TY - JOUR A1 - Villagomez, Gemma N. A1 - Nürnberger, Fabian A1 - Requier, Fabrice A1 - Schiele, Susanne A1 - Steffan-Dewenter, Ingo T1 - Effects of temperature and photoperiod on the seasonal timing of Western honey bee colonies and an early spring flowering plant JF - Ecology and Evolution N2 - Temperature and photoperiod are important Zeitgebers for plants and pollinators to synchronize growth and reproduction with suitable environmental conditions and their mutualistic interaction partners. Global warming can disturb this temporal synchronization since interacting species may respond differently to new combinations of photoperiod and temperature under future climates, but experimental studies on the potential phenological responses of plants and pollinators are lacking. We simulated current and future combinations of temperature and photoperiod to assess effects on the overwintering and spring phenology of an early flowering plant species (Crocus sieberi) and the Western honey bee (Apis mellifera). We could show that increased mean temperatures in winter and early spring advanced the flowering phenology of C. sieberi and intensified brood rearing activity of A. mellifera but did not advance their brood rearing activity. Flowering phenology of C. sieberi also relied on photoperiod, while brood rearing activity of A. mellifera did not. The results confirm that increases in temperature can induce changes in phenological responses and suggest that photoperiod can also play a critical role in these responses, with currently unknown consequences for real-world ecosystems in a warming climate. KW - Apis mellifera KW - climate change KW - rocus sieberi KW - phenology KW - plant–pollinator interaction KW - temporal mismatch Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-258770 VL - 11 IS - 12 ER - TY - JOUR A1 - Viljur, Mari‐Liis A1 - Abella, Scott R. A1 - Adámek, Martin A1 - Alencar, Janderson Batista Rodrigues A1 - Barber, Nicholas A. A1 - Beudert, Burkhard A1 - Burkle, Laura A. A1 - Cagnolo, Luciano A1 - Campos, Brent R. A1 - Chao, Anne A1 - Chergui, Brahim A1 - Choi, Chang‐Yong A1 - Cleary, Daniel F. R. A1 - Davis, Thomas Seth A1 - Dechnik‐Vázquez, Yanus A. A1 - Downing, William M. A1 - Fuentes‐Ramirez, Andrés A1 - Gandhi, Kamal J. K. A1 - Gehring, Catherine A1 - Georgiev, Kostadin B. A1 - Gimbutas, Mark A1 - Gongalsky, Konstantin B. A1 - Gorbunova, Anastasiya Y. A1 - Greenberg, Cathryn H. A1 - Hylander, Kristoffer A1 - Jules, Erik S. A1 - Korobushkin, Daniil I. A1 - Köster, Kajar A1 - Kurth, Valerie A1 - Lanham, Joseph Drew A1 - Lazarina, Maria A1 - Leverkus, Alexandro B. A1 - Lindenmayer, David A1 - Marra, Daniel Magnabosco A1 - Martín‐Pinto, Pablo A1 - Meave, Jorge A. A1 - Moretti, Marco A1 - Nam, Hyun‐Young A1 - Obrist, Martin K. A1 - Petanidou, Theodora A1 - Pons, Pere A1 - Potts, Simon G. A1 - Rapoport, Irina B. A1 - Rhoades, Paul R. A1 - Richter, Clark A1 - Saifutdinov, Ruslan A. A1 - Sanders, Nathan J. A1 - Santos, Xavier A1 - Steel, Zachary A1 - Tavella, Julia A1 - Wendenburg, Clara A1 - Wermelinger, Beat A1 - Zaitsev, Andrey S. A1 - Thorn, Simon T1 - The effect of natural disturbances on forest biodiversity: an ecological synthesis JF - Biological Reviews N2 - Disturbances alter biodiversity via their specific characteristics, including severity and extent in the landscape, which act at different temporal and spatial scales. Biodiversity response to disturbance also depends on the community characteristics and habitat requirements of species. Untangling the mechanistic interplay of these factors has guided disturbance ecology for decades, generating mixed scientific evidence of biodiversity responses to disturbance. Understanding the impact of natural disturbances on biodiversity is increasingly important due to human‐induced changes in natural disturbance regimes. In many areas, major natural forest disturbances, such as wildfires, windstorms, and insect outbreaks, are becoming more frequent, intense, severe, and widespread due to climate change and land‐use change. Conversely, the suppression of natural disturbances threatens disturbance‐dependent biota. Using a meta‐analytic approach, we analysed a global data set (with most sampling concentrated in temperate and boreal secondary forests) of species assemblages of 26 taxonomic groups, including plants, animals, and fungi collected from forests affected by wildfires, windstorms, and insect outbreaks. The overall effect of natural disturbances on α‐diversity did not differ significantly from zero, but some taxonomic groups responded positively to disturbance, while others tended to respond negatively. Disturbance was beneficial for taxonomic groups preferring conditions associated with open canopies (e.g. hymenopterans and hoverflies), whereas ground‐dwelling groups and/or groups typically associated with shady conditions (e.g. epigeic lichens and mycorrhizal fungi) were more likely to be negatively impacted by disturbance. Across all taxonomic groups, the highest α‐diversity in disturbed forest patches occurred under moderate disturbance severity, i.e. with approximately 55% of trees killed by disturbance. We further extended our meta‐analysis by applying a unified diversity concept based on Hill numbers to estimate α‐diversity changes in different taxonomic groups across a gradient of disturbance severity measured at the stand scale and incorporating other disturbance features. We found that disturbance severity negatively affected diversity for Hill number q = 0 but not for q = 1 and q = 2, indicating that diversity–disturbance relationships are shaped by species relative abundances. Our synthesis of α‐diversity was extended by a synthesis of disturbance‐induced change in species assemblages, and revealed that disturbance changes the β‐diversity of multiple taxonomic groups, including some groups that were not affected at the α‐diversity level (birds and woody plants). Finally, we used mixed rarefaction/extrapolation to estimate biodiversity change as a function of the proportion of forests that were disturbed, i.e. the disturbance extent measured at the landscape scale. The comparison of intact and naturally disturbed forests revealed that both types of forests provide habitat for unique species assemblages, whereas species diversity in the mixture of disturbed and undisturbed forests peaked at intermediate values of disturbance extent in the simulated landscape. Hence, the relationship between α‐diversity and disturbance severity in disturbed forest stands was strikingly similar to the relationship between species richness and disturbance extent in a landscape consisting of both disturbed and undisturbed forest habitats. This result suggests that both moderate disturbance severity and moderate disturbance extent support the highest levels of biodiversity in contemporary forest landscapes. KW - natural disturbance KW - diversity–disturbance relationship KW - disturbance severity KW - disturbance extent KW - intermediate disturbance hypothesis KW - forest communities KW - α‐diversity KW - β‐diversity Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-287168 VL - 97 IS - 5 SP - 1930 EP - 1947 ER - TY - JOUR A1 - Vikuk, Veronika A1 - Fuchs, Benjamin A1 - Krischke, Markus A1 - Mueller, Martin J. A1 - Rueb, Selina A1 - Krauss, Jochen T1 - Alkaloid Concentrations of Lolium perenne Infected with Epichloë festucae var. lolii with Different Detection Methods—A Re-Evaluation of Intoxication Risk in Germany? JF - Journal of Fungi N2 - Mycotoxins in agriculturally used plants can cause intoxication in animals and can lead to severe financial losses for farmers. The endophytic fungus Epichloë festucae var. lolii living symbiotically within the cool season grass species Lolium perenne can produce vertebrate and invertebrate toxic alkaloids. Hence, an exact quantitation of alkaloid concentrations is essential to determine intoxication risk for animals. Many studies use different methods to detect alkaloid concentrations, which complicates the comparability. In this study, we showed that alkaloid concentrations of individual plants exceeded toxicity thresholds on real world grasslands in Germany, but not on the population level. Alkaloid concentrations on five German grasslands with high alkaloid levels peaked in summer but were also below toxicity thresholds on population level. Furthermore, we showed that alkaloid concentrations follow the same seasonal trend, regardless of whether plant fresh or dry weight was used, in the field and in a common garden study. However, alkaloid concentrations were around three times higher when detected with dry weight. Finally, we showed that alkaloid concentrations can additionally be biased to different alkaloid detection methods. We highlight that toxicity risks should be analyzed using plant dry weight, but concentration trends of fresh weight are reliable. KW - Epichloë KW - Lolium perenne KW - toxicity KW - grasslands KW - HPLC/UPLC methods KW - endophyte KW - plant fresh/dry weight KW - alkaloid detection methods KW - mycotoxins KW - phenology Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213171 SN - 2309-608X VL - 6 IS - 3 ER - TY - THES A1 - Vikuk, Veronika T1 - Epichloë endophyte-grass symbioses in Germany – Infection rates, alkaloid concentrations and possible intoxication risks T1 - Epichloë Endophyt-Gras Symbiosen in Deutschland – Infektionsraten, Alkaloidkonzentrationen und mögliche Vergiftungsrisiken N2 - Endophytes live in partial symbiosis inside a plant and have been detected in all tested plants. They belong to the group of fungi or bacteria and their ecological function is mostly unknown. The fungal endophytes of the genus Epichloë belong to a special group of endophytes. Epichloë endophytes live symbiotically inside cool season grass species and some of them are able to produce alkaloids toxic to vertebrates and insects. Their symbiosis is seen as mutualistic for the following reasons: the fungus provides the plant herbivore resistance by producing alkaloids, and it increases the plant’s drought tolerance as well as its biomass production. In return, the grass provides the fungus shelter, nutrients and dispersal. Epichloë endophytes are host specific and the ability to produce alkaloids differs between species. In order to estimate intoxication risks in grasslands, it is necessary to detect infection rates of different grass species with Epichloë endophytes, and to determine the genotypes and chemotypes of the Epichloë species as well as the produced alkaloid concentrations. Factors like land-use intensity or season may have an influence on infection rates and alkaloid concentrations. Also, different methodological approaches may lead to different results. In this doctoral thesis my general aim was to evaluate intoxication risks in German grasslands caused by Epichloë endophytes. For that I investigated infection rates of different grass species and the genotypes and chemotypes of their Epichloë endophytes in German grasslands (Chapter II). Furthermore, I compared alkaloid concentrations detected with dry and fresh plant weight and different analytical methods. I also detected possible changes on the influence of season or land-use intensity (Chapter III). Additionally, I examined infections with Epichloë endophytes and alkaloid concentrations in commercially available grass seed mixtures and determined how that influences the intoxication risk of grazing animals in Europe (Chapter IV). It is of agricultural interest to estimate intoxication risks for grazing livestock on German grasslands due to Epichloë infected grass species. Therefore, it is important to investigate which grasses are infected with the Epichloë endophyte, if the endophytes have the ability to produce vertebrate and invertebrate toxic alkaloids and if the alkaloids are indeed produced. I showed that Epichloë festucae var. lolii infecting agriculturally important Lolium perenne lacked the starting gene for ergovaline biosynthesis. Hence, vertebrate toxic ergovaline was not detected in the majority of the collected L. perenne plants. The detection of alkaloid concentrations is an important tool to estimate intoxication risk for vertebrates, but also invertebrates. My studies showed that the usage of dry plant material is crucial to quantify the correct alkaloid concentrations, and that alkaloid concentrations can vary depending on the detection method. Hence, the usage of validated, similar detection methods is important to be able to compare alkaloid concentrations from different studies. Nevertheless, the trends of seasonal changes and the influence of land-use intensity stayed the same, regardless if dry or fresh plant weight was used. Also, alkaloid concentrations were below toxicity thresholds on population level, regardless of the method used. Two commercially available forage grass and two commercially available turf grass seed mixtures were infected with Epichloë endopyhtes and alkaloids were detected. This might contribute to the spreading of Epichloë endopyhtes in Germany, therefore seed mixtures should be tested for Epichloë infections. My results indicate that the intoxication risk is generally low in Germany at the moment, although that might change due to climate change, an increase of monocultural land-use, or the seeding of Epichloë infected grass seeds. N2 - Endophyten leben, zumindest zeitweise, symbiontisch in Pflanzen und sind bisher in allen untersuchten Pflanzen nachgewiesen worden. Es handelt sich dabei um Pilze oder Bakterien und ihre ökologische Funktion ist meistens unbekannt. Eine spezielle Gruppe der Endophyten sind Pilzendophyten der Gattung Epichloë. Diese leben symbiontisch innerhalb von kaltgemäßigten Grasarten und einige sind in der Lage vertebraten- und/oder insektentoxische Alkaloide herzustellen. Die Symbiose wird meist als mutualistisch bezeichnet, weil der Pilz der Pflanze einen Herbivorenschutz durch die Produktion der Alkaloide und eine gesteigerte Trockenresistenz und Biomassesteigerung bietet. Das Gras hingegen bietet dem Pilz Unterkunft, Nährstoffe und Verbreitung. Epichloë Endophyten sind wirtsspezifisch und die Fähigkeit Alkaloide zu produzieren schwankt zwischen den Arten. Um das Vergiftungsrisiko im Grünland einzuschätzen, ist es nötig Infektionsraten verschiedener Grasarten mit Epichloë Endophyten, die Geno- und Chemotypen der Epichloë Arten, und die produzierten Alkaloidkonzentrationen zu bestimmen. Faktoren wie Landnutzungsintensität oder die Jahreszeit können Infektionsraten und Alkaloidkonzentrationen beeinflussen. Ebenso können Alkaloidkonzentrationen von methodischen Faktoren abhängen. In dieser Doktorarbeit habe ich Infektionsraten verschiedener Grasarten in Deutschland und die Geno- und Chemotypen ihrer Epichloë Endophyten untersucht (Kapitel II). Außerdem habe ich Alkaloidkonzentrationen mit Frisch- bzw. Trockengewicht gemessen und mit verschiedenen analytischen Methoden verglichen, um mögliche Änderungen beim Einfluss von Jahreszeiten oder der Landnutzungsintensität zu detektieren. Des Weiteren habe ich das Vergiftungsrisiko auf deutschen Grasflächen abgeschätzt (Kapitel III). Zusätzlich habe ich kommerziell erhältliche Grassaatgutmischungen auf Epichloë Infektionen und Alkaloidgehalt untersucht und habe versucht einzuschätzen, wie sich das auf das Vergiftungsrisiko von Weidevieh in Europa auswirkt (Kapitel IV). Die Einschätzung von Vergiftungsrisiken für Weidevieh aufgrund von Epichloë infizierten Grasarten auf deutschen Graslandflächen ist von landwirtschaftlichem Interesse. Deshalb ist es wichtig zu untersuchen, welche Grasarten mit Epichloë Endophyten infiziert sind, ob der Endophyt in der Lage ist vertebraten- oder insektentoxische Alkaloide zu produzieren und ob diese tatsächlich produziert werden. Ich konnte zeigen, dass Epichloë festucae var. lolii, welches das landwirtschaflich wichtige Lolium perenne infiziert, das Startgen für die Ergovalinbiosynthese fehlt. Deshalb wurde das vertebraten-toxische Ergovalin in der Mehrheit der gesammelten L. perenne Pflanzen nicht nachgewiesen. Die Detektion von Alkaloidkonzentrationen ist ein wichtiges Werkzeug, um das Vergiftungsrisiko für Vertebraten aber auch Invertebraten einschätzen zu können. Ich konnte zeigen, dass die Verwendung von trockenem Pflanzenmaterial essenziell ist, um korrekte Alkaloidkonzentrationen zu quantifizieren und dass Alkaloidkonzentrationen in Abhängigkeit von der Detektionsmethode schwanken können. Deshalb ist die Verwendung von validierten, ähnlichen Detektionsmethoden wichtig, um die Alkaloidkonzentrationen von verschiedenen Studien vergleichen zu können. Dennoch blieben die jahreszeitlichen Trends und der Einfluss von Landnutzungsintensität gleich, egal ob Trocken- oder Frischgewicht der Pflanze verwendet wurde und Alkaloidkonzentrationen lagen unter der Toxizitätsschwelle auf Populationsebene. Ich konnte außerdem zeigen, dass zwei kommerziell erwerbliche Futtergrasmischungen, sowie zwei Rasengrasmischungen mit Epichloë Endophyten infiziert waren und auch Alkaloide detektiert werden konnten. Das könnte zu einer weiteren Ausbreitung von Epichloë-Endophyten in Deutschland beitragen, weshalb Saatgutmischungen auf Epichloë Infektionen getestet werden sollten. Meine Ergebnisse zeigen, dass das Vergiftungsrisiko in Deutschland im Moment generell eher niedrig ist. Allerdings kann sich das auf Grund von Klimawandel, zunehmenden Monokulturen in der Landnutzung, aber auch der Aussaat von Epichloë infiziertem Saatgut ändern. KW - Endophytische Pilze KW - HPLC-MS KW - Deutsches Weidelgras KW - Weidegräser KW - Alkaloide KW - intoxication risk KW - alkaloid concentrations KW - Epichloe endophytes KW - cool-season grass species KW - infection rates Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213895 ER - TY - JOUR A1 - Viera, Jonathan Trujillo A1 - El-Merahbi, Rabih A1 - Nieswandt, Bernhard A1 - Stegner, David A1 - Sumara, Grzegorz T1 - Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight JF - PLoS ONE N2 - Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1\(^{-/-}\) and Pld2\(^{-/-}\) mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes. KW - enzyme regulation KW - insulin resistance KW - body weight KW - mouse models KW - bioenergetics KW - insulin KW - hypothalamus KW - adipose tissue Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179729 VL - 11 IS - 6 ER - TY - JOUR A1 - Vieira, Jacqueline A1 - Jones, Alex R. A1 - Danon, Antoine A1 - Sakuma, Michiyo A1 - Hoang, Nathalie A1 - Robles, David A1 - Tait, Shirley A1 - Heyes, Derren J. A1 - Picot, Marie A1 - Yoshii, Taishi A1 - Helfrich-Förster, Charlotte A1 - Soubigou, Guillaume A1 - Coppee, Jean-Yves A1 - Klarsfeld, André A1 - Rouyer, Francois A1 - Scrutton, Nigel S. A1 - Ahmad, Margaret T1 - Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability JF - PLoS One N2 - Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism. KW - arabidopsi KW - dependent magnetosensitvity KW - protein KW - clock KW - gene KW - mechanism KW - rhythm KW - oscillator KW - circadian photoreception KW - mammalian CRY1 Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134513 VL - 7 IS - 3 ER - TY - JOUR A1 - Viebrock, A. A1 - Perz, A. A1 - Sebald, Walter T1 - The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA N2 - No abstract available KW - Biochemie Y1 - 1982 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62742 ER - TY - JOUR A1 - Viebrock, A A1 - Perz, A A1 - Sebald, Walter T1 - Molecular cloning of middle-abundant mRNAs from Neurospora crassa N2 - no abstract available KW - Neurospora crassa Y1 - 1983 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82033 ER - TY - JOUR A1 - Vey, Johannes A1 - Kapsner, Lorenz A. A1 - Fuchs, Maximilian A1 - Unberath, Philipp A1 - Veronesi, Giulia A1 - Kunz, Meik T1 - A toolbox for functional analysis and the systematic identification of diagnostic and prognostic gene expression signatures combining meta-analysis and machine learning JF - Cancers N2 - The identification of biomarker signatures is important for cancer diagnosis and prognosis. However, the detection of clinical reliable signatures is influenced by limited data availability, which may restrict statistical power. Moreover, methods for integration of large sample cohorts and signature identification are limited. We present a step-by-step computational protocol for functional gene expression analysis and the identification of diagnostic and prognostic signatures by combining meta-analysis with machine learning and survival analysis. The novelty of the toolbox lies in its all-in-one functionality, generic design, and modularity. It is exemplified for lung cancer, including a comprehensive evaluation using different validation strategies. However, the protocol is not restricted to specific disease types and can therefore be used by a broad community. The accompanying R package vignette runs in ~1 h and describes the workflow in detail for use by researchers with limited bioinformatics training. KW - bioinformatics tool KW - R package KW - machine learning KW - meta-analysis KW - biomarker signature KW - gene expression analysis KW - survival analysis KW - functional analysis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193240 SN - 2072-6694 VL - 11 IS - 10 ER - TY - THES A1 - Vershenya, Stanislav T1 - Quantitative and qualitative analyses of in-paralogs N2 - In our analysis I was interested in the gene duplications, with focus on in-paralogs. In-paralogs are gene duplicates which arose after species split. Here I analysed the in-paralogs quantitatively, as well as qualitatively. For quantitative analysis genomes of 21 species were taken. Most of them have vastly different lifestyles with maximum evolutionary distance between them 1100 million years. Species included mammals, fish, insects and worm, plus some other chordates. All the species were pairwised analysed by the Inparanoid software, and in-paralogs matrix were built representing number of in-paralogs in all vs. all manner. Based on the in-paralogs matrix I tried to reconstruct the evolutionary tree using in-paralog numbers as evolutionary distance. If all 21 species were used the resulting tree was very far from real one: a lot of species were misplaced. However if the number was reduced to 12, all of the species were placed correctly with only difference being wrong insect and fish clusters switched. Then to in-paralogs matrix the neighbour-net algorithm was applied. The resulting "net" tree showed the species with fast or slow duplications rates compared to the others. We could identify species with very high or very low duplications frequencies and it correlates with known occurrences of the whole genome duplications. As the next step I built the graphs for every single species showing the correlation between their in-paralogs number and evolutionary distance. As we have 21 species, graph for every species is built using 20 points. Coordinates of the points are set using the evolutionary distance to that particular species and in-paralogs number. In mammals with increasing the distance from speciation the in-paralogs number also increased, however not in linear fashion. In fish and insects the graph close to zero is just the same in mammals' case. However, after reaching the evolutionary distances more than 800 million years the number of inparalogs is beginning to decrease. We also made a simulation of gene duplications for all 21 species and all the splits according to the fossil and molecular clock data from literature. In our simulation duplication frequency was minimal closer to the past and maximum in the near-present time. Resulting curves had the same shape the experimental data ones. In case of fish and insect for simulation the duplication rate coefficient even had to be set negative in order to repeat experimental curve shape. To the duplication rate coefficient in our simulation contribute 2 criteria: gene duplications and gene losses. As gene duplication is stochastical process it should always be a constant. So the changing in the coefficient should be solely explained by the increasing gene loss of old genes. The processes are explained by the evolution model with high gene duplication and loss ratio. The drop in number of in-paralogs is probably due to the BLAST algorithm. It is observed in comparing highly divergent species and BLAST cannot find the orthologs so precisely anymore. In the second part of my work I concentrated more on the specific function of inparalogs. Because such analysis is time-consuming it could be done on the limited number species. Here I used three insects: Drosophila melanogaster (fruit y), Anopheles gambiae (mosquito) and Apis mellifera (honeybee). After Inparnoid analyses and I listed the cluster of orthologs. Functional analyses of all listed genes were done using GO annotations and also KEGG PATHWAY database. We found, that the gene duplication pattern is unique for each species and that this uniqueness is rejected through the differences in functional classes of duplicated genes. The preferences for some classes reject the evolutionary trends of the last 350 million years and allow assumptions on the role of those genes duplications in the lifestyle of species. Furthermore, the observed gene duplications allowed me to find connections between genomic changes and their phenotypic manifestations. For example I found duplications within carbohydrate metabolism rejecting feed pattern adaptation, within photo- and olfactory-receptors indicating sensing adaptation and within troponin indicating adaptations in the development. Despite these species specific differences, found high correlations between the independently duplicated genes between the species. This might hint for a "pool" of genes preferentially duplicated. Taken together, the observed duplication patterns reject the adaptational process and provide us another link to the field of genomic zoology. N2 - In unserer Analyse untersuchten wir Genduplikationen mit besonderem Fokus auf "Inparalogen". In-paraloge sind Genduplikationen die nach Speziazion enstehen. Diese betrachteten wir hier in einer quantitativen als auch qualitativen Messreihe. Die quantitative Analyse umfasste Genome aus insgesamt 21 Spezies. Der Großteil diese hat verschiedene Lebensgewonheiten mit eine maximalen Evolutionsdistanz von 1100 Millionen Jahren. Die Arten bestanden aus Säugetiere, Fischen, Insekten und Würmern, sowie weiteren Chordaten. Alle Arten wurden mittels der Inparanoid Software paarweise "all against all" analysiert und in in-paralog Matrizen gespeichert. Basierend auf der in-paralog Matrix versuchten wir den evolutionären Baum über die Anzahl der In-paraloge als Maß für die evolutionäre Distanz zu rekonstruiren. Bei der Betrachtung alle 21 Arten würde der Baum jedoch sehr unpräzise: viel Arten wurden falsch plaziert. Durch eine Reduktion der Anzahl auf nur 12 Spezies clusterten jedoch alle Arten richtig, nur Insekten und Fische waren vertauscht. Anschließend wurde auf die In-paralog Matrix der Neighbor-net Algorithmus angewandt. Der daraus resultierende "Netz"-Baum repräsentiert die Spezies mit schneller oder langsamer Duplikationsrate im Vergleich zu den Anderen. Wir konnten Spezies mit sehr niedriger oder sehr hoher Rate identifizieren. Dabei korrelieren die Genome mit der höheren Rate zu der Anzahl der auftauchenden Whole Genome Duplikationen. Im nächsten Schritt erstellten wir Graphen für jede einzelne Spezies die das Verhältnis zwischen der Anzahl ihrer In-paraloger zur evolutionäre Distanz anzeigen. Jeder der 21 Graphen enthält insgesamt 20 Punkte. Die Punktkoordianten repräsentiern die evolutionere Distanz auf der X-Achse zu der Anzahl In-paraloger auf der Y-Achse. Bei Säugertieren wächst mit steigender Distanz auch die Anzahl In-paraloger. Das Verhältnis ist jedoch nicht linear. Bei Fischen und Insekten ist der Graph in der Nähe des Nullpunkts gleich dem von Säugetieren. Beim Erreichen einer Distanz von mehr als 800 Millionen Jahren sinkt jedoch die Anzahl der In-paralogen. Wir haben nun zusätzlich eine Simulation der Genduplikationen für alle 21 Spezies und alle dazu gehörigen Splits durchgeführt. Die Splits wurden aus publizierten Fossilien und "Molecular Clock" Daten entnommen. In unsere Simulation stieg die Duplikationsrate mit Annäherung an die heutige Zeit. In Vergleich zu den Experimentellen Daten haben die simulierten Graphen das gleiche Aussehen. Bei Fischen und Insekten musste der Koeffizient der Duplikationsrate negiert werden um die experimentelle Kurve zu erhalten. Der Koeffizient der Duplikationsrate stützt sich dabei auf folgende 2 Kriterien: Gen-Duplikation und Gen-Verlust. Da Genduplikationen einem stochastischen Prozess folgen sollten sie immer konstant sein. Daher sind die erhöhten Genverluste alter Gene verantwortlich für die Veränderunrg dieses Koeffizienten. Die Erklärung für dieses Verhalten basiert auf dem Evolutionsmodel - mit hohem Gen-Verlust und hoher Gen Duplikation. Der Verlust der In-Paralogen enstehet wahrscheinlich durch den BLAST Algorithmus. Man beobachtet dies besonders bei sehr divergenten Arten bei dennen BLAST die Orthologen nicht mehr so präzise findet. Der zweite Teil meiner Arbeit bezieht sich auf die spezifische Funktion von In-paralogen. Da diese Analyse sehr zeitaufwendig ist konnte sie nur an einer begrenzten Anzahl von Spezies durchgeführt werden. Hier habe ich die folgenden drei Insekten verwendet: Drosophila melanogaster (Fruchtfliege), Anopheles gambiae (Moskito) und Apis mellifera (Honigbiene). Alle durch die Inparanoid-Software entstandenen Cluster wurden mit der GO Annotation und der KEGG Pathway Datenbank analyiert. Wir haben herausgefunden, dass das Gen-Duplikationsmuster für jede Spezies einzigartig ist, und dass diese Einzigartigkeit durch Funktionale Unterschiede in duplizierten Genen entsteht. Die Bevorzugung einiger Gene repräsentiert die Evolutionsgeschichte der letzten 350 Millionen Jahre und erlaubt Annahmen über die Auswirkung der Gen Duplikationen im Leben der Spezies zu treffen. Weiterhin fanden wir durch die beobachteten Genduplikationen Zusammenhänge zwischen der Genomveränderung und ihrer phenotypischen Manifestation. Beispielsweise haben wir Duplikationen innerhalb des Karbohydratestoffwechsels für die Anpassung des Essvehaltens, Photo- und Olifaktorisch Rezeptoren - für Seh- und Geruchsvermögen und Troponin - zuständig für die Muskelentwicklung gefunden. Trotz diese speziesspezifischen Unterschiede haben wir starke Korrelation zwischen unabhängig duplizierten Genen erkannt. Dies könnte ein Indikator für einen "Pool" von bevorzugt duplizierten Genen sein. Zusammengefasst stellen die beobachteten Duplikationsmuster den Evolvierungsprozess dar, und liefern eine weitere Verbindung zur genomischen Zoologie. KW - Duplikation KW - Evolution KW - Genetik KW - In-paralogs KW - Gene duplication KW - Inparanoid Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-51358 ER - TY - JOUR A1 - Vergho, Daniel A1 - Kneitz, Susanne A1 - Rosenwald, Andreas A1 - Scherer, Charlotte A1 - Spahn, Martin A1 - Burger, Maximilian A1 - Riedmiller, Hubertus A1 - Kneitz, Burkhard T1 - Combination of expression levels of miR-21 and miR-126 is associated with cancer-specific survival in clear-cell renal cell carcinoma N2 - Background Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs. Methods Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC). Results RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease. Conclusions A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis. KW - Renal cell carcinoma KW - RCC KW - Kidney cancer KW - miRNA KW - miR-21 KW - miR-126 KW - Prognosis KW - Profiling KW - Biomarker KW - Tumour markers Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110061 ER - TY - JOUR A1 - Vergho, Daniel Claudius A1 - Kneitz, Susanne A1 - Kalogirou, Charis A1 - Burger, Maximilian A1 - Krebs, Markus A1 - Rosenwald, Andreas A1 - Spahn, Martin A1 - Löser, Andreas A1 - Kocot, Arkadius A1 - Riedmiller, Hubertus A1 - Kneitz, Burkhard T1 - Impact of miR-21, miR-126 and miR-221 as Prognostic Factors of Clear Cell Renal Cell Carcinoma with Tumor Thrombus of the Inferior Vena Cava N2 - Clear cell renal cell carcinoma (ccRCC) characterized by a tumor thrombus (TT) extending into the inferior vena cava (IVC) generally indicates poor prognosis. Nevertheless, the risk for tumor recurrence after nephrectomy and thrombectomy varies. An applicable and accurate prediction system to select ccRCC patients with TT of the IVC (ccRCC/TT) at high risk after nephrectomy is urgently needed, but has not been established up to now. To our knowledge, a possible role of microRNAs (miRs) for the development of ccRCC/TT or their impact as prognostic markers in ccRCC/TT has not been explored yet. Therefore, we analyzed the expression of the previously described onco-miRs miR-200c, miR-210, miR-126, miR-221, let-7b, miR-21, miR-143 and miR-141 in a study collective of 74 ccRCC patients. Using the expression profiles of these eight miRs we developed classification systems that accurately differentiate ccRCC from non-cancerous renal tissue and ccRCC/TT from tumors without TT. In the subgroup of 37 ccRCC/TT cases we found that miR-21, miR-126, and miR-221 predicted cancer related death (CRD) accurately and independently from other clinico-pathological features. Furthermore, a combined risk score based on the expression of miR-21, miR-126 and miR-221 was developed and showed high sensitivity and specificity to predict cancer specific survival (CSS) in ccRCC/TT. Using the combined risk score we were able to classify ccRCC/TT patients correctly into high and low risk cases. The risk stratification by the combined risk score (CRS) will benefit from further cohort validation and might have potential for clinical application as a molecular prediction system to identify high- risk ccRCC/TT patients. KW - forecasting KW - metastasis KW - renal cancer KW - renal cell carcinoma KW - kidneys KW - surgical oncology KW - surgical and invasive medical procedures KW - regression analysis Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113633 ER - TY - JOUR A1 - Venjakob, Christine A1 - Leonhardt, Sara A1 - Klein, Alexandra-Maria T1 - Inter-individual nectar chemistry changes of field scabious, Knautia arvensis JF - Insects N2 - Nectar is crucial to maintain plant-pollinator mutualism. Nectar quality (nutritional composition) can vary strongly between individuals of the same plant species. The factors driving such inter-individual variation have however not been investigated closer. We investigated nectar quality of field scabious, Knautia arvensis in different grassland plant communities varying in species composition and richness to assess whether nectar quality can be affected by the surrounding plant community. We analyzed (with high performance liquid chromatography) the content of carbohydrates, overall amino acids, and essential amino acids. Amino acid and carbohydrate concentrations and proportions varied among plant individuals and with the surrounding plant community but were not related to the surrounding plant species richness. Total and individual carbohydrate concentrations were lowest, while proportions of the essential amino acids, valine, isoleucine, leucine (all phagostimulatory), and lysine were highest in plant species communities of the highest diversity. Our results show that K. arvensis nectar chemistry varies with the composition of the surrounding plant community, which may alter the taste and nutritional value and thus affect the plant’s visitor spectrum and visitation rate. However, the strong inter-individual variation in nectar quality requires additional studies (e.g., in semi-field studies) to disentangle different biotic and abiotic factors contributing to inter-individual nectar chemistry in a plant-community context. KW - amino acids KW - carbohydrates KW - flower-visiting insects KW - insect nutrition KW - Jena Experiment Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200866 SN - 2075-4450 VL - 11 IS - 2 ER - TY - JOUR A1 - Venjakob, C. A1 - Ruedenauer, F. A. A1 - Klein, A.‐M. A1 - Leonhardt, S. D. T1 - Variation in nectar quality across 34 grassland plant species JF - Plant Biology N2 - Floral nectar is considered the most important floral reward for attracting pollinators. It contains large amounts of carbohydrates besides variable concentrations of amino acids and thus represents an important food source for many pollinators. Its nutrient content and composition can, however, strongly vary within and between plant species. The factors driving this variation in nectar quality are still largely unclear. We investigated factors underlying interspecific variation in macronutrient composition of floral nectar in 34 different grassland plant species. Specifically, we tested for correlations between the phylogenetic relatedness and morphology of plants and the carbohydrate (C) and total amino acid (AA) composition and C:AA ratios of nectar. We found that compositions of carbohydrates and (essential) amino acids as well as C:AA ratios in nectar varied significantly within and between plant species. They showed no clear phylogenetic signal. Moreover, variation in carbohydrate composition was related to family‐specific structural characteristics and combinations of morphological traits. Plants with nectar‐exposing flowers, bowl‐ or parabolic‐shaped flowers, as often found in the Apiaceae and Asteraceae, had nectar with higher proportions of hexoses, indicating a selective pressure to decelerate evaporation by increasing nectar osmolality. Our study suggests that variation in nectar nutrient composition is, among others, affected by family‐specific combinations of morphological traits. However, even within species, variation in nectar quality is high. As nectar quality can strongly affect visitation patterns of pollinators and thus pollination success, this intra‐ and interspecific variation requires more studies to fully elucidate the underlying causes and the consequences for pollinator behaviour. KW - flower morphology KW - flowering grassland plants KW - Jena Experiment KW - nectar macronutrients KW - phylogeny Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262612 VL - 24 IS - 1 SP - 134 EP - 144 ER - TY - JOUR A1 - Vendelova, Emilia A1 - de Lima, Jeferson Camargo A1 - Lorenzatto, Karina Rodrigues A1 - Monteiro, Karina Mariante A1 - Mueller, Thomas A1 - Veepaschit, Jyotishman A1 - Grimm, Clemens A1 - Brehm, Klaus A1 - Hrčková, Gabriela A1 - Lutz, Manfred B. A1 - Ferreira, Henrique B. A1 - Nono, Justin Komguep T1 - Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions JF - PLoS Neglected Tropical Diseases N2 - Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts. KW - proteomic analysis KW - excretory-secretory KW - Mesocestoides corti Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166742 VL - 10 IS - 10 ER - TY - JOUR A1 - Velours, J. A1 - Esparza, M. A1 - Hoppe, J. A1 - Sebald, Walter A1 - Guerin, B. T1 - Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae N2 - The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the A TP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 8011/o fonnie acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 5011/o homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 genein Aspergillus nidulans. KW - Biochemie KW - ATP synthase KW - mitochondrially translated KW - proteolipid KW - sequence subunit Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62695 ER - TY - JOUR A1 - Vellmer, Tim A1 - Hartleb, Laura A1 - Fradera Sola, Albert A1 - Kramer, Susanne A1 - Meyer-Natus, Elisabeth A1 - Butter, Falk A1 - Janzen, Christian J. T1 - A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling JF - PLoS Pathogens N2 - A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed. KW - Trypanosoma KW - chromatin KW - histones KW - RNA interference KW - Trypanosoma brucei gambiense KW - luciferase KW - transcriptional control KW - nucleosomes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301372 VL - 18 IS - 6 ER - TY - THES A1 - Vellmer, Tim T1 - New insights into the histone variant H2A.Z incorporation pathway in \(Trypanosoma\) \(brucei\) T1 - Neue Erkenntnisse zum Einbau der Histonvariante H2A.Z in \(Trypanosoma\) \(brucei\) N2 - The histone variant H2A.Z is a key player in transcription regulation in eukaryotes. Histone acetylations by the NuA4/TIP60 complex are required to enable proper incorporation of the histone variant and to promote the recruitment of other complexes and proteins required for transcription initiation. The second key player in H2A.Z-mediated transcription is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant. By the time this project started little was known about H2A.Z in the unicellular parasite Trypanosoma brucei. Like in other eukaryotes H2A.Z was exclusively found in the transcription start sites of the polycistronic transcription units where it keeps the chromatin in an open conformation to enable RNA-polymerase II-mediated transcription. Previous studies showed the variant colocalizing with an acetylation of lysine on histone H4 and a methylation of lysine 4 on histone H3. Data indicated that HAT2 is linked to H2A.Z since it is required for acetylation of lyinse 10 on histone H4. A SWR1-like complex and a complex homologous to the NuA4/TIP60 could not be identified yet. This study aimed at identifying a SWR1-like remodelling complex in T. brucei and at identifying a protein complex orthologous to NuA4/TIP60 as well as at answering the question whether HAT2 is part of this complex or not. To this end, I performed multiple mass spectrometry-coupled co-Immunoprecipitation assays with potential subunits of a SWR1 complex, HAT2 and a putative homolog of a NuA4/TIP60 subunit. In the course of these experiments, I was able to identify the TbSWR1 complex. Subsequent cell fractionation and chromatin immunoprecipitation-coupled sequencing analysis experiments confirmed, that this complex is responsible for the incorporation of the histone variant H2A.Z in T. brucei. In addition to this chromatin remodelling complex, I was also able to identify two histone acetyltransferase complexes assembled around HAT1 and HAT2. In the course of my study data were published by the research group of Nicolai Siegel that identified the histone acetyltransferase HAT2 as being responsible for histone H4 acetylation, in preparation to promote H2A.Z incorporation. The data also indicated that HAT1 is responsible for acetylation of H2A.Z. According to the literature, this acetylation is required for proper transcription initiation. Experimental data generated in this study indicated, that H2A.Z and therefore TbSWR1 is involved in the DNA double strand break response of T. brucei. The identification of the specific complex composition of all three complexes provided some hints about how they could interact with each other in the course of transcription regulation and the DNA double strand break response. A proximity labelling approach performed with one of the subunits of the TbSWR1 complex identified multiple transcription factors, PTM writers and proteins potentially involved in chromatin maintenance. Overall, this work will provide some interesting insights about the composition of the complexes involved in H2A.Z incorporation in T. brucei. Furthermore, it is providing valuable information to set up experiments that could shed some light on RNA-polymerase II-mediated transcription and chromatin remodelling in T. brucei in particular and Kinetoplastids in general. N2 - Die Histonvariante H2A.Z ist ein Schlüsselelement bei der Transkriptionsregulation in Eukaryoten. Histonacetylierungen die vom NuA4/Tip60 Komplex prozessiert werden, sind für den korrekten Einbau der Variante unerlässlich. Darüber hinaus erlauben diese posttranslationellen Modifikationen die Rekrutierung weiterer Proteine und Komplexe die für die Transkription notwendig sind. Ein weiteres Schlüsselelement der mittels H2A.Z regulierten Transkription ist der Komplex zur Umstrukturierung des Chromatins SWR1, welcher das kanonische Histon H2A gegen seine Variante austauscht. Zu Beginn dieses Projektes war der Wissenstand bezüglich der Histonvariante H2A.Z in dem einzelligen Parasiten Trypanosoma brucei limitiert. Wie in anderen eukaryotischen Organismen wurde die Variante ausschließlich an den Startpunkten der polyzistronischen Transkriptionseinheiten gefunden, an denen es für die Öffnung des Chromatins verantwortlich ist und so die Transkription mittels RNAPolymerase II ermöglicht. Vorangegangene Studien konnten zeigen, dass die Variante mit einer Acetylierung des Lysins 10 im Histon H4 und einer Methylierung des Lysins 4 im Histon H3 co-lokalisiert. Einige Daten lieferten den Hinwies, dass die Histon-Acetyltransferase HAT2 mit H2A.Z in Zusammenhang steht, da diese die Acetylierung des Lysins 10 im Hinston H4 prozessiert. Komplexe die in ihrer Funktion dem SWR1 oder dem NuA4/TIP60 Komplex entsprechen, konnten bisher noch nicht gefunden werden. Die vorliegende Arbeit zielt darauf ab Komplexe zu identifizieren, die in ihrer Funktion dem SWR1 sowie dem NuA4/TIP60 Komplex entsprechen. Zudem soll die Frage geklärt werden ob HAT2 Teil eines möglichen NuA4/TIP60 Komplexes ist. In diesem Zusammenhang habe ich mehrere Massenspektrometrie gekoppelte Co-Immunopräzipitationen mit potenziellen Untereinheiten eines SWR1 Komplexes sowie HAT2 und einem Protein welches otholog zu einer NuA4/TIP60 Untereinheit ist, durchgeführt. Im Verlauf dieser Experimente konnte der SWR1 Komplex in T. brucei (TbSWR1) identifiziert werden. Anschließende Zellfraktionierungen sowie Chromatin Immunopräzipitationen gekoppelte Sequenzanalysen konnten bestätigen, dass der identifizierte Komplex für den Einbau der Histonvariante H2A.Z zuständig ist. Darüber hinaus konnten neben diesem Komplex noch zwei weitere Komplexe identifiziert werden, die jeweils die Histonacetyltransferasen HAT1 und HAT2 als Kernkomponenten enthalten. Im Verlauf meiner Arbeit wurden von der Arbeitsgruppe von Nicolai Siegel Daten publiziert die zeigten, dass die Histonacetyltransferase HAT2, in Vorbereitung auf den Einbau von H2A.Z, für die Acetylierung des Histons H4 verantwortlich ist. Im Gegenzug ist HAT1 für die Acetylierung von H2A.Z notwendig, welche wiederum für die korrekte Initiation der Transkription benötigt wird. Damit entspricht die Funktion der Acetylierung von H2A.Z in T. brucei der in der Literatur beschriebenen Funktion. Experimentelle Daten die im Verlauf dieser Arbeit generiert wurden, lieferten einen Hinweis darauf, dass H2A.Z auch an der Reparatur von DNS Doppelstrangbrüchen beteiligt ist. Die Aufschlüsselung der spezifischen Zusammensetzung aller drei Komplexe gab einige Hinweise darauf, wie sie sowohl während der Transkriptionsregulation als auch der Reparatur von DNS Doppelstrangbrüchen miteinander interagieren. Im Zuge einer molekularen Umgebungskartierung, die mit einer der Untereinheiten des TbSWR1 Komplexes durchgeführt wurde, konnten mehrere Transkriptionsfaktoren und Enzyme zur Histonmodifizierung identifiziert werden. Dabei wurden auch einige Proteine identifiziert, welche möglicherweise mit der Umformung des Chromatins in Zusammenhang stehen. Abschließend ist festzuhalten, dass diese Arbeit einige äußerst interessante Einsichten über die Zusammensetzung der Komplexe, die am H2A.Z Einbau in T. brucei beteiligt sind, liefern konnte. Darüber hinaus stellt sie einige wertvolle Informationen zur Verfügung. Diese könnten zur gezielten Planung von Experimenten genutzt werden, um mehr über RNA-Polymerase II vermittelte Transkription und Chromatin Umstrukturierung in T. brucei im speziellen und in Kinetoplastiden im Allgemeinen zu erfahren. KW - Chromatinremodelling KW - Histone KW - Transkription KW - Chromatinremodeling KW - Histones KW - Variants KW - Complexes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257960 ER - TY - JOUR A1 - Vaze, Koustubh M. A1 - Helfrich-Förster, Charlotte T1 - Drosophila ezoana uses an hour-glass or highly damped circadian clock for measuring night length and inducing diapause JF - Physiological Entomology N2 - Insects inhabiting the temperate zones measure seasonal changes in day or night length to enter the overwintering diapause. Diapause induction occurs after the duration of the night exceeds a critical night length (CNL). Our understanding of the time measurement mechanisms is continuously evolving subsequent to Bünning’s proposal that circadian systems play the clock role in photoperiodic time measurement (Bünning, 1936). Initially, the photoperiodic clocks were considered to be either based on circadian oscillators or on simple hour-glasses, depending on ‘positive’ or ‘negative’ responses in Nanda–Hamner and Bünsow experiments (Nanda & Hammer, 1958; Bünsow, 1960). However, there are also species whose responses can be regarded as neither ‘positive’, nor as ‘negative’, such as the Northern Drosophila species Drosophila ezoana, which is investigated in the present study. In addition, modelling efforts show that the ‘positive’ and ‘negative’ Nanda–Hamner responses can also be provoked by circadian oscillators that are damped to different degrees: animals with highly sustained circadian clocks will respond ‘positive’ and those with heavily damped circadian clocks will respond ‘negative’. In the present study, an experimental assay is proposed that characterizes the photoperiodic oscillators by determining the effects of non-24-h light/dark cycles (T-cycles) on critical night length. It is predicted that there is (i) a change in the critical night length as a function of T-cycle period in sustained-oscillator-based clocks and (ii) a fxed night-length measurement (i.e. no change in critical night length) in damped-oscillator-based clocks. Drosophila ezoana flies show a critical night length of approximately 7 h irrespective of T-cycle period, suggesting a damped-oscillator-based photoperiodic clock. The conclusion is strengthened by activity recordings revealing that the activity rhythm of D. ezoana flies also dampens in constant darkness. KW - photoperiodic time mesurement KW - wyeomyia smithii KW - protophormia terraenovae KW - immunoreactive neurons KW - geographical variation KW - reproductive diapause KW - rhythmic components KW - locomotor activity KW - circadian clock KW - damped-oscillator-model of photoperiodic clock KW - diapause KW - Drosophila KW - hour-glass KW - pitcher-plant mosquito KW - bug riptortus-pedestris KW - Nanda-Hamner KW - photoperiodism Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204278 VL - 41 IS - 4 ER - TY - THES A1 - Varagnolo, Linda T1 - PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem/progenitor cells T1 - Die Inhibition des PRC2 wirkt dem Kultur-bedingten Verlust des Repopulationspotenzials in humanen hämatopoetischen Stammzellen/Vorläuferzellen aus Nabelschnurblut entgegen N2 - Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for the treatment of a variety of malignant and non-malignant disorders. However, the low amount of cells collected per donor is often insufficient for treatment of adult patients. In order to make sufficient numbers of CB-HSCs available for adults, expansion is required. Different approaches were described for HSC expansion, however these approaches are impeded by the loss of engrafting potential during ex vivo culture. Little is known about the underlying molecular mechanisms. Epigenetic mechanisms play essential roles in controlling stem cell potential and fate decisions and epigenetic strategies are considered for HSC expansion. Therefore, this study aimed to characterize global and local epigenotypes during the expansion of human CB-CD34+, a well established CB progenitor cell type, to better understand the molecular mechanisms leading to the culture-associated loss of engrafting potential. Human CB-CD34+ cells were cultured using 2 different cytokine cocktails: the STF cocktail containing SCF, TPO, FGF-1 and the STFIA cocktail, which combines STF with Angiopoietin-like 5 (Angptl5) and Insulin-like growth factor-binding protein 2 (IGFBP2). The latter expands CB-HSCs ex vivo. Subsequently, the NOD-scid gamma (NSG) mouse model was used to study the engraftment potential of expanded cells. Engraftment potential achieved by fresh CB-CD34+ cells was maintained when CB-CD34+ cells were expanded under STFIA but not under STF conditions. To explore global chromatin changes in freshly isolated and expanded CB-CD34+ cells, levels of the activating H3K4me3 and the repressive H3K27me3 histone marks were determined by chromatin flow cytometry and Western blot analyses. For analysis of genome-wide chromatin changes following ex vivo expansion, transcriptome profiling by microarray and chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) were performed. Additionally, local chromatin transitions were monitored by ChIP analyses on promoter regions of developmental and self-renewal factors. On a global level, freshly isolated CD34+ and CD34- cells differed in H3K4me3 and H3K27me3 levels. After 7 days of expansion, CD34+ and CD34- cells adopted similar levels of active and repressive marks. Expanding the cells without IGFBP2 and Angptl5 led to a higher global H3K27me3 level. ChIP-seq analyses revealed a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Chemical inhibition of the H3K27 methyltransferase EZH2 counteracted the culture-associated loss of NSG engraftment potential. Collectively, the data presented in this study revealed that by adding epigeneticly active compounds in the culture media we observed changes on a chromatin level which counteracted the loss of engraftment potential. H3K27me3 rather than H3K4me3 may be critical to establish a specific engraftment supporting transcriptional program. Furthermore, I identified a critical function for the Polycomb repressive complex 2-component EZH2 in the loss of engraftment potential during the in vitro expansion of HPSCs. Taken together this thesis provides a better molecular understanding of chromatin changes upon expansion of CB-HSPCs and opens up new perspectives for epigenetic ex vivo expansion strategies. N2 - Hämatopoetische Stammzellen aus Nabelschnurblut (CB-HSCs) sind eine bedeutende Quelle für die Behandlung einer Vielzahl maligner und nicht-maligner Erkrankungen. Allerdings ist die geringe Anzahl an Stammzellen, die von einem Spender gewonnen werden kann, meist nicht ausreichend für die Rekonstitution des hämatopoetischen Systems erwachsener Patienten. Um eine ausreichende Menge an CB-HSCs zu gewinnen, ist eine Expansion der Zellen erforderlich. Verschiedene Ansätze zur ex vivo Expansion von HSCs wurden beschrieben, allerdings waren diese Ansätze durch den Verlust des Repopulationspotentials während der ex vivo Kultivierung nicht umsetzbar. Über die zugrundeliegenden Mechanismen ist wenig bekannt. Epigenetische Mechanismen spielen eine entscheidende Rolle in der Kontrolle von Selbsterneuerung und Differenzierung von Stammzellen. Aus diesem Grund werden epigenetische Strategien zur HSC-Expansion in Betracht gezogen. Das Ziel dieser Studie war, globale und lokale Epigenotypen während der Expansion humaner CB-CD34+-Zellen (CB-Vorläuferzellen) zu charakterisieren. Diese Studien sollten zu einem besseren Verständnis der molekularen Mechanismen, welche zum Kultivierungs-assoziierten Verlust des Repopulationspotentials führen. Humane CB-CD34+-Zellen wurden in zwei verschiedene Zytokin-Cocktails kultiviert: Der sogenannte STF-Cocktail, welcher SCF, TPO und FGF-1 enthält und der STFIA-Cocktail, welcher STF mit Angptl5 und IGFBP2 kombiniert. Aus der Literatur war zu Beginn dieser Doktorarbeit war bekannt, dass CB-HSCs ex vivo in STFIA, nicht aber in STF expandiert werden können. In Übereinstimmung mit diesem Befund zeigen die hier vorgestellten heterologen Transplantationsexperimente, dass das Repopulationspotential frischer CB-CD34+-Zellen nur erhalten blieb, wenn die Zellen unter STFIA, jedoch nicht, wenn sie unter STF-Bedingungen expandiert waren. Um die globalen Chromatinveränderungen frisch isolierter und expandierter Zellen zu untersuchen, wurden die Level der aktivierenden Histonmodifikation H3K4me3 und der repressiven H3K27me3-Modifikation durch Chromatin-Durchflusszytometrie und Western Blot Analyse bestimmt. Zur Analyse der genomweiten Chromatinveränderungen nach ex vivo Expansion wurden Transkriptomprofile durch Mikroarray und Chromatin-Immunpräzipitation, in Kombination mit Deep-Sequencing (ChiP-Seq) durchgeführt. Zusätzlich wurden lokale Chromatinveränderungen durch ChiP-Analysen an Promotorregionen von Entwicklungs- und Selbsterneuerungs-Faktoren analysiert. Auf globaler Ebene unterschieden sich frisch isolierte CD34+ und CD34- Zellen in ihren H3K4me3 und H3K27me3 Leveln. Nach siebentägiger Expansion nahmen CD34+ und CD34- Zellen ähnliche Level aktiver und repressiver Markierungen an. Die Expansion der Zellen ohne IGFBP2 und Angptl5 führte zu höheren globalen H3K27me3 Leveln. ChiP-seq Analysen zeigten eine Zytokin-Cocktail-abhängige Neuverteilung von H3K27me3 Mustern. Die chemische Inhibition der H3K27me-Transferase EZH2 wirkte dem Kultivierungs-assoziierten Verlust des NSG Repopulationspotentials entgegen. Zusammenfassend zeigen diese Daten, dass durch die Zugabe von spezifischen Zytokinen in das Kulturmedium Veränderungen auf Chromatinebene verbunden sind, die dem kultivierungs-assoziierten Verlust des Repopulationspotentials entgegen wirken. Diese Daten zeigen weiterhin, dass die durch die PRC2 Komponente EZH2 vermittelte H3K27me3, nicht jedoch die H3K4me3 Histonmodifikation ein kritischer Faktor für die Etablierung eines die Repopulation fördernden Transkriptionsprogrammes ist. Somit dient diese Arbeit einem besseren molekularen Verständnis der Chromatinveränderungen während der Expansion von CB-HSPCs und eröffnet eine Perspektive für neue epigenetische ex vivo Expansionsstrategien. KW - Epigenetik KW - Hämatopoese KW - PRC2 KW - Cord blood-derived hematopoietic stem and progenitor cells KW - Hematopoietic stem cell ex-vivo expansion Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-108073 ER - TY - JOUR A1 - Vansynghel, Justine A1 - Ocampo-Ariza, Carolina A1 - Maas, Bea A1 - Martin, Emily A. A1 - Thomas, Evert A1 - Hanf-Dressler, Tara A1 - Schumacher, Nils-Christian A1 - Ulloque-Samatelo, Carlos A1 - Tscharntke, Teja A1 - Steffan-Dewenter, Ingolf T1 - Cacao flower visitation: Low pollen deposition, low fruit set and dominance of herbivores JF - Ecological Solutions and Evidence N2 - 1. Pollination services of cacao are crucial for global chocolate production, yet remain critically understudied, particularly in regions of origin of the species. Notably, uncertainties remain concerning the identity of cacao pollinators, the influence of landscape (forest distance) and management (shade cover) on flower visitation and the role of pollen deposition in limiting fruit set. 2. Here, we aimed to improve understanding of cacao pollination by studying limiting factors of fruit set in Peru, part of the centre of origin of cacao. Flower visitors were sampled with sticky insect glue in 20 cacao agroforests in two biogeographically distinct regions of Peru, across gradients of shade cover and forest distance. Further, we assessed pollen quantities and compared fruit set between naturally and manually pollinated flowers. 3. The most abundant flower visitors were aphids, ants and thrips in the north and thrips, midges and parasitoid wasps in the south of Peru. We present some evidence of increasing visitation rates from medium to high shade (40%–95% canopy closure) in the dry north, and opposite patterns in the semi-humid south, during the wet season. 4. Natural pollination resulted in remarkably low fruit set rates (2%), and very low pollen deposition. After hand pollination, fruit set more than tripled (7%), but was still low. 5. The diversity and high relative abundances of herbivore flower visitors limit our ability to draw conclusions on the functional role of different flower visitors. The remarkably low fruit set of naturally and even hand pollinated flowers indicates that other unaddressed factors limit cacao fruit production. Such factors could be, amongst others, a lack of effective pollinators, genetic incompatibility or resource limitation. Revealing efficient pollinator species and other causes of low fruit set rates is therefore key to establish location-specific management strategies and develop high yielding native cacao agroforestry systems in regions of origin of cacao KW - agroforestry KW - cocoa KW - flower visitors KW - forest proximity KW - hand pollination KW - pollen KW - pollination services KW - shade cover Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-312722 SN - 2688-8319 VL - 3 IS - 2 ER - TY - THES A1 - Vansynghel, Justine T1 - Pollination and pest control along gradients of shade cover and forest distance in Peruvian cacao agroforestry landscapes T1 - Bestäubung und Schädlingsbekämpfung entlang von Beschattungs- und Waldentfernungsgradienten in peruanischen Kakao-Agroforstlandschaften N2 - Chapter I – Introduction Global trade of beans of the cacao tree (Theobroma cacao), of which chocolate is produced, contributes to the livelihoods of millions of smallholder farmers. The understorey tree is native to South America but is nowadays cultivated in many tropical regions. In Peru, a South American country with a particularly high cacao diversity, it is common to find the tree cultivated alongside non-crop trees that provide shade, in so-called agroforestry systems. Because of the small scale and low management intensity of such systems, agroforestry is one of the most wildlife-friendly land-use types, harbouring the potential for species conservation. Studying wildlife-friendly land-use is of special importance for species conservation in biodiversity-rich tropical regions such as Peru, where agricultural expansion and intensification are threatening biodiversity. Moreover, there is a growing body of evidence that shows co-occurrence of high biodiversity levels and high yield in wildlife-friendly cacao farming. Yet studies are restricted to non-native cacao countries, and since patterns might be different among continents, it is important to improve knowledge on wildlife-friendly agroforestry in native countries. Because studies of wildlife-friendly cultivation processes are still largely lacking for South America, we set out to study multiple aspects of cacao productivity in agroforests in Peru, part of cacao´s region of origin. The natural pollination process of cacao, which is critically understudied, was investigated by trapping flower visitors and studying pollen deposition from macrophotographs (Chapter II). Next, we excluded birds, bats, ants and flying insects and squirrels from cacao trees in a full-factorial field experiment and quantified these animals´ contribution to cacao fruit set, fruit loss and yield (Chapter III). Lastly, we aimed to assess whether fruit quantity and quality of native cacao increases through manually supplementing pollen (Chapter II and IV), and whether microclimatic conditions and the genetic background of the studied varieties limit fruit set (Chapter IV). Chapter II – Cacao flower visitation: Low pollen deposition, low fruit set and dominance of herbivores Given the importance of cacao pollination for the global chocolate production, it is remarkable that fruit set limitations are still understudied. Knowledge on flower visitation and the effect of landscape context and local management are lacking, especially in the crop’s region of origin. Moreover, the role of pollen deposition in limiting fruit set as well as the benefits of hand pollination in native cacao are unknown. In this chapter, we aimed to close the current knowledge gaps on cacao pollination biology and sampled flower visitors in 20 Peruvian agroforests with native cacao, along gradients of shade cover and forest distance. We also assessed pollen quantities and compared fruit set between manually and naturally pollinated flowers. We found that herbivores were the most abundant flower visitors in both northern and southern Peru, but we could not conclude which insects are effective cacao pollinators. Fruit set was remarkably low (2%) but improved to 7% due to pollen supplementation. Other factors such as a lack of effective pollinators, genetic pollen incompatibility or resource unavailability could be causing fruit set limitations. We conclude that revealing those causes and the effective pollinators of cacao will be key to improve pollination services in cacao. Chapter III – Quantifying services and disservices provided by insects and vertebrates in cacao agroforestry landscapes Pollination and pest control, two ecosystem services that support cacao yield, are provided by insects and vertebrates. However, animals also generate disservices, and their combined contribution is still unclear. Therefore, we excluded flying insects, ants, birds and bats, and as a side effect also squirrels from cacao trees and we assessed fruit set, fruit loss and final yield. Local management and landscape context can influence animal occurrence in cacao agroforestry landscapes; therefore, shade cover and forest distance were included in the analyses. Flying insects benefitted cacao fruit set, with largest gains in agroforests with intermediate shade cover. Birds and bats were also associated with improved fruit set rates and with a 114% increase in yield, potentially due to pest control services provided by these animals. The role of ants was complicated: these insects had a positive effect on yield, but only close to forest. We also evidenced disservices generated by ants and squirrels, causing 7% and 10% of harvest loss, respectively. Even though the benefits provided by animals outweighed the disservices, trade-offs between services and disservices still should be integrated in cacao agroforestry management. Chapter IV – Cross-pollination improves fruit set and yield quality of Peruvian native cacao Because yields of the cacao tree are restricted by pollination, hand pollination has been proposed to improve yield quantity and potentially, also quality. However, low self- and cross-compatibility of native cacao, and abiotic conditions could cancel out hand pollination benefits. Yet, the impact of genetic constraints and abiotic conditions on fruit set have not been assessed in native cacao so far. To increase our understanding of the factors that limit fruit set in native cacao, we compared manual self- and cross-pollination with five native genotypes selected for their sensorial quality and simultaneously tested for effects of soil water content, temperature, and relative air humidity. We also compared quality traits between manually and naturally pollinated fruits. Success rates of self-pollination were low (0.5%), but increased three- to eightfold due to cross-pollination, depending on the genotype of the pollen donor. Fruit set was also affected by the interaction between relative air humidity and temperature, and we found heavier and more premium seeds in fruits resulting from manual than natural pollination. Together, these findings show that reproductive traits of native cacao are constrained by genetic compatibility and abiotic conditions. We argue that because of the high costs of hand pollination, natural cross-pollination with native pollen donors should be promoted so that quality improvements can result in optimal economic gains for smallholder farmers. Chapter V – Discussion In this thesis, we demonstrated that the presence of flying insects, ants and vertebrates, local and landscape management practices, and pollen supplementation interactively affected cacao yield, at different stages of the development from flower to fruit. First, we showed that fruit set improved by intermediate shade levels and flower visitation by flying insects. Because the effective cacao pollinators remain unknown, we recommend shade cover management to safeguard fruit set rates. The importance of integrating trade-offs in wildlife-friendly management was highlighted by lower harvest losses due to ants and squirrels than the yield benefits provided by birds and bats. The maintenance of forest in the landscape might further promote occurrence of beneficial animals, because in proximity to forest, ants were positively associated with cacao yields. Therefore, an integrated wildlife-friendly farming approach in which shade cover is managed and forest is maintained or restored to optimize ecosystem service provision, while minimizing fruit loss, might benefit yields of native cacao. Finally, manual cross-pollination with native genotypes could be recommended, due to improved yield quantity and quality. However, large costs associated with hand pollination might cancel out these benefits. Instead, we argue that in an integrated management, natural cross-pollination should be promoted by employing compatible genotypes in order to improve yield quantity and quality of native cacao. N2 - Kapitel I – Einleitung Der weltweite Handel mit den Bohnen des Kakaobaums (Theobroma cacao) trägt zum Lebensunterhalt von Millionen von Kleinbauern bei. Der Unterholzbaum, aus dessen Bohnen Schokolade hergestellt wird, ist in Südamerika beheimatet, wird aber heute in vielen tropischen Regionen angebaut. In Peru, einem der Länder mit einer besonders hohen Kakaovielfalt, wird der Baum häufig zusammen mit schattenspendenden Bäumen in so genannten Agroforstsystemen angebaut. Aufgrund der Kleinräumigkeit und der geringen Bewirtschaftungsintensität solcher Systeme ist die Agroforstwirtschaft eine der wildtierfreundlichsten Landnutzungsformen, die ein großes Potenzial für den Artenschutz bietet. Die Erforschung wildtierfreundlicher Landnutzungsformen ist besonders wichtig für den Artenschutz in artenreichen tropischen Regionen wie Peru, in denen die Ausweitung und Intensivierung der Landwirtschaft die biologische Vielfalt bedroht. Darüber hinaus gibt es immer mehr Belege dafür, dass eine hohe Artenvielfalt mit hohen Erträgen im wildtierfreundlichen Kakaoanbau einhergeht. Die Studien beschränken sich jedoch auf nicht ursprüngliche Kakaoländer, und da die Muster auf den verschiedenen Kontinenten unterschiedlich sein könnten, ist es wichtig, das Wissen über wildtierfreundliche Agroforstwirtschaft in den Ursprungsländern zu verbessern. Da Studien über wildtierfreundliche Anbauprozesse in Südamerika noch weitgehend fehlen, haben wir uns vorgenommen, verschiedene Aspekte der Kakaoproduktivität in Agroforstbetrieben in Peru, einem Teil der Ursprungsregion des Kakaos, zu untersuchen. Der natürliche Bestäubungsprozess von Kakao, der wenig erforscht ist, wurde durch das Einfangen von Blütenbesuchern und die Untersuchung der Pollenablage anhand von Makrofotografien untersucht (Kapitel II). Als Nächstes haben wir gemeinsam Vögel, Fledermäuse, Ameisen und Fluginsekten und Eichhörnchen vom Zugang zu Kakaobäumen ausgeschlossen und den Beitrag dieser Tiere zum Fruchtansatz, Fruchtverlust und Ertrag von Kakao quantifiziert (Kapitel III). Schließlich wollten wir feststellen, ob sich die Fruchtmenge und -qualität des heimischen Kakaos durch die händische Zugabe von Pollen erhöht (Kapitel II und IV) und ob der genetische Hintergrund der untersuchten Sorten und die mikroklimatischen Bedingungen den Fruchtansatz limitieren (Kapitel IV). Kapitel II – Besuch der Kakaoblüten: Geringer Polleneintrag, geringer Fruchtansatz und Dominanz von Pflanzenfressern Angesichts der Bedeutung der Kakaobestäubung für die weltweite Schokoladenproduktion ist es bemerkenswert, dass der Fruchtansatz noch immer nicht ausreichend erforscht ist. Insbesondere in der Herkunftsregion der Pflanze fehlt es an Wissen über die Blütenbesucher und die Auswirkungen von Landschaft und Bewirtschaftung. Darüber hinaus sind die Rolle des Polleneintrags bei der Limitierung des Fruchtansatzes sowie die Vorteile der Handbestäubung bei einheimischem Kakao unbekannt. In diesem Kapitel wollten wir die derzeitigen Wissenslücken über die Bestäubungsbiologie von Kakao schließen und haben in 20 peruanischen Agroforsten mit einheimischem Kakao bei unterschiedlicher Beschattung und Waldentfernung Proben von Blütenbesuchern genommen. Wir untersuchten auch die Pollenmenge und verglichen den Fruchtansatz zwischen händisch und natürlich bestäubten Blüten. Wir stellten fest, dass Pflanzenfresser sowohl im Norden als auch im Süden Perus die häufigsten Blütenbesucher waren, konnten aber nicht feststellen, welche Insekten effektive Kakaobestäuber sind. Der Fruchtansatz war bemerkenswert niedrig (2 %), verbesserte sich aber durch die Pollenergänzung auf 7 %. Andere Faktoren wie ein Mangel an wirksamen Bestäubern, genetische Polleninkompatibilität oder die Nichtverfügbarkeit von Ressourcen könnten die Ursache für den geringen Fruchtansatz sein. Wir kommen zu dem Schluss, dass die Aufdeckung dieser Ursachen und der effektiven Bestäuber des Kakaos der Schlüssel zur Verbesserung der Bestäubungsleistungen im Kakao sein wird. Kapitel III – Quantifizierung der von Insekten und Wirbeltieren in agroforstwirtschaftlichen Kakaolandschaften erbrachten Ökosystemdienstleistungen und Gegenleistungen Bestäubung und Schädlingsbekämpfung, zwei Ökosystemleistungen, die den Kakaoertrag unterstützen, werden von Insekten und Wirbeltieren erbracht. Allerdings erbringen die Tiere auch andere Leistungen und ihr kombinierter Beitrag ist noch unklar. Daher haben wir Fluginsekten, Ameisen, Vögel und Fledermäuse und als Nebeneffekt auch Eichhörnchen vom Zugang zu den Kakaobäumen ausgeschlossen und den Fruchtansatz, den Fruchtverlust und den endgültigen Ertrag bewertet. Die Bewirtschaftung auf lokaler und Landschaftsebene kann das Vorkommen von Tieren in Kakao-Agroforstlandschaften erhöht werden; daher wurden auch die Beschattung und die Entfernung zum nächsten Wald in die Analysen einbezogen. Fluginsekten begünstigten den Fruchtansatz von Kakao, wobei die größten Zugewinne in Agroforsten mit mittlerer Beschattung zu verzeichnen waren. Vögel und Fledermäuse wurden ebenfalls mit verbesserten Fruchtansatzraten und einer 114%igen Ertragssteigerung in Verbindung gebracht, was möglicherweise auf die Schädlingsbekämpfung durch diese Tiere zurückzuführen ist. Die Rolle der Ameisen war kompliziert: Diese Insekten wirkten sich positiv auf den Ertrag aus, aber nur in Waldnähe. Wir haben auch negative Auswirkungen von Ameisen und Eichhörnchen festgestellt, die 7% bzw. 10 % der Ernteverluste verursachten. Auch wenn die Vorteile der Tiere die Nachteile überwiegen, sollte ein Ausgleich zwischen den Vor- und Nachteilen in die agroforstliche Bewirtschaftung von Kakao integriert werden. Kapitel IV – Kreuzbestäubung verbessert den Fruchtansatz und die Ertragsqualität von einheimischem peruanischem Kakao Da die Erträge des Kakaobaums durch die Bestäubung eingeschränkt werden, wurde die Handbestäubung vorgeschlagen, um die Ertragsmenge und möglicherweise auch die Qualität zu verbessern. Die geringe Selbst- und Kreuzkompatibilität der einheimischen Kakaosorten und die abiotischen Bedingungen könnten jedoch die Vorteile der Handbestäubung einschränken. Die Auswirkungen genetischer Limitierungen und abiotischer Bedingungen auf den Fruchtansatz wurden bei einheimischem Kakao bisher noch nicht untersucht. Um die Faktoren besser zu verstehen, die den Fruchtansatz bei einheimischem Kakao einschränken, verglichen wir die händische Selbst- und Kreuzbestäubung mit fünf einheimischen Genotypen, die aufgrund ihrer aromatischen Qualität ausgewählt wurden, und untersuchten gleichzeitig die Auswirkungen von Bodenwassergehalt, Temperatur und relativer Luftfeuchtigkeit. Außerdem verglichen wir die Qualitätsmerkmale zwischen händisch und natürlich bestäubten Früchten. Die Erfolgsrate der Selbstbestäubung war gering (0,5 %), stieg jedoch durch Kreuzbestäubung um das Drei- bis Achtfache, je nach Genotyp des Pollenspenders. Der Fruchtansatz wurde auch durch die Wechselwirkung zwischen relativer Luftfeuchtigkeit und Temperatur beeinflusst, und wir fanden schwerere und hochwertigere Samen in Früchten, die durch manuelle Bestäubung entstanden waren, als in den natürlich bestäubten. Diese Ergebnisse zeigen, dass die Fortpflanzungseigenschaften des einheimischen Kakaos durch genetische Kompatibilität und abiotische Bedingungen eingeschränkt werden. Wir argumentieren, dass aufgrund der hohen Kosten der Handbestäubung die natürliche Kreuzbestäubung mit heimischen Pollenspendern gefördert werden sollte, damit Qualitätsverbesserungen zu optimalen wirtschaftlichen Gewinnen für die Kleinbauern führen können. Kapitel V – Diskussion In dieser Arbeit haben wir gezeigt, dass die Anwesenheit von Fluginsekten, Ameisen und Wirbeltieren, die Bewirtschaftungspraktiken auf lokaler und Landschaftsebene sowie die Pollenergänzung den Kakaoertrag in verschiedenen Entwicklungsstadien von der Blüte bis zur Frucht interaktiv beeinflussen. Zunächst haben wir gezeigt, dass sich der Fruchtansatz durch eine mittlere Beschattung und den Blütenbesuch durch Fluginsekten verbessert. Da die effektiven Bestäuber des Kakaos noch nicht bekannt sind, empfehlen wir, die Beschattung so zu gestalten, dass der Fruchtansatz gesichert ist. Wie wichtig es ist, bei einer wildtierfreundlichen Bewirtschaftung Kompromisse einzugehen, zeigt sich daran, dass die Ernteverluste durch Ameisen und Eichhörnchen geringer sind als die Ertragsvorteile durch Vögel und Fledermäuse. Die Erhaltung des Waldes in der Landschaft könnte das Vorkommen von Nützlingen weiter fördern, da Ameisen in der Nähe von Wäldern positiv mit den Kakaoerträgen verbunden waren. Daher könnte ein integrativer, wildtierfreundlicher Anbauplan, bei dem die Beschattung und der Waldabstand so gesteuert werden, dass die Bereitstellung von Ökosystemleistungen optimiert und gleichzeitig der Verlust von Früchten minimiert wird, den Erträgen des heimischen Kakaos zugutekommen. Schließlich könnte die händische Kreuzbestäubung mit einheimischen Genotypen aufgrund der verbesserten Ertragsmenge und -qualität empfohlen werden. Die hohen Kosten der händischen Bestäubung könnten diese Vorteile jedoch zunichtemachen. Stattdessen sollte im Rahmen einer integrativen Bewirtschaftung die natürliche Kreuzbestäubung durch den Einsatz kompatibler Genotypen gefördert werden, um die Quantität und Qualität der Erträge von einheimischem Kakao zu verbessern. KW - Kakao KW - Bestäubung KW - Schädlingsbekämpfung KW - Landschaftspflege KW - landwirtschaftlicher Betrieb KW - landscape management KW - wildlife-friendly farming KW - local farm management Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-281574 ER - TY - JOUR A1 - van Heyningen, V. A1 - Bickmore, W. A. A1 - Seawright, A. A1 - Fletcher, J. M. A1 - Maule, J. A1 - Fekete, G. A1 - Gessler, Manfred A1 - Bruns, G. A. A1 - Huerre-Jeanpierre, C. A1 - Junien, C. T1 - Role for the Wilms tumor gene in genital development? N2 - No abstract available KW - Biochemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59238 ER - TY - JOUR A1 - Van den Hove, Daniel A1 - Jakob, Sissi Brigitte A1 - Schraut, Karla-Gerlinde A1 - Kenis, Gunter A1 - Schmitt, Angelika Gertrud A1 - Kneitz, Susanne A1 - Scholz, Claus-Jürgen A1 - Wiescholleck, Valentina A1 - Ortega, Gabriela A1 - Prickaerts, Jos A1 - Steinbusch, Harry A1 - Lesch, Klaus-Peter T1 - Differential Effects of Prenatal Stress in 5-Htt Deficient Mice: Towards Molecular Mechanisms of Gene x Environment Interactions N2 - Prenatal stress (PS) has been shown to influence the development of the fetal brain and to increase the risk for the development of psychiatric disorders in later life. Furthermore, the variation of human serotonin transporter (5-HTT, SLC6A4) gene was suggested to exert a modulating effect on the association between early life stress and the risk for depression. In the present study, we used a 5-Htt6PS paradigm to investigate whether the effects of PS are dependent on the 5-Htt genotype. For this purpose, the effects of PS on cognition, anxiety- and depression-related behavior were examined using a maternal restraint stress paradigm of PS in C57BL6 wild-type (WT) and heterozygous 5-Htt deficient (5-Htt +/2) mice. Additionally, in female offspring, a genome-wide hippocampal gene expression profiling was performed using the Affymetrix GeneChipH Mouse Genome 430 2.0 Array. 5-Htt +/2 offspring showed enhanced memory performance and signs of reduced anxiety as compared to WT offspring. In contrast, exposure of 5-Htt +/2 mice to PS was associated with increased depressive-like behavior, an effect that tended to be more pronounced in female offspring. Further, 5-Htt genotype, PS and their interaction differentially affected the expression of numerous genes and related pathways within the female hippocampus. Specifically, MAPK and neurotrophin signaling were regulated by both the 5-Htt +/2 genotype and PS exposure, whereas cytokine and Wnt signaling were affected in a 5-Htt genotype6PS manner, indicating a gene6environment interaction at the molecular level. In conclusion, our data suggest that although the 5-Htt +/2 genotype shows clear adaptive capacity, 5-Htt +/2 mice –particularly females– at the same time appear to be more vulnerable to developmental stress exposure when compared to WT offspring. Moreover, hippocampal gene expression profiles suggest that distinct molecular mechanisms mediate the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction. KW - Medizin Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75795 ER - TY - JOUR A1 - Vainshtein, Yevhen A1 - Sanchez, Mayka A1 - Brazma, Alvis A1 - Hentze, Matthias W. A1 - Dandekar, Thomas A1 - Muckenthaler, Martina U. T1 - The IronChip evaluation package: a package of perl modules for robust analysis of custom microarrays N2 - Background: Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results: The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions: ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see “Additional Files” section) and at: http://www.alice-dsl.net/evgeniy. vainshtein/ICEP/ KW - Microarray KW - ICEP KW - IronChip Evaluation Package Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67869 ER - TY - THES A1 - Vainshtein, Yevhen T1 - Applying microarray‐based techniques to study gene expression patterns: a bio‐computational approach T1 - Anwendung von Mikroarrayanalysen um Genexpressionsmuster zu untersuchen: Ein bioinformatischer Ansatz N2 - The regulation and maintenance of iron homeostasis is critical to human health. As a constituent of hemoglobin, iron is essential for oxygen transport and significant iron deficiency leads to anemia. Eukaryotic cells require iron for survival and proliferation. Iron is part of hemoproteins, iron-sulfur (Fe-S) proteins, and other proteins with functional groups that require iron as a cofactor. At the cellular level, iron uptake, utilization, storage, and export are regulated at different molecular levels (transcriptional, mRNA stability, translational, and posttranslational). Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5’- or 3‘- untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Methods In this project response to the iron treatment was examined under different conditions using bioinformatical methods. This would improve our understanding of an iron regulatory network. For these purposes we used microarray gene expression data. To identify novel IRE-containing mRNAs biochemical, biocomputational, and microarray-based experimental approaches were integrated. IRP/IRE messenger ribonucleoproteins were immunoselected and their mRNA composition was analysed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Analysis of IronChip microarray data requires specialized tool which can use all advantages of a customized microarray platform. Novel decision-tree based algorithm was implemented using Perl in IronChip Evaluation Package (ICEP). Results IRE-like motifs were identified from genomic nucleic acid databases by an algorithm combining primary nucleic acid sequence and RNA structural criteria. Depending on the choice of constraining criteria, such computational screens tend to generate a large number of false positives. To refine the search and reduce the number of false positive hits, additional constraints were introduced. The refined screen yielded 15 IRE-like motifs. A second approach made use of a reported list of 230 IRE-like sequences obtained from screening UTR databases. We selected 6 out of these 230 entries based on the ability of the lower IRE stem to form at least 6 out of 7 bp. Corresponding ESTs were spotted onto the human or mouse versions of the IronChip and the results were analysed using ICEP. Our data show that the immunoselection/microarray strategy is a feasible approach for screening bioinformatically predicted IRE genes and the detection of novel IRE-containing mRNAs. In addition, we identified a novel IRE-containing gene CDC14A (Sanchez M, et al. 2006). The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip, but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls (Vainshtein Y, et al., 2010). N2 - Die Regulierung und Aufrechterhaltung der Eisen-Homeostase ist bedeutend für die menschliche Gesundheit. Als Bestandteil des Hämoglobins ist es wichtig für den Transport von Sauerstoff, ein Mangel führt zu Blutarmut. Eukaryotische Zellen benötigen Eisen zum Überleben und zum Proliferieren. Eisen ist am Aufbau von Hämo- und Eisenschwefelproteinen (Fe-S) beteiligt und kann als Kofaktor dienen. Die Aufnahme, Nutzung, Speicherung und der Export von Eisen ist zellulär auf verschiedenen molekularen Ebenen reguliert (Transkription, mRNA-Level, Translation, Protein-Level). Die iron regulatory proteins (IRPs) 1 und 2 kontrollieren die Eisen-Homeostase in Säugetieren posttranslational durch die Bindung an Iron-responsive elements (IREs). IREs sind konservierte RNA stem-loop Strukturen in den 5' oder 3' untranslatierten Bereichen von Genen, die im Eisenmetabolismus involviert sind (z.B. FTH1, FTL und TFRC). In dieser Arbeit wurden biochemische und bioinformatische Methoden mit Microarray-Experimenten kombiniert, um neue mRNAs mit IREs zu identifizieren. Genexpressionsstudien verbessern unser Verständnis über die komplexen Zusammenhänge in genregulatorischen Netzwerken. Das komplexe Design von Microarrays, deren Produktion und Manipulation sind dabei die limitierenden Faktoren bezüglich der Datenqualität. Die Verwendung von angepassten DNA Microarrays verbessert häufig die Datenqualität, falls entsprechende Analysemöglichkeiten für diese Arrays existieren. Methoden Um unser Verständnis von eisenregulierten Netzwerken zu verbessern, wurde im Rahmen dieses Projektes die Auswirkung einer Behandlung mit Eisen bzw. von Knockout Mutation unter verschiedenen Bedingungen mittels bioinformatischer Methoden untersucht. Hierfür nutzen wir Expressionsdaten aus Microarray-Experimenten. Durch die Verknüpfung von biochemischen, bioinformatischen und Microarray Ansätzen können neue Proteine mit IREs identifiziert werden. IRP/IRE messenger Ribonucleoproteine wurden immunpräzipitiert. Die Zusammensetzung der enthaltenen mRNAs wurde mittels einem IronChip Microarray analysiert: Für diesen Chip wurden bioinformatisch Gene vorhergesagt, die IRE-like Motive aufweisen. Der Chip wurde mit solchen Oligonucleotiden beschichtet und durch Hybridisierung überprüft, ob die präzipitierten mRNA sich hieran binden. Die Analyse der erhaltenen Daten erfordert ein spezialisiertes Werkzeug um von allen Vorteilen der angepassten Microarrays zu profitieren. Ein neuer Entscheidungsbaum-basierter Algorithmus wurde in Perl im IronChip Evaluation Package (ICEP) implementiert. Ergebnisse Aus großen Sequenz-Datenbanken wurden IRE-like Motive identifiziert. Dazu kombiniert der Algorithmus, insbesondere RNA-Primärsequenz und RNA-Strukturdaten. Solche Datenbankanalysen tendieren dazu, eine große Anzahl falsch positiver Treffer zu generieren. Daher wurden zusätzliche Bedingungen formuliert, um die Suche zu verfeinern und die Anzahl an falsch positiven Treffer zu reduzieren. Die angepassten Suchkriterien ergaben 15 IRE-like Motive. In einem weiteren Ansatz verwendeten wir eine Liste von 230 IRE-like Sequenzen aus UTR-Datenbanken. Daraus wurden 6 Sequenzen ausgewählt, die auch im unteren Teil stabil sind (untere Helix über 6 bp stabil). Die korrespondierenden Expressed Sequence Tags (ESTs) wurden auf die humane oder murine Version des IronChips aufgetragen. Die Microarray Ergebnisse wurden mit dem ICEP Programm ausgewertet. Unsere Ergebnisse zeigen, dass die Immunpräzipitation mit anschließender Microarrayanalyse ein nützlicher Ansatz ist, um bioinformatisch vorhergesagte IRE-Gene zu identifizieren. Darüber hinaus ermöglicht uns dieser Ansatz die Detektion neuer mRNAs, die IREs enthalten, wie das von uns gefundene Gen CDC14A (Sanchez et al., 2006). ICEP ist ein optimiertes Programmpaket aus Perl Programmen (Vainshtein et al., BMC Bioinformatics, 2010). Es ermöglicht die einfache Auswertung von Microarray Daten mit dem Fokus auf selbst entwickelten Microarray Designs. ICEP diente für die statistische und bioinformatische Analyse von selbst entwickelten IronChips, kann aber auch leicht an die Analyse von oligonucleotidbasierten oder cDNA Microarrays adaptiert werden. ICEP nutzt einen Entscheidungsbaum-basierten Algorithmus um die Qualität zu bewerten und führt eine robuste Analyse basierend auf Chipeigenschaften, wie mehrfachen Wiederholungen, Signal/Rausch Verhältnis, Hintergrund und Negativkontrollen durch. KW - Microarray KW - Genexpression KW - Bioinformatik KW - geneexpression KW - microarrays KW - IronChip KW - ICEP Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-51967 ER - TY - JOUR A1 - Uttinger, Konstantin L. A1 - Riedmeier, Maria A1 - Reibetanz, Joachim A1 - Meyer, Thomas A1 - Germer, Christoph Thomas A1 - Fassnacht, Martin A1 - Wiegering, Armin A1 - Wiegering, Verena T1 - Adrenalectomies in children and adolescents in Germany – a diagnose related groups based analysis from 2009-2017 JF - Frontiers in Endocrinology N2 - Background Adrenalectomies are rare procedures especially in childhood. So far, no large cohort study on this topic has been published with data on to age distribution, operative procedures, hospital volume and operative outcome. Methods This is a retrospective analysis of anonymized nationwide hospital billing data (DRG data, 2009-2017). All adrenal surgeries (defined by OPS codes) of patients between the age 0 and 21 years in Germany were included. Results A total of 523 patient records were identified. The mean age was 8.6 ± 7.7 years and 262 patients were female (50.1%). The majority of patients were between 0 and 5 years old (52% overall), while 11.1% were between 6 and 11 and 38.8% older than 12 years. The most common diagnoses were malignant neoplasms of the adrenal gland (56%, mostly neuroblastoma) with the majority being younger than 5 years. Benign neoplasms in the adrenal gland (D350) account for 29% of all cases with the majority of affected patients being 12 years or older. 15% were not defined regarding tumor behavior. Overall complication rate was 27% with a clear higher complication rate in resection for malignant neoplasia of the adrenal gland. Bleeding occurrence and transfusions are the main complications, followed by the necessary of relaparotomy. There was an uneven patient distribution between hospital tertiles (low volume, medium and high volume tertile). While 164 patients received surgery in 85 different “low volume” hospitals (0.2 cases per hospital per year), 205 patients received surgery in 8 different “high volume” hospitals (2.8 cases per hospital per year; p<0.001). Patients in high volume centers were significant younger, had more extended resections and more often malignant neoplasia. In multivariable analysis younger age, extended resections and open procedures were independent predictors for occurrence of postoperative complications. Conclusion Overall complication rate of adrenalectomies in the pediatric population in Germany is low, demonstrating good therapeutic quality. Our analysis revealed a very uneven distribution of patient volume among hospitals. KW - pediatric KW - neuroblastoma – diagnosis KW - therapy KW - adrenocortical adenocarcinoma KW - outcome KW - volume KW - adrenalectomia Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-282280 SN - 1664-2392 VL - 13 ER - TY - JOUR A1 - Urban, Lara A1 - Remmele, Christian W. A1 - Dittrich, Marcus A1 - Schwarz, Roland F. A1 - Müller, Tobias T1 - covRNA: discovering covariate associations in large-scale gene expression data JF - BMC Reserach Notes N2 - Objective The biological interpretation of gene expression measurements is a challenging task. While ordination methods are routinely used to identify clusters of samples or co-expressed genes, these methods do not take sample or gene annotations into account. We aim to provide a tool that allows users of all backgrounds to assess and visualize the intrinsic correlation structure of complex annotated gene expression data and discover the covariates that jointly affect expression patterns. Results The Bioconductor package covRNA provides a convenient and fast interface for testing and visualizing complex relationships between sample and gene covariates mediated by gene expression data in an entirely unsupervised setting. The relationships between sample and gene covariates are tested by statistical permutation tests and visualized by ordination. The methods are inspired by the fourthcorner and RLQ analyses used in ecological research for the analysis of species abundance data, that we modified to make them suitable for the distributional characteristics of both, RNA-Seq read counts and microarray intensities, and to provide a high-performance parallelized implementation for the analysis of large-scale gene expression data on multi-core computational systems. CovRNA provides additional modules for unsupervised gene filtering and plotting functions to ensure a smooth and coherent analysis workflow. KW - Multivariate analysis KW - Fourthcorner analysis KW - RLQ analysis KW - Transcriptomics KW - High-throughput data KW - Visualization KW - Ordination methods KW - RNA-Seq analysis KW - Microarray analysis Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229258 VL - 13 ER - TY - JOUR A1 - Uppaluri, Sravanti A1 - Nagler, Jan A1 - Stellamanns, Eric A1 - Heddergott, Niko A1 - Herminghaus, Stephan A1 - Pfohl, Thomas A1 - Engstler, Markus T1 - Impact of Microscopic Motility on the Swimming Behavior of Parasites: Straighter Trypanosomes are More Directional JF - PLoS Computational Biology N2 - Microorganisms, particularly parasites, have developed sophisticated swimming mechanisms to cope with a varied range of environments. African Trypanosomes, causative agents of fatal illness in humans and animals, use an insect vector (the Tsetse fly) to infect mammals, involving many developmental changes in which cell motility is of prime importance. Our studies reveal that differences in cell body shape are correlated with a diverse range of cell behaviors contributing to the directional motion of the cell. Straighter cells swim more directionally while cells that exhibit little net displacement appear to be more bent. Initiation of cell division, beginning with the emergence of a second flagellum at the base, correlates to directional persistence. Cell trajectory and rapid body fluctuation correlation analysis uncovers two characteristic relaxation times: a short relaxation time due to strong body distortions in the range of 20 to 80 ms and a longer time associated with the persistence in average swimming direction in the order of 15 seconds. Different motility modes, possibly resulting from varying body stiffness, could be of consequence for host invasion during distinct infective stages. KW - African Trypanosomes KW - Cell Motility KW - Random-Walk KW - Brucei KW - Components KW - Flagellum KW - Biology KW - Motion KW - Chemotaxis KW - Movement Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140814 VL - 7 IS - 6 ER - TY - JOUR A1 - Uphus, Lars A1 - Lüpke, Marvin A1 - Yuan, Ye A1 - Benjamin, Caryl A1 - Englmeier, Jana A1 - Fricke, Ute A1 - Ganuza, Cristina A1 - Schwindl, Michael A1 - Uhler, Johannes A1 - Menzel, Annette T1 - Climate effects on vertical forest phenology of Fagus sylvatica L., sensed by Sentinel-2, time lapse camera, and visual ground observations JF - Remote Sensing N2 - Contemporary climate change leads to earlier spring phenological events in Europe. In forests, in which overstory strongly regulates the microclimate beneath, it is not clear if further change equally shifts the timing of leaf unfolding for the over- and understory of main deciduous forest species, such as Fagus sylvatica L. (European beech). Furthermore, it is not known yet how this vertical phenological (mis)match — the phenological difference between overstory and understory — affects the remotely sensed satellite signal. To investigate this, we disentangled the start of season (SOS) of overstory F.sylvatica foliage from understory F. sylvatica foliage in forests, within nine quadrants of 5.8 × 5.8 km, stratified over a temperature gradient of 2.5 °C in Bavaria, southeast Germany, in the spring seasons of 2019 and 2020 using time lapse cameras and visual ground observations. We explained SOS dates and vertical phenological (mis)match by canopy temperature and compared these to Sentinel-2 derived SOS in response to canopy temperature. We found that overstory SOS advanced with higher mean April canopy temperature (visual ground observations: −2.86 days per °C; cameras: −2.57 days per °C). However, understory SOS was not significantly affected by canopy temperature. This led to an increase of vertical phenological mismatch with increased canopy temperature (visual ground observations: +3.90 days per °C; cameras: +2.52 days per °C). These results matched Sentinel-2-derived SOS responses, as pixels of higher canopy height advanced more by increased canopy temperature than pixels of lower canopy height. The results may indicate that, with further climate change, spring phenology of F. sylvatica overstory will advance more than F. sylvatica understory, leading to increased vertical phenological mismatch in temperate deciduous forests. This may have major ecological effects, but also methodological consequences for the field of remote sensing, as what the signal senses highly depends on the pixel mean canopy height and the vertical (mis)match. KW - overstory KW - understory KW - Sentinel-2 KW - time lapse cameras KW - vertical mismatch KW - phenological escape KW - climate change KW - European beech Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248419 SN - 2072-4292 VL - 13 IS - 19 ER - TY - THES A1 - Ulrich, Tanja T1 - Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis T1 - Funktion von Lin9 in vivo und MAP3K4-p38 Signalweg reguliert einen p53-vermittelten Zellzyklus-Arrest nach fehlerhafte Mitose N2 - Eine genaue Kontrolle des Verlaufs durch die Mitose ist entscheidend für die Gewährleistung genomischer Stabilität und für die Vermeidung von Aneuploidy. Der DREAM Komplex ist ein wichtiger Regulator der Expression von mitotischen Genen. Die Depletion der DREAM-Untereinheit Lin9, führt zu einer verminderten Expression von G2/M Genen und beeinträchtigt die Proliferation. In konditionellen knockout Mauszellen (MEFs) verursacht das Ausschalten von Lin9 Defekte in Mitose und Zytokinese und löst vorzeitige Seneszenz aus, um eine weitere Zellproliferation zu verhindern. In dieser Arbeit konnte gezeigt werden, dass der seneszente Phänotyp in Lin9 knockout MEFs unabhängig von den beiden Tumorsuppressor-Signalwegen p53-p21 und p16-pRB induziert wird. Untersuchungen mit dem konditionellen Lin9 knockout Mausmodell verdeutlichten die wichtige Funktion von Lin9 in der Regulierung der mitotischen Genexpression und der Proliferation in vivo. Das Fehlen von Lin9 führte zu einer verringerten Proliferation in den Krypten des Dünndarms und verursachte eine Atrophie des Darmepithels und einen schnell eintretenden Tod der Tiere. Im zweiten Teil der Arbeit wurden Signalwege untersucht, die nach fehlerhafter Zytokinese zu einem p53 vermittelten G1-Arrest führen. Hierfür wurde ein chemischer Inhibitor der mitotischen Kinase Aurora B verwendet. Mit Hilfe eines Hochdurchsatz siRNA Screens wurde die MAP Kinase MAP3K4 als Aktivator des p53 Signalwegs identifiziert. Es konnte gezeigt werden, dass MAP3K4 die Stresskinase p38b aktiviert, um den p53 vermittelten Zellzyklusarrest in tetraploiden Zellen auszulösen. Dabei wurde p38b nach Hemmung von Aurora B für die transkriptionelle Aktivierung des p53 Zielgens p21 benötigt. Im Gegenteil dazu erfolgte die Phosphorylierung, Stabilisierung und die Rekrutierung von p53 an den p21 Promoter unabhängig von p38. Die teilweise Hemmung von Aurora B zeigte, dass fehlerhafte Segregation von Chromosomen auch den MAP3K4-p38-p53 Signalweg aktiviert und lässt darauf schließen, dass subtile Defekte in der Mitose ausreichen diesen Stress-Signalweg zu induzieren. Obwohl p38 für den G1 Zellzyklusarrest nach mitotischen Schäden erforderlich war, führte die gleichzeitige Inhibierung von p38 und Aurora B über einen längeren Zeitraum zu einer verringerten Proliferation, vermutlich aufgrund verstärkter Apoptose. Es ist anzunehmen, dass der MAP3K4-p38-p53 Signalweg generell nach Defekten in der Mitose oder Zytokinese aktiviert wird um Zellen in G1 zu arretieren und um chromosomale Instabilität zu vermeiden. N2 - Precise control of progression through mitosis is essential to maintain genomic stability and to prevent aneuploidy. The DREAM complex is an important regulator of mitotic gene expression. Depletion of Lin9, one core-subunit of DREAM, leads to reduced expression of G2/M genes and impaired proliferation. In conditional mouse knockout cells (MEFs) Lin9 deletion causes defects in mitosis and cytokinesis and cells undergo premature senescence in order to prevent further proliferation. In this work it could be shown that the senescence phenotype in Lin9 knockout MEFs is independently mediated by the two tumor suppressor pathways p53-p21 and p16-pRB. Studies using the conditional Lin9 knockout mouse model demonstrated an important function of Lin9 in the regulation of mitotic gene expression and proliferation in vivo. Deletion of Lin9 caused reduced proliferation in the intestinal crypts resulting in atrophy of the intestinal epithelium and in rapid death of the animals. In the second part of this work, the pathways leading to p53 mediated G1 arrest after failed cytokinesis were analyzed by using a chemical inhibitor of the mitotic kinase Aurora B. In a high throughput siRNA screen the MAP kinase MAP3K4 was identified as an upstream activator of p53. It could be shown that MAP3K4 activates the downstream stress kinase p38b to induce the p53 mediated cell cycle arrest of tetraploid cells. p38b was required for the transcriptional activation of the p53 target gene p21 in response to Aurora B inhibition. In contrast, phosphorylation, stabilization and recruitment of p53 to the p21 promoter occured independently of p38 signaling. Partial inhibition of Aurora B demonstrated that chromosome missegregation also activates the MAP3K4-p38-p53 pathway, suggesting that subtle defects in mitosis are sufficient for inducing this stress signaling pathway. Although p38 was required for the G1 cell cycle arrest after mitotic failures, long-term co-inhibition of p38 and Aurora B resulted in reduced proliferation probably due to increased apoptosis. Presumably, MAP3K4-p38-p53 signaling is a common pathway that is activated after errors in mitosis or cytokinesis to arrest cells in G1 and to prevent chromosomal instability. KW - Mitose KW - MAP-Kinase KW - Protein p53 KW - Aneuploidie KW - Lin9 KW - defective Mitosis KW - MAP3K4 KW - p53 KW - aneuploidy KW - fehlerhafte Mitose Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-73975 ER - TY - THES A1 - Ullrich, Melanie T1 - Identification of SPRED2 as a Novel Regulator of Hypothalamic-Pituitary-Adrenal Axis Activity and of Body Homeostasis T1 - SPRED2 - Ein neuer Regulator der Hypothalamus-Hypophysen-Nebennierenrindenachse und der Hormonbalance N2 - SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype. The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1. Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage. Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis. To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress. N2 - SPRED-Proteine sind Inhibitoren des hochkonservierten und in allen Geweben verbreiteten Ras/ERK/MAPK-Signalwegs, welcher Proliferation, Differenzierung und das Wachstum von Zellen reguliert. Um physiologische Konsequenzen der SPRED2-Defizienz im lebenden Modellorganismus aufzuklären, haben wir SPRED2-KO-Mäuse mithilfe der „gene trap“-Methode generiert. Eine erste Studie zur phänotypischen Charakterisierung mit KO-Mäusen bis zu einem Alter von fünf Monaten identifizierte SPRED2 als Regulator der Chondrozytendifferenzierung und des Knochenwachstums. So bewirkt der Verlust der SPRED2-Proteinfunktion eine erhöhte FGFR-vermittelte ERK-Aktivität, was wiederum einen Hypochondroplasie-ähnlichen Minderwuchs verursacht. Allerdings offenbarten Langzeitbeobachtungen älterer KO-Mäuse einen im Allgemeinen sehr schlechten Gesundheitszustand und weitere facettenreiche Symptome, darunter exzessives Putzverhalten mit schweren, selbst zugefügten Wunden, einen abnorm hohen täglichen Wasserkonsum, klare morphologische Anzeichen einer Nierenschädigung und eine reduzierte Überlebenswahrscheinlichkeit durch plötzlichen Tod. Ziel dieser Studie war es, basierend auf unseren Beobachtungen, einen Auslöser für diesen komplexen und vielseitigen Phänotyp zu finden. Die beobachtete Nierendegeneration in unseren SPRED2-KO-Mäusen war auf eine Hydronephrose zurückzuführen, welche durch schwere Atrophie des Nierengewebes und Apoptose von Nierentubuluszellen gekennzeichnet war. Aufgrund des Nierenschadens haben wir Trinkverhalten und gängige Serumparameter analysiert. Trotz der Polydipsie, die sich durch eine nahezu verdoppelte tägliche Wasseraufnahme manifestierte, konnten signifikant erhöhte Na+- und Cl--Werte und die daraus resultierende Hyperosmolalität im Serum der SPRED2-KOs nicht kompensiert werden. Weil Salz- und Wasserhaushalt zum größten Teil unter der hormonellen Kontrolle von Aldosteron und ADH stehen, haben wir beide Hormonspiegel untersucht. Während die ADH-Werte im Serum von WT- und KO-Mäusen vergleichbar waren, insbesondere nach experimentellem Wasserentzug und einem extremen Verlust von Körperflüssigkeit, waren die Serumspiegel von Aldosteron in den SPRED2-KO-Mäusen verdoppelt. Die systematische Untersuchung übergeordneter regulatorischer Hormonachsen ergab, dass sich der Hyperaldosteronismus unabhängig von einer erhöhten Aktivität des Renin-Angiotensin-Systems entwickelte, da die Serum-Ang II-Spiegel in den SPRED2-KOs etwa um die Hälfte reduziert waren. Die Expression der Aldosteronsynthase in der Nebenniere war jedoch wesentlich erhöht. Für Kortikosteron, das wie Aldosteron von der Nebennierenrinde freigesetzt wird, konnten wir ebenfalls mehr als doppelt so hohe Werte im Serum der KO-Tiere detektieren. Die Aldosteron-Produktion steht, ähnlich wie bei Kortikosteron, zumindest teilweise unter der Kontrolle des hypophysären Hormons ACTH, dessen Sekretion wiederum übergeordnet durch die Freisetzung von CRH aus dem Hypothalamus geregelt wird. Tatsächlich war die Stresshormon-Sekretion entlang dieser gesamten Hypothalamus-Hypophysen-Nebennierenrinden-Achse erhöht, da Serum-ACTH, das mittlere, hypophysäre Hormon, und hypothalamisches CRH, der übergeordnete hormonelle Induktor der HPA-Achse, in den SPRED2-KOs auch um 30% erhöht waren. Zusätzlich waren die ERK-Aktivität ebenso wie die CRH-mRNA-Spiegel im paraventrikulären Nukleus des Hypothalamus in unseren SPRED2-KO-Mäusen deutlich höher. In vitro Studien mit der Hypothalamus-Zelllinie mHypoE-44 zeigten weiterhin, dass sowohl SPRED1 als auch SPRED2 die Aktivität des CRH-Promotors, die CRH-Sekretion und die Ets-Faktor-abhängige CRH-Transkription reduzieren können. Passend dazu enthält die CRH-Promotorregion zahlreiche verschiedene Bindungsstellen für Transkriptionsfaktoren der Ets-Familie, speziell für Ets1. Somit zeigt diese Studie zum ersten Mal, dass die durch SPRED2-vermittelte Hemmung der Ras/ERK/MAPK-Signalkaskade mittels Unterdrückung der ERK-Aktivität zu einer Herunterregulation der Ets1-Faktor-abhängigen Transkription führt, was eine Hemmung der CRH-Promotoraktivität, der CRH-Transkription und der CRH-Freisetzung aus dem Hypothalamus zur Folge hat. Die daraus resultierende Hyperaktivität der gesamten HPA-Achse in unseren SPRED2-KO-Mäusen spiegelt eine erhöhte endogene Stress-Reaktion wider und äußert sich durch übermäßiges Putzverhalten und durch selbst zugefügte Hautläsionen auf der einen Seite; auf der anderen Seite erklärt dies, in Kombination mit der erhöhten Aldosteronsynthase-Expression, den Hyperaldosteronismus und das damit verbundene Ungleichgewicht in Salz- und Wasserhaushalt. Weiterhin tragen sowohl Hyperaldosteronismus als auch Polydipsie sehr wahrscheinlich zu den beobachteten Nierenschädigungen bei. Zusammengefasst ist diese Studie ein erster Hinweis, dass SPRED2 wesentlich an der adäquaten Regulation der HPA-Achsen-Aktivität beteiligt ist und essentiell ist für die Aufrechterhaltung der Homöostase im Körper. Um die Folgen von SPRED2-Defizienz in Mäusen und vor allem im Menschen weiter aufzuklären und zu vergleichen, erforschen wir in zwei Folgeprojekten die Funktion von SPRED2 speziell im Gehirn und im Herzen und führen parallel ein genetisches Screening zur Identifikation von funktionellen SPRED2-Mutationen im Menschen durch. KW - Renin-Angiotensin-System KW - Spred-Proteine KW - MAP-Kinase KW - Hypophysen-Zwischenhirn-System KW - Knockout KW - SPRED2 KW - ERK KW - MAP Kinase Signaling KW - HPA Axis KW - Renin Angiotensin System KW - Knockout mouse KW - Spred Protein KW - Hypothalamisch-hypophysäre Achse KW - Renin-Angiotensin-Aldosteron-System KW - MAP-Kinase KW - Gen-Knockout Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-107355 ER - TY - THES A1 - Ulbrich, Jannes T1 - Integrierung und biochemische Charakterisierung ektoper BMP Rezeptoren in Zellmembranen T1 - The integration and biochemical characterization of ectopic BMP receptors in cell membranes N2 - BMPs vermitteln ihre zellulären Effekte durch Rekrutierung und Aktivierung von zwei Typen spezifischer, membranständiger Rezeptoren. Die genauen Mechanismen der Rezeptorakivierung und die Komposition eines funktionellen, signalvermittelnden Komplexes auf der Zelloberfläche sind in den letzten Jahren genau untersucht worden. Die dimere Natur aller BMPs, die Promiskuitivität der BMPs sowie der entsprechenden Rezeptoren und die unterschiedlichen Rezeptorkonformationen (PFC, BISC) erschweren jedoch die experimentelle Zugänglichkeit dieser Proteinfamilie. Um den Einfluss der Membranverankerung der Rezeptoren auf deren Affinität zu einzelnen Liganden zu untersuchen, wurden verschiedene Methoden evaluiert, die eine quantitative Kopplung an Plasmamembranen ermöglichten. Die BMP Rezeptorektodomänen wurden u.a. mittels einer lysin-spezifischen Kopplung lipidiert, oder aber als His6-Ektodomänen an membranintegrierte Chelatlipide gekoppelt. N2 - BMPs elicit their cellular functions via recruitment and activation of specific receptor serin/threonine receptor kinases. The precise mechanisms leading to receptor activation and the composition of a functional signal transducing complex on the cell surface has been investigated intensively over the last decades. The dimeric nature of all BMPs, the promiscuity of both, the ligands and the receptors and the different receptor conformations on the cell surface (PFC, BISC) hamper the experimental accessibility of this protein family. To study the membrane anchorage's influence of the receptors on their affinity towards single ligands, different methods were evaluated that enabled us to couple the receptor ectodomains in a quantitative manner to plasma membranes. The BMP receptor ectodomains were, among other techniques, lipidated in a lysine specific way or coupled as hexahistidine fusion proteins to membrane integrated chelating lipids. KW - Knochen-Morphogenese-Proteine KW - Transforming Growth Factor KW - Transforming Growth Factor beta KW - BMPs KW - BMP Rezeptoren KW - Proteinmodifikationen KW - Protein Lipidierungen KW - NTA Lipide KW - BMP KW - BMP receptos KW - protein modifications KW - protein lipidations KW - NTA lipids Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-55462 ER - TY - JOUR A1 - Uhler, Johannes A1 - Redlich, Sarah A1 - Zhang, Jie A1 - Hothorn, Torsten A1 - Tobisch, Cynthia A1 - Ewald, Jörg A1 - Thorn, Simon A1 - Seibold, Sebastian A1 - Mitesser, Oliver A1 - Morinère, Jérôme A1 - Bozicevic, Vedran A1 - Benjamin, Caryl S. A1 - Englmeier, Jana A1 - Fricke, Ute A1 - Ganuza, Cristina A1 - Haensel, Maria A1 - Riebl, Rebekka A1 - Rojas-Botero, Sandra A1 - Rummler, Thomas A1 - Uphus, Lars A1 - Schmidt, Stefan A1 - Steffan-Dewenter, Ingolf A1 - Müller, Jörg T1 - Relationships of insect biomass and richness with land use along a climate gradient JF - Nature Communications N2 - Recently reported insect declines have raised both political and social concern. Although the declines have been attributed to land use and climate change, supporting evidence suffers from low taxonomic resolution, short time series, a focus on local scales, and the collinearity of the identified drivers. In this study, we conducted a systematic assessment of insect populations in southern Germany, which showed that differences in insect biomass and richness are highly context dependent. We found the largest difference in biomass between semi-natural and urban environments (-42%), whereas differences in total richness (-29%) and the richness of threatened species (-56%) were largest from semi-natural to agricultural environments. These results point to urbanization and agriculture as major drivers of decline. We also found that richness and biomass increase monotonously with increasing temperature, independent of habitat. The contrasting patterns of insect biomass and richness question the use of these indicators as mutual surrogates. Our study provides support for the implementation of more comprehensive measures aimed at habitat restoration in order to halt insect declines. KW - biodiversity KW - ecology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265058 VL - 12 IS - 1 ER - TY - JOUR A1 - Uhler, Johannes A1 - Haase, Peter A1 - Hoffmann, Lara A1 - Hothorn, Torsten A1 - Schmidl, Jürgen A1 - Stoll, Stefan A1 - Welti, Ellen A. R. A1 - Buse, Jörn A1 - Müller, Jörg T1 - A comparison of different Malaise trap types JF - Insect Conservation and Diversity N2 - Recent reports on insect decline have highlighted the need for long‐term data on insect communities towards identifying their trends and drivers. With the launch of many new insect monitoring schemes to investigate insect communities over large spatial and temporal scales, Malaise traps have become one of the most important tools due to the broad spectrum of species collected and reduced capture bias through passive sampling of insects day and night. However, Malaise traps can vary in size, shape, and colour, and it is unknown how these differences affect biomass, species richness, and composition of trap catch, making it difficult to compare results between studies. We compared five Malaise trap types (three variations of the Townes and two variations of the Bartak Malaise trap) to determine their effects on biomass and species richness as identified by metabarcoding. Insect biomass varied by 20%–55%, not strictly following trap size but varying with trap type. Total species richness was 20%–38% higher in the three Townes trap models compared to the Bartak traps. Bartak traps captured lower richness of highly mobile taxa but increased richness of ground‐dwelling taxa. The white roofed Townes trap captured a higher richness of pollinators. We find that biomass, total richness, and taxa group specific richness are all sensitive to Malaise trap type. Trap type should be carefully considered and aligned to match monitoring and research questions. Additionally, our estimates of trap type effects can be used to adjust results to facilitate comparisons across studies. KW - Bartak KW - biodiversity KW - insect communities KW - insect monitoring KW - Malaise trap KW - Townes KW - trap selectivity Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-293694 VL - 15 IS - 6 SP - 666 EP - 672 ER - TY - JOUR A1 - Tzagoloff, A. A1 - Macino, G. A1 - Sebald, Walter T1 - Mitochondrial genes and translation products N2 - No abstract available KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47408 ER - TY - THES A1 - Tyagi, Anu T1 - Role of SWI/SNF in regulating pre-mRNA processing in Drosophila melanogaster T1 - Funktion von SWI/SNF in der Regulation der prämRNA-Prozessierung in Drosophila melanogaster N2 - ATP dependent chromatin remodeling complexes are multifactorial complexes that utilize the energy of ATP to rearrange the chromatin structure. The changes in chromatin structure lead to either increased or decreased DNA accessibility. SWI/SNF is one of such complex. The SWI/SNF complex is involved in both transcription activation and transcription repression. The ATPase subunit of SWI/SNF is called SWI2/SNF2 in yeast and Brahma, Brm, in Drosophila melanogaster. In mammals there are two paralogs of the ATPase subunit, Brm and Brg1. Recent studies have shown that the human Brm is involved in the regulation of alternative splicing. The aim of this study was to investigate the role of Brm in pre-mRNA processing. The model systems used were Chironomus tentans, well suited for in situ studies and D. melanogaster, known for its full genome information. Immunofluorescent staining of the polytene chromosome indicated that Brm protein of C. tentans, ctBrm, is associated with several gene loci including the Balbiani ring (BR) puffs. Mapping the distribution of ctBrm along the BR genes by both immuno-electron microscopy and chromatin immunoprecipitation showed that ctBrm is widely distributed along the BR genes. The results also show that a fraction of ctBrm is associated with the nascent BR pre-mRNP. Biochemical fractionation experiments confirmed the association of Brm with the RNP fractions, not only in C. tentans but also in D. melanogaster and in HeLa cells. Microarray hybridization experiments performed on S2 cells depleted of either dBrm or other SWI/SNF subunits show that Brm affects alternative splicing and 3´ end formation. These results indicated that BRM affects pre-mRNA processing as a component of SWI/SNF complexes. 1 N2 - ATP abhängige Chromatin Remodelling Komplexe bestehen aus diversen Faktoren, welche die bei der Umsetzung von ATP freiwerdende Energie dazu nutzen, die Chromatinstruktur neu zu ordnen. Diese Veränderungen führen zu einer Zu- bzw. Abnahme in der Zugänglichkeit der DNA. Ein Beispiel dafür ist der SWI/SNF-Komplex, der sowohl in die Aktivierung als auch die Inhibierung der Transkription involviert ist. Die ATPase-Untereinheit von SWI/SNF heißt in Hefe SWI2/SNF2 und in Drosophila melanogaster Brahma (Brm). Im Gegensatz dazu besitzen Säuger zwei Paraloge der ATPase-Einheit, nämlich Brm und Brg1. Neueste Studien haben gezeigt, dass das humane Brm in der Regulation des Alternativen Spleißen beteiligt ist. Ziel dieser Arbeit ist es, die Rolle von Brm in der prä-mRNA-Prozessierung zu untersuchen. Als Versuchssysteme wurden Chironomus tentans und D. melanogaster herangezogen. Dabei eignete sich C. tentans vor allem für die in situ Studien während bei D. melanogaster das vollständig sequenzierte Genom von Vorteil war. Immunfluoreszenzfärbungen von Polytän-Chromosomen zeigen eine Assoziation von Brm von C. tentans, ctBrm; mit unterschiedlichen Genloci, einschließlich der Balbiani-Ringe (BR). Mit Hilfe von Immun-Elektronenmikroskopie und Chromatin-Immunpräzipitation (ChIP) wird die Verteilung von ctBrm entlang der BR-Gene untersucht. Dabei zeigt ctBrm eine weite Streuung. Die Ergebnisse lassen außerdem darauf schließen, dass ein Teil des ctBrm-Proteins mit naszierenden BRprä- mRNPs interagiert. Biochemische Fraktionierungs-experimente bestätigen die Assoziation von Brm mit RNP-Fraktionen nicht nur in C. tentans, sondern auch in D. melanogaster und in HeLa-Zellen. Microarray-Untersuchungen in S2-Zellen, in denen entweder dBrm oder eine andere Untereinheit von SWI/SNF depletiert war, zeigen, dass BRM als eine Komponente des SWI/SNF-Komplexes sowohl Alternatives Spleißen und die Formierung des 3´ Endes, als auch die prä-mRNA-Prozessierung beeinflusst. KW - Taufliege KW - Messenger-RNS KW - Prozessierung KW - SWI/SNF KW - mRNA processing KW - SWI/SNF KW - mRNA processing Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72253 ER - TY - THES A1 - Tulke, Moritz T1 - Grundlegende Arbeiten zum bio-artifiziellen renalen Tubulus aus ko-kultivierten adipozytären mesenchymalen Stammzellen und Endothelzellen auf einer synthetischen Kapillarmembran T1 - Fundamental work on a bio-artificial renal tubule consisting of co-cultivated adipose-derived mesenchymal stem cells and endothelial cells on a synthetic capillary membrane N2 - Mit fortschreitender chronischer Niereninsuffizienz kommt es zur Akkumulation von Urämietoxinen und im Endstadium unbehandelt zum Tod im sogenannten Urämischen Syndrom. Die Blutreinigung erfolgt bei der am häufigsten verwendeten Form der Nierenersatztherapie, der Hämodialyse, nur unzureichend. Die Folge ist eine erhöhte Morbidität und Mortalität der betroffenen Patienten. Bei der Hämodialyse werden nur Urämietoxine bis zu einer Größe von 20 kDa über die im Dialysator eingesetzten Hohlfaserdialysemembranen diffusiv und konvektiv semiselektiv nach Größenausschluss entfernt. Proteingebundene Urämietoxine, deren effektive Größe durch die Bindung an Transportproteine wie beispielsweise Albumin die Trennschärfe der Dialysemembranen übersteigt, werden retiniert. In-vivo werden proteingebundene Urämietoxine im proximalen Tubulus, einem Teil des tubulären Systems des Nephrons, sekretorisch eliminiert. Im Rahmen der vorliegenden Promotionsarbeit wurden die ersten Entwicklungsschritte auf dem Weg zu einem sogenannten bio-artifiziellen Tubulus evaluiert. Der angedachte biohybride Filter sollte aus einer Ko-Kultur funktionaler humaner proximaler Tubuluszellen und humaner Endothelzellen (HUVEC) auf synthetischen Hohlfasermembranen bestehen und könnte während der Hämodialyse als zusätzlicher Reinigungsschritt angewendet werden, um unter anderem proteingebundene Urämietoxine effektiv durch aktiven Transport aus dem Blut der Patienten zu entfernen. Die Differenzierung der proximalen Tubuluszellen erfolgte dabei aus adulten adipozytären mesenchymalen Stammzellen (ASC), deren Herkunft eine spätere autologe Behandlung ermöglicht. Die Ko-Kultur mit Endothelzellen wurde zur potentiellen Steigerung der Sekretion proteingebundener Urämietoxine verwendet. In der vorliegenden Arbeit konnten ASCs durch eine Kombination der löslichen Differenzierungsfaktoren All-Trans-Retinoinsäure (ATRA), Aktivin A und BMP-7 erfolgreich in Zytokeratin 18-exprimierende Zellen differenziert werden, wodurch die erwünschte epitheliale Differenzierung bestätigt wurde. Die Expression funktionaler Proteine, wie das für den Wassertransport relevante Aquaporin 1 oder auch der Na+-/K+-ATPase, konnte in dieser Arbeit bereits vor der Differenzierung nachgewiesen werden. Im nächsten Schritt wurde erfolgreich gezeigt, dass eine simultane, qualitativ hochwertige Ko-Kultur von ASCs und HUVECs auf der mit dem extrazellulären Matrixprotein Fibronektin modifizierten Innen- bzw. Außenseite von synthetischen Hohlfasermembranen aus Polypropylen bzw. Polyethersulfon möglich ist. Die Viabilität beider Zelltypen wurde dabei durch die Verwendung eines für die Ko-Kultur entwickelten Nährmediums erreicht, in welchem die Proliferation von ASCs bei gleichzeitiger Aufrechterhaltung ihrer Stammzelleigenschaften deutlich erhöht war. Die in dieser Arbeit erzielten Ergebnisse stellen eine aussichtsreiche Basis für einen bio-artifiziellen renalen Tubulus dar. Weitere Entwicklungsschritte, wie die Differenzierung der ASCs zu proximalen Tubuluszellen im 3D-Bioreaktor einschließlich ihrer funktionalen Charakterisierung anhand Tubulusepithel-spezifischer Transporter, sind erforderlich, be-vor erste funktionale Experimente vor dem „Upscaling“ auf klinisch verwendbare Module möglich sind. N2 - Progressing chronic kidney disease results in the accumulation of uremic toxins and, if left untreated in end-stage kidney disease, death due to the developing uremic syndrome. The most common renal replacement therapy is hemodialysis. It is a life-prolonging therapy but only delivering inadequate blood purification, which is associated with excess morbidity and mortality of the patients. In hemodialysis, only uremic toxins with a molecular size of up to 20 kDa are removed by diffusion or convection. Solutes are eliminated by semi-selective size exclusion across a hollow fiber dialysis membrane in a dialyzer. Binding of certain uremic toxins to carrier proteins, such as albumin, results in an increased effective size, which excludes them from passing through dialysis membranes. In the native kidney, these protein-bound uremic toxins are eliminated from blood by secretory transport in the proximal tubule, a specific part of the tubular filtration apparatus of the nephron. The present doctoral thesis evaluated the first steps towards a so-called bio-artificial tubule. The intended biohybrid filter was supposed to consist of a co-culture of functional human proximal tubule cells and human endothelial cells on synthetic hollow fiber membranes. In its final form, it would be implemented during hemodialysis as an additional purification step to more efficiently remove protein-bound uremic toxins from the patients’ blood by active transport. The proximal tubule cells were differentiated from adipose-derived mesenchymal stem cells, which facilitates a later autologous treatment. The co-culture with endothelial cells should further promote the expression of transporters for organic anions and, thereby, potentially increase the secretion of protein-bound uremic toxins. In the present study, the differentiation from ASCs to a CK18-expression lineage, which confirmed successful epithelial differentiation, was induced by a combination of the soluble differentiation factors all-trans-retinoic acid, activin A and BMP-7. The expression of functional proteins, i.e., of aquaporin 1, which is relevant for water transport, and Na+-/K+-ATPase, was shown already before differentiation. Additionally, the present work demon-strated a high-quality co-culture of ASCs and HUVECs on the inner- and outer membrane surfaces of synthetic polypropylene- or polyethersulfone-based hollow fiber membranes, which initially were surface-modified with the extracellular matrix protein fibronectin. The viability of both cell types was thereby ensured by the application of a specific co-culture medium, which further increased the proliferation of ASCs intensely while maintaining their stem-cell character. The results of the present approach represent a promising basis for a bio-artificial renal tubule. The further development requires the differentiation of ASCs into proximal tubule cells on the 3D-bioreactor membrane and their characterization by verifying tubulusepithel-specific transporters. Finally, subsequent functional experiments have to precede an upscaling to clinically applicable modules. KW - Hohlfaserreaktor KW - Stammzelle KW - Endothelzelle KW - Adipozytäre mesenchymale Stammzelle KW - Bio-artifizieller Tubulus KW - Ko-Kultur Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216896 ER - TY - JOUR A1 - Tuchscherr, Lorena A1 - Bischoff, Markus A1 - Lattar, Santiago M. A1 - Noto Llana, Mariangeles A1 - Pförtner, Henrike A1 - Niemann, Silke A1 - Geraci, Jennifer A1 - Van de Vyver, Hélène A1 - Fraunholz, Martin J. A1 - Cheung, Ambrose L. A1 - Herrmann, Mathias A1 - Völker, Uwe A1 - Sordelli, Daniel O. A1 - Peters, Georg A1 - Loeffler, Bettina T1 - Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, \(\Delta\)sigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections. KW - gene regulator agr KW - endothelial cells KW - modulates virulence KW - death pathway sar locus KW - factor B KW - small-colony variants KW - alpha-toxin KW - epithelial cells KW - in vitro Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143419 VL - 11 IS - 4 ER - TY - JOUR A1 - Tu, Xiaolin A1 - Chen, Jianquan A1 - Lim, Joohyun A1 - Karner, Courtney M. A1 - Lee, Seung-Yon A1 - Heisig, Julia A1 - Wiese, Cornelia A1 - Surendran, Kameswaran A1 - Kopan, Raphael A1 - Gessler, Manfred A1 - Long, Fanxin T1 - Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1 JF - PLoS Genetics N2 - Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo. KW - expression KW - axial skeletal defects KW - transcription factor KW - alagille syndrome KW - osteoblast differentiation KW - human jagged1 KW - aortic-valve KW - T cells KW - mutations KW - mice Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133490 VL - 8 IS - 3 ER - TY - THES A1 - Tsoneva, Desislava T1 - Humanized mouse model: a system to study the interactions of human immune system with vaccinia virus-infected human tumors in mice T1 - Humanisiertes Mausmodell: ein System, um die Wechselwirkungen des menschlichen Immunsystems mit Vaccinia-Virus-infizierten humanen Tumoren in Mäusen zu untersuchen N2 - Ein vielversprechender neuer Ansatz zur Behandlung von Krebs beim Menschen ist die Verwendung von onkolytischen Viren, die einen Tumor-spezifischen Tropismus aufweisen. Einer der Top-Kandidaten in diesem Bereich ist das onkolytische Vaccinia Virus (VACV), das bereits vielversprechende Ergebnisse in Tierversuchen und in klinischen Studien gezeigt hat. Aber die von den in vivo in tierischen Modellen erhaltenen Resultate könnten ungenaue Informationen wegen der anatomischen und physiologischen Unterschiede zwischen den Spezies liefern. Andererseits sind Studien in Menschen aufgrund ethischer Erwägungen und potenzieller Toxizität nur limitiert möglich. Die zahlreichen Einschränkungen und Risiken, die mit den Humanstudien verbunden sind, könnten mit der Verwendung eines humanisierten Mausmodells vermieden werden. Die LIVP-1.1.1, GLV-2b372, GLV-1h68, GLV-1h375, GLV-1h376 and GLV-1h377 VACV Stämmen wurden von der Genelux Corporation zur Verfügung gestellt. GLV-2b372 wurde durch Einfügen der TurboFP635 Expressionskassette in den J2R Genlocus des parentalen LIVP-1.1.1-Stammes konstruiert. GLV-1h375, -1h376 and -1h377 kodiert das Gen für den menschlichen CTLA4-blockierenden Einzelketten-Antikörper (CTLA4 scAb). Befunde aus Replikations- and Zytotoxizitätsstudien zeigten, dass alle sechs Viren Tumorzellen infizieren, sich in ihnen replizieren und sie in Zellkultur schließlich ebenso dosis- und zeitabhängig effizient abtöten konnten. CTLA4 scAb und β-Glucuronidase (GusA) Expression sowie Virus Titer in GLV-1h376-infizierten A549-Zellen wurde anhand von ELISA-, β-Glucuronidase- and Standard Plaque-Assays bestimmt. Hierbei zeigte sich eine ausgezeichnete Korrelation mit Korrelationskoeffizienten R2>0.9806. Der durch das GLV-1h376 kodierte CTLA4 scAb wurde erfolgreich aus Überständen von infizierten CV-1-Zellen gereinigt. CTLA4 scAb hat eine hohe in-vitro-Affinität zu seinem menschlichen CTLA4-Zielmolekül sowie abwesende Kreuzreaktivität gegenüber murine CTLA4 gezeigt. CTLA4 scAb Funktionalität wurde in Jurkat-Zellen bestätigt. LIVP-1.1.1, GLV-2b372, GLV-1h68 und GLV-1h376 wurden auch in nicht-tumorösen und/oder tumortragenden humanisierten Mäusen getestet. Zunächst wurde gezeigt, dass die Injektion von menschlichen CD34+ Stammzellen in die Leber von vorkonditionierten neugeborenen NSG Mäusen zu einer erfolgreichen systemische Rekonstitution mit menschlichen Immunzellen geführt hat. CD19+-B-Zellen, CD4+- und CD8+-CD3+-T-Zellen, NKp46+CD56- und NKp46+CD56+-NK-Zellen sowie CD33+-myeloischen Zellen wurden detektiert. Die Mehrheit der nachgewisenen humanen hämatopoetischen Zellen im Mäuseblut in den ersten Wochen nach der Humanisierung waren CD19+-B-Zellen, und nur ein kleiner Teil waren CD3+-T-Zellen. Mit der Zeit wurde eine signifikante Veränderung in CD19+/CD3+-Verhältnis beobachtet, die parallel zur Abnahme der B-Zellen und einem Anstieg der T-Zellen kam. Die Implantation von A549-Zellen unter die Haut dieser Mäuse führte zu einem progressiven Tumorwachstum. Bildgebende Verfahren zur Detektion von Virus-vermittelter TurboFP635- und GFP-Expression, Standard Plaque Assays sowie immunohistochemische Analysen bestätigten die erfolgreiche Invasion der Viren in die subkutanen Tumoren. Die humane CD45+-Zellpopulation in Tumoren wurde hauptsächlich durch NKp46+CD56bright-NK-Zellen und einen hohen Anteil von aktivierten CD4+- und zytotoxische CD8+-T-Zellen dargestellt. Es wurden jedoch keine signifikanten Unterschiede zwischen den Kontroll- und LIVP-1.1.1-infizierten Tumoren beobachtet, was darauf hindeutete, dass die Rekrutierung von NK- und aktivierten T-Zellen, mehr Tumorgewebe-spezifisch als Virus-abhängig waren. Die GLV-1h376-vermittelten CTLA4 scAb-Expression in den infizierten Tumoren war ebenfalls nicht in der Lage, die Aktivierung von Tumor-infiltrierenden T-Zellen im Vergleich zur Kontrolle und GLV-1h68-behandelten Mäusen, signifikant zu erhöhen. ELISA-, β-Glucuronidase- and Standard Plaque-Assays zeigten eine eindeutige Korrelation mit den Korrelationskoeffizienten R2>0,9454 zwischen CTLA4 scAb- und GusA-Konzentrationen und Virus Titer in Tumorproben von GLV-1h376-behandelten Mäusen. T-Zellen, die aus der Milz dieser Tumor-tragenden Mäuse isoliert wurden, waren funktionell und konnten erfolgreich mit Beads aktiviert werden. Mehr CD25+ und IFN-ɣ+ T-Zellen wurden in der GLV-1h376-Gruppe gefunden, wahrscheinlich aufgrund der CTLA4-Blockade durch die Virus-vermittelte CTLA4 scAb-Expression in den Mäusen. Außerdem wurde eine höhere Konzentration von IL-2 in dem Kulturüberstand von diesen Splenozyten im Vergleich zu Kontrollproben nachgewiesen. Im Gegensatz zu der Aktivierung mit Beads konnten T-Zellen von allen drei Maus-Gruppen nicht durch A549 Tumorzellen ex vivo aktiviert werden. Unser Mausmodell hat den besonderen Vorteil, dass sich Tumoren unter der Haut der humanisierten Mäuse entwickeln, was eine genaue Überwachung des Tumorwachstums und Auswertung der onkolytischen Virotherapie ermöglicht. N2 - A promising new approach for the treatment of human cancer is the use of oncolytic viruses, which exhibit tumor tropism. One of the top candidates in this area is the oncolytic vaccinia virus (VACV), which has already shown promising results in animal studies and in clinical trials. However, due to discrepancies in both innate and adaptive immunity between mice and men the evaluation of the vaccinia virus’ interactions with the host immune system in mice are not fully conclusive of what is actually happening in human cancer patients after systemic administration of vaccinia virus. Also, ethical and legal concerns as well as risk of potential toxicity limit research involving human patients. Therefore, a good in vivo model for testing interactions between vaccinia virus and human immune cells, avoiding the numerous limitations and risks associated with human studies, could be a humanized mouse model. LIVP-1.1.1, GLV-2b372, GLV-1h68, GLV-1h375, GLV-1h376 and GLV-1h377 VACVs were provided by Genelux Corporation. GLV-2b372 was constructed by inserting TurboFP635 expression cassette into the J2R locus of the parental LIVP-1.1.1. GLV-1h375, -1h376 and -1h377 VACVs encode the human CTLA4-blocking single-chain antibody (CTLA4 scAb). Performed replication and cytotoxicity assays demonstrated that all six viruses were able to infect, replicate in and kill human tumor cells in virus-dose- and time-dependent fashion. CTLA4 scAb and β-glucuronidase (GusA) expression as well as viral titers in GLV-1h376-infected cells were analyzed by ELISA, β-glucuronidase assay and standard plaque assay, respectively, and compared. An excellent correlation with correlation coefficients R2>0.9806 were observed. GLV-1h376-encoded CTLA4 scAb was successfully purified from supernatants of infected CV-1 cells and demonstrated in vitro affinity to its human CTLA4 target and lack of cross-reactivity to mouse CTLA4. CTLA4 scAb functionality was confirmed in Jurkat cells. LIVP-1.1.1, GLV-2b372, GLV-1h68 and GLV-1h376 were next studied in non-tumorous and/or tumor-bearing humanized mice. It was demonstrated that injection of human CD34+ stem cells into the liver of preconditioned newborn NSG mice let to a successful systemic reconstitution with human immune cells. CD19+ B cells, CD4 and CD8 single positive CD3+ T cell, NKp46+CD56- and NKp46+CD56+ NK cells as well as CD33+ myeloid cells developed. At early time points after engraftment, majority of the human hematopoietic cells detected in the mouse blood were CD19+ B cells and only a small portion were CD3+ T cells. With time a significant change in CD19+/CD3+ ratio was reported with a decrease of B cells and an increase of T cells. Implantation of A549 cells under the skin of those humanized NSG mice resulted in a progressive tumor growth, described for the first time in this thesis. Successful colonization of subcutaneous A549 tumors with VACVs was visualized and demonstrated by detection of virus-mediated TurboFP635 and GFP expression as well as by standard plaque assay and immunohistochemistry. The human CD45+ cell population in tumors was represented mainly by NKp46+CD56bright NK cells and a large portion of activated CD4+ and cytotoxic CD8+ T cells. However, no significant differences were observed between control and LIVP-1.1.1-infected tumors, suggesting that the recruitment of NK and activated T cells were more tumor tissue specific than virus-dependent. Unfortunately, virus-mediated CTLA4 scAb expression in the GLV-1h376-infected tumors was also not able to significantly increase activation of T cells compared to control and GLV-1h68-treated mice. Importantly, ELISA, β-glucuronidase and standard plaque assays showed an excellent correlation with correlation coefficients R2>0.9454 between CTLA4 scAb, GusA concentrations and viral titers in tumor samples from those GLV-1h376 treated mice. T cells isolated from the spleens of such control or GLV-1h68- or -1h376-treated A549 tumor-bearing mice were functional and could successfully be activated with human T cells activation beads. However, although no significant difference was observed between the three mouse groups, a slightly higher percentage of the GLV-1h376-treated mice-derived T cells were expressing CD25 and producing IFN-ɣ after ex vivo activation, probably due to the CTLA4 blockade by the virus-encoded CTLA4 scAb in the GLV-1h376-treated mice. Also, slightly higher levels of IL-2 were detected in the culture supernatant of those splenocytes compared to control samples. In contrast, T cells from all three mouse groups were not able be activated by A549 tumor cells ex vivo. Our model has the specific advantage that tumors develop under the skin of the humanized mice, which allows accurate monitoring of the tumor growth and evaluation of the oncolytic virotherapy. Therefore it is important to choose the right approaches for its further improvement. KW - Vaccinia virus KW - cancer KW - vaccinia virus KW - humanized mice Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118983 ER - TY - THES A1 - Tschäpe, Jakob-Andreas T1 - Molekulare und funktionelle Analyse der Drosophila-Mutante löchrig T1 - Molecular and Functional Analysis of the Drosophila mutant löchrig N2 - Neurodegenerative Erkrankungen des Menschen sind eines der Hauptfelder molekularer neurobiologischer Grundlagenforschung. Um generell molekulare, komplizierte Vorgänge in vivo untersuchen zu können, nutzt man seit geraumer Zeit Modellorganismen wie Caenorhabditis elegans oder Drosophila melanogaster. In der vorliegenden Arbeit wird die Drosophila-Neurodegenerationsmutante loe (löchrig) beschrieben, die als Modell für die Rolle des Cholesterinhaushalts im Bezug auf Neurodegeneration herangezogen werden kann. Die Fliegen dieser Mutante zeigen stark progressive, altersabhängige Degeneration von Neuronen, dabei unterlaufen diese Nervenzellen einen nekrotischenZelltod. Verantwortlich für diese Mutation ist die Insertion eines P-Elementes in einem Intron des Drosophila-g-5'-AMP-aktivierten Proteinkinase- (AMPK)-Gens. Die verschiedenen Spleißprodukte des loe Gens kodieren für die regulatorische g-Untereinheit des AMPK-Komplexes, der , aktiviert durch 5'AMP, energieintensive Prozesse negativ reguliert. Die Spleißform loeI ist durch die P-Element-Insertion betroffen, Anteile des P-Elementes werden in das loeI-Transkript hineingespleißt. Eine neuronale Expression von loeI im loe-Hintergrund führt zur Revertierung des loe-Phänotypes. Mit der Expression anderer Spleißformen kann dieser Effekt nicht erzielt werden. Das LOE I-Protein birgt in seinem N-Terminus eine Reihe möglicher Interaktionstellen mit anderen Proteinen, die den AMPK-Komplex in einen Kontext mit den Proteinen der APP (Amyloid Precursor Proteins) ?Familie stellen oder z. B. Interaktionen mit dem Cytoskelett herstellen können. Eine molekulare Interaktion mit NiPSNAP, einem Protein, dass vermutlich eine Rolle im Vesikelverkehr spielt, konnte nachgewiesen werden. Ein direktes humanes Homolog von LOE I ist nicht bekannt, wohlgleich es im Menschen drei AMPK-g-Untereinheiten gibt, von denen zwei ähnliche Funktionen übernehmen könnten wie LOE I. Die loe-Mutante interagiert genetisch mit der Mutante clb ? columbus, die einen Defekt im Gen der HMG-CoA-Reduktase trägt. Dieses Emzym ist das Schlüsselenzym der Cholesterinbiosynthese. Die Art der Interaktion belegt eine negative Regulierung der HMG-CoA-Reduktase durch die AMPK. So schwächt die clb-Mutation den neurodegenerativen loe-Phänotyp ab, eine Überexpression von clb verstärkt diesen. Eine Verminderung der Neurodegeneration kann auch mit Medikamenten erreicht werden: Statine, potente Hemmer der HMG-COA-Reduktase, reprimieren deutlich den loe-Phänotyp. In loe ist der Cholesterinester-Spiegel auf 40% abgesenkt. Eine weitere genetische Interaktion von loe konnte nachgewiesen werden: Die Mutante für das Drosophila-Homolog von APP (Appl) verstärkt den neurodegenerativen Phänotyp in loe stark, wogegen die Appl-Mutante selbst keine neurodegenerativen Defekte aufweist. Darüberhinaus zeigt die Doppelmutante Defekte, die keine der Einzelmutanten aufweist: Sterilität oder eine extrem kurze Lebensdauer von nur 3-4 Tagen. Diese Interaktion ließ sich auf molekularer Ebene charakterisieren. Die proteolytische Prozessierung von APPL durch Sekretasen ist in loe alteriert. In der vorliegenden Arbeit konnte gezeigt werden, dass durch die loe-Mutation die b-Sekretase aus Vertebraten (BACE) und eine bisher noch nicht beschriebene endogene Sekretase aus Drosophila negativ beeiflusst werden. Ein AMPK-Komplex mit LOE I als g-Untereinheit scheint über den Cholesterinester-Spiegel die Aktivität einer speziellen Untergruppe der Sekretasen zu beeinflussen. Die Missfunktion dieser Sekretasen ist ein kritischer Punkt in der Pathogenese der Alzheimer-Krankheit. Die loe-Mutation wirft neues Licht auf die bekannten Verbindungen zwischen Cholesterin-Stoffwechsel, Vesikelverkehr und Prozessierung von APP(L). Mit den großen Möglichkeiten, die die Drosophila-Genetik bietet, stellt diese neue Mutante ein weiteres Werkzeug zur Charakterisierung von Therapie-Ansätzen für die Alzheimer-Kankheit dar. Die vorliegende Arbeit belegt um ein weiteres Mal, dass Drosophila ein potentes Modellsystem zur Untersuchung humaner, neurodegenerativer Erkrankungen wie Chorea Huntington, Parkinson oder der Alzheimer Krankheit ist. N2 - Human neurodegenerative diseases are the main topic of molecular neurobiological basic research. To investigate detailed mechanisms in vivo one uses the tool of genetic model organisms like Caenorhabditis elegans or Drosophila melanogaster for quite a long while. This thesis describes the Drosophila neurodegenration mutant löchrig (loe), which can be used as a model for cholesterol metabolism in respect to neurodegeneration. Mutant loe flies show strong and progressive age-dependent degenration of neurons undergoing necrotic cell death. The P-element inserted in an intron of the gene coding for the Drosophila 5'-AMP activated protein kinase (AMPK) complex gamma subunit is responsible for the mutation in loe. The various splice forms of the loe gene code for different regulatory gamma subunits of this complex consisiting of three subunits. The splice form loeI is affected by the P-element insertion, parts of the P-element are spliced into the loeI transkript in the loe mutant. The neuronal expression of one copy of loeI in the mutant background revertes the neurodegenerative phenotype which can not be achieved by expression of one of the other splice forms. The LOE I protein contains in its N-terminus several putative interaction motifs and domaines. These could get a LOE I-containing AMPK complex in context with the APP (amyloid precursor protein) or the cytoskeletton. An interaction with NiPSNAP ? a protein with a putative function in the vesicular transport ? has been proved molecularly. A human homolog of LOE I is not yet known, although there are three different isoforms of a AMPK gamma subunit described in humans. The loe mutant interacts genetically with the columbus (clb) mutant, wich is affected in the gene of the HMG-CoA reductase, the key enzyme in cholesterol biosynthesis. This shown interaction verifies a negative regulation of the HMG-CoA reductase by the AMPK complex in Drosophila. Thus the clb mutation supresses the loe phenotype, an overexpression of clb enhances the neurodegeneration. A supression of the neurodegenerative phenotype can be also achieved by a statin treatment of loe flies. Statins are potent inhibitors of the HMG-CoA reductase. Another genetic interaction exists between loe and the Appl mutant. Appl d, the null mutant of the Drosophila APP homolog, enhances strongly the neurogenerative phenotype of loe, whereas the Appl mutant itself shows no neuronal defects. In addition the double mutant shows defects which none of the single mutants show: sterility of females and a dramatic shortened lifespan of only 3-4 days. This interaction could be characterized on the molecular level: The proteelytic processing of APPL by sectretases is altered in the loe mutant. Both the BACE sectretase from vertebrates and an so far uncharakterized endogenous sectretase in Drosophila are negatively influenced by the loe mutation. An AMPK complex containing LOE I as the gamma subunit seems to regulate the activity of a subgroup of the sectretases via the cholesterolester level. The misfunction of secretases is a crutial point in the pathogenesis of Alzheimer's disease. The loe mutation gives new insights in the already known links between cholesterol homeostasis, vesicular transport, and processing of APP(L). Together with the exstensive tools of Drosophila genetics this new mutant will supply new possibilities to characterize putative therapies to cure Alzheimer's disease. This thesis at another time presents Drosophila as an potent model system for the research on human neurodegenerative diseases like Huntington's disease, Parkinson or Alzheimer's disease. KW - Taufliege KW - Mutante KW - Cholesterin KW - Nervenzelle KW - Degeneration KW - Alzheimer-Krankheit KW - Neurodegeneration KW - Drosophila KW - APP KW - Cholesterin KW - Alzheimer Krankheit KW - AMPK KW - Neurodegeneration KW - Drosophila KW - APP KW - Cholesterol KW - Alzheimer's Disease KW - AMPK Y1 - 2002 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-2963 ER - TY - JOUR A1 - Tsai, Yu-Chen A1 - Grimm, Stefan A1 - Chao, Ju-Lan A1 - Wang, Shih-Chin A1 - Hofmeyer, Kerstin A1 - Shen, Jie A1 - Eichinger, Fred A1 - Michalopoulou, Theoni A1 - Yao, Chi-Kuang A1 - Chang, Chih-Hsuan A1 - Lin, Shih-Han A1 - Sun, Y. Henry A1 - Pflugfelder, Gert O. T1 - Optomotor-blind negatively regulates Drosophila eye development by blocking Jak/STAT signaling JF - PLoS ONE N2 - Organ formation requires a delicate balance of positive and negative regulators. In Drosophila eye development, wingless (wg) is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb) is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occur predominantly in the ventral margin. Our results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired. KW - morphogenetic furrow progression KW - cell fate KW - compartment boundary KW - reporter gene KW - compound eye KW - gene expression KW - retinal differentiation KW - acts downstream KW - imaginal disk KW - glial cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143577 VL - 10 IS - 3 ER - TY - THES A1 - Trunzer, Brigitte T1 - Paarungshäufigkeit und Aufteilung der Reproduktion bei Pachycondyla villosa T1 - Mating frequency and partitioning of reproduction in Pachycondyla villosa N2 - In Ameisensozietäten treten häufig Konflikte um die Reproduktion auf. Um dabei das soziale Verhalten der beteiligten Individuen und die Koloniestruktur zu verstehen ist es wichtig, die Verwandtschaftsstruktur innerhalb der Kolonien zu kennen. Diese wird durch die Paarungshäufigkeit der Königinnen, die Anzahl der Königinnen im Nest, deren Verwandtschaftsgrad zueinander, sowie der Aufteilung der Reproduktion zwischen ihnen bestimmt. Bei Pachycondyla villosa wurden durch die genetische Analyse dieser Faktoren mittels Multilokus-DNA- Fingerprinting das Paarungssystem und die Koloniestruktur genauer untersucht. Die Bestimmung der Paarungshäufigkeit ergab, daß sich P. villosa-Königinnen nur einmal paaren. Befanden sich mehrere Königinnen in einem Nest, so waren sie nicht miteinander verwandt und die Reproduktion war gleichmäßig zwischen ihnen aufgeteilt. Im Gegensatz zu den polygynen Kolonien von P. villosa traten in königinlosen Arbeiterinnengruppen zwischen den assoziierten Tieren heftige Konflikte um die Reproduktion auf. Diese führten zur Etablierung linearer Dominanzhierarchien und die Alpha-Tiere waren bei der Produktion von Männchen am erfolgreichsten. Betreuer Hölldobler, Berthold; Prof. Dr. Gutachter Hölldobler, Berthold; Prof. Dr. Gutachter Heinze, Jürgen; Prof. Dr. N2 - In ant societies there are often conflicts over reproduction. Therefore, to understand the social behavior and the structure of the colony, it is essential to know the kin structure within the colonies. Kin structure is affected by the mating frequency of queens, the number and relatedness of queens and the allocation of reproduction between them. In Pachycondyla villosa, the mating system and the colony structure was determined by analyzing these factores genetically with multilocus DNA fingerprinting. The examination of the mating frequency showed, that queens of P. villosa only mate once. In the presence of more than one queen in the nest, the associated queens were not related and reproduction was evenly shared. In contrast to the polygynous colonies overt conflicts over reproduction occured in queenless worker groups of P. villosa. By that linear dominance hierarchies were established and the alpha-workers were most successful in producing males. KW - Ponerinae KW - Fortpflanzungsverhalten KW - Formicidae KW - Ponerinae KW - DNA-Fingerprinting KW - Paarungshäufigkeit KW - Koloniegründung KW - reproductive skew KW - Dominanz KW - reproduktiver Erfolg KW - Formicidae KW - Ponerinae KW - DNA fingerprinting KW - mating frequency KW - colony founding KW - reproductive skew KW - dominance KW - reproductive success Y1 - 1999 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-2436 ER - TY - THES A1 - Triphan, Tilman T1 - The Central Control of Gap Climbing Behaviour in Drosophila melanogaster T1 - Die zentrale Kontrolle des Kletterverhaltens bei Drosophila melanogaster N2 - In this work, a behavioural analysis of different mutants of the fruit fly Drosophila melanogaster has been carried out. Primarily, the gap climbing behaviour (Pick & Strauss, 2005) has been assayed as it lends itself for the investigation of decision making processes and the neuronal basis of adaptive behaviour. Furthermore it shows how basic motor actions can be combined into a complex motor behaviour. Thanks to the neurogenetic methods, Drosophila melanogaster has become an ideal study object for neurobiological questions. Two different modules of climbing control have been examined in detail. For the decision making, the mutant climbing sisyphus was analysed. While wild-type flies adapt the initiation of climbing behaviour to the width of the gap and the probability for a successful transition. climbing sisyphus flies initiate climbing behaviour even at clearly insurmountable gap widths. The climbing success itself is not improved in comparison to the wild-type siblings. The mutant climbing sisyphus is a rare example of a hyperactive mutant besides many mutants that show a reduced activity. Basic capabilities in vision have been tested in an optomotor and a distance-estimation paradigm. Since they are not affected, a defect in decision making is most probably the cause of this behavioural aberration. A second module of climbing control is keeping up orientation towards the opposite side of the gap during the execution of climbing behaviour. Mutants with a structural defect in the protocerebral bridge show abnormal climbing behaviour. During the climbing attempt, the longitudinal body axis does not necessarily point into the direction of the opposite side. Instead, many climbing events are initiated at the side edge of the walking block into the void and have no chance to ever succeed. The analysed mutants are not blind. In one of the mutants, tay bridge1 (tay1) a partial rescue attempt used to map the function in the brain succeeded such that the state of the bridge was restored. That way, a visual targeting mechanism has been activated, allowing the flies to target the opposite side. When the visibility of the opposing side was reduced, the rescued flies went back to a tay1 level of directional scatter. The results are in accord with the idea that the bridge is a central constituent of the visual targeting mechanism. The tay1 mutant was also analysed in other behavioural paradigms. A reduction in walking speed and walking activity in this mutant could be rescued by the expression of UAS-tay under the control of the 007Y-GAL4 driver line, which concomitantly restores the structure of the protocerebral bridge. The separation of bridge functions from functions of other parts of the brain of tay1 was accomplished by rescuing the reduced optomotor compensation in tay1 by the mb247-GAL4>UAS-tay driver. While still having a tay1-like protocerebral bridge, mb247-GAL4 rescue flies are able to compensate at wild-type levels. An intact compensation is not depended on the tay expression in the mushroom bodies, as mushroom body ablated flies with a tay1 background and expression of UAS-tay under the control of mb247-GAL4 show wild-type behaviour as well. The most likely substrate for the function are currently unidentified neurons in the fan-shaped body, that can be stained with 007Y-GAL4 and mb247-GAL4 as well. N2 - In der vorliegenden Arbeit wurde eine Verhaltensanalyse verschiedener Mutanten der Fruchtfliege Drosophila melanogaster durchgeführt. Dazu wurde primär das Lücken-überwindungsparadigma (Pick & Strauss, 2005) herangezogen, das sich auf besondere Weise zur Erforschung von Entscheidungsfindung und adaptivem Verhalten anbietet. Weiterhin zeigt sich hier, wie einfache motorische Aktionen zu einem komplexen motorischen Verhalten zusammengefügt werden können. Dank der Möglichkeiten der Gentechnik bietet sich Drosophila hier als Studienobjekt an. Zwei Module der Kletterkontrolle wurden genauer untersucht. Im Bezug auf die Entscheidungsfindung wurde die Mutante climbing sisyphus getestet. Während der Wildtyp sein Kletterverhalten sehr genau an die Lückenbreite und die Wahrscheinlichkeit einer erfolgreichen Überquerung anpasst (Pick & Strauss, 2005), werden bei climbing sisyphus auch bei einer unmöglich zu überquerenden Lücke noch Kletteraktionen initiiert. Der Klettererfolg selbst ist im Vergleich zum Wildtyp nicht verbessert. Die Mutante climbing sisyphus ist ein seltenes Beispiel einer hyperaktiven Mutante neben vielen Mutanten die eine reduzierte Aktivität zeigen. Grundlegende Fähigkeiten im visuellen Bereich wurden in der Optomotorik und im Entfernungsschätzen getestet und sind in climbing sisyphus nicht beeinträchtigt, ein Defekt in der Entscheidungsfindung ist wahrscheinlich Ursache des gestörten Verhaltens. Ein zweites Modul der Kletterkontrolle betrifft die Aufrechterhaltung der Orientierung hin zur gegenüberliegenden Seite der Lücke. Mutanten mit einem Strukturdefekt in der Protozerebralbrücke des Zentralkomplexes zeigen ein abnormes Kletterverhalten. Die Körperlängsachse zeigt während des Klettervorgangs nicht in die Richtung der gegenüberliegenden Seite. Stattdessen werden oft Klettervorgänge am seitlichen Rand des Klettersteges initiiert, die keinerlei Aussicht auf Erfolg haben. Die untersuchten mutanten Fliegen sind nicht blind. In einem der Stämme, tay bridge1 (tay1), gelang zur funktionellen Kartierung eine partielle Rettung dieses Verhaltens durch die Expression des wildtypischen Gens in einem kleinen Teil des Nervensystems. Das Wiederherstellen der wildtypischen Brückenstruktur in tay1 aktiviert einen visuellen Zielmechanismus, der eine Ausrichtung der Fliegen auf die gegenüberliegende Seite ermöglicht. Wenn die Sichtbarkeit der gegenüberliegenden Seite reduziert wird, geht dieser Rettungseffekt verloren. Die Brücke ist nach diesen Befunden ein zentraler Bestandteil der visuell gesteuerten Zielmotorik. Die tay1 Mutante wurde auch in weiteren Verhaltensexperimenten untersucht. So konnte eine in dieser Mutante vorliegende Reduktion der Laufgeschwindigkeit und Laufaktivität durch die Expression von UAS-tay unter der Kontrolle des Treibers 007Y-GAL4 zusammen mit der Struktur der Brücke gerettet werden. Eine Rettung der reduzierten Kompensation für optomotorische Stimuli in tay1 durch den Treiber mb247-GAL4 erlaubte eine Trennung von tay1 Defekten in der Brücke von Defekten in anderen Teilen des Gehirns. Trotz einer tay1-typischen unterbrochenen Brücke sind mit mb247-GAL4>UAS-tay gerettete Fliegen in der Lage eine Stimulation mit optomotorischen Reizen auf wildtypischem Niveau zu kompensieren. Diese Kompensation hängt nicht von den Pilzkörpern ab, da auf chemischen Wege pilzkörperablatierte Fliegen mit einer Expression von UAS-tay unter der Kontrolle von mb247-GAL4 sich trotz tay1 Hintergrund ebenfalls wildtypisch verhalten. Die wahrscheinlichsten Träger für diese Rettung sind noch nicht identifizierte Neurone im Fächerförmigen Körper des Zentralkomplexes, die mit 007Y-GAL4 und mb247-GAL4 angefärbt werden können. KW - Taufliege KW - Drosophila KW - Bewegungsverhalten KW - Mutante KW - Verhaltensanalyse KW - Drosophila KW - Behaviour KW - Locomotion Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-43666 ER - TY - JOUR A1 - Trinks, Nora A1 - Reinhard, Sebastian A1 - Drobny, Matthias A1 - Heilig, Linda A1 - Löffler, Jürgen A1 - Sauer, Markus A1 - Terpitz, Ulrich T1 - Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy JF - Communications Biology N2 - Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells' lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution. KW - biological fluorescence KW - fluorescence imaging KW - imaging the immune system KW - infectious diseases KW - super-resolution microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-264996 VL - 4 IS - 1 ER - TY - JOUR A1 - Trinkl, Moritz A1 - Kaluza, Benjamin F. A1 - Wallace, Helen A1 - Heard, Tim A. A1 - Keller, Alexander A1 - Leonhardt, Sara D. T1 - Floral Species Richness Correlates with Changes in the Nutritional Quality of Larval Diets in a Stingless Bee JF - Insects N2 - Bees need food of appropriate nutritional quality to maintain their metabolic functions. They largely obtain all required nutrients from floral resources, i.e., pollen and nectar. However, the diversity, composition and nutritional quality of floral resources varies with the surrounding environment and can be strongly altered in human-impacted habitats. We investigated whether differences in plant species richness as found in the surrounding environment correlated with variation in the floral diversity and nutritional quality of larval provisions (i.e., mixtures of pollen, nectar and salivary secretions) composed by the mass-provisioning stingless bee Tetragonula carbonaria (Apidae: Meliponini). We found that the floral diversity of larval provisions increased with increasing plant species richness. The sucrose and fat (total fatty acid) content and the proportion and concentration of the omega-6 fatty acid linoleic acid decreased, whereas the proportion of the omega-3 fatty acid linolenic acid increased with increasing plant species richness. Protein (total amino acid) content and amino acid composition did not change. The protein to fat (P:F) ratio, known to affect bee foraging, increased on average by more than 40% from plantations to forests and gardens, while the omega-6:3 ratio, known to negatively affect cognitive performance, decreased with increasing plant species richness. Our results suggest that plant species richness may support T. carbonaria colonies by providing not only a continuous resource supply (as shown in a previous study), but also floral resources of high nutritional quality. KW - floral resources KW - plant-insect interactions KW - nutrition KW - biodiversity KW - bee decline Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200605 SN - 2075-4450 VL - 11 IS - 2 ER - TY - JOUR A1 - Trifault, Barbara A1 - Mamontova, Victoria A1 - Burger, Kaspar T1 - In vivo proximity labeling of nuclear and nucleolar proteins by a stably expressed, DNA damage-responsive NONO-APEX2 fusion protein JF - Frontiers in Molecular Biosciences N2 - Cellular stress can induce DNA lesions that threaten the stability of genes. The DNA damage response (DDR) recognises and repairs broken DNA to maintain genome stability. Intriguingly, components of nuclear paraspeckles like the non-POU domain containing octamer-binding protein (NONO) participate in the repair of DNA double-strand breaks (DSBs). NONO is a multifunctional RNA-binding protein (RBP) that facilitates the retention and editing of messenger (m)RNA as well as pre-mRNA processing. However, the role of NONO in the DDR is poorly understood. Here, we establish a novel human U2OS cell line that expresses NONO fused to the engineered ascorbate peroxidase 2 (U2OS:NONO-APEX2-HA). We show that NONO-APEX2-HA accumulates in the nucleolus in response to DNA damage. Combining viability assays, subcellular localisation studies, coimmunoprecipitation experiments and in vivo proximity labeling, we demonstrate that NONO-APEX2-HA is a stably expressed fusion protein that mimics endogenous NONO in terms of expression, localisation and bona fide interactors. We propose that in vivo proximity labeling in U2OS:NONO-APEX2-HA cells is capable for the assessment of NONO interactomes by downstream assays. U2OS:NONO-APEX2-HA cells will likely be a valuable resource for the investigation of NONO interactome dynamics in response to DNA damage and other stimuli. KW - APEX2 KW - proximity labeling KW - NONO KW - paraspeckles KW - nucleolus KW - DNA damage Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-276707 SN - 2296-889X VL - 9 ER - TY - CHAP A1 - Trendelenburg, Michael F. A1 - Spring, Herbert A1 - Scheer, Ulrich A1 - Franke, Werner W. T1 - Morphology of nucleolar cistrons in a plant cell, Acetabularia mediterranea N2 - The structural organization of transcriptionally active DNA that contains cistrons for precursor molecules of ribosomal RNA is described in positively stained spread preparations from nuclei and nucleoli isolated from the green alga, Acetabularia mediterranea Lmx. These nuclei contain large aggregates of nucleolar subunits in which fibril-covered regions, the putative active cistrons for precursors of ribosomal RNA, alternate with fibril-free intercepts, the "spacers". The length distribution of the different intercepts of this DNA is given, and the pattern is compared with those shown in animal cell systems. The data are discussed in relation to problems of transcription and of amplification of ribosomal RNA genes. KW - ribosomal RNA genes; electron microscopy; spread preparations Y1 - 1974 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-32213 ER - TY - JOUR A1 - Trendelenburg, Michael F. A1 - Scheer, Ulrich A1 - Zentgraf, Hanswalter A1 - Franke, Werner W. T1 - Heterogeneity of spacer lengths in circles of amplified ribosomal DNA of two insect species, Dytiscus marginalis and Acheta domesticus N2 - No abstract available Y1 - 1976 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33055 ER - TY - JOUR A1 - Trendelenburg, Michael F. A1 - Scheer, Ulrich A1 - Franke, W. W. T1 - Structural organization of the transcription of ribosomal DNA in oocytes of the house cricket N2 - No abstract available Y1 - 1973 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33113 ER - TY - JOUR A1 - Trendelenburg, Michael F. A1 - Franke, Werner W. A1 - Scheer, Ulrich T1 - Frequencies of circular units of nucleolar DNA in oocytes of two insects, Acheta domesticus and Dytiscus marginalis, and changes of nucleolar morphology during oogenesis N2 - The organization of the extrachromosomal nucleolar material in oocytes of two insect species with different ovary types, the house cricket Acheta domesticus (panoistic ovary) and the water beetle Dytiscus marginalis (meroistic ovary), was studied with light and electron microscopic techniques. Stages early in oogenesis were compared with fully vitellogenic stages (mid-to-Iate diplotene). The arrangement of the nucleolar material undergoes a marked change from a densely aggregated to a dispersed state. The latter was characterized by high transcriptional activity. In spread and positively stained preparations of isolated nucleolar material, a high frequency of small circular units of transcribed rDNA was observed and rings with small numbers (1-5) of pre-rRNA genes were predominant. The observations suggest that the "extra DNA body" observed in early oogenic stages of both species represents a dense aggregate of numerous short circular units of nucleolar chromatin, with morphological subcomponents identifiable in ultrathin sections. These apparently remain in close association with the chromosomal nucleolar organizer(s). The observations further indicate that the individual small nucleolar subunit circles dissociate and are dispersed as actively transcribed rDNA units later in diplotene. The results are discussed in relation to principles of the ultrastructural organization of nucleoli in other cell types as well as in relation to possible mechanisms of gene amplification. KW - Zelldifferenzierung Y1 - 1977 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-41370 ER - TY - CHAP A1 - Trendelenburg, M. F. A1 - Franke, Werner W. A1 - Spring, H. A1 - Scheer, Ulrich T1 - Ultrastructure of transcription in the nucleoli of the green algae Acetabularia major and A. mediterranea N2 - No abstract available Y1 - 1975 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33779 ER - TY - THES A1 - Treichel, Dieter T1 - Isolierung, evolutive Einordnung und funktionelle Charakterisierung von Knopfkopf, einem buttonhead-Ortholog in der Maus T1 - Isolation, evolutioniary analysis and functional characterization of Knopfkopf, a buttonhead ortholog in the mous. N2 - Isolierung des Sp1-verwandten Transkriptionsfaktors Knopfkopf mittels eines PCR-basierten Homologie-Screens in der Maus. Das Gen Knopfkopf wurde anschließend hinsichtlich der evolutiven Verwandtschaftsbeziehungen zum Drosophila-Gen buttonhead eingeordnet. Eine funktionelle Charakterisierung erfolgte mit Hilfe einer gezielten Geninaktivierung durch homologe Rekombination (knock out). Es konnte gezeigt werden, dass das Gen in der Embryogenese der Maus essentiell ist für die Entwicklung der Extremitäten, der Nase und des Zentralen Nervensystems sowie der sekundären Gastrulation. N2 - Isolation of the Sp1-related transkription factor Knopfkopf by a PCR-based homology screen in the mouse. The Knopfkopf gene was analysed regarding its evolutionary relationship with the Drosophila gene buttonhead. The functional characterization was done via a targeted gene inactivation by a homologous recombination (knock out). It was shown that the gene is necessary during the mouse embryogenesis for the development of limbs, nose, central nervous system, as well as the secondary gastrulation. KW - Maus KW - Gap-gen KW - Gastrulation KW - Genexpression KW - Knopfkopf KW - buttonhead KW - Knopfkopf KW - buttonhead Y1 - 2003 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-5867 ER - TY - THES A1 - Torlopp, Angela T1 - Die Rolle von FGF in der frühen Kardiogenese und Proepikardiogenese im Hühnerembryo T1 - The role of FGF signaling during early heart and proepicardium development in the chick embryo N2 - In dieser Arbeit sollte die Funktion von FGF-Signalen im Herzfeld und in der Entwicklung des Proepikards im Hühnerembryo untersucht werden. Fibroblasten-Wachstumsfaktoren (FGF) sind eine große Gruppe von Signalmolekülen und in eine Vielzahl von Entwicklungsprozessen involviert. Das Proepikard (PE), welches sich asymmetrisch auf dem rechten Sinushorn des Sinus venosus entwickelt, bildet die Grundlage des Koronargefäßsystems des Herzens. FGF-Liganden (FGF2, FGF10, FGF12) werden insbesondere in den epithelialen Zellen des Proepikards exprimiert, sowie an der sinomyokardialen Basis dieser embryonalen Progenitorpopulation. Die FGF-Rezeptoren (FGFR1, FGFR2, FGFR4) weisen ein ähnliches Expressionsmuster auf und deren Inhibition, durch spezifische Antagonisten, war der Ausgangspunkt für die funktionelle Analyse der proepikardialen FGF-Signalaktivität. Die Inhibition von FGF-Signalen in vitro führt zu einem verringerten Wachstum sowie einer erhöhten Apoptoserate in proepikardialen Explantaten, die unter serumfreien Bedingungen kultiviert wurden. Es konnte gezeigt werden, dass sowohl der Ras/MAPK- als auch der PI3-Kinase-Signalweg, beides Bestandteile der FGF-Signaltransduktion, für das Wachstum und Überleben proepikardialer Zellen verantwortlich sind. Dagegen sind FGF-Signale nicht in die Etablierung proepikardialer Identität involviert, wie die Analyse der Expression etablierter proepikardialer Markergene wie TBX18, WT1 und TBX5 nach FGF-Inhibition zeigte. Dies konnte gleichfalls durch in vivo-Experimente gezeigt werden, in denen die rechtsseitige Inhibition von FGF zu einem retardierten Proepikardwachstum führte. Weiterhin konnte gezeigt werden, dass die asymmetrische Apoptose in der sich transient entwickelnden linksseitigen Proepikardanlage auf eine frühe differentielle Expression von Apoptosegenen wie Caspase 2 zurückgeht. Diese asymmetrische Expression wird von FGF8 reguliert, wahrscheinlich als Teil eines frühen rechtsseitigen Signalweges, der Apoptose im rechten Sinushorn des kardialen Einflusstraktes verhindert. Im zweiten Teil der Arbeit wurde die Expression der Hyaluronansynthase 2 (HAS2) in Abhängigkeit von FGF in der Herzfeldregion analysiert. Hyaluronansynthasen produzieren Hyaluronsäure, welches eine essentielle Komponente der extrazellulären Matrix ist. Es wurde in vivo gezeigt, dass die Expression von HAS2 im primären Herzfeld in gleicher Weise von FGF reguliert wird wie die des kardialen Transkriptionsfaktors NKX2.5. Die Ergebnisse dieser Arbeit verdeutlichen, dass FGF während der frühen Entwicklung des Herzens und der Entstehung des Proepikards diverse Funktionen besitzt. N2 - The aim of this study was the functional analysis of FGF signaling during early heart field formation and proepicardial development in the chick embryo. Fibroblast growth factors (FGF) belong to a large group of signaling molecules and play crucial roles in many different developmental processes. The proepicardium (PE) develops asymmetrically on the right sinus horn of the cardiac inflow tract and is the source of the coronary vasculature of the heart. FGF ligands (FGF2, FGF10, and FGF12) are specifically expressed in epithelial cells of the proepicardium as well as in the underlying inflow tract myocardium. FGF receptors (FGFR1, FGFR2, and FGFR4) display similar expression patterns in the proepicardium and their inhibition by specific antagonists was the entry point into the functional analysis of FGF signaling in proepicardial cells. The inhibition of FGF signaling in vitro leads to retarded outgrowth as well as increased apoptosis in proepicardial explants, which were cultured under serum free conditions. It was shown that both Ras/MAPK and PI3 kinase signaling as integral parts of FGF signaling transduction are responsible for growth and survival of proepicardial cells in this context. However, FGF signaling is not involved in the establishment of proepicardial identity as shown by the maintenance of expression of well-established proepicardial marker genes such as TBX18, WT1 and TBX5 after FGF inhibition. These findings were verified by in vivo experiments, showing that inhibition of FGF leads to retarded outgrowth of the proepicardium. Furthermore it was shown that asymmetric apoptosis in a transiently established left-sided PE-anlage is based on an early differential expression of apoptosis-inducing genes like Caspase 2. This asymmetric expression is regulated by FGF8 probably as part of an early right-sided signaling pathway, which prevents apoptosis in the right sinus horn of the cardiac inflow tract. In a second topic of this thesis the expression of the hyaluronan synthase 2 (HAS2) in the control of FGF signaling during early heart field formation was analyzed. Hyaluronan synthases are involved in the production of hyaluronic acid, which is an essential component of the extracellular matrix. The role of FGF signaling was tested in vivo and it is shown here, that the expression of HAS2 in the primary heart field is dependent on FGF as well as other cardiac marker genes such as the transcription factor NKX2.5. This thesis demonstrates that FGF has multiple roles during early heart development and formation of the proepicardium. KW - Huhn KW - Embryonalentwicklung KW - Fibroblastenwachstumsfaktor KW - Epikard KW - Hyaluronsäure KW - FGF KW - Wnt KW - Has2 KW - Proepikard KW - Kardiogenese KW - Apoptose KW - Hühnerembryo KW - FGF KW - Wnt KW - Has2 KW - proepicardium KW - cardiogenesis KW - apoptosis KW - chick embryo Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47695 ER - TY - JOUR A1 - Tony, H. P. A1 - Shen, B. J. A1 - Reusch, P. A1 - Sebald, Walter T1 - Design of human interleukin-4 antagonists inhibiting interleukin-4-dependent and interleukin-13-dependent responses in T-cells and B-cells with high efficiency N2 - Human interleukin-4 possesses two distinct sites for receptor activation. A signaHing site, comprising residues near the C-terminus on helix D, determines the efficacy of interleukin-4 signal transduction without affecting the binding to the interleukin-4 receptor a subunit. A complete antagonist and a series of low-efficacy agonist variants of human interleukin-4 could be generated by introducing combinations of two or three negatively charged aspartic acid residues in this site at positions 121, 124, and 125. One of the double variants, designated [R121D,Y124D]interleukin-4, with replacements of böth Arg121 and Tyr124 by aspartic acid residues was completely inactive in all analysed cellular responses. The loss of efficacy in [R121D,Y124D]interleukin-4 is estimated to be larger than 2000-fold. Variant [R121D,Y124D]interleukin-4 was also a perfect antagonist for inhibition of interleukin-13-dependent responses in B-cells and the TF-1 cellline with a K\(_i\) value of approximately 100 pM. In addition, inhibition of both interleukin-4-induced and interleuk.in-13- induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin-4Rm the extracellular domain of the interleuk.in-4 receptor a subunit. These results indicate that efficient interleukin-4 antagonists can be designed on the basis of a sequential two-step activation model. In addition, the experiments indicate the functional participation of the interleukin-4 receptor a subunit in the interleukin-13 receptor system. KW - Biochemie Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62394 ER - TY - JOUR A1 - Tony, H. P. A1 - Lehrnbecher, T. A1 - Merz, H. A1 - Sebald, Werner A1 - Wilhelm, M. T1 - Regulation of IL-4 responsiveness in lymphoma B cells N2 - The responsiveness to IL-4 with and without costimulation with anti-IgM antibodies or phorbolester was studied in 35 cases of low grade non-Hodgkin Iymphoma by analyzing enhancement of CD23 and HLA dass li expression. The predominant phenotype responds directly to IL-4. Separate differentiation states can be distinguished according to coordinate or differential upregulation of CD23 and HLA dass II molecules by IL-4 alone, and differences in responsiveness to anti-IgM antibodies. A particular subgroup of B-lymphoma cells defines a separate stage of B-eeil differentiation. They fail to express high affinity binding sites for IL-4 and accordingly do not respond to IL-4- mediated signals. Cross-linking membrane lgM receptors or direct activation of protein kinase C via phorbolester induces IL-4 receptor expression and subsequent IL-4 reactivity. KW - Biochemie KW - B lymphocytes KW - CD23 KW - CLL KW - HLA class ll KW - IL-4 KW - IL-4-receptor KW - membrane immunoglobulin Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62520 ER - TY - JOUR A1 - Tomei, Sara A1 - Adams, Sharon A1 - Uccellini, Lorenzo A1 - Bedognetti, Davide A1 - De Giorgi, Valeria A1 - Erdenebileg, Narnygerel A1 - Libera Ascierto, Maria A1 - Reinboth, Jennifer A1 - Liu, Qiuzhen A1 - Bevilacqua, Generoso A1 - Wang, Ena A1 - Mazzanti, Chiara A1 - Marincola, Francesco M. T1 - Association between HRAS rs12628 and rs112587690 polymorphisms with the risk of melanoma in the North American population JF - Medical Oncology N2 - HRAS belongs to the RAS genes superfamily. RAS genes are important players in several human tumors and the single-nucleotide polymorphism rs12628 has been shown to contribute to the risk of bladder, colon, gastrointestinal, oral, and thyroid carcinoma. We hypothesized that this SNP may affect the risk of cutaneous melanoma as well. HRAS gene contains a polymorphic region (rs112587690), a repeated hexanucleotide -GGGCCT- located in intron 1. Three alleles of this region, P1, P2, and P3, have been identified that contain two, three, and four repeats of the hexanucleotide, respectively. We investigated the clinical impact of these polymorphisms in a case–control study. A total of 141 melanoma patients and 118 healthy donors from the North America Caucasian population were screened for rs12628 and rs112587690 polymorphisms. Genotypes were assessed by capillary sequencing or fragment analysis, respectively, and rs12628 CC and rs112587690 P1P1 genotypes significantly associated with increased melanoma risk (OR = 3.83, p = 0.003; OR = 11.3, p = 0.033, respectively), while rs112587690 P1P3 frequency resulted significantly higher in the control group (OR = 0.5, p = 0.017). These results suggest that rs12628 C homozygosis may be considered a potential risk factor for melanoma development in the North American population possibly through the linkage to rs112587690. KW - HRAS KW - polymorphism KW - melanoma KW - rs12628 KW - rs112587690 Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126834 VL - 29 IS - 5 ER - TY - JOUR A1 - Tomaszkiewicz, Marta A1 - Chalopin, Domitille A1 - Schartl, Manfred A1 - Galiana, Delphine A1 - Volff, Jean-Nicolas T1 - A multicopy Y-chromosomal SGNH hydrolase gene expressed in the testis of the platyfish has been captured and mobilized by a Helitron transposon JF - BMC Genetics N2 - Background: Teleost fish present a high diversity of sex determination systems, with possible frequent evolutionary turnover of sex chromosomes and sex-determining genes. In order to identify genes involved in male sex determination and differentiation in the platyfish Xiphophorus maculatus, bacterial artificial chromosome contigs from the sex-determining region differentiating the Y from the X chromosome have been assembled and analyzed. Results: A novel three-copy gene called teximY (for testis-expressed in Xiphophorus maculatus on the Y) was identified on the Y but not on the X chromosome. A highly related sequence called texim1, probably at the origin of the Y-linked genes, as well as three more divergent texim genes were detected in (pseudo) autosomal regions of the platyfish genome. Texim genes, for which no functional data are available so far in any organism, encode predicted esterases/lipases with a SGNH hydrolase domain. Texim proteins are related to proteins from very different origins, including proteins encoded by animal CR1 retrotransposons, animal platelet-activating factor acetylhydrolases (PAFah) and bacterial hydrolases. Texim gene distribution is patchy in animals. Texim sequences were detected in several fish species including killifish, medaka, pufferfish, sea bass, cod and gar, but not in zebrafish. Texim-like genes are also present in Oikopleura (urochordate), Amphioxus (cephalochordate) and sea urchin (echinoderm) but absent from mammals and other tetrapods. Interestingly, texim genes are associated with a Helitron transposon in different fish species but not in urochordates, cephalochordates and echinoderms, suggesting capture and mobilization of an ancestral texim gene in the bony fish lineage. RT-qPCR analyses showed that Y-linked teximY genes are preferentially expressed in testis, with expression at late stages of spermatogenesis (late spermatids and spermatozeugmata). Conclusions: These observations suggest either that TeximY proteins play a role in Helitron transposition in the male germ line in fish, or that texim genes are spermatogenesis genes mobilized and spread by transposable elements in fish genomes. KW - sex determination KW - testis KW - Y chromosome KW - rolling-circle transposons KW - factor acetylhydrolase activity KW - platelet activation factor KW - xiphophorus maculatus KW - oryzias-latipes KW - sequence alignment KW - DM-domain gene KW - sex-determining region KW - evolution KW - fish KW - SGNH hydrolase KW - helitron KW - transposition KW - platyfish KW - sex chromosomes Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116746 VL - 15 IS - 44 ER - TY - THES A1 - Tischner, Denise T1 - Mechanistische Untersuchungen zur Therapie von Multipler Sklerose am Beispiel der Experimentellen Autoimmunen Encephalomyelitis T1 - Investigation of therapy strategies of multiple sclerosis by using Experimental Autoimmune Encephalomyelitis N2 - No abstract available KW - Autoimmunität KW - Immunsystem KW - Multiple Sklerose KW - Glucocorticosteroide KW - Tiermodell KW - regulatorische T Zelle KW - CD28 KW - Antigentherapie KW - EAE KW - Superagonist KW - regulatory T cell KW - CD28 KW - antigen therapy KW - EAE KW - superagonist Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-25258 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 - THES A1 - Thum, Andreas Stephan T1 - Sugar reward learning in Drosophila : neuronal circuits in Drosophila associative olfactory learning T1 - Zucker-Belohnungslernen von Drosophila N2 - Genetic intervention in the fly Drosophila melanogaster has provided strong evidence that the mushroom bodies of the insect brain act as the seat of memory traces for aversive and appetitive olfactory learning (reviewed in Heisenberg, 2003). In flies, electroshock is mainly used as negative reinforcer. Unfortunately this fact complicates a comparative consideration with other inscets as most studies use sugar as positive reinforcer. For example, several lines of evidence from honeybee and moth have suggested another site, the antennal lobe, to house neuronal plasticity underlying appetitive olfactory memory (reviewed in Menzel, 2001; Daly et al., 2004). Because of this I focused my work mainly on appetitive olfactory learning. In the first part of my thesis, I used a novel genetic tool, the TARGET system (McGuire et al., 2003), which allows the temporally controlled expression of a given effector gene in a defined set of cells. Comparing effector genes which either block neurotransmission or ablate cells showed important differences, revealing that selection of the appropriate effector gene is critical for evaluating the function of neural circuits. In the second part, a new engram of olfactory memory in the Drosophila projection neurons is described by restoring Rutabaga adenlylate cyclase (rut-AC) activity specifically in these cells. Expression of wild-type rutabaga in the projection neurons fully rescued the defect in sugar reward memory, but not in aversive electric shock memory. No difference was found in the stability of the appetitive memories rescued either in projection neurons or Kenyon cells. In the third part of the thesis I tried to understand how the reinforcing signals for sugar reward are internally represented. In the bee Hammer (1993) described a single octopaminergic neuron – called VUMmx1 – that mediates the sugar stimulus in associative olfactory reward learning. Analysis of single VUM neurons in the fly (Selcho, 2006) identified a neuron with a similar morphology as the VUMmx1 neuron. As there is a mutant in Drosophila lacking the last enzymatic step in octopamine synthesis (Monastirioti et al., 1996), Tyramine beta Hydroxylase, I was able to show that local Tyramine beta Hydroxylase expression successfully rescued sugar reward learning. This allows to conclude that about 250 cells including the VUM cluster are sufficient for mediating the sugar reinforcement signal in the fly. The description of a VUMmx1 similar neuron and the involvement of the VUM cluster in mediating the octopaminergic sugar stimulus are the first steps in establishing a neuronal map for US processing in Drosophila. Based on this work several experiments are contrivable to reach this ultimate goal in the fly. Taken together, the described similiarities between Drosophila and honeybee regarding the memory organisation in MBs and PNs and the proposed internal representation of the sugar reward suggest an evolutionarily conserved mechanism for appetitive olfactory learning in insects. N2 - Arbeiten über das assoziative olfaktorische Lernen bei Drosophila, bei denen definierte Gruppen von Nerven genetisch verändert wurden, haben gezeigt, dass die Pilzkörper des Insektengehirns Gedächtnisspuren für aversives und appetitives Geruchslernen besitzen (Heisenberg, 2003). Hierzu wird bei der Fliege meistens Elektroschock als negativer Reiz bei der Pavlovschen Konditionierung benutzt. Leider erschwert dies einen Vergleich mit anderen Insekten, da in den meisten Studien Zucker als positiver Stimulus verwendet wird. Interessanterweise schlagen mehrere Arbeiten bei der Biene und der Motte zusätzlich zu den Pilzkörpern einen weiteren Bereich im Insektengehirn vor, der eine Gedächtnisspur des appetitiven Geruchslernens besitzt, die Antennalloben (Menzel, 2001; Daly et al., 2004). Aus diesen Gründen habe ich mich in meiner Arbeit intensiv mit dem appetitiven Geruchslernen beschäftigt. Im ersten Teil meiner Arbeit habe ich das TARGET System verwendet (McGuire et al., 2003), welches die zeitlich kontrollierte Expression eines beliebigen Reportergens in definierten Zellen erlaubt. Ein Vergleich verschiedener Effektoren zeigte, dass Proteine, die die Neurotransmission blocken (Shits; TNT, Kir2.1), besser geeignet sind, um die Funktion neuronaler Schaltkreise in Drosophila zu untersuchen. Effektoren, die Zellen abtöten, entfalten lediglich während der Entwicklung ihre volle Aktivität und eignen sich daher, z.B. um das larvale Verhalten zu analysieren. Im zweiten Teil beschreibe ich eine neue Gedächtnisspur für das Geruchslernen in den Projektionsneuronen. Die Expression des wildtypischen rutabaga Gens ausschließlich in diesen Zellen, rettete den Defekt im Zuckerlernen, nicht aber im Elektroschocklernen. Ferner scheinen die Gedächtnisspuren des appetitven Geruchslernens im Pilzkörper und den Projektionsneuronen gleich stabil zu sein. Im dritten Teil dieser Arbeit wurde die Frage gestellt, wie das Belohnungssignal des Zuckers im Fliegengehirn verarbeitet wird. Hammer (1993) beschrieb in der Biene ein einzelnes octopaminerges Neuron, das VUMmx1 Neuron, welches den Zuckerreiz beim assoziativen Geruchslernen vermittelt. Eine Einzelzellanalyse des VUM clusters von Drosophila zeigte ein ähnliches VUMmx1 Neuron erstmals bei der Fliege (M. Selcho, Diplomarbeit). Durch die lokale Expression der Tyramin beta Hydroxylase, das Oktopamin synthetisierende Enzym, im T-beta-H Mutanten Hintergrund, konnte gezeigt werden, dass ca. 250 Zellen (inklusive des VUM Clusters) ausreichen, das Belohnungssignal des Zuckers zu vermitteln. Beides, die Identifizierung eines VUMmx1 ähnlichen Neurons in der Fliege und die Eingrenzung der Neuronen, die das Belohnungssignal vermitteln, bilden die Basis für weitergehende Versuche. Diese erlauben es, neuronale Schaltkreise der US (Zucker)-Verarbeitung beim assoziativen olfaktorischen Lernen detailliert zu beschreiben. Insgesamt legen die übereinstimmenden Gedächtnisspuren im Pilzkörper und den Projektionsneuronen von Drosophila und der Honigbiene nahe, dass das olfaktorische Belohnungslernen einem in der Evolution konservierten Mechanismus entstammt. KW - Taufliege KW - Geruchswahrnehmung KW - Lernen KW - Neurologie KW - Zucker KW - Lernen KW - Gedächtnis KW - Dropsophila KW - sugar KW - learning KW - memory KW - drosophila Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-17930 ER - TY - JOUR A1 - Thorn, Simon A1 - Seibold, Sebastian A1 - Leverkus, Alexandro B A1 - Michler, Thomas A1 - Müller, Jörg A1 - Noss, Reed F A1 - Stork, Nigel A1 - Vogel, Sebastian A1 - Lindenmayer, David B T1 - The living dead: acknowledging life after tree death to stop forest degradation JF - Frontiers in Ecology and the Environment N2 - Global sustainability agendas focus primarily on halting deforestation, yet the biodiversity crisis resulting from the degradation of remaining forests is going largely unnoticed. Forest degradation occurs through the loss of key ecological structures, such as dying trees and deadwood, even in the absence of deforestation. One of the main drivers of forest degradation is limited awareness by policy makers and the public on the importance of these structures for supporting forest biodiversity and ecosystem function. Here, we outline management strategies to protect forest health and biodiversity by maintaining and promoting deadwood, and propose environmental education initiatives to improve the general awareness of the importance of deadwood. Finally, we call for major reforms to forest management to maintain and restore deadwood; large, old trees; and other key ecological structures. KW - forest degradation KW - biodiversity KW - deadwood Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-218575 VL - 18 IS - 9 SP - 505 EP - 512 ER - TY - JOUR A1 - Thorn, Simon A1 - Chao, Anne A1 - Georgiev, Konstadin B. A1 - Müller, Jörg A1 - Bässler, Claus A1 - Campbell, John L. A1 - Jorge, Castro A1 - Chen, Yan-Han A1 - Choi, Chang-Yong A1 - Cobb, Tyler P. A1 - Donato, Daniel C. A1 - Durska, Ewa A1 - Macdonald, Ellen A1 - Feldhaar, Heike A1 - Fontaine, Jospeh B. A1 - Fornwalt, Paula J. A1 - Hernández Hernández, Raquel María A1 - Hutto, Richard L. A1 - Koivula, Matti A1 - Lee, Eun-Jae A1 - Lindenmayer, David A1 - Mikusinski, Grzegorz A1 - Obrist, Martin K. A1 - Perlík, Michal A1 - Rost, Josep A1 - Waldron, Kaysandra A1 - Wermelinger, Beat A1 - Weiß, Ingmar A1 - Zmihorski, Michal A1 - Leverkus, Alexandro B. T1 - Estimating retention benchmarks for salvage logging to protect biodiversity JF - Nature Communications N2 - Forests are increasingly affected by natural disturbances. Subsequent salvage logging, a widespread management practice conducted predominantly to recover economic capital, produces further disturbance and impacts biodiversity worldwide. Hence, naturally disturbed forests are among the most threatened habitats in the world, with consequences for their associated biodiversity. However, there are no evidence-based benchmarks for the proportion of area of naturally disturbed forests to be excluded from salvage logging to conserve biodiversity. We apply a mixed rarefaction/extrapolation approach to a global multi-taxa dataset from disturbed forests, including birds, plants, insects and fungi, to close this gap. We find that 757% (mean +/- SD) of a naturally disturbed area of a forest needs to be left unlogged to maintain 90% richness of its unique species, whereas retaining 50% of a naturally disturbed forest unlogged maintains 73 +/- 12% of its unique species richness. These values do not change with the time elapsed since disturbance but vary considerably among taxonomic groups. Salvage logging has become a common practice to gain economic returns from naturally disturbed forests, but it could have considerable negative effects on biodiversity. Here the authors use a recently developed statistical method to estimate that ca. 75% of the naturally disturbed forest should be left unlogged to maintain 90% of the species unique to the area. KW - natural disturbance KW - bird communities KW - forest KW - management KW - beetle KW - conservation KW - windthrow KW - diversity KW - impact KW - fire Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230512 VL - 11 ER - TY - JOUR A1 - Thorn, Simon A1 - Chao, Anne A1 - Bernhardt-Römermann, Markus A1 - Chen, Yan-Han A1 - Georgiev, Kostadin B. A1 - Heibl, Christoph A1 - Müller, Jörg A1 - Schäfer, Hanno A1 - Bässler, Claus T1 - Rare species, functional groups, and evolutionary lineages drive successional trajectories in disturbed forests JF - Ecology N2 - Following natural disturbances, additional anthropogenic disturbance may alter community recovery by affecting the occurrences of species, functional groups, and evolutionary lineages. However, our understanding of whether rare, common, or dominant species, functional groups, or evolutionary lineages are most strongly affected by an additional disturbance, particularly across multiple taxa, is limited. Here, we used a generalized diversity concept based on Hill numbers to quantify the community differences of vascular plants, bryophytes, lichens, wood‐inhabiting fungi, saproxylic beetles, and birds in a storm‐disturbed, experimentally salvage logged forest. Communities of all investigated species groups showed dissimilarities between logged and unlogged plots. Most species groups showed no significant changes in dissimilarities between logged and unlogged plots over the first seven years of succession, indicating a lack of community recovery. In general, the dissimilarities of communities were mainly driven by rare species. Convergence of dissimilarities occurred more often than divergence during the early stages of succession for rare species, indicating a major role in driving decreasing taxonomic dissimilarities between logged and unlogged plots over time. Trends in species dissimilarities only partially match the trends in dissimilarities of functional groups and evolutionary lineages, with little significant changes in successional trajectories. Nevertheless, common and dominant species contributed to a convergence of dissimilarities over time in the case of the functional dissimilarities of wood‐inhabiting fungi. Our study shows that salvage logging following disturbances can alter successional trajectories in early stages of forest succession following natural disturbances. However, community changes over time may differ remarkably in different taxonomic groups and are best detected based on taxonomic, rather than functional or phylogenetic dissimilarities. KW - wood-inhabiting fungi KW - birds KW - bryophytes KW - climate change KW - forest succession KW - Hill numbers KW - natural disturbances KW - salvage logging KW - saproxylic beetles KW - vascular plants Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212378 VL - 101 IS - 3 ER - TY - JOUR A1 - Thormann, Birthe A1 - Raupach, Michael J. A1 - Wagner, Thomas A1 - Wägele, Johann W. A1 - Peters, Marcell K. T1 - Testing a Short Nuclear Marker for Inferring Staphylinid Beetle Diversity in an African Tropical Rain Forest JF - PLoS ONE N2 - Background: The use of DNA based methods for assessing biodiversity has become increasingly common during the last years. Especially in speciose biomes as tropical rain forests and/or in hyperdiverse or understudied taxa they may efficiently complement morphological approaches. The most successful molecular approach in this field is DNA barcoding based on cytochrome c oxidase I (COI) marker, but other markers are used as well. Whereas most studies aim at identifying or describing species, there are only few attempts to use DNA markers for inventorying all animal species found in environmental samples to describe variations of biodiversity patterns. Methodology/Principal Findings: In this study, an analysis of the nuclear D3 region of the 28S rRNA gene to delimit species-like units is compared to results based on distinction of morphospecies. Data derived from both approaches are used to assess diversity and composition of staphylinid beetle communities of a Guineo-Congolian rain forest in Kenya. Beetles were collected with a standardized sampling design across six transects in primary and secondary forests using pitfall traps. Sequences could be obtained of 99% of all individuals. In total, 76 molecular operational taxonomic units (MOTUs) were found in contrast to 70 discernible morphospecies. Despite this difference both approaches revealed highly similar biodiversity patterns, with species richness being equal in primary and secondary forests, but with divergent species communities in different habitats. The D3-MOTU approach proved to be an efficient tool for biodiversity analyses. Conclusions/Significance: Our data illustrate that the use of MOTUs as a proxy for species can provide an alternative to morphospecies identification for the analysis of changes in community structure of hyperdiverse insect taxa. The efficient amplification of the D3-marker and the ability of the D3-MOTUs to reveal similar biodiversity patterns as analyses of morphospecies recommend its use in future molecular studies on biodiversity. KW - DNA barcodes KW - Biological identifications KW - Species richness KW - Taxonomy KW - Conservation KW - Coleoptera KW - Parataxonomy KW - Assemblages KW - Madagascar Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142666 VL - 6 IS - 3 ER - TY - JOUR A1 - Thormann, Birthe A1 - Ahrens, Dirk A1 - Armijos, Diego Marín A1 - Peters, Marcell K. A1 - Wagner, Thomas A1 - Wägele, Johann W. T1 - Exploring the Leaf Beetle Fauna (Coleoptera: Chrysomelidae) of an Ecuadorian Mountain Forest Using DNA Barcoding JF - PLoS ONE N2 - Background Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates. Methodology/Principal Findings Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284–289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469–481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation. Conclusions/Significance Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing. KW - leaf beetle KW - Coleoptera: Chrysomelidae KW - Podocarpus National Park KW - DNA-based species delimitation KW - biodiversity Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167253 VL - 11 IS - 2 ER - TY - JOUR A1 - Thomas, Sarah A1 - Fiebig, Juliane E. A1 - Kuhn, Eva-Maria A1 - Mayer, Dominik S. A1 - Filbeck, Sebastian A1 - Schmitz, Werner A1 - Krischke, Markus A1 - Gropp, Roswitha A1 - Mueller, Thomas D. T1 - Design of glycoengineered IL-4 antagonists employing chemical and biosynthetic glycosylation JF - ACS Omega N2 - Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases. KW - Interleukin-4 (IL-4) KW - atopic diseases KW - IL-4 antagonists KW - glycoengineering KW - biosynthetic glycosylation KW - chemical glycosylation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350278 SN - 2470-1343 VL - 8 IS - 28 ER - TY - THES A1 - Thoma, Eva Christina T1 - Directed differentiation of pluripotent stem cells induced by single genes T1 - Gerichtete Differenzierung pluripotenter Stammzellen induziert durch einzelne Gene N2 - Pluripotency describes the ability of stem cells to form every cell type of the body.. Pluripotent stem cells are e.g. embryonic stem cells (ESCs), but also the so called induced pluripotent stem cells (IPS cells), that are generated by reprogramming differentiated somatic cells into a pluripotent state. Furthermore, it has been shown that spermatogonia (SG) derived from adult testes of mouse or human are pluripotent. Because of their ability to differentiate into every somatic cell type, pluripotent stem cells have a unique status in research and regenerative medicine. For the latter, they offer a valuable opportunity to replace destroyed tissues or organs. For basic research, stem cells represent a useful system to study differentiation or developmental processes that are difficult to access in the physiological situation e.g. during embryogenesis. Both applications, however, require methods that allow efficient and directed differentiation of stem cells into defined specialized cell types. This study first aims to investigate the differentiation potential of SG derived from the teleost fish medaka (Oryzias latipes). My results demonstrate that medaka SG are able to form different somatic cell types, namely adipocytes, melanocytes, osteoblasts, and neurons. This indicates that medake SG have retained a broad differentiation potential suggesting that pluripotency is not restricted to mouse and human SG but might be conserved among vertebrates. Next, I wanted to establish a differentiation method that is solely based on ectopic expression of genes known to be essential for the formation of certain somatic cell types – so called master regulators (MRs). My findings show that ectopic expression of the melanocyte-specific transcription factor mitf-m that has previously been shown to induce differentiation of medaka ESCs into pigment cells resulted in the formation of the same cell type in medaka SG. This approach could be used to generate other somatic cell types. Thus, ectopic expression of the MRs cbfa1 and mash1 in MF-SG was sufficient to induce differentiation into osteoblasts and neurons, respectively. Interestingly, these differentiation processes included the activation of genes that are expressed earlier during embryogenesis than the differentiation-inducing MR. Furthermore, my findings show that the approach of MR-induced differentiation can be transferred to mammalian stem cell systems. Ectopic expression of the neural transcription factor ngn2 was sufficient to induce efficient and rapid differentiation of neurons in mouse ESCs. This differentiation process also included the induction of genes that in vivo are activated at earlier stages that ngn2. By generating a transgenic cell line allowing induction of ectopic ngn2 expression, it was possible to obtain a relatively pure culture of functional neurons. Ngn2-induced differentiation did not require any additional signals and occurred even under pluripotency promoting conditions. Moreover, ectopic expression of ngn2 did also induce the formation of cells with neuronal morphology in IPS cells indicating that MR-induced differentiation is operative in different stem cell types. Furthermore, protein transduction of Ngn2 into mouse ESCs also resulted in a neuronal differentiation process up to the appearance of neural precursor cells. Last, my results show that MR-induced differentiation can also be used to generate other cell types than neurons from mouse ESCs. Myoblasts and macrophage-like cells were generated by ectopic expression of the MRs myoD and cebpa, respectively. Using transgenic cell lines enabling induction of MR expression it was possible to obtain mixed cultures with two different differentiation processes occurring in parallel. Altogether this study shows that ectopic expression of single genes is sufficient to induce directed differentiation of stem cells into defined cell types. The feasibility of this approach was demonstrated for different MRs and consequently different somatic cell types. Furthermore, MR induced differentiation was operative in different stem cell types from fish and mouse. Thus, one can conclude that certain genes are able to define cell fates in in vitro stem cell systems and that this cell fate defining potential appears to be a conserved feature in vertebrates. These findings therefore provide new insights in the role of MRs in cell commitment and differentiation processes. Furthermore, this study presents a new method to induce directed differentiation of stem cells that offers several advantages regarding efficiency, rapidness, and reproducibility. MR-induced differentiation therefore represents a promising tool for both stem cell research and regenerative medicine. N2 - Pluripotenz bezeichnet die Fähigkeit einer Stammzelle, jede Zelle des Körpers zu bilden. Zu den pluripotenten Stammzellen gehören embryonale Stammzellen (ESZ), aber auch so genannte induzierte pluripotente Stammzellen (IPS Zellen), die durch Rückprogrammierung ausdifferenzierter Körperzellen in einen pluripotenten Status gewonnen werden. Außerdem wurde gezeigt, dass adulte Spermatogonien (SG) in Maus und Mensch pluripotent sind. Pluripotente Stammzellen sind von großer Wichtigkeit für Forschung und regenerative Medizin. Für letztere bieten diese Zellen aufgrund ihrer Fähigkeit, jede Körperzellen zu bilden, eine vielversprechende Möglichkeit, zerstörte Gewebe oder Organe zu ersetzen. In der Forschung stellen sie ein nützliches System dar, um Entwicklungs- und Differenzierungsprozesse zu untersuchen, die in der physiologischen Situation z.B. der Embryonalentwicklung – schwer zugänglich sind. Eine wichtige Grundlage für diese Anwendungen sind jedoch Methoden, die die effiziente und gerichtete Differenzierung von Stammzellen in einen bestimmten Zelltyp erlauben. In dieser Arbeit wird zunächst das Differenzierungspotential von SG der Fischspezies Medaka (Oryzias latipes) untersucht, um festzustellen, ob Pluripotenz von SG, die bisher nur in Maus und Mensch gezeigt wurde, auch in anderen Wirbeltieren außerhalb der Säuger erhalten ist. Meine Ergebnisse zeigen, dass Medaka-SG fähig sind verschiedene somatische Zelltypen zu bilden. Das zweite Ziel dieser Studie ist die Entwicklung einer Differenzierungsmethode, die nur auf der Expression einzelner so genannter Masterregulatoren (MR) beruht – Gene, die als essentiell für die Entwicklung bestimmter Zelltypen bekannt sind. Meine Ergebnisse zeigen, dass der Pigmentzell-spezifische Transkriptionsfaktor Mitf-M, von dem gezeigt wurde, dass er die Differenzierung von Medaka-ESZ in Pigmentzellen induzieren kann, die Bildung desselben Zelltyps in Medaka-SG induziert. Dieser Ansatz ermöglichte auch die Bildung anderer somatischer Zelltypen. So führte Überexpression der MR cbfa1 und mash1 in Medaka SG zur Differenzierung in Osteoblasten bzw. Neuronen. Interessanterweise wurde bei diesen Differenzierungsprozessen die Aktivierung von Genen beobachtet, die während der Embryonalentwicklung vor dem Differenzierung-auslösenden MR aktiviert werden. Weiterhin zeigen meine Ergebnisse, dass der Ansatz einer gerichteten Differenzierung, ausgelöst durch einzelne MR, auch auf Säuger-Stammzellen übertragen werden kann. So wurde durch Überexpression des neuronalen Genes ngn2 in murinen ESZ die effiziente und schnelle Bildung von Nervenzellen induziert, wobei auch hier die Aktivierung von Genen beobachtet wurde, deren Expression in der Embryogenese der von ngn2 vorangeht. Die Herstellung einer transgenen Zelllinie, in der die Überexpression von ngn2 aktiviert werden kann, erlaubte die Entstehung einer fast reinen Kultur funktionaler Neuronen. Der durch ngn2 ausgelöste Differenzierungsprozess war unabhängig von zusätzlichen Faktoren und lief sogar unter Bedingungen ab, die normalerweise den pluripotenten Zustand unterstützen. Außerdem führte Überexpression von ngn2 auch in IPS Zellen zur Bildung von Zellen mit neuronalem Phenotyp. Weiterhin konnte auch durch Transduktion des Ngn2-Proteins in murine ESZ neuronale Differenzierung ausgelöst werden, und zwar die Bildung neuronaler Vorläuferzellen. Zuletzt wird bewiesen, dass gerichtete Differenzierung von murinen ESZ durch einzelne MR Gene neben neuronalen Zelltypen auch die Bildung anderer somatischer Zellen erlaubt: Überexpression der Gene myoD oder cebpa induzierte die Differenzierung in Muskelzellen bzw. Macrophagen-ähnliche Zellen. Unter Verwendung transgener Zelllinien, die die Aktivierung jeweils eines MRs erlauben, war es möglich, gemischte Kulturen zu erhalten, in denen zwei verschiedene Differenzierungsprozesse parallel abliefen. Diese Studie zeigt, dass die Überexpression einzelner Gene ausreichend ist, um gerichtete Differenzierungsprozesse in einen bestimmten Zelltyp auszulösen. Die erfolgreiche Durchführung dieses Ansatzes wird nicht nur mit verschiedenen Genen und somit verschiedenen resultierenden Zelltypen nachgewiesen, sondern auch in verschiedenen Stammzelltypen aus Fisch und Maus. Dies erlaubt die Schlussfolgerung, dass bestimmte Gene in vitro das Schicksal von Stammzellen festlegen können und dass diese Fähigkeit eine konservierte Eigenschaft in Wirbeltieren zu sein scheint. Somit präsentiert diese Arbeit neuen Erkenntnisse über die Rolle von MR bei der Festlegung von Zellidentitäten und in Differenzierungsprozessen. Weiterhin wird eine neue Methode zur Induktion gerichteter Differenzierung in Stammzellen aufgezeigt, die mehrere Vorteile in Bezug auf Effizienz, Geschwindigkeit und Reproduzierbarkeit hat. Auslösung von Differenzierung durch MR Gene bietet somit einen neuen vielversprechenden Ansatz mit potentieller Anwendung sowohl in Stammzellforschung, als auch in regenerativer Medizin. KW - Stammzelle KW - Zelldifferenzierung KW - Transkriptionsfaktor KW - Pluripotenz KW - Pluripotent stem cells KW - differentiation KW - transcription factors Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-54706 ER - TY - THES A1 - Thom, Corinna T1 - Dynamics and Communication Structures of Nectar Foraging in Honey Bees (Apis mellifera) T1 - Dynamik und Kommunikation beim Nektarsammeln der Honigbiene N2 - In this thesis, I examined honey bee nectar foraging with emphasis on the communication system. To document how a honey bee colony adjusts its daily nectar foraging effort, I observed a random sample of individually marked workers during the entire day, and then estimated the number and activity of all nectar foragers in the colony. The total number of active nectar foragers in a colony changed frequently between days. Foraging activity did not usually change between days. A honey bee colony adjusts its daily foraging effort by changing the number of its nectar foragers rather than their activity. I tested whether volatiles produced by a foraging colony activated nectar foragers of a non-foraging colony by connecting with a glass tube two colonies. Each colony had access to a different green house. In 50% of all experiments, volatile substances from the foraging colony stimulated nectar foragers of the non-foraging colony to fly to an empty feeder. The results of this study show that honey bees can produce a chemical signal or cue that activates nectar foragers. However, more experiments are needed to establish the significance of the activating volatiles for the foraging communication system. The brief piping signal of nectar foragers inhibits forager recruitment by stopping waggle dances (Nieh 1993, Kirchner 1993). However, I observed that many piping signals (approximately 43%) were produced off the dance floor, a restricted area in the hive where most waggle dances are performed. If the inhibition of waggle dances would be the only function of the brief piping signal, tremble dancers should produce piping signals mainly on the dance floor, where the probability to encounter waggle dancers is highest. To therefore investigate the piping signal in more detail, I experimentally established the foraging context of the brief piping signal, characterized its acoustic properties, and documented for the first time the unique behavior of piping nectar foragers by observing foragers throughout their entire stay in the hive. Piping nectar foragers usually began to tremble dance immediately upon their return into the hive, spent more time in the hive, more time dancing, had longer unloading latencies, and were the only foragers that sometimes unloaded their nectar directly into cells instead of giving it to a nectar receiver bee. Most of the brief piping signals (approximately 99%) were produced by tremble dancers, yet not all tremble dancers (approximately 48%) piped. This suggests that piping and tremble dancing have related, but not identical functions in the foraging system. Thus, the brief piping signals may not only inhibit forager recruitment, but have an additional function both on and off the dance floor. In particular, the piping signal might function 1. to stop the recruitment of additional nectar foragers, and 2. as a modulatory signal to alter the response threshold of signal receivers to the tremble dance. The observation that piping tremble dancers often did not experience long unloading delays before they started to dance gave rise to a question. A forager’s unloading delay provides reliable information about the relative work capacities of nectar foragers and nectar receivers, because each returning forager unloads her nectar to a nectar receiver before she takes off for the next foraging trip. Queuing delays for either foragers or receivers lower foraging efficiency and can be eliminated by recruiting workers to the group in shortage. Short unloading delays indicate to the nectar forager a shortage of foragers and stimulate waggle dancing which recruits nectar foragers. Long unloading delays indicate a shortage of nectar receivers and stimulate tremble dancing which recruits nectar receivers (Seeley 1992, Seeley et al. 1996). Because the short unloading delays of piping tremble dancers indicated that tremble dancing can be elicited by other factors than long unloading delays, I tested whether a hive-external stimulus, the density of foragers at the food source, stimulated tremble dancing directly. The experiments show that tremble dancing can be caused directly by a high density of foragers at the food source and suggest that tremble dancing can be elicited by a decrease of foraging efficiency either inside (e.g. shortage of receiver bees) or outside (e.g. difficulty of loading nectar) the hive. Tremble dancing as a reaction to hive-external stimuli seems to occur under natural conditions and can thus be expected to have some adaptive significance. The results imply that if the hive-external factors that elicit tremble dancing do not indicate a shortage of nectar receiver bees in the hive, the function of the tremble dance may not be restricted to the recruitment of additional nectar receivers, but might be the inhibition or re-organization of nectar foraging. N2 - In meiner Doktorarbeit habe ich die Charakteristika des Nektarsammelns bei Honigbienen mit spezieller Betonung des zugehörigen Kommunikationssytems untersucht. Im Einzelnen habe ich die täglichen Änderungen in der Aktivität und Anzahl der Nektarsammlerinnen einer nicht- manipulierten Kolonie verfolgt, habe getestet, ob Nektarsammlerinnen durch ein chemisches Signal aktiviert werden können, und habe die Auslöser und Charakteristika zweier Signale des Nektarsammelkommunikationssytems, dem kurzen Pipingsignal und dem Zittertanz der Nektarsammlerinnen untersucht. Um die täglichen Änderungen des Sammelaufwandes einer Kolonie zu dokumentieren, habe ich an verschiedenen Tagen die Anzahl und Aktivität (Anzahl Fouragierflüge pro Tag und Biene) der Nektarsammlerinnen einer Kolonie gemessen. Dafür beobachtete ich jeweils den ganzen Tag eine zufällig ausgewählte Gruppe von individuell markierten Arbeiterinnen. Aufgrund der so gewonnen Daten konnte ich die Anzahl und Aktivität aller Nektarsammlerinnen in der Kolonie schätzen. Die Ergebnisse zeigen, dass sich die absolute Anzahl von Nektarsammelerinnen regelmässig von Tag zu Tag änderte wahrscheinlich zurückzuführen auf die täglichen Änderungen im Nektarangebot, während sich die Aktivität der Sammlerinnen gewöhnlich nicht änderte. Die Ergebnisse zeigen, dass eine Arbeiterin eher die Entscheidung trifft zu sammeln oder nicht zu sammeln, statt eine abgestufte Entscheidung über die Anzahl ihrer Sammelflüge. Für eine Honigbienenkolonie bedeutet dies, das ihre Sammeleffizienz stärker durch die Anzahl der Sammlerinnen als durch deren Aktivität reguliert wird. Möglicherweise kann eine vergängliche Nektarquelle besser von vielen Sammlerinnen, die zeitgleich arbeiten, ausgebeutet werden als von weniger Sammlerinnen die zwar ihre Aktivität steigern, aber sequentielle Sammelflüge machen müssen und damit die Quelle vor ihrem Verschwinden nicht vollständig ausbeuten können. Es ist seit langem bekannt, das der Schwänzeltanz der Honigbienen Sammlerinnen aktivieren kann. Ich habe untersucht, ob die flüchtigen Substanzen einer fouragierenden Kolonie die Sammlerinnen einer nicht-fouragierenden Kolonie aktivieren können. Um dies zu testen, verband ich die Eingangsbereiche zweier Kolonien mit einer Glasröhre, so das flüchtige Substanzen von einer zur anderen Kolonie geleitet werden konnten. Jede Kolonie hatte Zugang zu einem separaten Gewächshaus. Während eine der Kolonien gefüttert wurde, wurde die Aktivität der nicht- gefütterten Kolonie gemessen. In 50% der Experimente wurden die Sammlerinnen der Kolonie, die kein Futter zur Verfügung hatte, durch die flüchtige Substanzen aus der fouragierenden Kolonie zu dem Besuch Ihrer leeren Futterstation aktiviert. Die Ergebnisse zeigen damit, dass Honigbienen eine flüchtige Substanz produzieren können, die Sammlerinnen aktiviert. Die Fragen, ob es sich bei dieser Substanz um ein ‘signal’ (speziell für die Situation entwickelt) oder einen ‘cue’ (nicht speziell für die Situtation entwickelt, wirft aber brauchbare Information als Nebenprodukt ab) handelt, sowie die Bedeutung der Substanz für die Sammeleffizienz einer Honigbienekolonie, müssen jedoch noch etabliert werden. Das Pipingsignal der Nektarsammlerinnen stoppt Schwänzeltänze (Nieh 1993, Kirchner 1993). Ich beobachtete, dass viele der kurzen Pipingsignale (ca. 43%) unerwartet nicht auf dem Tanzboden produziert wurden. Die Beobachtungen deuten darauf hin, dass das kurze Pipingsignal nicht nur Schwänzeltänze stopt, sondern auch die Reaktionsschwelle für den Zittertanz senkt. Pipende Zittertänzerinnen fingen sehr frueh nach ihrer Rückehr in den Stock an zu tanzen. Daher untersuchte ich, ob die Zustände an der Futterstelle Zittertänze auslösen kann. Die Experimente zeigen, dass Zittertänze eine direkte Reaktion auf eine hohe Dichte von Sammlerinnen an der Futterstelle sein können. Dies lässt vermuten, dass Zittertänze eine generelle Reaktion sind auf Faktoren, die entweder innerhalb (z.B. durch lange Wartezeit) oder ausserhalb (z.B. durch Schwierigkeiten beim Trinken) des Stockes die Sammeleffizienz senken. Unter natürlichen Umständen scheinen Zittertänze regelmässig eine direkte Reaktion auf Stock-externe Faktoren zu sein, und werden daher einige Bedeutung im Sammelkommunikationssytem haben. Sofern die Stock-externen Faktoren nicht einen Mangel an Nektarabnehmerinnen im Stock anzeigen, könnte es sein, dass der Zittertanz nicht nur Nektarabnehmerinnen rekruitiert, sondern, ähnlich wie die kurzen Pipingsignale der Zittertänzerinnen, der Hemmung oder Re-organisation der Sammelaktivität einer Honigbienen Kolonie dient. KW - Bienen KW - Kommunikation KW - Nahrungserwerb KW - Bienensprache KW - Biene KW - Nektar KW - Sammeln KW - Honigbiene KW - Kommunikation KW - Piping Signal KW - Flexibilität KW - Zittertanz KW - Honeybee KW - Nectar Foraging KW - tremble dance KW - worker piping KW - dynamics Y1 - 2002 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-3601 ER - TY - JOUR A1 - Thiry, Marc A1 - Scheer, Ulrich A1 - Goessens, Guy T1 - Immunoelectron microscopic study of nucleolar DNA during mitosis in Ehrlich tumour cells N2 - In order to investigate the DNA localization within Ehrlich tumor cell nucleoli during mitosis, two recent immunocytochemical methods using either an anti-DNA or an anti-bromodeoxyuridine (BrdU) monoclonal antibody have been applied. In both cases, the immunogold labeling has been performed on ultrathin sections of cells embedded either in Lowicryl K4M or in Epon, respectively. Identical results are observed with both immunocytochemical approaches. In the interphase nucleolus, besides the labeling of the perinucleolar chromatin shell and of its intranucleolar invaginations which penetrate into the nucleolar body and often terminate at the fibrillar centers, a few gold particles are also preferentially found towards the peripheral region of the fibrillar centers. In contrast, the dense fibrillar component and the granular component are never labeled. During mitosis, the fibrillar centers persist at the chromosomal nucleolus organizing regions (NOR's) and can be selectively stained by the silver method. However, these metaphase fibrillar centers are no longer decorated by the DNA- or BrdU antibodies. These results indicate that until the end of prophase, rRNA genes are present inside the fibrillar center material, disappear during metaphase and reappear in reconstituting nucleoli during telophase. Thus, fibrillar centers appear to represent structures sui generis, which are populated by rRNA genes only when the nucleolus is functionally active. In segregated nucleoli after actinomycin D treatment, the DNA labeling is exclusively restricted to the perinucleolar chromatin blocks. These findings also suggest that the DNA content of the fibrillar center material varies according to the rRNA transcription level of the cells. The results are discussed in the light of the present knowledge of the functional organization of the nucleolus. KW - Cytologie KW - Nucleolus KW - DNA KW - mitosis Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40745 ER - TY - JOUR A1 - Thiry, Marc A1 - Scheer, Ulrich A1 - Goessens, Guy T1 - Localization of DNA within Ehrlich tumour cells nucleoli by immunoelectron microscopy N2 - The distribution of DNA in Ehrlich tumour cell nucleoli was investigated by means of an immunocytochemical approach , involving a monoclonal antibody directed against double- and single-stranded DNA. Immunolabelling was performed . either before or after the embedding process. The postembedding labelling method allows better ultrastructural preservation than the preembedding labelling method. In particular, the various nucleolar components are well preserved and identifiable. In the nucleolus, labelling is particularly concentrated over the perinucleolar chromatin and over its intranucleolar invaginations, which penetrate the nucleolar body and often terminate at the fibrillar centres. In addition, aggregates of gold particles are found in the fibrillar centres, preferentially towards the peripheral regions. By contrast, the dense fibrillar component is completely devoid of labelling. The results seem to indicate that DNA containing the rDNA genes is located in the fibrillar centres, with a preference for the peripheral regions. This finding suggests that transcription of the rDNA genes should occur within the confines of the fibrillar centre, probably close to the boundary region of the surrounding dense fibrillar component. The results are discussed in the light of present knowledge of the functional organization of the nucleolus. KW - nucleolus KW - DNA KW - monoclonal antibody Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-39327 ER - TY - JOUR A1 - Thiry, Marc A1 - Scheer, Ulrich A1 - Goessens, Guy T1 - Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level N2 - Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure. Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA to po isomerase I are located together. Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component. Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-39289 ER - TY - JOUR A1 - Thiem, Alexander A1 - Hesbacher, Sonja A1 - Kneitz, Hermann A1 - di Primio, Teresa A1 - Heppt, Markus V. A1 - Hermanns, Heike M. A1 - Goebeler, Matthias A1 - Meierjohann, Svenja A1 - Houben, Roland A1 - Schrama, David T1 - IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression JF - Journal of Experimental & Clinical Cancer Research N2 - Background Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma. Methods We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway. Results For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53\(^{L22Q,W23S}\), a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53\(^{L22Q,W23S}\) in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. Conclusions While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy. KW - Melanoma KW - PD-L1 KW - CD274 KW - p53 KW - TP53 KW - JAK2 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201016 VL - 38 ER - TY - JOUR A1 - Thiele, Jonas A. A1 - Richter, Aylin A1 - Hilger, Kirsten T1 - Multimodal brain signal complexity predicts human intelligence JF - eNeuro N2 - Spontaneous brain activity builds the foundation for human cognitive processing during external demands. Neuroimaging studies based on functional magnetic resonance imaging (fMRI) identified specific characteristics of spontaneous (intrinsic) brain dynamics to be associated with individual differences in general cognitive ability, i.e., intelligence. However, fMRI research is inherently limited by low temporal resolution, thus, preventing conclusions about neural fluctuations within the range of milliseconds. Here, we used resting-state electroencephalographical (EEG) recordings from 144 healthy adults to test whether individual differences in intelligence (Raven’s Advanced Progressive Matrices scores) can be predicted from the complexity of temporally highly resolved intrinsic brain signals. We compared different operationalizations of brain signal complexity (multiscale entropy, Shannon entropy, Fuzzy entropy, and specific characteristics of microstates) regarding their relation to intelligence. The results indicate that associations between brain signal complexity measures and intelligence are of small effect sizes (r ∼ 0.20) and vary across different spatial and temporal scales. Specifically, higher intelligence scores were associated with lower complexity in local aspects of neural processing, and less activity in task-negative brain regions belonging to the default-mode network. Finally, we combined multiple measures of brain signal complexity to show that individual intelligence scores can be significantly predicted with a multimodal model within the sample (10-fold cross-validation) as well as in an independent sample (external replication, N = 57). In sum, our results highlight the temporal and spatial dependency of associations between intelligence and intrinsic brain dynamics, proposing multimodal approaches as promising means for future neuroscientific research on complex human traits. KW - brain signal complexity KW - cognitive ability KW - EEG KW - intelligence KW - microstates KW - resting-state Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-312949 VL - 10 IS - 2 ER - TY - JOUR A1 - Thielcke, Gerhard A1 - Linsenmair, Karl Eduard T1 - Zur geographischen Variation des Gesanges des Zilpzalps, Phylloscopus collybita, in Mittel- und Südwesteuropa mit einem Vergleich des Gesanges de Fitis, Phylloscopus trochilus N2 - No abstract available Y1 - 1963 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-44657 ER - TY - THES A1 - Thelen, David T1 - Erstellung eines genregulatorischen Netzwerkes zur Simulation der Entstehung von Zahnhartsubstanz T1 - Construction of a gene regulatory network to simulate the formation of dental hard tissue N2 - In this dissertation, the author describes the creation of a basic bioinformatic model of human enamel maturation. Supported by the interactions found in the KEGG Pathway database, we were able to establish a gene regulatory network (GRN) that focuses primarily on the signal transduction pathways apoptosis, cell cycle, hedgehog signaling pathway, MAP kinase pathway, mTOR signaling pathway, Notch signaling pathway, TGF-β signaling pathway and Wnt signaling pathway. We extended this through further verified interactions and implicated the tooth-specific genes AMELX, AMELY, AMBN, ENAM and DSPP. In the subsequent simulation of the network by the simulation tool Jimena, six stable states could be identified. These are examined in more detail and juxtaposed with results of a GEO dataset. The long-term goal is to draw conclusions about the odontogenesis of humans through consistent optimization of the bioinformatics network. N2 - In dieser Dissertation beschreibt der Autor die Erstellung eines grundlegenden bioinformatischen Modelles der menschlichen Zahnschmelzreifung. Mithilfe der KEGG Pathway-Datenbank wurde ein genregulatorisches Netzwerk (GRN) erstellt, welches maßgeblich auf den Signaltransduktionswegen Apoptose, Zellzyklus, Hedgehog-Signalweg, MAP-Kinase-Weg, mTOR-Signalweg Notch-Signalweg Signalweg, TGF-β-Signalweg und Wnt-Signalweg basiert. Im Weiteren wurde dieses Netzwerk durch zahlreiche verifizierte Wechselwirkungen erweitert und die zahnspezifischen Gene AMELX, AMELY, AMBN, ENAM und DSPP implementiert. In der anschließenden Simulation des Netzwerks mit dem Simulations-Tool Jimena konnten sechs stabile Zustände identifiziert werden. Diese wurden genauer untersucht und den Erkenntnissen eines GEO-Datensatzes gegenübergestellt. Langfristiges Ziel ist es, durch konsequente Optimierung des bioinformatischen Netzwerks Rückschlüsse auf die Odontogenese des Menschen zu ziehen. KW - Universität Würzburg. Lehrstuhl für Bioinformatik KW - Boolesches Netz KW - Zahnentwicklung KW - Amelogenese KW - Genregulation KW - genregulatorisches Netzwerk Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204068 ER - TY - THES A1 - Thakar, Juilee T1 - Computational models for the study of responses to infections T1 - Bioinformatische Modelle zur Analyse der Immunantwort auf Infektionen N2 - In diesem Jahrhundert haben neue experimentelle Techniken und Computer-Verfahren enorme Mengen an Information erzeugt, die bereits viele biologische Rätsel enthüllt haben. Doch die Komplexität biologischer Systeme wirft immer weitere neue Fragen auf. Um ein System zu verstehen, bestand der Hauptansatz bis jetzt darin, es in Komponenten zu zerlegen, die untersucht werden können. Ein neues Paradigma verknüpft die einzelnen Informationsteile, um sie auf globaler Ebene verstehen zu können. In der vorgelegten Doktorarbeit habe ich deshalb versucht, infektiöse Krankheiten mit globalen Methoden („Systembiologie“) bioinformatisch zu untersuchen. Im ersten Teil wird der Apoptose-Signalweg analysiert. Apoptose (Programmierter Zelltod) wird bei verschiedenen Infektionen, zum Beispiel bei Viruserkrankungen, als Abwehrmaßnahme eingesetzt. Die Interaktionen zwischen Proteinen, die ‚death’ Domänen beinhalten, wurden untersucht, um folgende Fragen zu klären: i) wie wird die Spezifität der Interaktionen erzielt? –sie wird durch Adapter erreicht, ii) wie werden Proliferation/ Überlebenssignale während der Aktivierung der Apoptose eingeleitet? – wir fanden Hinweise für eine entscheidende Rolle des RIP Proteins (Rezeptor-Interagierende Serine/Threonine-Proteinkinase 1). Das Modell erlaubte uns, die Interaktions-Oberflächen von RIP vorherzusagen. Der Signalweg wurde anschließend auf globaler Ebene mit Simulationen für verschiedene Zeitpunkte analysiert, um die Evolution der Aktivatoren und Inhibitoren des Signalwegs und seine Struktur besser zu verstehen. Weiterhin wird die Signalverarbeitung für Apoptosis-Signalwege in der Maus detailliert modelliert, um den Konzentrationsverlauf der Effektor-Kaspasen vorherzusagen. Weitere experimentelle Messungen von Kaspase-3 und die Überlebenskurven von Zellen bestätigen das Modell. Der zweite Teil der Resultate konzentriert sich auf das Phagosom, eine Organelle, die eine entscheidende Rolle bei der Eliminierung von Krankheitserregern spielt. Dies wird am Beispiel von M. tuberculosis veranschaulicht. Die Fragestellung wird wiederum in zwei Aspekten behandelt: i) Um die Prozesse, die durch M. tuberculosis inhibiert werden zu verstehen, haben wir uns auf das Phospholipid-Netzwerk konzentriert, das bei der Unterdrückung oder Aktivierung der Aktin-Polymerisation eine große Rolle spielt. Wir haben für diese Netzwerkanalyse eine Simulation für verschiedene Zeitpunkte ähnlich wie in Teil eins angewandt. ii) Es wird vermutet, dass Aktin-Polymere bei der Fusion des Phagosoms mit dem Lysosom eine Rolle spielen. Um diese Hypothese zu untersuchen, wurde ein in silico Modell von uns entwickelt. Wir fanden heraus, dass in der Anwesenheit von Aktin-Polymeren die Suchzeit für das Lysosom um das Fünffache reduziert wurde. Weiterhin wurden die Effekte der Länge der Aktin-Polymere, die Größe der Lysosomen sowie der Phagosomen und etliche andere Modellparameter analysiert. Nach der Untersuchung eines Signalwegs und einer Organelle führte der nächste Schritt zur Untersuchung eines komplexen biologischen Systems der Infektabwehr. Dies wurde am Beispiel der Wirt-Pathogen Interaktion bei Bordetella pertussis und Bordetella bronchiseptica dargestellt. Die geringe Menge verfügbarer quantitativer Daten war der ausschlaggebende Faktor bei unserer Modellwahl. Für die dynamische Simulation wurde ein selbst entwickeltes Bool’sches Modell verwendet. Die Ergebnisse sagen wichtige Faktoren bei der Pathologie von Bordetellen hervor, besonders die Bedeutung der Th1 assoziierten Antworten und dagegen nicht der Th2 assoziierten Antworten für die Eliminierung des Pathogens. Einige der quantitativen Vorhersagen wurden durch Experimente wie die Untersuchung des Verlaufs einer Infektion in verschiedenen Mutanten und Wildtyp-Mäusen überprüft. Die begrenzte Verfügbarkeit kinetischer Daten war der kritische Faktor bei der Auswahl der computer-gestützten Modelle. Der Erfolg unserer Modelle konnte durch den Vergleich mit experimentellen Beobachtungen belegt werden. Die vergleichenden Modelle in Kapitel 6 und 9 können zur Untersuchung neuer Wirt-Pathogen Interaktionen verwendet werden. Beispielsweise führt in Kapitel 6 die Analyse von Inhibitoren und inhibitorischer Signalwege aus drei Organismen zur Identifikation wichtiger regulatorischer Zentren in komplexen Organismen und in Kapitel 9 ermöglicht die Identifikation von drei Phasen in B. bronchiseptica und der Inhibition von IFN-γ durch den Faktor TTSS die Untersuchung ähnlicher Phasen und die Inhibition von IFN-γ in B. pertussis. Eine weitere wichtige Bedeutung bekommen diese Modelle durch die mögliche Identifikation neuer, essentieller Komponenten in Wirt-Pathogen Interaktionen. In silico Modelle der Effekte von Deletionen zeigen solche Komponenten auf, die anschließend durch experimentelle Mutationen weiter untersucht werden können. N2 - In this century new experimental and computational techniques are adding an enormous amount of information, revealing many biological mysteries. The complexities of biological systems still broach new questions. Till now the main approach to understand a system has been to divide it in components that can be studied. The upcoming new paradigm is to combine the pieces of information in order to understand it at a global level. In the present thesis we have tried to study infectious diseases with such a global ‘Systems Biology’ approach. In the first part the apoptosis pathway is analyzed. Apoptosis (Programmed cell death) is used as a counter measure in different infections, for example viral infections. The interactions between death domain containing proteins are studied to address the following questions: i) How specificity is maintained - showing that it is induced through adaptors, ii) how proliferation/ survival signals are induced during activation of apoptosis – suggesting the pivotal role of RIP. The model also allowed us to detect new possible interacting surfaces. The pathway is then studied at a global level in a time step simulation to understand the evolution of the topology of activators and inhibitors of the pathway. Signal processing is further modeled in detail for the apoptosis pathway in M. musculus to predict the concentration time course of effector caspases. Further, experimental measurements of caspase-3 and viability of cells validate the model. The second part focuses on the phagosome, an organelle which plays an essential role in removal of pathogens as exemplified by M. tuberculosis. Again the problem is addressed in two main sections: i) To understanding the processes that are inhibited by M. tuberculosis; we focused on the phospholipid network applying a time step simulation in section one, which plays an important role in inhibition or activation of actin polymerization on the phagosome membrane. ii) Furthermore, actin polymers are suggested to play a role in the fusion of the phagosome with lysosome. To check this hypothesis an in silico model was developed; we find that the search time is reduced by 5 fold in the presence of actin polymers. Further the effect of length of actin polymers, dimensions of lysosome, phagosome and other model parameter is analyzed. After studying a pathway and then an organelle, the next step was to move to the system. This was exemplified by the host pathogen interactions between Bordetella pertussis and Bordetella bronchiseptica. The limited availability of quantitative information was the crucial factor behind the choice of the model type. A Boolean model was developed which was used for a dynamic simulation. The results predict important factors playing a role in Bordetella pathology especially the importance of Th1 related responses and not Th2 related responses in the clearance of the pathogen. Some of the quantitative predictions have been counterchecked by experimental results such as the time course of infection in different mutants and wild type mice. All these computational models have been developed in presence of limited kinetic data. The success of these models has been validated by comparison with experimental observations. Comparative models studied in chapters 6 and 9 can be used to explore new host pathogen interactions. For example in chapter 6, the analysis of inhibitors and inhibitory paths in three organism leads to the identification of regulatory hotspots in complex organisms and in chapter 9 the identification of three phases in B. bronchiseptica and inhibition of IFN-γ by TTSS lead us to explore similar phases and inhibition of IFN-γ in B. pertussis. Further an important significance of these models is to identify new components playing an essential role in host-pathogen interactions. In silico deletions can point out such components which can be further analyzed by experimental mutations. KW - Bordetella pertussis KW - Infektion KW - Apoptosis KW - Signaltransduktion KW - Bioinformatik KW - Tuberkelbakterium KW - Biologische Kaskaden KW - Bordetellae KW - M. tuberculosis KW - Apoptose KW - Biological cascades KW - Bordetellae KW - M. tuberculosis KW - Apoptosis Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-17266 ER - TY - JOUR A1 - Teutschbein, Janka A1 - Haydn, Johannes M. A1 - Samans, Birgit A1 - Krause, Michael A1 - Eilers, Martin A1 - Schartl, Manfred A1 - Meierjohann, Svenja T1 - Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins N2 - Background: Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods: Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results: Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion: Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes. KW - Melanoma Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67900 ER - TY - THES A1 - Teutschbein, Janka T1 - Identifizierung und Charakterisierung von Genen und Proteinen in der Xmrk-induzierten Entwicklung von Melanomen T1 - Identification and characterization of genes and proteins in Xmrk-induced melanomagenesis N2 - Melanome stellen die gefährlichste Form von Hautkrebs mit der höchsten Mortalitätsrate dar. Der Transformation normaler Melanozyten zu malignen Melanomen liegen komplexe molekulare und biochemische Veränderungen zu Grunde. Im Xiphophorus-Melanom-Modell ist die onkogene Rezeptortyrosinkinase "Xiphophorus melanoma receptor kinase" (Xmrk) der alleinige Auslöser der Melanominitiation und -progression. Die Aufklärung der Xmrk-vermittelten Signaltransduktion kann zum besseren Verständnis von Ereignissen, die auch bei der humanen Melanomentwicklung eine Rolle spielen, beitragen. In der vorliegenden Arbeit wurde mit Hilfe der Microarray-Technologie die Regulation der Genexpression durch Xmrk analysiert. Zu den nach Rezeptoraktivierung am stärksten herabregulierten Genen gehörten "son of sevenless homolog 1" (Sos1) und "ubiquitin-conjugating enzyme E2I" (Ube2i); stark hochreguliert waren "early growth response 1" (Egr1), "cysteine-rich protein 61" (Cyr61), "dual-specificity phosphatase 4" (Dusp4), "fos-like antigen 1" (Fosl1), "epithelial membrane protein" (Emp1), Osteopontin (Opn), "insulin-like growth factor binding protein 3" (Igfbp3) und "tumor-associated antigen L6" (Taal6). Die für die Regulation dieser Gene verantwortlichen Signalwege wurden durch die Anwendung von niedermolekularen Inhibitoren und siRNA identifiziert, wobei für die SRC-Kinase FYN eine zentrale Bedeutung bei der Xmrk-abhängigen Regulation der Genexpression festgestellt wurde. Darüber hinaus wurde die Expression der Gene in humanen Melanomzelllinien im Vergleich zu normalen humanen Melanozyten untersucht. Als besonders vielversprechende Kandidaten stellten sich dabei DUSP4 und TAAL6 heraus, deren Rolle in der humanen Melanominduktion und -progression Gegenstand zukünftiger Studien sein wird. In einem anderen Ansatz zur Aufklärung des Signalnetzwerkes sollten Zielproteine von Xmrk durch Protein-Protein-Interaktionsstudien mit Hilfe des Split-Ubiquitin-Systems ermittelt werden. Aufgrund ungünstiger Expressions- oder Faltungseigenschaften von Xmrk in diesem System war es aber nicht möglich, den Rezeptor als Köderprotein einzusetzen. Das für die Xmrk-vermittelte Melanomentstehung zentrale Protein FYN konnte jedoch als Köder etabliert und seine Wechselwirkung mit der Tyrosinkinase FAK analysiert werden. Es wurde gezeigt, dass der phosphorylierte Tyrosinrest an Position 397 von FAK für die Interaktion einer N-terminal trunkierten FAK-Variante mit FYN notwendig ist und dass diese Phosphorylierung in Hefe gewährleistet zu sein scheint. Die Suche nach neuen Interaktionspartnern von FYN mittels der Split-Ubiquitin-Technologie könnte Einblicke in weitere FYN-abhängige Ereignisse bieten, die zur Aufklärung seiner zentralen Rolle bei der Tumorentstehung dienen könnte. N2 - Melanoma is the most aggressive type of skin cancer with the highest mortality rate. The transformation of melanocytes to malignant melanoma is based on complex molecular and biochemical alterations. In the Xiphophorus melanoma model, the oncogenic receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) is the sole trigger of melanoma initiation and progression. Elucidating Xmrk-dependent signaling pathways may contribute to a better understanding of processes that play a role in human melanomagenesis, too. Here, the regulation of gene expression by Xmrk was analyzed using a microarray approach. The genes with the strongest down-regulation in response to receptor activation included son of sevenless homolog 1 (Sos1) and ubiquitin-conjugating enzyme E2I (Ube2i), whereas early growth response 1 (Egr1), cysteine-rich protein 61 (Cyr61), dual-specificity phosphatase 4 (Dusp4), fos-like antigen 1 (Fosl1), epithelial membrane protein (Emp1), Osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), and tumor-associated antigen L6 (Taal6) were strongly up-regulated. The pathways regulating expression of these genes were identified by applying small molecule inhibitors and siRNA. Interestingly, the SRC-family kinase FYN was found to be a key-player in Xmrk-dependent gene regulation. Furthermore, expression of the genes in human melanoma cell lines compared to normal human melanocytes was investigated. The most promising candidates, which might be important for melanoma induction and progression, were DUSP4 and TAAL6. Their potential suitability as diagnostic and prognostic melanoma markers will be addressed in future studies. In addition to gene expression analysis, protein-protein interactions were to be assayed by the split-ubiquitin-system in order to identify novel Xmrk targets. Unfortunately, inappropriate expression or folding of the receptor in this system precluded it from working as bait. However, the FYN protein, which has a central role in Xmrk-mediated signaling, was established as bait and its association with FAK was analyzed in more detail. A phosphorylated tyrosine residue at position 397 of FAK was demonstrated to be necessary for the interaction of an N-terminally truncated FAK variant with FYN, and this phosphorylation event seems to be feasible in yeast. In future, a split-ubiquitin based screen for novel interaction partners of FYN might provide insights into FYN-dependent processes and help to understand its central role in tumor development. KW - Melanom KW - Schwertkärpfling KW - Microarray KW - Onkogen KW - Epidermaler Wachstumsfaktor-Rezeptor KW - Yeast-Two-Hybrid-System KW - Xiphophorus KW - Rezeptortyrosinkinase KW - Xmrk KW - Protein-Protein-Interaktion KW - Split-Ubiquitin-System KW - Microarray KW - EGFR KW - oncogene KW - melanoma KW - protein-protein interaction Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-27516 ER - TY - JOUR A1 - Temme, Sebastian A1 - Friebe, Daniela A1 - Schmidt, Timo A1 - Poschmann, Gereon A1 - Hesse, Julia A1 - Steckel, Bodo A1 - Stühler, Kai A1 - Kunz, Meik A1 - Dandekar, Thomas A1 - Ding, Zhaoping A1 - Akhyari, Payam A1 - Lichtenberg, Artur A1 - Schrader, Jürgen T1 - Genetic profiling and surface proteome analysis of human atrial stromal cells and rat ventricular epicardium-derived cells reveals novel insights into their cardiogenic potential JF - Stem Cell Research N2 - Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73,CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases. KW - Biology KW - Epicardium-derived cells KW - Human atrial stromal cells KW - Cell surface proteomics Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172716 VL - 25 ER - TY - JOUR A1 - Tauscher, Sabine A1 - Nakagawa, Hitoshi A1 - Völker, Katharina A1 - Werner, Franziska A1 - Krebes, Lisa A1 - Potapenko, Tamara A1 - Doose, Sören A1 - Birkenfeld, Andreas L. A1 - Baba, Hideo A. A1 - Kuhn, Michaela T1 - β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice JF - Cardiovascular Diabetology N2 - Background: The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells. Methods: Mice with a floxed GC-A gene were bred to Rip-CreTG mice, thereby deleting GC-A selectively in β-cells (β GC-A KO). Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion were monitored in normal diet (ND)- and high-fat diet (HFD)-fed mice. β-cell size and number were measured by immunofluorescence-based islet morphometry. Results: In vitro, the insulinotropic and proliferative actions of ANP were abolished in islets isolated from β GC-A KO mice. Concordantly, in vivo, infusion of BNP mildly enhanced baseline plasma insulin levels and glucose-induced insulin secretion in control mice. This effect of exogenous BNP was abolished in β GC-A KO mice, corroborating the efficient inactivation of the GC-A receptor in β-cells. Despite this under physiological, ND conditions, fasted and fed insulin levels, glucose-induced insulin secretion, glucose tolerance and β-cell morphology were similar in β GC-A KO mice and control littermates. However, HFD-fed β GC-A KO animals had accelerated glucose intolerance and diminished adaptative β-cell proliferation. Conclusions: Our studies of β GC-A KO mice demonstrate that the cardiac hormones ANP and BNP do not modulate β-cell's growth and secretory functions under physiological, normal dietary conditions. However, endogenous NP/GC-A signaling improves the initial adaptative response of β-cells to HFD-induced obesity. Impaired β-cell NP/GC-A signaling in obese individuals might contribute to the development of type 2 diabetes. KW - cylic GMP KW - guanylyl cyclase-A KW - insulin KW - natriuretic peptides KW - obesity KW - β-cells Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176322 VL - 17 IS - 103 ER - TY - JOUR A1 - Tamihardja, Jörg A1 - Zehner, Leonie A1 - Hartrampf, Philipp A1 - Lisowski, Dominik A1 - Kneitz, Susanne A1 - Cirsi, Sinan A1 - Razinskas, Gary A1 - Flentje, Michael A1 - Polat, Bülent T1 - Salvage nodal radiotherapy as metastasis-directed therapy for oligorecurrent prostate cancer detected by positron emission tomography shows favorable outcome in long-term follow-up JF - Cancers N2 - Simple Summary Patients, who suffer from oligorecurrent prostate cancer with limited nodal involvement, may be offered positron emission tomography (PET)-directed salvage nodal radiotherapy to delay disease progression. This current analysis aimed to access salvage radiotherapy for nodal oligorecurrent prostate cancer with simultaneous integrated boost to PET-involved lymph nodes as metastasis-directed therapy. A long-term oncological outcome was favorable after salvage nodal radiotherapy and severe toxicity rates were low. Androgen deprivation therapy plays a major role in recurrent prostate cancer management and demonstrates a positive influence on the rate of biochemical progression in patients receiving salvage nodal radiotherapy. The present long-term analysis may help clinicians identify patients who would benefit from salvage nodal radiotherapy and androgen deprivation therapy, as a multimodal treatment strategy for oligorecurrent prostate cancer. Abstract Background: The study aimed to access the long-term outcome of salvage nodal radiotherapy (SNRT) in oligorecurrent prostate cancer. Methods: A total of 95 consecutive patients received SNRT for pelvic and/or extrapelvic nodal recurrence after prostate-specific membrane antigen (PSMA) or choline PET from 2010 to 2021. SNRT was applied as external beam radiotherapy with simultaneous integrated boost up to a median total dose of 62.9 Gy (EQD2\(_{1.5Gy}\)) to the recurrent lymph node metastases. The outcome was analyzed by cumulative incidence functions with death as the competing risk. Fine–Gray regression analyses were performed to estimate the relative hazards of the outcome parameters. Genitourinary (GU)/gastrointestinal (GI) toxicity evaluation utilized Common Toxicity Criteria for Adverse Events (v5.0). The results are as follows: the median follow-up was 47.1 months. The five-year biochemical progression rate (95% CI) was 50.1% (35.7–62.9%). Concomitant androgen deprivation therapy (ADT) was adminstered in 60.0% of the patients. The five-year biochemical progression rate was 75.0% (42.0–90.9%) without ADT versus 35.3% (19.6–51.4%) with ADT (p = 0.003). The cumulative five-year late grade 3 GU toxicity rate was 2.1%. No late grade 3 GI toxicity occured. Conclusions: Metastasis-directed therapy through SNRT for PET-staged oligorecurrent prostate cancer demonstrated a favorable long-term oncologic outcome. Omittance of ADT led to an increased biochemical progression. KW - metastasis-directed therapy KW - long-term outcome KW - oligorecurrence KW - prostate cancer KW - salvage radiotherapy KW - PSMA Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286064 SN - 2072-6694 VL - 14 IS - 15 ER - TY - THES A1 - Subota, Ines T1 - Switches in trypanosome differentiation: ALBA proteins acting on post-transcriptional mRNA control T1 - Steuerungsmechanismen der Differenzierung in Trypanosomen: die Rolle von ALBA Proteinen in post-transkriptioneller mRNA Kontrolle N2 - Trypanosoma brucei is a digenetic eukaryotic parasite that develops in different tissues of a mammalian host and a tsetse fly. It is responsible for sleeping sickness in sub-saharan Africa. The parasite cycle involves more than nine developmental stages that can be clearly distinguished by their general morphology, their metabolism and the relative positioning of their DNA-containing organelles. During their development, trypanosomes remain exclusively extracellular and encounter changing environments with different physico-chemical properties (nutritional availability, viscosity, temperature, etc.). It has been proposed that trypanosomes use their flagellum as a sensing organelle, in agreement with the established role of structurally-related cilia in metazoa and ciliates. Recognition of environmental triggers is presumed to be at the initiation of differentiation events, leading to the parasite stage that is the best suited to the new environment. These changes are achieved by the modification of gene expression programmes, mostly underlying post-transcriptional control of mRNA transcripts. We first demonstrate that the RNA-binding proteins ALBA3/4 are involved in specific differentiation processes during the parasite development in the fly. They are cytosolic and expressed throughout the parasite cycle with the exception of the stages found in the tsetse fly proventriculus, as shown by both immunofluorescence and live cell analysis upon endogenous tagging with YFP. Knock-down of both proteins in the developmental stage preceding these forms leads to striking modifications: cell elongation, cell cycle arrest and relocalization of the nucleus in a posterior position, all typical of processes acting in parasites found in the proventriculus region. When ALBA3 is over-expressed from an exogenous copy during infection, it interferes with the relocalization of the nucleus in proventricular parasites. This is not observed for ALBA4 over-expression that does not visibly impede differentiation. Both ALBA3/4 proteins react to starvation conditions by accumulating in cytoplasmic stress granules together with DHH1, a recognized RNA-binding protein. ALBA3/4 proteins also partially colocalize with granules formed by polyA+ RNA in these conditions. We propose that ALBA are involved in trypanosome differentiation processes where they control a subset of developmentally regulated transcripts. These processes involving ALBA3/4 are likely to result from the specific activation of sensing pathways. In the second part of the thesis, we identify novel flagellar proteins that could act in sensing mechanisms. Several protein candidates were selected from a proteomic analysis of intact flagella performed in the host laboratory. This work validates their flagellar localization with high success (85% of the proteins examined) and defines multiple different patterns of protein distribution in the flagellum. Two proteins are analyzed during development, one of them showing down-regulation in proventricular stages. The functional analysis of one novel flagellar membrane protein reveals its rapid dynamics within the flagellum but does not yield a visible phenotype in culture. This is coherent with sensory function that might not be needed in stable culture conditions, but could be required in natural conditions during development. In conclusion, this work adds new pieces to the puzzle of identifying molecular switches involved in developmental mRNA control and environmental sensing in trypanosome stages in the tsetse fly. N2 - Trypanosoma brucei ist ein digenetischer, eukaryotischer Parasit, der zwischen Säugetier und Tsetsefliege alterniert, in welchen er unterschiedliche Gewebe besiedelt. Er ist die Ursache für die Schlafkrankheit in Afrika südlich der Sahara. Der Lebenszyklus der Trypanosomen besteht aus mehr als neun Parasitenstadien, die eindeutig anhand ihrer Morphologie, ihres Metabolismus und der Positionierung ihrer DNA Organellen unterschieden werden können. Trypanosomen bleiben ausschließlich extrazellulär und kommen im Laufe ihres Infektionszyklus mit sich verändernden Umwelteinflüssen in Berührung, z. B. Temperaturschwankungen, Variation in vorhandenen Energiequellen, erhöhte Viskosität usw. In Übereinstimmung mit der anerkannten sensorischen Funktion die Cilien in Vielzellern ausüben, wurde für diese Rolle das strukturverwandte Flagellum in Trypanosomen vorgeschlagen. Die Erkennung wechselnder Umweltparameter ist der vermutliche Auslöser für Differenzierungsprozesse, die ein Entwicklungsstadium hervorbringen, welches am besten an die neue Umgebung angepasst ist. Dies wird durch eine Modifizierung der Genexpression erreicht, die in Trypanosomen fast ausschließlich auf posttranskriptioneller Ebene erfolgt. Diese Arbeit zeigt, dass die RNA bindenden Proteine ALBA3 und ALBA4 an der Differenzierung von Trypanosomen in der Tsetsefliege beteiligt sind. Immunfluoreszenzanalyse und Lebendvideomikroskopie von Zellen, die eine an YFP gekoppelte Variante der Proteine enthalten, haben gezeigt, dass sich ALBA3/4 im Zytosol befinden und dass sie in jedem Parasitenstadium exprimiert sind, mit Ausnahme derer, die im Proventrikel der Tsetsefliege zu finden sind. Das Herunterregulieren der Proteine in vorangehenden Stadien, führt zu markanten Veränderungen, die mit denjenigen, die in Parasiten im Proventrikel zu finden sind, vergleichbar sind: z. B. Verlängerung der Zelle, Zellzyklusarrest und Lokalisierung des Zellkerns in eine posteriore Position. Im Gegenteil dazu findet die Umpositionierung des Zellkerns nicht statt, wenn ALBA3 während der Entwicklung des Parasiten in der Tsetsefliege überexprimiert wird. Ein vergleichbarer Effekt wird mit ALBA4 Überexpression nicht erreicht, welches die Entwicklung nicht negativ zu beeinflussen scheint. Wenn Trypanosomen Hungerstress ausgesetzt sind, reichern sich beide ALBA Proteine zusammen mit DHH1, einem anerkannten RNA bindenden Protein, in zytoplasmatischen Aggregaten an, die nur teilweise mit denjenigen kolokalisieren, die durch polyA+ RNA in diesen Bedingungen verursacht werden. Diese Arbeit zeigt, dass ALBA Proteine eine wichtige Rolle in der Entwicklung von Trypanosomen spielen und legt nahe, dass sie an der entwicklungsbedingten Kontrolle eines Teils der mRNA Expression beteiligt sind. Der zweite Teil dieser Arbeit handelt von der Identifizierung neuer flagellarer Proteine, die eine sensorische Funktion haben könnten. Hierfür wurden mehrere Proteinkandidaten aus einer durchgeführten Proteomanalyse intakter Flagellen gewählt. Die vorliegende Arbeit bestätigt die flagellare Lokalisierung der Proteine mit großem Erfolg (85% der untersuchten Proteine) und zeigt, dass sie unterschiedliche Verteilungsmuster vorweisen. Zwei der Proteine werden während der Infektion des Parasiten in der Tsetsefliege untersucht, was aufdeckt, dass eines davon in den Stadien im Proventrikel herunterreguliert ist. Die Funktionsstudie eines neu identifizierten flagellaren Membranproteins weist seine schnelle Dynamik im Flagellum auf, führt jedoch zu keinem sichtbaren Phänotyp in Laborbedingungen. Diese Beobachtung passt zu der Annahme, dass Proteine mit sensorischer Funktion in stabilen Laborverhältnissen nicht essentiell sind aber eine wichtige Rolle während der Entwicklung des Parasiten in natürlichen Bedingungen spielen. Zusammenfassend fügt diese Arbeit Teile zum Puzzle der Identifizierung molekularer Schalter, die in Trypanosomenstadien in der Tsetsefliege an der mRNA Kontrolle und der Erkennung der Umwelt beteiligt sind. KW - Trypanosoma brucei KW - Parasit KW - Entwicklung KW - Tsetsefliege KW - Trypanosomen KW - parasitärer Entwicklungszyklus KW - Differenzierung KW - Tsetse Fliege KW - ALBA Proteine KW - Kontrolle der Genexpression KW - trypanosomes KW - parasite cycle KW - differentiation KW - tsetse fly KW - ALBA proteins KW - gene expression control KW - flagellar sensing proteins KW - FLAMM KW - Genexpression Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85707 N1 - Durchführung der Experimente am Institut Pasteur, Arbeitsgruppe Trypanosome Cell Biology Unit, Paris, Frankreich ER - TY - JOUR A1 - Subbarayal, Prema A1 - Karunakaran, Karthika A1 - Winkler, Ann-Cathrin A1 - Rother, Marion A1 - Gonzalez, Erik A1 - Meyer, Thomas F. A1 - Rudel, Thomas T1 - EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis JF - PLoS Pathogens N2 - The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance. KW - membrane proteins KW - chlamydia infection KW - chlamydia trachomatis KW - chlamydia KW - HeLa cells KW - apoptosis KW - host cells KW - membrane receptor signaling Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125566 VL - 11 IS - 4 ER - TY - THES A1 - Stübs, Dorothee T1 - Identifizierung und Regulation von kälteinduzierbaren Faktoren aus B. bronchiseptica T1 - Identification and regulation of cold induced factors in B. bronchiseptica N2 - Kälteschockproteine werden in Bakterien, gleichermaßen wie die gut charakterisierten Hitzeschockproteine, bei hohen Temperaturschwankungen stark induziert und ermöglichen der Zelle durch unterschiedliche Funktionen ein Wachstum in der Kälte. In dieser Promotionsarbeit wurde begonnen, die Kälteschock-Antwort von Bakterien des Genus Bordetella zu charakterisieren. Sowohl B. bronchiseptica als auch B. pertussis codieren für fünf Kälteschockproteine, die als CspA, CspB, CspC, CspD und CspE bezeichnet werden. Die fünf Proteine weisen eine signifikante Homologie zum Haupt-Kälteschockprotein CspA aus E. coli auf. Während in den Modellorganismen E. coli und B. subtilis mindestens vier (E. coli) bzw. alle drei (B. subtilis) csp-Gene deletiert sein müssen, um einen Wachstumsdefizit zu erkennen, genügt im Falle von B. bronchiseptica eine einzige Insertionsmutation im Gen cspB, um einen temperaturunabhängigen Wachstumsdefekt zu beobachten. Nach einem Kälteschock werden in B. bronchiseptica drei der fünf csp-Gene, cspA, cspB und cspC, deutlich induziert. Betrachtet man das Expressionsmuster der fünf csp-Gene unter verschiedenen Stressbedingungen, wie Zugabe von translationshemmenden Antibiotika, Hitzeschock oder osmotischer Stress, so lässt sich ein komplexes Expressionsmuster aufzeichnen. Außerdem besitzen die drei kälteinduzierbaren Gene cspA, cspB und cspC mehrere Transkriptionsstartpunkte, deren Transkriptmengen unter den verschiedenen Schockbedingungen stark variieren. Es stellte sich heraus, dass eine Überexpression von CspB aus B. bronchiseptica für die E. coli – Zelle toxisch ist, daher wurde das CspB-Protein als GST-Fusionsprotein exprimiert und über Glutathion-Sepharose aufgereinigt. Um eine potentielle Funktion von CspB in der Zelle zu untersuchen, wurden Filterbindeassays mit CspB::GST durchgeführt. Es wurde eine hochaffine, aber unspezifische Bindung an ssDNA festgestellt, was auf eine mögliche Funktion von CspB als Chaperon hindeutet. Nach Synthese eines CspB-spezifischen Antikörpers wurde die Kälteinduktion von CspB auch auf Proteinebene nachgewiesen. Durch 2D-Gelelektrophorese und massenspektrometrische Charakterisierung konnten 17 weitere kälteinduzierbare Proteine aus B. bronchiseptica identifiziert werden. Darunter waren u. a. ein Chaperon mit Ähnlichkeit zu GroES, ein Translationsinhibitor BB2940 und das CspB. Diese kälteinduzierbaren Proteine ähneln den CIPs aus E. coli. Weiterhin konnten noch das UspA und mehrere am Metabolismus beteiligte Proteine als CIPs aus B. bronchiseptica identifiziert werden, was signifikante Unterschiede in Bezug auf die Kälteadaptation zwischen den beiden Organismen aufzeigt. Betrachtet man die Promotorbereiche aller identifizierten csp-Gene, so fällt eine für diese Gene typische sehr lange 5’UTR auf. Innerhalb dieser upstream Region findet man in vier der fünf csp-Gene einen 9 bp langen Consensus mit der Sequenz TCCTTGATT, der in nahezu gleichem Abstand vom postulierten Startcodon vorkommt. Diese identifizierte 9bp-box ist für eine effiziente Transkription in der Kälte jedoch nicht von Bedeutung. Auf posttranskriptioneller Ebene wird die lange 5’UTR für die Stabilisierung der cold-shock mRNA in der Kälte verantwortlich gemacht. Außerdem ist das Vorhandensein der kompletten 5’UTR essentiell für eine effiziente Translation bei niedriger Temperatur, wobei eine Mutation der 9bp-box einen geringen, aber signifikanten negativen Effekt auf die Translation ausübt. Sechs Gene, der neu identifizierten CIPs, beinhalten ebenfalls eine 9bp-box in ihrer upstream Region. Interessanterweise werden zwei der fünf csp-Gene, cspC und cspD, vom BvgAS Zweikomponentensystem, dem Haupttranskriptionsregulator der Virulenzgene im Genus Bordetella, reguliert. Die beiden Gene gehören zu den Bvg-negativ regulierten Genen, die in der Bvg-minus-Phase exprimiert werden. Weiterhin beeinflusst eine leichte Überexpression von CspB aus B. pertussis die Expression der Adenylatzyklase sowohl in B. pertussis, als auch in B. bronchiseptica negativ. Dieser für das CspB spezifische Effekt erinnert an das strukturell verwandte Tex-Protein (Fuchs et al, 1996; König et al, 2002). Beide Proteine beeinflussen die Expression der Virulenzfaktoren negativ, wobei für CspB gezeigt werden konnte, dass es einen direkten Einfluss auf die verminderte cyaA-Expression auf Transkriptionsebene besitzt. Dies zeigt eine Verbindung der Kälteschockantwort mit dem Virulenz-Regulon der Bordetellen, deren Rolle im Infektionszyklus bislang ungeklärt ist. N2 - Bacterial cold shock proteins (CSPs), like the well characterized heat shock proteins (HSPs) are highly induced in response to strong variation in temperature and cell growth at lower temperatures could be attributed to the different functions of CIPs. In this work we have studied the cold shock response of bacteria of the genus Bordetella. Both B. bronchiseptica and B. pertussis code for five CSPs (termed CspA to CspE) with significant amino acid homology to the major CspA of Escherichia coli. Mutations of a single csp gene (cspB) strongly affected the growth of B. bronchiseptica independent of temperature while a similar effect was observed in E. coli when four out of nine csp genes and in B. subtilis when all three csp genes were deleted. Transcription of cspA, cspB and cspC increased strongly after cold shock. The exposure to other stress conditions including translational inhibitors, heat shock and osmotic stress resulted in a complex pattern of changes in the transcription of the five cold shock genes. In the case of three csp genes (cspA, cspB, cspC), more than one specific transcript could be detected. To investigate the function of one of the cold shock proteins, CspB was purified as GSTfusion over a glutathion-sepharose column, because overexpression of pure CspB was shown to be toxic for the E. coli cell. Due to its high affinity but rather unspecific binding to ssDNA as tested by filter binding assays, it is possible that CspB functions as a chaperone. Induction of CspB was confirmed using a specific antibody and subsequently 17 other cold inducible proteins (CIPs) were identified by 2D-gelelectrophoresis and mass spectrometric characterization. Among these CIPs are some proteins which resemble the cold shock response of E. coli, like CspB, a chaperone with similarities to GroES and a translation inhibitor protein. Furthermore, interesting examples are the universal stress protein UspA and some proteins that are involved in the amino acid metabolism indicating signficant differences in the cold shock response of the two organism. The coding regions of all cold shock genes are preceeded by a long non-translated upstream region. Within this 5’UTR of four of the csp genes an identical sequence of 9 nucleotides with the consensus TCCTTGATT (9bp box) was identified which is located at similar positions with respect to their start codons. This identified 9bp-box was found to be irrelevant for transcription in the cold. Furthermore by in silico analysis a putative 54- binding site in the upstream region of cspB could be identified which has a regulatory function on cspB transcription. The long 5’ UTR itself seems to be important for transcript stabilization and efficient translation under cold shock conditions. Furthermore mutation or deletion of the 9bp box has a negative effect on translation. Six of the new identified CIPs are encoded by genes that contain the 9bp box in their 5’-UTR. Using bioinformatic tools (HMMR search) we identified 131 genes in the B. bronchiseptica RB50 genome that contain such a 9mer, but only 17 of the genes contain these consensus at appropriate position. Using this approach, infB, encoding for IF-2, could be identified as cold inducible. A connection between the occurence of the 9bp box and the cold induction could not be shown yet. Interestingly, two cold shock genes (cspC and cspD) were found to be under the negative control of the BvgAS system, the main transcriptional regulator of Bordetella virulence genes. Morover, a negative effect of a slight overexpression of CspB, but not of the other CSPs, on the transcription of the adenylate cyclase toxin CyaA in both B. pertussis and B. bronchiseptica was observed. Like the overexpression of previously described Tex protein (Fuchs et al, 1996; König et al, 2002), both proteins have a negative effect on the expression of the virulence factors. In this work, a direct influence of CspB on the cyaA transcription could be confirmed, suggesting a cross talk between the CSP mediated stress response stimulon and the Bordetella virulence regulon. KW - Bordetella bronchiseptica KW - Kälteschock-Proteine KW - Mikrobiologie KW - Bordetella KW - Regulation KW - Kälteschock KW - Bordetella KW - regulation KW - cold-shock Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-12704 ER - TY - JOUR A1 - Sturm, Julia B. A1 - Hess, Michael A1 - Weibel, Stephanie A1 - Chen, Nanhei G. A1 - Yu, Yong A. A1 - Zhang, Quian A1 - Donat, Ulrike A1 - Reiss, Cora A1 - Gambaryan, Stepan A1 - Krohne, Georg A1 - Stritzker, Jochen A1 - Szalay, Aladar A. T1 - Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy N2 - Background: Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. Methods: Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. Results: We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. Conclusion: Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects. KW - Biologie KW - vaccinia virus KW - cancer KW - cytokine KW - hyper-IL-6 KW - oncolysis KW - chemotherapy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75224 ER - TY - JOUR A1 - Strube-Bloss, Martin F. A1 - Brown, Austin A1 - Spaethe, Johannes A1 - Schmitt, Thomas A1 - Rössler, Wolfgang T1 - Extracting the Behaviorally Relevant Stimulus: Unique Neural Representation of Farnesol, a Component of the Recruitment Pheromone of Bombus terrestris JF - PLoS One N2 - To trigger innate behavior, sensory neural networks are pre-tuned to extract biologically relevant stimuli. Many male-female or insect-plant interactions depend on this phenomenon. Especially communication among individuals within social groups depends on innate behaviors. One example is the efficient recruitment of nest mates by successful bumblebee foragers. Returning foragers release a recruitment pheromone in the nest while they perform a ‘dance’ behavior to activate unemployed nest mates. A major component of this pheromone is the sesquiterpenoid farnesol. How farnesol is processed and perceived by the olfactory system, has not yet been identified. It is much likely that processing farnesol involves an innate mechanism for the extraction of relevant information to trigger a fast and reliable behavioral response. To test this hypothesis, we used population response analyses of 100 antennal lobe (AL) neurons recorded in alive bumblebee workers under repeated stimulation with four behaviorally different, but chemically related odorants (geraniol, citronellol, citronellal and farnesol). The analysis identified a unique neural representation of the recruitment pheromone component compared to the other odorants that are predominantly emitted by flowers. The farnesol induced population activity in the AL allowed a reliable separation of farnesol from all other chemically related odor stimuli we tested. We conclude that the farnesol induced population activity may reflect a predetermined representation within the AL-neural network allowing efficient and fast extraction of a behaviorally relevant stimulus. Furthermore, the results show that population response analyses of multiple single AL-units may provide a powerful tool to identify distinct representations of behaviorally relevant odors. KW - instinct KW - plant-insect interactions KW - pheromones KW - bumblebees KW - odorants KW - principal component analysis KW - neurons KW - action potentials Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125875 VL - 10 IS - 9 ER - TY - THES A1 - Streit, Sebastian T1 - Automatische Identifizierung bei sozialen Insekten : Design und Praxistest T1 - Automatic insect identification: Design and test N2 - Design und Implementierung eines RFID basierten Systems für soziale Insekten (Hummeln, Bienen) N2 - Design and implementation of a rfid based system for identifying social insects (honeybees, bumblebees) KW - Soziale Insekten KW - Individuum KW - Identifikation KW - Methode KW - rfid KW - Identifizierung KW - Honigbiene KW - rfid KW - insect identification KW - honeybee Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-8962 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 - JOUR A1 - Streinzer, Martin A1 - Brockmann, Axel A1 - Nagaraja, Narayanappa A1 - Spaethe, Johannes T1 - Sex and Caste-Specific Variation in Compound Eye Morphology of Five Honeybee Species JF - PLoS ONE N2 - Ranging from dwarfs to giants, the species of honeybees show remarkable differences in body size that have placed evolutionary constrains on the size of sensory organs and the brain. Colonies comprise three adult phenotypes, drones and two female castes, the reproductive queen and sterile workers. The phenotypes differ with respect to tasks and thus selection pressures which additionally constrain the shape of sensory systems. In a first step to explore the variability and interaction between species size-limitations and sex and caste-specific selection pressures in sensory and neural structures in honeybees, we compared eye size, ommatidia number and distribution of facet lens diameters in drones, queens and workers of five species (Apis andreniformis, A. florea, A. dorsata, A. mellifera, A. cerana). In these species, male and female eyes show a consistent sex-specific organization with respect to eye size and regional specialization of facet diameters. Drones possess distinctly enlarged eyes with large dorsal facets. Aside from these general patterns, we found signs of unique adaptations in eyes of A. florea and A. dorsata drones. In both species, drone eyes are disproportionately enlarged. In A. dorsata the increased eye size results from enlarged facets, a likely adaptation to crepuscular mating flights. In contrast, the relative enlargement of A. florea drone eyes results from an increase in ommatidia number, suggesting strong selection for high spatial resolution. Comparison of eye morphology and published mating flight times indicates a correlation between overall light sensitivity and species-specific mating flight times. The correlation suggests an important role of ambient light intensities in the regulation of species-specific mating flight times and the evolution of the visual system. Our study further deepens insights into visual adaptations within the genus Apis and opens up future perspectives for research to better understand the timing mechanisms and sensory physiology of mating related signals. KW - eyes KW - foraging KW - honey bees KW - insect flight KW - physiological parameters KW - sensory systems KW - vision KW - visual system Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96412 ER - TY - THES A1 - Streinzer, Martin T1 - Sexual dimorphism of the sensory systems in bees (Hymenoptera, Apoidea) and the evolution of sex-specific adaptations in the context of mating behavior T1 - Sensorischer Geschlechtsdimorphismus bei Bienen (Hymenoptera, Apoidea) und die Evolution geschlechtsspezifischer Anpassungen im Kontext des Paarungsverhaltens N2 - Bees have had an intimate relationship with humans for millennia, as pollinators of fruit, vegetable and other crops and suppliers of honey, wax and other products. This relationship has led to an extensive understanding of their ecology and behavior. One of the most comprehensively understood species is the Western honeybee, Apis mellifera. Our understanding of sex-specific investment in other bees, however, has remained superficial. Signals and cues employed in bee foraging and mating behavior are reasonably well understood in only a handful of species and functional adaptations are described in some species. I explored the variety of sensory adaptations in three model systems within the bees. Females share a similar ecology and similar functional morphologies are to be expected. Males, engage mainly in mating behavior. A variety of male mating strategies has been described which differ in their spatiotemporal features and in the signals and cues involved, and thus selection pressures. As a consequence, males’ sensory systems are more diverse than those of females. In the first part I studied adaptations of the visual system in honeybees. I compared sex and caste-specific eye morphology among 5 species (Apis andreniformis, A. cerana, A. dorsata, A. florea, A. mellifera). I found a strong correlation between body size and eye size in both female castes. Queens have a relatively reduced visual system which is in line with the reduced role of visual perception in their life history. Workers differed in eye size and functional morphology, which corresponds to known foraging differences among species. In males, the eyes are conspicuously enlarged in all species, but a disproportionate enlargement was found in two species (A. dorsata, A. florea). I further demonstrate a correlation between male visual parameters and mating flight time, and propose that light intensities play an important role in the species-specific timing of mating flights. In the second study I investigated eye morphology differences among two phenotypes of drones in the Western honeybee. Besides normal-sized drones, smaller drones are reared in the colony, and suffer from reduced reproductive success. My results suggest that the smaller phenotype does not differ in spatial resolution of its visual system, but suffers from reduced light and contrast sensitivity which may exacerbate the reduction in reproductive success caused by other factors. In the third study I investigated the morphology of the visual system in bumblebees. I explored the association between male eye size and mating behavior and investigated the diversity of compound eye morphology among workers, queens and males in 11 species. I identified adaptations of workers that correlate with distinct foraging differences among species. Bumblebee queens must, in contrast to honeybees, fulfill similar tasks as workers in the first part of their life, and correspondingly visual parameters are similar among both female castes. Enlarged male eyes are found in several subgenera and have evolved several times independently within the genus, which I demonstrate using phylogenetic informed statistics. Males of these species engage in visually guided mating behavior. I find similarities in the functional eye morphology among large-eyed males in four subgenera, suggesting convergent evolution as adaptation to similar visual tasks. In the remaining species, males do not differ significantly from workers in their eye morphology. In the fourth study I investigated the sexual dimorphism of the visual system in a solitary bee species. Males of Eucera berlandi patrol nesting sites and compete for first access to virgin females. Males have enlarged eyes and better spatial resolution in their frontal eye region. In a behavioral study, I tested the effect of target size and speed on male mate catching success. 3-D reconstructions of the chasing flights revealed that angular target size is an important parameter in male chasing behavior. I discuss similarities to other insects that face similar problems in visual target detection. In the fifth study I examined the olfactory system of E. berlandi. Males have extremely long antennae. To investigate the anatomical grounds of this elongation I studied antennal morphology in detail in the periphery and follow the sexual dimorphism into the brain. Functional adaptations were found in males (e.g. longer antennae, a multiplication of olfactory sensilla and receptor neurons, hypertrophied macroglomeruli, a numerical reduction of glomeruli in males and sexually dimorphic investment in higher order processing regions in the brain), which were similar to those observed in honeybee drones. The similarities and differences are discussed in the context of solitary vs. eusocial lifestyle and the corresponding consequences for selection acting on males. N2 - Bienen und Menschen verbindet eine lange andauernde und enge Beziehung. Diese enge Beziehung hat zu einem ausgeprägten Wissen über die Ökologie und das Verhalten geführt. Die am besten untersuchte Bienenart ist die westliche Honigbiene, Apis mellifera. Der ausgeprägte Kasten- und Sexualdimorphismus hat das Studium der Geschlechterunterschiede vereinfacht und vorangetrieben. Unser Wissen über geschlechtsspezifische Investitionen bei Bienen ist jedoch in vielerlei Hinsicht lückenhaft geblieben. Die Signale und Achtungssignale die im Paarungsverhalten eine Rolle spielen sind nur bei einer Handvoll Arten hinreichend bekannt und funktionelle Anpassungen an diese sind in wenigen Arten beschrieben. In dieser Arbeit habe ich sensorische Anpassungen an geschlechtsspezifische Verhaltensweisen in drei Bienengruppen genauer untersucht. Weibchen und Arbeiterinnen haben generell eine ähnliche Lebensweise. Männchen beschäftigen sich fast ausschließlich mit der Partnersuche. Infolgedessen, zeigt die Sensorik der Männchen eine größere Vielfalt an morphologischen und funktionellen Anpassungen als die der Weibchen. Im ersten Abschnitt dieser Arbeit habe ich Anpassungen des visuellen Systems von 5 Honigbienenarten (Apis andreniformis, A. cerana, A. dorsata, A. florea, A. mellifera) untersucht. Ich finde eine deutliche Korrelation zwischen Körper- und Augengröße bei beiden weiblichen Kasten. Königinnen haben relativ kleinere Augen als Arbeiterinnen, was der verringerten Rolle visueller Wahrnehmung im Lebenszyklus dieser Kaste entspricht. Die Arbeiterinnen unterschieden sich sowohl in ihrer Augengröße als auch in der funktionellen Morphologie. Die Unterschiede passen jeweils zu der artspezifischen Ökologie. Drohnen aller Arten haben auffällig vergrößerte Augen, jedoch sind sie in zwei Arten (A. dorsata, A. florea) überproportional vergrößert. Zusätzlich zeige ich, dass bestimmte Augenparameter mit dem artspezifischen Paarungszeitpunkt korrelieren, und schlage vor, dass die Lichtintensität eine Rolle bei der Feststellung des richtigen Paarungszeitpunktes spielen könnte. In der zweiten Untersuchung habe ich die Augen von zwei Drohnenphänotypen von A. mellifera untersucht. Neben normalen Drohen werden in der Kolonie auch kleinere Drohnen aufgezogen, die unter einem geringeren Fortpflanzungserfolg leiden. Meine Ergebnisse zeigen, dass sich die Phänotypen vermutlich nicht in der räumlichen Auflösungsfähigkeit, jedoch in der Lichtempfindlichkeit der Augen von normalen Drohnen unterscheiden. In der dritten Untersuchung habe ich die Augenmorphologie bei 11 Hummelarten untersucht. Ich beschreibe in dieser Studie Anpassungen der Arbeiterinnen, die vermutlich mit der Habitatwahl im Zusammenhang stehen. Hummelköniginnen sind, im Gegensatz zu Königinnen der Honigbiene, in der ersten Zeit nach der Koloniegründung auf sich allein gestellt und müssen alle Aufgaben, die später von den Arbeiterinnen übernommen werden, selbst ausführen. Dementsprechend sind die Augen beider Weibchenkasten ähnlich in ihrer relativen Größe und funktionellen Morphologie. Vergrößerte Augen der Männchen können in Arten verschiedener Untergattungen gefunden werden und der Phänotyp ist im Laufe der Evolution mehrfach unabhängig entstanden, was ich mit phylogenetisch vergleichenden Methoden zeige. Die Augenmorphologie der vier untersuchten großäugigen Arten ist sehr ähnlich, was auf konvergente Evolution hinweist. Die Augenmorphologie der restlichen Arten unterscheidet sich hingegen nicht deutlich von jener der Weibchen. In der vierten Untersuchung habe ich mich dem Sexualdimorphismus der Solitärbienenart Eucera berlandi gewidmet. Männchen haben größere Augen und sowohl größere Facetten als auch eine höhere räumliche Auflösung im frontalen Gesichtsfeld als Weibchen. In einem Verhaltensversuch habe ich die Auswirkungen der Größe von Weibchendummies auf die Detektion getestet. In 3-D Rekonstruktionen der Weibchenverfolgung zeigte sich dass die Winkelgröße des Objektes, eine von der Distanz unabhängige Größe, eine wichtige Rolle spielt. Im Zusammenhang mit den gefundenen Daten diskutiere ich die Parallelen zu anderen Insektenarten. In der fünften Studie untersuche ich das olfaktorische System von E. berlandi. Männchen haben extreme lange Antennen. Um die anatomischen Grundlagen der geschlechtsspezifischen Antennenmorphologie zu untersuchen habe ich die Antennen beider Geschlechter im Detail studiert. Zusätzlich bin ich dem Dimorphismus entlang der olfaktorischen Bahn bis ins Gehirn gefolgt. Männchen zeige funktionelle Anpassungen (z.B. längere Antennen, eine höhere Anzahl an olfaktorischen Sensillen und Rezeptorneuronen, stark vergrößerte Glomeruli im Antennallobus, eine zahlenmäßige Reduktion der Glomeruli und geschlecherspezifische Investition in höhere Integrationszentren im Gehirn) an die Weibchendetektion. KW - Biene KW - Sinne KW - Verhalten KW - Neurobiologie KW - Geschlechtsunterschied KW - Biene KW - Hummel KW - Sinnesphysiologie KW - Evolution KW - bees KW - sensory ecology KW - evolution KW - visual system Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78689 ER - TY - THES A1 - Strehl, Christoph-Peter T1 - Evolution of colony characteristics in the harvester ant genus Pogonomyrmex T1 - Evolution von Kolonie-Charakteristika in der Ernteameisen-Gattung Pogonomyrmex N2 - Die Gattung Pogonomyrmex ist besonders gut geeignet, um die Evolution der Charakteristika von Ameisenkolonien allgemein zu untersuchen, und insbesondere deren soziogenetische Struktur, da die Biologie für einige ihrer Arten sehr gut bekannt ist, und eine Diversität an Paarungshäufigkeiten und Könniginnenzahlen vorkommt. Diese Variation in der soziogenetischen Struktur der Kolonien erzeugt eine hohe Varianz an Verwandschaftsgraden innerhalb von Kolonien, und kann eine Hauptkomponente darstellen, welche die Evolution verschiedenster Koloniecharakteristika vorantreibt. Um die Variabilität intrakolonialer Verwandschaftsgrade innerhalb der Gattung Pogonomyrmex genau zu bestimmen, wurde für ausgewählte Mitglieder der Gattung, nämlich für P. (sensu stricto) rugosus, P. (sensu stricto) badius and P. (Ephebomyrmex) pima, mit Hilfe der Technik des DNA-Fingerabdruckes die Anzahl an Matrilinien und Patrilinien bestimmt. Es wurde versucht die Evolution dieser Koloniecharakteristika vor dem Hintergrund einer Phylogenie zu erklären. Zu diesem Zweck wurde ein Gen-Stammbaum für 39 Arten der Gattung Pogonomyrmex erstellt. Die Artabdeckung betrug 83% bei den Nord-Amerikanischen, und 43% bei den Süd-Amerikanischen Arten. Effektive Mehrfachpaarung von Königinnen wurde für P. rugosus (me=4.1) und P. badius (me=6.7) bestätigt. Zusätzlich wurde gezeigt, dass beide Arten monogyn sind. Diese Ergebnisse bestätigen Verhaltensbeobachtungen von Mehrfachpaarungen in diesen Arten. Mittlerweile ist Mehrfachpaarung in 9 Pogonomyrmex Arten bekannt (bei 3 Arten durch Verhaltensbeobachtungen – bei 6 Arten durch genetischen Nachweis). In P. (E.) pima hingegen waren alle der untersuchten Königinnen einfach gepaart (me=1.0). Daher könnte es sein, dass Mehrfachpaarung entweder früh in der Evolution der Gattung Pogonomyrmex entstand und nachträglich in der Untergattung Ephebomyrmex verloren wurde (Plesiomorphie-Hypothese), oder sie entstand zum ersten mal in der Untergattung Pogonomyrmex sensu stricto (Apomorphie-Hypothese). In P. huachucanus, einer Art, die basal zu dem Nord-Amerikanischen sensu stricto Komplex ist, könnten die im Vergleich zu ihren sensu stricto Verwandten geringeren effektiven Paarungshäufigkeiten der Königinnen (J. Gadau and C.-P. Strehl, unveröffentlicht) einen Wechsel von Monandrie zu Polyandrie im Verlauf der Entstehung der fortschrittlicheren sensu stricto Arten widerspiegeln, was die Apomorphie-Hypothese unterstützen würde. Die intrakolonialen Verwandtschaftsgrade sind dennoch in P. (E.) pima niedrig. Dies ist möglicherweise auf mehrere reproduktive Königinnen (Polygynie) zurückzuführen. Polygynie ist ebenfalls dokumentiert für mindestens vier weitere Arten der Untergattung Ephebomyrmex, mit genetischer Evidenz allerdings bisher nur für P. (E.) pima. Es könnte sein, dass es einen evolutionären Ausgleich (trade-off) zwischen Polyandrie und Polygynie innerhalb der Untergattung Ephebomyrmex gab, und daher beide Untergattungen eine hohe genetische Vielfalt innerhalb der Kolonien behielten. Diese hohe genetische Vielfalt könnte einer der Gründe sein für den Erfolg und die Radiation der Gattung Pogonomyrmex in Trockengebieten. Evolution könnte eine hohe genetische Vielfalt von Pogonomyrmex Kolonien begünstigt haben, da sie den Kolonien hilft die Organisation der Kolonie und die Effizienz mit der externe Aufgaben ausgeführt werden zu verbessern. Wenigstens in P. badius konnte eine Verknüpfung zwischen Patrilinien und physischem Polyethismus gefunden werden, was auf eine Verbesserung der Kolonieorganisation mit Hilfe von Polyandrie hindeutet. Darüber hinaus könnten die dargelegten extremen Polyandrie- Werte den P. badius-Weibchen helfen die Möglichkeit der Inzucht aufgrund eingeschränkter Ausbreitung zu bewältigen. Eine eingeschränkte Ausbreitung wird auch in P. (E.) pima durch flügellose, intermorphe Königinnen beobachtet. Jedoch wird bei P. (E.) pima die Inzucht durch Auskreuzen mittels Männchen möglicherweise verhindert, da keine signifikante Inzucht gefunden wurde. In den vorliegenden Gen-Stammbäumen war die Untergattung Pogonomyrmex Ephebomyrmex von der Untergattung Pogonomyrmex sensu stricto getrennt. Daher könnte es sein, dass P. Ephebomyrmex in den Status einer Gattung erhoben wird, auch aufgrund distinkter morphologischer und lebensgeschichtlicher Charaktere. Für eine präzise taxonomische Revision müsste allerdings eine breite Ergänzung an Arten vorgenommen werden. Es wurde in P. rugosus Kolonien normalerweise eine geringe Anzahl von unverwandten Arbeiterinnen vorgefunden, die möglicherweise aus Brutraub ausgewachsener Kolonien auf Gündungs-Kolonien stammen. Es ist allseits bekannt, dass die meisten Gründungskolonien von benachbarten, ausgewachsenen Kolonien der eigenen Art zerstört werden, aber es wurde bisher angenommen, dass die Brut dieser Kolonien ebenfalls zerstört wurde. Dieser oft vernachlässigte Aspekt könnte einen wichtigen Stärke-Bonus für ausgewachsene Kolonien darstellen. N2 - The genus Pogonomyrmex is predisposed for analyzing the evolution of ant colony characteristics in general and the sociogenetic structure in particular, due to the renowned biology of several species and the diversity of mating frequency and queen number. This variation in the sociogenetic structure of colonies produces a high variance in intracolonial relatedness which can be a major component driving the evolution of various colony characteristics. To exactly determine the variability of the intracolonial relatedness in the genus Pogonomyrmex both were analyzed, the number of matrilines and patrilines, in selected members of Pogonomyrmex, namely P. (sensu stricto) rugosus, P. (sensu stricto) badius and P. (Ephebomyrmex) pima using DNA fingerprint techniques. The evolution of these colony characteristics were tried to be explained within a phylogenetic framework. For that purpose we constructed a gene-tree of 39 species of the genus Pogonomyrmex. The taxon sampling covered about 83 % of the North American species and 43 % of the South American species. Effective multiple mating of queens was confirmed for P. rugosus (me=4.1) and P. badius (me=6.7). Additionally, both species are monogynous. These results corroborate behavioral observations of multiple mating for these species. Multiple mating is now known from 9 Pogonomyrmex species (behavioral evidence for 3 species – genetic evidence for 6 species). However, in P. (E.) pima all queens that were analyzed were single mated (me=1.0). Therefore, multiple mating may have either evolved early during the evolution of the genus Pogonomyrmex and has subsequently been lost in the subgenus Ephebomyrmex (plesiomorphic hypothesis), or it has first been evolved in the subgenus Pogonomyrmex sensu stricto (apomorphic hypothesis). In P. huachucanus, a species basal to the North- American sensu stricto complex, smaller effective mating number of queens compared to its sensu stricto relatives (J. Gadau and C.-P. Strehl, unpublished) probably do mirror a change from monandry to polyandry during the evolution of more advanced sensu stricto species, which would support the apomorphic hypothesis. The intracolonial relatedness in P. (E.) pima is however rather low. This is probably the result of multiple reproducing queens (polygyny). Polygyny is also documented for at least four other species of the subgenus Ephebomyrex, but so far P. (E.) pima is the only species with genetic evidence. It might be that there was an evolutionary trade-off within the subgenus Ephebomyrmex between polyandry and polygyny. Therefore, both subgenera retained a high intracolonial genetic diversity. This high genetic diversity might be one cause for the success and radiation of the genus Pogonomyrmex in arid environments. Evolution might have favored high genetic diversity of Pogonomyrmex colonies, because it helps colonies to improve their colonial organization and efficiency in performing external tasks. At least in P. badius a link between patrilines and physical polyethism was found, indicative of an improvement of colonial organization via polyandry. Furthermore, the documented extreme levels of polyandry might help P. badius females to overcome the possibility of inbreeding due to restricted dispersal. Restricted dispersal is also found in P. (E.) pima due to wingless, intermorphic queens. However, in P. (E.) pima inbreeding is probably prevented by outcrossing via males because no significant inbreeding is found. In the presented gene trees the subgenus Pogonomyrmex Ephebomyrmex was separated from the subgenus Pogonomyrmex sensu stricto. Therefore, P. Ephebomyrmex might be elevated to generic status, also due to its distinct morphological and life history characters. Nevertheless, for a precise taxonomic revision a broader complement of species has to be applied. Regularly a low number of unrelated workers was found in P. rugosus colonies, which probably stem from brood raids between mature and founding colonies. It is well known that most founding colonies are destroyed by neighboring conspecific mature colonies, but so far it was assumed that the brood of these colonies was also destroyed. This often neglected aspect might be an important fitness token for mature colonies. KW - Pogonomyrmex KW - Phylogenie KW - Koloniebildung KW - Phylogenie KW - Mehrfachpaarung KW - Pogonomyrmex rugosus KW - Pogonomyrmex badius KW - Ephebomyrmex pima KW - phylogeny KW - polyandry KW - Pogonomyrmex rugosus KW - Pogonomyrmex badius KW - Ephebomyrmex pima Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-14324 ER - TY - JOUR A1 - Storms, Mona A1 - Jakhar, Aryan A1 - Mitesser, Oliver A1 - Jechow, Andreas A1 - Hölker, Franz A1 - Degen, Tobias A1 - Hovestadt, Thomas A1 - Degen, Jacqueline T1 - The rising moon promotes mate finding in moths JF - Communications Biology N2 - To counteract insect decline, it is essential to understand the underlying causes, especially for key pollinators such as nocturnal moths whose ability to orientate can easily be influenced by ambient light conditions. These comprise natural light sources as well as artificial light, but their specific relevance for moth orientation is still unknown. We investigated the influence of moonlight on the reproductive behavior of privet hawkmoths (Sphinx ligustri) at a relatively dark site where the Milky Way was visible while the horizon was illuminated by distant light sources and skyglow. We show that male moths use the moon for orientation and reach females significantly faster with increasing moon elevation. Furthermore, the choice of flight direction depended on the cardinal position of the moon but not on the illumination of the horizon caused by artificial light, indicating that the moon plays a key role in the orientation of male moths. KW - animal behaviour KW - biodiversity KW - conservation biology KW - entomology KW - urban ecology Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301365 VL - 5 ER - TY - THES A1 - Stolzenberger, Sascha T1 - Spezifische Hemmung der allergieassoziierten Interleukin-4 Signaltransduktion N2 - Das Cytokin Interleukin-4 (IL-4) ist ein essentieller Faktor bei der Entstehung von Sofort-Typ Allergien. Die Bindung von IL-4 an seinen Rezeptor und die anschließende Phosphorylierung des IL-4 aktivierten Transkriptionsfaktors Stat6 ist ein Schlüsselereignis bei der allergischen Immunantwort. In der vorliegenden Arbeit werden Ergebnisse zur Hemmung der Stat6 vermittelten Signaltransduktion des IL-4 Rezeptors vorgestellt. Dazu wurde ein Vektorsystem etabliert, bei dem ein von dem Drosophila-Transkriptionsfaktor Antennapedia abgeleitetes 16 AS langes Peptid benutzt wird. Dieses Antennapediapeptid kann Plasmamembranen lebender Zellen energie- und rezeptorunabhängig durchqueren und dabei andere hydrophile Moleküle mittransportieren. Stat6 bindet über eine SH2 Domäne an phosphorylierte Reste von IL4Ra und bildet, nachdem es selbst phosphoryliert ist, mit anderen Stat6-Molekülen aktive Dimere. Ein aus der Stat6-Bindestelle des IL-4Ra abgeleitetes phosphoryliertes Peptid (Stat6BP) wurde mit Hilfe des Antennapediapeptids in verschiedene humane und murine Zellinien transportiert. Für Stat6BP konnte mit Hilfe von spezifischer Immunpräzipitation und Western-Blot gezeigt werden, dass es IL-4 induzierte Phosphorylierung und Aktivierung von Stat6 transient hemmen kann. Durch zusätzliche Applikation des Tyrosinphosphataseinhibitors Natriumpervanadat gelang es, die hemmende Wirkung von Stat6BP zu verlängern. Unter gleichen Bedingungen konnte auch gezeigt werden, dass Stat6BP spezifisch die Aktivierung von Stat6 hemmt, da die durch IL-4 oder IL-3 induzierte Phosphorylierung des eng verwandten Stat5 völlig unbeeinträchtigt bleibt. Ferner wurde durch das Peptid die Expression eines Stat6 kontrollierten Reportergens gehemmt. Im Rahmen dieser Arbeit wurde außerdem die Rolle der Src-Typ Kinasen p56lck und p59fyn in der IL-4 Signaltransduktion in unterschiedlichen T-Zellinien untersucht. Es zeigte sich, dass die Aktivierung der beide Kinasen stark von der getesteten Zellinie abhängt. In einigen T-Zellinien aktiviert IL-4 eher p56lck, in anderen eher p59fyn. N2 - Interleukin-4 (IL-4) is the major factor in the development of allergic diseases like hay fever or asthma. The most important cytoplasmic event following stimulation with IL-4 is the activation of the transcription factor Stat6 (signal transducer and activator of transcription 6). Stat6 binds via a single SH2 domain first to tyrosine-phosphorylated motifs in the IL-4Ra-chain, and then to another Stat6 molecule, which results in the formation of active dimers. Since Stat6 is exclusively used by the IL-4 receptor, it is a promising approach to specifically disrupt IL-4 signal- transduction by inhibiting Stat6 activation. A vector system was established for the delivery of hydrophilic agents into living cells. To this purpose, a 16 amino acid membrane-permeable peptide derived from the Drosophila transcription factor Antennapedia was used. The Antennapedia peptide has been shown to internalize into living cell in a receptor- and energy-independent manner. In this thesis it is shown that a peptide derived from the Stat6-binding region of IL-4Ra (Stat6BP) is an effective inhibitor when it is delivered into cells by coupling with the Antennapedia peptide. Stat6BP completely inhibited IL-4 dependent phosphorylation of Stat6 in different human and murine cell lines, while IL-3 and IL-4 dependent phosphorylation of Stat5 was not affected. The inhibitory effect of Stat6BP was transient, but could be prolonged by treating the cells with the phospatase inhibitor sodium pervanadate. Transcription from a reporter gene construct with a Stat6-dependent promoter was inhibited by Stat6BP as well, indicating that the peptide is a suitable inhibitor for cellular responses downstream from Stat6 phosphorylation. Another aim of this study was to investigate the role of the src-kinases p56lck and p59fyn in IL-4 signaltransduction. The results indicate, that the activation of both kinases is celline dependent. In some T-cellines p56lck was activated dominantly, in others p59fyn. KW - Interleukin 4 KW - Allergie KW - Signaltransduktion KW - Molekularbiologie KW - IL-4 KW - Interleukin-4 KW - Allergie KW - Peptide KW - STAT6 KW - Signaltransduktion KW - IL-4 KW - Interleukin-4 KW - Allergy KW - Peptid KW - Stat6 KW - Signaltransduction Y1 - 2000 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-2375 ER - TY - JOUR A1 - Stoll, Sascha A1 - Feldhaar, Heike A1 - Fraunholz, Martin J. A1 - Gross, Roy T1 - Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus N2 - Background: The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C.floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals. Results: We investigated the distribution and dynamics of endosymbiotic bacteria and bacteriocytes at different stages during development of the animals from larva to imago by confocal laser scanning microscopy. The number of bacteriocytes in relation to symbiont-free midgut cells varied strongly over different developmental stages. Especially during metamorphosis the relative number of bacteria-filled bacteriocytes increased strongly when the larval midgut epithelium is shed. During this developmental stage the midgut itself became a huge symbiotic organ consisting almost exclusively of cells harboring bacteria. In fact, during this phase some bacteria were also found in midgut cells other than bacteriocytes indicating a cell-invasive capacity of Blochmannia. In adult animals the number of bacteriocytes generally decreased. Conclusions: During the life cycle of the animals the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic. Our data show how the endosymbiont is retained within the midgut tissue during metamorphosis thereby ensuring the maintenance of the intracellular endosymbiosis despite a massive reorganization of the midgut tissue. The transformation of the entire midgut into a symbiotic organ during pupal stages underscores the important role of Blochmannia for its host in particular during metamorphosis. KW - Camponotus floridanus KW - carpenter ant Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67950 ER - TY - THES A1 - Stoll, Sascha T1 - Funktionelle Analyse von Blochmannia floridanus, dem primären Endosymbionten der Rossameise Camponotus floridanus T1 - Functional analysis of Blochmannia floridanus, the primary endosymbiont of the carpenter ant Camponotus floridanus N2 - Ameisen der Gattung Camponotus beherbergen bakterielle Symbionten der Gattung Blochmannia in spezialisierten Zellen des Mitteldarms (Blochmann, 1882; Buchner, 1965; Sauer, 2000; Schröder et al., 1996). Die Genomsequenzierung dieser Symbionten zeigte, dass Blochmannia, ähnlich den Symbionten von Blattläusen, hauptsächlich Gene der Aminosäurebiosynthese beibehalten hat (Degnan et al., 2005; Gil et al., 2003). Die Relevanz dieser nahrungsaufwertenden Funktion konnte experimentell bestätigt werden (Feldhaar et al., 2007). Ein Schwerpunkt der vorliegenden Arbeit war die Aufklärung der dynamischen Interaktion der beiden Partner während des komplexen Lebenszyklus des holometabolen Wirtes. Frühere Studien deuteten darauf hin, dass die Symbiose vor allem während der Larven- und Puppenphasen von Bedeutung sein könnte (Feldhaar et al., 2007; Wolschin et al., 2004; Zientz et al., 2006). Mit fluoreszenter in situ Hybridisierung (FISH) und konfokaler Laserscanning Mikroskopie konnte in der vorliegenden Arbeit die Lokalisierung von B. floridanus während der wichtigsten Entwicklungsstadien aufgeklärt werden. Hierbei konnte gezeigt werden, dass die Symbionten schon im ersten Larvenstadium in spezialisierten Zellen um den Darm angeordnet sind, aber in späteren Stadien nicht, wie bisher angenommen, auf diese Bakteriozyten beschränkt sind, sondern bis zum Schlupf der jungen Arbeiterinnen massiv andere Darmzellen infizieren. Übereinstimmend mit Bestimmungen der Zellzahl in den verschiedenen Wirtsstadien ist die Anzahl der Symbionten gegen Ende der Metamorphose am höchsten. Die Symbiose degeneriert in sehr alten Arbeiterinnen, gut gefüllte Bakteriozyten werden jedoch noch monatelang beibehalten. Mit Macroarray- und qRT- PCR- basierten Transkriptomanalysen wurde die Expression der bakteriellen Gene in charakteristischen Entwicklungsstadien des Wirtes untersucht. Allgemein zeigen vor allem Gene für molekulare Chaperons und bestimmte bakterielle Grundfunktionen eine hohe Expression. Aber auch viele Gene, die möglicherweise wichtige Funktionen in der Symbiose besitzen, wie die Biosynthese essentieller Aminosäuren und das Recycling von Stickstoffverbindungen, zeigen ein hohes absolutes Transkriptlevel. Zudem besteht eine positive Korrelation zwischen dem Expressionsniveau und dem GC- Gehalt der Gene, die in dem höheren Selektionsdruck und damit einer geringeren Mutationsrate der essentiellen Gene begründet liegt (Schaber et al., 2005). Durch Proteinanalysen konnte bestätigt werden, dass die Faktoren mit der höchsten absoluten Transkription die dominanten Proteine der Symbionten darstellen. In den unterschiedlichen Entwicklungsstadien zeigen viele Gene eine deutliche Dynamik, deren Ausmaß aber, verglichen mit freilebenden Bakterien, gering ist. Aus den Expressionsprofilen aufeinanderfolgender Gene lassen sich mögliche Transkriptionseinheiten ableiten, die teilweise auch experimentell bestätigt wurden. Oftmals zeigen auch Gene, die nicht in Transkriptionseinheiten angeordnet sind, aber verwandten Stoffwechselwegen angehören, ähnliche Muster. Dies deutet auf das Vorhandensein grundlegender Genregulations-mechanismen hin, obwohl im Genom von B. floridanus nur noch sehr wenige Transkriptionsfaktoren codiert sind (Gil et al., 2003). Auf übergeordneter Ebene zeigt sich, dass bei Symbionten aus späten Puppenstadien viele symbioserelevante Gene im Vergleich zu Genen des Grundmetabolismus eine erhöhte Expression zeigen. Dies betrifft besonders die Biosynthese aromatischer und verzweigter Aminosäuren, die in diesen Stadien vom Wirt in hoher Menge benötigt werden, während die internen Reserven gleichzeitig zur Neige gehen. Dies äußert sich auch im deutlichen Abfallen der Speicherproteinmenge des Wirts gegen Ende der Puppenphase. Die festgestellte Veränderung der Symbiontenzahl übertrifft das geringe Ausmaß der Genregulation um ein Vielfaches. Die Bakterien liegen in jedem Stadium polyploid mit bis zu 100 Genomkopien vor, dieser Polyploidiegrad bleibt jedoch während der gesamten Wirtsentwicklung weitestgehend konstant. Somit scheint die Kontrolle des Wirts über die bakterielle Vermehrung der entscheidende Faktor dieser Symbiose zu sein. Die verbleibenden regulatorischen Fähigkeiten der Bakterien stellen möglicherweise eine Feinjustierung von optimierten Produktionseinheiten dar, deren Anzahl nach den Bedürfnissen des Wirtes verändert wird. Insgesamt konnten in der vorliegenden Arbeit neue Einblicke in das komplexe Zusammenleben von Blochmannia und Camponotus gewonnen werden, die zu einem besseren Verständnis der biologischen Funktion und der grundlegenden Mechanismen dieser Symbiose führen. Eine der wichtigsten Fragestellungen nach dem Sinn einer nahrungsaufwertenden Symbiose für einen Nahrungsgeneralisten konnte mit starken Hinweisen auf eine stadienabhängige Relevanz der Symbiose beantwortet werden, die den enormen evolutionären Erfolg dieser Ameisengattung erklären könnte.  N2 - Ants of the genus Camponotus harbor bacterial endosymbionts of the genus Blochmannia in specialized cells of their midgut (Blochmann, 1882; Buchner, 1965; Sauer, 2000; Schröder et al., 1996). The complete sequencing of the symbiont’s genome revealed, that Blochmannia, comparable to the symbionts of aphids, mainly retained genes involved in the biosynthesis of essential amino acids (Degnan et al., 2005; Gil et al., 2003). The biological relevance of a nutritional upgrading by Blochmannia could be confirmed experimentally (Feldhaar et al., 2007). One focus of this thesis was the elucidation of the dynamic interactions between the two partners during the complex life cycle of the holometabolic host animal. Previous studies pointed towards a temporal relevance of this symbiosis especially during larval and pupal development (Feldhaar et al., 2007; Wolschin et al., 2004; Zientz et al., 2006). In this thesis the localization of B. floridanus could be documented throughout all life stages of the host by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy. A layer of densely filled bacteriocytes surrounding the gut could already be identified in first instar larvae. In contrast to previous assumptions, the bacteria are not restricted to these cells in later stages, as until the eclosion of the young adult workers bacteria massively infect other midgut cells. Concordant with previous findings, bacterial load is highest at the end of metamorphosis and symbiont numbers decrease in older workers, yet densely filled bacteriocytes are still visible after several months. The expression of the bacterial genes during characteristic life stages of the C. floridanus was assessed by macroarray and qRT- PCR- based experiments. In general, especially molecular chaperones, central basic metabolism and may putative symbiosis related factors like pathways leading to essential amino acids or nitrogen recycling show highest absolute expression levels. A positive correlation between expression level and GC- content of the genes can be observed, which is caused by a higher selection pressure and lower mutation rate of these essential factors (Schaber et al., 2005). Protein analyses confirmed the correlation between gene expression and translation of the most abundant factors. Many B. floridanus genes exhibit a dynamic expression during the different host stages but the extent of this gene regulation is modest as compared to free living bacteria. Expression profiles of genes located next to each other on the genome allow proposal of local transcription units, which were confirmed experimentally in several cases. Often genes that are not clustered locally but belong to related metabolic functions also exhibit similar expression patterns. This indicates the existence of basic mechanisms of gene regulation despite the low number of transcription factors annotated in the B. floridanus genome (Gil et al., 2003). In late pupal stages symbiosis related genes often show a higher expression compared to basic metabolic functions. This especially includes biosynthetic pathways for aromatic and branched amino acids, which are needed by the host at this stage in increased amounts, while internal storages are depleted. This could be demonstrated by the significant decrease in storage proteins of the host at the end of the pupal phase. The observed change in bacterial numbers per host exceeds the extent of bacterial gene regulation by far. The symbionts are polyploid in each host stage with up to 100 genome copies per cell. The degree of polyploidy is largely constant during host development. Thus the control over bacterial reproduction seems to be the decisive factor in this symbiosis. The residual regulatory capacities of the symbionts might represent a mechanism of fine tuning of a production unit that has been streamlined by evolution and whose numbers are adjusted according to the host’s needs. In conclusion, this thesis delivers new insights into the complex symbiosis of Blochmannia and Camponotus leading to a better understanding of its biological function and the underlying mechanisms. One of the central mysteries concerning the need of a symbiont for nutritional upgrading for an omnivorous host could be explained by a temporal, stage- dependent relevance of this symbiosis, possibly being the reason for the enormous evolutionary success of this ant genus. KW - Intrazelluläre Symbiose KW - Symbiose KW - Ameisen KW - Mikrobiologie KW - Gram-negative Bakterien KW - Bakterien KW - Differentielle Genexpression KW - Genexpression KW - Entwicklung KW - Blochmannia KW - Camponotus KW - symbiosis KW - endosymbiosis KW - ants KW - bacteria KW - gene expression Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-37238 ER - TY - THES A1 - Stoll, Regina T1 - Einfluss der Phosphoenolpyruvat-Phosphotransferasesysteme auf die Aktivität des Virulenzgenregulators PrfA von Listeria monocytogenes T1 - Impact of the phosphoenolpyruvate phosphotransferasesystems on the activity of the virulence gene regulator PrfA of Listeria monocytogenes N2 - Die PrfA-Aktivität im L. monocytogenes Stamm EGD sowie dessen prfA Deletionsmutante mit dem prfA- bzw. prfA*-Gen unter Kontrolle des prfA-Promotors auf dem High-Copy Plasmid pERL3 wurde nach Wachstum in BHI, LB (Luria-Bertani Medium) und definiertem MM untersucht. Die Medien waren versetzt mit 50 mM der PTS-Kohlenstoffquellen Glucose, Mannose oder Cellobiose oder mit der Nicht-PTS-Kohlenstoffquelle Glycerin. Mit dem Wildtyp EGD konnte in BHI und LB mit allen genannten Kohlenstoffquellen nur eine geringe PrfA-Aktivität beobachtet werden. In MM dagegen war die PrfA-Aktivität in Anwesenheit von Glycerin stark erhöht und mit Cellobiose als einziger Kohlenstoffquelle stark reprimiert. Mit dem PrfA*-überexprimierenden Stamm wurden unter allen Bedingungen hohe PrfA-Aktivität gefunden. EGDΔprfApPrfA zeigte dagegen trotz gleicher PrfA-Menge wie EGDΔprfApPrfA* nur in BHI eine hohe PrfA-Aktivität. Die Zugabe des Amberlites XAD4 in LB erhöht die reduzierte PrfA-Aktivität in EGDΔprfApPrfA und in MM verstärkt XAD4-Zugabe die PrfA-Aktivität des Wildtyps. Eine ptsH-Mutante ist in LB und MM unabhängig von der Zugabe einer der vier Kohlenstoffquellen nicht in der Lage zu wachsen (Stoll et al., 2008), was darauf hin deutet, dass die Aufnahme der verwendeten Kohlenstoffquelle und auch der Glycerinstoffwechsel von einem intakten PTS-Weg abhängig sind. In BHI stehen dagegen offensichtlich noch PTS-unabhängige Kohlenstoffquellen zur Verfügung, da die ptsH-Mutante in BHI noch wachsen kann. Dies unterstützt auch die Beobachtung, dass die Generationszeiten von L. monocytogenes in LB und vor allem MM im Vergleich zu BHI wesentlich länger sind. Expressionsdaten der PTS-Gene wurden von allen drei Stämmen unter verschiedenen Wachstumsbedingungen erstellt. Die Daten deuten darauf hin, dass die PrfA-Aktivität mit der Expressionsstärke und dem Phosphorylierungsstatus bestimmter PTS-Permeasen zusammenhängt. PTS-Permeasen bestehen immer aus mindestens drei Domänen, der Membran überspannenden Zucker transportierenden Domäne EIIC (und EIID im Falle von Mannose spezifischen PTS) und den zwei im Zytosol löslichen Komponenten EIIA und EIIB. EIIA wird direkt von HPr-His-P phosphoryliert, welches sein Phosphat von dem von PEP phosphorylierten EI empfängt. Das PTS spielt neben der Zuckeraufnahme eine Rolle in vielen regulatorischen Vorgängen in der Bakterienzelle, unter anderem in der Pathogenese (Barabote and Saier, 2005; Deutscher et al., 2006; Postma et al., 1993). Listerien codieren für alle sieben bekannten PTS-Familien, 86 Gene codieren für 29 komplette und einige unvollständige PTS. Trotz der großen Anzahl an PTS-Genen besitzt L. monocytogenes kein vollständiges PtsG, welches homolog zu E. coli oder B. subtilis ist, sondern nur ein EIIAGlc. Um die an der Glucoseaufnahme involvierten PTS-Permeasen zu identifizieren und einen möglichen Zusammenhang zwischen diesen PTS-Permeasen und der PrfA-Aktivität zu untersuchen, wurden in dieser Arbeit systematisch PTS-Permeasen deletiert, welche für putative Beta-Glucosid-PTS (PTSGlc), Mannose-PTS (PTSMan) und Cellobiose-PTS (PTSLac) codieren. Diese Deletionsmutanten wurden bezüglich ihres Wachstumes in Gegenwart der entsprechenden PTS-Zucker und die PrfA-Aktivität untersucht. Deletionen von in L. monocytogenes EGD-e nur schwach exprimierten PTSGlc haben keinen Einfluss auf das Wachstum in MM mit 10 mM Glucose oder Cellobiose. Von den vier exprimierten PTSMan sind zumindest zwei eindeutig in der Lage, Glucose zu transportieren, und die Deletion dieser PTS-Permeasen, codiert von lmo0096-0098 und lmo0781-0784, erhöht sehr deutlich die Expression des im Wildtyp wenig exprimierten Gens für die PTS-Permease PTSGlc(lmo0027). Für den Cellobiose-Transport scheint von den sechs vollständigen PTSLac-Permeasen vor allem PTSLac(lmo2683-2685) und nach Deletion dieses Operons, ebenfalls die PTSGlc(lmo0027)-Permease wichtig zu sein. Obwohl die multiple Deletion dieser für die Glucose/Mannose- bzw. Cellobiose-Aufnahme in L. monocytogenes wichtigen PTS-Permeasen das Wachstum in definiertem MM drastisch reduziert, haben diese Deletionen offensichtlich keine Auswirkung auf das intrazelluläre Wachstum, da die Infektionsrate so effizient ist wie die des Wildtyps. Auf PrfA hat die schrittweise Deletion der Glucose/Mannose-spezifischen PTS-Permeasen nach Wachstum in MM mit Glucose als einziger Kohlenstoffquelle eine aktivierende Wirkung, jedoch keine Auswirkung nach Wachstum in Cellobiose-haltigem MM. Umgekehrt verhält es sich mit den PTSLac-Deletionsmutanten. In vitro Transkriptionsstudien mit (teilweise phosphoryliert) aufgereinigten Lmo0096 (EIIABMan) und Lmo1017 (EIIAGlc) -Proteinen deuten auf eine direkte Interaktion zwischen PrfA und bestimmten EII-Proteinen hin. Dies konnte für Lmo0096 auch in Immunpräzipitationsassays gezeigt werden. Eine Überexpression von Lmo0096 führte zudem zu einer sehr deutlichen Reduktion der PrfA-Aktivität nach Wachstum in MM mit Glucose. N2 - In this study the PrfA activity was assessed in L. monocytogenes strain EGD and its isogenic deletion mutant (EGDΔprfA) with the prfA or prfA* gene under the control of the prfA promoter located on the high copy plasmid pERL3 (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in BHI, LB (Luria-Bertani broth) and defined minimal medium. Media were supplemented with 50 mM of the PTS carbon sources glucose, mannose or cellobiose or with the non-PTS carbon source glycerol. In the wild type EGD grown in BHI and LB a low PrfA activity was observed with all of the above carbon sources. In MM PrfA activity was strongly increased in the presence of glycerol and strongly decreased with cellobiose as sole carbon source. In the PrfA* overexpressing strain EGDΔprfApPrfA* high PrfA activity was detected under all conditions. EGDΔprfApPrfA exhibited a high activity only in BHI, though PrfA amounts were equally high as in EGDΔprfApPrfA*. Addition of the amberlite XAD4 to LB increases the reduced PrfA activity in EGDΔprfApPrfA and the activity of the wildtype in MM. A ptsH mutant is unable to grow in LB and MM irrespective of the supplementation with the four carbon sources (Stoll et al., 2008), indicating that the uptake of the carbon source used as well as the glycerol metabolism are dependent on an intact PTS pathway. In contrast, BHI obviously possesses PTS independent carbon sources, as the ptsH mutant is still able to grow in BHI. This is also confirmed by the fact that L. monocytogenes generation times are significantly longer in LB and even more in MM as compared to BHI. Expression of the PTS genes was assessed in all three strains upon different growth conditions. The data suggest that PrfA activity is correlated with the expression level and the phosphorylation state of specific PTS permeases. PTS permeases always consist of at least three domains, the membrane crossing sugar transporting domain EIIC (and EIID in mannose specific PTS) and the two cytosolic components EIIA and EIIB. EIIA is directly phosphorylated by HPr-His-P which receives its phosphate group from EI which is phosphorylated by PEP. Aside from sugar transport PT Systems are involved in a variety of regulatory processes in the bacterial cell, e.g. in pathogenesis (Barabote and Saier, 2005; Deutscher et al., 2006; Postma et al., 1993). Listeria code for all of the seven known PTS families with 86 genes coding for 29 complete and several incomplete PTS. Despite the large number of PTS genes L. monocytogenes does not possess a complete PtsG homologue to E. coli or B. subtilis but only an orphan EIIAGlc. To identify the PTS permeases involved in glucose uptake and to investigate a possible role in PrfA regulation, mutants with deletions of beta glucoside PTS (PTSGlc), mannose PTS (PTSMan) and cellobiose PTS (PTSLac) permeases have been analyzed systematically in this study. These deletion mutants were analyzed in respect to their growth upon the respective PTS sugars and to their PrfA activity. Deletion of the five PTSGlc permeases only weakly expressed in L. monocytogenes EGD-e had no impact on growth in MM with 10 mM glucose or cellobiose. At least two out of the four expressed PTSMan permeases are able to transport glucose and the deletion of these PTS (encoded by lmo0096-0098 and lmo0781-0784) causes a significant increase in expression of the PTSGlc(lmo0027) permease, which is expressed at a low level in the wildtype. For transport of cellobiose, only PTSLac(lmo2683-2685) out of the six complete PTSLac permeases and, after deletion of this operon, PTSGlc(lmo0027) seem to be of importance. Although multiple deletions of the PTS important for glucose/mannose and cellobiose uptake have severe consequences on growth in defined MM, obviously intracellular life is not affected, as infection rates resemble those of the wild type. PrfA is activated by the stepwise deletion of the glucose/mannose specific PTS upon growth in MM supplemented with glucose, but no effect is seen upon growth in cellobiose supplemented MM. The behavior of the PTSLac deletion mutants is conversely. In vitro transcription studies with (partially phosphorylated) purified Lmo0096 (EIIABMan) and Lmo1017 (EIIAGlc) proteins suggest a direct interaction between PrfA and specific EII proteins. This could be confirmed for Lmo0096 in immuno precipitation assays. An overexpression of lmo0096 lead to a significant reduction of PrfA activity upon growth in MM supplemented with glucose. KW - Listeria monocytogenes KW - Phosphotransferasesystem KW - Virulenz KW - PrfA KW - PrfA Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-32072 ER - TY - THES A1 - Stoll, Georg T1 - Identification of the mRNA-associated TOP3β- TDRD3-FMRP (TTF) -complex and its implication for neurological disorders T1 - Identifikation des mRNA-assoziierten TOP3β-TDRD3-FMRP (TTF) -Komplex und seine Bedeutung für neurologische Störungen N2 - The propagation of the genetic information into proteins is mediated by messenger- RNA (mRNA) intermediates. In eukaryotes mRNAs are synthesized by RNA- Polymerase II and subjected to translation after various processing steps. Earlier it was suspected that the regulation of gene expression occurs primarily on the level of transcription. In the meantime it became evident that the contribution of post- transcriptional events is at least equally important. Apart from non-coding RNAs and metabolites, this process is in particular controlled by RNA-binding proteins, which assemble on mRNAs in various combinations to establish the so-called “mRNP- code”. In this thesis a so far unknown component of the mRNP-code was identified and characterized. It constitutes a hetero-trimeric complex composed of the Tudor domain-containing protein 3 (TDRD3), the fragile X mental retardation protein (FMRP) and the Topoisomerase III beta (TOP3β) and was termed TTF (TOP3β-TDRD3-FMRP) -complex according to its composition. The presented results also demonstrate that all components of the TTF-complex shuttle between the nucleus and the cytoplasm, but are predominantly located in the latter compartment under steady state conditions. Apart from that, an association of the TTF-complex with fully processed mRNAs, not yet engaged in productive translation, was detected. Hence, the TTF-complex is a component of „early“ mRNPs. The defined recruitment of the TTF-complex to these mRNPs is not based on binding to distinct mRNA sequence-elements in cis, but rather on an interaction with the so-called exon junction complex (EJC), which is loaded onto the mRNA during the process of pre-mRNA splicing. In this context TDRD3 functions as an adapter, linking EJC, FMRP and TOP3β on the mRNP. Moreover, preliminary results suggest that epigenetic marks within gene promoter regions predetermine the transfer of the TTF-complex onto its target mRNAs. Besides, the observation that TOP3β is able to catalytically convert RNA-substrates disclosed potential activities of the TTF-complex in mRNA metabolism. In combination with the already known functions of FMRP, this finding primarily suggests that the TTF-complex controls the translation of bound mRNAs. In addition to its role in mRNA metabolism, the TTF-complex is interesting from a human genetics perspective as well. It was demonstrated in collaboration with researchers from Finland and the US that apart from FMRP, which was previously linked to neurocognitive diseases, also TOP3β is associated with neurodevelopmental disorders. Understanding the function of the TTF-complex in mRNA metabolism might hence provide important insight into the etiology of these diseases. N2 - Die Umwandlung der genetischen Information in Proteine erfolgt über Boten-RNA (mRNA) -Intermediate. Diese werden in Eukaryonten durch die RNA-Polymerase II gebildet und nach diversen Prozessierungs-Schritten der Translationsmaschinerie zugänglich gemacht. Während man früher davon ausging, dass die Genexpression primär auf der Ebene der Transkription reguliert wird, ist heute klar, dass post- transkriptionelle Prozesse einen ebenso wichtigen Beitrag hierzu leisten. Neben nicht-kodierenden RNAs und Metaboliten tragen insbesondere RNA- Bindungsproteine zur Kontrolle dieses Vorgangs bei. Diese finden sich in unterschiedlichen Kombinationen auf den mRNAs zusammen und bilden dadurch den sog. „mRNP-Code“ aus. Im Rahmen dieser Dissertation wurde eine bislang unbekannte Komponente des mRNP-Codes identifiziert und charakterisiert. Es handelt es sich dabei um einen hetero-trimeren Komplex, welcher aus dem Tudor Domänen Protein 3 (TDRD3) dem Fragilen X Mentalen Retardations-Protein (FMRP) sowie der Topoisomerase III beta (TOP3β) besteht. Aufgrund seiner Zusammensetzung wurde dieser TTF (TOP3β-TDRD3-FMRP) -Komplex genannt. In der vorliegenden Arbeit konnte der Nachweis geführt werden, dass sämtliche Komponenten des TTF-Komplexes zwischen Zellkern und Cytoplasma pendeln, unter Normalbedingungen jedoch vornehmlich im Cytoplasma lokalisiert sind. Des Weiteren ließ sich eine Assoziation des TTF-Komplexes mit mRNAs nachweisen, die zwar vollständig prozessiert, jedoch noch nicht Teil der produktiven Phase der Translation sind. Der TTF-Komplex ist somit eine Komponente „früher“ mRNPs. Die Rekrutierung des TTF-Komplexes an definierte mRNPs wird nicht durch Bindung an spezifische mRNA-Sequenzelemente bedingt, sondern basiert auf einer Interaktion mit dem sog. Exon Junction Complex (EJC), welcher im Kontext des pre-mRNA Spleißens auf die mRNA geladen wird. Hierbei spielt TDRD3 als Adapter zwischen dem EJC, FMRP und TOP3β die entscheidende Rolle. Präliminäre Experimente legen darüber hinaus den Schluss nahe, dass epigenetische Markierungen im Promotor-Bereich distinkter Gene von entscheidender Bedeutung für den Transfer des TTF-Komplexes auf dessen Ziel-mRNAs sind. Einen wichtigen ersten Hinweis auf die potentielle Funktion des TTF-Komplexes im Kontext des mRNA Metabolismus erbrachte die Beobachtung, dass TOP3β in der Lage ist RNA katalytisch umzusetzen. Dieser Befund lässt in Verbindung mit den bereits beschriebenen Aktivitäten von FMRP vermuten, dass der TTF-Komplex die Translation gebundener mRNAs kontrolliert. Zusätzlich zu seiner Rolle im mRNA Metabolismus ist der TTF-Komplex auch aus humangenetischer Sicht hoch interessant. So konnte in Zusammenarbeit mit finnischen und US-amerikanischen Forschern gezeigt werden, dass neben FMRP, einem bekannten Krankheitsfaktor neurokognitiver Syndrome, auch TOP3β mit neurologischen Entwicklungsstörungen assoziiert ist. Das Verständnis der Funktion des TTF-Komplexes im mRNA Metabolismus könnte daher wichtige Einblicke in die Etiologie dieser Krankheiten liefern. KW - Messenger-RNS KW - Messenger-RNP KW - RNA binding proteins KW - mRNA metabolism KW - eukaryotic gene expression Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111440 ER - TY - THES A1 - Stojic, Jelena T1 - Cloning and functional characterization of novel genes expressed preferentially in the human retina N2 - The human retina is a multi-layered neuronal tissue specialized for the reception and processing of visual information. The retina is composed of a great diversity of neuronal cell types including rod and cone photoreceptors, bipolar cells, ganglion cells, amacrine cells, horizontal cells and Müller glia. In response to light, a coordinated series of molecular events, the so-called phototransduction cascade, is triggered in photoreceptor cells and the signals from the photoreceptors are further processed by the bipolar and ganglion cells to the higher centers of the brain. The retina as highly complex system may be greatly susceptible to genetic defects which can lead to a wide range of disease phenotypes. Therefore, isolation and characterisation of the genes active in the human retina will facilitate our deeper understanding of retinal physiology and mechanisms underlying retinal degeneration and provide novel candidates for the retinal disease genes. To identify novel genes that are specifically or predominantly expressed in the human retina, a cDNA library enriched for retina specific transcripts was generated using suppression subtractive hybridization (SSH) technique. In total, 1113 clones were randomly isolated from the retina SSH cDNA library and partially sequenced. On the basis of BLASTN algorithm analysis these clones were classified into four categories including those with I) significant homology to known human genes (766/1113), II) significant homology to partial transcripts and hypothetical gene predictions (162/1113), III) no homology to known mRNAs (149/1113), and IV) vector sequences and clones derived from mitochondrial genes (36/1113). After correcting for redundancy, category I represented 234 known human genes and category II a total of 92unknown transcripts. Clones from category I, were selected for expression analysis by RT-PCR in a great number of human tissues. This resulted in the identification of 16 genes which were expressed exclusively in the retina, 13 which were highly expressed in the retina compared to other tissues, 12 genes which were specifically expressed in neuronal tissues and 48 ubiquitously expressed genes. Thus, our expression analysis resulted in the identification of 29 genes exclusively or abundantly transcribed in the human retina. Of those, retina specific genes L25,L33, L35, L37, L38 and L40 were selected for further analysis. To characterize the complete mRNA sequences of these transcripts a full-length human retina cDNA library was constructed. The analysis of the L25 gene revealed three splicing variants of the ABCC5 gene, consequently named ABCC5_SV1 (SV1), ABCC5_SV2 (SV2) and ABCC5_SV3 (SV3).These isoforms comprise the first five exons of ABCC5 and additional novel exons named 5a, 5b and 5c, generated by differential exon usage. The determined lengths of the three transcripts are 2039 bp, 1962 bp, and 1887 bp in size, respectively. RT-PCR, real-time PCR and Northern blot analysis of ABCC5 as well as the isoforms SV1, SV2 and SV3demonstrated high levels of expression for all transcripts in the retina compared to other tissues. Analysis of their nucleotide sequences revealed that inclusion of exon 5a in splicing variant SV1 produced a frame shift and premature termination codon (PTC). Our data show that this splice variant is the target of nonsense mediated mRNA decay (NMD). This was shown by inhibition of protein synthesis with antibiotics puromycin and anisomycin in human cell lines A-RPE 19 and Y79. Our analysis resulted in an increase of the PTC containing transcript and a decrease of the ABCC5 transcript. Conversely, the amount of both transcripts (SV1 and ABCC5) returned to pre-treatment levels after removal of the inhibitors. Together, our results suggest that alternative splicing of the ubiquitously expressed ABCC5 gene in addition to NMD is involved in retina-specific transcriptional regulation of the mRNA level of ABCC5. In contrast, additional experiments demonstrated that the levels of expression ofSV2 and SV3 isoforms do not appear to influence ABCC5 transcription. Several of the cloned genes were selected for additional genotyping of single nucleotide polymorphisms (SNPs) in order to construct their SNP maps which are going to be used for future association studies of complex disease AMD. Thus, identification of novel retinal genes and their functional characterization will further our elucidation of retinal physiology in general and in the diseased state in particular, by providing candidate retinal disease genes. N2 - Die menschliche Retina ist ein vielschichtiges neuronales Gewebe, das auf die Aufnahme und Verarbeitung visueller Informationen spezialisiert ist. Sie besteht aus einergroßen Vielfalt neuronaler Zelltypen, inklusive der Stäbchen und Zapfen (Photorezeptoren), Bipolar- und Ganglionzellen, Amakrinen Zellen sowie Horizontalzellen und Müllerglia. Als Antwort auf einen Lichteinfall wird eine koordinierte Serie von molekularen Ereignissen, die so genannte Phototransduktionskaskade, in den Photorezeptoren ausgelöst, die von den Photorezeptoren kommenden Signale von den Bipolar- und Ganglionzellen weiterverarbeitetund an höhere Hirnzentren gesandt. Als äußerst komplexes System kann die Retina hochanfällig für genetische Defektesein, die zu einem breiten Spektrum an Krankheitsphänotypen führen können. Deshalb wird die Isolation und Charakterisierung von in der Retina aktiven Genen unser tieferes Verständnis für die Physiologie der Netzhaut, sowie den einer Retinadegeneration zu Grundeliegenden Mechanismen, erleichtern. Um neue Gene, die spezifisch oder überwiegend in der Netzhaut exprimiert werden, identifizieren zu können wurde, unter Nutzung der subtractive hybridization technique (SSH) eine cDNA Bibliothek, angereichert mit retinaspezifischen Genen, generiert. Insgesamt wurden 1113 Klone zufällig aus der Retina SSH cDNA Bibliothek ausgewählt und teilweise sequenziert. Auf der Basis einer BLASTN Algorithmus Analyse wurden diese Klone in 4 Kategorien eingeteilt, wobei I) Klone mit signifikanten Homologien zu bekannten menschlichen Genen (766/1113) beinhaltet, II) Klone mitsignifikanten Homologien zu partiellen Transkripten und hypothetischen Genvorhersagen (162/1113), III) Klone ohne Homologie zu bekannten mRNAs (149/1113) und IV)Vektorsequenzen und Klone, erhalten von mitochondrialen Genen (36/1113). Nach Redundanzkorrektur repräsentierte Kategorie I 234 bekannte menschliche Gene undKategorie II insgesamt 92 unbekannte Transkripte. Klone der Kategorie I wurden für eine Expressionsanalyse mittels RT-PCR in einer Vielzahl menschlicher Gewebe ausgewählt. Dies führte zur Identifikation von 16 exklusiv inder Retina exprimierten Genen, weiteren 13 mit, im Vergleich zu anderen Geweben, hoher Expression in der Netzhaut, 12 Genen, die spezifisch in neuronalen Geweben exprimiertwerden, sowie 48 ubiquitär exprimierten Genen. Somit resultierte unsere Expressionsanalysen der Identifikation von 29 exklusiv oder reichlich in der menschlichen Retina transkribierten Genen. Aus diesen wurden die retinaspezifischen Gene L25, L33, L35, L38 und L40 für weitere Analysen ausgewählt. Um die kompletten mRNA Sequenzen dieser Transkripte zucharakterisieren wurde eine full-length human retina cDNA Bibliothek konstruiert. Die Analyse des L25 Genes erbrachte 3 Splicevarianten des ABCC5 Genes, die konsequent mit ABCC5_SV1 (SV1), ABCC5_SV2 (SV2) und ABCC5_SV3 (SV3) benannt wurden. Diese Isoformen bestehen aus den ersten 5 Exonen von ABCC5 und zusätzlichenneuen 5a, 5b und 5c genannten Exonen, die durch unterschiedliche Exonnutzung entstehen. Die ermittelten Längen der drei Transkripte sind 2039 bp, 1962 bp, und 1887 bp. RT-PCR, real time PCR und Northern Blot Analyse des ABCC5 Genes sowie der Isoformen SV1, SV2und SV3, konnten hohe Expressionslevel all dieser Transkripte in der Retina im Vergleich zu anderen Geweben zeigen. Die Analyse ihrer Nukleotidsequenzen enthüllte, dass der Einschluss von Exon 5a in Splicevariante SV1 eine Leserahmenverschiebung sowie die Entstehung eines premature termination Kodons (PTC) bewirkt. Unsere Daten zeigen, dass diese Splicevariante Ziel eines nonsense mediated mRNA decay (NMD) ist. Dies wurde durch Hemmung der Proteinsynthese mittels Gabe der Antibiotika Puromycin und Anisomycin in den humanen Zelllinien ARPE-19 und Y79 gezeigt. Es kam bei diesem Versuch zu einem Anstieg des PTC enthaltenden Transkripts und einem Abfall des ABCC5Transkripts. Umgekehrt ging die Menge beider Transkripte (SV1 und ABCC5) nach Wegnahme der Inhibitoren auf die Ausgangswerte vor Antibiotikagabe zurück. Zusammengefasst weisen unsere Ergebnisse darauf hin, dass alternatives Splicing des ubiqitärexprimierten ABCC5 Genes zusätzlich zu NMD in der retinaspezifischen Transkriptionsregulation der mRNA Level des ABCC5 Genes involviert ist. Im Gegensatzdazu zeigten zusätzliche Experimente, dass die Expressionslevel der SV2 und SV3 Isoformendie ABCC5 Transkription scheinbar nicht beeinflussen. Einige dieser klonierten Gene wurden zur zusätzliche Genotypisierungen ihrer single nucleotide Polymorphismen (SNPs) ausgewählt, mit dem Ziel SNP Karten zu erstellen, die für spätere Assoziationsstudien komplexer AMD Erkrankungen verwendet werden sollen.Auf diese Weise wird die Identifizierung neuer retinaler Gene sowie deren Funktionscharakterisierung die Aufklärung der Netzhautphysiologie im Allgemeinen und deren krankhafter Veränderungen im besonderen fördern, indem sie Kandidatengene für Retinaerkrankungen zu Verfügung stellt. KW - Netzhaut KW - cDNS KW - Senile Makuladegeneration KW - Genanalyse KW - retina KW - AMD KW - cDNA library KW - NMD KW - ncRNA genes Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-13746 ER -