2 - MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients. KW - genetic engineering KW - oncogenes KW - small-cell lung cancer KW - targeted therapies Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223569 VL - 10 ER - TY - JOUR A1 - Coelho, Luis Pedro A1 - Alves, Renato A1 - Monteiro, Paulo A1 - Huerta-Cepas, Jaime A1 - Freitas, Ana Teresa A1 - Bork, Peer T1 - NG-meta-profiler: fast processing of metagenomes using NGLess, a domain-specific language JF - Microbiome N2 - Background Shotgun metagenomes contain a sample of all the genomic material in an environment, allowing for the characterization of a microbial community. In order to understand these communities, bioinformatics methods are crucial. A common first step in processing metagenomes is to compute abundance estimates of different taxonomic or functional groups from the raw sequencing data. Given the breadth of the field, computational solutions need to be flexible and extensible, enabling the combination of different tools into a larger pipeline. Results We present NGLess and NG-meta-profiler. NGLess is a domain specific language for describing next-generation sequence processing pipelines. It was developed with the goal of enabling user-friendly computational reproducibility. It provides built-in support for many common operations on sequencing data and is extensible with external tools with configuration files. Using this framework, we developed NG-meta-profiler, a fast profiler for metagenomes which performs sequence preprocessing, mapping to bundled databases, filtering of the mapping results, and profiling (taxonomic and functional). It is significantly faster than either MOCAT2 or htseq-count and (as it builds on NGLess) its results are perfectly reproducible. Conclusions NG-meta-profiler is a high-performance solution for metagenomics processing built on NGLess. It can be used as-is to execute standard analyses or serve as the starting point for customization in a perfectly reproducible fashion. NGLess and NG-meta-profiler are open source software (under the liberal MIT license) and can be downloaded from https://ngless.embl.de or installed through bioconda. KW - metagenomics KW - next-generation sequencing KW - domain-specific language Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223161 VL - 7 IS - 84 ER - TY - JOUR A1 - Hupp, Sabrina A1 - Rosenkranz, Maaria A1 - Bonfig, Katharina A1 - Pandey, Chandana A1 - Roitsch, Thomas T1 - Noninvasive Phenotyping of Plant–Pathogen Interaction: Consecutive In Situ Imaging of Fluorescing Pseudomonas syringae, Plant Phenolic Fluorescence, and Chlorophyll Fluorescence in Arabidopsis Leaves JF - Frontiers in Plant Science N2 - Plant–pathogen interactions have been widely studied, but mostly from the site of the plant secondary defense. Less is known about the effects of pathogen infection on plant primary metabolism. The possibility to transform a fluorescing protein into prokaryotes is a promising phenotyping tool to follow a bacterial infection in plants in a noninvasive manner. In the present study, virulent and avirulent Pseudomonas syringae strains were transformed with green fluorescent protein (GFP) to follow the spread of bacteria in vivo by imaging Pulse-Amplitude-Modulation (PAM) fluorescence and conventional binocular microscopy. The combination of various wavelengths and filters allowed simultaneous detection of GFP-transformed bacteria, PAM chlorophyll fluorescence, and phenolic fluorescence from pathogen-infected plant leaves. The results show that fluorescence imaging allows spatiotemporal monitoring of pathogen spread as well as phenolic and chlorophyll fluorescence in situ, thus providing a novel means to study complex plant–pathogen interactions and relate the responses of primary and secondary metabolism to pathogen spread and multiplication. The study establishes a deeper understanding of imaging data and their implementation into disease screening. KW - green fluorescence protein (GFP) KW - plant–pathogen interaction KW - imaging PAM KW - chlorophyll fluorescence imaging KW - phenolic compounds Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189425 SN - 1664-462X VL - 10 IS - 1239 ER - TY - JOUR A1 - Blümel, Rabea A1 - Zink, Miriam A1 - Klopocki, Eva A1 - Liedtke, Daniel T1 - On the traces of tcf12: Investigation of the gene expression pattern during development and cranial suture patterning in zebrafish (Danio rerio) JF - PLoS ONE N2 - The transcription factor 12 (tcf12) is a basic Helix-Loop-Helix protein (bHLH) of the E-protein family, proven to play an important role in developmental processes like neurogenesis, mesoderm formation, and cranial vault development. In humans, mutations in TCF12 lead to craniosynostosis, a congenital birth disorder characterized by the premature fusion of one or several of the cranial sutures. Current research has been primarily focused on functional studies of TCF12, hence the cellular expression profile of this gene during embryonic development and early stages of ossification remains poorly understood. Here we present the establishment and detailed analysis of two transgenic tcf12:EGFP fluorescent zebrafish (Danio rerio) reporter lines. Using these transgenic lines, we analyzed the general spatiotemporal expression pattern of tcf12 during different developmental stages and put emphasis on skeletal development and cranial suture patterning. We identified robust tcf12 promoter-driven EGFP expression in the central nervous system (CNS), the heart, the pronephros, and the somites of zebrafish embryos. Additionally, expression was observed inside the muscles and bones of the viscerocranium in juvenile and adult fish. During cranial vault development, the transgenic fish show a high amount of tcf12 expressing cells at the growth fronts of the ossifying frontal and parietal bones and inside the emerging cranial sutures. Subsequently, we tested the transcriptional activity of three evolutionary conserved non-coding elements (CNEs) located in the tcf12 locus by transient transgenic assays and compared their in vivo activity to the expression pattern determined in the transgenic tcf12:EGFP lines. We could validate two of them as tcf12 enhancer elements driving specific gene expression in the CNS during embryogenesis. Our newly established transgenic lines enhance the understanding of tcf12 gene regulation and open up the possibilities for further functional investigation of these novel tcf12 enhancer elements in zebrafish. KW - Zebrafish KW - Neurons KW - Skull KW - Enhancer elements KW - Hindbrain KW - Cranial sutures KW - Embryos KW - Somites Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201428 VL - 14 IS - 6 ER - TY - JOUR A1 - Panzer, Sabine A1 - Brych, Annika A1 - Batschauer, Alfred A1 - Terpitz, Ulrich T1 - Opsin 1 and Opsin 2 of the corn smut fungus ustilago maydis are green light-driven proton pumps JF - Frontiers in Microbiology N2 - In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis. KW - Ustilago maydis KW - patch-clamp KW - fungal rhodopsins KW - microbial rhodopsins KW - acetate KW - indole-3-acetic acid KW - structured illumination microscopy KW - sporidia Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201453 VL - 10 ER - TY - JOUR A1 - Kim, Brandon J. A1 - Shusta, Eric V. A1 - Doran, Kelly S. T1 - Past and current perspectives in modeling bacteria and blood–brain barrier interactions JF - Frontiers in Microbiology N2 - The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future. KW - bacteria KW - blood–brain barrier KW - meningitis KW - stem cells KW - brain endothelial cell Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201766 VL - 10 IS - 1336 ER - TY - THES A1 - König, Eva-Maria T1 - Pathogenese von Kraniosynostosen T1 - Pathogenesis of Craniosynostoses N2 - Das humane Schädeldach besteht aus fünf Schädelplatten, die durch intramembranöse Ossifikation entstehen. Wenn diese in der Embryonalentwicklung aufeinandertreffen, bilden sich Schädelnähte aus, die eine Fusion der Schädelplatten verhindern und damit ein Schädelwachstum parallel zu Gehirnentwicklung ermöglichen. Für diesen Prozess ist eine Balance aus Zellproliferation und Differenzierung nötig, deren Aufrechterhaltung wiederum durch eine komplexe Regulation von verschiedenen Signalwegen gewährleistet wird. Störungen in diesem regulatorischen System können zu einer vorzeitigen Fusion der Schädelplatten, Kraniosynostose genannt, führen. Die Kraniosynostose ist eine der häufigsten kraniofazialen Fehlbildungen beim Menschen. Durch kompensatorisches Wachstum an den nicht fusionierten Suturen entstehen charakteristische Schädeldeformationen, die sekundär einen erhöhten intrakranialen Druck zur Folge haben können. Eine vorzeitige Fusion der Suturen kann sowohl isoliert als auch syndromal zusammen mit weiteren klinischen Auffälligkeiten vorliegen. Bisher sind über 150 verschiedene Kraniosynostose Syndrome beschrieben und insgesamt 25-30% aller Kraniosynostose Patienten sind von einer syndromalen Form betroffen. Da die klinischen Merkmale der Kraniosynostose Syndrome variabel sind und zum Teil überlappen, ist eine klare klinische Diagnose häufig erschwert. Sowohl Umwelteinflüsse als auch genetische Veränderungen können die Ursache für Kraniosynostosen sein. Vor allem bei syndromalen Kraniosynostosen wurden genetische Veränderungen, wie beispielsweise Mutationen in den Genen FGFR2, FGFR3, TWIST1 und EFNB1, identifiziert. Darüber hinaus wurden chromosomale Veränderungen wie partielle Monosomien von 7p, 9p oder 11p sowie partielle Trisomien von 5q, 13q oder 15q mit Kraniosynostose assoziiert. Trotzdem ist in über 50% der Fälle die genetische Ursache unbekannt und die Pathogenese von Kraniosynostosen noch nicht vollständig geklärt. Ziel dieser Arbeit war es neue genetische Ursachen bei Kraniosynostose Patienten zu identifizieren und so zur Aufklärung der Pathogenese beizutragen. Es wurde die genomische DNA von 83 Patienten molekulargenetisch durch Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) oder durch ein speziell entworfenes Next Generation Sequencing (NGS) Genpanel untersucht. Bei 30% der Patienten konnte eine potentiell pathogene Veränderung identifiziert werden. Davon waren 23% chromosomale Aberrationen wie unbalancierte Translokationen, isolierte interstitielle Verluste und ein Zugewinn an genomischen Material. Bei zwei Patienten wurden unbalancierte Translokationen mit partieller 5q Trisomie nachgewiesen. Das Gen MSX2 liegt innerhalb des duplizierten Bereichs, sodass möglicherweise eine MSX2 Überexpression vorliegt. Für ein normales Schädelwachstum ist jedoch die richtige Menge an MSX2 kritisch. Des Weiteren wurde eine partielle Deletion von TCF12 detektiert, die in einer Haploinsuffizienz von TCF12 resultiert. TCF12 Mutationen sind mit Koronarnahtsynosten assoziiert. In einem anderen Fall lag das Gen FGF10 innerhalb der duplizierten 5p15.1-p12 Region. Das Gen kodiert für einen Liganden des FGF Signalwegs und wurde bisher noch nicht mit Kraniosynostose assoziiert. Aufgrund dessen wurden Analysen im Tiermodell Danio rerio durchgeführt. Eine simulierte Überexpression durch Injektion der fgf10a mRNA in das 1-Zell Stadium führte zu schweren Gehirn-, Herz- und Augendefekten. Mittels NGS wurden 77% der potentiell pathogenen genetischen Veränderungen identifiziert. Hierfür wurde in dieser Arbeit ein Genpanel erstellt, das 68 Gene umfasst. Es wurden sowohl bekannte Kraniosynostose- als auch Kandidaten-Gene sowie Gene, die mit der Ossifikation assoziiert sind, in die Analyse eingeschlossen. Das Genpanel wurde durch die Sequenzierung von fünf Kontrollproben mit bekannten Mutationen erfolgreich validiert. Anschließend wurde die genomische DNA von 66 Patienten analysiert. Es konnten 20 (potentiell) pathogene Varianten identifiziert werden. Neben bereits bekannten Mutationen in den Genen FGFR1, FGFR2, FGFR3 und TWIST1, konnten zusätzlich 8 neue, potentiell pathogene Varianten in den Genen ERF, MEGF8, MSX2, PTCH1 und TCF12 identifiziert werden. Die Ergebnisse dieser Arbeit tragen dazu bei das Mutationsspektrum dieser Gene zu erweitern. Bei zwei der Varianten handelte es sich um potentielle Spleißvarianten. Für diese konnte in einem in vitro Spleißsystem gezeigt werden, dass sie eine Änderung des Spleißmusters bewirken. Der Nachweis von zwei seltenen Varianten in den Genen FGFR2 und HUWE1 hat außerdem dazu beigetragen die Pathogenität dieser spezifischen Varianten zu bekräftigen. Eine Variante in POR, die aufgrund bioinformatischer Analysen als potentiell pathogen bewertet wurde, wurde nach der Segregationsanalyse als wahrscheinlich benigne eingestuft. Zusammenfassend konnten bei etwa einem Drittel der Patienten, die mit dem NGS Genpanel analysiert wurden, eine genetische Ursache identifiziert werden. Dieses Genpanel stellt somit ein effizientes diagnostisches Tool dar, das zukünftig in der genetischen Routine-Diagnostik von Kraniosynostose-Patienten eingesetzt werden kann. Die Ergebnisse dieser Arbeit zeigen, dass sowohl eine Untersuchung auf CNVs als auch auf Sequenzänderungen bei Kraniosynostose Patienten sinnvoll ist. N2 - Cranial bones are formed by intramembranous ossification. During development, the cranial bones are separated by fibrous sutures, which function as bone growth sites and therefore, the cranial sutures need to remain patent to allow the expansion of the skull during brain development. Thus, there must be a balance of cell proliferation and differentiation within the suture. This complex process requires a tight regulation of gene expression and interacting signal pathways. Imbalances or dysfunction of the involved factors can result in abnormal skull growth. One of the most common congenital craniofacial disorders by affecting approximately one in 2500 newborns is craniosynostosis. It is defined as the premature ossification of one or more calvarial sutures. Compensatory growth of the skull leads to a characteristic dysmorphic cranial vault and facial asymmetry. Premature ossification of the cranial sutures can occur either as isolated malformation or as part of a syndrome. Isolated craniosynostoses are more frequent, nevertheless, 25-30% of all cases are syndromic craniosynostoses with more than 150 syndromes reported. There is a high intra- and interfamilial variability and clinical overlap of the different syndromes. Environmental influences as well as genetic defects like mutations and chromosomal aberrations are known to cause craniosynostosis. So far genetic causes have been identified mainly for syndromic craniosynostoses, i.e. mutations in FGFR2, FGFR3, TWIST1, and EFNB1. Furthermore, chromosomal rearrangements like i.e. partial monosomy of 7p, 9p, and 11p as well as partial trisomy of 5q, 13q, and 15q, have been reported in 11-15% of the syndromic craniosynostosis cases. However, in more than 50% of the cases the underlying genetic cause remains unknown. Furthermore, the pathogenesis of craniosynostoses is still not fully understood. In this project 83 craniosynostosis patients were analysed either by microarray-based comparative genomic hybridisation (array-CGH) or gene panel based next generation sequencing (NGS) to further investigate the pathogenesis of craniosynostosis. In a total of 30% of the patients a potential genetic cause was identified. Among those 23% had chromosomal rearrangements which are likely to cause the observed phenotypes, i.e. unbalanced translocations affecting several genes as well as interstitial deletions and an isolated duplication have been detected. Two patients had unbalanced translocations with partial 5q trisomies encompassing MSX2. MSX2 gene dosage is critical for normal growth of the cranial bone plates as loss-of-function mutations lead to delayed and incomplete ossification of the parietal bones. Furthermore, we identified a partial TCF12 deletion which is likely to result in TCF12 haploinsufficiency. TCF12 mutations frequently lead to premature fusion of the coronal sutures, although its pathogenesis is still not fully understood. In another case isolated duplication of 5p15.1-p12 includes FGF10 which is a known ligand of the FGF signalling pathway. So far, no association of FGF10 with craniosynostosis has been made. To investigate its potential role during development and in the pathogenesis of craniosynostosis functional experiments were performed in Danio rerio, an animal model for craniosynostosis. Simulation of fgf10a overexpression by injection of fgf10a RNA at 1-cell stage resulted in severe anomalies of the brain, heart and eyes. In addition, 77% of the identified genetic causes were detected by NGS. For this study a gene panel was designed comprising 68 genes of known and candidate craniosynostosis genes as well as genes associated with bone development. Performance of the NGS gene panel was validated by sequencing five control patients with known mutations. Subsequently, genomic DNA of 66 patients was analysed by the designed craniosynostosis panel. 20 (potential) pathogenic variants were detected. Although, in most of the cases hot spot sequencing of one or more common craniosynostosis genes was performed prior to including the patients in the study, we determined 9 known mutations in the genes FGFR1, FGFR2, FGFR3, and TWIST1. In addition, 8 novel, potentially disease-causing variants in the genes ERF, MEGF8, MSX2, PTCH1, and TCF12 were identified. This work contributed to extend the mutational spectrum within those genes. Two of those variants were predicted to affect splice sites. Analysis by an in vitro splice assay revealed that those variants result in aberrant splicing. Furthermore, the detection of two rare variants of FGFR2 and HUWE1 adds support to their pathogenicity. An additional variant within POR had to be classified as likely benign after segregation analysis. Overall, in nearly one third of the analysed cases an underlying genetic cause could be identified by the designed gene panel. Thus, the NGS panel presents as an efficient tool for genetic diagnostics of craniosynostoses. The data of this work clearly show both copy number variant and single nucleotide variant analysis should be considered in genetic diagnostics of craniosynostosis patients. KW - Kraniosynostose KW - Genetik KW - Next Generation Sequencing (NGS) KW - Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175181 ER - TY - THES A1 - Njovu, Henry Kenneth T1 - Patterns and drivers of herbivore diversity and invertebrate herbivory along elevational and land use gradients at Mt. Kilimanjaro, Tanzania T1 - Muster und Determinanten von Herbivorendiversität, von Herbivorieraten durch Invertebraten sowie die Diversität und Gesamtbiomasse von Säugetieren entlang von Höhen- und Landnutzungsgradienten am Kilimandscharo (Tansania) untersucht N2 - This thesis elucidates patterns and drivers of invertebrate herbivory, herbivore diversity, and community-level biomass along elevational and land use gradients at Mt. Kilimanjaro, Tanzania. Chapter I provides background information on the response and predictor variables, study system, and the study design. First, I give an overview of the elevational patterns of species diversity/richness and herbivory published in the literature. The overview illuminates existing debates on elevational patterns of species diversity/richness and herbivory. In connection to these patterns, I also introduce several hypotheses and mechanisms put forward to explain macroecological patterns of species richness. Furthermore, I explain the main variables used to test hypotheses. Finally, I describe the study system and the study design used. Chapter II explores the patterns of invertebrate herbivory and their underlying drivers along extensive elevational and land use gradients on the southern slopes of Mt. Kilimanjaro. I recorded standing leaf herbivory from leaf chewers, leaf miners and gall-inducing insects on 55 study sites located in natural and anthropogenic habitats distributed from 866 to 3060 meters above sea level (m asl) on Mt. Kilimanjaro. Standing leaf herbivory was related to climatic variables [mean annual temperature - (MAT) and mean annual precipitation - (MAP)], net primary productivity (NPP) and plant functional traits (leaf traits) [specific leaf area (SLA), carbon to nitrogen ratio (CN), and nitrogen to phosphorous ratio (NP)]. Results revealed an unimodal pattern of total leaf herbivory along the elevation gradient in natural habitats. Findings also revealed differences in the levels and patterns of herbivory among feeding guilds and between anthropogenic and natural habitats. Changes in NP and CN ratios which were closely linked to NPP were the strongest predictors of leaf herbivory. Our study uncovers the role of leaf nutrient stoichiometry and its linkages to climate in explaining the variation in leaf herbivory along climatic gradients. Chapter III presents patterns and unravels direct and indirect effects of resource (food) abundance (NPP), resource (food) diversity [Functional Dispersion (FDis)], resource quality (SLA, NP, and CN rations), and climate variables (MAT and MAP) on species diversity of phytophagous beetles. Data were collected from 65 study sites located in natural and anthropogenic habitats distributed from 866 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Sweep net and beating methods were used to collect a total of 3,186 phytophagous beetles representing 21 families and 304 morphospecies. Two groups, weevils (Curculionidae) and leaf beetles (Chrysomelidae) were the largest and most diverse families represented with 898 and 1566 individuals, respectively. Results revealed complex (bimodal) and dissimilar patterns of Chao1-estimated species richness (hereafter referred to as species diversity) along elevation and land use gradients. Results from path analysis showed that temperature and climate-mediated changes in NPP had a significant positive direct and indirect effect on species diversity of phytophagous beetles, respectively. The results also revealed that the effect of NPP (via beetles abundance and diversity of food resources) on species diversity is stronger than that of temperature. Since we found that factors affecting species diversity were intimately linked to climate, I concluded that predicted climatic changes over the coming decades will likely alter the species diversity patterns which we observe today. Chapter IV presents patterns and unravels the direct and indirect effects of climate, NPP and anthropogenic disturbances on species richness and community-level biomass of wild large mammals which represent endothermic organisms and the most important group of vertebrate herbivores. Data were collected from 66 study sites located in natural and anthropogenic habitats distributed from 870 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Mammals were collected using camera traps and used path analysis to disentangle the direct and indirect effects of climatic variables, NPP, land use, land area, levels of habitat protection and occurrence of domesticated mammals on the patterns of richness and community-level biomass of wild mammals, respectively. Results showed unimodal patterns for species richness and community-level biomass of wild mammals along elevation gradients and that the patterns differed depending on the type of feeding guild. Findings from path analysis showed that net primary productivity and levels of habitat protection had a strong direct effect on species richness and community-level biomass of wild mammals whereas temperature had an insignificant direct effect. Findings show the importance of climate-mediated food resources in determining patterns of species richness of large mammals. While temperature is among key predictors of species richness in several ectotherms, its direct influence in determining species richness of wild mammals was insignificant. Findings show the sensitivity of wild mammals to anthropogenic influences and underscore the importance of protected areas in conserving biodiversity. In conclusion, despite a multitude of data sets on species diversity and ecosystem functions along broad climatic gradients, there is little mechanistic understanding of the underlying causes. Findings obtained in the three studies illustrate their contribution to the scientific debates on the mechanisms underlying patterns of herbivory and diversity along elevation gradients. Results present strong evidence that plant functional traits play a key role in determining invertebrate herbivory and species diversity along elevation gradients and that, their strong interdependence with climate and anthropogenic activities will shape these patterns in future. Additionally, findings from path analysis demonstrated that herbivore diversity, community-level biomass, and herbivory are strongly influenced by climate (either directly or indirectly). Therefore, the predicted climatic changes are expected to dictate ecological patterns, biotic interactions, and energy and nutrient fluxes in terrestrial ecosystems in the coming decades with stronger impacts probably occurring in natural ecosystems. Furthermore, findings demonstrated the significance of land use effects in shaping ecological patterns. As anthropogenic pressure is advancing towards more pristine higher elevations, I advocate conservation measures which are responsive to and incorporate human dimensions to curb the situation. Although our findings emanate from observational studies which have to take several confounding factors into account, we have managed to demonstrate global change responses in real ecosystems and fully established organisms with a wide range of interactions which are unlikely to be captured in artificial experiments. Nonetheless, I recommend additional experimental studies addressing the effect of top-down control by natural enemies on herbivore diversity and invertebrate herbivory in order to deepen our understanding of the mechanisms driving macroecological patterns along elevation gradients.   N2 - In dieser Dissertation werden Muster und Determinanten von Herbivorendiversität, von Herbivorieraten durch Invertebraten sowie die Diversität und Gesamtbiomasse von Säugetieren entlang von Höhen- und Landnutzungsgradienten am Kilimandscharo (Tansania) untersucht. Kapitel I liefert Hintergrundinformationen zu den betrachteten Variablen, dem Untersuchungssystem und dem generellen Studiendesign: Zuerst fasse ich den aktuellen Kenntnisstand über die Muster des Artenreichtums und der Herbivorie entlang von Höhengradienten zusammen und erläutere in diesem Zusammenhang verschiedene Hypothesen, die zur Erklärung von Gradienten des Artenreichtum herangezogen werden. Ich erkläutere verschiedene Variablen, die zum Testen dieser Hypothesen erhoben wurden und stelle dar, wie diese den Artenreichtum, die Herbivorieraten und die Biomasse beeinflussen könnten. Anschließend beschreibe ich das Untersuchungssystem, sowie das generelle Design der Studie. In Kapitel II werden die Muster und Determinanten der Invertebratenherbivorie entlang von Höhen- und Landnutzungsgradienten an den südlichen Hängen des Kilimandscharos präsentiert. Auf insgesamt 55 Untersuchungsflächen, die sowohl natürliche als auch anthropogen genutzte Habitate am Kilimandscharo in Höhenlagen zwischen 866 und 3060 Meter über Normalnull (m ü. NN) umfassten, wurden die Herbivorieraten ektophager, minierender und gallbildener Insekten an Blättern erfasst. Die Blattherbivorie war sowohl mit klimatischen Variablen [Jahresmitteltemperatur und mittlere Jahresniederschlagsmenge], der Nettoprimärproduktivität (NPP) und mit funktionellen Blattmerkmalen von Pflanzen [spezifische Blattfläche (SLA), Kohlenstoff (C) / Stickstoff (N)-Verhältnis, sowie N / Phosphor (P)-Verhältnis] assoziiert. Die Gesamtherbivorie zeigte eine unimodale Verteilung über den Höhengradienten, wurde aber sowohl von der Herbivorengilde, als auch vom Habitattyp (natürlich versus anthropogen) beeinflusst. Das C/N-Verhältnis von Blättern war die stärkste Determinante der Blattherbivorie und wurde selbst stark durch die NPP bestimmt. Herbivorieraten sanken mit steigendem C/N-Verhältnis. Das C/N Verhältnis nahm mit steigender NPP zu.- Letztere konnte fast vollständig durch Änderungen der mittleren Jahrestemperatur (MAT) und des Jahresniederschlags (MAP) entlang des Höhengradienten erklärt werden. Damit zeigt unsere Studie, dass sich durch klimatische Faktoren und Energie, welche ihrerseits die Blattchemie beeinflussen und so Variationen in der Blattherbivorie entlang großer Klimagradienten ergeben. In Kapitel III werden die Muster im Artenreichtum phytophager Käfer entlang der Höhen- und Landnutzungsgradienten untersucht und die direkten und indirekten Effekte von klimatischen Faktoren (MAT, MAP), NPP und funktionellen Pflanzenmerkmalen (funktionelle Dispersion, SLA, C/N - und N/P - Verhältnisse) auf diese Muster analysiert. Die entsprechenden Daten wurden auf 65 Untersuchungsflächen, die sowohl natürliche als auch anthropogene Habitate entlang eines Höhengradienten am Kilimandscharo von 866 bis 4550 m ü. NN abdeckten, erhoben. Mittels Kescher wurden insgesamt 3186 phytophage Käfer aus 21 Familien gesammelt und in 304 Morphospezies eingeteilt. Der Artenreichtum phytophager Käfer zeigte eine komplexe, zweigipflige Verteilung entlang der Höhen- und Landnutzungsgradienten. Eine Pfadanalyse ergab, dass sowohl die MAT, als auch NPP positiven direkte bzw. indirekte Effekt auf die Artendiversität phytophager Käfer hatte. Die NPP war positiv mit der funktionellen Dispersion von Blattmerkmalen, ein Maß für die Diversität der Nahrungsressourcen, korreliert. Letztere hatte einen positiven Effekt auf die Diversität der Käfer. Die starken direkten und indirekten Effekte von Klima auf die Diversität und Abundanz von phytophagen Käfern, lassen vermuten dass der Klimawandel in den nächsten Dekaden großen Änderungen der Struktur von phytophagen Käfergemeinschaften bewirken wird. In Kapitel IV untersuchen wir den Effekt von Klima, NPP und anthropogener Störung auf den Artenreichtum und die Gesamtbiomasse von Großwild. Dazu wurden auf 66 Untersuchungsflächen, welche natürliche und anthropogene Habitate in Höhenstufen zwischen 870 und 4550m ü. NN umfassten, Daten zum Artenreichtum un der Abundanz von Großwild mittels Kamerafallen erfasst. Mittels einer Pfadanalyse wurden die direkten und indirekten Effekte von klimatischen Variablen, NPP, Landnutzung, Größe und Schutzstatus der Flächen, sowie der Präsenz von domestizierten Säugetieren auf den Artenreichtum und die Biomasse von Großwild untersucht. Artenreichtum und Gesamtbiomasse dieser endothermen Organismen zeigten eine unimodale Verteilung über den Höhengradienten. Verschiedene Nahrungsgilden zeigten unterschiedliche Muster. Es konnte gezeigt werden, dass NPP und der Schutzstatus der Fläche, aber nicht die Temperatur einen direkten, positiven Einfluss auf den Artenreichtum und die Gesamtbiomasse des Großwildes hatte. Die vom Klima abhängige Nahrungsressourcenverfügbarkeit ist also eine wichtige Determinante im Artenreichtum von Großwild. Die Temperatur hingegen, die den Artenreichtum verschiedener ektothermer Organismen entscheidend prägt, hatte keinen direkten Einfluss auf den Artenreichtum des Großwildes Dafür reagiert das Großwild besonders sensibel auf anthropogene Einflüsse, was wiederum die Wichtigkeit von Schutzgebieten unterstreicht. Obwohl die Muster im Artenreichtum und in Ökosystemfunktionen entlang großer klimatischer Gradienten bereits gut dokumentiert sind, ist das Wissen über die zu Grunde liegenden Prozesse nach wie vor unzureichend. Mit meinen drei Studien über die Muster und Determinanten der Herbivorendiversität, der Herbivorieraten und der Großwildbiomasse trage ich somit zur Verbesserung des mechanistischen Verständnisses solcher makroökologischer Muster bei. Wie die Pfadanalysen zeigten, wurden sowohl der Artenreichtum die Biomasse als auch ökologische Prozesse direkt oder indirekt vom Klima beeinflusst. Es ist somit zu erwarten, dass der vorhergesagte Klimawandel ökologische Muster, biotische Interaktionen, Energie- und Nährstoffkreisläufe in terrestrischen Ökosystemen wesentlich umstrukturieren wird, wobei natürliche Systeme wahrscheinlich besonders sensibel auf den Klimawandel reagieren werden. Meine Ergebnisse demonstrieren auch den Einfluss von Landnutzung auf Artenreichtum und ökologische Prozesse. Da der anthropogene Druck auf die natürlichen Ökosysteme des Kilimandscharos immer weiter zunimmt, sollten objektive Biodiversitätsmaße implementiert werden mit denen man Veränderungen in den Ökosystemen und in Ökosystemldienstleistungen schnell detektieren kann. Meine Ergebnisse basieren auf Beobachtungsdaten, die von bestimmten Nebenfaktoren im Feld beeinflusst werden können. Dennoch ist es mir gelungen mit korrelativen Methoden, Organismen in ihrem biotischen und abiotischen Interaktionsumfeld zu untersuchen – ein Szenario, welches in einem rein experimentellen Aufbau in dieser Form wahrscheinlich nicht geschaffen werden kann. Über weiterführende Experimente könnte jedoch zum Beispiel der Einfluss von Prädatoren auf die Herbivorendiversität und Herbivorieraten quantifiziert werden, welches unser Verständnis über die Determinanten makroökologischer Muster noch vertiefen würde.   KW - Species richness KW - Invertebrate herbivory KW - Leaf traits KW - drivers and patterns of diversity and herbivory KW - Patterns and drivers of invertebrate herbivory KW - Patterns and drivers of species diversity of phytophagous beetles KW - Patterns and drivers of species richness and community biomass of large mammals Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172544 ER - TY - JOUR A1 - Duque, Laura A1 - Poelman, Erik H. A1 - Steffan-Dewenter, Ingolf T1 - Plant-mediated effects of ozone on herbivores depend on exposure duration and temperature JF - Scientific Reports N2 - Abiotic stress by elevated tropospheric ozone and temperature can alter plants’ metabolism, growth, and nutritional value and modify the life cycle of their herbivores. We investigated how the duration of exposure of Sinapis arvensis plants to high ozone and temperature levels affect the life cycle of the large cabbage white, Pieris brassicae. Plants were exposed to ozone-clean (control) or ozone-enriched conditions (120 ppb) for either 1 or 5 days and were afterwards kept in a greenhouse with variable temperature conditions. When given the choice, P. brassicae butterflies laid 49% fewer eggs on ozone-exposed than on control plants when the exposure lasted for 5 days, but showed no preference when exposure lasted for 1 day. The caterpillars took longer to hatch on ozone-exposed plants and at lower ambient temperatures. The ozone treatment had a positive effect on the survival of the eggs. Ozone decreased the growth of caterpillars reared at higher temperatures on plants exposed for 5 days, but not on plants exposed for 1 day. Overall, longer exposure of the plants to ozone and higher temperatures affected the life cycle of the herbivore more strongly. With global warming, the indirect impacts of ozone on herbivores are likely to become more common. KW - Ecology KW - Environmental impact Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202805 VL - 9 ER - TY - THES A1 - Letschert, Sebastian T1 - Quantitative Analysis of Membrane Components using Super-Resolution Microscopy T1 - Quantitative Analyse von Membrankomponenten mittels hochauflösender Fluoreszenzmikroskopie N2 - The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules. An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed. Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and –lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates. In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60–1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed. N2 - Die Plasmamembran gehört zu den am meisten untersuchten, gleichzeitig aber auch zu den komplexesten, vielfältigsten und am wenigsten verstandenen biologischen Strukturen. Ihre Funktion wird nicht nur durch die molekulare Zusammensetzung bestimmt, sondern auch durch die räumliche Anordnung ihrer Bestandteile. Selbst nach Jahrzehnten intensiver Forschung und der Veröffentlichung dutzender Membranmodelle und Theorien bleibt die genaue strukturelle Organisation der Plasmamembran ein Rätsel. Moderne Bildgebungsverfahren wie etwa die hochauflösende Fluoreszenzmikroskopie gehören mittlerweile zu den effizientesten Techniken der Lebenswissenschaften und werden immer öfter verwendet, um die räumliche Anordnung als auch die Anzahl von Biomolekülen in fixierten und lebenden Zellen zu studieren. Im Rahmen dieser Arbeit wurde die hochauflösende Mikroskopie-Methode dSTORM (direct stochastic optical reconstruction microscopy) angewendet, um die räumliche Verteilung von Membranmolekülen mit annähernd molekularer Auflösung zu untersuchen. Schwerpunkte dieser Arbeit sind dabei verschiedene Präparations- und Färbemethoden für die mikroskopische Untersuchung von Zellmembranen sowie lokalisationsbasierte quantitative Analysemethoden von Membranmolekülen. Eine Voraussetzung für die räumliche als auch quantitative Analyse von Membranmolekülen ist die Vermeidung von Photoschalt-Artefakten in rekonstruierten Lokalisationsmikroskopie-Bildern. Um dies genauer zu demonstrieren, wurden die Auswirkungen von Anregungsintensität, Markierungsdichte und verändertem Photoschalten auf die räumliche Verteilung von Proteinen der Plasma- und Mitochondrienmembran in dSTORM-Bildern analysiert. Es wird gezeigt, dass eine dicht markierte Plasmamembran in Kombination mit ungeeigneten Photoschaltraten zu artifiziellen Clustern in der Membran führt. Es sind vor Allem oft die Projektionen dreidimensionaler Membranstrukturen wie etwa Mikrovilli und Vesikel dafür verantwortlich, dass lokale Unterschiede in der Lokalisationsdichte entstehen, wodurch unter Umständen Bildartefakte generiert werden können. Darüber hinaus werden alternative Mikroskopie-Methoden und Möglichkeiten, Artefakte in Einzelmolekül-Lokalisationsmikroskopie-Bildern zu verhindern, präsentiert und ausführlich diskutiert. Ein weiteres zentrales Thema dieser Arbeit ist die räumliche Anordnung von glykosylierten Membranmolekülen. Es wird demonstriert, wie ein bioorthogonales chemisches Reportersystem bestehend aus modifizierten Monosacchariden und organischen Fluorophoren für die spezifische Markierung von Membran-assoziierten Glykoproteinen und –lipiden eingesetzt werden kann. Mittels dSTORM wird gezeigt, dass die Verteilung von Glykanen in der Plasmamembran unterschiedlicher Zelllinien homogen und frei von Clustern ist. Des Weiteren zeigt eine quantitative Analyse, dass sich in etwa fünf Millionen Glykane auf einer einzigen Zelle befinden. Die Ergebnisse demonstrieren, dass die Kombination aus metabolisch markierten Zielmolekülen, Click-Chemie und Einzelmolekül-Lokalisationsmikroskopie effizient genutzt werden kann, um Glykokonjugate auf Zelloberflächen zu untersuchen. In einem dritten Projekt wurde dSTORM zur Untersuchung von Rezeptormolekülen auf Krebszellen verwendet. Die Expression dieser Oberflächenproteine ist so gering, dass sich nur wenige Moleküle auf einer Zelle befinden, die jedoch als Zielmoleküle in der personalisierten Immuntherapie dienen könnten. Dafür wurden primäre Tumorzellen aus dem Knochenmark von Patienten, die am Multiplen Myelom erkrankt sind, auf die Expression des CD19-Oberflächenproteins als potentielles Ziel für CAR-modifizierte T-Zellen (chimeric antigen receptor) untersucht. Es wird gezeigt, dass sich, abhängig vom untersuchten Patienten, auf einer Zelle 60 bis 1600 CD19-Moleküle befinden. Funktionale in-vitro-Experimente demonstrieren, dass weniger als 100 CD19 Moleküle ausreichen, um CD19-CAR-T-Zellen zu aktivieren. Diese Ergebnisse werden mit Durchflusszytometrie-Daten verglichen und die wichtige Rolle von Lebendzellfärbung und geeigneten Kontrollexperimenten wird diskutiert. KW - Fluoreszenzmikroskopie KW - Quantifizierung KW - Polysaccharide KW - Immuntherapie KW - Antigen CD19 KW - super-resolution fluorescence microscopy KW - dSTORM KW - click chemistry KW - plasma membrane organization KW - localization microscopy KW - artifacts KW - Hochauflösende Fluoreszenzmikroskopie KW - Plasmamembranorganisation KW - Click Chemie KW - Glykane Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162139 ER - TY - JOUR A1 - Hickl, Oskar A1 - Heintz-Buschart, Anna A1 - Trautwein-Schult, Anke A1 - Hercog, Rajna A1 - Bork, Peer A1 - Wilmes, Paul A1 - Becher, Dörte T1 - Sample preservation and storage significantly impact taxonomic and functional profiles in metaproteomics studies of the human gut microbiome JF - Microorganisms N2 - With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at −80 °C should be chosen wherever possible. KW - proteomics KW - metaproteomics KW - metagenomics KW - microbiome KW - microbiota KW - flash freezing KW - RNAlater KW - sample storage Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195976 SN - 2076-2607 VL - 7 IS - 9 ER - TY - JOUR A1 - Thölken, Clemens A1 - Thamm, Markus A1 - Erbacher, Christoph A1 - Lechner, Marcus T1 - Sequence and structural properties of circular RNAs in the brain of nurse and forager honeybees (Apis mellifera) JF - BMC Genomics N2 - Background The honeybee (Apis mellifera) represents a model organism for social insects displaying behavioral plasticity. This is reflected by an age-dependent task allocation. The most protruding tasks are performed by young nurse bees and older forager bees that take care of the brood inside the hive and collect food from outside the hive, respectively. The molecular mechanism leading to the transition from nurse bees to foragers is currently under intense research. Circular RNAs, however, were not considered in this context so far. As of today, this group of non-coding RNAs was only known to exist in two other insects, Drosophila melanogaster and Bombyx mori. Here we complement the state of circular RNA research with the first characterization in a social insect. Results We identified numerous circular RNAs in the brain of A. mellifera nurse bees and forager bees using RNA-Seq with exonuclease enrichment. Presence and circularity were verified for the most abundant representatives. Back-splicing in honeybee occurs further towards the end of transcripts and in transcripts with a high number of exons. The occurrence of circularized exons is correlated with length and CpG-content of their flanking introns. The latter coincides with increased DNA-methylation in the respective loci. For two prominent circular RNAs the abundance in worker bee brains was quantified in TaqMan assays. In line with previous findings of circular RNAs in Drosophila, circAmrsmep2 accumulates with increasing age of the insect. In contrast, the levels of circAmrad appear age-independent and correlate with the bee's task. Its parental gene is related to amnesia-resistant memory. Conclusions We provide the first characterization of circRNAs in a social insect. Many of the RNAs identified here show homologies to circular RNAs found in Drosophila and Bombyx, indicating that circular RNAs are a common feature among insects. We find that exon circularization is correlated to DNA-methylation at the flanking introns. The levels of circAmrad suggest a task-dependent abundance that is decoupled from age. Moreover, a GO term analysis shows an enrichment of task-related functions. We conclude that circular RNAs could be relevant for task allocation in honeybee and should be investigated further in this context. KW - circRNA KW - circular transcriptome sequencing KW - honeybee KW - brain KW - neuronal KW - Methylation KW - CpG KW - alternative splicing KW - behavioral plasticity Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241302 VL - 20 ER - TY - JOUR A1 - Dechaud, Corentin A1 - Volff, Jean-Nicolas A1 - Schartl, Manfred A1 - Naville, Magali T1 - Sex and the TEs: transposable elements in sexual development and function in animals JF - Mobile DNA N2 - Transposable elements are endogenous DNA sequences able to integrate into and multiply within genomes. They constitute a major source of genetic innovations, as they can not only rearrange genomes but also spread ready-to-use regulatory sequences able to modify host gene expression, and even can give birth to new host genes. As their evolutionary success depends on their vertical transmission, transposable elements are intrinsically linked to reproduction. In organisms with sexual reproduction, this implies that transposable elements have to manifest their transpositional activity in germ cells or their progenitors. The control of sexual development and function can be very versatile, and several studies have demonstrated the implication of transposable elements in the evolution of sex. In this review, we report the functional and evolutionary relationships between transposable elements and sexual reproduction in animals. In particular, we highlight how transposable elements can influence expression of sexual development genes, and how, reciprocally, they are tightly controlled in gonads. We also review how transposable elements contribute to the organization, expression and evolution of sexual development genes and sex chromosomes. This underscores the intricate co-evolution between host functions and transposable elements, which regularly shift from a parasitic to a domesticated status useful to the host. KW - Transposable element KW - Sex determination KW - Sexual development and function KW - Germline KW - piRNA KW - Sex chromosome Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202510 VL - 10 ER - TY - JOUR A1 - Streinzer, Martin A1 - Chakravorty, Jharna A1 - Neumayer, Johann A1 - Megu, Karsing A1 - Narah, Jaya A1 - Schmitt, Thomas A1 - Bharti, Himender A1 - Spaethe, Johannes A1 - Brockmann, Axel T1 - Species composition and elevational distribution of bumble bees (Hymenoptera, Apidae, Bombus Latreille) in the East Himalaya, Arunachal Pradesh, India JF - ZooKeys N2 - The East Himalaya is one of the world’s most biodiverse ecosystems. However, very little is known about the abundance and distribution of many plant and animal taxa in this region. Bumble bees are a group of cold-adapted and high elevation insects that fulfil an important ecological and economical function as pollinators of wild and agricultural flowering plants and crops. The Himalayan mountain range provides ample suitable habitats for bumble bees. Systematic study of Himalayan bumble bees began a few decades ago and the main focus has centred on the western region, while the eastern part of the mountain range has received little attention and only a few species have been verified. During a three-year survey, more than 700 bumble bee specimens of 21 species were collected in Arunachal Pradesh, the largest of the north-eastern states of India. The material included a range of species that were previously known from a limited number of collected specimens, which highlights the unique character of the East Himalayan ecosystem. Our results are an important first step towards a future assessment of species distribution, threat, and conservation. Clear elevation patterns of species diversity were observed, which raise important questions about the functional adaptations that allow bumble bees to thrive in this particularly moist region in the East Himalaya. KW - Alpine habitats KW - Apidae KW - conservation KW - global change KW - insect collection KW - pollination Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201937 VL - 851 ER - TY - THES A1 - Röser [geb. Aßmus], Benjamin T1 - SPRED2 (Sprouty-related EVH1 domain containing 2) reguliert die Autophagie in Kardiomyozyten T1 - SPRED2 (Sprouty-related EVH1 domain containing 2) regulates autophagy in cardiomyocytes N2 - Das Sprouty-related, EVH1 domain containing protein 2 (SPRED2) ist ein inhibitorisches, downstream von Ras wirkendes Protein des MAP-Kinase Signalwegs, welches entscheidenden Einfluss auf die Regulation von Proliferation, Expression von Proteinen und der zellulären Homöostase hat. Der kardiale Phänotyp von SPRED2- defizienten Mäusen zeigt nicht nur eine deutliche linksventrikuläre Hypertrophie, sondern auch eine erhöhte Fibrosierung des Herzgewebes. Zellulär wird die SPRED2- Defizienz durch die Akkumulation von vesikulären Strukturen innerhalb der Zelle, sowie eine markant erhöhte Anzahl von Vesikeln entlang der longitudinalen Reihen der Mitochondrien gekennzeichnet. Ziel dieser Arbeit war es, den Charakter dieser vesikulären Strukturen näher zu beleuchten und festzustellen, in welchem Zusammenhang die subzellulär veränderte Architektur mit der Hypertrophie der SPRED2-defizienten Tiere steht. Um diese Fragestellung zu beantworten, wurde zunächst nach einem vesikulären Degradationsmechanismus gesucht, der in SPRED2-/--Cardiomyocyten betroffen sein könnte. Die Macroautophagie, im folgenden Autophagie bezeichnet, ist ein solcher Degradationsmechanismus, bei dem selektiv langlebige Proteine und Zellorganellen abgebaut werden. Es konnten signifikante Veränderung der Protein-Level an Schlüsselpositionen der Autophagie identifiziert werden. Das Ubiquitin-aktivierende (E1) Enzym Homolog Atg7 sowie die Cystein-Protease Atg4B zeigen sich im SPRED2- KO deutlich reduziert. Ebenso Atg16L, das als essentieller Bestandteil des Atg5- Atg12-Atg16-Konjugationssystems bei der Konjugation von MAPLC3-II an das Phospholipid Phosphatidylethanolamin beteiligt ist. Die Autophagie-Rate als Verhältnis von konjugiertem zu unkonjugiertem MAPLC3 ist ebenfalls reduziert. Die Akkumulation der autophagischen Vesikel zeigt sich kongruent zu dem erhöhten Protein-Level der autophagischen Cargo-Rezeptoren SQSTM1 und NBR1, sowie des lysosomalen Markers CathepsinD. Außer der verringerten Autophagie-Rate zeigt sich in Einklang mit der Fibrosierung des Herzgewebes eine erhöht aktive Caspase-3 als Marker für Apoptose. Um die mitochondriale Integrität näher zu beleuchten, wurde die Menge an reaktiven Sauerstoffspezies (ROS) in Wildtyp und SPRED2-KO untersucht. Hierbei zeigte sich eine erhöhte Menge an ROS im KO, was ein Hinweis auf eine Beeinträchtigung der Mitochondrien darstellt. Letztlich wurde die Hypothese überprüft, ob ein gestörter Transport der Vesikel durch eine Beeinträchtigung der Motorproteine Dynein und Kinesin vorliegt. In der Tat zeigte sich die Aktivität der Dynein-ATPase verringert in der Abwesenheit von SPRED2. Diese Beobachtung wird durch die erhöhten Mengen des vSNARE-Proteins VTI1b unterstützt, was letztlich die Akkumulation der autophagischen Vesikel mit einer verringerten Fähigkeit zur Membranfusion und dem ineffizienteren Transport der Vesikel in Einklang bringt. Da die gesamten Experimente in einem globalen SPRED2-KO System durchgeführt wurden, können eventuelle Auswirkungen der beeinflussten hormonellen Situation der SPRED2-KO Tiere auf den Herzphänotyp nicht final ausgeschlossen werden. Um die genaue Wirkung einer SPRED2-Defizienz auf das Herzgewebe und das Herz als Organ zu untersuchen, wurde im Rahmen dieser Arbeit eine SPRED2- defiziente knockout Mauslinie mit konditionalem Potential generiert, die eine gesteuerte Deletion von SPRED2 im Herzgewebe erlaubt. N2 - The Sprouty-related, EVH1 domain containing protein 2 (SPRED2) is a MAP kinase signaling inhibitor working downstream of Ras. It has a critical influence on regulating proliferation, differentiation, expression of proteins and cellular hemostasis. The cardiac phenotype of SPRED2 deficient mice not only shows a significant left ventricular hypertrophy but also a hightened fibrosis of the heart tissue. On the cellular level the SPRED2 deficiency is marked by an accumulation of ventricular structures within the cell, as well as a decisive number of vesicles along the longitudinal rows of mitochondria. The aim of this work was to elucidate the properties of these vesicular structures and to determine in which context the subcellularly modified architecture and the hypertrophy of the SPRED2 deficient animals stand to each other. To answer this question, a protein degradation mechanism that could be changed within the SPRED2 deficient cardiomyocytes was identified. Macroautophagy, further called autophagy, is such a degradation mechanism, which degrades long-lived proteins and cell organelles. This work identified significant changes made to the protein level of key regulators of autophagy. The ubiquitin-activating (E1) enzyme homolog Atg7 as well as the cystein protease Atg4B are reduced in the SPRED2 KO. Similarly, Atg16L, which acts as an essential part of the Atg5-Atg12-Atg16 conjugation system in the process of conjugating MAPLC3 to the phospholipid phosphatidylethanolamine. The autophagic flux, as the relation between conjugated and unconjugated MAPLC3, is reduced in the knockout as well. The accumulation of autophagic vesicles is in accordance with the elevated protein levels of the cargo receptors SQSTM1 and NBR1 as well as the lysosomal marker CathepsinD. Besides the reduced autophagic flux there is an elevated protein level of activated caspase-3 as a marker of apoptosis. To further elucidate the mitochondrial integrity, the endogenous levels of reactive oxygen species were determined in wildtype and knockout individuals. It was shown that the SPRED2 knockout contains an elevated level of ROS which could be a sign of reduced mitochondrial survival. Finally, it was investigated whether the disturbed transport of vesicles was due to impaired motor protein efficiency. It was shown that the activity of the dynein ATPase was reduced when SPRED2 was absent. This observation is supported by the elevated levels of the vSNARE protein VTI1b, which connects the accumulation of autophagic vesicles with the reduced ability to membrane fusion and a less efficient transport of vesicles. The experiments of this work were conducted in a global SPRED2-KO system. Possible effects of the changed hormonal situation of the SPRED2 deficient animals to the heart phenotype cannot be excluded. For that reason a conditional SPRED2 knockout mouse line with conditional potential was created capable of further elucidating the effect of a SPRED2 deficiency to the heart. KW - Spred-Proteine KW - Autophagie KW - Herzmuskelzelle KW - Autophagozytose KW - Autophagosom KW - autophagocytosis KW - autophagosome KW - Kardiomyozyt KW - Vesikel KW - Lysosom Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-182700 ER - TY - THES A1 - Baig, Ayesha Anjum T1 - Studies on platelet interactions with the coagulation system and on modulators of platelet (hem)ITAM signaling in genetically modified mice T1 - Studien zur Thrombozyteninteraktion mit der Gerinnungskaskade und Modulation des (hem)ITAM Signalwegs in genetisch veränderaten Mäusen N2 - Activated platelets and coagulation jointly contribute to physiological hemostasis. However, pathological conditions can also trigger unwanted platelet activation and initiation of coagulation resulting in thrombosis and precipitation of ischemic damage of vital organs such as the heart or brain. The specific contribution of procoagulant platelets, positioned at the interface of the processes of platelet activation and coagulation, in ischemic stroke had remained uninvestigated. The first section of the thesis addresses this aspect through experiments conducted in novel megakaryocyte- and platelet-specific TMEM16F conditional KO mice (cKO). cKO platelets phenocopied defects in platelets from Scott Syndrome patients and had severely impaired procoagulant characteristics. This led to decelerated platelet-driven thrombin generation and delayed fibrin formation. cKO mice displayed prolonged bleeding times and impaired arterial thrombosis. However, infarct volumes in cKO mice were comparable to wildtype (WT) mice in an experimental model of ischemic stroke. Therefore, while TMEM16F-regulated platelet procoagulant activity is critical for hemostasis and thrombosis, it is dispensable for cerebral thrombo-inflammation in mice. The second section describes the generation and initial characterization of a novel knockin mouse strain that expresses human coagulation factor XII (FXII) instead of endogenous murine FXII. These knockin mice had normal occlusion times in an experimental model of arterial thrombosis demonstrating that human FXII is functional in mice. Therefore, these mice constitute a valuable tool for testing novel pharmacological agents against human FXII – an attractive potential target for antithrombotic therapy. Glycoprotein (GP)VI and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represent a major pathway for platelet activation. The last section of the thesis provides experimental evidence for redundant functions between the two members of the Grb2 family of adapter proteins - Grb2 and Gads that lie downstream of GPVI and CLEC-2 stimulation. In vitro and in vivo studies in mice deficient in both Grb2 and Gads (DKO) revealed that DKO platelets had defects in (hem)ITAM-stimulation-specific activation, aggregation and signal transduction that were more severe than the defects observed in single Grb2 KO or Gads KO mice. Furthermore, the specific role of these adapters downstream of (hem)ITAM signaling was essential for maintenance of hemostasis but dispensable for the known CLEC-2 dependent regulation of blood-lymphatic vessel separation. N2 - Aktivierte Thrombozyten und die Gerinnungskaskade bilden gemeinsam die Grundlage der physiologischen Hämostase. Daneben können jedoch auch pathologische Bedingungen Thrombozytenaktivierung herbeiführen und die Gerinnungskaskade auslösen und somit zum Gefäßverschluss führen, was häufig ischämische Schäden lebenswichtiger Organe wie beispielsweise des Herzens oder des Gehirns verursachen kann. Prokoagulante Thrombozy-ten befinden sich an der Schnittstelle zwischen Thrombozytenaktivierung und der Gerin-nungskaskade, ihre Funktion bei der Pathogenese des ischämischen Schlaganfalls wurde jedoch bisher nicht im Detail untersucht. Der erste Teil dieser Doktorarbeit widmet sich dieser Fragestellung durch die Analyse von neu generierten konditionalen, Megakaryozyten- und Thrombozyten-spezifischen Tmem16f Knockout Mäusen. TMEM16F-defiziente Thrombozy-ten wiesen ähnliche Defekte wie die Thrombozyten von Scott-Syndrom-Patienten sowie stark beeinträchtigte prokoagulante Eigenschaften auf. Diese Defekte gingen mit signifikant verlangsamter thrombozytenabhängiger Thrombingenerierung und verzögerter Fibrinbildung einher. TMEM16F-Defizienz führte zu verlängerter Blutungszeit und beeinträchtigte in einem experimentellen Modell die Bildung arterieller Thromben. TMEM16F-defiziente Mäuse wiesen jedoch im Vergleich zu wildtypischen Mäusen keinerlei Unterschiede im experimentellen ischämischen Schlaganfall auf. TMEM16F-gesteuerte prokoagulante Thrombozytenfunktion ist demnach kritisch für Hämostase und Thrombose, während sie eine untergeordnete Rolle in zerebraler Thrombo-Inflammation spielt. Der zweite Teil dieser Arbeit befasst sich mit der Generierung und Erstbeschreibung einer neuen Mauslinie, welche den humanen Hageman-Faktor (FXII) anstelle des endogenen murinen FXII exprimiert. In den resultierenden Knock-in Mäusen war die Bildung okklusiver arterieller Thromben nach chemisch induzierter Gefäßverletzung nicht beeinträchtigt, was zeigt, dass der humane FXII im Maussystem voll funktionstüchtig ist. Somit können diese Mäuse in der Zukunft als ein wertvolles Werkzeug zum Testen neuer pharmakologischer Ansätze zur Herabsetzung der FXII-Aktivität eingesetzt werden, welche einen vielver-sprechenden Targets neuartiger antithrombotischer Behandlungsansätze darstellt. Die thrombozytären Rezeptoren Glykoprotein (GP)VI und C-type lectin-like receptor 2 (CLEC-2) lösen (hem)immunoreceptor tyrosine-based activation motif (ITAM)-gekoppelte Signalwege aus, welche eine eine Schlüsselrolle in der Thrombozytenaktivierung spielen. Der dritte Teil dieser Doktorarbeit liefert experimentelle Hinweise für überlappende Funkti-onen der Adapterproteine Grb2 und Gads in der (hem)ITAM-anhängigen Signalkaskade. In vitro und in vivo Studien zeigten, dass Grb2/Gads-doppeldefiziente Thrombozyten (hem)ITAM-spezifische Defekte in der Aktivierung, Aggregation und Signaltransduktion aufweisen, die im Vergleich zu einzeldefizienten Thrombozyten deutlich ausgeprägter sind und somit eine redundante Rolle der Adapterproteine offenbaren. Während Grb2 und Gads gemeinsam an der Aufrechterhaltung physiologischer Hämostase beteiligt sind, tragen sie nicht entscheidend zur bekannten CLEC-2-abhängigen Regulation der Trennung von Blut- und Lymphgefäßen bei. KW - Blutgerinnung KW - Thrombozyt KW - Signaltransduktion KW - Maus KW - Thrombosis KW - Thrombo-inflammation KW - TMEM16F KW - (hem)ITAM signaling Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164888 ER - TY - THES A1 - Schmitt [geb. Wolf], Karen T1 - Studies on the role of platelet serotonin in platelet function, hemostasis, thrombosis and stroke T1 - Studien zur Rolle des Serotonins aus Thrombozyten für die Thrombozytenfunktion, Hämostase, Thrombose und Schlaganfall N2 - Platelet activation and aggregation are important processes in hemostasis resulting in reduction of blood loss upon vessel wall injury. However, platelet activation can lead to thrombotic events causing myocardial infarction and stroke. A more detailed understanding of the regulation of platelet activation and the subsequent formation of thrombi is essential to prevent thrombosis and ischemic stroke. Cations, platelet surface receptors, cytoskeletal rearrangements, activation of the coagulation cas-cade and intracellular signaling molecules are important in platelet activation and thrombus formation. One such important molecule is serotonin (5 hydroxytryptamin, 5 HT), an indolamine platelet agonist, biochemically derived from tryptophan. 5 HT is secreted from the enterochromaffin cells into the gastrointestinal tract (GI) and blood. Blood borne 5 HT has been proposed to regulate hemostasis by acting as a vaso-constrictor and by triggering platelet signaling through 5 HT2A receptor. Although platelets do not synthetize 5 HT, they take it up from the blood and store it in their dense granules which are secreted upon platelet activation. To identify the molecu-lar composite of the 5 HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke, 5 HT transporter knock out mice (5Htt / ) were analyzed in different in vitro and in vivo assays and in a model of is-chemic stroke. In 5Htt / platelets, 5 HT uptake from the blood was completely abol-ished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein (GP) VI and C type lectin-like receptor 2 (CLEC 2) were reduced. These observed in vitro defects in 5Htt / platelets could be normalized by the addition of exogenous 5 HT. Moreover, reduced 5 HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt / mice. Surprisingly, in the transient middle cerebral artery occlusion model (tMCAO) of ischemic stroke 5Htt / mice showed near-ly normal infarct volumes and a neurological outcome comparable to control mice. Although secreted platelet 5 HT does not appear to play a crucial role in the devel-opment of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and thus plays an important role in thrombus stabilization. To further investigate the role of cations, granules and their contents and regulation of integrin activation in the process of thrombus formation, genetically modified mice were analyzed in the different in vivo thrombosis models. Whereas Tph1 / mice (lacking the enzyme responsible for the production of 5 HT in the periphery), Trpm7KI (point mu-tation in the kinase domain of Trpm7 channel, lacking kinase activity) and Unc13d / /Nbeal2 / mice (lacking α granules and the release machinery of dense granules) showed a delayed thrombus formation in vivo, MagT1y/ mice (lacking a specific Mg2+ transporter) displayed a pro thrombotic phenotype in vivo. Trpm7fl/fl Pf4Cre (lacking the non specific Mg2+ channel) and RIAM / mice (lacking a potential linker protein in integrin “inside out” signaling) showed no alterations in thrombus formation upon injury of the vessel wall. N2 - Thrombozytenaktivierung und Aggregation sind wichtige Schritte der Hämostase, die zur Reduktion des Blutverlustes bei Gefäßwandverletzung führen. Jedoch kann die Aktivierung von Thrombozyten zur Thrombose führen, wodurch Herzinfarkt und Schlaganfall entstehen kann. Ein besseres Verständnis der Regulierung der Throm-bozytenaktivierung und die darauf folgende Thrombusbildung sind notwendig, um Thrombose und Hirninfarkte zu vermeiden. Kationen, Thrombozy-ten Oberflächenrezeptoren, Zytoskelett Reorganisation, Aktivierung der Koagulati-onskaskade und intrazellulare Signalmoleküle sind wichtig in der Thrombozytenakti-vierung und Thrombusbildung. Solch ein wichtiges Molekül ist Serotonin (5 hydroxytryptamin, 5 HT), ein Indolamin Thrombozyten-Agonist, welcher aus Tryp-tophan synthetisiert wird. 5 HT wird aus den Enterochromaffinzellen in den Gastroin-testinaltrakt (GI) und das Blut abgegeben. 5 HT aus dem Blut wirkt als Regulator der Hämostase durch die Wirkung als Vasokonstriktor und die Auslösung der Throm-bozyten-Signalwege durch den 5 HT2A Rezeptor. Thrombozyten synthetisieren kein 5 HT, sondern nehmen es aus dem Blut auf und speichern es in den dichten Granu-la, die nach der Thrombozyten-Aktivierung freigesetzt werden. Um die molekulare Zusammensetzung des 5 HT Aufnahmesystems in Thrombozyten zu identifizieren und die Rolle des 5 HT aus Thrombozyten in Thrombose und ischämischem Schlag-anfalls zu klären, wurde eine 5 HT Transporter-defiziente Mauslinie (5Htt / ) in ver-schiedenen in vitro und in vivo Untersuchungen und im Model des ischämischen Schlaganfalls analysiert. In 5Htt / Thrombozyten ist die Aufnahme von 5 HT aus dem Blut vollständig geblockt und Agonisten-induzierter Ca2+ Fluss durch Speicher-abhängigen Ca2+ Einstrom (SOCE), Integrinaktivierung, Degranulierung und Aggre-gation abhängig von Glykoprotein (GP) VI und C type lectin-like receptor 2 (CLEC 2) waren reduziert. Diese in vitro beobachteten Defekte in 5Htt / Thrombozyten konnten durch Zugabe von 5 HT normalisiert werden. Zudem wurden reduzierte 5 HT Werte im Plasma, eine erhöhte Blutungszeit und die Bildung von instabilen Thromben ex vivo unter Fluss und in vivo in der abdominalen Aorta und der Carotis von 5Htt / Mäusen beobachtet. Überraschenderweise zeigten die 5Htt / Mäuse nach transientem Verschluss der A. cerebri media (tMCAO), einem Modell des ischämi-schen Schlaganfalls, ein normales Infarktvolumen und einen unveränderten neurolo-gischen Endzustand im Vergleich zu Kontrollmäusen. Obwohl sekretiertes 5 HT aus Thrombozyten keine wesentliche Rolle in der Entwicklung eines Reperfusionsscha-dens nach einem Schlaganfall spielt, ist es essentiell in der Verstärkung der zweiten Phase der Thrombozytenaktivierung durch SOCE und spielt eine wichtige Rolle in der Thrombusstabilität. Um die Rolle von Kationen, Granula und deren Bestandteile und der Regulierung in der Integrinaktivierung im Prozess der Thrombusbildung zu untersuchen, wurden genetisch veränderte Mäuse in den verschiedenen in vivo Thrombosemodellen ge-testet. Während Tph1 / Mäuse (denen das Enzym zur Produktion von 5 HT in der Peripherie fehlt), Trpm7KI (Punktmutation in der Kinasedomäne des Trpm7 Kanals, Fehlen der Kinase Aktivität) und Unc13d / /Nbeal2 / Mäuse (denen die α Granula und die Freisetzungsmaschinerie der dichten Granula fehlt) und keine oder eine verlang-samte Thrombusbildung zeigten, wiesen MagT1y/ Mäuse (denen der spezifische Mg2+ Transporter fehlt) einen prothrombotischen Phänotyp auf. Trpm7fl/fl Pf4Cre Mäuse (denen der nicht spezifische Mg2+ Kanal fehlt) und RIAM / Mäuse (denen ein potenti-elles Linker Protein im Integrin “inside out” Signal fehlt) zeigten keine Veränderung in der Thrombus Bildung nach Verletzung der Gefäßwand. KW - Biomedicine KW - Serotonin KW - Blutgerinnung Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134711 ER - TY - THES A1 - Gulve, Nitish T1 - Subversion of Host Genome Integrity by Human Herpesvirus 6 and \(Chlamydia\) \(trachomatis\) T1 - Störung der Integrität des Wirts Genoms durch das Human Herpesvirus 6 und \(Chlamydia\) \(trachomatis\) N2 - Ovarian cancer is one of the most common gynecological malignancies in the world. The prevalence of a microbial signature in ovarian cancer has been reported by several studies till date. In these microorganisms, Human herpesvirus 6 (HHV-6) and Chlamydia trachomatis (C.tr) are especially important as they have significantly high prevalence rate. Moreover, these pathogens are directly involved in causing DNA damage and thereby disrupting the integrity of host genome which is the underlying cause of any cancer. This study focuses on how the two pathogens, HHV-6 and C. trachomatis can affect the genome integrity in their individual capacities and thereby may drive ovarian epithelial cells towards transformation. HHV-6 has unique tendency to integrate its genome into the host genome at subtelomeric regions and achieve a state of latency. This latent virus may get reactivated during the course of life by stress, drugs such as steroids, during transplantation, pregnancy etc. The study presented here began with an interesting observation wherein the direct repeat (DR) sequences flanking the ends of double stranded viral genome were found in unusually high numbers in human blood samples as opposed to normal ratio of two DR copies per viral genome. This study was corroborated with in vitro data where cell lines were generated to mimic the HHV-6 status in human samples. The same observation of unusually high DR copies was found in these cell lines as well. Interestingly, fluorescence in situ hybridization (FISH) and inverse polymerase chain reaction followed by southern blotting showed that DR sequences were found to be integrated in nontelomeric regions as opposed to the usual sub-telomeric integration sites in both human samples and in cell lines. Sanger sequencing confirmed the non-telomeric integration of viral DR sequences in the host genome. Several studies have shown that C. trachomatis causes DNA damage and inhibits the signaling cascade of DNA damage response. However, the effect of C. trachomatis infection on process of DNA repair itself was not addressed. In this study, the effect of C. trachomatis infection on host base excision repair (BER) has been addressed. Base excision repair is a pathway which is responsible for replacing the oxidized bases with new undamaged ones. Interestingly, it was found that C. trachomatis infection downregulated polymerase β expression and attenuated polymerase β- mediated BER in vitro. The mechanism of the polymerase β downregulation was found to be associated with the changes in the host microRNAs and downregulation of tumor suppressor, p53. MicroRNA-499 which has a binding site in the polymerase β 3’UTR was shown to be upregulated during C. trachomatis infection. Inhibition of miR-499 using synthetic miR-499 inhibitor indeed improved the repair efficiency during C. trachomatis infection in the in vitro repair assay. Moreover, p53 transcriptionally regulates polymerase β and stabilizing p53 during C. trachomatis infection enhanced the repair efficiency. Previous studies have shown that C. trachomatis can reactivate latent HHV-6. Therefore, genomic instability due to insertions of unstable ‘transposon-like’ HHV-6 DR followed by compromised BER during C. trachomatis infection cumulatively support the hypothesis of pathogenic infections as a probable cause of ovarian cancer N2 - Diese Studie fokussiert sich darauf, wie die beiden Pathogene HHV-6 und C. trachomatis die Genom Integrität beeinflussen und dadurch die Transformation ovarialer Epithelzellen zu Tumorzellen antreiben können. Das latente Virus HHV-6 kann sich in Subtelomer-Regionen des Genoms integrieren und zu jeder Lebensphase (z.B. durch Stress oder Pharmaka) reaktiviert werden. Zu Beginn dieser Studie wurde die Beobachtung gemacht, dass in menschlichen Blutproben eine ungewöhnlich hohe Anzahl an sogenannten direct repeat Sequnzen, die die Enden des doppelsträngigen Virus Genoms flankieren, aufwiesen. Bestätigt wurde diese Beobachtung durch in vitro Daten, wofür Zelllinien generiert wurden, um den HHV-6 Wert in menschlichen Proben zu imitieren. Außerdem konnte durch Sanger Sequenzierung die Integration der viralen DR Sequenzen außerhalb von Telomer Regionen in das Genom nachgewiesen werden. Verschiedene Studien konnten zeigen, dass C. trachomatis DNA Schäden verursacht und die Signal Kaskade von Antworten auf DNA-Schäden inhibiert. Bisher wurde die Auswirkung einer C. trachomatis Infektion auf den Prozess der DNA Reparatur selbst noch nicht behandelt. In dieser Studie wird die Auswirkung einer C. trachomatis Infektion auf Basen-Exzisionsreparatur (BER) thematisiert. Interessanterweise wurde herausgefunden, dass während einer C. trachomatis Infektion die Expression von Polymerase β herunterreguliert ist und dadurch die Polymerase β-vermittelte Basen-Exzisionsreparatur in vitro gestoppt wird. Diese Herunterregulierung konnte mit einer verminderten Expression des Tumorsuppressor p53 assoziiert werden. Darüber hinaus reguliert p53 auf transkriptioneller Ebene Polymerase β und eine Stabilisierung von p53 während einer C. trachomatis Infektion verbesserte die Reparatur-Effizienz. Vorangegangene Studien haben außerdem gezeigt, dass C. trachomatis die latente Form von HHV-6 reaktivieren kann. Deshalb unterstützt die genomische Instabilität aufgrund einer Insertion von HHV-6 DR, gefolgt von komprimierter BER während einer C. trachomatis Infektion, zunehmend die Hypothese, dass eine pathogene Infektion ein vermutlicher Auslöser von Eierstockkrebs sein könnte. KW - Chlamydia trachomatis KW - Host Genome Integrity KW - Chlamydia trachomatis KW - Human Herpesvirus 6 KW - Humanes Herpesvirus 6 KW - Eierstockkrebs KW - Molekulargenetik Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162026 ER - TY - JOUR A1 - Schlegel, Jan A1 - Peters, Simon A1 - Doose, Sören A1 - Schubert-Unkmeir, Alexandra A1 - Sauer, Markus T1 - Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites JF - Frontiers in Cell and Developmental Biology N2 - Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection. KW - Neisseria meningitidis KW - sphingolipids KW - gangliosides and lipid rafts KW - super-resolution microscopy KW - single-molecule tracking Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201639 VL - 7 IS - 194 ER - TY - THES A1 - Hieke, Marie T1 - Synaptic arrangements and potential communication partners of \(Drosophila’s\) PDF-containing clock neurons within the accessory medulla T1 - Synaptische Konstellationen und potentielle Kommunikationspartner von \(Drosophila’s\) PDF-enthaltenden Uhrneuronen innerhalb der akzessorischen Medulla N2 - Endogenous clocks regulate physiological as well as behavioral rhythms within all organisms. They are well investigated in D. melanogaster on a molecular as well as anatomical level. The neuronal clock network within the brain represents the center for rhythmic activity control. One neuronal clock subgroup, the pigment dispersing factor (PDF) neurons, stands out for its importance in regulating rhythmic behavior. These neurons express the neuropeptide PDF (pigment dispersing factor). A small neuropil at the medulla’s edge, the accessory medulla (AME), is of special interest, as it has been determined as the main center for clock control. It is not only highly innervated by the PDF neurons but also by terminals of all other clock neuron subgroups. Furthermore, terminals of the photoreceptors provide light information to the AME. Many different types of neurons converge within the AME and afterward spread to their next target. Thereby the AME is supplied with information from a variety of brain regions. Among these neurons are the aminergic ones whose receptors’ are expressed in the PDF neurons. The present study sheds light onto putative synaptic partners and anatomical arrangements within the neuronal clock network, especially within the AME, as such knowledge is a prerequisite to understand circadian behavior. The aminergic neurons’ conspicuous vicinity to the PDF neurons suggests synaptic communication among them. Thus, based on former anatomical studies regarding this issue detailed light microscopic studies have been performed. Double immunolabellings, analyses of the spatial relation of pre- and postsynaptic sites of the individual neuron populations with respect to each other and the identification of putative synaptic partners using GRASP reenforce the hypothesis of synaptic interactions within the AME between dopaminergic/ serotonergic neurons and the PDF neurons. To shed light on the synaptic partners I performed first steps in array tomography, as it allows terrific informative analyses of fluorescent signals on an ultrastructural level. Therefore, I tested different ways of sample preparation in order to achieve and optimize fluorescent signals on 100 nm thin tissue sections and I made overlays with electron microscopic images. Furthermore, I made assumptions about synaptic modulations within the neuronal clock network via glial cells. I detected their cell bodies in close vicinity to the AME and PDFcontaining clock neurons. It has already been shown that glial cells modulate the release of PDF from s-LNvs’ terminals within the dorsal brain. On an anatomical level this modulation appears to exist also within the AME, as synaptic contacts that involve PDF-positive dendritic terminals are embedded into glial fibers. Intriguingly, these postsynaptic PDF fibers are often VIIAbstract part of dyadic or even multiple-contact sites in opposite to prolonged presynaptic active zonesimplicating complex neuronal interactions within the AME. To unravel possible mechanisms of such synaptic arrangements, I tried to localize the ABC transporter White. Its presence within glial cells would indicate a recycling mechanism of transmitted amines which allows their fast re-provision. Taken together, synapses accompanied by glial cells appear to be a common arrangement within the AME to regulate circadian behavior. The complexity of mechanisms that contribute in modulation of circadian information is reflected by the complex diversity of synaptic arrangements that involves obviously several types of neuron populations N2 - Endogene Uhren steuern sowohl physiologische als auch verhaltensbedingte Rhythmen bei allen Organismen. In D. melanogaster sind sie nicht nur auf molekularer sondern auch auf anatomischer Ebene bereits gut erforscht. Das neuronale Uhrnetzwerk im Gehirn stellt das Zentrum der Steuerung der rhythmischen Aktivität dar. Eine Uhrneuronengruppe sticht allein schon durch ihre besonderen anatomischen Eigenschaften hervor. Diese Neurone exprimieren das Neuropeptid PDF (pigment dispersing factor), welches zudem besonderen Einfluss auf die Lokomotionsaktivität der Fliege hat. Ein kleines Neuropil am Rande der Medulla, die akzessorische Medulla (AME) ist von besonderem Interesse, da neben seiner intensiven Innervation durch die PDF-Neurone auch Terminale aller anderen Uhrneuronengruppen zu finden sind. Zudem wird sie durch Terminale der Photorezeptoren mit Informatonen über die Lichtverhätnisse versorgt. Die AME erreichen des Weiteren Informationen aus vielen anderen Hirnregionen. Eine Vielzahl von Neuronentypen laufen in ihr zusammen, um sich anschließend wieder in verschiedenste Hirnareale zu verteilen. So wird die AME auch durchzogen von Fasern mit aminergem Inhalt, dessen Rezeptoren wiederum auf den PDF-Neuronen zu finden sind. Die vorliegende Arbeit gibt Aufschluss über vermutliche synaptische Partner und anatomische Anordnungen innerhalb des neuronalen Uhrnetzwerkes, insbesondere innerhalb der AME. Solch Wissen stellt eine Grundvoraussetzung dar, um zirkadianes Verhalten verstehen zu können. Die auffällige Nähe der aminergen Neurone zu den PDF Neuronen lässt eine synaptische Interaktion zwischen ihnen vermuten. Deshalb wurden basierend auf vorangegangen Studien detailiertere Untersuchungen dieser Thematik durchgeführt. So wird die Hypothese über synaptische Interaktionen innerhalb der AME zwischen dopaminergen/ serotonergen Neuronen und den PDF Neuronen bestärkt mittels Doppelimmunofärbungen, gegenüberstellende Analysen über die räumlichen Nähe von prä- und postsynaptischen Stellen der jeweiligen Neuronenpopulationen und durch die Identifikation vermutlicher synaptischer Partner unter Verwendung von GRASP. Zur möglichen Identifikation der synaptischen Partner unternahm ich erste Schritte in der Array Tomographie, welche hochinformative Analysen von fluoreszierenden Signalen auf einem ultrastrukturellen Level ermöglicht. Dazu testete ich verschieden Wege der Gewebepräparation, um Flureszenzsignale zu erhalten bzw. zu optimieren und bildete erste Überlagerungen der Fluoreszenz- und Elektronenmikrskopbilder. Die Auswertung der elektronenmikroskopischen Bilder erlaubten Mutmaßungen über mö- gliche synaptische Modulationen innerhalb des neuronalen Uhrnetzwerkes durch Gliazellen. Ihre Zellkörper fand ich in unmittelbarer Nähe zu den PDF Neuronen. Im dorsalen Hirn wurden neuronale Modulationen an den kleinen PDF Neuronen durch Gliazellen bereits festgestellt. Auf anatomischer Ebene scheint diese Modulation auch innerhalb der AME zu erfolgen, da synaptische Kontakte, welche PDF-positive Dendriten involvieren, von Gliafasern umgeben sind. Interessanterweise sind diese postsynaptischen PDF Fasern dabei oftmals Teil dyadischer oder sogar multipler Kontakte, die sich gegenüber einer ausgedehnten aktiven Zone befinden. Um mögliche Mechanismen solcher synaptischer Anordnungen zu erklären, versuchte ich den ABC Transporter White im Hirn von Drosophila zu lokalisieren. Seine Präsenz in Gliazellen würde auf einen Recyclingmechanismus hindeuten, welcher eine schnelle Wiederbereitstellung des Transmiters ermöglichen würde. Zusammengefasst scheinen Synapsen mit postsynaptischen PDF-Neuronen in Begleitung von Gliazellen, ein gebräuchliches synaptisches Arrangement innerhalb der AME dazustellen. Diese komplexe Diversität der synaptischen Anordnung reflektiert die komplexen Mechanismen, welche der Verarbeitung der zirkadianen Informationen zugrunde liegen KW - Taufliege KW - Chronobiologie KW - Endogene Rhythmik KW - PDF neurons KW - glia cells KW - circadian clock KW - accessory medulla KW - sleep KW - aminergic neurons KW - synapses KW - Gliazelle KW - Aminerge Nervenzelle KW - Pigmentdispergierender Faktor KW - Drosophila melanogaster Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175988 ER - TY - JOUR A1 - Grebinyk, Anna A1 - Prylutska, Svitlana A1 - Chepurna, Oksana A1 - Grebinyk, Sergii A1 - Prylutskyy, Yuriy A1 - Ritter, Uwe A1 - Ohulchanskyy, Tymish Y. A1 - Matyshevska, Olga A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - Synergy of chemo- and photodynamic therapies with C\(_{60}\) Fullerene-Doxorubicin nanocomplex JF - Nanomaterials N2 - A nanosized drug complex was explored to improve the efficiency of cancer chemotherapy, complementing it with nanodelivery and photodynamic therapy. For this, nanomolar amounts of a non-covalent nanocomplex of Doxorubicin (Dox) with carbon nanoparticle C\(_{60}\) fullerene (C\(_{60}\)) were applied in 1:1 and 2:1 molar ratio, exploiting C\(_{60}\) both as a drug-carrier and as a photosensitizer. The fluorescence microscopy analysis of human leukemic CCRF-CEM cells, in vitro cancer model, treated with nanocomplexes showed Dox’s nuclear and C\(_{60}\)'s extranuclear localization. It gave an opportunity to realize a double hit strategy against cancer cells based on Dox's antiproliferative activity and C\(_{60}\)'s photoinduced pro-oxidant activity. When cells were treated with 2:1 C\(_{60}\)-Dox and irradiated at 405 nm the high cytotoxicity of photo-irradiated C\(_{60}\)-Dox enabled a nanomolar concentration of Dox and C\(_{60}\) to efficiently kill cancer cells in vitro. The high pro-oxidant and pro-apoptotic efficiency decreased IC\(_{50}\) 16, 9 and 7 × 10\(^3\)-fold, if compared with the action of Dox, non-irradiated nanocomplex, and C\(_{60}\)'s photodynamic effect, correspondingly. Hereafter, a strong synergy of therapy arising from the combination of C\(_{60}\)-mediated Dox delivery and C\(_{60}\) photoexcitation was revealed. Our data indicate that a combination of chemo- and photodynamic therapies with C\(_{60}\)-Dox nanoformulation provides a promising synergetic approach for cancer treatment. KW - photodynamic chemotherapy KW - synergistic effect KW - C\(_{60}\) fullerene KW - Doxorubicin KW - nanocomplex KW - leukemic cells KW - apoptosis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193140 SN - 2079-4991 VL - 9 IS - 11 ER - TY - JOUR A1 - Villalobos, Alvaro S. A1 - Wiese, Jutta A1 - Imhoff, Johannes F. A1 - Dorador, Cristina A1 - Keller, Alexander A1 - Hentschel, Ute T1 - Systematic affiliation and genome analysis of Subtercola vilae DB165T with particular emphasis on cold adaptation of an isolate from a high-altitude cold volcano lake JF - Microorganisms N2 - Among the Microbacteriaceae the species of Subtercola and Agreia form closely associated clusters. Phylogenetic analysis demonstrated three major phylogenetic branches of these species. One of these branches contains the two psychrophilic species Subtercola frigoramans and Subtercola vilae, together with a larger number of isolates from various cold environments. Genomic evidence supports the separation of Agreia and Subtercola species. In order to gain insight into the ability of S. vilae to adapt to life in this extreme environment, we analyzed the genome with a particular focus on properties related to possible adaptation to a cold environment. General properties of the genome are presented, including carbon and energy metabolism, as well as secondary metabolite production. The repertoire of genes in the genome of S. vilae DB165\(^T\) linked to adaptations to the harsh conditions found in Llullaillaco Volcano Lake includes several mechanisms to transcribe proteins under low temperatures, such as a high number of tRNAs and cold shock proteins. In addition, S. vilae DB165\(^T\) is capable of producing a number of proteins to cope with oxidative stress, which is of particular relevance at low temperature environments, in which reactive oxygen species are more abundant. Most important, it obtains capacities to produce cryo-protectants, and to combat against ice crystal formation, it produces ice-binding proteins. Two new ice-binding proteins were identified which are unique to S. vilae DB165\(^T\). These results indicate that S. vilae has the capacity to employ different mechanisms to live under the extreme and cold conditions prevalent in Llullaillaco Volcano Lake. KW - cold adaptation KW - Subtercola vilae KW - genome analysis KW - systematic affiliation KW - Llullaillaco Volcano Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197394 SN - 2076-2607 VL - 7 IS - 4 ER - TY - THES A1 - Tiwarekar, Vishakha Rakesh T1 - The APOBEC3G-regulated host factors REDD1 and KDELR2 restrict measles virus replication T1 - Die durch APOBEC3G-regulierten Wirtsfaktoren REDD1 und KDELR2 restringieren die Masernvirus Replikation N2 - Measles is an extremely contagious vaccine-preventable disease responsible for more than 90000 deaths worldwide annually. The number of deaths has declined from 8 million in the pre-vaccination era to few thousands every year due to the highly efficacious vaccine. However, this effective vaccine is still unreachable in many developing countries due to lack of infrastructure, while in developed countries too many people refuse vaccination. Specific antiviral compounds are not yet available. In the current situation, only an extensive vaccination approach along with effective antivirals could help to have a measles-free future. To develop an effective antiviral, detailed knowledge of viral-host interaction is required. This study was undertaken to understand the interaction between MV and the innate host restriction factor APOBEC3G (A3G), which is well-known for its activity against human immunodeficiency virus (HIV). Restriction of MV replication was not attributed to the cytidine deaminase function of A3G, instead, we identified a novel role of A3G in regulating cellular gene functions. Among two of the A3G regulated host factors, we found that REDD1 reduced MV replication, whereas, KDELR2 hampered MV haemagglutinin (H) surface transport thereby affecting viral release. REDD1, a negative regulator of mTORC1 signalling impaired MV replication by inhibiting mTORC1. A3G regulated REDD1 expression was demonstrated to inversely correlate with MV replication. siRNA mediated silencing of A3G in primary human blood lymphocytes (PBL) reduced REDD1 levels and simultaneously increased MV titres. Also, direct depletion of REDD1 improved MV replication in PBL, indicating its role in A3G mediated restriction of MV. Based on these finding, a new role of rapamycin, a pharmacological inhibitor of mTORC1, was uncovered in successfully diminishing MV replication in Vero as well as in human PBL. The ER and Golgi resident receptor KDELR2 indirectly affected MV by competing with MV-H for cellular chaperones. Due to the sequestering of chaperones by KDELR2, they can no longer assist in MV-H folding and subsequent surface expression. Taken together, the two A3G-regulated host factors REDD1 and KDELR2 are mainly responsible for mediating its antiviral activity against MV. N2 - Masern ist eine extrem ansteckende, durch Impfung verhinderbare Infektionskrankheit, die für mehr als 90000 Todesfälle jährlich weltweit verantwortlich ist. Die Zahl der Todesfälle nahm von ca. 8 Millionen in der Prä- Impf-Ära auf wenige Tausend pro Jahr aufgrund dieses effizienten Impfstoffs ab. Dieser ist jedoch aufgrund mangelnder Infrastruktur in vielen Entwicklungsländern nicht ausreichend verfügbar, oder die Impfung wird – vor allem in entwickelten Ländern – verweigert. Spezifische antivirale Substanzen sind noch nicht verfügbar. So könnte nur eine extensive Impfkampagne zu einer Masern-freien Zukunft führen. Um antivirale Substanzen zu generieren wird detailiertes Wissen über Virus-Wirt-Interaktionen benötigt. Diese Studie wurde unternommen um Interaktionen zwischen Masernviren (MV) und dem zellulären Restriktionsfaktor APOBEC3G (A3G), der allgemein bekannt für seine antivirale Wirkung gegen das humane Immundefizienzvirus (HIV) ist, zu charakterisieren. A3G hemmt die MV-Replikation nicht aufgrund seiner Cytidin-Desaminase-Funktion, sondern wir entdeckten eine neue Funktion des A3G, nämlich dass es die Expression zellulärer Faktoren reguliert. Wir fanden, dass unter den A3G-regulierten Wirtszellfaktoren REDD1 die MV-Replikation reduzierte, während KDELR2 den Transport des MV-Hämagglutinins (H) zur Zelloberfläche, und somit die Virusfreisetzung, inhibierte. REDD1, ein negativer Regulator des mTORC1-Signalübertragungswegs, reduzierte die MV-Replikation indem es mTORC1 inhibiert. Die Expression des durch A3G regulierten REDD1 korrelierte umgekehrt mit der MV Replikation. SiRNA-vermittelte Reduktion des A3G in primären humanen Lymphozyten des Bluts (PBL) führte zu einer Abnahme des REDD1 und gleichzeitig zu einer Zunahme des MV-Titers. Ebenso führte direktes Silencing des REDD1 zu einer verstärkten MV-Replikation in PBL, was seine Rolle bei der A3G-vermittelten Restriktion der MV-Replikation unterstreicht. Aufgrund dieser Befunde wurde auch eine neue Funktion des mTORC1-Inhibitors Rapamycin als Inhibitor der MV-Replikation in Vero-Zellen und primären PBL aufgedeckt. Der ER- und Golgi-residente Rezeptor KDELR2 wirkte sich indirekt auf die MV-Replikation aus, indem er mit dem MV-H um die Interaktion mit Chaperonen kompetiert. KDELR2 bindet Chaperone und verhindert so deren Interaktion mit MV-H und den Transport zur Zelloberfläche. Zusammenfassend lässt sich sagen, dass die beiden A3G-regulierten Wirtszellfaktoren REDD1 und KDELR2 hauptsächlich für die antivirale Aktivität des A3G gegen MV verantwortlich sind. KW - measles virus KW - restriction factors KW - APOBEC3G KW - REDD1 KW - KDELR2 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179526 ER - TY - JOUR A1 - Horn, Melanie A1 - Mitesser, Oliver A1 - Hovestadt, Thomas A1 - Yoshii, Taishi A1 - Rieger, Dirk A1 - Helfrich-Förster, Charlotte T1 - The circadian clock improves fitness in the fruit fly, Drosophila melanogaster JF - Frontiers in Physiology N2 - It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. We raised each of the three classical Drosophila period mutants for >50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant’s natural cycle, i.e., a 19-h day in the case of pers mutants and a 29-h day for perl mutants. The arrhythmic per0 mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. We found that wildtype flies quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but perl mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast, pers and per0 mutants did not win against wildtype flies under the 19-h day and constant light, respectively. Our results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the period mutants and – as revealed for perl mutants – this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that perl and pers persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants. KW - competition KW - mutants KW - resonance theory KW - mating preference KW - fertility Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195738 SN - 1664-042X VL - 10 IS - 1374 ER - TY - JOUR A1 - Beer, Katharina A1 - Schenk, Mariela A1 - Helfrich-Förster, Charlotte A1 - Holzschuh, Andrea T1 - The circadian clock uses different environmental time cues to synchronize emergence and locomotion of the solitary bee Osmia bicornis JF - Scientific Reports N2 - Life on earth adapted to the daily reoccurring changes in environment by evolving an endogenous circadian clock. Although the circadian clock has a crucial impact on survival and behavior of solitary bees, many aspects of solitary bee clock mechanisms remain unknown. Our study is the first to show that the circadian clock governs emergence in Osmia bicornis, a bee species which overwinters as adult inside its cocoon. Therefore, its eclosion from the pupal case is separated by an interjacent diapause from its emergence in spring. We show that this bee species synchronizes its emergence to the morning. The daily rhythms of emergence are triggered by temperature cycles but not by light cycles. In contrast to this, the bee’s daily rhythms in locomotion are synchronized by light cycles. Thus, we show that the circadian clock of O. bicornis is set by either temperature or light, depending on what activity is timed. Light is a valuable cue for setting the circadian clock when bees have left the nest. However, for pre-emerged bees, temperature is the most important cue, which may represent an evolutionary adaptation of the circadian system to the cavity-nesting life style of O. bicornis. KW - Behavioural ecology KW - Evolutionary developmental biology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202721 VL - 9 ER - TY - JOUR A1 - Westermann, Alexander J. A1 - Venturini, Elisa A1 - Sellin, Mikael E. A1 - Förstner, Konrad U. A1 - Hardt, Wolf-Dietrich A1 - Vogel, Jörg T1 - The major RNA-binding protein ProQ impacts virulence gene expression in Salmonella enterica serovar Typhimurium JF - mBio N2 - FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs. IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria. KW - Hfq KW - noncoding RNA KW - ProQ KW - RNA-seq KW - bacterial pathogen KW - posttranscriptional control Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177722 VL - 10 IS - 1 ER - TY - JOUR A1 - Pattschull, Grit A1 - Walz, Susanne A1 - Gründl, Marco A1 - Schwab, Melissa A1 - Rühl, Eva A1 - Baluapuri, Apoorva A1 - Cindric-Vranesic, Anita A1 - Kneitz, Susanne A1 - Wolf, Elmar A1 - Ade, Carsten P. A1 - Rosenwald, Andreas A1 - von Eyss, Björn A1 - Gaubatz, Stefan T1 - The Myb-MuvB complex is required for YAP-dependent transcription of mitotic genes JF - Cell Reports N2 - YAP and TAZ, downstream effectors of the Hippo pathway, are important regulators of proliferation. Here, we show that the ability of YAP to activate mitotic gene expression is dependent on the Myb-MuvB (MMB) complex, a master regulator of genes expressed in the G2/M phase of the cell cycle. By carrying out genome-wide expression and binding analyses, we found that YAP promotes binding of the MMB subunit B-MYB to the promoters of mitotic target genes. YAP binds to B-MYB and stimulates B-MYB chromatin association through distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. The cooperation between YAP and B-MYB is critical for YAP-mediated entry into mitosis. Furthermore, the expression of genes coactivated by YAP and B-MYB is associated with poor survival of cancer patients. Our findings provide a molecular mechanism by which YAP and MMB regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways. KW - YAP KW - B-MYB KW - Myb-MuvB KW - mitotic genes KW - enhancer KW - transcription Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202039 VL - 27 IS - 12 ER - TY - JOUR A1 - El-Mesery, Mohamed A1 - Rosenthal, Tina A1 - Rauert-Wunderlich, Hilka A1 - Schreder, Martin A1 - Stühmer, Thorsten A1 - Leich, Ellen A1 - Schlosser, Andreas A1 - Ehrenschwender, Martin A1 - Wajant, Harald A1 - Siegmund, Daniela T1 - The NEDD8-activating enzyme inhibitor MLN4924 sensitizes a TNFR1+ subgroup of multiple myeloma cells for TNF-induced cell death JF - Cell Death & Disease N2 - The NEDD8-activating enzyme (NAE) inhibitor MLN4924 inhibits cullin-RING ubiquitin ligase complexes including the SKP1-cullin-F-box E3 ligase βTrCP. MLN4924 therefore inhibits also the βTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines (e.g., RPMI-8226, MM.1S, KMS-12BM) and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1+ myeloma cells and a TNFhigh tumor microenvironment. KW - cancer therapy KW - tumour-necrosis factors Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226666 VL - 10 ER - TY - JOUR A1 - Schartl, Manfred A1 - Kneitz, Susanne A1 - Volkoff, Helene A1 - Adolfi, Mateus A1 - Schmidt, Cornelia A1 - Fischer, Petra A1 - Minx, Patrick A1 - Tomlinson, Chad A1 - Meyer, Axel A1 - Warren, Wesley C. T1 - The piranha genome provides molecular insight associated to its unique feeding behavior JF - Genome Biology and Evolution N2 - The piranha enjoys notoriety due to its infamous predatory behavior but much is still not understood about its evolutionary origins and the underlying molecular mechanisms for its unusual feeding biology. We sequenced and assembled the red-bellied piranha (Pygocentrus nattereri) genome to aid future phenotypic and genetic investigations. The assembled draft genome is similar to other related fishes in repeat composition and gene count. Our evaluation of genes under positive selection suggests candidates for adaptations of piranhas’ feeding behavior in neural functions, behavior, and regulation of energy metabolism. In the fasted brain, we find genes differentially expressed that are involved in lipid metabolism and appetite regulation as well as genes that may control the aggression/boldness behavior of hungry piranhas. Our first analysis of the piranha genome offers new insight and resources for the study of piranha biology and for feeding motivation and starvation in other organisms. KW - whole-genome sequencing KW - genome annotation KW - comparative genomics KW - RNA-seq transcriptome KW - energy homeostasis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202218 VL - 11 IS - 8 ER - TY - THES A1 - Kremer, Antje T1 - Tissue Engineering of a Vascularized Meniscus Implant T1 - Tissue Engineering eines vaskularisierten Meniskus-Implantates N2 - The knee joint is a complex composite joint containing the C-shaped wedge-like menisci composed of fibrocartilage. Due to their complex composition and structure, they provide mechanical resilience to the knee joint protecting the articular cartilage. Because of the limited repair potential, meniscal injuries do not only affect the meniscus itself but also lead to altered joint homeostasis and inevitably to secondary osteoarthritis. The meniscus was characterized focusing on its anatomy, structure and meniscal markers such as aggrecan, collagen type I (Col I) and Col II. The components relevant for meniscus tissue engineering, namely cells, Col I scaffolds, biochemical and biomechanical stimuli were studied. Meniscal cells (MCs) were isolated from meniscus, mesenchymal stem cells (MSCs) from bone marrow and dermal microvascular endothelial cells (d-mvECs) from foreskin biopsies. For the human (h) meniscus model, wedge-shape compression of a hMSC-laden Col I gel was successfully established. During three weeks of static culture, the biochemical stimulus transforming growth factor beta-3 (TGF beta-3) led to a compact collagen structure. On day 21, this meniscus model showed high metabolic activity and matrix remodeling as confirmed by matrix metalloproteinases detection. The fibrochondrogenic properties were illustrated by immunohistochemical detection of meniscal markers, significant GAG/DNA increase and increased compressive properties. For further improvement, biomechanical stimulation systems by compression and hydrostatic pressure were designed. As one vascularization approach, direct stimulation with ciclopirox olamine (CPX) significantly increased sprouting of hd-mvEC spheroids even in absence of auxiliary cells such as MSCs. Second, a cell sheet composed of hMSCs and hd-mvECs was fabricated by temperature triggered cell sheet engineering and transferred onto the wedge-shaped meniscus model. Third, a biological vascularized scaffold (BioVaSc-TERM) was re-endothelialized with hd-mvECs providing a viable vascularized network. The vascularized BioVaSc-TERM was suggested as wrapping scaffold of the meniscus model by using two suture techniques, the all-inside-repair (AIR) for the posterior horn, and the outside-in-refixation (OIR) for the anterior horn and the middle part. This meniscus model for replacing torn menisci is a promising approach to be further optimized regarding vascularization, biochemical and biomechanical stimuli. N2 - Das Knie ist ein komplex zusammengesetztes Gelenk mit zwei C-förmigen Keilen aus Bindegewebsknorpel, die Menisken. Sie sorgen für die mechanische Belastbarkeit des Knies, wodurch der Gelenksknorpel geschützt wird. Aufgrund des limitierten Heilungspotentials beeinträchtigen Meniskusverletzungen nicht nur den Meniskus selbst, sondern schädigen auch das Gelenksgleichgewicht und führen zu sekundärer Osteoarthritis. Der Meniskus wurde in seiner Anatomie, Struktur und Meniskusmarkern wie Aggrekan, Kollagen I und Kollagen II charakterisiert. Die Komponenten von Meniskus Tissue Engineering, Zellen, Kollagen I Materialien, biochemische und biomechanische Stimuli wurden untersucht. Meniskuszellen (MCs) wurden aus Meniskus isoliert, mesenchymale Stammzellen (MSCs) aus Knochenmark und dermale mikrovaskuläre Endothelzellen (d-mvECs) aus Vorhautbiopsien. Für das humane (h) Meniskus-Modell wurde die keilförmige Kompression eines hMSC-beladenen Kollagen I Gels erfolgreich etabliert. Während drei Wochen statischer Kultur führte der biochemische Stimulus transformierender Wachs-tumsfaktor beta-3 (TGF beta-3) zu einer kompakten Kollagenstruktur. An Tag 21 zeigte dieses Meniskus-Modell eine hohe metabolische Aktivität und Matrixumbau durch die Detektion von Matrix-Metalloproteasen. Der Bindegewebsknorpel wurde durch immunhistochemische Detektion der Meniskusmarker, einem signifikanten GAG/DNA Anstieg und erhöhter Kompressionseigenschaften bestätigt. Für weitere Verbesserungen wurden biomechanische Stimulierungssysteme mittels Kompression und hydrostatischen Druck aufgebaut. Als Vaskularisierungsansatz führte die direkte Stimulierung mit Ciclopirox Olamine (CPX) sogar in Abwesenheit von Helferzellen wie MSCs zu einem erhöhten Sprouting der hd-mvEC Spheroide. Zweitens wurde ein hMSC/hd-mvEC Sheet mithilfe eines Temperatur-abhängigen Verfahrens produziert und auf das keilförmige Meniskus-Modell transferiert. Drittens wurde ein vaskularisiertes Biomaterial (BioVaSc-TERM) mit hd-mvECs besiedelt, wodurch ein vitales Gefäßystem bereitgestellt wurde. Die vaskularisierte BioVaSc-TERM wurde als Hülle des Meniskus-Modells unter der Verwendung von zwei Nahttechniken vorgeschlagen: die All-Inside-Repair (AIR) für das Hinterhorn und die Outside-In-Refixation (OIR) für das Vorderhorn und den mittleren Teil. Dieses Meniskus-Modell ist ein vielversprechender Ansatz für den Meniskusersatz, um in Vaskularisierung, biochemischer und biomechanischer Stimuli weiter optimiert zu werden. KW - Meniskus KW - Tissue Engineering KW - Regenerative Medizin KW - Meniskusimplantat KW - meniscus implant KW - Tissue Engineering KW - tissue engineering KW - vascularization KW - Vaskularisierung Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-184326 ER - TY - THES A1 - Griffoni, Chiara T1 - Towards advanced immunocompetent skin wound models for in vitro drug evaluation T1 - Auf dem Weg zu fortschrittlichen immunkompetenten Hautwundmodellen für die in vitro-Medikamentenbewertung N2 - Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models. N2 - Aktuelle präklinische Modelle zur Bewertung neuartiger Therapien für eine verbesserte Heilung um- fassen sowohl in vitro als auch in vivo Methoden. Ethische Bedenken im Zusammenhang mit der Ver- wendung von Tieren sowie die schlechte physiologische Übersetzung zwischen tierischer und mensch- licher Hautwundheilung bezeichnen In-vitro-Modelle jedoch als hochrelevante und vielversprechende Plattformen für die Heilungsforschung. Während die aktuellen in vitro 3D-Hautmodelle ein reifes Ge- webe mit heilenden Eigenschaften rekapitulieren, stellen sie dennoch eine Vereinfachung der in vivo- Bedingungen dar, bei denen beispielsweise die nach der Wundbildung entstehende Entzündungsreak- tion den Beitrag von Immunzellen beinhaltet. Makrophagen gehören zu den Hauptverursachern der Entzündungsreaktion und regulieren ihren Verlauf durch ihre Plastizität. Daher könnte ihre Implemen- tierung in die in vitro Haut die physiologische Relevanz der Modelle deutlich erhöhen. Da bisher kein volldickes, immunkompetentes Hautmodell mit Makrophagen berichtet wurde, wurden hier die für eine erfolgreiche Dreifach-Cokultur von Fibroblasten, Keratinozyten und Makrophagen notwendigen Parameter untersucht. Zuerst wurden die Zellquelle und die Kultur zeitlich festgelegt, aber auch eine Implementierungsstrategie für Makrophagen festgelegt. Die Implementierung von Makrophagen in das Hautmodell konzentrierte sich auf die Minimierung der Kultivierungszeit, um die Lebensfähigkeit und den Phänotyp der Immunzellen zu erhalten, da die Umgebung einen großen Einfluss auf die Zell- polarisation und Zytokinproduktion hat. Zu diesem Zweck wurde die Integration von Makrophagen in 3D-Gelen vor der Kombination mit Hautmodellen ausgewählt, um die in vivo-Umgebung besser nach- ahmen zu können. Eingebettet in Kollagenhydrogele zeigten Makrophagen eine homogene Zellvertei- lung im Gel, die die Zelllebensfähigkeit bewahrt, auf Reize reagiert und durch die Matrix wandert, die alle bei der Beteiligung von Makrophagen an der Entzündungsreaktion benötigt werden. Nachdem festgestellt worden war, wie Makrophagen in Hautmodelle eingeführt werden können, wurden ver- schiedene Kulturmedien hinsichtlich ihrer Auswirkungen auf Primärfibroblasten, Keratinozyten und Makrophagen untersucht, um eine geeignete Medienzusammensetzung für die Kultur immunkompe- tenter Haut zu identifizieren. Die vorliegende Arbeit bestätigte, dass jeder Zelltyp eine andere Supple- mentkombination zur Aufrechterhaltung der Funktionsmerkmale benötigt und zeigte erstmals, dass Medien, die eine reife Hautstruktur fördern und aufrechterhalten, negative Auswirkungen auf die pri- mären Makrophagen haben. Hautdifferenzierungsmedien wirkten sich negativ auf die Makrophagen in Bezug auf Lebensfähigkeit, Morphologie, Fähigkeit, auf pro- und antiinflammatorische Reize zu rea- gieren und durch ein Kollagengel zu wandern aus. Die Kombination aus verwundeten Hautäquivalen- ten und makrophagenhaltigen Gelen bestätigte, dass das Kulturmedium die Teilnahme der Makro- phage an der Entzündungsreaktion, die nach der Wunde auftritt, hemmt. Die beschriebene Makrophagen-Einschlussmethode zur immunkompetenten Hautbildung ist ein vielversprechender An- satz zur Generierung relevanterer Hautmodelle. Eine weitere Optimierung des Co-Kulturmediums wird es möglicherweise ermöglichen, eine physiologische Entzündungsreaktion nachzuahmen und die Aus- wirkungen neuartiger Medikamente zur verbesserten Heilung auf verbesserte In-vitro-Modelle zu be- werten. KW - skin model KW - macrophages KW - wound healing KW - immunocompetent skin KW - Haut KW - In vitro KW - Wundheilung Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192125 ER - TY - JOUR A1 - Goos, Carina A1 - Dejung, Mario A1 - Wehman, Ann M. A1 - M-Natus, Elisabeth A1 - Schmidt, Johannes A1 - Sunter, Jack A1 - Engstler, Markus A1 - Butter, Falk A1 - Kramer, Susanne T1 - Trypanosomes can initiate nuclear export co-transcriptionally JF - Nucleic Acids Research N2 - The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes. KW - molecular biology KW - nuclear export KW - trypanosomes KW - mRNA KW - nuclear envelope Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177709 VL - 47 IS - 1 ER - TY - JOUR A1 - Dörk, Thilo A1 - Peterlongo, Peter A1 - Mannermaa, Arto A1 - Bolla, Manjeet K. A1 - Wang, Qin A1 - Dennis, Joe A1 - Ahearn, Thomas A1 - Andrulis, Irene L. A1 - Anton-Culver, Hoda A1 - Arndt, Volker A1 - Aronson, Kristan J. A1 - Augustinsson, Annelie A1 - Beane Freeman, Laura E. A1 - Beckmann, Matthias W. A1 - Beeghly-Fadiel, Alicia A1 - Behrens, Sabine A1 - Bermisheva, Marina A1 - Blomqvist, Carl A1 - Bogdanova, Natalia V. A1 - Bojesen, Stig E. A1 - Brauch, Hiltrud A1 - Brenner, Hermann A1 - Burwinkel, Barbara A1 - Canzian, Federico A1 - Chan, Tsun L. A1 - Chang-Claude, Jenny A1 - Chanock, Stephen J. A1 - Choi, Ji-Yeob A1 - Christiansen, Hans A1 - Clarke, Christine L. A1 - Couch, Fergus J. A1 - Czene, Kamila A1 - Daly, Mary B. A1 - dos-Santos-Silva, Isabel A1 - Dwek, Miriam A1 - Eccles, Diana M. A1 - Ekici, Arif B. A1 - Eriksson, Mikael A1 - Evans, D. Gareth A1 - Fasching, Peter A. A1 - Figueroa, Jonine A1 - Flyger, Henrik A1 - Fritschi, Lin A1 - Gabrielson, Marike A1 - Gago-Dominguez, Manuela A1 - Gao, Chi A1 - Gapstur, Susan M. A1 - García-Closas, Montserrat A1 - García-Sáenz, José A. A1 - Gaudet, Mia M. A1 - Giles, Graham G. A1 - Goldberg, Mark S. A1 - Goldgar, David E. A1 - Guenél, Pascal A1 - Haeberle, Lothar A1 - Haimann, Christopher A. A1 - Håkansson, Niclas A1 - Hall, Per A1 - Hamann, Ute A1 - Hartman, Mikael A1 - Hauke, Jan A1 - Hein, Alexander A1 - Hillemanns, Peter A1 - Hogervorst, Frans B. L. A1 - Hooning, Maartje J. A1 - Hopper, John L. A1 - Howell, Tony A1 - Huo, Dezheng A1 - Ito, Hidemi A1 - Iwasaki, Motoki A1 - Jakubowska, Anna A1 - Janni, Wolfgang A1 - John, Esther M. A1 - Jung, Audrey A1 - Kaaks, Rudolf A1 - Kang, Daehee A1 - Kapoor, Pooja Middha A1 - Khusnutdinova, Elza A1 - Kim, Sung-Won A1 - Kitahara, Cari M. A1 - Koutros, Stella A1 - Kraft, Peter A1 - Kristensen, Vessela N. A1 - Kwong, Ava A1 - Lambrechts, Diether A1 - Le Marchand, Loic A1 - Li, Jingmei A1 - Lindström, Sara A1 - Linet, Martha A1 - Lo, Wing-Yee A1 - Long, Jirong A1 - Lophatananon, Artitaya A1 - Lubiński, Jan A1 - Manoochehri, Mehdi A1 - Manoukian, Siranoush A1 - Margolin, Sara A1 - Martinez, Elena A1 - Matsuo, Keitaro A1 - Mavroudis, Dimitris A1 - Meindl, Alfons A1 - Menon, Usha A1 - Milne, Roger L. A1 - Mohd Taib, Nur Aishah A1 - Muir, Kenneth A1 - Mulligan, Anna Marie A1 - Neuhausen, Susan L. A1 - Nevanlinna, Heli A1 - Neven, Patrick A1 - Newman, William G. A1 - Offit, Kenneth A1 - Olopade, Olufunmilayo I. A1 - Olshan, Andrew F. A1 - Olson, Janet E. A1 - Olsson, Håkan A1 - Park, Sue K. A1 - Park-Simon, Tjoung-Won A1 - Peto, Julian A1 - Plaseska-Karanfilska, Dijana A1 - Pohl-Rescigno, Esther A1 - Presneau, Nadege A1 - Rack, Brigitte A1 - Radice, Paolo A1 - Rashid, Muhammad U. A1 - Rennert, Gad A1 - Rennert, Hedy S. A1 - Romero, Atocha A1 - Ruebner, Matthias A1 - Saloustros, Emmanouil A1 - Schmidt, Marjanka K. A1 - Schmutzler, Rita K. A1 - Schneider, Michael O. A1 - Schoemaker, Minouk J. A1 - Scott, Christopher A1 - Shen, Chen-Yang A1 - Shu, Xiao-Ou A1 - Simard, Jaques A1 - Slager, Susan A1 - Smichkoska, Snezhana A1 - Southey, Melissa C. A1 - Spinelli, John J. A1 - Stone, Jennifer A1 - Surowy, Harald A1 - Swerdlow, Anthony J. A1 - Tamimi, Rulla M. A1 - Tapper, William J. A1 - Teo, Soo H. A1 - Terry, Mary Beth A1 - Toland, Amanda E. A1 - Tollenaar, Rob A. E. M. A1 - Torres, Diana A1 - Torres-Mejía, Gabriela A1 - Troester, Melissa A. A1 - Truong, Thérèse A1 - Tsugane, Shoichiro A1 - Untch, Michael A1 - Vachon, Celine M. A1 - van den Ouweland, Ans M. W. A1 - van Veen, Elke M. A1 - Vijai, Joseph A1 - Wendt, Camilla A1 - Wolk, Alicja A1 - Yu, Jyh-Cherng A1 - Zheng, Wei A1 - Ziogas, Argyrios A1 - Ziv, Elad A1 - Dunnig, Alison A1 - Pharaoh, Paul D. P. A1 - Schindler, Detlev A1 - Devilee, Peter A1 - Easton, Douglas F. T1 - Two truncating variants in FANCC and breast cancer risk JF - Scientific Reports N2 - Fanconi anemia (FA) is a genetically heterogeneous disorder with 22 disease-causing genes reported to date. In some FA genes, monoallelic mutations have been found to be associated with breast cancer risk, while the risk associations of others remain unknown. The gene for FA type C, FANCC, has been proposed as a breast cancer susceptibility gene based on epidemiological and sequencing studies. We used the Oncoarray project to genotype two truncating FANCC variants (p.R185X and p.R548X) in 64,760 breast cancer cases and 49,793 controls of European descent. FANCC mutations were observed in 25 cases (14 with p.R185X, 11 with p.R548X) and 26 controls (18 with p.R185X, 8 with p.R548X). There was no evidence of an association with the risk of breast cancer, neither overall (odds ratio 0.77, 95%CI 0.44–1.33, p = 0.4) nor by histology, hormone receptor status, age or family history. We conclude that the breast cancer risk association of these two FANCC variants, if any, is much smaller than for BRCA1, BRCA2 or PALB2 mutations. If this applies to all truncating variants in FANCC it would suggest there are differences between FA genes in their roles on breast cancer risk and demonstrates the merit of large consortia for clarifying risk associations of rare variants. KW - oncology KW - risk factors Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222838 VL - 9 ER - TY - JOUR A1 - Figueiredo, Ludmilla A1 - Krauss, Jochen A1 - Steffan-Dewenter, Ingolf A1 - Cabral, Juliano Sarmento T1 - Understanding extinction debts: spatio-temporal scales, mechanisms and a roadmap for future research JF - Ecography N2 - Extinction debt refers to delayed species extinctions expected as a consequence of ecosystem perturbation. Quantifying such extinctions and investigating long‐term consequences of perturbations has proven challenging, because perturbations are not isolated and occur across various spatial and temporal scales, from local habitat losses to global warming. Additionally, the relative importance of eco‐evolutionary processes varies across scales, because levels of ecological organization, i.e. individuals, (meta)populations and (meta)communities, respond hierarchically to perturbations. To summarize our current knowledge of the scales and mechanisms influencing extinction debts, we reviewed recent empirical, theoretical and methodological studies addressing either the spatio–temporal scales of extinction debts or the eco‐evolutionary mechanisms delaying extinctions. Extinction debts were detected across a range of ecosystems and taxonomic groups, with estimates ranging from 9 to 90% of current species richness. The duration over which debts have been sustained varies from 5 to 570 yr, and projections of the total period required to settle a debt can extend to 1000 yr. Reported causes of delayed extinctions are 1) life‐history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions. Other potential factors that may extend survival time such as microevolutionary dynamics, or delayed extinctions of interaction partners, have rarely been analyzed. Therefore, we propose a roadmap for future research with three key avenues: 1) the microevolutionary dynamics of extinction processes, 2) the disjunctive loss of interacting species and 3) the impact of multiple regimes of perturbation on the payment of debts. For their ability to integrate processes occurring at different levels of ecological organization, we highlight mechanistic simulation models as tools to address these knowledge gaps and to deepen our understanding of extinction dynamics. KW - Anthropocene KW - biotic interaction KW - extinction dynamics KW - mechanistic modelling KW - time lag KW - transient dynamics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204859 VL - 42 IS - 12 ER - TY - THES A1 - Pomper [geb. Müller], Laura Dorothea T1 - Unterschiede in Frontaler Kortex Oxygenierung in zweierlei Risikogruppen der Alzheimer Demenz T1 - Differences in Frontal Lobe Oxygenation in Two Risk Groups for Alzheimer's Disease N2 - Die verbesserte medizinische Versorgung führt zu einer zunehmenden Lebenserwartung unserer Gesellschaft. Damit steigt auch die sozioökonomische Relevanz neurodegenerativer Erkrankungen kontinuierlich. Für die Alzheimer Demenz (AD), die dabei die häufigste Ursache darstellt, stehen bisher keine krankheitsmodifizierenden Behandlungsoptionen zur Verfügung. Die lange präklinische Phase der Erkrankung birgt jedoch großes Potential für die Entwicklung neuer Behandlungsoptionen. Das Untersuchen von Risikogruppen ist für die Identifikation von Prädiktoren einer späteren AD Manifestation von besonderem Interesse. In diesem Zusammenhang werden insbesondere das Vorliegen genetischer Risikokonstellationen, wie dem Apolipoprotein E (APOE) Ɛ4-Allel, sowie kognitiver Risikofaktoren, wie der „leichten kognitiven Beeinträchtigung“ (MCI), diskutiert. Die Identifikation präklinischer Aktivierungsunterschiede in relevanten Gehirnregionen von Risikogruppen kann als Basis für die Entwicklung neurofunktioneller Früherkennungs-Marker dienen. Der präfrontale Kortex (PFC), welcher mit der Steuerung von Exekutivfunktionen assoziiert wird, hat sich in diesem Zusammenhang in bisherigen Studien als eine relevante Schlüsselregion manifestiert. Aufgrund der aufwendigen und kostenintensiven bildgebenden Untersuchungsmethoden, sind die genauen Prozesse jedoch noch unklar. Ziel der vorliegenden Arbeit war es daher, Unterschiede in der PFC Oxygenierung in zweierlei Risikogruppen der AD mit einer kostengünstigeren Bildgebungsmethode, der funktionellen Nahinfrarot Spektroskopie (fNIRS), zu untersuchen. Dafür wurde in einem ersten Schritt, der Trailmaking Test (TMT), ein weitverbreiteter neuropsychologischer Test zur Erfassung exekutiver Funktionen, für fNIRS implementiert. Als Grundlage für die Untersuchung frühpathologischer Prozesse, wurden zunächst gesunde Alterungsprozesse betrachtet. Der Vergleich von jungen und älteren Probanden (n = 20 pro Gruppe) wies neben der Eignung der Testimplementierung für fNIRS auf eine spezifische bilaterale PFC Oxygenierung hin, welche bei jungen Probanden rechtshemisphärisch lateralisiert war. Ältere Probanden hingegen zeigten bei vergleichbaren Verhaltensdaten insgesamt mehr signifikante Kanäle sowie eine Abnahme der Lateralisierung. Dies kann als zusätzlicher Bedarf an Ressourcen in gesunden Alterungsprozessen interpretiert werden. Im Rahmen der Hauptstudie wurden anschließend insgesamt 604 ältere Probanden im Alter von 70 bis 76 Jahren untersucht. Zunächst wurde die genetische Risikogruppe der Ɛ4-Allel-Träger (n = 78) mit den neutralen Ɛ3-Allel-Trägern (n = 216) und den Trägern des als protektiv geltenden Ɛ2-Allels (n = 50) verglichen. Hierbei zeigte sich eine geringere Oxygenierung der Risikogruppe bei geringer Aufgabenschwierigkeit, während sich ein erhöhter Oxygenierungsanstieg im medialen PFC mit steigender Aufgabenschwierigkeit zeigte. Dies deutet auf einen erhöhten Bedarf an neuronalen Kontrollmechanismen der Risikogruppe zur Bewältigung der steigenden Aufgabenschwierigkeit hin. Die protektive Gruppe zeigte hingegen eine erhöhte Oxygenierung im ventralen PFC mit steigender Aufgabenschwierigkeit, was möglicherweise auf einen präventiven Effekt hindeuten könnte. Weiterführend wurden MCI-Patienten mit gesunden Probanden (n = 57 pro Gruppe) hinsichtlich des kognitiven Risikofaktors verglichen. Hierbei zeigte sich ein punktuell reduzierter Oxygenierunganstieg der MCI Patienten mit steigender Aufgabenschwierigkeit vor allem im ventralen PFC bei ebenfalls stabiler Verhaltensleistung. Die gefundene Reduktion könnte ein Zeichen für eine aufgebrauchte kognitive Reserve sein, welche Einbußen auf Verhaltensebene voranzugehen scheint. Diese charakteristischen Unterschiede in den frontalen Oxygenierungsmustern von Risikogruppen (APOE, MCI) könnten als Biomarker zur Früherkennung von AD noch vor dem Auftreten kognitiver Einbußen dienen. Die fNIRS-Untersuchung während der Durchführung des TMT hat sich in diesem Zusammenhang als potentielles Instrument zur Frühdiagnose der präklinischen Phase der AD als geeignet erwiesen. Die Ergebnisse werden unter Einbezug des wissenschaftlichen Kontexts interpretiert und Implikationen für weitere notwendige Studien sowie die klinische Anwendbarkeit diskutiert. N2 - Due to the improved medical care, the life expectancy of the society steadily rises. Consequently, the socioeconomic relevance of neurodegenerative disorders increases. In order to treat the Alzheimer’s Disease (AD), as the most frequent cause, disease-modulating treatment options are desperately awaited. The extensive preclinical phase of the disease has the potential for gaining new insights for the development of effective treatment strategies. The investigation of risk groups for AD is of great importance for the identification of preclinical prediction markers for the manifestation of a subsequent AD. Especially the presence of genetic risk factors like the Apolipoprotein E (APOE) Ɛ4-allele and cognitive risk factors such as the “mild cognitive impairment” (MCI) are being discussed in this context. Differences in brain activation patterns of risk groups based on functional brain imaging methods have been shown to be beneficial as potential biomarkers for early AD detection. As such, the prefrontal cortex (PFC) which is important for executive control mechanisms has been identified as a key structure of interest. However, many of the involved processes are still not sufficiently understood since most imaging methods are time-consuming and rather expensive. The aim of the present dissertation was to identify differences in PFC oxygenation in two different risk groups for AD by applying a cost-effective and easy-conductible imaging method, the functional Nearinfrared Spectroscopy (fNIRS). In a first step, the Trailmaking Test (TMT), which is a commonly used neuropsychological test for the investigation of executive functioning, was implemented for fNIRS. The neural subtracts were investigated as a basis for the subsequent examination of pre-pathological processes. Besides the usability of the suggested TMT implementation for fNIRS, the comparison of young and elderly subjects (n = 20 per group) showed a specific bilateral PFC oxygenation pattern which was right lateralized for the young group. Elderly adults on the other hand showed a decreased lateralization and more significant channels, pointing towards a need for additional resources in healthy aging. Subsequently the main study examined 604 elderly subjects aged between 70 and 76 years divided in two risk groups (APOE, MCI). In the first step, the genetic risk group of the Ɛ4-allele carriers (n = 78) was compared with the neutral Ɛ3-allele carriers (n = 216) and the carriers of the possibly protective Ɛ2-allele (n = 50). Thereby a reduced oxygenation of the risk group at low task difficulty has been shown, while a raised level of oxygenation increase in the medial PFC was found with growing task difficulty. This points towards a higher demand for neuronal control mechanisms in the genetic risk group in order to keep the performance level stable while task difficulty is increased. The protective group however showed a higher oxygenation in the ventral PFC with increasing task difficulty, which could indicate a higher cognitive reserve. In the second step, the MCI patients were compared with matched healthy control subjects (n = 57 per group). The result showed a reduced increase of oxygenation with increasing task difficulty limited to specific channels mostly within the central PFC while the performance was stable. This reduction could be a sign for the limit of the cognitive reserve, which becomes apparent before the decline of the cognitive performance. The characteristic differences of frontal oxygenation patterns in risk groups (APOE, MCI) could possibly serve as biomarkers for the early AD detection even before task performance declines. The investigation of neural oxygenation with fNIRS during the completion of the TMT has been shown to be suitable as a potential early diagnosis method in the preclinical phase of AD. The results are embedded in the scientific context and implications for future research as well as the clinical applicability are being discussed. KW - Alzheimerkrankheit KW - Apolipoprotein E KW - Leichte kognitive Beeinträchtigung KW - NIR-Spektroskopie KW - Präfrontaler Cortex KW - Trailmaking Test Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-156757 ER - TY - THES A1 - Beck, Sebastian T1 - Using optogenetics to influence the circadian clock of \(Drosophila\) \(melanogaster\) T1 - Die Verwendung der Optogenetik zur Beeinflussung der circadianen Uhr von \(Drosophila\) \(melanogaster\) N2 - Almost all life forms on earth have adapted to the most impactful and most predictable recurring change in environmental condition, the cycle of day and night, caused by the axial rotation of the planet. As a result many animals have evolved intricate endogenous clocks, which adapt and synchronize the organisms’ physiology, metabolism and behaviour to the daily change in environmental conditions. The scientific field researching these endogenous clocks is called chronobiology and has steadily grown in size, scope and relevance since the works of the earliest pioneers in the 1960s. The number one model organism for the research of circadian clocks is the fruit fly, Drosophila melanogaster, whose clock serves as the entry point to understanding the basic inner workings of such an intricately constructed endogenous timekeeping system. In this thesis it was attempted to combine the research on the circadian clock with the techniques of optogenetics, a fairly new scientific field, launched by the discovery of Channelrhodopsin 2 just over 15 years ago. Channelrhodopsin 2 is a light-gated ion channel found in the green alga Chlamydomonas reinhardtii. In optogenetics, researches use these light-gated ion channels like Channelrhodopsin 2 by heterologously expressing them in cells and tissues of other organisms, which can then be stimulated by the application of light. This is most useful when studying neurons, as these channels provide an almost non-invasive tool to depolarize the neuronal plasma membranes at will. The goal of this thesis was to develop an optogenetic tool, which would be able to influence and phase shift the circadian clock of Drosophila melanogaster upon illumination. A phase shift is the adaptive response of the circadian clock to an outside stimulus that signals a change in the environmental light cycle. An optogenetic tool, able to influence and phase shift the circadian clock predictably and reliably, would open up many new ways and methods of researching the neuronal network of the clock and which neurons communicate to what extent, ultimately synchronizing the network. The first optogenetic tool to be tested in the circadian clock of Drosophila melanogaster was ChR2-XXL, a channelrhodopsin variant with dramatically increased expression levels and photocurrents combined with a prolonged open state. The specific expression of ChR2-XXL and of later constructs was facilitated by deploying the three different clock-specific GAL4-driver lines, clk856-gal4, pdf-gal4 and mai179-gal4. Although ChR2-XXL was shown to be highly effective at depolarizing neurons, these stimulations proved to be unable to significantly phase shift the circadian clock of Drosophila. The second series of experiments was conducted with the conceptually novel optogenetic tools Olf-bPAC and SthK-bPAC, which respectively combine a cyclic nucleotide-gated ion channel (Olf and SthK) with the light-activated adenylyl-cyclase bPAC. These tools proved to be quite useful when expressed in the motor neurons of instar-3 larvae of Drosophila, paralyzing the larvae upon illumination, as well as affecting body length. This way, these new tools could be precisely characterized, spawning a successfully published research paper, centered around their electrophysiological characterization and their applicability in model organisms like Drosophila. In the circadian clock however, these tools caused substantial damage, producing severe arrhythmicity and anomalies in neuronal development. Using a temperature-sensitive GAL80-line to delay the expression until after the flies had eclosed, yielded no positive results either. The last series of experiments saw the use of another new series of optogenetic tools, modelled after the Olf-bPAC, with bPAC swapped out for CyclOp, a membrane-bound guanylyl-cyclase, coupled with less potent versions of the Olf. This final attempt however also ended up being unsuccessful. While these tools could efficiently depolarize neuronal membranes upon illumination, they were ultimately unable to stimulate the circadian clock in way that would cause it to phase shift. Taken together, these mostly negative results indicate that an optogenetic manipulation of the circadian clock of Drosophila melanogaster is an extremely challenging subject. As light already constitutes the most impactful environmental factor on the circadian clock, the combination of chronobiology with optogenetics demands the parameters of the conducted experiments to be tuned with an extremely high degree of precision, if one hopes to receive positive results from these types of experiments at all. N2 - Nahezu alle Lebewesen der Erde haben sich an den Tag-Nacht-Zyklus angepasst, die einflussreichste und verlässlichste wiederkehrende Veränderung der Umwelt-bedingungen, verursacht durch die axiale Rotation des Planeten. Daraus resultierend haben viele Tiere komplizierte innere Uhren entwickelt, welche ihre Physiologie, ihren Stoffwechsel und ihr Verhalten an die tägliche Veränderung der natürlichen Bedingungen anpassen. Das Wissenschaftsfeld, das sich der Erforschung dieser inneren Uhren widmet, wird Chronobiologie genannt und hat seit der Arbeit der ersten Pioniere ab 1960 stetig an Größe und Relevanz gewonnen. Der prominenteste Modellorganismus für die Erforschung der circadianen Uhr ist Drosophila melanogaster, deren Uhr als Ansatzpunkt dient, die grundlegenden Vorgänge eines derart komplexen, endogenen Taktsystems zu verstehen. In dieser Thesis wurde versucht die Forschung an der circadianen Uhr mit den Techniken der Optogenetik zu kombinieren, eines jungen Forschungsfeldes, welches durch die Entdeckung von Channelrhodpsin 2 vor über 15 Jahren eröffnet wurde. Channelrhodopsin 2 ist ein Licht-gesteuerter Ionenkanal, der in der Grünalge Chlamydomonas reinhardtii entdeckt wurde. In der Optogenetik nutzen Forscher diese Licht-gesteuerten Ionenkanäle, indem sie sie in den Zellen anderer Organismen exprimieren, welche dann durch Licht stimuliert werden können. Dies ist besonders nützlich bei der Untersuchung von Neuronen, da diese Kanäle ein nahezu nicht-invasives Werkzeug zur Depolarisation neuronaler Membranen bieten. Das Ziel dieser Thesis war es, ein optogenetisches Werkzeug zu entwickeln, welches die circadiane Uhr von Drosophila melanogaster durch Licht manipulieren und deren Phase verschieben kann. Eine Phasenverschiebung ist die adaptive Antwort der circadianen Uhr auf einen äußeren Reiz, welcher eine Veränderung des natürlichen Lichtzyklus signalisiert. Ein optogenetisches Werkzeug, das die Phase der inneren Uhr verlässlich verschieben kann, würde viele neue Möglichkeiten zur Erforschung des neuronalen Uhrnetzwerks eröffnen und wie die Neuronen miteinander kommunizieren um das Netzwerk zu synchronisieren. Das erste optogenetische Werkzeug das in der circadianen Uhr von Drosophila melanogaster getestet wurde war „ChR2-XXL“, eine Channelrhodopsin-Variante mit erhöhter Expression und Photoströmen, gepaart mit einem verlängerten geöffneten Zustand. Die spezifische Expression von ChR2-XXL und auch die späterer Konstrukte wurde durch die Verwendung der drei Uhr-spezifischen GAL4-Treiberlinien clk856-gal4, pdf-gal4 und mai179-gal4 bewerkstelligt. Obwohl bereits gezeigt wurde, dass ChR2-XXL höchst effektiv die Depolarisierung von Neuronen bewirkt, waren diese Stimulationen jedoch nicht in der Lage die Phase der circadianen Uhr von Drosophila signifikant zu verschieben. Die zweite Serie an Versuchen wurde mit den konzeptionell neuartigen optogenetischen Werkzeugen Olf-bPAC und SthK-bPAC durchgeführt, welche jeweils einen durch zyklische Nukleotide gesteuerten Ionenkanal (Olf und SthK) mit der Licht-gesteuerten Adenylatcyclase bPAC kombinieren. Diese Werkzeuge erwiesen sich als äußert nützlich, solange sie in den Motoneuronen von Drosophila-Larven im dritten Larvenstadium exprimiert wurden, wo sie bei Beleuchtung die Larven sowohl paralysierten, als auch deren Körperlänge beeinflussten. Auf diese Weise konnten diese neuen Werkzeuge präzise charakterisiert werden, was in der erfolgreichen Veröffentlichung eines Forschungsartikels mündete, welcher hauptsächlich von der elektrophysiologischen Charakterisierung der Werkzeuge handelte und von deren Anwendungsmöglichkeiten in Modellorganismen wie Drosophila. In der circadianen Uhr verursachten diese Werkzeuge jedoch substantielle Schäden und produzierten schwere Arrhythmie und Anomalien in der neuronalen Entwicklung. Die Verwendung einer temperatur-sensitiven GAL80-Linie um die Expression zu verzögern, erzeugte ebenfalls keinerlei positive Ergebnisse. Für die letzte Serie an Experimenten wurde eine weitere Reihe neuer optogenetischer Werkzeuge verwendet, orientiert an Olf-bPAC und SthK-bPAC, wobei bPAC durch die membrangebundene Guanylatcyclase „CyclOp“ ausgetauscht wurde, welche wiederrum mit weniger wirkstarken Olf-Varianten kombiniert wurde. Dieser letzte Ansatz scheiterte jedoch ebenfalls. Obwohl diese neuen Werkzeuge in der Lage waren die Neuronenmembran bei Beleuchtung effektiv zu depolarisieren, vermochten sie es letztendlich nicht eine Phasenverschiebung zu bewirken. Zusammengenommen zeigen diese überwiegend negativen Ergebnisse, dass die optogenetische Manipulation der circadianen Uhr von Drosophila melanogaster ein extrem anspruchsvolles Thema ist. Da Licht bereits ohnehin den einflussreichsten Umweltfaktor für die circadiane Uhr darstellt, verlangt die Kombination von Chronobiologie und Optogenetik eine extrem präzise Feinabstimmung der Versuchsparameter, um überhaupt darauf hoffen zu dürfen, positive Ergebnisse mit derlei Versuchen zu erzeugen. KW - Chronobiologie KW - Optogenetik KW - Taufliege KW - Optogenetics KW - Chronobiology KW - Channelrhodopsin KW - Drosophila melanogaster Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-184952 ER - TY - JOUR A1 - Jaślan, Dawid A1 - Dreyer, Ingo A1 - Lu, Jinping A1 - O'Malley, Ronan A1 - Dindas, Julian A1 - Marten, Irene A1 - Hedrich, Rainer T1 - Voltage-dependent gating of SV channel TPC1 confers vacuole excitability JF - Nature Communications N2 - In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K\(^+\)-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca\(^{2+}\) levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca\(^{2+}\), can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K\(^{+}\) transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K\(^+\)-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca\(^{2+}\)- and voltage-induced electrical excitability to the central organelle of plant cells. KW - Biophysics KW - Plant signalling Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202029 VL - 10 ER - TY - JOUR A1 - Kehrberger, Sandra A1 - Holzschuh, Andrea T1 - Warmer temperatures advance flowering in a spring plant more strongly than emergence of two solitary spring bee species JF - PLoS ONE N2 - Climate warming has the potential to disrupt plant-pollinator interactions or to increase competition of co-flowering plants for pollinators, due to species-specific phenological responses to temperature. However, studies focusing on the effect of temperature on solitary bee emergence and the flowering onset of their food plants under natural conditions are still rare. We studied the effect of temperature on the phenology of the two spring bees Osmia cornuta and Osmia bicornis, by placing bee cocoons on eleven grasslands differing in mean site temperature. On seven grasslands, we additionally studied the effect of temperature on the phenology of the red-list plant Pulsatilla vulgaris, which was the first flowering plant, and of co-flowering plants with later flowering. With a warming of 0.1°C, the abundance-weighted mean emergence of O. cornuta males advanced by 0.4 days. Females of both species did not shift their emergence. Warmer temperatures advanced the abundance-weighted mean flowering of P. vulgaris by 1.3 days per 0.1°C increase, but did not shift flowering onset of co-flowering plants. Competition for pollinators between P. vulgaris and co-flowering plants does not increase within the studied temperature range. We demonstrate that temperature advances plant flowering more strongly than bee emergence suggesting an increased risk of pollinator limitation for the first flowers of P. vulgaris. KW - Flowering plants KW - Bees KW - Proteus vulgaris KW - Evolutionary emergence KW - Plants KW - Species delimitation KW - Flowers KW - Insect flight Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201165 VL - 14 IS - 6 ER - TY - JOUR A1 - Mammadova-Bach, Elmina A1 - Braun, Attila T1 - Zinc homeostasis in platelet-related diseases JF - International Journal of Molecular Sciences N2 - Zn\(^{2+}\) deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn\(^{2+}\)-deficient diets, accounting for 1–4% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn\(^{2+}\) deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn\(^{2+}\) status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn\(^{2+}\) uptake in the gut using different nutritional supplementation of Zn\(^{2+}\) could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn\(^{2+}\) diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn\(^{2+}\) in hemostasis. Storage protein metallothionein maintains or releases Zn\(^{2+}\) in the cytoplasm, and the dynamic change of this cytoplasmic Zn\(^{2+}\) pool is regulated by the redox status of the cell. An increase of labile Zn\(^{2+}\) pool can be toxic for the cells, and therefore cytoplasmic Zn\(^{2+}\) levels are tightly regulated by several Zn\(^{2+}\) transporters located on the cell surface and also on the intracellular membrane of Zn\(^{2+}\) storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn\(^{2+}\) is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn\(^{2+}\) transport and the physiological role of Zn\(^{2+}\) store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn\(^{2+}\) to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn\(^{2+}\) homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases. KW - Zinc KW - platelets KW - hemostasis KW - thrombosis KW - ischemic stroke KW - storage-pool diseases Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285554 SN - 1422-0067 VL - 20 IS - 21 ER -