TY - JOUR A1 - Ono, Mitsuaki A1 - Sonoyama, Wataru A1 - Nema, Kazuki A1 - Hara, Emilio Satoshi A1 - Oida, Yasutaka A1 - Pham, Hai Thanh A1 - Yamamoto, Katushi A1 - Hirota, Kazuo A1 - Sugama, Kazushige A1 - Sebald, Walter A1 - Kuboki, Takuo T1 - Regeneration of calvarial defects with Escherichia coli-derived rhBMP-2 adsorbed in PLGA membrane JF - Cells Tissues Organs N2 - Objective: Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E-BMP-2) has been shown to be as effective as mammalian cell-derived BMP-2. However, several in vitro and in vivo experiments are still necessary to validate the effectiveness of E-BMP-2 due to the difference in synthesis process, mainly related to protein nonglycosylation. The objective of this study was to investigate whether biodegradable polylactide-co-glycolide (PLGA) membrane is a suitable carrier for E-BMP-2 delivery for bone regeneration of critical-sized defects in rat calvaria. Materials and Methods: First, the osteoinductive effect of E-BMP-2 was confirmed in vitro in mouse bone marrow stromal cells by analysis of osteocalcin mRNA levels, and calcium deposition was detected by alizarin red staining. Before in vivo experiments, the release profile of E-BMP-2 from PLGA membranes was determined by ELISA. E-BMP-2 (0, 1, 5 and 10 μg/μl) was applied for ectopic and orthotopic bone formation and was analyzed by X-ray, micro-CT and histology. Results: Release-profile testing showed that PLGA membrane could retain 94% of the initially applied E-BMP-2. Ectopic bone formation assay revealed that combination of E-BMP-2/PLGA membrane strongly induced bone formation. Stronger osteoinductivity with complete repair of critical-sized defects was observed only with PLGA membranes adsorbed with 5 and 10 μg/μl of E-BMP-2, whereas no bone formation was observed in the groups that received no membrane or 0-μg/μl dose of E-BMP-2. Conclusion: PLGA membrane was shown to be a suitable carrier for sustained release of E-BMP-2, and the E-BMP-2/PLGA membrane combination was demonstrated to be efficient in bone regeneration in a model of critical-sized defects. KW - Escherichia coli-derived recombinant human bone morphogenetic protein-2 KW - Bone regeneration KW - Polylactide-co-glycolide KW - Ectopic bone formation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-196680 SN - 1422-6405 SN - 1422-6421 N1 - This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. VL - 198 IS - 5 SP - 367 EP - 376 ER - TY - THES A1 - Fischer, Peter T1 - Untersuchungen zum Einfluss der Anzahl primordialer Keimzellen auf die Geschlechtsbestimmung von Medaka, Oryzias latipes T1 - Investigations on the influence of Primordial Germ Cell number on the sex determination of Medaka, Oryzias latipes N2 - Die primordialen Keimzellen (PGCs) sind die einzigen Zellen des Embryos, die die genetische Information von einer Generation an die nächste weiter geben können. Es wurde gezeigt, dass in allen bislang untersuchten Knochenfischen die Anzahl der Urgeschlechtszellen während der Embryonalentwicklung der erste sichtbare Unterschied zwischen Männchen und Weibchen ist. Daraus ergibt sich die Frage, ob die Anzahl der primordialen Keimzellen das Geschlecht bestimmt, oder ob die somatischen Zellen je nach sexueller Identität die Urgeschlechtszellen zur Proliferation anregen. Um zu untersuchen, wie die Anzahl der Urgeschlechtszellen mit der Geschlechtsdetermination zusammenhängt, habe ich in dieser Arbeit die Anzahl der Urgeschlechtszellen manipuliert und deren Schicksal im Verlauf der Embryonalentwicklung verfolgt. Weiterhin untersuchte ich, in wieweit die Temperatur einen Einfluss auf die Geschlechtsbestimmung hat und ob sie Auswirkungen auf die Anzahl und die Wanderung der Urgeschlechtszellen hat beim Medaka hat. Durch meine Experimente, in denen ich die Fische während der Embryonalentwicklung bei verschiedenen Temperaturen hielt, konnte ich zeigen, dass beim Medaka der genetische Geschlechtsbestimmungsmechanismus durch erhöhte Temperatur überschrieben werden kann. Die Temperaturerhöhung in der Embryonalentwicklung führt zu einer Weibchen­‐zu­‐Männchen Geschlechtsumkehr. Dabei wird die Anzahl der primordialen Keimzellen im Vergleich zu den Kontrollen reduziert. Zudem wird durch die höhere Temperatur das autosomale dmrt1a viel früher angeschaltet, wa sauf einen alternativenSignalweg deutet, der die männliche Geschlechtsentwicklung in XX geschlechtsumgewandelten Tieren steuert. N2 - Primordial Germ Cells (PGCs) are the only cells, which can transport genetic information from one generation to the next one. In all investigated teleost fish the different number of germ cells is the foist recognizable difference between mal and female. Therefore the question arises, if the number of germ cells is the sex determining mechanism, or if the somatic cells of the gonad stimulate the PGCs to proliferate. To investigate the connection between germ cell number and sex determination in medaka, i manipulated the number of germ calls and followed their fate through embryonic development. Furthermore i investigated the influence of temperature on sex determination and behavior and number of germ cells. I could show, that it is possible to ablate all PGCs by DND morpholino injection. While total absence of PGCs led to infertile male development with string like gonads, increasing the number of PGCs by bucky ball mRNA injection showed no influence on sexual development. Interestingly I observed that even when I increased the number early in embryonic development by more than two-­fold, PGC number after some time went down to the untreated embryo level. This points to a non‐cell autonomous mechanism, which limits PGC number according to the somatic SD process. KW - Geschlechtsbestimmung KW - Gamet KW - Sex determination KW - Sexual development KW - Japankärpfling KW - Gonadenentwicklung Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-106846 ER - TY - THES A1 - Zdzieblo, Daniela T1 - Das Polycomb group Protein PCGF6 ist ein neuer und essentieller Faktor der iPS Reprogrammierung und kann in Kombination mit Oct4, Klf4 und c-Myc den Transkriptionsfaktor Sox2 ersetzen T1 - The Polycomb group protein PCGF6 is a new and essential factor for iPS reprogramming that can replace the transcription factor Sox2 in combination with Oct4, Klf4 and c-Myc N2 - Embryonale Stammzellen (ESCs) sind durch zwei charakteristische Eigenschaften definiert. Neben einer kontinuierlichen Selbsterneuerungskapazität weisen ESCs die Fähigkeit auf, in alle Zelltypen der drei Keimblätter differenzieren zu können. Diese Eigenschaften werden unter anderem durch ein Netzwerk wichtiger Pluripotenzfaktoren als auch durch epigenetische Mechanismen reguliert, welche die Transkription von Pluripotenz- und Differenzierungsgenen kontrollieren. In murinen ESCs sind an der Repression von Differenzierungsgenen auch Polycomb group (PcG) Proteine beteiligt. Diese Proteine bauen zwei Chromatin-modifizierende Komplexe auf, die als Polycomb repressive complex 1 bzw. 2 (PRC1 bzw. PRC2) bezeichnet werden. Nach dem klassischen Modell der Polycombfunktion, katalysieren PRC1 und PRC2 gemeinsam zwei charakteristische Histonmodifikationen, die zur Repression PRC-spezifischer Zielgene beitragen. Zahlreiche Studien in den letzten Jahren belegen, dass der Proteinaufbau der PRC1 Komplexe stark variieren kann, wobei die Familie der Polycomb group RING finger (Pcgf) Proteine eine wichtige Rolle spielt. In diesem Zusammenhang definieren einzelne Pcgf Paraloge (Pcgf1 – 6) verschiedene PRC1 Varianten (PRC1.1 – 1.6), die Komplex-spezifische Bindestellen im Genom aufweisen. Diese Erkenntnisse lassen auf unterschiedliche Mechanismen der PRC1 Varianten und Pcgf Paralog-spezifische Funktionen schließen, die zum jetzigen Zeitpunkt nur wenig erforscht sind. Für manche Pcgf Paraloge sind wichtige Rollen in verschiedenen Stammzelltypen und während der iPS Reprogrammierung bekannt. Pcgf1 (Nspc1), Pcgf2 (Mel18) und Pcgf4 (Bmi1) zeigen eine Funktion in verschiedenen adulten Stammzellen. Pcgf4 spielt darüber hinaus eine wichtige Rolle in der murinen iPS Reprogrammierung. Für Pcgf6 (Mblr) wird eine Pluripotenz-assoziierte Funktion angenommen, denn Pcgf6 ist das einzige Pcgf Paralog, das eine erhöhte Expression in murinen ESCs aufweist, die jedoch im Verlauf der ESC-Differenzierung absinkt. Außerdem zeigen murine Pcgf6 KD ESCs eine verminderte Expression der Pluripotenzgene Oct4, Sox2 und Nanog, eine De-Repression mesodermaler und Testes-spezifischer Gene als auch eine erhöhte Tendenz zur hämatopoetischen Differenzierung. Wie genau Pcgf6 an der Regulation dieser Prozesse in murinen ESCs beteiligt ist, ist nicht bekannt. In der hier vorliegenden Dissertation wurde die Funktion von Pcgf6 in der murinen iPS Reprogrammierung untersucht. Da bereits für Pcgf4 eine Rolle in der Reprogrammierung somatischer Zellen gezeigt wurde und Pcgf6 eine erhöhte Expression in ESCs aufweist, wurde auch für Pcgf6 eine Funktion in der iPS Reprogrammierung angenommen. Zunächst konnte in dieser Arbeit gezeigt werden, dass Pcgf6 während der iPS Reprogrammierung verstärkt exprimiert wird und in iPS Zellen eine ESC-ähnliche Expression aufweist. Darüber hinaus konnte Pcgf6 in Kombination mit Oct4, Klf4 und c-Myc spezifisch den Transkriptionsfaktor Sox2 in der iPS Reprogrammierung ersetzen. Zudem wurden für OPKM-induzierte iPS Zellen charakteristische Eigenschaften pluripotenter Zellen nachgewiesen. Außerdem konnte eine Rolle von Pcgf6 als Enhancer-Faktor für die iPS Reprogrammierung ausgeschlossen werden, da die Überexpression von Pcgf6 zusammen mit den OSKM Faktoren keine additiven Effekte auf die Reprogrammierungseffizienz erzielte. Im Gegensatz dazu führte der Knockdown (KD) von Pcgf6 in embryonalen Mausfibroblasten (MEFs) zu verminderten Effizienzen nach OSKM Reprogrammierung. Darüber hinaus handelte es sich bei der Mehrheit der AP+ Kolonien, die unter Pcgf6 KD Konditionen entstanden, um partiell-reprogrammierte iPS Zellen. Zusammengefasst zeigen die Ergebnisse der hier vorliegenden Arbeit, dass Pcgf6 ein neuer und essentieller Faktor der iPS Reprogrammierung ist, der in Kombination mit Oct4, Klf4 und c-Myc spezifisch den Transkriptionsfaktor Sox2 ersetzen kann. N2 - Embryonic stem cells (ESCs) are characterized by their ability for continuous self-renewal maintaining the undifferentiated cell state and their capacity to generate all differentiated cell types of the three germ layers. Regulators of these characteristics include a protein interaction network of pluripotency-specific factors and epigenetic mechanisms that together control the transcription of pluripotency- and differentiation-associated genes. Among the interaction partners of the pluripotency-specific protein network in murine ESCs are Polycomb group (PcG) proteins. These proteins assemble in two complexes termed Polycomb group repressive complex 1 and 2 (PRC1 and PRC2). In the classical model, PRC1 and PRC2 act hierarchically together in catalyzing two characteristic histone modifications thereby contributing to the repression of PRC-specific target genes like differentiation-associated genes in ESCs. Recently, it has been shown that there are numerous PRC1 variants that differ in their protein composition. In this context, the family of Polycomb group RING finger (Pcgf) proteins (Pcgf1 – 6) defines distinct PRC1 variants (PRC1.1 – 1.6) that exhibit complex-specific genomic binding sites. Together, this indicates diverse mechanisms of PRC1 variants and Pcgf Paralog-specific functions that are not well understood. Pcgf Paralogs are known to play essential roles in various stem cell types and during iPS reprogramming. Pcgf1 (Nspc1), Pcgf2 (Mel18) and Pcgf4 (Bmi1) function in diverse adult stem cells. Further, Pcgf4 plays a role in murine iPS Reprogramming. For Pcgf6 (Mblr), a pluripotency-associated function is assumed. Pcgf6 is the only Pcgf paralog with an elevated expression in murine ESCs and when ESCs differentiate Pcgf6 expression decreases. In addition, following Pcgf6 KD in ESCs the expression of Oct4, Sox2 and Nanog declines while mesodermal and testes-specific genes become de-repressed. Furthermore, Pcgf6 KD ESCs are more prone for hematopoietic lineage differentiation. The precise mechanisms by which Pcgf6 controls these processes in murine ESCs are not known. In this work, I investigated the function of Pcgf6 in murine iPS reprogramming. I assumed a role for Pcgf6 in iPS reprogramming because it is highly expressed in ESCs and it was recently shown that Pcgf4 plays a role in the reprogramming of somatic cells. The data of my work show that Pcgf6 expression is increased in iPS reprogramming and that Pcgf6 exhibits an ESC-like gene expression pattern in iPS cells. Furthermore, Pcgf6 was able to replace the transcription factor Sox2 in combination with Oct4, Klf4 and c-Myc. In addition, OPKM-induced iPS cells showed pluripotency-specific characteristics. The overexpression of Pcgf6 together with the OSKM factors did not result in significantly increased reprogramming efficiencies indicating that Pcgf6 does not function as an enhancer-factor. However, Pcgf6 knockdown (KD) in mouse embryonic fibroblasts (MEFs) resulted in decreased efficiencies after iPS reprogramming. Additionally, the majority of AP+ colonies formed after OSKM reprogramming of Pcgf6 KD MEFs represented partially reprogrammed iPS cells, as they did not exhibit ESC-like morphologies and reduced expression levels of Oct4, Sox2 and Nanog. Together, the data of my work show that Pcgf6 is a new and essential factor in iPS reprogramming that can specifically replace the transcription factor Sox2 in combination with Oct4, Klf4 and c-Myc. KW - Stammzelle KW - iPS Reprogrammierung KW - Differenzierung KW - Repression KW - Reprogramming Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-106870 ER - TY - THES A1 - Kaiser, Dorkas T1 - Termites and ants in BURKINA FASO (WEST AFRICA): taxonomic and functional diversity along land-use gradients; ecosystem services of termites in the traditional ZAÏ SYSTEM T1 - Termiten und Ameisen in Burkina Faso (West Africa): Taxonomische und funktionelle Diversität entlang Landnutzungs-Gradienten, Ökosystemdienste von Termiten im traditionellen Zai system N2 - The consequences of habitat change for human well-being are assumed to be especially extreme in Burkina Faso. The country is located in a highly drought-sensitive zone of West Africa, and small‐scale subsistence farmers may be especially affected if losses of biodiversity lead to changes in ecosystem functioning; many depend on more or less degraded lands for agricultural production. The overall aim of the present thesis consequently was to characterize the functional traits of soil-organisms which are crucial for a productive and balanced soil environment in the study region – termites and ants. They are true ecosystem engineers whose activity alters the habitat. Through soil-turnover in the course of constructing biogenic structures of varying size and nature (mounds, nests, galleries, soil-sheetings, foraging-holes), they bioturbate huge amounts of soil masses and exert massive effects on soil structure, positively influencing the fertility, stability, aeration and water infiltration rate into soils; and they provide habitats for other species. In sub-Saharan Africa, ants and termites are the only active soil macrofauna during the long dry season; in the sub-Sahel zone of Burkina Faso, termites even represent the only active, quantitatively remarkable decomposers all year round. Since no information was available about the actual diversity of the focal arthropods, I divided the thesis in two main parts: In the first part, a baseline study, I assessed the local termite and ant fauna, and investigated their quantitative and qualitative response to changing habitat parameters resulting from increasing human impact (‘functional response traits’). In the second and applied part, I addressed the impact of the biogenic structures which are important for the restoration of degraded soils (‘functional effect traits’). Two traditional agricultural systems characteristic for the study region were selected. Each system represented a land-use intensification gradient comprising four distinct habitats now differing in the magnitude of human intervention but formerly having the same initial state. The first disturbance gradient, the temporal cross-section of a traditional soil water conservation technique to restore degraded heavily encrusted, barren soil named Zaï in Ouahigouya (Yatenga province, sub-Sahel zone); the second disturbance gradient, an agriculture type using crop rotation and fallow as nutrient management techniques near Fada N’Gourma (Gourma province, North-Sudanese zone). No standard protocol existed for the assessment of termite and ant diversity in semi-arid (agro-) ecosystems; two widely accepted standard protocols provided the basis for the newly revised and combined rapid assessment protocol ‘RAP’: the ALL protocol for leaf litter ants of Agosti and Alonso (2000), and the transect protocol for termites in tropical forests of Jones and Eggleton (2000). In each study site, three to four replicate transects were conducted during the rainy seasons (2004—2008). The RAP-protocol turned out to be very effective to characterize, compare and monitor the taxonomic and functional diversity of termites and ants; between 70% and 90% of the estimated total species richness were collected on all levels (transects, habitats, regions). Together in both regions, 65 ant species (25 genera) and 39 termite species (13 genera) were collected. These findings represent the first records for Burkina Faso. The data indicate a high sensitivity of termites and ants to land-use intensification. The diversity strongly decreased with increasing anthropogenic impact in the North-Sudan region. In total, 53 ant species (23 genera) and 31 termite species (12 genera) were found. Very promising results concerning the recovery potential of the soil-arthropods’ diversity were gathered in the Zaï system. The diversity of both taxa strongly increased with increasing habitat rehabilitation – in total, 41 ant species (16 genera) and 33 termite species (11 genera) were collected. For both taxa significant differences could be noted in the shape of the density variations along the gradient. For instance termites: Fungus-growers showed the greatest adaptability to different management practices. The greatest variations between the habitats were observed in soil and grass-feeding termites. Whole functional groups were missing in heavily impacted habitats, e.g. soil-, grass-, and wood-feeders were absent in the degraded site in the sub-Sahel zone. Several environmental parameters could be identified which significantly explained a great part of the variations in the composition of the arthropods’ communities; they indicate the importance of the habitats’ structural complexity (vegetation structure) and concomitant effects on diurnal temperature and moisture fluctuations, the availability of food sources, and the soil-structure. The diversity of termites in the sub-Sahel region was strongly correlated with the crown-cover percentages, the topsoils’ sand-content, and the availability of litter; in the North-Sudan region with the cumulated woody plant basal area, the topsoils’ clay- and organic matter-content. The parameters identified for ant communities in the Zaï system, were the height of trees, the topsoils’ clay-content and air humidity; in the North-Sudan region the habitats’ crown-cover percentages, the quantity of litter and again the height of trees. In the second part of the thesis, I first rapidly assessed the (natural) variations in the amount of epigeal soil-structures along the two disturbance gradients in order to judge the relative importance of termites and ants for soil-turnover. The results illustrated impressively that a) in all study sites, termites were the main bioturbators while ant structures were of minor importance for soil turn-over; b) earthworms and grass-feeding termites contributed significantly to soil turn-over in the more humid North-Sudan region; and c) the bioturbated soil mass varied between seasons and years, however, the relative importance of the different taxa seemed to be fairly constant. In the sub-Sahel zone, fungus-growing Odontotermes and Macrotermes species fully take over the important function of bioturbation, leading to the transport of huge amounts of fine-textured soil material to the surface; with increasing habitat restoration, coarse fragments decreased in the upper horizons and became concentrated deeper along the soil profile. Consequently, in the applied part, I concentrated on the bioturbation activity of fungus-growing termites in the four main stages of the Zaï system: crusted bare soil (initial stage), millet field, young and old forest. In each of the four Zaï sites nine experimental blocks (each comprising four plots of 1m2) were used to stimulate the foraging activity of fungus-growing termites with different, locally available organic materials (Aristida kerstingii hay, Bombax costatum wooden blocks, compost and a control without any organic amendment). The experiment was conducted twice for the duration of four weeks (rainy season 2005, dry season 2006). The plots were regularly checked and the increase of the area covered by sheetings chronologically followed. After four weeks a) all sheeting-soil was collected, air dried and separately weighed according to the different genera, and b) the foraging-holes were counted and their diameter measured. Additionally, c) ponded water infiltration was measured in selected plots, and d) the physicochemical properties of sheeting-soil were analyzed. In case of complete consumption of the offered hay during the experimental 4-weeks-duration, the same procedure (a, b) was followed before adding new hay to the respective plot. The comparison between the different plots, sites and seasons revealed clearly that hay was the most attractive bait; for each gram of hay removed, Odontotermes brought about 12 g soil to the surface, Macrotermes 4 g. Odontotermes was the only genus attracted by organic material to the degraded area, and was therefore the decisive primary physical ecosystem engineer in the Zaï system, initiating the restoration process. The mass of soil bioturbated in the course of foraging increased strongly from the degraded, barren towards the most rehabilitated reforested site. Combining all 36 experimental plots per Zaï stage, Odontotermes bioturbated 31.8 tons of soil per hectare and month dry season in the degraded area, and 32.4 tons ha-1 mon-1 in the millet fields; both genera moved 138.9 tons ha-1 mon-1 in the young and 215.5 tons ha-1 mon-1 in the old Zaï forest. Few comparable figures were found in the literature. In northern Burkina Faso, both genera constructed 20 tons of sheetings ha-1 mon-1 after mulching with a straw-wood mixture (Mando & Miedema 1997), and in Senegal, around 10 tons ha-1 mon-1 were moved in heavily foraged plots (Rouland et al. 2003). Within a site, soil turn-over and the number of foraging holes created was always highest in hay, followed by compost, then by wood and in the end control. The fungus-growers’ foraging-activity was leading to an enormous increase in surface pore space – after one month of induced foraging activity in hay-plots, the median number of foraging-holes increased from 142 m-2 in the degraded site up to 921 m-2 in the old Zaï forest. The creation of subterranean galleries and macropores significantly increased the water infiltration rate by a mean factor 2–4. Laboratory analyses revealed that sheeting-soil differed strongly from the respective control soil as well as between the seasons, the food-type covered, and the two genera. Odontotermes-sheetings differed in more parameters than Macrotermes-sheetings, and dry season sheetings differed in more parameters (and more strongly) than rainy season sheetings. In the present study, soil organic matter, carbon and nitrogen contents were significantly increased in all dry season sheetings; in the rainy season mainly in those built on compost. Texture analysis pointed out that both genera used topsoil and soil from deeper horizons in varying mixture ratios, thereby supporting findings of Jouquet et al. (2006). To summarize, the present thesis contributes to a better understanding of the functional response traits of termites and ants to changing environmental parameters resulting from increasing human impact. The RAP-protocol represents an easy-to-learn and very effective method to representatively characterize, compare and monitor the taxonomic and functional diversity of termites and ants. The experiment has provided conclusive evidence of the importance of the consideration of fungus-growing termites (particularly Odontotermes and Macrotermes species) when aiming to restore infertile, degraded and crusted soils and to maintain a sustainable agricultural production in the Sahel‐Sudanese zone of West Africa. N2 - Die Folgen von Lebensraumveränderungen für die Lebensqualität der Bevölkerung sind vermutlich besonders extrem in Burkina Faso. Das Land liegt in einem für Dürren sehr anfälligen Gebiet von Westafrika. Die Kleinbauern, welche die Hauptproduzenten für Lebensmittel der Region sind, können besonders betroffen sein, wenn Verluste der biologischen Vielfalt zu Veränderungen in den Ökosystemfunktionen führen, da viele von degradierten Flächen für die landwirtschaftliche Produktion abhängen. Das Hauptziel der vorliegenden Arbeit war daher, die funktionellen Merkmale derjenigen Bodenorganismen zu charakterisieren, die im Untersuchungsgebiet von entscheidender Bedeutung für ertragreiche und ausgewogene Böden sind: Termiten und Ameisen. Sie sind wahre Ökosystem‐Ingenieure, deren Aktivität den Lebensraum verändert. Durch Bodenumwälzung während des Baus von biogenen Strukturen unterschiedlicher Größe und Natur (Hügel, Nester, unterirdischer Gänge, Schutzschichten aus Erde, sogenannte „soil-sheetings“, Furagierlöcher, etc.) bewegen sie riesige Bodenmassen und haben enorme Auswirkungen auf die Bodenstruktur. Dies wirkt sich wiederrum positiv auf die Bodenfruchtbarkeit, die Stabilität, die Bodenbelüftung und die Wasserinfiltration aus, und bietet so auch Lebensraum für andere Arten. In den afrikanischen Ländern südlich der Sahara sind Ameisen und Termiten die einzige nennenswerte aktive Bodenmakrofauna während des gesamten Jahres. In der Sub-Sahelregion von Burkina Faso sind während der Trockenzeit Termiten die einzigen aktiven Primärzersetzer. Da keine Informationen über den Artenreichtum der Termiten- und Ameisen-Fauna in Burkina Faso vorlagen, setzte ich in der Dissertation zwei Schwerpunkte: Im ersten Teil, einer Grundlagenstudie, erfasste ich die lokale Termiten und Ameisenfauna in verschiedenen Landnutzungssystemen und untersuchte deren quantitative und qualitative Reaktion auf sich ändernde Umweltbedingungen, die aus dem zunehmenden Einfluss des Menschen resultieren ("funktionelle Reaktionsmerkmale"). Im zweiten und anwendungsbezogenen Teil befasste ich mich mit dem Einfluss der für die Regeneration degradierter Böden wichtigen biogenen Strukturen ("funktionelle Wirkungsmerkmale"). Es wurden zwei für die Untersuchungsregion typische traditionelle Landwirtschafts-formen ausgewählt. Jede stellte einen Landnutzungsgradienten dar, der vier verschiedene Habitate umfasste, welche sich in der Stärke des anthropogenen Einflusses unterschieden, ursprünglich aber den gleichen Anfangszustand hatten. Der erste Nutzungsgradient in Ouahigouya (Provinz Yatenga, Sub-Sahel-Zone) – namens Zaï – war ein zeitlicher Querschnitt durch eine traditionelle Boden- und Wasserschutztechnik zur Regeneration stark verkrusteter, degradierter Böden. Der zweite Nutzungsgradient nahe der Stadt Fada N’Gourma (Provinz Gourma, Nord-Sudan Region) war ein Landwirtschaftstyp, der Fruchtfolge und Brachzeiten für das Nährstoffmanagement nutzte. Zur Erhebung der Termiten- und Ameisen-Diversität in semi-ariden (Agrar-) Ökosystemen existierte kein Standardprotokoll; zwei international akzeptierte Protokolle bildeten die Grundlage für das neu überarbeitete und kombinierte Protokoll „RAP“ zur schnellen Erhebung der Termiten- und Ameisenfauna: Das ALL-Protokoll für Ameisen der Laubstreuschicht von Agosti and Alonso (2000) und das Transektprotokoll für Termiten in tropischen Wäldern von Jones and Eggleton (2000). In meiner Untersuchung wurden zwischen 2004 und 2008 während der Regenzeit in jedem der Untersuchungsgebiete drei bis vier Transekte abgesammelt. Das RAP-Protokoll erwies sich als sehr effektive Methode, um die taxonomische und funktionelle Vielfalt von Termiten und Ameisen zu beschreiben, zu vergleichen und zu überwachen. Zwischen 70% und 90% der geschätzten Gesamtartenzahl wurden auf allen Ebenen (Transekte, Lebensräume, Regionen) gesammelt. Insgesamt wurden in beiden Regionen 65 Ameisenarten (25 Gattungen) und 39 Termitenarten (13 Gattungen) gesammelt. Dies sind bislang die ersten Nachweise für Burkina Faso. Die Daten weisen auf eine hohe Sensitivität von Termiten und Ameisen gegenüber einer Landnutzungsintensivierung hin. Mit zunehmendem anthropogenem Einfluss nahm die Artenvielfalt in der Nord-Sudanregion stark zu. Insgesamt wurden 53 Ameisenarten (23 Gattungen) und 31 Termitenarten (12 Gattungen) gefunden. Sehr vielversprechende Ergebnisse wurden bezüglich des Erholungspotenzials der Bodenarthropoden-Diversität im Zaï-System gesammelt; die Vielfalt beider Taxa nahm in mit zunehmender Lebensraumsanierung stark zu: Insgesamt wurden 41 Ameisenarten (16 Gattungen) und 33 Termitenarten (11 Gattungen) dieser Region gefunden. Entlang der Landnutzungsgradienten zeigten sich signifikante Unterschiede im Vorkommen von Termiten und Ameisen. So bewiesen bei Termiten beispielsweise die Pilzzüchter die größte Anpassungsfähigkeit an die unterschiedlichen Bewirtschaftungspraktiken. Die größten Unterschiede zwischen den Lebensräumen wurden bei den boden- und den grasfressenden Termiten beobachtet. In stark vom Menschen beeinflussten Lebensräumen fehlten ganze funktionelle Gruppen, beispielsweise kamen in der degradierten Fläche der Sub-Sahelregion weder Bodenfresser noch Grasfresser oder Holzfresser vor. Mehrere Umweltparameter wurden identifiziert, welche einen großen Teil der Veränderungen in der Zusammensetzung der Arthropoden-Gemeinschaften entlang der Gradienten signifikant erklärten; sie lassen auf eine große Bedeutung der strukturellen Habitat-Komplexität (Vegetationsstruktur) und den damit verbundenen mikroklimatischen Schwankungen (Temperatur- und Feuchtigkeit), der Nahrungsverfügbarkeit und der Bodenstruktur schließen. Die Termitenvielfalt in der Sub-Sahelzone korrelierte stark mit dem Überschirmungsgrad, dem Sandgehalt im Oberboden und der Verfügbarkeit von Streu. Ihre Vielfalt in der Nord-Sudanregion korrelierte stark mit der kumulierten Gehölzpflanzen-Grundfläche, dem Tongehalt und dem organischen Material im Oberboden. Die identifizierten Parameter für die Ameisen-Gemeinschaften im Zaï-System waren die Höhe der Bäume, der Sandgehalt im Oberboden und die Luftfeuchte. Ihre Vielfalt in der Nord-Sudanregion korrelierte stark mit dem Überschirmungsgrad, dem Trockengewicht der verfügbaren Streu und der Baumhöhe. Um die relative Bedeutung von Termiten und Ameisen für die Bodenumwälzung beurteilen zu können, erfasste ich im zweiten Teil der Arbeit zunächst die natürlichen Schwankungen im Trockengewicht der in jedem Untersuchungsgebiet oberirdisch vorhandenen biogenen Strukturen. Die Ergebnisse veranschaulichen eindrucksvoll, dass: 1. Termiten in allen Untersuchungsgebieten die Hauptumwälzer, Ameisenstrukturen dagegen von untergeordneter Bedeutung für die Bioturbation waren; 2. Regenwürmer und grasfressende Termiten in der regenreicheren Nord-Sudanregion wesentlich zur Bodenumwälzung beitrugen; 3. die Gesamtmasse der umgewälzten Erde von Jahr zu Jahr schwankte, die relative Bedeutung beider Taxa für die Bioturbation jedoch ziemlich konstant war; 4. in der Sub-Sahelzone die wichtige Funktion der Bodenumwälzung vollständig von pilzzüchtenden Macrotermes- und Odontotermes-Arten übernommen wird, die zusammen große Mengen von feinkörnigem Bodenmaterial an die Oberfläche transportieren. Dadurch sank mit zunehmender Habitat-Rehabilitation der Gehalt an grobkörnigem Bodenmaterial in den oberen Bodenschichten und reicherte sich zunehmend in den tieferen Horizonten an. Im anwendungsbezogen Teil der Arbeit konzentrierte ich mich daher auf die Bioturbationsleistung pilzzüchtender Termiten in den vier Hauptstadien des Zaï-Systems: der degradierten Fläche (Ausgangsstadium der vier Sukzessionsstadien), dem Hirsefeld, dem jungen und dem alten Zaï-Wald. In jedem dieser vier Sukzessionsstadien wurde die Furagiertätigkeit von pilzzüchtenden Termiten folgendermaßen angeregt: Es wurden neun Versuchsblöcke installiert, mit je vier Unterquadraten mit einer Fläche von 1 m2. Drei der Unterquadrate wurde mit unterschiedlichen, lokal verfügbaren organischen Materialien bedeckt (mit Aristida kerstingii Stroh, Bombax costatum Holz, Kompost), eines blieb als Kontrolle ohne organisches Material. Das vierwöchige Experiment wurde zweimal durchgeführt (Regenzeit 2005, Trockenzeit 2006). Dabei wurden die Untersuchungsflächen regelmäßig auf Termitenaktivität überprüft und die Zunahme der “soil-sheetings“ kartiert. Nach vier Wochen wurde: i. Die gesamte Termitenerde gesammelt, luftgetrocknet und für jede Gattung getrennt gewogen; ii. die Furagierlöcher gezählt und ihr Durchmesser vermessen; iii. in ausgewählten Flächen die Wasserinfiltrationsrate gemessen; iv. die physikalisch-chemischen Eigenschaften der Sheeting-Erde analysiert. Sobald das Stroh in einem Unterquadrat abgetragen war, wurde nach den betreffenden Messungen (s. oben i. und ii.) neues aufgebracht. Der Vergleich der Ergebnisse beider Durchläufe zeigte deutlich, dass Stroh der attraktivste Köder war. Für jedes Gramm abgetragenes Stroh wurden von Odontotermes etwa 12 g, von Macrotermes etwa 4 g Erde an die Oberfläche gebracht. Odontotermes war die einzige Gattung, die in der degradierten Fläche von organischem Material angelockt wurde. Sie ist damit der entscheidende primäre physikalische Ökosystem-Ingenieur im Zaï-System, der den Restaurierungsprozess anstößt. Mit zunehmender Habitatsanierung nahm die Menge der umgewälzten Erde stark zu: In den 36 Unterquadraten der degradierten Fläche bewegte Odontotermes insgesamt 31,8 Tonnen Erde pro Hektar und Monat Trockenzeit, in denen der Hirsefelder insgesamt 32,4 Tonnen. Beide Gattungen zusammen bewegten im jungen Zaï-Wald insgesamt 138,9 Tonnen, im alten Wald 215,5 Tonnen Erde pro Hektar und Monat Trockenzeit. In jedem Sukzessionsstadium waren sowohl die Bodenumwälzung als auch die Anzahl der Furagierlöcher in den Versuchsflächen mit Stroh am größten, gefolgt von denen mit Kompost, dann denen mit Holz und zuletzt den Kontrollflächen. Die Furagiertätigkeit der pilzzüchtenden Termiten führte zu einer starken Zunahme der Makroporosität des Oberbodens. Nach einem Monat induzierter Fraßaktivität stieg im Mittel die Anzahl der Furagierlöcher pro Quadratmeter von 142 in der degradierten Fläche auf 921 Löcher im alten Zaï Wald. Die bei der Nahrungssuche gegrabenen Gänge und Löcher führten zu einem signifikanten Anstieg der Wasserinfiltrationsrate, im Mittel um den Faktor 2–4. Nur wenige vergleichbare Zahlen konnten in der Literatur gefunden werden. Bei den Untersuchungen von Mando and Miedema (1997) im Norden von Burkina Faso wälzten die beiden Gattungen nach Mulchen mit einem Holz-Stroh-Gemisch Sheetings mit einem Trockengewicht von insgesamt 20 Tonnen je Hektar und Monat Trockenzeit um. Im Senegal wurden in Versuchsflächen mit starker Furagieraktivität rund 10 Tonnen Erde bewegt (Rouland et al. 2003). Laboranalysen ergaben, dass sich Sheeting-Erde stark sowohl von der entsprechenden Kontroll-Erde unterschied als auch dem überdeckten Futtertyp und ebenso zwischen den beiden Gattungen. Dabei unterschied sich Sheeting-Erde von Odontotermes in mehr Parametern als Sheeting-Erde von Macrotermes, und Sheetings aus der Trockenzeit unterschieden sich in mehreren Parametern und in stärkerem Maße als Sheetings aus der Regenzeit. Der Gehalt an organischem Material, an Kohlenstoff und Stickstoff war in allen Trockenzeit-Sheetings deutlich erhöht, in der Regenzeit vor allem in Sheetings, die über Kompost gebaut wurden. Die Analyse der Korngrößenverteilung ließ darauf schließen, dass beide Gattungen Erde aus dem Oberboden und aus tieferen Horizonten in unterschiedlichen Mischungsverhältnissen nutzten. Dies bestätigt Beobachtungen von Jouquet et al. (2006). Zusammenfassend lässt sich sagen, dass die vorliegende Untersuchung zu einem besseren Verständnis der funktionellen Reaktionsmerkmale von Termiten und Ameisen auf sich ändernde Umweltparameter beiträgt, die aus dem zunehmenden Einfluss des Menschen resultieren. Das RAP-Protokoll erwies sich als eine einfach zu erlernende und sehr effektive Methode, um die taxonomische und funktionelle Vielfalt von Termiten und Ameisen in semi-ariden Savannen und Agrarökosystemen repräsentativ zu charakterisieren, zu vergleichen und zu überwachen. Das Experiment erbrachte schlüssige Beweise für die Bedeutung pilzzüchtender Termiten (insbesondere Odontotermes und Macrotermes-Arten) für die Sanierung vollständig degradierter und verkrusteter Böden, und für eine nachhaltige landwirtschaftliche Produktion in der Sub-Sahelzone in Westafrika. KW - Termiten KW - termites KW - Ameisen KW - Biodiversität KW - menschlicher Einfluss KW - ants KW - diversity KW - human impact Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-107001 ER - TY - THES A1 - Andronic, Joseph T1 - Volumenregulatorische Transportwege von anorganischen und organischen Osmolyten in Säugetierzellen T1 - Volume ragulatory pathways of anorganic and organic osmolytes in mammalian cells N2 - Die Aufrechterhaltung des Zellvolumens unter variablen osmotischen Bedingungen stellt für nahezu alle tierischen Zellen eine essenzielle Aufgabe dar. Um regulatorische Volumenanpassungen vorzunehmen besitzen sie daher effektive Mechanismen, mit deren Hilfe der zelluläre Gehalt an organischen und anorganischen Osmolyten erhöht (= regulatorische Volumenzunahme; RVI) oder gesenkt (= regulatorische Volumenabnahme; RVD) werden kann. Trotz langjähriger Forschung auf diesem Gebiet konnten die hieran beteiligten Transportwege für Osmolyte bisher nur unvollständig aufgeklärt werden. Insbesondere bei T-Lymphozyten sind wichtige Zellfunktionen wie die Proliferation, Migration und die T-Zell-Aktivierung eng mit volumenregulatorischen Mechanismen verbunden. Bei all diesen Prozessen sind u. a. unterschiedliche Kaliumkanäle beteiligt, die insbesondere für die pharmakologische Manipulation von Immunsystemprozessen von wissenschaftlichem Interesse sind. Bisherige Modelle der hypotonen Volumenregulation von T-Lymphozyten berücksichtigen lediglich den spannungsabhängigen KV1.3 sowie den Ca2+-aktivierten IKCa1-Kanal, die zur Klasse der 6TM/P-K+-Kanäle gehören. Im ersten Teil der vorliegenden Arbeit wurde eine potentielle Rolle von kürzlich entdeckten Zwei-Poren Domänen Kaliumkanälen (K2P) am RVD von murinen und humanen primären CD4+-T-Lymphozyten untersucht. In einem kombinierten genetischen und pharmakologischen Ansatz mittels knockout-Tiermodellen und dem Einsatz kanalspezifischer Inhibitoren konnte mithilfe zellvolumetrischer Analysen gezeigt werden, dass die K2P-Vertreter TASK1, TASK2, TASK3 und TRESK maßgeblich am schwellungsaktivierten Efflux von K+ beteiligt sind. Beurteilt an den Ergebnissen dieser Untersuchung sind der spannungsabhängige TASK2- und der Ca2+-aktivierte TRESK-Kanal für die hypotone Volumenregulation in T-Zellen deutlich bedeutender als TASK1 und TASK3. Der Beitrag der Kanäle TASK2 und TRESK am RVD-Prozess war über dies vergleichbar mit dessen des bisher bekannten KV1.3-Kanals. In dieser Arbeit wurde damit erstmals eine Beteiligung der K2P-Kanäle am RVD muriner und humaner CD4+-Lymphozyten identifiziert. Aufgrund der engen Verbindung zwischen T-Zell-Funktion und der Volumenregulation können Zwei-Poren Domänen K+-Kanäle damit in den engeren Kreis potentieller immunmodulierende Angriffspunkte aufgefasst werden. Im zweiten und umfangreicheren Teil dieser Arbeit wurden darüber hinaus die schwellungsaktivierten Transportwege für organische Osmolyte (small organic osmolytes; SOOs) untersucht. SOOs stellen chemisch inerte Verbindungen dar, zu denen vor allem Polyole (Sorbitol, myo-Inositol), Methylamine (Betain, α-Glycerophosphocholin) sowie Aminosäuren (α- bzw. β-Alanin und Prolin) und deren Derivate (Taurin) zählen. Da SOOs weder die zelluläre Struktur noch die Funktion von Makromolekülen beeinträchtigen, sind sie wichtige Instrumente der Volumenregulation, die sich in hohen Konzentrationen im Zytosol nahezu aller Zellen wiederfinden. Werden tierische Zellen mit hypotonen Bedingungen konfrontiert, dann ist bei nahezu allen Zellen die Freisetzung organischer Osmolyte zu beobachten, wodurch die zelluläre Osmolarität unabhängig von Elektrolyten angepasst werden kann. Trotz der wichtigen Funktion der SOOs in der Osmoregulation tierischer Zellen konnte die molekulare Identität beteiligter Effluxwege (Kanäle bzw. Transporter) bisher nicht aufgeklärt werden. Ungeachtet der molekularen Identität der SOO-Effluxwege war es aus zahlreichen biotechnologischen Anwendungen zu Beginn dieser Arbeit bekannt, dass die schwellungsaktivierten Transportwege für organische Osmolyte eine größenselektive Permeabilität für eine Reihe monomerer Zucker und verwandter Verbindungen aufweisen. Um diese Größenselektivität näher zu charakterisieren, wurde im ersten Schritt die schwellungsaktivierte Membranpermeabilität für eine Reihe strukturell homogener Polyethylenglykole unterschiedlicher Polymerlänge (PEG200–1500; hydrodynamische Radien zwischen ~0,5-1,5 nm) unter iso- und hypotonen Bedingungen in Jurkat-Lymphozyten untersucht. Unter milden hypotonen Bedingungen (200 mOsm) war die Plasmamembran der untersuchten Lymphozyten für PEG300-1500 undurchlässig, was aus der Fähigkeit der Zellen zur hypotonen Volumenregulation geschlossen werden konnte. Darüber hinaus wurde RVD in stark hypotonen Lösungen (100 mOsm) mit PEG600-1500 beobachtet, während PEG300-400 unter vergleichbaren osmotischen Bedingungen die Volumenregulation der Zellen inhibierten. Dieses Ergebnis deutet darauf hin, dass starkes hypotones Zellschwellen der Lymphozyten zur Permeabilisierung der Plasmamembran für PEG300-400, nicht jedoch für PEG600-1500, führt. Anhand der hydrodynamischen Radien Rh der verwendeten PEGs konnte ein cutoff-Radius von ~0,74 nm für schwellungsaktivierte Transportwege organischer Osmolyte bestimmt werden. Da diese schwellungsaktivierten Transportwege vielfältig für Zellbeladungstechniken verwendet werden, könnte dieses Ergebnis für zahlreiche biotechnologische und biomedizinische Anwendungen von Interesse sein. Im zweiten Schritt wurde der Versuch unternommen, potentielle Transportwege für organische Osmolyte im RVD-Prozess molekular zu identifizieren. Da es grundlegend ungeklärt war, wie viele unterschiedliche Transporter bzw. Kanäle am Efflux der zahlreichen organischen Osmolyte beteiligt sind, erfolgte zunächst die vergleichende Analyse des schwellungsaktivierten Membrantransports strukturell verschiedener SOOs einschließlich der Aminosulfonsäure Taurin und des Polyols myo-Inositol. Hierbei wurde erstmals gezeigt, dass die schwellungsaktivierten Transportwege für Taurin und myo-Inositol deutlich unterschiedliche Aktivitätsprofile aufweisen. Während der Taurintransport bereits unter milden hypotonen Bedingungen, d.h. nach einer geringen Absenkung der Osmolalität von 300 auf ~230 mOsm, aktiviert wurde, erfolgte die Aktivierung der Membranpermeabilität für myo-Inositol bei einer viel niedrigeren Osmolalität von ~150 mOsm. Darüber hinaus wiesen die beiden Transportwege unter vergleichbarem hypotonen Stress von 100 mOsm deutlich unterschiedliche Aktivitätsdauern auf (Transport von Taurin ~95 min und myo-Inositol ~40 min). Somit deuteten diese Ergebnisse erstmals auf substrat-spezifische Transportwege für SOOs hin, die voneinander stark abweichende osmotische Aktivierungsprofile besitzen. Als aussichtsreiche Kandidaten für diese Transportwege wurden zwei Mitglieder der Gruppe der Solute Carrier (SLC) untersucht, die klare Übereinstimmungen mit den gesuchten Transportern für SOOs aufweisen. Daher wurde im Weiteren eine RVD-Beteiligung dieser Transportergruppe mit einer Kombination aus molekularbiologischer und konventioneller bzw. hochaufgelöster mikroskopischen Techniken überprüft. Die semiqantitativen RT-PCR-Ergebnisse dieser Arbeit zeigen dabei, dass die Gentranskription der potentiellen SOO-Transporter SLC5A3 und SLC6A6 in den untersuchten Zelllinien Jurkat, HEK wie auch HepG2-Zellen durch hypotone Bedingungen deutlich verstärkt wird. Hierbei nimmt der zelluläre mRNA-Gehalt der Gene SLC5A3 zwischen 20-60% und SLC6A6 um 30-100% innerhalb von 10-20 min zu, was auf eine potentielle RVD-Beteiligung von SLC-Transportern hindeutet. Ausgehend von diesem Ergebnis wurde daraufhin die zelluläre Lokalisation des SLC5A3-Transporters unter isotonen und hypotonen Bedingungen mikroskopisch untersucht. Wie anhand der konfokalen lasermikroskopischen Untersuchung zu erkennen ist, findet unter hypotoner Stimulation eine zelluläre Umverteilung des mit EGFP fluoreszenzmarkierten Proteins SLC5A3 statt. Innerhalb von 10 min wird der Transporter dabei von intrazellulären Regionen in Richtung Plasmamembran verlagert. Darüber hinaus konnte mit Hilfe der hochauflösenden Mikroskopie-Technik dSTORM gezeigt werden, dass der Transporter SLC5A3 unter hypotoner Stimulation verstärkt mit der Plasmamembran assoziiert vorliegt. Diese verstärkte Membranassoziation des SLC5A3-Proteins deutet damit auf einen schwellungsinduzierten exozytotischen Einbau des Transporters hin. Die Ergebnisse dieser Arbeit zeigen damit erstmals, dass SLC-Transporter wie SLC5A3, SLC6A6 und vermutlich andere Vertreter der SLC-Superfamilie potentiell am Mechanismus der hypotonen Volumenregulation beteiligt sind. Da SLC-Transporter als wichtige Transportsysteme für Therapeutika angesehen werden und die Mechanismen der Volumenregulation bereits in zahlreichen biotechnologischen Anwendungen implementiert sind, könnte der hier aufgedeckte Zusammenhang einen Erkenntnisgewinn für zahlreiche biomedizinische Forschungsgebiete darstellen. N2 - Cell volume homeostasis is critically important for the functional and structural integrity of mammalian cells. To counteract osmotically induced volume perturbations, cells possess efficient mechanisms that control the intracellular osmolyte composition. The volume regulatory mechanisms operating under hyper- and hypotonic conditions are known, respectively, as regulatory volume increase (RVI) and decrease (RVD). During both, RVI and RVD, cells adjust the cellular content of inorganic ions (most notably Na+, K+ and Cl-) and organic solutes in order to gain or lose osmotically obligated water. These mechanisms counteract osmotic cell damage and enable the adaptation of cells to a wide range of extracellular osmolarities. Despite decades of research in this field, many aspects of the mechanisms underlying RVD and RVI remain poorly understood. In case of T lymphocytes, various cellular functions, including proliferation, migration and T cell activation are closely associated with the cell volume regulatory machinery. Among other mechanisms, all these processes are tightly linked by a network of potassium channels. The identification of this network is of great biomedical interest as it provides a key to pharmacological manipulation of the immune system. Current models of hypotonic volume regulation (RVD) in T-lymphocytes consider primarily the voltage-gated KV1.3 and the calcium-activated IKCa1 channel. The first part of this thesis explores the potential role of two-pore domain (K2P) potassium channels in RVD in murine and human primary CD4+-T lymphocytes. Using a combined genetic and pharmacological approach, time-resolved cell volume analysis revealed an important role of the K2P channels TASK1, TASK2, TASK3 and TRESK in swelling activated K+ efflux from hypotonically swollen T cells. Based on the analysis carried out here, the voltage-gated TASK2 as well as the calcium-activated TRESK channel were found as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The importance of TASK2 and TRESK in the RVD process was comparable to that of KV1.3. In summary, the data provide first evidence that hypotonic volume regulation of murine and human CD4+-T lymphocytes relies on K2P channels. With respect to the close relationship of T-cell function and volume regulatory mechanisms K2P channels may thus be considered as potential targets for immunomodulation. In the second and major part of this thesis, the swelling-activated transport pathways for small organic osmolytes (SOOs) were investigated. Nearly all eukaryotic cells possess a considerable reservoir of SOOs, such as polyols (e.g. sorbitol, myo-inositol), methylamines (e.g. betaine, α-glycerophosphoryl choline) and small amino acids (e.g. α-/β- alanine, proline and the derivate taurine), which are synthesized within the cells or accumulated from the extracellular medium. Since SOOs do not interfere with the integrity of macromolecules and the membrane potential, cells tolerate great cytosolic fluctuations of these solutes without negative effects on cellular structure or function. Due to these properties, small organic osmolytes are important tools for cell volume regulatory mechanisms, by which the intracellular osmolarity can be adjusted independently of electrolytes. Although the importance of SOOs for hypotonic volume regulation has been known for long time, the molecular identity of participating membrane efflux pathways is far from being clear. Regardless of the involved transporters, swelling-activated pathways have been reported to exhibit a size selective permeability for a wide range of sugars and related compounds. To gain a deeper insight into this issue, in a first step the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200–1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions was analyzed. Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane was found to remain impermeable to PEG300–1500, which allowed the cells to accomplish regulatory volume decrease. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600–1500, whereas 100 mOsm solutions of PEG300–400 inhibited RVD. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300–400, but not to PEG600–1500. Using the values of hydrodynamic radii Rh for PEGs, the observed size-selectivity of membrane permeation yielded an estimate of ∼0.74 nm for the cut-off radius of the swelling-activated pathway for organic osmolytes. This result may be of interest for many biotechnological and biomedical applications, where swelling-activated SOO-pathways are widely used for cell-loading techniques. As a second step, an attempt was made to elucidate the molecular identity of transporters for organic osmolytes potentially involved in RVD. Since it was not clear whether RVD-related efflux of SOOs is mediated by one common or several distinct transporter(s), at first, the plasma membrane permeability profiles for two structurally dissimilar SOOs, including the amino sulfonic acid taurine and the polyol myo-inositol were analyzed. The results of the time resolved volumetric measurements clearly showed that the membrane permeability to taurine was activated upon moderate cell swelling (by ~15%) in mildly hypotonic solutions (~230 mOsm). In sharp contrast, the membrane permeability to myo-inositol was activated after a much larger swelling (~50%) in strongly hypotonic media (<150 mOsm). Moreover, the swelling-activated permittivity to taurine during RVD in 100 mOsm medium persisted for about twice as long as that for myo-inositol (taurine ~95 min, myo-inositol ~40 min). These findings clearly showed that, taurine and myo-inositol utilized separate, apparently substrate-specific pathways, which were activated at different hypotonic thresholds. Since many members of SLC-family proteins (Solute Carrier) are known for their substrate selectivity and also for their contribution to osmoregulatory mechanisms a participation of SLCs was investigated in the context of RVD. To this end, a combination of molecular biological (semiquantitative RT-PCR) and fluorescence microscopy techniques (confocal and super-resolution microscopy) was used. The semiquantitative RT-PCR data showed a transcriptional upregulation for the SLC proteins SLC5A3 (myo-inositol transporter; SMIT) and SLC6A6 (taurine transporter TauT) in hypotonically stressed Jurkat lymphocytes, HEK293, and HepG2 cells. In all three human cell lines strongly hypotonic solutions (100 mOsm) increased the mRNA level of the genes SLC5A3 and SLC6A6 between 20-60% and 30-100%, respectively, suggesting a potential participation of SLC transporters in RVD. In addition, confocal microscopy images clearly showed the intracellular displacement of EGFP-tagged SLC5A3 expressed in HEK293 cells following strongly hypotonic stress (100 mOsm). Within 10 min the fluorescence of EGFP was shifted from intracellular regions towards the plasma membrane. Furthermore, super-resolution microscopy by means of dSTORM revealed a considerably increased membrane association of SLC5A3 in strongly hypotonic stressed (100 mOsm) HEK293 and Jurkat cells. This finding suggests that SLC5A3 is integrated into the plasma membrane by swelling-induced exocytosis. Taken together, the results of this investigation provided first evidence that transporters such as SLC5A3, SLC6A6 and probably other SLC-proteins participate in the mechanism of hypotonic volume regulation. Due to the relevance of SLC-proteins as potential drug delivery systems the possible role of these transporters might be of great interest for many biomedical research areas. KW - Säugetiere KW - Osmoregulation KW - Zelle KW - Regulatory Volume Decrease KW - Transporter SLC5A3 KW - Transporter SLC6A6 KW - small organic osmolytes KW - Zwei-Poren Domänen Kaliumkanäle KW - two-pore domain potassium channels KW - Zellvolumen KW - Volumenregulation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-103255 ER - TY - THES A1 - Fackler, Marc T1 - Biochemical characterization of GAS2L3, a target gene of the DREAM complex T1 - Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex N2 - GAS2L3 was identified recently as a target gene of the DREAM complex (Reichert et al., 2010; Wolter et al., 2012). It was shown that GAS2L3 is expressed in a cell cycle specific manner and that depletion of the protein leads to defects in cytokinesis and genomic instability (Wolter et al., 2012). Major aim of this thesis was, to further characterize the biochemical properties and physiological function of GAS2L3. By in vitro co-sedimentation and bundling assays, GAS2L3 was identified as a cytoskeleton associated protein which bundles, binds and crosslinks F-actin and MTs. GST pulldown assays and co-immunoprecipitation experiments revealed that GAS2L3 interacts in vitro and in vivo with the chromosomal passenger complex (CPC), a very important regulator of mitosis and cytokinesis, and that the interaction is mediated by the GAR domain of GAS2L3 and the C-terminal part of Borealin and the N-terminal part of Survivin. Kinase assays showed that GAS2L3 is not a substrate of the CPC but is strongly phosphorylated by CDK1 in vitro. Depletion of GAS2L3 by shRNA influenced protein stability and activity of the CPC. However pharmacological studies showed that the decreased CPC activity is not responsible for the observed cytokinesis defects upon GAS2L3 depletion. Immunofluorescence experiments revealed that GAS2L3 is localized to the constriction zone by the CPC in a GAR dependent manner and that the GAR domain is important for proper protein function. New interacting proteins of GAS2L3 were identified by stable isotope labelling by amino acids in cell culture (SILAC) in combination with tandem affinity purification and subsequent mass spectrometrical analysis. Co-immunoprecipitation experiments further confirmed the obtained mass spectrometrical data. To address the physiological function of GAS2L3 in vivo, a conditional and a non-conditional knockout mouse strain was established. The non-conditional mouse strain showed a highly increased mortality rate before weaning age probably due to heart failure. The physiological function of GAS2L3 in vivo as well as the exact reason for the observed heart phenotype is not known at the moment. N2 - GAS2L3 wurde vor kurzem als Zielgen des DREAM Komplex identifiziert (Reichert et al., 2010; Wolter et al., 2012). Es konnte gezeigt werden, dass die Expression von GAS2L3 Zellzyklus abhängig reguliert wird und dass Depletion des Proteins zu Fehlern in der Zytokinese und genomischer Instabilität führt (Wolter et al., 2012). Hauptziel dieser Doktorarbeit war es, GAS2L3 hinsichtlich seiner biochemischen Eigenschaften und physiologischer Funktion näher zu charakterisieren. Unter Verwendung verschiedener in vitro Experimente konnte gezeigt werden, dass GAS2L3 sowohl F-Aktin als auch Mikrotubuli binden, bündeln und quervernetzen kann. In vitro und in vivo Protein-Protein Interaktionsexperimente zeigten, dass GAS2L3 mit dem „chromosomal passenger complex“ (CPC), einem wichtigen Mitose- und Zytokineseregulator, interagiert und dass diese Interaktion durch die GAR Domäne von GAS2L3 und den C-Terminus von Borealin beziehungsweise den N-terminus von Survivin vermittelt wird. Phosphorylierungsexperimente zeigten deutlich, dass GAS2L3 kein Substrat des CPC ist, jedoch von CDK1 phosphoryliert wird. Zellbiologische Experimente belegten, dass Depletion von GAS2L3 mittels shRNA die Proteinstabilität und Aktivität des CPC beeinflusst. Experimente mit einem chemischen Aurora B Inhibitor dokumentierten, dass die verringerte CPC Aktivität nicht die Ursache der beobachteten Zytokinesefehler nach GAS2L3 Depletion ist. Immunfluoreszenzexperimente machten deutlich, dass GAS2L3 mit Hilfe des CPC an der Abschnürungszone lokalisiert wird und dass die Lokalisation abhängig von der GAR Domäne erfolgt. Mit Hilfe von SILAC in Kombination mit Tandem-Affinitätsaufreinigung und anschließender massenspektrometrischer Auswertung wurden neue Proteininteraktoren von GAS2L3 identifiziert. Protein-Protein Interaktionsexperimente bestätigten die massenspektrometrisch ermittelten Daten. Um die physiologische Funktion von GAS2L3 in vivo näher analysieren zu können, wurden verschiedene Knockout Mauslinien etabliert. Die nicht-konditionelle Mauslinie zeigte erhöhte Sterblichkeit vor dem Absetzalter wahrscheinlich verursacht durch Herzversagen. Die genaue physiologische Funktion von GAS2L3 und der Grund für den beobachteten Herzphänotyp sind momentan noch unbekannt. KW - Zellzyklus KW - Zellteilung KW - Cytoskeleton Chromosomal Passenger Complex Interaction GAR Domain KW - Regulation KW - Molekulargenetik KW - GAS2L3 KW - Chromosomal Passenger Complex Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-103394 ER - TY - THES A1 - Hofstetter, Christine T1 - Inhibition of H3K27me-Specific Demethylase Activity During Murine ES cell Differentiation Induces DNA Damage Response T1 - Inhibierung der H3K27me-Spezifischen Demethylase Aktivität in Murin Differenzierenden ES Zellen Induziert die DNA Schadensantwort N2 - Stem cells are defined by their capacity to self-renew and their potential to differentiate into multiple cell lineages. Pluripotent embryonic stem (ES) cells can renew indefinitely while keeping the potential to differentiate into any of the three germ layers (ectoderm, endoderm or mesoderm). For decades, ES cells are in the focus of research because of these unique features. When ES cells differentiate they form spheroid aggregates termed “embryoid bodies” (EBs). These EBs mimic post- implantation embryonic development and therefore facilitate the understanding of developmented mechanisms. During ES cell differentiation, de-repression or repression of genes accompanies the changes in chromatin structure. In ES cells, several mechanisms are involved in the regulation of the chromatin architecture, including post-translational modifications of histones. Post-translational histone methylation marks became one of the best- investigated epigenetic modifications, and they are essential for maintaining pluripotency. Until the first histone demethylase KDM1A was discovered in 2004 histone modifications were considered to be irreversible. Since then, a great number of histone demethylases have been identified. Their activity is linked to gene regulation as well as to stem cell self-renewal and differentiation. KDM6A and KDM6B are H3K27me3/2-specific histone demethylases, which are known to play a central role in the regulation of posterior development by regulating HOX gene expression. So far less is known about the molecular function of KDM6A or KDM6B in undifferentiated and differentiating ES cells. In order to completely abrogate KDM6A and KDM6B demethylase activity in undifferentiated and differentiating ES cells, a specific inhibitor (GSK-J4) was employed. Treatment with GSK-J4 had no effect on the viability or proliferation on ES cells. However, in the presence of GSK-J4 ES cell differentiation was completely abrogated with cells arrested in G1-phase and an increased rate of apoptosis. Global transcriptome analyses in early-differentiating ES cells revealed that only a limited set of genes were differentially regulated in response to GSK-J4 treatment with more genes up- regulated than down-regulated. Many of the up-regulated genes are linked to DNA damage response (DDR). In agreement with this, DNA damage was found in EBs incubated with GSK-J4. A co-localization of H3K27me3 or KDM6B with γH2AX foci, marking DNA breaks, could be excluded. However, differentiating Eed knockout (KO) ES cells, which are devoid of the H3K27me3 mark, showed an attenuated GSK-J4- induced DDR. Finally, hematopoietic differentiation in the presence of GSK-J4 resulted in a reduced colony-forming potential. This leads to the conclusion that differentiation in the presence of GSK-J4 is also restricted to hematopoietic differentiation. In conclusion, my results show that the enzymatic activity of KDM6A and KDM6B is not essential for maintaining the pluripotent state of ES cells. In contrast, the enzymatic activity of both proteins is indispensable for ES cell and hematopoietic differentiation. Additionally KDM6A and KDM6B enzymatic inhibition in differentiating ES cells leads to increased DNA damage with an activated DDR. Therefore, KDM6A and KDM6B are associated with DNA damage and in DDR in differentiating ES cells. N2 - Stammzellen sind definiert durch ihre Fähigkeit zur Selbsterneuerung und dem Potential in multiple Zellinien zu differenzieren. Pluripotente embryonale Stammzellen (ES Zellen) können sich fortlaufend erneuern und besitzen zudem das Potential, in alle drei Keimblätter (Ektoderm, Endoderm oder Mesoderm) zu differenzieren. Auf Grund dieser einzigartigen Eigenschaften sind ES Zellen seit Jahrzehnten im Focus der Wissenschaft. Wenn ES Zellen differenzieren, sind sie in der Lage, sphäroid-förmige Aggregate zu bilden, welche als embryoide Körperchen (EBs) bezeichnet werden. In EBs finden sich Zellen aller 3 Keimblätter und daher dienen sie als in vitro Modell für frühe embryonale Entwicklung. Während der ES Zell Differenzierung verändert die De-repression oder Repression von Genen die Struktur des Chromatins. ES Zellen besitzen eine Vielzahl von Mechanismen, die mit der Regulation des Chromatins assoziiert sind, einschließlich post-translationale Modifikationen an Histonen. Post-translationale Histon- methylierung gehören zu den am häufigsten untersuchten epigenetischen Modifikationen und spielen z.B. ein wichtige Rolle bei der Aufrechterhaltung der Pluripotenz. Bis zur Entdeckung der ersten Histon-Demethylase KDM1A im Jahre 2004 glaubte man, dass Modifikationen an Histonen irreversible sind. Bislang wurden jedoch eine Vielzahl an Histon-Demethylasen identifiziert, welche mit der Genregulation, sowie der Selbsterneuerung und Differenzierung von Stammzelle in Verbindung gebracht werden konnten. KDM6A und KDM6B sind H3K27me3/2-spezifische Histon-Demethylasen, welche bei der posterioren Entwicklung durch Regulation der Hox Gene eine wichtige Rolle spielen. Bislang ist über die molekulare Funktion von KDM6A und KDM6B in nicht differenzierten und differenzierenden ES Zellen wenig bekannt. Um die KDM6A und KDM6B Demethylase Aktivität in nicht differenzierten und differenzierenden ES Zellen außer Kraft zu setzten kam ein spezifischer Inhibitor (GSK-J4) zum Einsatz. Die Behandlung mit GSK-J4 zeigte keine Auswirkungen auf die Viabilität oder Proliferation von nicht differenzierten ES Zellen. Jedoch war die Differenzierung von ES Zellen in Gegenwart von GSK-J4 inhibiert und zeigte einen erhöhten G1-Phase Arrest sowie eine erhöhte Rate an apoptotischen Zellen. Eine globale Transkriptionsanalyse in frühen differenzierenden ES Zellen, in Gegenwart von GSK- J4 zeigte, dass lediglich eine relativ geringe Zahl von Genen differenziell reguliert war. Dabei waren mehr Gene hochreguliert als herunterreguliert. Viele der hochregulierten Gene konnten mit der DNA Schadensantwort in Verbindung gebracht werden. In Übereinstimmung damit konnte in Gegenwart von GSK-J4 in differenzierenden ES Zellen DNA Schaden nachgewiesen werden. Eine Kolokalisation von H3K27me3 oder KDM6B mit γH2AX markierten Foci, welche DNA Schaden markieren, konnte nicht nachgewiesen werden. Nichts desto trotz zeigten GSK-J4 behandelte, differenzierende Eed KO ES Zellen, welche keine H3K27me3 Modifikation besitzen, eine abgemilderte DNA Schadensantwort. In Anwesenheit von GSK-J4 konnte während der hämatopoetischen Differenzierung eine reduzierte Kolonie-Bildung beobachtet werden. Daraus lässt sich schließen, dass in Anwesenheit von GSK-J4 ebenfalls auch die hämatopoetische Differenzierung inhibiert wird. Zusammenfassend zeigen meine Ergebnisse, dass die enzymatische Aktivität von KDM6A und KDM6B für die Aufrechterhaltung des pluripotenten Zustands nicht essenziell ist. Im Gegensatz dazu ist die enzymatische Aktivität von beiden Proteinen unabdingbar für die ES Zell sowie die hämatopoetische Differenzierung. Die enzymatische Inhibierung von KDM6A und KDM6B führt während der Differenzierung zu einem erhöhten DNA Schaden, wodurch die DNA Schadensantwort aktiviert wird. Somit sind KDM6A und KDM6B mit DNA Schaden und der DNA Schadensantwort assoziiert. KW - Embryonale Stammzelle KW - Epigenetic KW - Maus KW - Histone KW - Demethylierung KW - DNS-Schädigung KW - Epigenetik Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-107023 ER - TY - THES A1 - Wende, Beate T1 - Diversity of saproxylic beetles and host tree specialisation in differently managed forests across Germany T1 - Artenvielfalt und Wirtsbaumartenspezialiserung totholzbewohnender Käfer in verschiedenen Wäldern Deutschlands N2 - Chapter I The gradual turnover of dead organic material into mineral nutrients is a key ecological function, linking decomposition and primary production, the essential parts of the nutrient-energy cycle. However, disturbances in terms of species or resource losses might impair the equilibrium between production and decomposition. Humanity has converted large proportions of natural landscapes and intensified land-use activity for food production. Globally, only very few areas are totally unaffected by human activity today. To ensure the maintenance of both essential ecosystem services, knowledge about the interplay of biodiversity and ecosystem functioning as well as effects of intensified management on both is crucial. The vast majority of terrestrial biomass production as well as decomposition take place in forest ecosystems. Though forestry has a long sustainable history in Europe, its intensification during the last century has caused severe impacts on forest features and, consequently, on the associated biota, especially deadwood dependent organisms. Among these, saproxylic beetles are the most diverse group in terms of species numbers and functional diversity, but also most endangered due to habitat loss. These features classify them as ideal research organisms to study effects of intensified forestry on ecosystem services. The BELONGDEAD project located in Germany aimed to investigate deadwood decay and functional consequences of diversity changes in the associated fauna on the decomposition process from the initialisation of deadwood decay to complete degradation. As part of the BeLongDead project, this dissertation focussed on saproxylic beetle species, thereby evaluating (1) regionally effects of tree species identity of fresh deadwood and (2) forest management of varying intensities on the diversity, abundance and community composition of saproxylic beetles (chapter II); (3) the specialisation degree of different trophic guilds of saproxylic beetles, and thus the stability and robustness of their interaction networks against disturbances (chapter III); (4) the impact of environmental features of local to regional spatial scales on species richness of saproxylic beetles differing in their habitat niche in terms of deadwood decay stages (chapter IV). Chapter II The vast majority of European forest ecosystems have been anthropogenically affected, leaving less than 1% of the about 1 milliard hectare as natural forests. A long history of forestry and especially the technological progress during the last century have caused massive habitat fragmentation as well as substantial loss of essential resources in European forest ecosystems. Due to this, the substrate-dependent group of saproxylic beetles has experienced severe species losses. Thus, investigations concerning saproxylic diversity and deadwood volume were badly needed. However, the importance of different deadwood in terms of tree species identity for the colonization by saproxylic beetles under different local and regional management regimes is poorly understood. Therefore, we studied possible regional differences in colonization patterns of saproxylic beetle species in a total of 688 fresh deadwood logs of 13 tree species in 9 sites of managed conifer and beech forests, and unmanaged beech forests, respectively. We found that tree species identity was an important driver in determining saproxylic species composition and abundance within fresh deadwood. However, saproxylic species showed different colonization patterns of deadwood items of the same tree species among the study regions. Regionally consistent, conifer forests were most diverse. We attribute the latter result to the historically adaption of saproxylic beetle species to semi-open forests, which conditions are actually best reflected by conifer forests. To preserve a diverse local species pool of early successional saproxylic beetles, we suggest an equal high degree of deadwood diversity in a tree species context in due consideration of regional differences. Chapter III The extinction risk of a particular species corresponds with its species-specific requirements on resources and habitat conditions, in other words with the width of the species` ecological niche. Species with a narrow ecological niche are defined as specialists. Members of this group experience higher extinction risk by resource limitation than generalists, which are able to utilize a variety of resources. For the classification of species as specialists or generalists, thus evaluating possible extinction risks, ecologists use the concept of interaction networks. This method has often been applied for mutualistic or antagonistic plant-animal interactions, but information for networks of detritivores is scarce. Therefore, saproxylic beetle species sampled as described in chapter II were categorised according to their larval diet; additionally their interaction networks (N=108) with 13 dead host tree species were analysed. Specialisation degree was highest for wood-digesting beetles and decreased with increasing trophic level. Also the network indices evaluating robustness and generality indicated a higher susceptibility to species extinctions for xylophagous than for mycetophagous and predatory beetles. The specialisation of xylophagous species on specific tree species might be an adaption to tree species specific ingredients stored for defence against pathogens and pests. However, we conclude that the high specialisation degree of xylophages and thus their higher extinction risk by resource loss harbours certain dangers for ecosystem function and stability as species diversity is positively linked to both. Chapter IV Populations depend on individual emigration and immigration events to ensure genetic exchange. For successful migration it is of utmost importance that spatially separated populations are obtainable by specimen. Migratory success depends on the one hand on the species dispersal abilities and on the other on the availability of suitable habitats in the surrounding landscape in which the distinct host populations exist. However, consequences of intensive forest management correspond not only to severe reduction of local deadwood amount, but, among others, also a change in tree species composition and high levels of fragmentation in the surrounding forest area. Saproxylic beetle species differ in their dispersal behaviour according to the temporal availability of their preferred habitat. Generally, early successional saproxylic beetles are able to disperse over large distances, whereas beetles inhabiting advanced decayed wood often remain close to their larval habitat. Due to this, environmental factors might affect saproxylic beetle guilds differently. We classified the saproxylic beetles sampled as described in chapter II according to their calculated habitat niche as early, intermediate or late successional saproxylic beetles. For the different guilds the effects of 14 environmental factors on different spatial scales (stand factors at 0.1 km radius, landscape composition at 2 km radius, and regionally differing abiotic factors in 400 km to 700 km distance) were investigated. Consistently for all guilds, species richness decreased with fragmentation at local and landscape scale, and increased in warmer climate. However, we found contradictory results between the guilds to some extent. We relate this to guild specific habitat requirements of the saproxylic beetles. Therefore, for the development of appropriate conservation practices guild-specific requirements saproxylic beetles have to be considered not only locally but on larger spatial scales. Chapter V In conclusion, this dissertation identified main drivers of early successional saproxylic beetle species richness on various spatial scales. Our results emphasize the importance to develop management schemes meeting species-specific and guild-specific habitat requirements of the saproxylic beetle fauna at relevant spatial and temporal scales. Therefore, short-term actions suggested for sustainable forest management should be the focus on a diverse tree species composition consisting of indigenous tree species with respect to regional differences. Moreover, senescent trees, fallen and standing deadwood should remain in the forests, and some tree individuals should be allowed to grow old. Long-term actions should involve the reduction of forest fragmentation and the connection of spatial widely separated forest fragments. Furthermore, to fully understand the effects of forest management long-term research should be conducted to compare habitat requirements of intermediate and late successional beetles with the results presented in this dissertation. N2 - Kapitel I Die Mineralisierung von toter organischer Materie nimmt eine Schlüsselfunktion innerhalb eines Ökosystems ein, da sie die beiden essentiellen Komponenten des Energie-Nährstoff-Zyklus - Zersetzung und Primärproduktion - miteinander verbindet. Anthropogen bedingte Störungen wie z.B. Arten-, oder Ressourcenverluste können jedoch das Gleichgewicht zwischen den beiden wichtigen Ökosystemdienstleistungen Produktion und Abbau aus der Balance bringen. Um die Nahrungsversorgung der Menschheit zu gewährleisten, wurde bereits ein großer Teil der Natur in Agrarflächen umgewandelt und die Produktion durch intensive Bewirtschaftungsformen gesteigert. Weltweit gibt es nur noch wenige Gebiete ohne menschliche Beeinflussung. Um dauerhaft essentielle Ökosystemdienstleistungen zu gewährleisten, sind Kenntnisse über die Auswirkungen von intensiver Bewirtschaftung auf das Zusammenspiel zwischen Artenvielfalt und Ökosystemfunktionen unabdingbar. Der größte Teil der terrestrischen Biomasseproduktion wird von Wäldern geleistet. Obwohl die Waldbewirtschaftung in Europa lange Zeit nachhaltig war, wirkte sich deren Intensivierung während des letzten Jahrhunderts massiv auf die Waldstruktur und die damit assoziierte Fauna aus. Besonders betroffen sind die obligatorisch an Totholz gebundenen Organismen. Innerhalb der Totholzfauna sind xylobionte Käfer eine artenreiche und funktional hoch diverse Gruppe, doch aufgrund von Lebensraumverlusten sind viele Arten stark bedroht. All diese Eigenschaften klassifizieren Totholzkäfer zu idealen Forschungsobjekten, um die Auswirkungen von intensiver Waldbewirtschaftung auf Ökosystemfunktionen zu untersuchen. Das BELONGDEAD-Projekt hat als Ziel, die funktionalen Auswirkungen von Veränderungen in der Artengemeinschaft auf die Abbauraten von Totholz zu analysieren. Der Untersuchungszeitraum des in Deutschland beheimateten Projekts umfasst die Initialisierung des Zersetzungsprozesses bis zum vollständigen Abbau von experimentell ausgelegten Totholzstämmen unterschiedlicher Baumarten. Als Teil des BELONGDEAD-Projekts lag der Fokus der vorliegenden Dissertation auf der Totholzkäferfauna. Wir untersuchten (1) regionale Effekte der Baumartenzugehörigkeit von frischem Totholz und (2) die Auswirkungen von Waldbewirtschaftung unterschiedlicher Intensität auf die Artenvielfalt, Abundanz und Struktur der Artengemeinschaften von totholzbewohnenden Käfern (Kapitel II); (3) den Spezialisierungsgrad verschiedener trophischer Gilden von Totholzkäfern, sowie die Stabilität und Robustheit ihrer jeweiligen Netzwerke gegen Störungen (Kapitel III); (4) den Einfluss von Umweltfaktoren auf die Artenvielfalt xylobionter Käfergilden auf mehreren räumlichen Skalen. Kapitel II Der Großteil der europäischen Wälder ist anthropogen beeinflusst. In Europa bilden Naturwälder weniger als 1% der gesamten Waldfläche von ca. 1 Mrd. Hektar. Traditionelle Waldbewirtschaftung und vor allem der technologische Fortschritt des letzten Jahrhunderts fragmentierten die Waldfläche in hohem Maß, und verursachten beträchtliche Verluste an lebensnotwendigen Ressourcen. Besonders innerhalb der obligatorisch an Totholz gebundenen Gruppe der xylobionten Käfer verzeichnete man einen rasanten Artenrückgang. Daher gab es einen großen Bedarf an Studien, die Untersuchungen zur Mindestmenge an lokal vorhandenem Totholz zur Sicherung der xylobionten Artenvielfalt durchführten. Wenig beachtet wurde bisher jedoch die Bedeutung der Baumartenzugehörigkeit von Totholz für die Besiedlung durch xylobionter Käfer in verschiedenen Waldbewirtschaftungssystemen auf lokaler und regionaler Ebene. Wir untersuchten daher mögliche Unterschiede zwischen 3 Regionen im Besiedlungsmuster xylobionter Käfer bei insgesamt 651 experimentell ausgelegten Baumstämmen in einem frühen Sukzessionsstadium von 13 Baumarten auf jeweils 9 Untersuchungsflächen in bewirtschafteten Buchen- und Nadelwäldern, sowie in unbewirtschafteten Buchenwäldern. Bei den ausgelegten Totholzstämmen war die Baumartenzugehörigkeit ausschlaggebend für die Struktur der Artengemeinschaften und Abundanzen xylobionter Käfer. Aufgrund der unterschiedlichen regionalen Artenpools divergierten die Besiedlungsmuster xylobionter Käferarten von Totholz der gleichen Baumart in den verschiedenen Regionen stark voneinander. In allen Regionen zeigten die Totholzkäfer in Nadelwäldern die höchste Artenvielfalt. Dieses Ergebnis lässt sich auf die - historisch bedingte - Anpassung der Totholzkäferfauna an eine halboffene Waldstruktur zurückführen, die derzeit am besten durch Nadelwälder widergespiegelt wird. Um eine diverse lokale Artengemeinschaft xylobionter Käfer zu gewährleisten ist eine große Variabilität vom baumartspezifischen Totholz unabdingbar, wobei regionale Unterschiede in Betracht gezogen werden müssen. Kapitel III Das Aussterberisiko einer Art ist abhängig von den artspezifischen Ansprüchen an ihre Umwelt und den dort vorkommenden Ressourcen –auch definiert als die ökologische Nische der betrachteten Art. Arten mit geringer Nischenbreite sind per definitionem Spezialisten. Mitglieder dieser Gruppe stehen durch Verarmung ihres Ressourcenangebots unter einem höheren Aussterberisiko als Generalisten, die eine größere Variabilität in ihrem Ressourcenspektrum aufweisen. Interaktionsnetzwerke dienen in der Ökologie als wichtiges Werkzeug um das Aussterberisiko spezifischer Arten zu bewerten und eine Einteilung hinsichtlich Spezialist oder Generalist vorzunehmen. Bei mutualistischen oder antagonistischen Tier-Pflanzen-Interaktionen ist diese Methode etabliert, doch für die Gruppe der Zersetzer ist das Netzwerk-Konzept bisher nur sporadisch angewandt worden. Daher teilten wir die xylobionten Käferarten, die im Rahmen des in Kapitel II beschriebenen Experiments gesammelt wurden, anhand ihres larvalen Ernährungstyps in drei trophische Gilden (Xylophage, Mycetophage und Räuber) ein; anschließend wurden ihre Interaktionsnetzwerke (N= 108) mit den 13 Wirtsbaumarten analysiert. Rein xylophage Arten wiesen den höchsten Spezialisierungsgrad auf, der mit zunehmendem trophischem Grad geringer wurde. Die Netzwerkparameter Robustheit und Generalität ließen ebenfalls auf eine höhere Anfälligkeit für Artenverluste bei xylophagen als bei mycetophagen oder räuberischen Arten schließen. Die Spezialisierung xylophager Arten auf spezifische Baumarten ist möglicherweise eine Adaption an artenspezifische sekundäre Inhaltsstoffe, die als Schutz vor Schädlingen und Krankheitserregern in Holz und Rinde gespeichert werden. Der hohe Spezialisierungsgrad xylophager Käfer bedingt ein höheres Aussterberisiko bei Ressourcenverlust. Dies würde die Stabilität des Ökosystems und dessen Ökosystemfunktionen nachhaltig schwächen da eine hohe Artenvielfalt Garant für ein funktionierendes Ökosystem ist. Kapitel IV Individuelle Immigrations- und Emigrationsereignisse sind für die Sicherstellung des genetischen Austauschs zwischen Populationen essentiell. Daher ist von größter Wichtigkeit, dass die räumliche Distanz zwischen Populationen von den zu- oder abwandernden Individuen überwunden werden kann. Der Migrationserfolg ist dabei zum einen von der artspezifischen Ausbreitungsfähigkeit, und zum anderen von der Verfügbarkeit an geeigneten Habitaten in der Umgebung der Populationen abhängig. Die Folgen intensiver Waldbewirtschaftung sind jedoch nicht nur ein drastische Verminderung des lokalen Totholzvolumens, sondern unter anderem auch die Veränderung der Baumartengesellschaften, sowie hochgradige Fragmentierung der Waldflächen und der umgebenden Landschaft. Xylobionte Käferarten unterscheiden sich in ihrem Ausbreitungsverhalten hinsichtlich der zeitlichen Verfügbarkeit ihres bevorzugten Habitats. Im Allgemeinen können Besiedler früher Sukzessionsstadien weite Strecken überwinden, wohingegen Bewohner von Alttotholzstrukturen meist nahe ihrem Ursprungshabitat verbleiben. Dies legt die Vermutung nahe, dass Umweltparameter verschieden auf unterschiedliche Habitatgilden einwirken. Um diese Vermutung zu überprüfen, wurde für die gesammelten xylobionten Käferarten ihre jeweilige Habitatnische berechnet. Wir klassifizierten die Arten als Besiedler von entweder frühen, mittleren oder alten Totholzstrukturen. Für jede Gilde wurde der Einfluss von 14 Umweltparametern auf verschiedenen räumlichen Skalen - Standortfaktoren der Untersuchungsfläche (Radius: 100 m), Landschaftsparameter im Umkreis von 2 km der Untersuchungsfläche, sowie regionenspezifische abiotische Faktoren (Distanz zwischen den Regionen: 400 – 700 km) - untersucht. Bei starker lokaler und landschaftlicher Fragmentierung nahmen die Artenzahlen in den xylobionten Gilden ab, während sich höhere Jahresdurchschnittstemperatur positiv auf die Artenvielfalt auswirkte. Jedoch gab es hatten nicht alle Umweltfaktoren den gleichen Effekt auf die Gilden. Wir führen dies auf die unterschiedlichen Habitatansprüche der xylobionten Gilden zurück. Um adäquate Schutzmaßnahmen für Totholzkäfer zu entwickeln, müssen die spezifischen Habitatansprüche der verschiedenen xylobionten Gilden, nicht nur auf lokaler, sondern auch auf größeren räumlichen Ebenen in die Planungen miteinbezogen werden. Kapitel V In der vorliegenden Dissertation konnte ich wichtige Triebfedern der Artenvielfalt xylobionter Käfer identifizieren. Unsere Ergebnisse unterstreichen die Notwendigkeit der Entwicklung von nachhaltigen Waldbewirtschaftungskonzepten, die den art- und gildenspezifischen Anforderungen xylobionter Käfer an den Lebensraum auf den relevanten räumlichen und zeitlichen Skalen gerecht werden. Kurzfristige Maßnahmepläne für eine nachhaltige Forstwirtschaft sollte die Förderung von Mischwäldern mit einer vielfältigen Baumartengemeinschaft mit standortgemäßen einheimischen Hölzern unter Berücksichtigung regionaler Besonderheiten beinhalten. Alte Bäume, sowie liegendes und stehendes Totholz sollten im Wald verbleiben und einzelne Bäume aus der Nutzung genommen werden um die Strukturen altgewachsener Bäume langfristig zu gewährleisten. Langfristige Ziele sind die Verringerung der Waldfragmentierung und das Anlegen von Biotopverbundsystemen, um weit auseinanderliegende Waldflächen wieder miteinander zu verbinden. Um die Auswirkungen kommerzieller Forstwirtschaft im vollen Umfang zu erfassen, sind Langzeitstudien notwendig die die Habitatansprüche xylobionter Käfer aus mittleren und alten Totholzsukzessionsstadien mit den Ergebnissen der vorliegenden Dissertation vergleichen. KW - Saproxylophage KW - Käfer KW - Ökosystem KW - Saproxylic beetles KW - temperate forests KW - Deutschland KW - Wald KW - saproxylic Coleoptera KW - Forest management KW - Diversity Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-107049 ER - TY - THES A1 - Schwarze, Simone T1 - Untersuchung von Faltungs- und Funktionsdynamik isolierter Proteindomänen mittels Fluoreszenzlöschung T1 - Investigation of folding and function dynamics of isolated Protein Domains using fluorescence quenching N2 - Proteine bestehen aus einer spezifischen Sequenz verschiedener Aminosäuren, die ihre charakteristische Funktion bestimmt. Die große Variabilität an Aminosäuresequenzen ermöglichte die Evolution einer nahezu unbegrenzten Anzahl an Proteinen. Meistens nehmen diese Schlüsselpositionen ein, von robusten Baustoffen bis hin zu molekularen Maschinen. Daher kann eine Fehlfunktion gravierende Auswirkungen auf das Leben haben, z.B. Krankheiten wie Alzheimer oder Epilepsi. Um die Funktionen und Fehlfunktionen zu verstehen, ist eine umfassende Kenntnis der Proteinfaltung, der Protein-Protein Assoziation, sowie den Dynamiken innerhalb von Proteinen erforderlich. Diese Vorgänge wurden in dieser Arbeit an drei isolierten Proteindomänen durch die Anwendung der Fluoreszenzlöschmechanismen der H-Dimerbildung und des photoinduzierten Elektronentransfers untersucht. Der entfaltete Zustand der Bindungsdomäne BBL, das Teil des 2-oxo-acid Dehydrogenasekomplexes ist, wurde unter physiologischen Bedingungen mit Zirkulardichroismus (CD) und einer Kombination aus photoinduziertem Elektronentransfer und Fluoreszenzkorrelationsspektroskopie analysiert. Beide Methoden zeigten übereinstimmend anhand von 20 in BBL einzeln eingefügten konservativen Punktmutationen, dass Seitenketteninteraktionen keine Auswirkungen auf die Sekundärstruktur des denaturierten Zustandes, den Ausgangspunkt der Faltung, haben. Mit Hilfe der Dekonvolation der CD-Spektren wurde zudem gezeigt, dass die Reststruktur im denaturierten Zustand der helikalen Proteindomäne von β-Strängen und β-Kehren dominiert wird, die eine entscheidende Funktion bei der Faltung in den nativen Zustand haben könnten. Die N-terminale Domäne (NTD), der für die Materialforschung hochinteressanten Spinnen-seidenfaser, ist für die Polymerisation des Spinnenseidenfadens auf den pH-Wechsel von pH 7 auf pH 6 hin verantwortlich. Dieser für die Proteinfunktion wichtige Prozess wurde durch die Einbringung eines extrinsischen Fluoreszenzschalters, basierend auf der H-Dimerbildung, mit der Stopped-Flow-Technik untersucht. Es wurde gezeigt, dass die NTDs 104 mit einer Rate von 3 x 10^8 M-1 s-1 assoziieren und somit nahezu das Geschwindigkeitslimit der Protein-Protein Assoziation erreicht wird. Zwei geladenen Seitenketten, der D39 und D40, kommt eine entscheidende Funktion in dem Prozess zu, da eine Mutation dieser die Assoziation verhindert. Des Weiteren wurde gezeigt, dass sich die NTD auf eine Erhöhung der Ionenstärke entgegengesetzt zu anderen Proteinen verhält: die Dissoziation wird beschleunigt, die Assoziation nicht beeinflusst. Gleiches Verhalten wurde auf den einzelnen Austausch der übrigen protonierbaren Aminosäureseitenketten hin beobachtet, ausgenommen die Mutation der E119, welche die Dissoziation verlangsamt. Daher scheint der makromolekulare Dipol, der auf Grund der Ladungsverteilung in der NTD entsteht, die Assoziation maßgeblich zu beeinflussen. Glutamatrezeptoren sind an der schnellen synaptischen Signalweiterleitung im Nervensys-tem von Vertebraten beteiligt. Die Konformationen der Ligandenbindungsdomäne (LBD) haben dabei entscheidende Auswirkungen auf die Funktion des Gesamtrezeptors. Diese wurden mit einer Kombination aus photoinduziertem Elektronentransfer und Fluoreszenzkorrelationsspektroskopie untersucht. Mit dieser Methode wurde ein dynamisches Bild der gebundenen sowie ungebundenen Form der AMPA-spezifischen Glutamatrezeptor 2-LBD gezeigt. Es wurde zudem gezeigt, dass sich die Dynamiken in Abhängigkeit der Bindung von den Agonisten Glutamat und AMPA, dem partiellen Agonisten Kainate oder Cyclothiazid (CTZ), welches eine Dimerisierung der LBDs bewirkt, unterschiedlich verändern. Dies könnte eine Auswirkung auf die Funktion der Rezeptoren haben. Die Anwendung der Fluoreszenzlöschmechanismen der H-Dimerbildung und des photoinduzierten Elektronentransfers in dieser Arbeit hat gezeigt, dass diese die Möglichkeit bieten, unterschiedlichste Fragestellungen zu beantworten und so Einblicke in dynamische Funktionsweisen von Proteinen eröffnen. Kombiniert mit etablierten Fluoreszenzmethoden ist es so möglich quantitativ Kinetiken auf unterschiedlichen Zeitskalen zu untersuchen. N2 - Proteins are composed of a specific sequence of variable amino acids that specify their specific function. The vast variability of possible amino acid sequences allowed for the evolution of a nearly infinite number of different proteins. Mostly these will have key positions, reaching from strong building material to molecular machines. Therefore a malfunction will have an immense effect on life, i.e. diseases like Alzheimer disease or epilepsy. In order to understand the function and malfunction an intense understanding of the protein folding, the protein-protein association, as well as the protein dynamics is essential. These processes have been investigated in this thesis with three isolated protein domains by the application of the fluorescence quenching mechanisms of the H-dimer-formation and by the photoinduced electron transfer. The unfolded state of the binding domain BBL, which is part of the 2-oxo-acid-dehydrogenase-complex, was analysed under physiological conditions by circular dichroism (CD), and a combination of photoinduced electron transfer and fluorescence correlation spectroscopy. Both methods showed accordingly, using 20 singly implanted conservative point mutations in BBL, that side-chain interactions have no effect on the secondary struc-ture of the denatured condition, the initial state of the folding. Using deconvolution on the CD spectra it was shown in addition, that the remaining structure of the helical protein domain in the denatured state is dominated by β-strands and β-turns. These may have a decisive function during the folding in the native state. The N-terminal domain of the spider silk fibre, which is of high interest for the material research, is responsible for the polymerisation of the spider silk fibre during the pH-change from pH 7 to pH 6. This important process for the protein function was investigated with the Stopped-Flow technique by the application of an extrinsic fluorescence switch, based on the H-dimer formation. It was shown, that NTDs associate with a rate of 3 x 10^8 M-1 s-1, and so nearly reach the speed limit of the protein-protein association. In this process two charged side chains, the D39 and D40, have a decisive function, as a mutation of these will 106 avoid the association. Furthermore it was shown, that the NTD will react in opposition to other proteins on the increase of the ionic strength: the dissociation will be speeded up, and the association is not influenced. The identical behaviour was observed with the single ex-change of the other protonable amino acid side chains, except for the mutation of E119, which retards dissociation. Therefore the macromolecular dipol, that is formed by the charge distribution of the NTD, is obviously influencing the association. Glutamate receptors participate in the fast synaptic signal transfer in vertebrates. Here dif-ferent conformations of the ligand binding domain (LBD) have decisive effects on the function of the entire receptor. These have been investigated with a combination of photoin-duced electron transfer and fluorescence correlation spectroscopy. Using this method a dynamic behaviour of the bound and unbound form of the AMPA specific glutamate receptor 2 LBD was shown. Furthermore it was shown, that the dynamics will change differently in dependence of the binding of the agonist glutamate and AMPA, the partial agonist kainate or Cyclothiazide (CTZ), which effects a dimerization of the LBDs. This may have an effect on the function of the receptors. The use of the fluorescence quenching mechanisms of the H-dimer formation and the pho-toinduced electron transfer in this thesis has shown, that these offer the opportunity to an-swer a large range of questions and open a view to the dynamic functionality of proteins. Combined with established fluorescence methods it is possible to quantitatively investigate kinetic rates at different time scales. KW - Protein-Protein-Wechselwirkung KW - Fluoreszenzlöschung KW - Domäne KW - Proteindynamiken KW - H-Dimerbildung KW - Photoinduzierter Elektronentransfer KW - Spinnenseide KW - Glutamatrezeptor KW - BBL KW - protein dynamics KW - fluorescence quenching KW - Proteindomänen Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-107080 ER - TY - THES A1 - Schulze, Katja T1 - Automatisierte Klassifizierung und Viabilitätsanalyse von Phytoplankton T1 - Automated classification and viability analysis for phytoplankton N2 - Zentrales Ziel dieser Arbeit war es, Methoden der Mikroskopie, Bildverarbeitung und Bilderkennung für die Charakterisierungen verschiedener Phyotplankter zu nutzen, um deren Analyse zu verbessern und zu vereinfachen. Der erste Schwerpunkt der Arbeit lag auf der Analyse von Phytoplanktongemeinschaften, die im Rahmen der Überprüfung der Süßwasserqualität als Marker dienen. Die konventionelle Analyse ist dabei sehr aufwendig, da diese noch immer vollständig von Hand durchgeführt wird und hierfür speziell ausgebildetes Personal eingesetzt werden muss. Ziel war es, ein System zur automatischen Erkennung aufzubauen, um die Analyse vereinfachen zu können. Mit Hilfe von automatischer Mikroskopie war es möglich Plankter unterschiedlicher Ausdehnung durch die Integration mehrerer Schärfeebenen besser in einem Bild aufzunehmen. Weiterhin wurden verschiedene Fluoreszenzeigenschaften in die Analyse integriert. Mit einem für ImageJ erstellten Plugin können Organismen vom Hintergrund der Aufnahmen abgetrennt und eine Vielzahl von Merkmalen berechnet werden. Über das Training von neuralen Netzen wird die Unterscheidung von verschieden Gruppen von Planktontaxa möglich. Zudem können weitere Taxa einfach in die Analyse integriert und die Erkennung erweitert werden. Die erste Analyse von Mischproben, bestehend aus 10 verschiedenen Taxa, zeigte dabei eine durchschnittliche Erkennungsrate von 94.7% und eine durchschnittliche Falsch-Positiv Rate von 5.5%. Im Vergleich mit bestehenden Systemen konnte die Erkennungsrate verbessert und die Falsch Positiv Rate deutlich gesenkt werde. Bei einer Erweiterung des Datensatzes auf 22 Taxa wurde darauf geachtet, Arten zu verwenden, die verschiedene Stadien in ihrem Wachstum durchlaufen oder höhere Ähnlichkeiten zu den bereits vorhandenen Arten aufweisen, um evtl. Schwachstellen des Systemes erkennen zu können. Hier ergab sich eine gute Erkennungsrate (86.8%), bei der der Ausschluss von nicht-planktonischen Partikeln (11.9%) weiterhin verbessert war. Der Vergleich mit weiteren Klassifikationsverfahren zeigte, dass neuronale Netze anderen Verfahren bei dieser Problemstellung überlegen sind. Ähnlich gute Klassifikationsraten konnten durch Support Vektor Maschinen erzielt werden. Allerdings waren diese bei der Unterscheidung von unbekannten Partikeln dem neuralen Netz deutlich unterlegen. Der zweite Abschnitt stellt die Entwicklung einer einfachen Methode zur Viabilitätsanalyse von Cyanobakterien, bei der keine weitere Behandlung der Proben notwendig ist, dar. Dabei wird die rote Chlorophyll - Autofluoreszenz als Marker für lebende Zellen und eine grüne unspezifische Fluoreszenz als Marker für tote Zellen genutzt. Der Assay wurde mit dem Modellorganismus Synechocystis sp. PCC 6803 etabliert und validiert. Die Auswahl eines geeigeneten Filtersets ermöglicht es beide Signale gleichzeitig anzuregen und zu beobachten und somit direkt zwischen lebendenden und toten Zellen zu unterscheiden. Die Ergebnisse zur Etablierung des Assays konnten durch Ausplattieren, Chlorophyllbestimmung und Bestimmung des Absorbtionsspektrums bestätigt werden. Durch den Einsatz von automatisierter Mikroskopie und einem neu erstellten ImageJ Plugin wurde eine sehr genaue und schnelle Analyse der Proben möglich. Der Einsatz beim Monitoring einer mutagenisierten Kultur zur Erhöhung der Temperaturtoleranz ermöglichte genaue und zeitnahe Einblicke in den Zustand der Kultur. Weitere Ergebnisse weisen darauf hin, dass die Kombination mit Absorptionsspektren es ermöglichen können bessere Einblicke in die Vitalität der Kultur zu erhalten. N2 - Central goal of this work was to improve and simplify the characterization of different phytoplankter by the use of automated microscopy, image processing and image analysis. The first part of the work dealt with the analysis of pytoplankton communities, which are used as a marker for the determination of fresh water quality. The current routine analysis, is very time consuming and expensive, as it is carried out manually by trained personnel. Thus the goal of this work was to develop a system for automating the analysis. With the use of automated microscopy different focal planes could be integrated into one image, which made it possible to image plankter of different focus levels simultaneously. Additionally it allowed the integration of different fluorescence characteristics into the analysis. An image processing routine, developed in ImageJ, allows the segmentation of organisms from the image background and the calculation of a large range of features. Neural networks are then used for the classification of previously defined groups of plankton taxa. The program allows easy integration of additional taxa and expansion of the recognition targets. The analysis of samples containing 10 different taxa showed an average recognition rate of 94.7% and an average error rate of 5.5%. The obtained recognition rate was better than those of existing systems and the exclusion of non-plankton particles could be greatly improved. After extending the data set to 22 different classes of (more demanding) taxa a still good recognition (86.9 %) and still improved error rate (11.9 %) were obtained. This extended set was specifically selected in order to target potential weaknesses of the system. It contained mainly taxa that showed strong similarities to each other or taxa that go through various different morphological stages during their growth. The obtained recognition rates were comparable or better than those of existing systems and the exclusion of non-plankton particles could be greatly improved. A comparison of different classification methods showed, that neural networks are superior to all other investigated methods when used for this specific task. While similar recognition rates could be achieved with the use of support vector machines they were vastly inferior for the differentiation of unknown particles. The second part focused on the development of a simple live - dead assay for unicellular cyanobacteria without the need of sample preparation. The assay uses red chlorophyll fluorescence, corresponding to viable cells, and an unspecific green autofluorescence, that can only be observed in non viable cells. The assay was established and validated for the model organism Synechocystis sp. PCC 6803. With the selection of a suitable filter-set both signals could be excited and observed simultaneously, allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and an ImageJ based image analysis plugin allows a very precise and fast analysis. The monitoring of a random mutagenized culture undergoing selection for improved temperature tolerance allowed an accurate and prompt insight into the condition of the culture. Further results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells. KW - Bilderkennnung KW - Bioinformatik KW - Phytoplankton KW - Bilderkennung KW - Phytoplankton KW - Viabilität KW - Mikroskopie KW - Bioinformatik Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-107174 ER - TY - JOUR A1 - Chen, Yi-chun A1 - Gerber, Bertram T1 - Generalization and discrimination tasks yield concordant measures of perceived distance between odours and their binary mixtures in larval Drosophila JF - The Journal of Experimental Biology N2 - Similarity between odours is notoriously difficult to measure. Widely used behavioural approaches in insect olfaction research are cross-adaptation, masking, as well as associative tasks based on olfactory learning and the subsequent testing for how specific the established memory is. A concern with such memory-based approaches is that the learning process required to establish an odour memory may alter the way the odour is processed, such that measures of perception taken at the test are distorted. The present study was therefore designed to see whether behavioural judgements of perceptual distance are different for two different memory-based tasks, namely generalization and discrimination. We used odour-reward learning in larval Drosophila as a study case. In order to challenge the larvae's olfactory system, we chose to work with binary mixtures and their elements (1-octanol, n-amyl acetate, 3-octanol, benzaldehyde and hexyl acetate). We determined the perceptual distance between each mixture and its elements, first in a generalization task, and then in a discrimination task. It turns out that scores of perceptual distance are correlated between both tasks. A re-analysis of published studies looking at element-to-element perceptual distances in larval reward learning and in adult punishment learning confirms this result. We therefore suggest that across a given set of olfactory stimuli, associative training does not grossly alter the pattern of perceptual distances. KW - discrimination KW - drosophila melanogaster KW - generalization KW - memory KW - olfaction KW - perception Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121625 VL - 217 IS - 12 ER - TY - THES A1 - Kirscher, Lorenz T1 - Melanogene rekombinante Vaccinia-Viren als diagnostisches und therapeutisches Agenz zur Tumorbehandlung T1 - Melanogenic recombinant Vaccina Viruses as diagnostic and therapeutic agent for tumor treatment N2 - Die gängigen therapeutischen Behandlungsmethoden für die verschiedensten Krebserkrankungen zeigen nach wie vor Mängel bezüglich der Effizienz sowie zahlreiche Nebenwirkungen während und nach der Behandlung. Maßgeblich für diese Defizite ist die teilweise geringe Sensitivität der meisten konventionellen diagnostischen Systeme und damit einhergehend die oftmals zu späte Identifikation entarteter Gewebsbereiche. Zur Lösung dieser Problematik bieten onkolytische Vaccinia-Viren einen Ansatz, sowohl die Effizienz der Therapie wie auch die Diagnostik zu verbessern. In beiden Fällen sind die Tumorzell-spezifische Vermehrung der Viren und die Möglichkeit entscheidend, die Viren als Vektorsystem zur Expression therapeutischer oder diagnostischer Fremdgenkassetten zu nutzen. Um ein auf Vaccinia-Virus-basierendes Reportersystem zum diagnostischen Nachweis von Krebszellen mittels Tiefengewebs-Tomographie bereit zu stellen, wurden die für die murine Tyrosinase (mTyr) und das Tyrosinase-Helferprotein 1 (Tyrp1) kodierenden Gene in das Genom eines onkolytischen Vaccinia-Virus inseriert. Die Tyrosinase ist das Schlüsselenzym der Melaninsynthese. Bereits die solitäre Expression der Tyrosinase führt in der transformierten Zelle zur Melaninproduktion. Das Tyrosinase-Helferprotein 1 ist an der Prozessierung und Stabilisierung der Tyrosinase beteiligt. Bereits in verschiedenen Studien konnte gezeigt werden, dass Melanin als Reportermolekül für die Magnetresonanz sowie für die multispektrale optoakustische Tomographie einsetzbar ist. Es wurde deswegen angestrebt, die Kombination aus dem therapeutischen Potential des onkolytischen Vaccinia-Virus und der diagnostischen Anwendung des Melanins als Reporter auszunutzen. Sämtliche in dieser Arbeit aufgeführten rekombinanten Vaccinia-Viren (rVACV) wurden von der Firma Genelux Corporation zur Verfügung gestellt und in dieser Arbeit hinsichtlich der therapeutischen Effizienz und des diagnostischen Potentials untersucht. In ersten Zellkultur-Versuchen wurde anhand verschiedener konstitutiv melanogener rVACV-Konstrukte festgestellt, dass die Kombination aus dem Vaccinia-Virus-spezifischen synthetic early/late Promotor und dem Enzym Tyrosinase (GLV-1h327) bzw. den Enzymen Tyrosinase und Tyrosinase-Helferprotein 1 (GLV-1h324) die höchste Melaninsynthese-Rate zeigte. Anschließend wurde mittels der Bestimmung der spektralen Absorption und der Enzymaktivität der viral kodierten Melanin synthetisierenden Enzyme sowie mikroskopischer Analysen gezeigt, dass es mit diesen auf 8 Vaccinia-Virus-basierenden melanogenen Reportersystemen möglich ist, die Melaninsynthese in nicht-melanogenen Zellen zu induzieren. Anhand elektronenmikroskopischer Untersuchungen in Zellkultur und ex vivo konnte gezeigt werden, dass die nach rVACV-Infektion stattfindende Melaninsynthese in den Lysosomen der Wirtszelle abläuft. Eine Analyse der atomaren Zusammensetzung des viral vermittelten Melanins ergab, dass es sich um eine Mischform aus Eu- und Phäomelanin handelt. Dieser Melanin-Mix ähnelte dem Melanin aus Haut und Augen, jedoch lagen an Melanin-gebundene Metallionen in erhöhtem Maß vor... N2 - The common therapeutic approaches available to treat various cancers still have deficiencies concerning their efficiency and side effects. Decisive for these deficiencies is the poor sensitivity of most of the conventional diagnostic systems and associated with that the late, sometimes too late, identification of tumorous tissue. The oncolytic vaccinia virus is an opportunity to enhance the efficiency of both the therapy and the diagnosis of tumor tissue. In both cases, the specificity for tumor cells and the simplicity of inserting foreign gene cassettes into the viral genome are key factors. The genes encoding murine tyrosinase (mTyr) and tyrosinase related protein 1 (Tyrp1) were inserted into the genome of an oncolytic vaccinia virus. Tyrosinase is the key enzyme during melanogenesis and expression of this enzyme alone can lead to melanin production. Tyrp1 is involved in processing and stabilizing tyrosinase. Various studies have demonstrated melanin to be an excellent reporter molecule for MRI (magnetic resonance imaging) and MSOT (multispectral optoacoustic tomography). Therefore, melanogenic oncolytic vaccinia viruses were constructed to combine the therapeutic potential of this virus with diagnostic abilities of melanin. All recombinant vaccinia viruses (rVACV) mentioned were kindly provided by Genelux Corporation. In this thesis, we tested for their therapeutic efficiency and diagnostic potential. Initial cell culture experiments have shown that the combination of vaccinia virus-specific synthetic early/late promoter and the melanogenic enzyme tyrosinase (GLV-1h327) or the enzymes mTyr and Tyrp1 (GLV-1h324) have the highest melanin synthesis rate. It was observed that the infection of non-melanin producing cells with an rVACV-based melanogenic reporter system resulted in melanogenesis. Electron microscopy showed that the viral associated melanogenesis is localized in the lysosomes of the infected host cell. The atomic composition analysis of the virus-mediated melanin molecules revealed a mixture of eu- and pheomelanin. The virus-associated melanin is comparable to that in the skin and eye but showed higher amounts of melanin-bound metal ions. ... KW - Melanin KW - Vaccinia Virus KW - Onkolyse KW - MSOT KW - MRT KW - Tyrosinase Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112074 ER - TY - THES A1 - Vona, Barbara C. T1 - Molecular Characterization of Genes Involved in Hearing Loss T1 - Molekulare Charakterisierung der in Hörstörungen involvierten Genen N2 - The auditory system is an exquisitely complex sensory organ dependent upon the synchronization of numerous processes for proper function. The molecular characterization of hereditary hearing loss is complicated by extreme genetic heterogeneity, wherein hundreds of genes dispersed genome-wide play a central and irreplaceable role in normal hearing function. The present study explores this area on a genome-wide and single gene basis for the detection of genetic mutations playing critical roles in human hearing. This work initiated with a high resolution SNP array study involving 109 individuals. A 6.9 Mb heterozygous deletion on chromosome 4q35.1q35.2 was identified in a syndromic patient that was in agreement with a chromosome 4q deletion syndrome diagnosis. A 99.9 kb heterozygous deletion of exons 58-64 in USH2A was identified in one patient. Two homozygous deletions and five heterozygous deletions in STRC (DFNB16) were also detected. The homozygous deletions alone were enough to resolve the hearing impairment in the two patients. A Sanger sequencing assay was developed to exclude a pseudogene with a high percentage sequence identity to STRC from the analysis, which further solved three of the six heterozygous deletion patients with the hemizygous, in silico predicted pathogenic mutations c.2726A>T (p.H909L), c.4918C>T (p.L1640F), and c.4402C>T (p.R1468X). A single patient who was copy neutral for STRC and without pathogenic copy number variations had compound heterozygous mutations [c. 2303_2313+1del12 (p.G768Vfs*77) and c.5125A>G (p.T1709A)] in STRC. It has been shown that STRC has been previously underestimated as a hearing loss gene. One additional patient is described who does not have pathogenic copy number variation but is the only affected member of his family having hearing loss with a paternally segregating translocation t(10;15)(q26.13;q21.1). Twenty-four patients without chromosomal aberrations and the above described patient with an USH2A heterozygous deletion were subjected to a targeted hearing loss gene next generation sequencing panel consisting of either 80 or 129 hearing-relevant genes. The patient having the USH2A heterozygous deletion also disclosed a second mutation in this gene [c.2276G>T (p.C759F)]. This compound heterozygous mutation is the most likely cause of hearing loss in this patient. Nine mutations in genes conferring autosomal dominant hearing loss [ACTG1 (DFNA20/26); CCDC50 (DFNA44); EYA4 (DFNA10); GRHL2 (DFNA28); MYH14 (DFNA4A); MYO6 (DFNA22); TCF21 and twice in MYO1A (DFNA48)] and four genes causing autosomal recessive hearing loss were detected [GJB2 (DFNB1A); MYO7A (DFNB2); MYO15A (DFNB3), and USH2A]. Nine normal hearing controls were also included. Statistical significance was achieved comparing controls and patients that revealed an excess of mutations in the hearing loss patients compared to the control group. The family with the GRHL2 c.1258-1G>A mutation is only the second family published worldwide with a mutation described in this gene to date, supporting the initial claim of this gene causing DFNA28 hearing loss. Audiogram analysis of five affected family members uncovered the progressive nature of DFNA28 hearing impairment. Regression analysis predicted the annual threshold deterioration in each of the five family members with multiple audiograms available over a number of years. N2 - Das Gehör als komplexes Sinnesorgan ist für eine einwandfreie Funktion abhängig von der Synchronisation zahlreicher Prozesse. Durch die extreme genetische Heterogenität wird die molekulare Charakterisierung einer erblich bedingten Schwerhörigkeit erschwert, da hunderte genomweit verteilter Gene eine zentrale und unersetzliche Rolle beim Hören spielen. Die vorliegende Studie untersucht dieses Forschungsgebiet auf genomweiter Ebene und auf der Basis von Einzelgenen, um genetische Mutationen zu ermitteln, die eine entscheidende Rolle bei der menschlichen auditiven Wahrnehmung besitzen. Diese Arbeit beginnt mit einer Studie an 109 Personen unter Zuhilfenahme von hochauflösenden SNP-Arrays. In dieser Studie wurde eine 6,9 Mb heterozygote Deletion auf Chromosom 4q35.1q35.2 bei einem syndromalen Patienten identifiziert, die eine Übereinstimmung mit einem Chromosom 4q-Deletionssyndrom aufwies. Bei einem weiteren Patienten wurde eine 99,9 kb heterozygote Deletion der Exons 58-64 in USH2A nachgewiesen. Zwei homozygote Deletionen und fünf heterozygote Deletionen in STRC (DFNB16) wurden ebenfalls detektiert. Die homozygoten Deletionen waren ausreichend, um die Schwerhörigkeit bei beiden Patienten zu klären. Ein Sanger-Sequenzierungs-Assay wurde entwickelt, um ein Pseudogen mit einer hohen prozentualen Sequenzidentität zu STRC von der Analyse auszuschließen. Dadurch konnten drei der sechs heterozygoten Deletionspatienten mit hemizygot in silico vorhergesagten pathogenen Mutationen, c.2726A>T (p.H909L), c.4918 C>T (p.L1640F) und c.4402C>T (p.R1468X), aufgeklärt werden. Ein Patient, der eine kopieneutrale STRC Variation und keine pathogenen Kopienzahlvariationen besaß, zeigte eine compound heterozygote Mutation [c.2303_2313+1del12 (p.G768Vfs*77) und c.5125A>G (p.T1709A)] in STRC. Es wurde gezeigt, daß die Beurteilung von STRC als Hörstörungsgen bisher unterschätzt wurde. Zusätzlich wird ein Patient beschrieben, der keine pathogenen Kopienzahlvariationen aufwies, aber das einzige Familienmitglied mit einer Schwerhörigkeit und einer paternalen segregierten Translokation t(10;15)(q26.13;q21.1) war. Vierundzwanzig Patienten ohne Chromosomenstörungen und der oben beschriebene Patient mit einer USH2A heterozygoten Deletion wurden mit einem Next Generation Sequencing Panel bestehend aus entweder 80 oder 129 für das Hören relevanter Gene untersucht. Der Patient mit einer USH2A heterozygoten Deletion zeigte eine zweite Mutation in diesem Gen [c.2276G>T (p.C759F)]. Diese compound heterozygote Mutation ist die wahrscheinlichste Ursache für die Schwerhörigkeit des Patienten. Neun Mutationen in Genen, die zu einem autosomal dominanten Hörverlust führen [ACTG1 (DFNA20/26); CCDC50 (DFNA44); EYA4 (DFNA10); GRHL2 (DFNA28); MYH14 (DFNA4A); MYO6 (DFNA22); TCF21], sowie zwei MYO1A (DFNA48) Mutationen und Mutationen in vier weiteren Genen, verantwortlich für autosomal rezessive Schwerhörigkeit [GJB2 (DFNB1A); MYO7A (DFNB2); MYO15A (DFNB3) und USH2A], konnten identifiziert werden. Neun normal hörende Kontrollen waren ebenfalls in diese Studie einbezogen worden. Durch einen Vergleich der Kontrollen mit den Patienten konnte eine statistische Signifikanz erreicht werden, die einen Überschuss an Mutationen bei der Patientengruppe gegenüber der Kontrollgruppe aufzeigte. Die Familie mit einer GRHL2 c.1258-1G>A Mutation ist die erst zweite Familie weltweit, die mit einer Mutation in diesem Gen publiziert worden ist. Dies unterstützt die initiale Behauptung, dass dieses Gen für eine DFNA28 Schwerhörigkeit verantwortlich ist. Die Audiogrammanalyse von fünf der betroffenen Familienmitglieder lässt eine voranschreitende Natur der DFNA28 Hörschädigung erkennen. Eine jährliche Verschlechterung der Hörschwelle bei jedem der fünf Familienmitglieder konnte eine Regressionsanalyse anhand von Audiogrammen, die über eine Anzahl von Jahren zur Verfügung standen, vorhersagen. KW - Molekularbiologie KW - Hearing loss KW - Hörverlust Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112170 N1 - Dieses Dokument wurde aus Datenschutzgründen - ohne inhaltliche Änderungen - erneut veröffentlicht. Die ursprüngliche Veröffentlichung war am: 09.07.2014 ER - TY - THES A1 - Kubisch (geb. Wiegand), Franziska T1 - Learning in botanical gardens: Investigating educational methods during an instruction about plants and water T1 - Lernen in Botanischen Gärten: Die Untersuchung von Lehrmethoden während einer Intervention über Pflanzen und Wasser N2 - The contribution of botanical gardens to out-of-school education should be larger than it is currently in Germany. In the curricula of all school types botany plays only a minor role, although plants form the base for all animal life on earth. To increase the attractiveness of botanical gardens for teachers, offers and programs should be created and conducted in didactically sensible manners and allow students an emotional approach towards the topics through trial and experiments. Therefore it is insufficient to conduct guided tours, which are still most common. Student-centered methods, like learning at workstations, or experimental courses, can lead to an improved retention of the contents learned at the out-of-school learning setting. There are, however, methodological differences even within learning at workstations. In the first part of my study I compared a student- (S) and a teacher-centered (T) type of learning at workstations (chapter III). My intention was to find out, which of both methods results in more positive emotions at the out-of-school learning location and a higher sustainable knowledge increase. Like in all three parts of my study, 8th grade students from so-called “Mittelschulen” and “Realschulen” from Lower Franconia participated in the programs. I evaluated them by using multiple-choice tests assessing the students' knowledge regarding the topic 'plants and water' (see Appendix), following a before-after / control-impact study design. The students' emotions were assessed using the intrinsic motivation inventory directly after the garden visit. Using generalized linear mixed models, I did not find a significant difference between either of the two approaches. A reason for this could be that the students could be practically active in both methods, which made them fairly similar. Given that there was a significant knowledge increase in both methods, and the effort to develop the teacher-centered learning at workstations was much lower, I would suggest to follow that method for educational work in botanical gardens. Students already have many predefined concepts regarding many topics, especially when these are important in everyday life. These concepts do often not match the scientific state-of-the-art. Still, students bring their so-called 'alternative conceptions' into visits to the botanical garden. According to theory, confronting them with their own conceptions in the light of scientific facts, should foster updating their concepts with scientifically correct additions. To investigate this method regarding my topic 'plants and water', I developed an intervention with experiments on the lotus effect, which also plays a role in everyday life (chapter IV). Topics like the surface tension of the water, which is also found in 6th grade curricula in German schools, were included. Prior to the intervention, I assessed the students' conceptions using questionnaires and used the three most frequent alternative conceptions to develop a multiple-choice test, which was also used in a before-after / control-impact design. A group of students was also confronted with their conceptions during an introductory talk (AC), whereas another was not (NAC). This was conducted in a way, that likely led to dissatisfaction of the students with their own concepts. The analysis of the questionnaires with the Mann-Whitney U test showed, however, no difference between the two groups directly following the treatment. Over longer time, however, the NAC group retained significantly more knowledge. Probably the students confronted with the alternative conceptions remembered the illustrations of these more easily than the scientifically correct view. For some botanical topics it is certainly helpful to include this conceptual change approach, but apparently not for the lotus effect. In this case it is most sensible to focus on the surface structure of water-repellent leaves and fruits, as we describe it in a publication in 'Unterricht Biologie'. For the practical work in botanical gardens I would suggest to rather assess the students' concepts and assumptions in the beginning of an intervention in a botanical garden, especially with respect to feasibility. In the third part of my study I concentrate on the application of concept maps (chapter V). This method of cross-linking old and newly acquired knowledge is effective, but not very common in Germany, neither in schools, nor in botanical gardens. One group of students followed exclusively a teacher-centered learning at workstations regarding 'plants and water' (NCM), a second group created concept maps directly after the treatment and a second directly before the retention test (CM). The first map was intended to be a means of consolidation, whereas the late map was rather focused on recapitulation of what was learned about six weeks ago. To evaluate that I used the same multiple-choice tests as I did for the first part. The CM group showed a significantly higher knowledge increase, over short and long time-scales, although these students did significantly worse in the pretest than those of the NCM group. Regarding genders, female students profited especially from the first concept map (consolidation), males rather from the second (recapitulation). From the results one can conclude that prominently weaker students benefit from this method. Additionally the gender-related results show that using concept maps multiple times can be beneficial for different types of learners. In every study there also was a control group (C), which only had to fill out the questionnaires at the same time as the participating students, to account for external factors (like media, etc.). Especially learning at workstations and concept maps are very appropriate to be conducted at the out-of-school learning location botanical garden and are likely to strongly increase learning success. It is beneficial to mix several methods to achieve the best results in different types of learners. Additionally, when methods in school are mixed with those of out-of-school learning, the education gets more open, practical and colorful. That all resulted in a substantial long-term knowledge gain of all participating students. N2 - Der Beitrag botanischer Gärten zur außerschulischen Bildung sollte größer sein, als er momentan in Deutschland ist, denn in den Lehrplänen aller Schularten spielt die Botanik eine sehr geringe Rolle, obwohl Pflanzen die Grundlage allen tierischen Lebens sind. Doch um diesen Lernort für Lehrer attraktiver zu machen, sollten die Programme und Angebote didaktisch aufbereitet sein und den Schülern durch Ausprobieren und Experimentieren einen emotionalen Zugang bieten. Hierfür genügt es nicht, Führungen und Lehrervorträge durchzuführen, welche noch immer zu hohen Prozentsätzen stattfinden. Schülerzentrierte Methoden, wie das Lernen an Stationen, oder experimentelle Praktika, können dazu führen, dass das am außerschulischen Lernort (ASL) Gelernte besser im Gedächtnis bleibt. Jedoch gibt es auch beim Lernen an Stationen methodische Unterschiede. Im ersten Teil meiner Studie habe ich eine schülerzentrierte (S) gegen eine lehrerzentrierte (T) Form des Lernens an Stationen gegeneinander getestet (siehe Kapitel III), um herauszufinden, welche der beiden Methoden zu positiveren Emotionen am ASL und einem erhöhten, anhaltenden Wissenszuwachs führt. Wie bei allen drei Teilen meiner Studie nahmen Schüler und Schülerinnen der 8. Jahrgangstufe von Mittel-und Realschulen aus Unterfranken teil. Evaluiert wurde mithilfe eines selbst entwickelten Multiple-Choice-Tests zum Wissen der Schüler und Schülerinnen zum Thema Wasser und Pflanzen (siehe Appendix 5). Dieser Test erfolgte als Vor- und Nachtest sowie als verzögerter Behaltenstest (retention). Die Emotionen der Schüler und Schülerinnen wurden über den IMI-Fragebogen (intrinsic motivation inventory) direkt nach dem Besuch im botanischen Garten einmalig erfragt. Weder beim Wissenstest, noch bei den Emotionen, ergab sich nach Auswertung mittels generalisierter gemischter linearer Modelle (GLMM) ein klares Signal für eine der beiden Methoden. Ein Grund könnte sein, dass bei beiden Formen des Lernens an Stationen die Schüler und Schülerinnen auch praktisch aktiv werden konnten, sich die Methoden somit sehr ähnelten. Da bei beiden Methoden insgesamt signifikant dazu gelernt wurde und das eher lehrerzentierte Lernen an Stationen nicht so aufwändig war in der Entwicklung wie das schülerzentrierte, würde ich den botanischen Gärten erstere Methode für die Bildungsarbeit empfehlen. Schüler und Schülerinnen haben zu vielen Themen, vor allem des Alltags, bereits ganz eigene Konzepte und Vorstellungen, die nicht unbedingt denen das aktuellen wissenschaftlichen Standes entsprechen. So kommen die Schüler und Schülerinnen natürlich auch mit diesen individuellen Konzepten in den botanischen Garten. Hier sollte nun auch auf diese sogenannten “alternativen Konzepte” eingegangen werden, da diese Konfrontation, laut Theorie, die Übernahme neuer, wissenschaftlich korrekter Bausteine in das bereits vorhandene Konzept fördern soll. Um bei dem Thema Wasser und Pflanzen zu bleiben und gleichzeitig ein alltagsrelevantes Thema anzusprechen, habe ich ein Praktikum und Experimente zum Lotuseffekt entwickelt und die Vorstellungen der Schüler und Schülerinnen dazu erfragt (siehe chapter IV). Hierbei spielten auch Themen wie die Oberflächenspannung von Wasser eine Rolle, was in Deutschland in der 6. Klasse angesprochen wird. Aus nicht korrekten Vorstellungen wurden die drei häufigsten ausgesucht und daraus ein Multiple-Choice-Test entwickelt, der ebenfalls als Vor-, Nach- und Behaltenstest fungierte. In einem einführenden Vortrag zum Lotuseffekt wurde ein Teil der Schüler und Schülerinnen (AC) zusätzlich mit diesen alternativen Vorstellungen konfrontiert, über Bilder und im Unterrichtsgespräch. Dies erfolgte in einer Art und Weise, sodass die Schüler und Schülerinnen mit der eigenen Vorstellung unzufrieden wurden. Eine zweite Gruppe wurde während des Vortrags nicht mit ihren alternativen Vorstellungen konfrontiert (NAC). Die Auswertung der Fragebögen über den Mann-Whitney U Test ergab keinen Unterschied zwischen den beiden Gruppen im Hinblick auf den kurzfristigen Wissenserwerb. Die Gruppe jedoch, welche nicht mit ihren alternativen Vorstellungen konfrontiert wurde (NAC), lernte langfristig im Vergleich zu der AC-Gruppe signifikant mehr dazu. Womöglich erinnerten sich die Schüler und Schülerinnen der AC-Gruppe nur noch an die Bilder der falschen und nicht an die der wissenschaftlich korrekten Vorstellung und wurden somit irritiert. Bei einigen botanischen Themen ist es sicherlich von Vorteil, die alternativen Vorstellungen der Schüler und Schülerinnen einzubringen, vielleicht nicht unbedingt beim Lotuseffekt. Hier sollte man sich, wie in einem von uns dazu verfassten Artikel in der Unterricht Biologie beschrieben sein wird, auf die Oberflächenstruktur von wasserabweisenden Blättern und Früchten beschränken. Für die pädagogische Arbeit in botanischen Gärten würde ich die mündliche Abfrage der Vorstellungen und Vermutungen der Schüler und Schülerinnen zu Beginn eines Programmes aus Gründen der besseren und schnelleren Umsetzbarkeit empfehlen. Im dritten Teil meiner Studie beschäftigte ich mich mit der Anwendung von Concept Maps (siehe Kapitel V). Diese Methode des Vernetzens von altem und neu erworbenem Wissen ist effektiv, aber weder in deutschen Schulen, noch in botanischen Gärten weit verbreitet. Eine Gruppe folgte ausschließlich dem lehrerzentrierten Lernen an Stationen zu Wasser und Pflanzen (NCM), eine zweite Gruppe erstellte direkt im Anschluss an das lehrerzentrierte Lernen an Stationen sowie direkt vor dem Behaltenstest eine Concept Map (CM). Die erste Map diente hierbei als Sicherungsform des gerade Gelernten und die späte Map als Wiederholung des vor circa sechs Wochen Gelernten. Als Evaluationsinstrument diente erneut der eigens entwickelte Multiple-Choice-Wissenstest aus der ersten Teilstudie. Die CM-Gruppe zeigte einen signifikant größeren Lernzuwachs, kurz- wie auch langfristig, im Vergleich zur NCM-Gruppe, obwohl die CM-Gruppe im Vortest signifikant schlechter war. Im Hinblick auf die Geschlechter haben die Mädchen vor allem von der ersten Sicherungs-Map und die Jungen mehr von der Wiederholungs-Map profitiert. Anhand der Ergebnisse kann man schlussfolgern, dass vor allem schwächere Schüler und Schülerinnen von dieser Methode profitieren. Außerdem zeigen die Ergebnisse zu den Geschlechtern, dass das mehrmalige Anwenden von Concept Maps unterschiedliche Lerntypen fördern kann. Bei jeder der drei Studien gab es eine Kontrollgruppe (C), die ausschließlich die Fragebögen im Abstand von sechs bis acht Wochen in der Schule beantworten musste. Dies diente dem Ausschließen von Vorkomnissen in der Öffentlichkeit und dem Umfeld der Schüler und Schülerinnen, was deren Wissen zu Wasser und Pflanzen und dem Lotuseffekt hätte erheblich steigern können. Vor allem das Lernen an Stationen sowie die Concept Maps lassen sich sehr gut am ASL botanischer Garten durchführen und können zu einem größeren Lernerfolg führen. Am besten spricht man hier die meisten Lerntypen an, wenn man während der Programme möglichst viele verschiedene Methoden anwendet. Dazu kommt, dass man neben den Methoden aus der Schule natürlich auch die des ASL einbringt und so der Unterricht automatisch anschaulicher, offener und praktischer wird. Dies alles hat dazu geführt, das alle Gruppen langfristig signifikant dazu gelernt haben. KW - Konstruktive Didaktik KW - Out-of-school learning settings KW - botanical gardens KW - conceptual change KW - learning at workstations KW - concept maps KW - Botanischer Garten KW - Lernort KW - Stationenarbeit Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111620 ER - TY - THES A1 - Hondke, Sylvia T1 - Elucidation of WISP3 function in human mesenchymal stem cells and chondrocytes T1 - Aufklärung der WISP3 Funktion in humanen mesenchymalen Stammzellen und Chondrozyten N2 - WISP3 is a member of the CCN family which comprises six members found in the 1990’s: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes. WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis. Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells. N2 - WISP3 ist ein Mitglied der CCN-Familie, die aus sechs Familienmitgliedern besteht und in den 1990er Jahren endeckt wurde: Cysteine-rich, angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) und den Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6). Die CCN-Proteine sind durch ihre Interaktion mit verschiede- nen Integrinen und Heparansulfaten involviert in die Regulation der Adhäsion, der Migration, der Mi- togenese, der Chemotaxis, der Proliferation, des Zellüberlebens, der Angiogenese, der Tumorgenese und der Wundheilung. WISP3 ist momentan das einzige Mitglied, das direkt mit einer muskuloskelettalen Erkrankung, der Progressiven Pseudorheumatoiden Dysplasie (PPD), assoziiert wird. PPD ist charakter- isiert durch eine normale embryonale Entwicklung mit fortschreitender Knorpeldegeneration beginnend im Alter von drei bis acht Jahren. Tierversuche mit knock out oder Überexpression von WISP3 in Mäusen waren nicht in der Lage die Symptome der Erkrankung nachzustellen, da keine Unterschiede im Vergleich zu den Wurfgeschwistern beobachtbar waren. In vitro und in vivo Studien offenbarten eine antagonisierende Rolle für WISP3 im BMP, IGF und Wnt Signalweg. Da die meisten Informationen über WISP3 jedoch in Epithel- und Krebszellen sowie immortalisierten Chondrozytenzelllinien generiert wurden, untersuchten wir die Rolle von WISP3 in primären humanen mesenchymalen Stammzellen (hMSZs) und primären Chondrozyten. Der WISP3 knock down wurde mit drei short hairpin RNAs in beiden Zelltypen etabliert und wies eine veränderte Zellmorphologie sowie eine reduzierte Zellzahl auf. Knock down mit gleichzeitiger rekombi- nanter WISP3-Behandlung konnte den beobachteten Phänotyp sowie den Zellverlust nicht retten und auch eine Änderung der endogenen Genexpression von WISP3 war nicht zu detektieren. Schlussfolgernd muss WISP3 ein wichtiger Überlebensfaktor sein, dessen Verlust zur Überschreitung von Zellzyklus- Kontrollpunkten führt, was in Apoptose mündet. Apoptosenachweise wie Annexin V-Cy3 Färbung, Immunfluoreszenzfärbung und Western blot für aktive Caspasen lieferten keine positiven Beweise für diese Form des Zelltodes. Auch die Genexpression der Todesrezeptoren TRAILR1 und TRAILR2, wichtig für die extrinsische Aktivierung der Apoptose, zeigte kein verändertes Expressionsmuster in WISP3-defizienten hMSZs. Autophagie als Zelltod wurde ebenfalls ausgeschlossen, nachdem im West- ern Blot kein gespaltene Form des Autophagiemarkers LC3 A/B zu detektieren war. Um die Rolle von WISP3 beim Zelltod weiter zu entschlüsseln, wurden Genom-Expressionsanalysen von WISP3-defizienten hMSZs im Vergleich zu Kontroll-hMSZs angefertigt. Die Analysen ergaben unterschiedlich regulierte Gene vor allem in den Bereichen Zellzyklus-Regulation, Adhäsion, Zytoskelett und Zelltod. Der durch WISP3-Verlust ausgelöste Zelltod kann möglicherweise durch die Aktivierung der Nektroptose über den BMP/TAK1/RIPK1 Signalweg erklärt werden. Es ist bekannt, dass WISP3 BMP4 bindet und so dessen Bindung an den Rezeptor verhindert. Bei WISP3 Verlust bindet BMP4 an seinen Rezeptor und aktiviert den Smad 2/3/4 Komplex der wiederum TAK1 phosphoryliert, wie zuvor in Epithelzellen demonstriert. TAK1 ist in der Lage die Caspase-induzierte Apoptose zu blockieren und auf diese Weise die Bildung des Nekrosomes auszulösen, welches zum Zelluntergang durch Nekroptose führt. Zusammen mit seiner Rolle in der Zellzyklus-Kontrolle und der extrazellulären Matrixadhäsion, die in humanen Brustepithelialzellen nachgewiesen wurden, unterstützen diese Daten eine Rolle für WISP3 als Tumorsuppressor und Überlebensfaktor in Zellen des Epithel und des muskuloskelettalen Systems. KW - Knorpelzelle KW - PPD KW - mesenchymal stem cells KW - cell death KW - chondrocytes KW - Mesenchymzelle KW - Dysplasie KW - Genexpression KW - Werk Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-109641 ER - TY - JOUR A1 - Wiegering, Armin A1 - Korb, Doreen A1 - Thalheimer, Andreas A1 - Kämmerer, Ulrike A1 - Allmanritter, Jan A1 - Matthes, Niels A1 - Linnebacher, Michael A1 - Schlegel, Nicolas A1 - Klein, Ingo A1 - Ergün, Süleyman A1 - Germer, Christoph-Thomas A1 - Otto, Christoph T1 - E7080 (Lenvatinib), a Multi-Targeted Tyrosine Kinase Inhibitor, Demonstrates Antitumor Activities Against Colorectal Cancer Xenografts N2 - Clinical prognosis of metastasized colorectal carcinoma (CRC) is still not at desired levels and novel drugs are needed. Here, we focused on the multi-tyrosine kinase inhibitor E7080 (Lenvatinib) and assessed its therapeutic efficacy against human CRC cell lines in vitro and human CRC xenografts in vivo. The effect of E7080 on cell viability was examined on 10 humanCRCcell lines and humanendothelial cells (HUVEC). The inhibitory effect of E7080 on VEGF-induced angiogenesis was studied in an ex vivo mouse aortic ring angiogenesis assay. In addition, the efficacy of E7080 against xenografts derived fromCRC cell lines and CRC patient resection specimenswithmutated KRASwas investigated in vivo. Arelatively low cytotoxic effect of E7080 on CRC cell viabilitywas observed in vitro. Endothelial cells (HUVEC)weremore susceptible to the incubation with E7080. This is in line with the observation that E7080 demonstrated an anti-angiogenic effect in a three-dimensional ex vivo mouse aortic ring angiogenesis assay. E7080 effectively disrupted CRC cell-mediated VEGF-stimulated growth of HUVEC in vitro. Daily in vivo treatment with E7080 (5 mg/kg) significantly delayed the growth of KRAS mutated CRC xenografts with decreased density of tumor-associated vessel formations and without tumor regression. This observation is in line with results that E7080 did not significantly reduce the number of Ki67-positive cells in CRC xenografts. The results suggest antiangiogenic activity of E7080 at a dosage thatwas well tolerated by nudemice. E7080 may provide therapeutic benefits in the treatment of CRC with mutated KRAS. KW - Chirurgie Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111165 ER - TY - THES A1 - Bettaga, Noomen T1 - Bedeutung der NO-sensitiven Guanylyl Cyclase bei der Angiogenese und der Arteriogenese in der Maus T1 - Role of NO-sensitive guanylyl cyclase in angiogenesis and arteriogenesis in mice N2 - Stickstoffmonoxid (NO) spielt eine wichtige Rolle bei Gefäßremodelling-Prozessen wie Angiogenese und Arteriogenese. Die NO-Synthese im Gefäßsystem wird hauptsächlich durch die endotheliale NO-Synthase (eNOS) gewährleistet. Sie kann durch verschiedene Faktoren wie Scherkräfte und Zytokine wie der vaskuläre endotheliale Wachstumsfaktor (VEGF) reguliert werden. VEGF ist ein wichtiger Stimulator der Angiogenese und wird während dieses Prozesses hochreguliert. Die meisten physiologischen Effekte von NO werden durch die NO-sensitive Guanylyl-Cyclase (NO-GC) vermittelt. Als Hauptrezeptor für NO produziert die NO-GC den sekundären Botenstoff cyklisches Guanosinmonophosphat (cGMP) und führt dadurch zur Stimulation der verschiedenen Effektoren wie z.B. der PKG. Ob die Wirkung von NO in Angiogenese und Arteriogenese ebenfalls durch NO-GC vermittelt wird, war bis zum Beginn dieser Arbeit noch unklar. Die NO-GC besteht aus zwei Untereinheiten (α und ß). Die Deletion der ß1-Untereinheit in Mäusen resultiert in einer vollständigen Knockout Maus (GCKO). Mithilfe des Cre-LoxP-Systems wurden zusätzlich zellspezifische Knockout-Mäuse für glatte Muskelzellen (SMC-GCKO) und Endothelzellen (EC-GCKO) generiert. Um die Rolle der NO-GC in der Angiogenese und Arteriogenese zu untersuchen, wurden drei gut etablierte Methoden benutzt. Im ersten Teil des Projekts sollte die Expression der NO-GC in Endothelzellen untersucht werden. Zu diesem Zweck wurde die reverse Transkriptase-Polymerase-Kettenreaktion (RT-PCR) benutzt. Die Ergebnisse zeigen, dass die NO-GC in Endothelzellen der Lunge nur äußerst gering wenig exprimiert ist. Durch den Aortenring-Assay wurde eine Rolle der NO-GC bei der VEGF-vermittelten Angiogenese festgestellt. Dabei zeigte sich eine stärkere Angiogeneserate bei globaler Abwesenheit der NO-GC. Bei Fehlen der NO-GC ausschließlich in Endothelzellen zeigte sich kein Unterschied in den aussprossenden Aorten im Vergleich zu den Kontroll-Tieren. Dies zeigt, dass die NO-GC in Endothelzellen sehr wahrscheinlich keine Rolle bei der VEGF-vermittelten Angiogenese spielt. Im zweiten Teil wurde die Rolle der NO-GC bei der Angiogenese in einem in vivo-Modell untersucht. In dem Modell der Sauerstoff-induzierten-Retinopathie zeigten die GCKO-Mäuse eine verringerte Vaso-Obliteration, eine verlangsamte Angiogenese und eine erhöhte Tuft-Bildung. Ähnliche Ergebnisse wurden bei den SMC-GCKO-Tieren beobachtet. EC-GCKO-Mäuse zeigten eine gegenüber den Kontroll-Tieren unveränderte Vaso-Obliteration, Angiogeneserate und Tuft-Bildung. Diese Ergebnisse lassen darauf schließen, dass die NO-GC in Endothelzellen keine Rolle spielt. Immunfluoreszenz-Aufnahmen zeigten die Expression von NO-GC in Perizyten der Gefäßkapillaren der Mausretina. Daher könnte die NO-GC in diesem Zelltyp letztendlich für die Effekte bei den GCKO- und SMC-GCKO-Tieren verantwortlich sein. Im letzten Teil dieser Arbeit wurde eine Versuchsreihe unter Anwendung des Hinterlauf-Ischämie-Modells durchgeführt. Hierbei entwickelten die Pfoten aller GCKO- und teilweise der SMC-GCKO-Tiere nach der Ligation der Femoralarterie eine Nekrose. Die Regeneration der Hinterläufe der EC-GCKO-Tiere nach der Operation verlief normal. Diese Ergebnisse schließen eine bedeutende Rolle der NO-GC in Endothelzellen aus, zeigen allerdings, dass die NO-GC in den glatten Muskelzellen essentiell für den Arteriogenese-Prozess ist. Zusammengefasst führt die Deletion der NO-GC in glatten Muskelzellen und wahrscheinlich auch in Perizyten zur einer verlangsamten Angiogenese und Inhibierung der Arteriogenese. N2 - Nitric oxide (NO) plays an important role in vascular remodelling processes such as angiogenesis and arteriogenesis. The synthesis of NO in the vascular system is ensured mainly by endothelial NO synthase (eNOS). It can be regulated by a number of factors, such as shear stress and cytokines like the vascular endothelial growth factor (VEGF). VEGF is an important stimulator of angiogenesis and is upregulated during this process. Most of the physiological effects of NO are mediated by the NO-sensitive guanylyl cyclase (NO-GC). As the main receptor for NO, NO-GC produces the second messenger cyclic guanosine monophosphate (cGMP) and thereby leads to a variety of physiological effects. However, whether the effects of NO in angiogenesis and arteriogenesis are also mediated by NO-GC is still unclear. NO-GC consists of two subunits (α and ß). The deletion of the ß1 subunit in mice results in a global knockout mouse (GCKO). Using the Cre-LoxP system we also generated smooth muscle cell-specific (SMC GCKO) and endothelial cell-specific knockout mice (EC GCKO). To investigate the role of NO-GC in angiogenesis and arteriogenesis, three well-established methods have been used. In the first part of the project, the expression of the NO-GC in endothelial cells should be investigated. By using the reverse transcription polymerase chain reaction method (RT-PCR), the results show a very weak expression of the NO-GC in endothelial cells of the lung. A role for NO-GC in the VEGF-mediated angiogenesis was ascertained by the aortic ring assay. The results show an increased angiogenesis in the global absence of NO-GC. However, the EC-GCKO shows no difference compared to control mice. This indicates that NO-GC in endothelial cells is unlikely to play a major role in VEGF-mediated angiogenesis. In the second part of the project, the role of NO-GC in angiogenesis was investigated in an in vivo model. In the oxygen induced-retinopathy model (OIR), GCKO mice showed reduced vaso-obliteration, slowed angiogenesis and increased tuft formation. Similar results were observed in the SMC-GCKO animals. In contrast, EC-GCKO mice showed vaso-obliteration as well as angiogenesis rate and tuft formation similar to those seen in control animals. The results of this experiment suggest that NO-GC in endothelial cells is not involved in vaso-obliteration, physiological angiogenesis and tuft formation. Immunhistochemical analyses showed the expression of NO-GC in pericytes of the vascular capillaries of the mouse retina. Therefore, NO-GC in this cell type could be responsible for the effects in GCKO- and SMC-GCKO animals. In the last part of this thesis, hindlimb-ischemia experiments were performed. For this purpose, the paws of all GCKO- and some SMC-GCKO animals showed necrosis after ligation of the femoral artery. The regeneration of legs from EC-GCKO animals after the operation was normal. These results exclude a major role of NO-GC in endothelial cells, but show that NO-GC in smooth muscle cells is essential in the arteriogenesis process. In summary, the deletion of NO-GC in smooth muscle cells and probably also in pericytes leads to a slowed angiogenesis and inhibits arteriogenesis. KW - Guanylylcyclase KW - cGMP KW - Stickstoffmonoxid KW - Angiogenese KW - Arteriogenese KW - Maus Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111284 ER - TY - JOUR A1 - Floren, Andreas A1 - Mupepele, Anne-Christine A1 - Müller, Tobias A1 - Dittrich, Marcus T1 - Are Temperate Canopy Spiders Tree-Species Specific? N2 - Arboreal spiders in deciduous and coniferous trees were investigated on their distribution and diversity. Insecticidal knock-down was used to comprehensively sample spiders from 175 trees from 2001 to 2003 in the Białowieża forest and three remote forests in Poland. We identified 140 species from 9273 adult spiders. Spider communities were distinguished between deciduous and coniferous trees. The richest fauna was collected from Quercus where beta diversity was also highest. A tree-species-specific pattern was clearly observed for Alnus, Carpinus, Picea and Pinus trees and also for those tree species that were fogged in only four or three replicates, namely Betula and Populus. This hitherto unrecognised association was mainly due to the community composition of common species identified in a Dufrene-Legendre indicator species analysis. It was not caused by spatial or temporal autocorrelation. Explaining tree-species specificity for generalist predators like spiders is difficult and has to involve physical and ecological tree parameters like linkage with the abundance of prey species. However, neither did we find a consistent correlation of prey group abundances with spiders nor could differences in spider guild composition explain the observed pattern. Our results hint towards the importance of deterministic mechanisms structuring communities of generalist canopy spiders although the casual relationship is not yet understood. KW - trees KW - spiders KW - conifers KW - forests KW - predation KW - oaks KW - community structures KW - pines Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111413 ER - TY - JOUR A1 - Haydn, Johannes M. A1 - Hufnagel, Anita A1 - Grimm, Johannes A1 - Maurus, Katja A1 - Schartl, Manfred A1 - Meierjohann, Svenja T1 - The MAPK pathway as an apoptosis enhancer in melanoma JF - Oncotarget N2 - Inhibition of RAF/MEK/ERK signaling is beneficial for many patients with BRAFV600E–mutated melanoma. However, primary and secondary resistances restrict long-lasting therapy success. Combination therapies are therefore urgently needed. Here, we evaluate the cellular effect of combining a MEK inhibitor with a genotoxic apoptosis inducer. Strikingly, we observed that an activated MAPK pathway promotes in several melanoma cell lines the pro-apoptotic response to genotoxic stress, and MEK inhibition reduces intrinsic apoptosis. This goes along with MEK inhibitor induced increased RAS and P-AKT levels. The protective effect of the MEK inhibitor depends on PI3K signaling, which prevents the induction of pro-apoptotic PUMA that mediates apoptosis after DNA damage. We could show that the MEK inhibitor dependent feedback loop is enabled by several factors, including EGF receptor and members of the SPRED family. The simultaneous knockdown of SPRED1 and SPRED2 mimicked the effects of MEK inhibitor such as PUMA repression and protection from apoptosis. Our data demonstrate that MEK inhibition of BRAFV600E-positive melanoma cells can protect from genotoxic stress, thereby achieving the opposite of the intended anti-tumorigenic effect of the combination of MEK inhibitor with inducers of intrinsic apoptosis. KW - PI3K KW - melanoma KW - RAS KW - chemotherapy resistance KW - crosstalk Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120649 SN - 1949-2553 VL - 5 IS - 13 ER - TY - JOUR A1 - Dandekar, Thomas A1 - Fieselmann, Astrid A1 - Fischer, Eva A1 - Popp, Jasmin A1 - Hensel, Michael A1 - Noster, Janina T1 - Salmonella—how a metabolic generalist adopts an intracellular lifestyle during infection JF - Frontiers in Cellular and Infection Microbiology N2 - The human-pathogenic bacterium Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, “-omics” data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology. KW - regulation KW - virulence KW - "-omics" KW - metabolism KW - Salmonella-containing vacuole (SCV) Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120686 SN - 2235-2988 VL - 4 IS - 191 ER - TY - JOUR A1 - Klatt, Björn K. A1 - Holzschuh, Andrea A1 - Westphal, Catrin A1 - Clough, Yann A1 - Smit, Inga A1 - Pawelzik, Elke A1 - Tscharntke, Teja T1 - Bee pollination improves crop quality, shelf life and commercial value JF - Proceedings of the Royal Society B: Biological Sciences N2 - Pollination improves the yield of most crop species and contributes to one-third of global crop production, but comprehensive benefits including crop quality are still unknown. Hence, pollination is underestimated by international policies, which is particularly alarming in times of agricultural intensification and diminishing pollination services. In this study, exclusion experiments with strawberries showed bee pollination to improve fruit quality, quantity and market value compared with wind and self-pollination. Bee-pollinated fruits were heavier, had less malformations and reached higher commercial grades. They had increased redness and reduced sugar–acid–ratios and were firmer, thus improving the commercially important shelf life. Longer shelf life reduced fruit loss by at least 11%. This is accounting for 0.32 billion US$ of the 1.44 billion US$ provided by bee pollination to the total value of 2.90 billion US$ made with strawberry selling in the European Union 2009. The fruit quality and yield effects are driven by the pollination-mediated production of hormonal growth regulators, which occur in several pollination-dependent crops. Thus, our comprehensive findings should be transferable to a wide range of crops and demonstrate bee pollination to be a hitherto underestimated but vital and economically important determinant of fruit quality. KW - commercial grades KW - ecosystem services KW - post-harvest quality KW - shelf life KW - strawberry KW - crop yield KW - ecology Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120797 VL - 281 IS - 1775 ER - TY - JOUR A1 - Stefanovic, Sonia A1 - Barnett, Phil A1 - van Duijvenboden, Karel A1 - Weber, David A1 - Gessler, Manfred A1 - Christoffels, Vincent M. T1 - GATA-dependent regulatory switches establish atrioventricular canal specificity during heart development JF - Nature Communications N2 - The embryonic vertebrate heart tube develops an atrioventricular canal that divides the atrial and ventricular chambers, forms atrioventricular conduction tissue and organizes valve development. Here we assess the transcriptional mechanism underlying this localized differentiation process. We show that atrioventricular canal-specific enhancers are GATA-binding site-dependent and act as switches that repress gene activity in the chambers. We find that atrioventricular canal-specific gene loci are enriched in H3K27ac, a marker of active enhancers, in atrioventricular canal tissue and depleted in H3K27ac in chamber tissue. In the atrioventricular canal, Gata4 activates the enhancers in synergy with Bmp2/Smad signalling, leading to H3K27 acetylation. In contrast, in chambers, Gata4 cooperates with pan-cardiac Hdac1 and Hdac2 and chamber-specific Hey1 and Hey2, leading to H3K27 deacetylation and repression. We conclude that atrioventricular canal-specific enhancers are platforms integrating cardiac transcription factors, broadly active histone modification enzymes and localized co-factors to drive atrioventricular canal-specific gene activity. KW - biological sciences KW - developmental biology KW - molecular biology Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121437 SN - 2041-1723 VL - 5 IS - 3680 ER - TY - JOUR A1 - Zhan, Hong A1 - Stanciauskas, Ramunas A1 - Stigloher, Christian A1 - Dizon, Kevin K. A1 - Jospin, Maelle A1 - Bessereau, Jean-Luis A1 - Pinaud, Fabien T1 - In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans JF - Nature Communications N2 - Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals. Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121125 VL - 5 IS - 4974 ER - TY - JOUR A1 - Dusik, Verena A1 - Senthilan, Pingkalai R. A1 - Mentzel, Benjamin A1 - Hartlieb, Heiko A1 - Wülbeck, Corina A1 - Yoshii, Taishi A1 - Raabe, Thomas A1 - Helfrich-Förster, Charlotte T1 - The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock JF - PLoS Genetics N2 - All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways. KW - in vitro kinase assay KW - biological locomotion KW - circadian oscillators KW - MAPK signaling cascades KW - circadian rhythms KW - drosophila melanogaster KW - neurons KW - phosphorylation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119433 SN - 1553-7404 VL - 10 IS - 8 ER - TY - JOUR A1 - Wirth, Christine C. A1 - Glushakova, Svetlana A1 - Scheuermayer, Matthias A1 - Repnik, Urska A1 - Garg, Swatl A1 - Schaack, Dominik A1 - Kachman, Marika M. A1 - Weißbach, Tim A1 - Zimmerberg, Joshua A1 - Dandekar, Thomas A1 - Griffiths, Gareth A1 - Chitnis, Chetan E. A1 - Singh, Shallja A1 - Fischer, Rainer A1 - Pradel, Gabriele T1 - Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes JF - Cellular Microbiology N2 - Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(−)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(−) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(−) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(−) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(−) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(−) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm. Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120895 VL - 16 IS - 5 ER - TY - JOUR A1 - McCarthy, Michael A. A1 - Moore, Alana L. A1 - Krauss, Jochen A1 - Morgan, John W. A1 - Clements, Christopher F. T1 - Linking Indices for Biodiversity Monitoring to Extinction Risk Theory T1 - Conectando Índices para el Monitoreo de la Biodiversidad con la Teoría de Riesgo de Extinción JF - Conservation Biology N2 - Biodiversity indices often combine data from different species when used in monitoring programs. Heuristic properties can suggest preferred indices, but we lack objective ways to discriminate between indices with similar heuristics. Biodiversity indices can be evaluated by determining how well they reflect management objectives that a monitoring program aims to support. For example, the Convention on Biological Diversity requires reporting about extinction rates, so simple indices that reflect extinction risk would be valuable. We developed 3 biodiversity indices that are based on simple models of population viability that relate extinction risk to abundance. We based the first index on the geometric mean abundance of species and the second on a more general power mean. In a third index, we integrated the geometric mean abundance and trend. These indices require the same data as previous indices, but they also relate directly to extinction risk. Field data for butterflies and woodland plants and experimental studies of protozoan communities show that the indices correlate with local extinction rates. Applying the index based on the geometric mean to global data on changes in avian abundance suggested that the average extinction probability of birds has increased approximately 1% from 1970 to 2009. N2 - Los índices de biodiversidad combinan frecuentemente los datos de diferentes especies cuando se usan en los programas de monitoreo. Las propiedades heurísticas pueden sugerir índices preferidos, pero carecemos de medios objetivos para discriminar a los índices con propiedades heurísticas similares. Los índices de biodiversidad pueden evaluarse al determinar qué tan bien reflejan los objetivos de manejo que un programa de monitoreo busca apoyar. Por ejemplo, la Convención sobre la Diversidad Biológica requiere reportar las tasas de extinción, así que los índices que reflejan el riesgo de extinción serían valiosos. Desarrollamos 3 índices de biodiversidad que se basan en modelos sencillos de viabilidad de población y que relacionan el riesgo de extinción con la abundancia. Basamos el primer índice en la media geométrica de la abundancia de especies, y el segundo en una media de poder m´as general. En el tercer índice integramos la media geométrica y la tendencia. Estos índices requieren los mismos datos que índices previos, pero también se relacionan directamente con el riesgo de extinci´on. La información de campo sobre mariposas y plantas de bosque, y los estudios experimentales de comunidades protozoarias, muestran que los índices se correlacionan con las tasas locales de extinción. Al aplicar el índice basado en la media geométrica sobre los datos globales de los cambios en la abundancia de aves, sugirió que la probabilidad de extinción promedio de aves ha incrementado aproximadamente 1% desde 1970 hasta 2009. KW - biodiversity index KW - biodiversity measure KW - extinction risk KW - geometric mean KW - riesgo de extinción KW - medida de la biodiversidad KW - media geométrica KW - índice de biodiversidad Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121218 VL - 28 IS - 6 ER - TY - JOUR A1 - Naseem, Muhammad A1 - Kunz, Meik A1 - Dandekar, Thomas T1 - Probing the unknowns in cytokinin-mediated immune defense in Arabidopsis with systems biology approaches JF - Bioinformatics and Biology Insights N2 - Plant hormones involving salicylic acid (SA), jasmonic acid (JA), ethylene (Et), and auxin, gibberellins, and abscisic acid (ABA) are known to regulate host immune responses. However, plant hormone cytokinin has the potential to modulate defense signaling including SA and JA. It promotes plant pathogen and herbivore resistance; underlying mechanisms are still unknown. Using systems biology approaches, we unravel hub points of immune interaction mediated by cytokinin signaling in Arabidopsis. High-confidence Arabidopsis protein-protein interactions (PPI) are coupled to changes in cytokinin-mediated gene expression. Nodes of the cellular interactome that are enriched in immune functions also reconstitute sub-networks. Topological analyses and their specific immunological relevance lead to the identification of functional hubs in cellular interactome. We discuss our identified immune hubs in light of an emerging model of cytokinin-mediated immune defense against pathogen infection in plants. KW - plant hormones KW - systems biology KW - interaction networks KW - gene expression KW - cytokinin Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120199 SN - 1177-9322 VL - 8 ER - TY - JOUR A1 - Leonhardt, Sara D. A1 - Kaltenpoth, Martin T1 - Microbial Communities of Three Sympatric Australian Stingless Bee Species JF - PLoS ONE N2 - Bacterial symbionts of insects have received increasing attention due to their prominent role in nutrient acquisition and defense. In social bees, symbiotic bacteria can maintain colony homeostasis and fitness, and the loss or alteration of the bacterial community may be associated with the ongoing bee decline observed worldwide. However, analyses of microbiota associated with bees have been largely confined to the social honeybees (Apis mellifera) and bumblebees (Bombus spec.), revealing – among other taxa – host-specific lactic acid bacteria (LAB, genus Lactobacillus) that are not found in solitary bees. Here, we characterized the microbiota of three Australian stingless bee species (Apidae: Meliponini) of two phylogenetically distant genera (Tetragonula and Austroplebeia). Besides common plant bacteria, we find LAB in all three species, showing that LAB are shared by honeybees, bumblebees and stingless bees across geographical regions. However, while LAB of the honeybee-associated Firm4–5 clusters were present in Tetragonula, they were lacking in Austroplebeia. Instead, we found a novel clade of likely host-specific LAB in all three Australian stingless bee species which forms a sister clade to a large cluster of Halictidae-associated lactobacilli. Our findings indicate both a phylogenetic and geographical signal of host-specific LAB in stingless bees and highlight stingless bees as an interesting group to investigate the evolutionary history of the bee-LAB association. KW - bacteria KW - lactic acid bacteria KW - sequence alignment KW - insects KW - lactobacillus KW - sequence databases KW - honey bees Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119341 VL - 9 IS - 8 ER - TY - JOUR A1 - Adelfinger, Marion A1 - Gentschev, Ivaylo A1 - de Guibert, Julio Grimm A1 - Weibel, Stephanie A1 - Langbein-Laugwitz, Johanna A1 - Härtl, Barbara A1 - Escobar, Hugo Murua A1 - Nolte, Ingo A1 - Chen, Nanhai G. A1 - Aguilar, Richard J. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Frentzen, Alexa A1 - Szalay, Aladar A. T1 - Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy JF - PLoS ONE N2 - Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis. In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model. KW - antibodies KW - cancer treatment KW - carcinomas KW - vaccinia virus KW - oncolytic viruses KW - viral replication KW - cell cultures KW - enzyme-linked immunoassays Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119387 VL - 9 IS - 8 ER - TY - JOUR A1 - Siegl, Christine A1 - Prusty, Bhupesh K. A1 - Karunakaran, Karthika A1 - Wischhusen, Jörg A1 - Rudel, Thomas T1 - Tumor Suppressor p53 Alters Host Cell Metabolism to Limit Chlamydia trachomatis Infection JF - Cell Reports N2 - Obligate intracellular bacteria depend entirely on nutrients from the host cell for their reproduction. Here, we show that obligate intracellular Chlamydia downregulate the central tumor suppressor p53 in human cells. This reduction of p53 levels is mediated by the PI3K-Akt signaling pathway, activation of HDM2, and subsequent proteasomal degradation of p53. The stabilization of p53 in human cells severely impaired chlamydial development and caused the loss of infectious particle formation. DNA-damage-induced p53 interfered with chlamydial development through downregulation of the pentose phosphate pathway (PPP). Increased expression of the PPP key enzyme glucose-6-phosphate dehydrogenase rescued the inhibition of chlamydial growth induced by DNA damage or stabilized p53. Thus, downregulation of p53 is a key event in the chlamydial life cycle that reprograms the host cell to create a metabolic environment supportive of chlamydial growth. KW - chlamydia trachomatis KW - tumor Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118200 SN - 2211-1247 VL - 9 IS - 3 ER - TY - JOUR A1 - Schülein-Völk, Christina A1 - Wolf, Elmar A1 - Zhu, Jing A1 - Xu, Wenshan A1 - Taranets, Lyudmyla A1 - Hellmann, Andreas A1 - Jänicke, Laura A. A1 - Diefenbacher, Markus E. A1 - Behrens, Axel A1 - Eilers, Martin A1 - Popov, Nikita T1 - Dual Regulation of Fbw7 Function and Oncogenic Transformation by Usp28 JF - CELL REPORTS N2 - Fbw7, the substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, mediates the turnover of multiple proto-oncoproteins and promotes its own degradation. Fbw7-dependent substrate ubiquitination is antagonized by the Usp28 deubiquitinase. Here, we show that Usp28 preferentially antagonizes autocatalytic ubiquitination and stabilizes Fbw7, resulting in dose-dependent effects in Usp28 knockout mice. Monoallelic deletion of Usp28 maintains stable Fbw7 but drives Fbw7 substrate degradation. In contrast, complete knockout triggers Fbw7 degradation and leads to the accumulation of Fbw7 substrates in several tissues and embryonic fibroblasts. On the other hand, overexpression of Usp28 stabilizes both Fbw7 and its substrates. Consequently, both complete loss and ectopic expression of Usp28 promote Ras-driven oncogenic transformation. We propose that dual regulation of Fbw7 activity by Usp28 is a safeguard mechanism for maintaining physiological levels of proto-oncogenic Fbw7 substrates, which is equivalently disrupted by loss or overexpression of Usp28. KW - Fbw7 KW - oncogenic transformation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118219 SN - 2211-1247 N1 - The sequencing data have been submitted to the GEO repository under accession number GSE59354. VL - 9 IS - 3 ER - TY - JOUR A1 - Naseem, Muhammad A1 - Srivastava, Mugdha A1 - Dandekar, Thomas T1 - Stem-cell-triggered immunity safeguards cytokinin enriched plant shoot apexes from pathogen infection JF - Frontiers in Plant Science N2 - Intricate mechanisms discriminate between friends and foes in plants. Plant organs deploy overlapping and distinct protection strategies. Despite vulnerability to a plethora of pathogens, the growing tips of plants grow bacteria free. The shoot apical meristem (SAM) is among three stem cells niches, a self-renewable reservoir for the future organogenesis of leaf, stem, and flowers. How plants safeguard this high value growth target from infections was not known until now. Recent reports find the stem cell secreted 12-amino acid peptide CLV3p (CLAVATA3 peptide) is perceived by FLS2 (FLAGELLIN SENSING 2) receptor and activates the transcription of immunity and defense marker genes. No infection in the SAM of wild type plants and bacterial infection in clv3 and fls2 mutants illustrate this natural protection against infections. Cytokinins (CKs) are enriched in the SAM and regulate meristem activities by their involvement in stem cell signaling networks. Auxin mediates plant susceptibility to pathogen infections while CKs boost plant immunity. Here, in addition to the stem-cell-triggered immunity we also highlight a potential link between CK signaling and CLV3p mediated immune response in the SAM. KW - auxin KW - stem cell niche KW - FLS2 receptor KW - CLAVATA3 KW - cytokinins Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118247 SN - 1664-462X VL - 5 ER - TY - JOUR A1 - Schartl, Manfred T1 - Beyond the zebrafish: diverse fish species for modeling human disease JF - Disease Models & Mechanisms N2 - In recent years, zebrafish, and to a lesser extent medaka, have become widely used small animal models for human diseases. These organisms have convincingly demonstrated the usefulness of fish for improving our understanding of the molecular and cellular mechanisms leading to pathological conditions, and for the development of new diagnostic and therapeutic tools. Despite the usefulness of zebrafish and medaka in the investigation of a wide spectrum of traits, there is evidence to suggest that other fish species could be better suited for more targeted questions. With the emergence of new, improved sequencing technologies that enable genomic resources to be generated with increasing efficiency and speed, the potential of non-mainstream fish species as disease models can now be explored. A key feature of these fish species is that the pathological condition that they model is often related to specific evolutionary adaptations. By exploring these adaptations, new disease-causing and disease-modifier genes might be identified; thus, diverse fish species could be exploited to better understand the complexity of disease processes. In addition, non-mainstream fish models could allow us to study the impact of environmental factors, as well as genetic variation, on complex disease phenotypes. This Review will discuss the opportunities that such fish models offer for current and future biomedical research. KW - evolutionary mutant model KW - natural variation KW - cancer KW - fish model Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119919 SN - 1754-8411 VL - 7 IS - 2 ER - TY - JOUR A1 - Andreska, Thomas A1 - Aufmkolk, Sarah A1 - Sauer, Markus A1 - Blum, Robert T1 - High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons JF - Frontiers in Cellular Neuroscience N2 - In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity. KW - hippocampal neurons KW - synapse structure KW - presynapse KW - synaptic localization KW - BDNF Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119793 SN - 1662-5102 VL - 8 IS - 107 ER - TY - JOUR A1 - Yilmaz, Ayse A1 - Aksoy, Volkan A1 - Camlitepe, Yilmaz A1 - Giurfa, Martin T1 - Eye structure, activity rhythms, and visually-driven behavior are tuned to visual niche in ants JF - Frontiers in Behavioral Neuroscience N2 - Insects have evolved physiological adaptations and behavioral strategies that allow them to cope with a broad spectrum of environmental challenges and contribute to their evolutionary success. Visual performance plays a key role in this success. Correlates between life style and eye organization have been reported in various insect species. Yet, if and how visual ecology translates effectively into different visual discrimination and learning capabilities has been less explored. Here we report results from optical and behavioral analyses performed in two sympatric ant species, Formica cunicularia and Camponotus aethiops. We show that the former are diurnal while the latter are cathemeral. Accordingly, F. cunicularia workers present compound eyes with higher resolution, while C. aethiops workers exhibit eyes with lower resolution but higher sensitivity. The discrimination and learning of visual stimuli differs significantly between these species in controlled dual-choice experiments: discrimination learning of small-field visual stimuli is achieved by F. cunicularia but not by C. aethiops, while both species master the discrimination of large-field visual stimuli. Our work thus provides a paradigmatic example about how timing of foraging activities and visual environment match the organization of compound eyes and visually-driven behavior. This correspondence underlines the relevance of an ecological/evolutionary framework for analyses in behavioral neuroscience. KW - visual learning KW - ant KW - activity rhythm KW - camponotus aethiops KW - formica cunicularia KW - compound eye Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119595 VL - 8 ER - TY - JOUR A1 - Batram, Christopher A1 - Jones, Nivola G. A1 - Janzen, Christian J. A1 - Markert, Sebastian M. A1 - Engstler, Markus T1 - Expression site attenuation mechanistically links antigenic variation and development in Trypanosoma brucei JF - eLife N2 - We have discovered a new mechanism of monoallelic gene expression that links antigenic variation, cell cycle, and development in the model parasite Trypanosoma brucei. African trypanosomes possess hundreds of variant surface glycoprotein (VSG) genes, but only one is expressed from a telomeric expression site (ES) at any given time. We found that the expression of a second VSG alone is sufficient to silence the active VSG gene and directionally attenuate the ES by disruptor of telomeric silencing-1B (DOT1B)-mediated histone methylation. Three conserved expression-site-associated genes (ESAGs) appear to serve as signal for ES attenuation. Their depletion causes G1-phase dormancy and reversible initiation of the slender-to-stumpy differentiation pathway. ES-attenuated slender bloodstream trypanosomes gain full developmental competence for transformation to the tsetse fly stage. This surprising connection between antigenic variation and developmental progression provides an unexpected point of attack against the deadly sleeping sickness. KW - antigenic variation KW - expression site attenuation KW - developmental reprogramming KW - cell biology KW - genes and chromosomes KW - Trypanosoma brucei KW - variant surface glycoprotein (VSG) KW - monoallelic expression Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119727 SN - 2050-084X VL - 3 IS - e02324 ER - TY - JOUR A1 - Proppert, Sven A1 - Wolter, Steve A1 - Holm, Thorge A1 - Klein, Theresa A1 - van de Linde, Sebastian A1 - Sauer, Markus T1 - Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging JF - Optics Express N2 - In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity. KW - three-dimensional microscopy KW - fluorescence microscopy KW - medical and biological imaging KW - superresolution Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119730 SN - 1094-4087 VL - 22 IS - 9 ER - TY - JOUR A1 - Ahmed, Zeeshan A1 - Zeeshan, Saman A1 - Huber, Claudia A1 - Hensel, Michael A1 - Schomburg, Dietmar A1 - Münch, Richard A1 - Eylert, Eva A1 - Eisenreich, Wolfgang A1 - Dandekar, Thomas T1 - ‘Isotopo’ a database application for facile analysis and management of mass isotopomer data JF - Database N2 - The composition of stable-isotope labelled isotopologues/isotopomers in metabolic products can be measured by mass spectrometry and supports the analysis of pathways and fluxes. As a prerequisite, the original mass spectra have to be processed, managed and stored to rapidly calculate, analyse and compare isotopomer enrichments to study, for instance, bacterial metabolism in infection. For such applications, we provide here the database application ‘Isotopo’. This software package includes (i) a database to store and process isotopomer data, (ii) a parser to upload and translate different data formats for such data and (iii) an improved application to process and convert signal intensities from mass spectra of \(^{13}C\)-labelled metabolites such as tertbutyldimethylsilyl-derivatives of amino acids. Relative mass intensities and isotopomer distributions are calculated applying a partial least square method with iterative refinement for high precision data. The data output includes formats such as graphs for overall enrichments in amino acids. The package is user-friendly for easy and robust data management of multiple experiments. KW - stable-isotope Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120102 VL - 2014 IS - bau077 ER - TY - JOUR A1 - Bensaad, Karim A1 - Favaro, Elena A1 - Lewis, Caroline A. A1 - Peck, Barrie A1 - Lord, Simon A1 - Collins, Jennifer M. A1 - Pinnick, Katherine E. A1 - Wigfield, Simon A1 - Buffa, Francesca M. A1 - Li, Ji-Liang A1 - Zhang, Qifeng A1 - Wakelam, Michael J. O. A1 - Karpe, Fredrik A1 - Schulze, Almut A1 - Harris, Adrian L. T1 - Fatty Acid Uptake and Lipid Storage Induced by HIF-1 alpha Contribute to Cell Growth and Survival after Hypoxia-Reoxygenation JF - Cell Reports N2 - An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1 alpha (HIF-1 alpha)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O-2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via beta-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo. KW - inducible factor-I KW - binding protein KW - triglyceride accumulation KW - cancer cell KW - complex-III KW - beta-oxidation KW - metabolism KW - lipogenesis KW - proliferation KW - resistance Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115162 SN - 2211-1247 VL - 9 IS - 1 ER - TY - JOUR A1 - Daniel, Katrin A1 - Tränkner, Daniel A1 - Wojtasz, Lukasz A1 - Shibuya, Hiroki A1 - Watanabe, Yoshinori A1 - Alsheimer, Manfred A1 - Toth, Attila T1 - Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein JF - BMC Cell Biology N2 - Background: Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. Results: We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. Conclusion: CCDC79 is a meiosis-specific telomere associated protein. Based on our findings we propose that CCDC79 plays a role in meiosis-specific telomere functions. In particular, we favour the possibility that CCDC79 is involved in telomere-nuclear envelope attachment and/or the stabilization of meiotic telomeres. These conclusions are consistent with the findings of an independently initiated study that analysed CCDC79/TERB1 functions. KW - SUN1 KW - meiosis KW - telomeres KW - telomere attachment KW - CCDC79 KW - TERB1 KW - DNA-binding domain KW - meiotic chromosome dynamics KW - fission yeast KW - cohesin SMC1-Beta Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116248 SN - 1471-2121 VL - 15 IS - 17 ER - TY - JOUR A1 - Baur, Stefanie A1 - Rautenberg, Maren A1 - Faulstich, Manuela A1 - Grau, Timo A1 - Severin, Yannik A1 - Unger, Clemens A1 - Hoffmann, Wolfgang H. A1 - Rudel, Thomas A1 - Autenrieth, Ingo B. A1 - Weidenmaier, Christopher T1 - A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization JF - PLOS PATHOGENS N2 - Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization. KW - SREC-I KW - clumping factor-B KW - scavender receptor KW - teichoic acids KW - surface proteins KW - cotton rats KW - carriage KW - determinant KW - infections KW - expression Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116280 SN - 1553-7374 VL - 10 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 - JOUR A1 - Klein, Barett Anthony A1 - Stiegler, Martin A1 - Klein, Arno A1 - Tautz, Jürgen T1 - Mapping Sleeping Bees within Their Nest: Spatial and Temporal Analysis of Worker Honey Bee Sleep JF - PLOS ONE N2 - Patterns of behavior within societies have long been visualized and interpreted using maps. Mapping the occurrence of sleep across individuals within a society could offer clues as to functional aspects of sleep. In spite of this, a detailed spatial analysis of sleep has never been conducted on an invertebrate society. We introduce the concept of mapping sleep across an insect society, and provide an empirical example, mapping sleep patterns within colonies of European honey bees (Apis mellifera L.). Honey bees face variables such as temperature and position of resources within their colony's nest that may impact their sleep. We mapped sleep behavior and temperature of worker bees and produced maps of their nest's comb contents as the colony grew and contents changed. By following marked bees, we discovered that individuals slept in many locations, but bees of different worker castes slept in different areas of the nest relative to position of the brood and surrounding temperature. Older worker bees generally slept outside cells, closer to the perimeter of the nest, in colder regions, and away from uncapped brood. Younger worker bees generally slept inside cells and closer to the center of the nest, and spent more time asleep than awake when surrounded by uncapped brood. The average surface temperature of sleeping foragers was lower than the surface temperature of their surroundings, offering a possible indicator of sleep for this caste. We propose mechanisms that could generate caste-dependent sleep patterns and discuss functional significance of these patterns. KW - apis mellifera KW - age polyethism KW - waggle dance KW - colony KW - hive KW - thermoregulation KW - deprivation KW - dynamics KW - rhythms KW - comb Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115857 SN - 1932-6203 VL - 9 IS - 7 ER - TY - JOUR A1 - Ioakeimidis, Fotis A1 - Ott, Christine A1 - Kozjak-Pavlovic, Vera A1 - Violitzi, Foteini A1 - Rinotas, Vagelis A1 - Makrinou, Eleni A1 - Eliopoulos, Elias A1 - Fasseas, Costas A1 - Kollias, George A1 - Douni, Eleni T1 - A Splicing Mutation in the Novel Mitochondrial Protein DNAJC11 Causes Motor Neuron Pathology Associated with Cristae Disorganization, and Lymphoid Abnormalities in Mice JF - PLOS ONE N2 - Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases. KW - dominant optic atrophy KW - amyotrophic-lateral-sclerosis KW - nervous system KW - membrane organization KW - mitofilin KW - data-bank KW - model KW - biogenesis KW - morphology KW - reveals Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115581 VL - 9 IS - 8 ER - TY - JOUR A1 - Volceanov, Larisa A1 - Herbst, Katharina A1 - Biniossek, Martin A1 - Schilling, Oliver A1 - Haller, Dirk A1 - Nölke, Thilo A1 - Subbarayal, Prema A1 - Rudel, Thomas A1 - Zieger, Barbara A1 - Häcker, Georg T1 - Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion JF - MBIO N2 - Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. IMPORTANCE Chlamydia trachomatis is a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections. C. trachomatis can develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal "coats" or "cages," whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell. KW - mammalian septins KW - host-cells KW - binding KW - proteins KW - organization KW - cytoskeleton KW - cytokinesis KW - mechanisms KW - expression KW - protease Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115421 SN - 2150-7511 VL - 5 IS - 5 ER - TY - JOUR A1 - Morris, E. Kathryn A1 - Caruso, Tancredi A1 - Buscot, Francois A1 - Fischer, Markus A1 - Hancock, Christine A1 - Maier, Tanja S. A1 - Meiners, Torsten A1 - Müller, Caroline A1 - Obermaier, Elisabeth A1 - Prati, Daniel A1 - Socher, Stephanie A. A1 - Sonnemann, Ilja A1 - Wäschke, Nicola A1 - Wubet, Tesfaye A1 - Wurst, Susanne A1 - Rillig, Matthias C. T1 - Choosing and using diversity indices: insights for ecological applications from the German Biodiversity Exploratories JF - Ecology and Evolution N2 - Biodiversity, a multidimensional property of natural systems, is difficult to quantify partly because of the multitude of indices proposed for this purpose. Indices aim to describe general properties of communities that allow us to compare different regions, taxa, and trophic levels. Therefore, they are of fundamental importance for environmental monitoring and conservation, although there is no consensus about which indices are more appropriate and informative. We tested several common diversity indices in a range of simple to complex statistical analyses in order to determine whether some were better suited for certain analyses than others. We used data collected around the focal plant Plantago lanceolata on 60 temperate grassland plots embedded in an agricultural landscape to explore relationships between the common diversity indices of species richness (S), Shannon's diversity (H'), Simpson's diversity (D-1), Simpson's dominance (D-2), Simpson's evenness (E), and Berger-Parker dominance (BP). We calculated each of these indices for herbaceous plants, arbuscular mycorrhizal fungi, aboveground arthropods, belowground insect larvae, and P.lanceolata molecular and chemical diversity. Including these trait-based measures of diversity allowed us to test whether or not they behaved similarly to the better studied species diversity. We used path analysis to determine whether compound indices detected more relationships between diversities of different organisms and traits than more basic indices. In the path models, more paths were significant when using H', even though all models except that with E were equally reliable. This demonstrates that while common diversity indices may appear interchangeable in simple analyses, when considering complex interactions, the choice of index can profoundly alter the interpretation of results. Data mining in order to identify the index producing the most significant results should be avoided, but simultaneously considering analyses using multiple indices can provide greater insight into the interactions in a system. KW - molecular diversity KW - plant diversity KW - plantago lanceolata KW - shannon index KW - simpson's index KW - arbuscular mycorrhizal fungi KW - Hill's powers KW - chemical diversity KW - Berger-Parker KW - arthropods Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115462 SN - 2045-7758 VL - 4 IS - 18 ER - TY - JOUR A1 - Bartomeus, Ignasi A1 - Potts, Simon G. A1 - Steffan-Dewenter, Ingolf A1 - Vaissiere, Bernard E. A1 - Woyciechowski, Michal A1 - Krewenka, Kristin M. A1 - Tscheulin, Thomas A1 - Roberts, Stuart P. M. A1 - Szentgyoergyi, Hajnalka A1 - Westphal, Catrin A1 - Bommarco, Riccardo T1 - Contribution of insect pollinators to crop yield and quality varies with agricultural intensification JF - PEERJ N2 - Background. Up to 75% of crop species benefit at least to some degree from animal pollination for fruit or seed set and yield. However, basic information on the level of pollinator dependence and pollinator contribution to yield is lacking for many crops. Even less is known about how insect pollination affects crop quality. Given that habitat loss and agricultural intensification are known to decrease pollinator richness and abundance, there is a need to assess the consequences for different components of crop production. Methods. We used pollination exclusion on flowers or inflorescences on a whole plant basis to assess the contribution of insect pollination to crop yield and quality in four flowering crops (spring oilseed rape, field bean, strawberry, and buckwheat) located in four regions of Europe. For each crop, we recorded abundance and species richness of flower visiting insects in ten fields located along a gradient from simple to heterogeneous landscapes. Results. Insect pollination enhanced average crop yield between 18 and 71% depending on the crop. Yield quality was also enhanced in most crops. For instance, oilseed rape had higher oil and lower chlorophyll contents when adequately pollinated, the proportion of empty seeds decreased in buckwheat, and strawberries' commercial grade improved; however, we did not find higher nitrogen content in open pollinated field beans. Complex landscapes had a higher overall species richness of wild pollinators across crops, but visitation rates were only higher in complex landscapes for some crops. On the contrary, the overall yield was consistently enhanced by higher visitation rates, but not by higher pollinator richness. Discussion. For the four crops in this study, there is clear benefit delivered by pollinators on yield quantity and/or quality, but it is not maximized under current agricultural intensification. Honeybees, the most abundant pollinator, might partially compensate the loss of wild pollinators in some areas, but our results suggest the need of landscape-scale actions to enhance wild pollinator populations. KW - biodiversity KW - pollination KW - honeybee KW - wild bees KW - agroecosystems KW - native pollinators KW - species richness KW - bee pollinators KW - wild KW - ecosystemservices KW - fruit-quality KW - oilseed rape KW - land-use KW - honey KW - patterns Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116928 SN - 2167-9843 VL - 2 IS - e328 ER - TY - JOUR A1 - Baalbergen, Els A1 - Helwerda, Renate A1 - Schelfhorst, Rense A1 - Castillo Cajas, Ruth F. A1 - van Moorsel, Coline H. M. A1 - Kundrata, Robin A1 - Welter-Schultes, Francisco W. A1 - Giokas, Sinos A1 - Schilthuizen, Menno T1 - Predator-Prey Interactions between Shell-Boring Beetle Larvae and Rock-Dwelling Land Snails JF - PLOS ONE N2 - Drilus beetle larvae (Coleoptera: Elateridae) are specialized predators of land snails. Here, we describe various aspects of the predator-prey interactions between multiple Drilus species attacking multiple Albinaria (Gastropoda: Clausiliidae) species in Greece. We observe that Drilus species may be facultative or obligate Albinaria-specialists. We map geographically varying predation rates in Crete, where on average 24% of empty shells carry fatal Drilus bore holes. We also provide first-hand observations and video-footage of prey entry and exit strategies of the Drilus larvae, and evaluate the potential mutual evolutionary impacts. We find limited evidence for an effect of shell features and snail behavioral traits on inter-and intraspecifically differing predation rates. We also find that Drilus predators adjust their predation behavior based on specific shell traits of the prey. In conclusion, we suggest that, with these baseline data, this interesting predator-prey system will be available for further, detailed more evolutionary ecology studies. KW - clausiliidae KW - evolution KW - pulmonata KW - albinaria KW - behavior KW - species gastropoda Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115963 SN - 1932-6203 VL - 9 IS - 6 ER - TY - JOUR A1 - Garcia, Tzintzuni I. A1 - Matos, Isa A1 - Shen, Yingjia A1 - Pabuwal, Vagmita A1 - Coelho, Maria Manuela A1 - Wakamatsu, Yuko A1 - Schartl, Manfred A1 - Walter, Ronald B. T1 - Novel Method for Analysis of Allele Specific Expression in Triploid Oryzias latipes Reveals Consistent Pattern of Allele Exclusion JF - PLOS ONE N2 - Assessing allele-specific gene expression (ASE) on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types) and diseased tissues (trisomies, non-disjunction events, cancerous tissues). In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82%) shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18%) displayed a wide range of ASE levels. Interestingly the majority of genes (78%) displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression. KW - RNA-SEQ data KW - copy-number alteration KW - squalius alburnoides KW - gene expression KW - medaka KW - variant detection KW - transplantation KW - genome KW - generation KW - evolution Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116000 SN - 1932-6203 VL - 9 IS - 6 ER - TY - JOUR A1 - Wäschke, Nicole A1 - Hardge, Kerstin A1 - Hancock, Christine A1 - Hilker, Monika A1 - Obermaier, Elisabeth A1 - Meiners, Torsten T1 - Odour Environments: How Does Plant Diversity Affect Herbivore and Parasitoid Orientation? JF - PlOS ONE N2 - Plant diversity is known to affect success of host location by pest insects, but its effect on olfactory orientation of non-pest insect species has hardly been addressed. First, we tested in laboratory experiments the hypothesis that non-host plants, which increase odour complexity in habitats, affect the host location ability of herbivores and parasitoids. Furthermore, we recorded field data of plant diversity in addition to herbivore and parasitoid abundance at 77 grassland sites in three different regions in Germany in order to elucidate whether our laboratory results reflect the field situation. As a model system we used the herb Plantago lanceolata, the herbivorous weevil Mecinus pascuorum, and its larval parasitoid Mesopolobus incultus. The laboratory bioassays revealed that both the herbivorous weevil and its larval parasitoid can locate their host plant and host via olfactory cues even in the presence of non-host odour. In a newly established two-circle olfactometer, the weevils capability to detect host plant odour was not affected by odours from non-host plants. However, addition of non-host plant odours to host plant odour enhanced the weevils foraging activity. The parasitoid was attracted by a combination of host plant and host volatiles in both the absence and presence of non-host plant volatiles in a Y-tube olfactometer. In dual choice tests the parasitoid preferred the blend of host plant and host volatiles over its combination with non-host plant volatiles. In the field, no indication was found that high plant diversity disturbs host (plant) location by the weevil and its parasitoid. In contrast, plant diversity was positively correlated with weevil abundance, whereas parasitoid abundance was independent of plant diversity. Therefore, we conclude that weevils and parasitoids showed the sensory capacity to successfully cope with complex vegetation odours when searching for hosts. KW - dentichasmias busseolae KW - nonhost plant KW - volatiles KW - selection KW - invertebrate herbivory KW - location behavior KW - foraging behavior KW - background odor KW - natural enemies Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117687 SN - 1932-6203 VL - 9 IS - 1 ER - TY - JOUR A1 - Breeze, Tom D. A1 - Vaissiere, Bernhard E. A1 - Bommarco, Riccardo A1 - Petanidou, Theodora A1 - Seraphides, Nicos A1 - Kozak, Lajos A1 - Scheper, Jeroen A1 - Biesmeijer, Jacobus C. A1 - Kleijn, David A1 - Gyldenkærne, Steen A1 - Moretti, Marco A1 - Holzschuh, Andrea A1 - Steffan-Dewenter, Ingolf A1 - Stout, Jane C. A1 - Pärtel, Meelis A1 - Zobel, Martin A1 - Potts, Simon G. T1 - Agricultural Policies Exacerbate Honeybee Pollination Service Supply-Demand Mismatches Across Europe JF - PLOS ONE N2 - Declines in insect pollinators across Europe have raised concerns about the supply of pollination services to agriculture. Simultaneously, EU agricultural and biofuel policies have encouraged substantial growth in the cultivated area of insect pollinated crops across the continent. Using data from 41 European countries, this study demonstrates that the recommended number of honeybees required to provide crop pollination across Europe has risen 4.9 times as fast as honeybee stocks between 2005 and 2010. Consequently, honeybee stocks were insufficient to supply >90% of demands in 22 countries studied. These findings raise concerns about the capacity of many countries to cope with major losses of wild pollinators and highlight numerous critical gaps in current understanding of pollination service supplies and demands, pointing to a pressing need for further research into this issue. KW - economy services KW - fruit set KW - sequential introduction KW - enhance KW - biodiversity KW - abundance KW - declines KW - crops KW - colonies KW - density Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117692 SN - 1932-6203 VL - 9 IS - 1 ER - TY - JOUR A1 - Koetschan, Christian A1 - Kittelmann, Sandra A1 - Lu, Jingli A1 - Al-Halbouni, Djamila A1 - Jarvis, Graeme N. A1 - Müller, Tobias A1 - Wolf, Matthias A1 - Janssen, Peter H. T1 - Internal Transcribed Spacer 1 Secondary Structure Analysis Reveals a Common Core throughout the Anaerobic Fungi (Neocallimastigomycota) JF - PLOS ONE N2 - The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments. KW - profile distances KW - ITS2 KW - phylogenetic trees KW - RNA sequence KW - reconstruction KW - diversity KW - populations KW - tool KW - systematics KW - herbivores Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117058 VL - 9 IS - 3 ER - TY - JOUR A1 - Heller, Klaus-Gerhard A1 - Hemp, Claudia T1 - Fiddler on the Tree - A Bush-Cricket Species with Unusual Stridulatory Organs and Song JF - PLOS ONE N2 - Insects of the order Orthoptera are well-known for their acoustic communication. The structures used for this purpose show a high diversity which obviously relates to differences in song parameters and to the physics of sound production. Here we describe song and morphology of the sound producing organs of a tropical bush-cricket, Ectomoptera nepicauda, from East Africa. It has a very unusual calling song consisting of frequency-modulated, pure-tone sounds in the high ultrasonic range of 80 to 120 kHz and produced by extremely fast wing movements. Concerning morphology, it represents the most extreme state in the degree of left-right fore-wing differentiation found among Orthoptera: the acoustic parts of the left fore-wing consist exclusively of the stridulatory file, comparable in function to the bow of a violin, while the right wing carries only the plectrum (= string) and mirror (= soundbox). KW - constraints KW - sound production KW - katydids orthoptera KW - mole crickets KW - tettigoniidae KW - mechanics Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117068 VL - 9 IS - 3 ER - TY - JOUR A1 - Groeneweg, Femke L. A1 - van Royen, Martin E. A1 - Fenz, Susanne A1 - Keizer, Veer I. P. A1 - Geverts, Bart A1 - Prins, Jurrien A1 - de Kloet, E. Ron A1 - Houtsmuller, Adriaan B. A1 - Schmidt, Thomas S. A1 - Schaaf, Marcel J. M. T1 - Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP JF - PLOS ONE N2 - Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (similar to 0.7 s) and the other half for longer time periods (similar to 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (<= 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences. KW - NF-KAPPA-B KW - image correlation spectroscopy KW - human mineralocorticoid receptor KW - nuclear-pore complexes KW - in-vivo KW - living cells KW - mobility KW - transcription KW - protein KW - reveals Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117085 VL - 9 IS - 3 ER - TY - JOUR A1 - Oervoessy, Noemi A1 - Koroesi, Adam A1 - Batary, Peter A1 - Vozar, Agnes A1 - Peregovits, Laszlo T1 - Habitat Requirements of the Protected Southern Festoon (Zerynthia Polysena); Adult, Egg and Larval Distribution in a Highly Degraded Habitat Complex JF - Acta Zoologica Academiae Scientiarum Hungaricae N2 - Habitat quality affects the presence and size of butterfly populations. Resources for all life stages must be found in a given or few habitat patches. Southern festoon (Zerynthia polyxena) is a vulnerable, but locally abundant species in Hungary. The larva requires birthwort (Aristolochia clematitis) as food plant. We examined the small scale habitat use of adults and distribution of eggs and larvae among different vegetation types to reveal the requirements of the species in all life stages. Transect counts were conducted in a tree plantation complex comprising four types of vegetation. Number (+/- SE) of adults, eggs and larvae were lowest in poplar plantation (adult 0.3 +/- 0.2, egg 1.1 +/- 1.1, larva 0.6 +/- 0.3). Medium amount of butterflies were observed in open (adult 8.3 +/- 2.9, egg 3.1 +/- 2.6, larva 3.1 +/- 1.9) and black-locust (adult 9.4 +/- 4.2, egg 12.7 +/- 4.9, larva 4.1 +/- 1.1) habitat. Number of butterflies was highest in hummocks (adult 13.5 +/- 1.5, egg 12.9 +/- 5.7, larva 8.4 +/- 2.1). Adults avoided bare ground. We encountered most eggs in dense food plant patches with high plants. Food plant height also positively influenced the occurrence of the larvae. Although distribution of adults and juvenile forms showed quite similar patterns, we could also reveal some differences that caused by different environmental conditions in distinct vegetation types. Our study stresses the importance of habitat quality, which affects population size of butterflies even in a highly degraded habitat complex. KW - habitat quality KW - resource use KW - life stage KW - butterfly euphydryas-aurinia KW - ecology KW - metapopulation KW - conservation KW - quality KW - management KW - population KW - nympahlidae KW - fragmented landscapes KW - lepidoptera KW - tree plantations KW - habitat patch Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117810 VL - 60 IS - 4 ER - TY - JOUR A1 - Grözinger, Franziska A1 - Thein, Jürgen A1 - Feldhaar, Heike A1 - Rödel, Mark-Oliver T1 - Giants, Dwarfs and the Environment - Metamorphic Trait Plasticity in the Common Frog JF - PLOS ONE N2 - In order to understand adaptation processes and population dynamics, it is central to know how environmental parameters influence performance of organisms within populations, including their phenotypes. The impact of single or few particular parameters in concert was often assessed in laboratory and mesocosm experiments. However, under natural conditions, with many biotic and abiotic factors potentially interacting, outcomes on phenotypic changes may be different. To study the potential environmental impact on realized phenotypic plasticity within a natural population, we assessed metamorphic traits (developmental time, size and body mass) in an amphibian species, the European common frog Rana temporaria, since a) larval amphibians are known to exhibit high levels of phenotypic plasticity of these traits in response to habitat parameters and, b) the traits' features may strongly influence individuals' future performance and fitness. In 2007 we studied these metamorphic traits in 18 ponds spread over an area of 28 km 2. A subset of six ponds was reinvestigated in 2009 and 2010. This study revealed locally high variances in metamorphic traits in this presumed generalist species. We detected profound differences between metamorphing froglets (up to factor ten); both between and within ponds, on a very small geographic scale. Parameters such as predation and competition as well as many other pond characteristics, generally expected to have high impact on development, could not be related to the trait differences. We observed high divergence of patterns of mass at metamorphosis between ponds, but no detectable pattern when metamorphic traits were compared between ponds and years. Our results indicate that environment alone, i.e. as experienced by tadpoles sharing the same breeding pond, can only partly explain the variability of metamorphic traits observed. This emphasizes the importance to assess variability of reaction norms on the individual level to explain within-population variability. KW - rana temporaria populations KW - prey growth rate KW - phenotypic plasticity KW - larval density KW - amphibian metamorphosis KW - ambystoma opacum KW - predation risk KW - life history KW - developmental plasticity KW - adaptive plasticity Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117203 SN - 1932-6203 VL - 9 IS - 3 ER - TY - JOUR A1 - Pascoalino, Bruno A1 - Dindar, Gülcin A1 - Vieira-da-Rocha, João P. A1 - Machado, Carlos Renato A1 - Janzen, Christian J. A1 - Schenkman, Sergio T1 - Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei JF - Nucleic Acids Research N2 - The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes. KW - chromatin assembly factors KW - DNA-damage checkpoint KW - tousled-like kinases KW - saccharomyes cerevisiae KW - gene expression KW - acetyltransferase RTT109 KW - african trypanosomes KW - antigenetic variation KW - cycle regulation KW - nuclear import Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117220 SN - 1362-4962 VL - 42 IS - 5 ER - TY - JOUR A1 - Sanz-Moreno, Adrian A1 - Fuhrmann, David A1 - Wolf, Elmar A1 - von Eyss, Björn A1 - Eilers, Martin A1 - Elsässer, Hans-Peter T1 - Miz1 Deficiency in the Mammary Gland Causes a Lactation Defect by Attenuated Stat5 Expression and Phosphorylation JF - PLOS ONE N2 - Miz1 is a zinc finger transcription factor with an N-terminal POZ domain. Complexes with Myc, Bcl-6 or Gfi-1 repress expression of genes like Cdkn2b (p15(Ink4)) or Cd-kn1a (p21(Cip1)). The role of Miz1 in normal mammary gland development has not been addressed so far. Conditional knockout of the Miz1 POZ domain in luminal cells during pregnancy caused a lactation defect with a transient reduction of glandular tissue, reduced proliferation and attenuated differentiation. This was recapitulated in vitro using mouse mammary gland derived HC11 cells. Further analysis revealed decreased Stat5 activity in Miz1 Delta POZ mammary glands and an attenuated expression of Stat5 targets. Gene expression of the Prolactin receptor (PrlR) and ErbB4, both critical for Stat5 phosphorylation (pStat5) or pStat5 nuclear translocation, was decreased in Miz1 Delta POZ females. Microarray, ChIP-Seq and gene set enrichment analysis revealed a down-regulation of Miz1 target genes being involved in vesicular transport processes. Our data suggest that deranged intracellular transport and localization of PrlR and ErbB4 disrupt the Stat5 signalling pathway in mutant glands and cause the observed lactation phenotype. KW - C-MYC KW - transcription factor MIZ-1 KW - breast-cancer cells KW - gene expression KW - epithelial cells KW - prolactin KW - transgenic mice KW - growth KW - differentiation KW - proliferation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117286 VL - 9 IS - 2 ER - TY - JOUR A1 - Benz, Roland A1 - Maier, Elke A1 - Bauer, Susanne A1 - Ludwig, Albrecht T1 - The Deletion of Several Amino Acid Stretches of Escherichia coli Alpha-Hemolysin (HlyA) Suggests That the Channel-Forming Domain Contains Beta-Strands JF - PLOS ONE N2 - Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71–110, 158–167, 180–203, and 264–286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71–110 and HlyAΔ264–286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158–167 and HlyAΔ180–203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71–110 and HlyAΔ264–286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71–110, and HlyAΔ264–286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures. KW - membrane potential KW - molecular mass KW - cations KW - membrane structures KW - membrane proteins KW - lipid bilayer KW - red blood cells KW - toxins Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118115 SN - 1932-6203 VL - 9 IS - 12 ER - TY - JOUR A1 - Sommerlandt, F. M. J. A1 - Huber, W. A1 - Spaethe, J. T1 - Social Information in the Stingless Bee, Trigona corvina Cockerell (Hymenoptera: Apidae): The Use of Visual and Olfactory Cues at the Food Site JF - Sociobiology N2 - For social insects, colony performance is largely dependent on the quantity and quality of food intake and thus on the efficiency of its foragers. In addition to innate preferences and previous experience, foragers can use social information to decide when and where to forage. In some stingless bee (Meliponini) species, individual foraging decisions are shown to be influenced by the presence of social information at resource sites. In dual choice tests, we studied whether visual and/or olfactory cues affect individual decision-making in rigona corvina Cockerell and if this information is species-specific. We found that T. corvina foragers possess local enhancement: they are attracted by olfactory and visual cues released by conspecifics but avoid feeders associated with heterospecific individuals of the species Tetragona ziegleri (Friese). Overall, olfactory cues seem to be more important than visual cues, but information by visual cues alone is sufficient for discrimination. KW - visual cues KW - recruitment KW - local enhancement KW - odor marks KW - communication Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118120 VL - 61 IS - 4 ER - TY - JOUR A1 - Peter, Stefanie A1 - Bultinck, Jennyfer A1 - Myant, Kevin A1 - Jaenicke, Laura A. A1 - Walz, Susanne A1 - Müller, Judith A1 - Gmachl, Michael A1 - Treu, Matthias A1 - Boehmelt, Guido A1 - Ade, Casten P. A1 - Schmitz, Werner A1 - Wiegering, Armin A1 - Otto, Christoph A1 - Popov, Nikita A1 - Sansom, Owen A1 - Kraut, Norbert A1 - Eilers, Martin T1 - H Tumor cell-specific inhibition of MYC function using small molecule inhibitors of the HUWE1 ubiquitin ligase JF - EMBO Molecular Medicine N2 - Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. KW - colorectal cancer KW - HUWE1 KW - MIZ1 KW - MYC KW - ubiquitination KW - cancer KW - digestive system KW - pharmacology KW - drug discovery Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118132 SN - 1757-4684 VL - 6 IS - 12 ER - TY - THES A1 - Axmacher, Franz T1 - Die SVM-gestützte Prädiktabilität der Bindungsspezifität ‎von SH3-Domänen anhand ihrer Aminosäuresequenz T1 - The SVM-based predictability of SH3-domain binding specificity by means of its amino-acid-‎sequence. ‎ N2 - Die Identifikation der Bindungsspezifitäten von Proteininteraktionsdomänen und damit letztlich auch ‎die Fähigkeit potentielle Bindungspartner dieser in vivo vorherzusagen bildet ein grundlegendes ‎Element für das Verständnis der biologischen Funktionen dieser Domänen. In dieser Arbeit wurde ‎untersucht, inwieweit solche Vorhersagen bezüglich der SH3-Domäne – als Beispiel für eine ‎Proteininteraktionsdomäne – mithilfe von Support-Vector-Machines (SVMs) möglich sind, wenn ‎diesen als Informationsquelle ausschließlich die innerhalb der Aminosäuresequenz der Domäne ‎konservierten Informationen zur Verfügung stehen. Um den SVM-basierten Klassifikator zu ‎trainieren und zu validieren, wurde ein Satz aus 51 SH3-Domänen verwendet, die zuvor ‎entsprechend ihrer Ligandenpräferenz in ein System aus acht verschiedenen Klassen eingeteilt ‎worden waren. Da die innerhalb der Aminosäuresequenzen konservierten Informationen in ‎abstrakte Zahlenwerte konvertiert werden mussten (Voraussetzung für mathematisch basierte ‎Klassifikatoren wie SVMs), wurde jede Aminosäuresequenz durch ihren jeweiligen Fisher-Score-‎Vektor ausgedrückt. Die Ergebnisse erbrachten einen Klassifikationserror, welcher weit unterhalb des ‎Zufallsniveaus lag, was darauf hindeutet, dass sich die Bindungsspezifität (Klasse) einer SH3-Domäne ‎in der Tat von seiner Aminosäuresequenz ableiten lassen dürfte. Mithilfe klassenspezifisch ‎emittierter, artifizieller Sequenzen, implementiert in den Trainingsprozess des Klassifikators, um ‎etwaigen nachteiligen Auswirkungen von Overfitting zu entgegenzuwirken, sowie durch ‎Berücksichtigung taxonomischer Informationen des Klassensystems während Training und ‎Validierung, ließ sich der Klassifikationserror sogar noch weiter senken und lag schließlich bei lediglich ‎‎35,29% (vergleiche Zufall: 7/8 = 87.50%). Auch die Nutzung von Feature Selections zur Abmilderung ‎Overfitting-bedingter, negativer Effekte lieferte recht vielversprechende Ergebnisse, wenngleich ihr ‎volles Potential aufgrund von Software-Beschränkungen nicht ausgenutzt werden konnte.‎ Die Analyse der Positionen im Sequence-Alignment, welche für den SVM- basierten Klassifikator am ‎relevantesten waren, zeigte, dass diese häufig mit Positionen korrelierten, von denen angenommen ‎wird auch in vivo eine Schlüsselrolle bei der Determination der Bindungsspezifität (Klasse) zu spielen. ‎Dies unterstreicht nicht nur die Reliabilität des präsentierten Klassifikators, es gibt auch Grund zur ‎Annahme, dass das Verfahren möglicherweise auch als Supplement anderer Ansätze genutzt werden ‎könnte, welche zum Ziel haben die Positionen zu identifizieren, die die Ligandenpräferenz in vivo ‎determinieren. Informationen, die nicht nur für ein besseres Verständnis der SH3-Domäne (und ‎möglicherweise auch anderer Proteininteraktionsdomänen) von grundlegender Bedeutung sind, ‎sondern auch aus pharmakologischer Sicht von großem Interesse sein dürften.‎ N2 - Regarding protein-interaction-domains the identification of their binding specificities and ‎eventually ‎also the ability to predict potential binding partners for them in vivo constitutes a fundamental ‎element for the understanding of the biological functions of these domains. In this study it ‎was ‎investigated to what extent such predictions could be made for the SH3-domain – as an ‎example ‎for a protein-interaction-domain – when using support-vector-machines (SVMs) trained ‎exclusively ‎with the information conserved within the amino-acid-sequence of the domain. A set of ‎‎51 SH3-‎domains, pre-classified into a system of eight different classes according to their ligand ‎preference, was used to train and cross-validate the SVM-based classifier. To convert the ‎information ‎conserved within the amino-acid-sequences into abstract numeric values (a ‎prerequisite for a ‎mathematics-based classifier like SVMs) each sequence was represented by its ‎respective Fisher-‎score-vector. The results revealed a classification error level way below chance ‎level, indicating the ‎binding specificity (class) of an SH3-domain can indeed be inferred from its ‎amino-acid-sequence. ‎With the help of class-specific emitted, artificial sequences introduced into ‎the training process of the ‎classifier to counter adverse overfitting effects and by additionally ‎considering taxonomic ‎information of the class system during training and cross-validation, the ‎classification error level of ‎the classifier could be lowered even farther, eventually reaching a level ‎as low as 35.29% (compare ‎chance level: 7/8 = 87.50%). The use feature selections to counter ‎overfitting returned quite ‎promising results, too, however couldn't be exploited to its full potential ‎due to software limitations. ‎ The analysis of those positions in the sequence-alignment being most relevant for the SVM-‎based ‎classifier showed, they frequently correlated with positions considered to also play in vivo a ‎pivotal ‎role in binding specificity (class) determination of the SH3-domain. Not only does this ‎underline the ‎reliability of the presented classifier, it also gives reason to believe, the method could ‎possibly be ‎used as a supplement for other approaches trying to identify positions that determine ‎ligand ‎preference in vivo. Information, not only fundamental for a better understanding of the SH3-‎‎domain (and maybe also other protein-interaction-domains), but also likely to be of great interest ‎from a pharmacological point of view.‎ KW - Support-Vektor-Maschine KW - Alignment KW - Hidden-Markov-Modell KW - Kreuzvalidierung KW - Taxonomie KW - SH3-Domäne KW - Fisher-Score KW - Regularisierung KW - Feature-Selection KW - PyMOL KW - WebLogo KW - e1071 Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113349 ER - TY - JOUR A1 - Hudson, Lawrence N. A1 - Newbold, Tim A1 - Contu, Sara A1 - Hill, Samantha L. L. A1 - Lysenko, Igor A1 - De Palma, Adriana A1 - Phillips, Helen R. P. A1 - Senior, Rebecca A. A1 - Bennett, Dominic J. A1 - Booth, Hollie A1 - Choimes, Argyrios A1 - Correia, David L. P. A1 - Day, Julie A1 - Echeverria-Londono, Susy A1 - Garon, Morgan A1 - Harrison, Michelle L. K. A1 - Ingram, Daniel J. A1 - Jung, Martin A1 - Kemp, Victoria A1 - Kirkpatrick, Lucinda A1 - Martin, Callum D. A1 - Pan, Yuan A1 - White, Hannah J. A1 - Aben, Job A1 - Abrahamczyk, Stefan A1 - Adum, Gilbert B. A1 - Aguilar-Barquero, Virginia A1 - Aizen, Marcelo A1 - Ancrenaz, Marc A1 - Arbelaez-Cortes, Enrique A1 - Armbrecht, Inge A1 - Azhar, Badrul A1 - Azpiroz, Adrian B. A1 - Baeten, Lander A1 - Báldi, András A1 - Banks, John E. A1 - Barlow, Jos A1 - Batáry, Péter A1 - Bates, Adam J. A1 - Bayne, Erin M. A1 - Beja, Pedro A1 - Berg, Ake A1 - Berry, Nicholas J. A1 - Bicknell, Jake E. A1 - Bihn, Jochen H. A1 - Böhning-Gaese, Katrin A1 - Boekhout, Teun A1 - Boutin, Celine A1 - Bouyer, Jeremy A1 - Brearley, Francis Q. A1 - Brito, Isabel A1 - Brunet, Jörg A1 - Buczkowski, Grzegorz A1 - Buscardo, Erika A1 - Cabra-Garcia, Jimmy A1 - Calvino-Cancela, Maria A1 - Cameron, Sydney A. A1 - Cancello, Eliana M. A1 - Carrijo, Tiago F. A1 - Carvalho, Anelena L. A1 - Castro, Helena A1 - Castro-Luna, Alejandro A. A1 - Cerda, Rolando A1 - Cerezo, Alexis A1 - Chauvat, Matthieu A1 - Clarke, Frank M. A1 - Cleary, Daniel F. R. A1 - Connop, Stuart P. A1 - D'Aniello, Biagio A1 - da Silva, Pedro Giovani A1 - Darvill, Ben A1 - Dauber, Jens A1 - Dejean, Alain A1 - Diekötter, Tim A1 - Dominguez-Haydar, Yamileth A1 - Dormann, Carsten F. A1 - Dumont, Bertrand A1 - Dures, Simon G. A1 - Dynesius, Mats A1 - Edenius, Lars A1 - Elek, Zoltán A1 - Entling, Martin H. A1 - Farwig, Nina A1 - Fayle, Tom M. A1 - Felicioli, Antonio A1 - Felton, Annika M. A1 - Ficetola, Gentile F. A1 - Filgueiras, Bruno K. C. A1 - Fonte, Steve J. A1 - Fraser, Lauchlan H. A1 - Fukuda, Daisuke A1 - Furlani, Dario A1 - Ganzhorn, Jörg U. A1 - Garden, Jenni G. A1 - Gheler-Costa, Carla A1 - Giordani, Paolo A1 - Giordano, Simonetta A1 - Gottschalk, Marco S. A1 - Goulson, Dave A1 - Gove, Aaron D. A1 - Grogan, James A1 - Hanley, Mick E. A1 - Hanson, Thor A1 - Hashim, Nor R. A1 - Hawes, Joseph E. A1 - Hébert, Christian A1 - Helden, Alvin J. A1 - Henden, John-André A1 - Hernández, Lionel A1 - Herzog, Felix A1 - Higuera-Diaz, Diego A1 - Hilje, Branko A1 - Horgan, Finbarr G. A1 - Horváth, Roland A1 - Hylander, Kristoffer A1 - Horváth, Roland A1 - Isaacs-Cubides, Paola A1 - Ishitani, Mashiro A1 - Jacobs, Carmen T. A1 - Jaramillo, Victor J. A1 - Jauker, Birgit A1 - Jonsell, Matts A1 - Jung, Thomas S. A1 - Kapoor, Vena A1 - Kati, Vassiliki A1 - Katovai, Eric A1 - Kessler, Michael A1 - Knop, Eva A1 - Kolb, Annette A1 - Körösi, Àdám A1 - Lachat, Thibault A1 - Lantschner, Victoria A1 - Le Féon, Violette A1 - LeBuhn, Gretchen A1 - Légaré, Jean-Philippe A1 - Letcher, Susan G. A1 - Littlewood, Nick A. A1 - López-Quintero, Carlos A. A1 - Louhaichi, Mounir A1 - Lövei, Gabor L. A1 - Lucas-Borja, Manuel Esteban A1 - Luja, Victor H. A1 - Maeto, Kaoru A1 - Magura, Tibor A1 - Mallari, Neil Aldrin A1 - Marin-Spiotta, Erika A1 - Marhall, E. J. P. A1 - Martínez, Eliana A1 - Mayfield, Margaret M. A1 - Mikusinski, Gregorz A1 - Milder, Jeffery C. A1 - Miller, James R. A1 - Morales, Carolina L. A1 - Muchane, Mary N. A1 - Muchane, Muchai A1 - Naidoo, Robin A1 - Nakamura, Akihiro A1 - Naoe, Shoji A1 - Nates-Parra, Guiomar A1 - Navarerete Gutierrez, Dario A. A1 - Neuschulz, Eike L. A1 - Noreika, Norbertas A1 - Norfolk, Olivia A1 - Noriega, Jorge Ari A1 - Nöske, Nicole M. A1 - O'Dea, Niall A1 - Oduro, William A1 - Ofori-Boateng, Caleb A1 - Oke, Chris O. A1 - Osgathorpe, Lynne M. A1 - Paritsis, Juan A1 - Parrah, Alejandro A1 - Pelegrin, Nicolás A1 - Peres, Carlos A. A1 - Persson, Anna S. A1 - Petanidou, Theodora A1 - Phalan, Ben A1 - Philips, T. Keith A1 - Poveda, Katja A1 - Power, Eileen F. A1 - Presley, Steven J. A1 - Proença, Vânia A1 - Quaranta, Marino A1 - Quintero, Carolina A1 - Redpath-Downing, Nicola A. A1 - Reid, J. Leighton A1 - Reis, Yana T. A1 - Ribeiro, Danilo B. A1 - Richardson, Barbara A. A1 - Richardson, Michael J. A1 - Robles, Carolina A. A1 - Römbke, Jörg A1 - Romero-Duque, Luz Piedad A1 - Rosselli, Loreta A1 - Rossiter, Stephen J. A1 - Roulston, T'ai H. A1 - Rousseau, Laurent A1 - Sadler, Jonathan P. A1 - Sáfián, Szbolcs A1 - Saldaña-Vásquez, Romeo A. A1 - Samnegård, Ulrika A1 - Schüepp, Christof A1 - Schweiger, Oliver A1 - Sedlock, Jodi L. A1 - Shahabuddin, Ghazala A1 - Sheil, Douglas A1 - Silva, Fernando A. B. A1 - Slade, Eleanor A1 - Smith-Pardo, Allan H. A1 - Sodhi, Navjot S. A1 - Somarriba, Eduardo J. A1 - Sosa, Ramón A. A1 - Stout, Jane C. A1 - Struebig, Matthew J. A1 - Sung, Yik-Hei A1 - Threlfall, Caragh G. A1 - Tonietto, Rebecca A1 - Tóthmérész, Béla A1 - Tscharntke, Teja A1 - Turner, Edgar C. A1 - Tylianakis, Jason M. A1 - Vanbergen, Adam J. A1 - Vassilev, Kiril A1 - Verboven, Hans A. F. A1 - Vergara, Carlos H. A1 - Vergara, Pablo M. A1 - Verhulst, Jort A1 - Walker, Tony R. A1 - Wang, Yanping A1 - Watling, James I. A1 - Wells, Konstans A1 - Williams, Christopher D. A1 - Willig, Michael R. A1 - Woinarski, John C. Z. A1 - Wolf, Jan H. D. A1 - Woodcock, Ben A. A1 - Yu, Douglas W. A1 - Zailsev, Andreys A1 - Collen, Ben A1 - Ewers, Rob M. A1 - Mace, Georgina M. A1 - Purves, Drew W. A1 - Scharlemann, Jörn P. W. A1 - Pervis, Andy T1 - The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts JF - Ecology and Evolution N2 - Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species' threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project - and avert - future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups - including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems - ). We make site-level summary data available alongside this article. The full database will be publicly available in 2015. KW - urban-rural gradient KW - instensively managed farmland KW - Mexican coffee plantations KW - Bombus Spp. Hymenoptera KW - bumblebee nest density KW - data sharing KW - land use KW - habitat destruction KW - global change KW - land-use change KW - plant community composition KW - Northeastern Costa Rica KW - dung beetle coleoptera KW - bird species richness Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114425 VL - 4 IS - 24 ER - TY - JOUR A1 - Akhoon, Bashir A. A1 - Singh, Krishna P. A1 - Varshney, Megha A1 - Gupta, Shishir K. A1 - Shukla, Yogeshwar A1 - Gupta, Shailendra K. T1 - Understanding the Mechanism of Atovaquone Drug Resistance in Plasmodium falciparum Cytochrome b Mutation Y268S Using Computational Methods JF - PLOS ONE N2 - The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance. KW - molecular-dynamics simulations KW - HIV-1 protease KW - structure prediction KW - saccharomyces cerevisiae KW - I-tasser KW - inhibitors KW - binding KW - malaria KW - complex KW - protein-protein interactions Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114882 VL - 9 IS - 10 ER - TY - THES A1 - Wurster, Sebastian T1 - Die Bedeutung von LIN9 für die Regulation der Genexpression, die genomische Stabilität und die Tumorsuppression T1 - The significance of LIN9 for gene regulation, genomic stability and tumor suppression N2 - Pocket proteins and E2F transcription factors regulate the expression of cell cycle associated genes and play a central role in the coordination of cell division, differentiation, and apoptosis. Disorders of these pathways contribute to the development of various human tumor entities. Despite intensive research in the field of cell cycle regulation many details are not yet understood. The LIN complex (LINC / DREAM) is a recently discovered human multiprotein complex, which dynamically interacts with pocket proteins and E2F transcription factors. An essential component of the LIN complex is the LIN9 protein. In order to obtain a better insight into the function of this protein in cell cycle regulation and tumorigenesis, a conditional Lin9 knockout mouse model was established in our laboratory. The primary objective of this study was the phenotypic characterization of embryonic fibroblasts (MEFs) from these mice. Shortly after inactivation of Lin9 cell proliferation was massively impaired. Multiple types of mitotic defects such as structural abnormalities of the spindle apparatus, aberrant nuclei, failed nuclear segregation and cytokinesis failure have been observed in Lin9-depleted cells leading to a dramatic increase in polyploid and aneuploid cells. Ultimately these serious aberrations result in premature cellular senescence. If the senescence of Lin9-deficient cells is overcome by the Large T antigen the cells can adhere to the loss of Lin9, but show severe genomic instability and grow anchorage-independently in soft-agar as a sign of oncogenic transformation. In the second part of the thesis the gene expression of Lin9-deficient cells was assessed by quantitative real time PCR analyses to determine, whether the mitotic abnormalities are caused by transcriptional defects. Here a significant reduction of mitotic gene expression was observed in Lin9-depleted cells. Additionally chromatin immunoprecipitation experiments were performed to clarify the underlying molecular mechanisms. Compared to control cells epigenetic alterations at the promoters of mitotic target genes with regard to activating histone modifications were found in Lin9-deficient MEFs. In the last section of this study, the effects of Lin9 heterozygosity were analyzed. Lin9 heterozygous MEFs showed normal proliferation, although expression of different mitotic genes was slightly reduced. It appeared, however, that the mitotic spindle checkpoint of Lin9 heterozygous MEFs is weakened and thus over several cell generations an increase in polyploid cells was observed. Soft-agar assays showed that Lin9 heterozygosity contributes to oncogenic transformation. Taken together, these results document a crucial role of LIN9 in the regulation of cell cycle-associated gene expression. LIN9 is an essential factor for cell proliferation on one hand, while at the same time it functions as a tumor suppressor. N2 - Pocket-Proteine und E2F-Transkriptionsfaktoren regulieren die Expression von Zellzyklus-assoziierten Genen und spielen eine zentrale Rolle bei der Koordination der Zellteilung, Differenzierung und Apoptose. Störungen dieser Signalwege tragen zur Entstehung zahlreicher Tumorentitäten beim Menschen bei. Trotz der intensiven Untersuchung der Zellzyklusregulation sind viele Details noch unverstanden. Der LIN-Komplex (LINC / DREAM) ist ein kürzlich entdeckter humaner Multiprotein-komplex, welcher dynamisch mit Pocket-Proteinen und E2F-Transkriptionsfaktoren interagiert. Eine essentielle Komponente des LIN-Komplexes ist das LIN9-Protein. Um die Funktion dieses Proteins bei der Zellzyklusregulation und Tumorentstehung genauer untersuchen zu können, wurde in unserer Arbeitsgruppe ein konditionelles Lin9-Knockout-Mausmodell etabliert. Primäres Ziel der Arbeit war es, den Phänotyp embryonaler Fibroblasten (MEFs) aus diesen Mäusen zu charakterisieren. Bereits kurz nach Inaktivierung von Lin9 konnte ein stark verlangsamtes Zellwachstums beobachtet werden. In Lin9-depletierten MEFs wurden multiple mitotische Defekte detektiert, die u. a. strukturelle Auffälligkeiten des Spindelapparates, aberrante Zellkerne, Störungen der Chromosomensegregation sowie zytokinetische Defekte umfassen und in einer dramatischen Zunahme polyploider und aneuploider Zellen resultieren. Im Langzeitverlauf führen diese erheblichen Aberrationen zu einer vorzeitigen zellulären Seneszenz. Wird diese durch das Large T-Protoonkogen durchbrochen, können sich MEFs an den Verlust von Lin9 adaptieren, zeigen dann jedoch eine hochgradige genomische Instabilität und Substrat-unabhängiges Wachstum im Weichagar als Zeichen onkogener Transformation. Im zweiten Abschnitt der vorliegenden Arbeit wurde die Genexpression in Lin9-defizienten MEFs mittels quantitativer Real Time-PCR untersucht um zu klären, ob die beschriebenen Defekte auf Veränderungen der transkriptionellen Aktivität zurück-zuführen sind. Dabei wurde eine erhebliche Reduktion der Expressionslevel mitotischer Gene nach Verlust von Lin9 beobachtet. Des Weiteren wurden zur Klärung der zu Grunde liegenden molekularen Mechanismen Chromatin-Immunpräzipitations-Experimente (ChIP) durchgeführt. Im Vergleich zu Kontrollzellen wurden dabei in Lin9-defizienten Zellen signifikante epigenetische Veränderungen bezüglich aktivierender Histon-Modifikationen an den Promotoren mitotischer Lin9-Zielgene festgestellt. Im letzten Abschnitt der Arbeit sollten die Auswirkungen des heterozygoten Verlustes von Lin9 analysiert werden. Dabei zeigte sich, dass Lin9-haploinsuffiziente Zellen normal proliferieren, obwohl die Expression verschiedener G2/M-Gene leicht vermindert war. Es wurde jedoch eine Schwächung des mitotischen Spindelkontrollpunktes und in der Folge über mehrere Zellgenerationen eine Zunahme polyploider Zellen beobachtet. Mit Weichagar-Assays konnte gezeigt werden, dass bereits der heterozygote Verlust des Lin9-Gens zur onkogenen Transformation beiträgt. Zusammengenommen dokumentieren diese Studien, dass LIN9 eine entscheidende Bedeutung bei der Regulation von Zellzyklus-assoziierten Genen spielt und sowohl einen essentiellen Faktor für die Zellproliferation darstellt als auch durch die Gewährleistung genomischer Stabilität tumorsuppressive Eigenschaften aufweist. KW - Zellzyklus KW - Genexpression KW - Mitose KW - Knock-Out KW - LIN9 KW - Mausmodell KW - konditioneller Knockout Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114967 ER - TY - JOUR A1 - Stellamanns, Eric A1 - Uppaluri, Sravanti A1 - Hochstetter, Axel A1 - Heddergott, Niko A1 - Engstler, Markus A1 - Pfohl, Thomas T1 - Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei JF - Scientific Reports N2 - Unicellular parasites have developed sophisticated swimming mechanisms to survive in a wide range of environments. Cell motility of African trypanosomes, parasites responsible for fatal illness in humans and animals, is crucial both in the insect vector and the mammalian host. Using millisecond-scale imaging in a microfluidics platform along with a custom made optical trap, we are able to confine single cells to study trypanosome motility. From the trapping characteristics of the cells, we determine the propulsion force generated by cells with a single flagellum as well as of dividing trypanosomes with two fully developed flagella. Estimates of the dissipative energy and the power generation of single cells obtained from the motility patterns of the trypanosomes within the optical trap indicate that specific motility characteristics, in addition to locomotion, may be required for antibody clearance. Introducing a steerable second optical trap we could further measure the force, which is generated at the flagellar tip. Differences in the cellular structure of the trypanosomes are correlated with the trapping and motility characteristics and in consequence with their propulsion force, dissipative energy and power generation. KW - African Trypanosomes KW - components KW - bacteria KW - brain Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115348 SN - 2045-2322 VL - 4 IS - 6515 ER - TY - JOUR A1 - Pamir, Evren A1 - Szyszka, Paul A1 - Scheiner, Ricarda A1 - Nawrot, Martin P. T1 - Rapid learning dynamics in individual honeybees during classical conditioning JF - Frontiers in Behavioral Neuroscience N2 - Associative learning in insects has been studied extensively by a multitude of classical conditioning protocols. However, so far little emphasis has been put on the dynamics of learning in individuals. The honeybee is a well-established animal model for learning and memory. We here studied associative learning as expressed in individual behavior based on a large collection of data on olfactory classical conditioning (25 datasets, 3298 animals). We show that the group-averaged learning curve and memory retention score confound three attributes of individual learning: the ability or inability to learn a given task, the generally fast acquisition of a conditioned response (CR) in learners, and the high stability of the CR during consecutive training and memory retention trials. We reassessed the prevailing view that more training results in better memory performance and found that 24 h memory retention can be indistinguishable after single-trial and multiple-trial conditioning in individuals. We explain how inter-individual differences in learning can be accommodated within the Rescorla Wagner theory of associative learning. In both data-analysis and modeling we demonstrate how the conflict between population-level and single-animal perspectives on learning and memory can be disentangled. KW - sucrose sensitivity KW - sucrose responsiveness KW - learning curve KW - bees KW - apis mellifera KW - single-trial learning KW - classical conditioning KW - Rescorla-Wagner model KW - proboscis extension response (PER) Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115365 SN - 1662-5153 VL - 8 IS - 313 ER - TY - JOUR A1 - Muthalagu, Nathiya A1 - Junttila, Melissa R. A1 - Wiese, Kathrin E. A1 - Wolf, Elmar A1 - Morton, Jennifer A1 - Bauer, Barbara A1 - Evan, Gerard I. A1 - Eilers, Martin A1 - Murphy, Daniel J. T1 - BIM Is the Primary Mediator of MYC-Induced Apoptosis in Multiple Solid Tissues JF - Cell Reports N2 - MYC is one of the most frequently overexpressed oncogenes in human cancer, and even modestly deregulated MYC can initiate ectopic proliferation in many postmitotic cell types in vivo. Sensitization of cells to apoptosis limits MYC's oncogenic potential. However, the mechanism through which MYC induces apoptosis is controversial. Some studies implicate p19ARF-mediated stabilization of p53, followed by induction of proapoptotic BH3 proteins NOXA and PUMA, whereas others argue for direct regulation of BH3 proteins, especially BIM. Here, we use a single experimental system to systematically evaluate the roles of p19ARF and BIM during MYC-induced apoptosis, in vitro, in vivo, and in combination with a widely used chemotherapeutic, doxorubicin. We find a common specific requirement for BIM during MYC-induced apoptosis in multiple settings, which does not extend to the p53-responsive BH3 family member PUMA, and find no evidence of a role for p19ARF during MYC-induced apoptosis in the tissues examined. KW - ARF tumor-suppressor induced lymphomagenes KW - BCL-X-L P53 KW - C-MYC PUMA KW - in-vivo expression KW - cell-cycle arrest cancer therapy Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115370 VL - 8 IS - 5 ER - TY - JOUR A1 - Kern, Selina A1 - Agarwal, Shruti A1 - Huber, Kilian A1 - Gehring, Andre P. A1 - Strödke, Benjamin A1 - Wirth, Christine C. A1 - Brügl, Thomas A1 - Abodo, Liane Onambele A1 - Dandekar, Thomas A1 - Doerig, Christian A1 - Fischer, Rainer A1 - Tobin, Andrew B. A1 - Alam, Mahmood M. A1 - Bracher, Franz A1 - Pradel, Gabriele T1 - Inhibition of the SR Protein-Phosphorylating CLK Kinases of Plasmodium falciparum Impairs Blood Stage Replication and Malaria Transmission JF - PLOS ONE N2 - Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-beta-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs. KW - parasite KW - expression KW - mosquito KW - splicing factors KW - lactate dehydrogenase KW - xanthurenic acid KW - in-vitro KW - RNA-SEQ KW - identification KW - culture Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115405 SN - 1932-6203 VL - 9 IS - 9 ER - TY - JOUR A1 - Senecal, Jean-Luc A1 - Isabelle, Catherine A1 - Fritzler, Marvin J. A1 - Targoff, Ira N. A1 - Goldstein, Rose A1 - Gagne, Michel A1 - Raynauld, Jean-Pierre A1 - Joyal, France A1 - Troyanov, Yves A1 - Dabauvalle, Marie-Christine T1 - An Autoimmune Myositis-Overlap Syndrome Associated With Autoantibodies to Nuclear Pore Complexes Description and Long-Term Follow-up of the Anti-Nup Syndrome JF - Medicine N2 - Autoimmune myositis encompasses various myositis-overlap syndromes, each being identified by the presence of serum marker autoantibodies. We describe a novel myositis-overlap syndrome in 4 patients characterized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes. The clinical phenotype was characterized by prominent myositis in association with erosive, anti-CCP, and rheumatoid factor-positive arthritis, trigeminal neuralgia, mild interstitial lung disease, Raynaud phenomenon, and weight loss. The myositis was typically chronic, relapsing, and refractory to corticosteroids alone, but remitted with the addition of a second immuno-modulating drug. There was no clinical or laboratory evidence for liver disease. The prognosis was good with 100% long-term survival (mean follow-up 19.5 yr). By indirect immunofluorescence on HEp-2 cells, sera from all 4 patients displayed a high titer of antinuclear autoantibodies (ANA) with a distinct punctate peripheral (rim) fluorescent pattern of the nuclear envelope characteristic of nuclear pore complexes. Reactivity with nuclear pore complexes was confirmed by immunoelectron microscopy. In a cohort of 100 French Canadian patients with autoimmune myositis, the nuclear pore complex fluorescent ANA pattern was restricted to these 4 patients (4%). It was not observed in sera from 393 adult patients with systemic sclerosis (n = 112), mixed connective tissue disease (n = 35), systemic lupus (n = 94), rheumatoid arthritis (n = 45), or other rheumatic diseases (n = 107), nor was it observed in 62 normal adults. Autoantibodies to nuclear pore complexes were predominantly of IgG isotype. No other IgG autoantibody markers for defined connective tissue diseases or overlap syndromes were present, indicating a selective and highly focused immune response. In 3 patients, anti-nuclear pore complex autoantibody titers varied in parallel with myositis activity, suggesting a pathogenic link to pathophysiology. The nuclear pore complex proteins, that is, nucleoporins (nup), recognized by these sera were heterogeneous and included Nup358/RanBP2 (n = 2 patients), Nup90 (n = 1), Nup62 (n = 1), and gp210 (n = 1). Taken together the data suggest that nup autoantigens themselves drive the anti-nup autoimmune response. Immunogenetically, the 4 patients shared the DQA1*0501 allele associated with an increased risk for autoimmune myositis. In conclusion, we report an apparent novel subset of autoimmune myositis in our population of French Canadian patients with connective tissue diseases. This syndrome is recognized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes that react with nups, consistent with an "anti-nupsyndrome.'' KW - idiopathic inflammatory myopathies KW - primary biliary-cirrhosis KW - transfer RNA-synthetases KW - major histocompatibility complex KW - systemic sclerosis KW - French-Canadian patients KW - protein KW - predictive factors KW - envelope KW - antibodies Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114829 SN - 0025-7974 VL - 93 IS - 24 ER - TY - JOUR A1 - Rozycka, Miroslawa A1 - Wojtas, Magdalena A1 - Jakob, Michal A1 - Stigloher, Christian A1 - Grzeszkowiak, Mikolaj A1 - Mazur, Maciej A1 - Ozyhar, Andrzej T1 - Intrinsically Disordered and Pliable Starmaker-Like Protein from Medaka (Oryzias latipes) Controls the Formation of Calcium Carbonate Crystals JF - PLOS ONE N2 - Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed. KW - circular-dichroism KW - unstructured proteins KW - olyelectrolyte domains KW - modulating KW - biominarlization proteins KW - nacreous layer formation KW - alpha-helical structure KW - dye stains-all KW - polyelectrolyte domains KW - phosphorylation sites KW - procambarus-clarkii KW - secondary structure Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114251 SN - 1932-6203 VL - 9 IS - 12 ER - TY - THES A1 - Sibilski, Claudia T1 - Identification and characterization of the novel mKSR1 phosphorylation site Tyr728 and its role in MAPK signaling T1 - Identifizierung und Charakterisierung der neuartigen mKSR1-Phosphorylierungsstelle Tyr728 und deren Rolle in der MAPK-Signalkaskade N2 - In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work. Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases. Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity. Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1. Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses. N2 - KSR1 fungiert bei Säugetieren als zentrales Gerüstprotein, welches die Anordnung von RAF/MEK/ERK-Komplexen koordiniert und die intrazelluläre Signalweiterleitung nach extrazellulärer Stimulation reguliert. Eine abweichende Aktivierung des entsprechenden MAPK-Signalwegs wurde mit vielen humanen Krebsformen und einigen Entwicklungsstörungen in Verbindung gebracht. Der Mechanismus der KSR1-Regulierung ist hochgradig komplex und involviert mehrfach Schritte der Phosphorylierung/Dephosphorylierung. In der vorliegenden Studie wurden etliche neue in-vivo-Phosphorylierungsstellen in mKSR1 mittels massenspektrometrischer Analyse entdeckt. Neben anderen wurde Tyr728 als besonderer regulatorischer Rest identifiziert, welcher durch LCK, einem Mitglied der Src-Kinase-Familie, phosphoryliert wird. Um zu verstehen wie die Phosphorylierung von Tyr728 die Funktion von KSR1 innerhalb der Signalweiterleitung und zellulärer Prozesse regulieren könnte, wurden strukturelle Modellierungen und biochemische Untersuchungen in diese Arbeit integriert. Die Computermodellierung der mKSR1(KD)-Proteinstruktur zeigte starke Wasserstoff- brückenbindungen zwischen Phospho-Tyr728 und den Resten in der Umgebung von Arg649 auf. Dieses Muster war auffällig verändert, wenn Tyr728 nicht phosphoryliert oder substituiert war. Wie anhand biochemischer Analyse untermauert wurde, könnte Arg649 für phospho-Tyr728 als Hauptankerpunkt dienen, um interne Strukturen in KSR1 zu stabilisieren. In Übereinstimmung mit den Ergebnissen der Proteinmodellierung enthüllten die Mutationsstudien, dass die Substitution von Tyr728 mit Phenylalanin zu einer weniger kompakten Interaktion zwischen KSR1 und MEK, einer erleichterten KSR1/B-RAF-Bindung und einer ansteigenden Phosphorylierung von MEK im Komplex mit KSR1 führt. Anhand dieser Erkenntnisse kann man rückschließen, dass Phospho-Tyr728 in die Verstärkung der Interaktionen innerhalb der KSR1/MEK-Grenzfläche und in die Regulierung der Phosphorylierung von KSR1-gebundenem MEK durch entweder RAF- oder KSR1-Kinasen involviert ist. Neben Tyr728 wurde Ser722 als eine neuartige regulatorische Phosphorylierungsstelle identifiziert. Aminosäureaustausche an der betreffenden Position demonstrierten, dass Ser722 die Phosphorylierung von KSR1-gebundenem MEK reguliert ohne die KSR1/MEK-Bindung selbst zu beeinträchtigen. Bedingt durch seine Lokalisierung könnte Ser722 folglich die katalytische Aktivität von KSR1 kontrollieren, indem es den Zugang des Substrates (möglicherweise MEK) zur aktiven Seite der KSR1-Kinase behindert. Zusammen mit Ser722 könnte phosphoryliertes Tyr728 ferner die Kinaseaktivität von KSR1 positiv beeinflussen, infolge von dessen Nähe zur Aktivierungs- und katalytischen Schleife in der KSR1(KD). Wie mittels Strukturmodellierung offengelegt wurde, bildet Phospho-Tyr728 eine Wasserstoffbrücke mit dem hochgradig konservierten Lys685 aus. Folglich hat Phospho-Tyr728 einen stabilisierenden Effekt auf interne Strukturen, welche in die katalytische Reaktion involviert sind, und erleichtert möglicherweise den Phosphattransfer innerhalb der katalytischen Spalte in KSR1. In Anbetracht dieser Fakten scheint es sehr wahrscheinlich, dass die LCK-abhängige Phosphorylierung von Tyr728 eine äußerst wichtige Rolle in der Regulierung der katalytischen Aktivität von KSR1 spielt. Die Ergebnisse der Fraktionierungs- und Morphologieanalysen enthüllten, dass KSR1 für die Phosphorylierung an Tyr728 LCK zum Zytoskelett rekrutiert, was darauf hindeutet, dass dieser Rest die Dynamik des Zytoskeletts und folglich Zellmotilität regulieren könnte. Darüber hinaus ist die Phosphorylierung von Tyr728 in die Regulierung der Zellproliferation involviert, wie anhand einer bedeutend reduzierten Populationsverdopplungszeit von KSR1-Y728F-Zellen im Vergleich zu Zellen, welche wildtypisches KSR1 exprimieren, gezeigt wurde. Zusammenfassend lässt sich sagen, dass die Tyrosin-Phosphorylierung in KSR1 eine neue Verknüpfung zwischen Kinasen der Src-Familie und der MAPK-Signalwirkung enthüllt. Tyr728, die neuartige regulatorische Phosphorylierungsstelle in Maus-KSR1, könnte den Übergang zwischen der Gerüst- und der katalytischen Funktion von KSR1 koordinieren und damit als Kontrollpunkt dienen, um zelluläre Reaktionen fein abzustimmen. KW - MAP-Kinase KW - Signaltransduktion KW - Regulation KW - tyrosine phosphorylation KW - KSR1 KW - LCK KW - MAPK KW - phosphorylation KW - signaling Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114672 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 - Linder, Bastian A1 - Hirmer, Anja A1 - Gal, Andreas A1 - Rüther, Klaus A1 - Bolz, Hanno Jörn A1 - Winkler, Christoph A1 - Laggerbauer, Bernhard A1 - Fischer, Utz T1 - Identification of a PRPF4 Loss-of-Function Variant That Abrogates U4/U6.U5 Tri-snRNP Integration and Is Associated with Retinitis Pigmentosa N2 - Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina. KW - zebrafish KW - embryos KW - immunoprecipitation KW - arginine KW - messenger RNA KW - spliceosomes KW - mutation KW - RNA splicing Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113663 ER - TY - JOUR A1 - Remmele, Christian W. A1 - Xian, Yibo A1 - Albrecht, Marco A1 - Faulstich, Michaela A1 - Fraunholz, Martin A1 - Heinrichs, Elisabeth A1 - Dittrich, Marcus T. A1 - Müller, Tobias A1 - Reinhardt, Richard A1 - Rudel, Thomas T1 - Transcriptional landscape and essential genes of Neisseria gonorrhoeae N2 - The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes. KW - Neisseria gonorrhoeae Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113676 ER - TY - THES A1 - Gjorgjevikj, Maja T1 - IL-4 analogues with site-specific chemical modification as screening tools for foldamers T1 - IL-4-Muteine mit ortsspezifische chemische Modifikation als Screening-Tools für Foldamere N2 - The cytokine Interleukin-4 (IL-4) plays a crucial role in the pathophysiology and progression of asthma and other atopic diseases. Its activities are signaled into the cells upon binding to and signaling through a shared receptor complex composed of the subunits IL-4Rα and common γc. Another cytokine, Interleukin-13 shares many functions with IL-4. This can be explained by the fact that both, IL-4 and IL-13, can signal via a shared receptor complex comprising the IL-4R and the IL-13R1 subunit. Therefore, the IL-4Rα receptor subunit has become a highly promising drug target, since it mediates IL-4 and IL-13 responses and blocking IL-4Rα will abrogate IL-4 as well as IL-13 effector functions. Currently, an IL-4 based mutein (Pitrakinra), acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. This work describes the generation and production of biologically active IL-4 muteins, which contain a single additional engineered cysteine. The introduction of a free thiol group allows site-specific chemical modification. The muteins were expressed in E. coli in insoluble form, refolded and purified. The thiol group of the mutein was protected as mixed disulfide with the tripeptide glutathione. A first attempt to chemically reduce the engineered cysteine residue failed, because the three native disulfide bonds of IL-4 exhibit a similar reactivity and chemical reduction of the native disulfide resulted in full deactivation and precipitation of the IL-4 protein. Therefore, an enzymatic approach was developed which specifically reduces the mixed disulfide bonds with an attached glutathion moiety and thus leaves the native structurally essential disulfide bonds unaltered. For optimization, four different IL-4 cysteine muteins with four cysteine residues introduced at positions close to the IL-4Rα binding site were tested and their reduction rates by glutaredoxin was determined. The enzymatic reduction occured at different rates for all four muteins indicating that accessibility is an important influence and must be determined individually for each mutant protein. After optimization of the pH value and particularly the reaction time, all muteins could be prepared with the engineered thiol group being released in reasonable yield. The proteins exhibiting the free thiol group were then modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effects of these modifications at different positions on binding to IL-4R were measured employing SPR biosensor technology. In the second project of this study, foldamers, which represent a new class of stable, compactly folded biomolecules and can specifically interact with proteins and nucleic acids, were examined to identify their potential as new drugs to interfere with IL-4 activities. Fragment-based drug discovery offers great promise for providing new starting points for drug discovery and facilitates the lead optimization. As foldamers equipped with a thiol-group for tethering could not to be produced; only the effect of foldamers present in a synthesized foldamer library on the binding to IL-4R could be tested. Two libraries containing different foldamers based on aromatic amide were synthesized by Michael Grotz and Dr. Michael Deligny and tested in our lab for their capability to disrupt the ligand-receptor interaction of IL-4 and its receptor IL-4Rα [ECD] using surface plasmon resonance technology. None of the studied foldamers could specifically inhibit the IL-4/IL-4Rα interaction. Some foldamers showed non-specific binding. The study presented here shows the design and production of a potentially new type of IL-4 antagonists, which employ site-specific chemical modification to exert their antagonistic function. N2 - Das Zytokin Interleukin-4 (IL-4) spielt eine entscheidende Rolle in der Entstehung und Pathophysiologie von Asthma und anderen atopischen Krankheiten. Seine Aktivitäten können in die Zelle durch die Bindung an einen Rezeptorkomplex übertragen werden, welcher aus den Untereinheiten IL-4Rα und γc besteht. Interleukin-13 (IL-13), ein verwandtes Zytokin, und IL-4 besitzen viele gemeinsame Funktionen. Das kann dadurch erklärt werden, dass IL-4 wie auch IL-13 ihre Signale über einen gemeinsamen Rezeptorkomplex übertragen können, der aus der IL-4R und der IL-13R1 Untereinheit besteht. Die IL-4R Untereinheit ist ein vielversprechendes Zielmolekül für die Entwicklung von Pharmaka, da sie IL-4 und IL-13 Reaktionen vermittelt. Durch Blockieren von IL-4R werden die Aktivitäten von IL-4 sowie IL-13 unterdrückt. Ein IL-4 basiertes Doppelmutein (Pitrakinra), welches als Gegenspieler zu IL-4 und IL-13 Rezeptoren fungiert, befindet sich derzeit in der klinischen Entwicklung. In dieser Arbeit wird die Bildung und Produktion von biologisch aktiven IL-4 Muteinen mit einem einzelnen zusätzlich eingefügten Cysteinrest beschrieben. Die Einführung einer freien Thiol-Gruppe ermöglicht ortsspezifische chemische Modifizierungen. Ein „Tethering“ Ansatz sollte dann auch eine sehr schwach Bindung von thiol-reaktiven Verbindungen an IL-4 messbar machen. Die Muteine wurden in unlöslicher Form in E. coli exprimiert, zurückgefaltet und auf gereinigt. Dabei wurde die Thiolgruppe des Muteins als Disulfid mit dem Tripeptid Glutathion geschützt. Erste Versuche gezielt den eingeführten Cysteinrest selektiv chemisch zu reduzieren schlugen fehl, da die drei proteineigenen Disulfidbrücken von IL-4 eine ähnliche Reaktivität zeigten, und die Reduktion zur vollständigen Desaktivierung und Fällung des IL-4 Proteins führte. Daher wurde ein enzymatischer Ansatz entwickelt, der gezielt die Disulfidbrücke zum Glutathionrest reduziert und die proteineigenen strukturell essentiellen Disulfidbrücken unverändert lässt. Zur Optimierung wurden vier verschiedene IL-4 Cystein-Muteine mit Cysteinresten an verschiedenen Positionen nahe der IL-4Rα Bindungsstelle getestet und die Reduktionsgeschwindigkeit in Gegenwart von Glutaredoxin bestimmt. Die enzymatische Reduktion verlief für alle vier Muteine mit verschiedenen Geschwindigkeiten. Dies deutet darauf hin, dass die Zugänglichkeit der Disulfidgruppe einen wichtigen Einfluss besitzt. Die Reduktionsbedingungen mussten daher für jedes Mutein neu bestimmt werden. Nach Optimierung des pH Wertes und insbesondere der Reaktionszeit konnten alle Muteine mit einer freien Thiolgruppe in angemessener Ausbeute erhalten werden. Die Proteine mit jeweils einer freien Thiolgruppe wurden daraufhin mit N-Ethylmaleinimid (NEM) oder Maleimido-PEG modifiziert. Die Effekte der Modifizierung an verschiedenen Positionen des IL-4 auf die Bindung an IL-4R wurden mit Hilfe der SPR-Spektroskopie (Oberflächen Plasmon Resonanz Spektroskopie) gemessen. Im zweiten Teil dieser Arbeit wurde die Interaktion von Foldameren mit der IL-4Ra Rezeptorkette untersucht. Foldamere stellen eine neue Klasse von stabilen, kompakt gefalteten Biomolekülen dar, die möglicherweise spezifisch mit Proteinen und Nukleinsäuren wechselwirken können. Es sollten Vorversuche durchgeführt werden um zu sondieren, ob aus Foldameren Hemmstoffe für IL-4 und IL-13 entwickelt werden können. Da Foldamere mit einer Thiolgruppe zur Anbindung (Tethering) an IL-4 nicht hergestellt werden konnten, wurden zunächst nur nichtreaktive Foldamare aus einer synthetisierten Foldamer-Bibliothek getestet. Zwei Bibliotheken mit verschiedenen auf aromatischen Amiden basierenden Foldameren wurden von Michael Grotz und Dr. Michael Deligny synthetisiert und von mir mit Hilfe der SPR Spektroskopie auf ihre Fähigkeit getestet, die Ligand-Rezeptor Wechselwirkung von IL-4 und derIL-4Rα Rezeptoruntereinheit zu unterbinden. Keines der untersuchten Foldamere konnte die IL-4/IL-4Rα Wechselwirkung spezifisch hemmen. Einige Foldamere zeigten eine unspezifische Bindung. Die hier dargestellten Studien zeigen das Design und die Herstellung eines potentiell neuen Typs von Gegenspieler zu IL-4, welcher ortsspezifische chemische Modifikationen ausnutzt um seine antagonistische Funktion zu erfüllen. KW - Il 4 KW - Foldamere KW - Modifizierung KW - Foldamers KW - PEG chemical modification KW - Zutokin KW - chemische Modifizierung KW - SPR-Spektroskopie Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113531 ER - TY - JOUR A1 - Römer, Daniela A1 - Roces, Flavio T1 - Nest Enlargement in Leaf-Cutting Ants: Relocated Brood and Fungus Trigger the Excavation of New Chambers N2 - During colony growth, leaf-cutting ants enlarge their nests by excavating tunnels and chambers housing their fungus gardens and brood. Workers are expected to excavate new nest chambers at locations across the soil profile that offer suitable environmental conditions for brood and fungus rearing. It is an open question whether new chambers are excavated in advance, or will emerge around brood or fungus initially relocated to a suitable site in a previously-excavated tunnel. In the laboratory, we investigated the mechanisms underlying the excavation of new nest chambers in the leaf-cutting ant Acromyrmex lundi. Specifically, we asked whether workers relocate brood and fungus to suitable nest locations, and to what extent the relocated items trigger the excavation of a nest chamber and influence its shape. When brood and fungus were exposed to unfavorable environmental conditions, either low temperatures or low humidity, both were relocated, but ants clearly preferred to relocate the brood first. Workers relocated fungus to places containing brood, demonstrating that subsequent fungus relocation spatially follows the brood deposition. In addition, more ants aggregated at sites containing brood. When presented with a choice between two otherwise identical digging sites, but one containing brood, ants' excavation activity was higher at this site, and the shape of the excavated cavity was more rounded and chamber-like. The presence of fungus also led to the excavation of rounder shapes, with higher excavation activity at the site that also contained brood. We argue that during colony growth, workers preferentially relocate brood to suitable locations along a tunnel, and that relocated brood spatially guides fungus relocation and leads to increased digging activity around them. We suggest that nest chambers are not excavated in advance, but emerge through a self-organized process resulting from the aggregation of workers and their density-dependent digging behavior around the relocated brood and fungus. KW - fungi KW - ants KW - fungal structure KW - fungal pathogens KW - foraging KW - humidity KW - pupae KW - fungal diseases Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112860 ER - TY - JOUR A1 - Hopfenmueller, Sebastian A1 - Steffan-Dewenter, Ingolf A1 - Holzschuh, Andrea T1 - Trait-Specific Responses of Wild Bee Communities to Landscape Composition, Configuration and Local Factors N2 - Land-use intensification and loss of semi-natural habitats have induced a severe decline of bee diversity in agricultural landscapes. Semi-natural habitats like calcareous grasslands are among the most important bee habitats in central Europe, but they are threatened by decreasing habitat area and quality, and by homogenization of the surrounding landscape affecting both landscape composition and configuration. In this study we tested the importance of habitat area, quality and connectivity as well as landscape composition and configuration on wild bees in calcareous grasslands. We made detailed trait-specific analyses as bees with different traits might differ in their response to the tested factors. Species richness and abundance of wild bees were surveyed on 23 calcareous grassland patches in Southern Germany with independent gradients in local and landscape factors. Total wild bee richness was positively affected by complex landscape configuration, large habitat area and high habitat quality (i.e. steep slopes). Cuckoo bee richness was positively affected by complex landscape configuration and large habitat area whereas habitat specialists were only affected by the local factors habitat area and habitat quality. Small social generalists were positively influenced by habitat area whereas large social generalists (bumblebees) were positively affected by landscape composition (high percentage of semi-natural habitats). Our results emphasize a strong dependence of habitat specialists on local habitat characteristics, whereas cuckoo bees and bumblebees are more likely affected by the surrounding landscape. We conclude that a combination of large high-quality patches and heterogeneous landscapes maintains high bee species richness and communities with diverse trait composition. Such diverse communities might stabilize pollination services provided to crops and wild plants on local and landscape scales. KW - habitats KW - bees KW - grasslands KW - species diversity KW - biodiversity KW - pollination KW - flowers KW - foraging Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112872 ER - TY - JOUR A1 - Morton, Charles Oliver A1 - Fliesser, Mirjam A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Bauer, Ruth A1 - Kneitz, Susanne A1 - Hope, William A1 - Rogers, Thomas Richard A1 - Einsele, Hermann A1 - Löffler, Jürgen T1 - Gene Expression Profiles of Human Dendritic Cells Interacting with Aspergillus fumigatus in a Bilayer Model of the Alveolar Epithelium/Endothelium Interface N2 - The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA. KW - aspergillus fumigatus KW - gene expression KW - immune receptors KW - immune response KW - denritic cells KW - B cell receptors KW - gene regulation KW - RNA extraction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112893 ER - TY - JOUR A1 - Albert, Štefan A1 - Spaethe, Johannes A1 - Grübel, Kornelia A1 - Rössler, Wolfgang T1 - Royal jelly-like protein localization reveals differences in hypopharyngeal glands buildup and conserved expression pattern in brains of bumblebees and honeybees N2 - Royal jelly proteins (MRJPs) of the honeybee bear several open questions. One of them is their expression in tissues other than the hypopharyngeal glands (HGs), the site of royal jelly production. The sole MRJP-like gene of the bumblebee, Bombus terrestris (BtRJPL), represents a pre-diversification stage of the MRJP gene evolution in bees. Here we investigate the expression of BtRJPL in the HGs and the brain of bumblebees. Comparison of the HGs of bumblebees and honeybees revealed striking differences in their morphology with respect to sex- and caste-specific appearance, number of cells per acinus, and filamentous actin (F-actin) rings. At the cellular level, we found a temporary F-actin-covered meshwork in the secretory cells, which suggests a role for actin in the biogenesis of the end apparatus in HGs. Using immunohistochemical localization, we show that BtRJPL is expressed in the bumblebee brain, predominantly in the Kenyon cells of the mushroom bodies, the site of sensory integration in insects, and in the optic lobes. Our data suggest that a dual glandbrain function preceded the multiplication of MRJPs in the honeybee lineage. In the course of the honeybee evolution, HGs dramatically changed their morphology in order to serve a food-producing function. KW - Hypopharyngeal glands KW - Bumblebee KW - Bombus KW - Brain KW - Labial glands KW - Immunohistochemistry KW - Kenyon cells KW - Mushroom bodies KW - Honeybee Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112733 ER - TY - JOUR A1 - Rudel, Thomas A1 - Prusty, Bhupesh K. A1 - Siegl, Christine A1 - Gulve, Nitish A1 - Mori, Yasuko T1 - GP96 Interacts with HHV-6 during Viral Entry and Directs It for Cellular Degradation N2 - CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus. KW - host cells KW - immunoprecipitation KW - HeLa cells KW - antibodies KW - cell binding KW - viral transmission and infection KW - viral entry KW - flow cytometry Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111068 ER - TY - JOUR A1 - Shityakov, Sergey A1 - Förster, Carola A1 - Rethwilm, Axel A1 - Dandekar, Thomas T1 - Evaluation and Prediction of the HIV-1 Central Polypurine Tract Influence on Foamy Viral Vectors to Transduce Dividing and Growth-Arrested Cells N2 - Retroviral vectors are potent tools for gene delivery and various biomedical applications. To accomplish a gene transfer task successfully, retroviral vectors must effectively transduce diverse cell cultures at different phases of a cell cycle. However, very promising retroviral vectors based on the foamy viral (FV) backbone lack the capacity to efficiently transduce quiescent cells. It is hypothesized that this phenomenon might be explained as the inability of foamy viruses to form a pre-integration complex (PIC) with nuclear import activity in growth-arrested cells, which is the characteristic for lentiviruses (HIV-1). In this process, the HIV-1 central polypurine tract (cPPT) serves as a primer for plus-strand synthesis to produce a “flap” element and is believed to be crucial for the subsequent double-stranded cDNA formation of all retroviral RNA genomes. In this study, the effects of the lentiviral cPPT element on the FV transduction potential in dividing and growth-arrested (G1/S phase) adenocarcinomic human alveolar basal epithelial (A549) cells are investigated by experimental and theoretical methods. The results indicated that the HIV-1 cPPT element in a foamy viral vector background will lead to a significant reduction of the FV transduction and viral titre in growth-arrested cells due to the absence of PICs with nuclear import activity. KW - Evaluation KW - Prognose KW - HIV KW - Spumaviren KW - Einfluss Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112763 ER - TY - JOUR A1 - Leingärtner, Annette A1 - Hoiss, Bernhard A1 - Krauss, Jochen A1 - Steffan-Dewenter, Ingolf T1 - Combined Effects of Extreme Climatic Events and Elevation on Nutritional Quality and Herbivory of Alpine Plants N2 - Climatic extreme events can cause the shift or disruption of plant-insect interactions due to altered plant quality, e.g. leaf carbon to nitrogen ratios, and phenology. However, the response of plant-herbivore interactions to extreme events and climatic gradients has been rarely studied, although climatic extremes will increase in frequency and intensity in the future and insect herbivores represent a highly diverse and functionally important group. We set up a replicated climate change experiment along elevational gradients in the German Alps to study the responses of three plant guilds and their herbivory by insects to extreme events (extreme drought, advanced and delayed snowmelt) versus control plots under different climatic conditions on 15 grassland sites. Our results indicate that elevational shifts in CN (carbon to nitrogen) ratios and herbivory depend on plant guild and season. CN ratios increased with altitude for grasses, but decreased for legumes and other forbs. In contrast to our hypotheses, extreme climatic events did not significantly affect CN ratios and herbivory. Thus, our study indicates that nutritional quality of plants and antagonistic interactions with insect herbivores are robust against seasonal climatic extremes. Across the three functional plant guilds, herbivory increased with nitrogen concentrations. Further, increased CN ratios indicate a reduction in nutritional plant quality with advancing season. Although our results revealed no direct effects of extreme climatic events, the opposing responses of plant guilds along elevation imply that competitive interactions within plant communities might change under future climates, with unknown consequences for plant-herbivore interactions and plant community composition. KW - Plant-herbivore interactions KW - Herbivory KW - Leaves KW - Grasses KW - Legumes KW - Insects KW - Drought KW - Climate Change Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112812 ER - TY - JOUR A1 - Keller, Daniela Barbara A1 - Schultz, Jörg T1 - Word Formation Is Aware of Morpheme Family Size N2 - Words are built from smaller meaning bearing parts, called morphemes. As one word can contain multiple morphemes, one morpheme can be present in different words. The number of distinct words a morpheme can be found in is its family size. Here we used Birth-Death-Innovation Models (BDIMs) to analyze the distribution of morpheme family sizes in English and German vocabulary over the last 200 years. Rather than just fitting to a probability distribution, these mechanistic models allow for the direct interpretation of identified parameters. Despite the complexity of language change, we indeed found that a specific variant of this pure stochastic model, the second order linear balanced BDIM, significantly fitted the observed distributions. In this model, birth and death rates are increased for smaller morpheme families. This finding indicates an influence of morpheme family sizes on vocabulary changes. This could be an effect of word formation, perception or both. On a more general level, we give an example on how mechanistic models can enable the identification of statistical trends in language change usually hidden by cultural influences. KW - linguistic morphology KW - language KW - death rates KW - psycholinguistics KW - chi square tests KW - vocabulary KW - birth rates KW - culture Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112848 ER - TY - JOUR A1 - Roces, Flavio A1 - Pielström, Steffen T1 - Soil Moisture and Excavation Behaviour in the Chaco Leaf-Cutting Ant (Atta vollenweideri): Digging Performance and Prevention of Water Inflow into the Nest N2 - The Chaco leaf-cutting ant Atta vollenweideri is native to the clay-heavy soils of the Gran Chaco region in South America. Because of seasonal floods, colonies are regularly exposed to varying moisture across the soil profile, a factor that not only strongly influences workers' digging performance during nest building, but also determines the suitability of the soil for the rearing of the colony's symbiotic fungus. In this study, we investigated the effects of varying soil moisture on behaviours associated with underground nest building in A. vollenweideri. This was done in a series of laboratory experiments using standardised, plastic clay-water mixtures with gravimetric water contents ranging from relatively brittle material to mixtures close to the liquid limit. Our experiments showed that preference and group-level digging rate increased with increasing water content, but then dropped considerably for extremely moist materials. The production of vibrational recruitment signals during digging showed, on the contrary, a slightly negative linear correlation with soil moisture. Workers formed and carried clay pellets at higher rates in moist clay, even at the highest water content tested. Hence, their weak preference and low group-level excavation rate observed for that mixture cannot be explained by any inability to work with the material. More likely, extremely high moistures may indicate locations unsuitable for nest building. To test this hypothesis, we simulated a situation in which workers excavated an upward tunnel below accumulated surface water. The ants stopped digging about 12 mm below the interface soil/water, a behaviour representing a possible adaptation to the threat of water inflow field colonies are exposed to while digging under seasonally flooded soils. Possible roles of soil water in the temporal and spatial pattern of nest growth are discussed. KW - ants KW - fungi KW - surface water KW - vibration KW - acoustic signals KW - physical properties KW - analysis of variance KW - fungal structure Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111298 ER - TY - JOUR A1 - Djuzenova, Cholpon S. A1 - Memmel, Simon A1 - Sukhorukov, Vladimir L. A1 - Höring, Marcus A1 - Westerling, Katherine A1 - Fiedler, Vanessa A1 - Katzer, Astrid A1 - Krohne, Georg A1 - Flentje, Michael T1 - Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status N2 - Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types. KW - cell membranes KW - hypotonic KW - capacitance KW - isotonic KW - microvilli KW - membrane characteristics KW - membrane proteins KW - scanning electron microscopy Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111322 ER - TY - JOUR A1 - Alsheimer, Manfred A1 - Link, Jana A1 - Leubner, Monika A1 - Schmitt, Johannes A1 - Göb, Eva A1 - Benavente, Ricardo A1 - Jeang, Kuan-Teh A1 - Xu, Rener T1 - Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function N2 - LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun12/2 meiocytes attached telomeres retained the capacity to form bouquetlike clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun12/2 mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional. Author summary: Correct genome haploidization during meiosis requires tightly regulated chromosome movements that follow a highly conserved choreography during prophase I. Errors in these movements cause subsequent meiotic defects, which typically lead to infertility. At the beginning of meiotic prophase, chromosome ends are tethered to the nuclear envelope (NE). This attachment of telomeres appears to be mediated by well-conserved membrane spanning protein complexes within the NE (LINC complexes). In mouse meiosis, the two main LINC components SUN1 and SUN2 were independently described to localize at the sites of telomere attachment. While SUN1 has been demonstrated to be critical for meiotic telomere attachment, the precise role of SUN2 in this context, however, has been discussed controversially in the field. Our current study was targeted to determine the factual capacity of SUN2 in telomere attachment and chromosome movements in SUN1 deficient mice. Remarkably, although telomere attachment is impaired in the absence of SUN1, we could find a yet undescribed SUN1-independent telomere attachment, which presumably is mediated by SUN2 and KASH5. This SUN2 mediated telomere attachment is stable throughout prophase I and functional in moving telomeres within the NE. Thus, our results clearly indicate that SUN1 and SUN2, at least partially, fulfill redundant meiotic functions. KW - telomeres KW - spermatocytes KW - Oocytes KW - meiosis KW - protein domains KW - cytoskeleton KW - synapsis KW - homologous chromosomes Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111355 ER - TY - THES A1 - Riedinger, Verena T1 - Landscape-scale spillover of pollinators from oil-seed rape to crop and semi-natural habitats on different temporal scales T1 - Landschafts-Spillover von Bestäubern aus Raps auf ackerbaulich genutzte und naturnahe Habitate auf verschiedenen zeitlichen Ebenen N2 - Organisms use different resources in different habitat types during their life cycle. Thereby, they connect habitats and provide ecosystem services or disservices in several habitat types. In agricultural landscapes, the spillover of organisms, i.e. movement of an organism and its function from one habitat to another, especially from semi-natural to managed habitats, is one of the most important processes that influence population dynamics and community composition. Importantly, spillover connects habitats not only spatially, but also on different temporal scales, because availability of resources changes over time in agricultural landscapes, e.g. by mass-flowering events of crops, harvesting or crop rotation. Most often, semi-natural habitats are seen as beneficial source of organisms, but also managed habitats can provide valuable resources, and thereby initiate spillover to other habitats. Mass-flowering crops, like oil-seed rape, are such valuable feeding resources for pollinators, and pollinators might spillover from oil-seed rape to other habitats which provide alternative foraging resources. The focus of this dissertation was to evaluate the influence of oil-seed rape on pollinators in agricultural landscapes by studying effects (1) on different temporal scales (from effects during the flowering period of oil-seed rape, Chapter II & IV, to intermediate effects on a second mass-flowering crop, Chapter III, to spillover effects to the flowering period in the next year, Chapter IV), (2) semi-natural (Chapter II) and crop (Chapter III, IV) habitats, and (3) on various pollinator groups which differ in their life cycle (Chapter II, III, IV). In this dissertation effects from oil-seed rape on all temporal scales – in the short term during mass-flowering and in the long term on a late-flowering crop and even in the next year on oil-seed rape fields ─ were found. These effects might be important for crop and wild plant pollination, and pollinator conservation. Importantly, the effects on different temporal scales depend on the considered habitat (managed or different semi-natural habitats) and on the investigated pollinator group. The more pollinators match the flowering period of oil-seed rape in their activity period and the more dependent they are on flowering resources in their life cycle, the more pronounced are their responses. Effects were found for wild bees, but not for hoverflies and honey bees. Moreover, the availability of semi-natural habitats in the landscape is important and may modulate effects from oil-seed rape. The longevity of effects of oil-seed rape shows the importance of including several temporal scales into ecosystem-service studies, not only for pollinators, but also for other ecosystem-service providing species groups. N2 - Organismen nutzen während ihres Lebens verschiedene Ressourcen in unterschiedlichen Habitaten. Dabei verbinden sie Habitate miteinander und erbringen positive oder negative Ökosystemdienstleistungen in verschiedenen Habitattypen. In Agrarlandschaften ist der „Spillover“ von Organismen, d.h. die Bewegung von Organismen und die gleichzeitige Verschiebung ihrer Funktion von einem Habitat in ein anderes, insbesondere von naturnahen Habitaten hin zu landwirtschaftlich genutzten Habitaten, einer der wichtigsten Prozesse, die die Populationsdynamik und Zusammensetzung von Gemeinschaften beeinflussen. Zu betonen ist, dass Spillover Habitate nicht nur räumlich, sondern auch auf unterschiedlichen zeitlichen Skalen verbindet, da sich die Verfügbarkeit von Ressourcen in Agrarlandschaften über die Zeit, z.B. durch die Massenblüte von Feldfrüchten, die Ernte oder die Fruchtfolge, verändert. Meist werden naturnahe Habitate als wertvolle Quelle von Organismen betrachtet, aber auch landwirtschaftlich genutzte Habitate können wertvolle Ressourcen zur Verfügung stellen und damit den Spillover von Organismen in andere Habitate initiieren. Massentrachten, wie Raps, sind solche wertvollen Ressourcen für Bestäuber. Bestäuber können von Rapsfeldern auf andere Habitate überschwappen, die ihnen alternative Futterquellen bieten. Der Schwerpunkt dieser Dissertation liegt auf der Beurteilung des Einflusses von Raps auf Bestäuber in Agrarlandschaften anhand der Betrachtung von Effekten (1) auf unterschiedlichen zeitlichen Ebenen (von Effekten während der Blühperiode des Rapses, Kapitel II & IV, über mittelfristige Effekte auf eine zweite blühende Feldfrucht, Kapitel III, bis hin zu Spillover-Effekten in die Blühperiode des nächsten Jahres, Kapitel IV), (2) in naturnahen (Kapitel II) und landwirtschaftlich genutzten (Kapitel III, IV) Habitaten und (3) für unterschiedliche Bestäubergruppen, die sich in ihrem Lebenszyklus unterscheiden (Kapitel II, III, IV). In dieser Dissertation wurden Effekte von Raps auf allen untersuchten zeitlichen Ebenen gefunden – sowohl kurzfristig während der Rapsblüte als auch langfristig in einer spätblühenden Massentracht und sogar im nächsten Jahr während der Rapsblüte. Diese Effekte können Folgen für die Bestäubung von Kultur- und Wildpflanzen und auch für den Schutz von Bestäubern haben. Es ist wichtig, darauf hinzuweisen, dass die Effekte auf unterschiedlichen zeitlichen Ebenen abhängig von dem untersuchten Habitat (landwirtschaftlich genutzte, verschiedene naturnahe Habitate), sowie der untersuchten Bestäubergruppe sind. Je mehr die Bestäuber in ihrer Aktivitätsperiode mit der Blütezeit von Raps übereinstimmen und je mehr sie auf Blütenressourcen in ihrem Lebenszyklus angewiesen sind, umso größer scheint ihre Reaktion. Effekte wurden für Wildbienen, nicht aber für Schwebfliegen und Honigbienen gefunden. Darüber hinaus ist auch die Verfügbarkeit von naturnahen Habitaten in der Landschaft wichtig und kann die Effekte des Rapses beeinflussen. Die Langlebigkeit der Effekte von Raps zeigt die Bedeutung der Integration von unterschiedlichen zeitlichen Ebenen in die Untersuchung von Ökosystem-Dienstleistungen, nicht nur für Bestäuber, sondern auch für andere Artengruppen, die Ökosystem-Dienstleistungen erbringen. KW - Raps KW - Bestäuber KW - Bienen <Überfamilie> KW - Ackerrandstreifen KW - Schwebfliegen KW - mass-flowering crops KW - semi-natural habitats KW - temporal spillover KW - Massentrachten KW - naturnahe Habitate KW - zeitlicher Spillover KW - sunflowers KW - Sonnenblumen Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96844 N1 - Kapitel IV. ist mittlerweile in einer überarbeiteten Version veröffentlicht als: V. Riedinger, O. Mitesser, T. Hovestadt, I. Steffan-Dewenter, A. Holzschuh (2015). Annual dynamics of wild bee densities: attractiveness and productivity effects of oilseed rape. Ecology 96:1351-1360. ER - TY - THES A1 - Huber, Annette T1 - Chlamydial deubiquitinase ChlaDUB1 as regulator of host cell apoptosis and new target for anti-chlamydial therapy T1 - Die chlamydiale Deubiquitinase ChlaDUB1 als Regulator der Wirtszellapoptose und neue Zielstruktur in der anti-chlamydialen Therapie N2 - Chlamydia trachomatis is an obligate intracellular pathogen that replicates inside a vacuole, the so-called inclusion. During replication by a biphasic life-cycle Chlamydia secrete via their type 3 secretion system various effector proteins into the inclusion lumen, the inclusion membrane or the host cell cytosol to form their favored replication niche. Chlamydia-infected cells are highly resistant against apoptosis since the replicative form of Chlamydia is non-infectious and premature cell death would cause complete loss of one Chlamydia generation. The bacteria block apoptosis by preventing mitochondrial outer membrane permeabilization. Various proteins with anti-apoptotic function are enriched in Chlamydia-infected cells such as Mcl-1, cIAP2, Survivin or HIF1α. The accumulation of these proteins is a result of increased gene expression and direct protein stabilization. However, the molecular mechanisms and involved bacterial effector proteins are mostly unknown. With this work the molecular mechanisms of Mcl-1 stabilization and the participation of chlamydial factors were investigated. Mcl-1 is a member of the Bcl-2 protein family and has an extremely short half-life causing its permanent ubiquitination and subsequent degradation by the 26S proteasome under normal homeostasis whilst Mcl-1 accumulation results in apoptosis inhibition. It was shown that during C. trachomatis infection Mcl-1 ubiquitination is reduced causing its stabilization albeit no cellular ubiquitin-proteasome-system components are involved in this process. However, C. trachomatis express the two deubiquitinases ChlaDUB1 and ChlaDUB2 which are mostly uncharacterized. With this work the expression profile, subcellular localization, substrates and function of the deubiquitinases were investigated. It was shown that ChlaDUB1 is secreted to the surface of the inclusion where it interacts with Mcl-1 which is accumulated in the proximity of this compartment. By utilization of infection experiments, heterologous expression systems and in vitro experiments a direct interaction of ChlaDUB1 and Mcl-1 was demonstrated. Furthermore, it was shown that Mcl-1 is deubiquitinated by ChlaDUB1 causing its stabilization. During replicative phase of infection, ChlaDUB2 seems to be accumulated in the chlamydial particles. However, ChlaDUB2 substrates could not be identified which would give an indication for the physiological role of ChlaDUB2. Since 2011, a protocol to transform C. trachomatis with artificial plasmid DNA is available. As part of this work the transformation of C. trachomatis with plasmid DNA suitable for the permanent or inducible protein overexpression on a routinely basis was established. In addition, the first targeted homologous recombination into the chlamydial genome to replace the ChlaDUB1 gene by a modified one was performed and validated. The targeted homologous recombination was also used to create a ChlaDUB1 knock-out mutant; however deletion of ChlaDUB1 seems to be lethal for C. trachomatis. Due to the fact that ChlaDUB1-lacking Chlamydia could not be obtained an inhibitor screen was performed and identified CYN312 as a potential ChlaDUB1 inhibitor. Application of CYN312 during infection interfered with chlamydial growth and reduced Mcl-1 quantity in infected cells. Furthermore, CYN312 treated Ctr-infected cells were significantly sensitized for apoptosis. Taken together, C. trachomatis secretes the deubiquitinase ChlaDUB1 to the surface of the inclusion where it deubiquitinates Mcl-1 causing its accumulation in infected cells resulting in apoptosis resistance. Application of the ChlaDUB1 inhibitor CYN312 interferes with Mcl-1 stabilization sensitizing infected cells for apoptosis. N2 - Chlamydia trachomatis ist ein obligat intrazelluläres Bakterium, welches sich in einer Vakuole, der sogenannten Inclusion vermehrt. Chlamydien durchlaufen einen zweiphasigen Entwicklungszyklus während welchem sie zu bestimmten Zeitpunkten der Infektion Effektorproteine mittels ihres Typ 3 Sekretionssystems in das Inclusionslumen, die Inclusionsmembran oder das Wirtszellzytoplasma sekretieren. Durch die Aktivität der Effektorproteine schaffen die Chlamydien die für sie favorisierten Bedingungen. Zusätzlich zeigen infizierte Zellen eine hohe Resistenz gegenüber Apoptose. Ein vorzeitiger Zelltod der Wirtszelle würde zum Verlust einer vollständigen Generation an Chlamydien führen, da die replizierende Form der Chlamydien nicht infektiös ist. Chlamydien hemmen die Wirtszellapoptose indem sie die Permeabilisierung der äußeren Mitochondrienmembran verhindern. Es ist bekannt, dass mehrere anti-apoptotische Proteine wie Mcl-1, cIAP2, Survivin oder HIF1α während der Infektion mit Chlamydien zu bestimmten Zeitpunkten angereichert werden und für die Apoptoseinhibition wichtig sind. Allerdings sind die molekularen Mechanismen sowie die beteiligten bakteriellen Proteine weitestgehend unbekannt. Mit dieser Arbeit wurden die molekularen Mechanismen der Mcl-1 Stabilisierung sowie die darin involvierten chlamydialen Proteine untersucht. Mcl-1, ein Mitglied der Bcl-2 Proteinfamilie, ist ein extrem instabiles Protein welches unter normalen Bedingungen permanent ubiquitiniert und vom 26S Proteasom abgebaut wird; eine Anreicherung von Mcl-1 hingegen führt zur Apoptoseinhibierung. In dieser Arbeit konnte gezeigt werden, dass während der Chlamydieninfektion Mcl-1 weniger ubiquitiniert wird was dessen Stabilisierung zur Folge hat. Es konnte jedoch keine Beteiligung von Komponenten des zellulären Ubiquitin-Proteasom-Systems festgestellt werden. C. trachomatis exprimiert zwei Deubiquitinasen welche weitestgehend uncharakterisiert sind. Ein weiteres Ziel dieser Arbeit war es das Expressionsprofil, die Lokalisierung, Substrate und die Funktion der Deubiquitinasen zu untersuchen. Es konnte gezeigt werden, dass ChlaDUB1 zur Oberfläche der Inclusion sekretiert wird und dort mit Mcl-1 interagiert, welches in diesem Kompartiment angereichert vorliegt. Unter Verwendung von Infektionsmodellen, heterologen Expressionssystemen sowie in vitro Experimenten konnte eine direkte Bindung beider Proteine sowie die spezifische Deubiquitinierung von Mcl-1 durch ChlaDUB1 gezeigt werden. Durch die permanente Deubiquitinierung mittels ChlaDUB1 wird Mcl-1 stabilisiert und im Bereich der Inclusionsoberfläche angereichert. Im Gegensatz zu ChlaDUB1 konnte ChlaDUB2 während der replikativen Phase der Infektion nicht im Zytoplasma sondern lediglich innerhalb der Bakterien detektiert werden. Außerdem konnten bislang keine Substrate für ChlaDUB2 identifiziert werden, welche auf die physiologische Funktion dieses Effektors schließen lassen könnten. Seit 2011 ist ein Protokoll für die Transformation von Chlamydien mit artifizieller Plasmid-DNA verfügbar. Als Teil dieser Arbeit wurde die routinemäßige Transformation von Chlamydien mit Plasmid-DNA zur permanenten und induzierbaren Proteinüberexpression etabliert. Außerdem konnte die erste gezielte homologe Rekombination ins chlamydiale Genom durchgeführt werden. Hierbei wurde das ChlaDUB1-Gen durch eine modifizierte Form ersetzt. Die Herstellung einer ChlaDUB1-Deletionsmutante mittels homologer Rekombination war jedoch nicht erfolgreich, da ChlaDUB1 vermutlich essentiell für C. trachomatis ist. Da ChlaDUB1-defiziente Chlamydien nicht generiert werden konnten, wurde ein Inhibitorscreen durchgeführt und CYN312 als ChlaDUB1-Inhibitor identifiziert. Die Anwendung von CYN312 während Infektionsversuchen zeigte eine deutliche Reduktion des Chlamydienwachstums sowie eine verminderte Mcl-1 Stabilisierung. Als Folge dessen waren Chlamydien-infizierte und mit CYN312 behandelte Zellen signifikant für die Apoptoseinduktion sensibilisiert. Mit der vorliegenden Arbeit konnte gezeigt werden, dass C. trachomatis die Deubiquitinase ChlaDUB1 während der Infektion an die Oberfläche der Inclusion sekretiert. Dort katalysiert ChlaDUB1 die Deubiquitinierung von Mcl-1 was dessen Anreicherung in infizierten Zellen und somit eine erhöhte Apoptoseresistenz zur Folge hat. Die Verwendung des ChlaDUB1-Inhibitors CYN312 verhindert die Mcl-1 Stabilisierung und sensibilisiert somit infizierte Zellen für Apoptose. KW - Chlamydia trachomatis KW - Apoptosis KW - Chlamydia trachomatis KW - Apoptosis KW - secreted effector protein Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110013 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 - Rost, Simone A1 - Müller, Elisabeth A1 - Keller, Alexander A1 - Fregin, Andreas A1 - Müller, Clemens R. T1 - Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling N2 - Background VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1. Results In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme. Conclusions The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism. KW - VKORC1 KW - Vitamin K epoxide reductase KW - Anticoagulants KW - Warfarin KW - Coumarin KW - Coexpression KW - Coagulation factor IX Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110095 ER - TY - JOUR A1 - Brehm, Klaus A1 - Koziol, Uriel A1 - Rauschendorfer, Theresa A1 - Rodríguez, Luis Zanon A1 - Krohne, Georg T1 - The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis N2 - Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae. KW - Cestoda KW - Echinococcus KW - Neoblast KW - Germinative cell KW - Stem cell KW - Nanos KW - Argonaute KW - Mucin KW - Alkaline phosphatase Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110315 ER - TY - JOUR A1 - Wiegering, Armin A1 - Isbert, Christoph A1 - Dietz, Ulrich A. A1 - Kunzmann, Volker A1 - Ackermann, Sabine A1 - Kerscher, Alexander A1 - Maeder, Uwe A1 - Flentje, Michael A1 - Schlegel, Nicolas A1 - Reibetanz, Joachim A1 - Germer, Christoph-Thomas A1 - Klein, Ingo T1 - Multimodal therapy in treatment of rectal cancer is associated with improved survival and reduced local recurrence - a retrospective analysis over two decades N2 - Background The management of rectal cancer (RC) has substantially changed over the last decades with the implementation of neoadjuvant chemoradiotherapy, adjuvant therapy and improved surgery such as total mesorectal excision (TME). It remains unclear in which way these approaches overall influenced the rate of local recurrence and overall survival. Methods Clinical, histological and survival data of 658 out of 662 consecutive patients with RC were analyzed for treatment and prognostic factors from a prospectively expanded single-institutional database. Findings were then stratified according to time of diagnosis in patient groups treated between 1993 and 2001 and 2002 and 2010. Results The study population included 658 consecutive patients with rectal cancer between 1993 and 2010. Follow up data was available for 99.6% of all 662 treated patients. During the time period between 2002 and 2010 significantly more patients underwent neoadjuvant chemoradiotherapy (17.6% vs. 60%) and adjuvant chemotherapy (37.9% vs. 58.4%). Also, the rate of reported TME during surgery increased. The rate of local or distant metastasis decreased over time, and tumor related 5-year survival increased significantly with from 60% to 79%. Conclusion In our study population, the implementation of treatment changes over the last decade improved the patient’s outcome significantly. Improvements were most evident for UICC stage III rectal cancer. KW - Rectal cancer KW - Improved survival KW - TME Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110606 ER - TY - JOUR A1 - Schultz, Jörg A1 - Baier, Herbert T1 - ISAAC - InterSpecies Analysing Application using Containers N2 - Background Information about genes, transcripts and proteins is spread over a wide variety of databases. Different tools have been developed using these databases to identify biological signals in gene lists from large scale analysis. Mostly, they search for enrichments of specific features. But, these tools do not allow an explorative walk through different views and to change the gene lists according to newly upcoming stories. Results To fill this niche, we have developed ISAAC, the InterSpecies Analysing Application using Containers. The central idea of this web based tool is to enable the analysis of sets of genes, transcripts and proteins under different biological viewpoints and to interactively modify these sets at any point of the analysis. Detailed history and snapshot information allows tracing each action. Furthermore, one can easily switch back to previous states and perform new analyses. Currently, sets can be viewed in the context of genomes, protein functions, protein interactions, pathways, regulation, diseases and drugs. Additionally, users can switch between species with an automatic, orthology based translation of existing gene sets. As todays research usually is performed in larger teams and consortia, ISAAC provides group based functionalities. Here, sets as well as results of analyses can be exchanged between members of groups. Conclusions ISAAC fills the gap between primary databases and tools for the analysis of large gene lists. With its highly modular, JavaEE based design, the implementation of new modules is straight forward. Furthermore, ISAAC comes with an extensive web-based administration interface including tools for the integration of third party data. Thus, a local installation is easily feasible. In summary, ISAAC is tailor made for highly explorative interactive analyses of gene, transcript and protein sets in a collaborative environment. KW - Teamwork KW - Gene sets KW - Explorative analyses KW - Cross-species analyses Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110124 ER - TY - JOUR A1 - Brehm, Klaus A1 - Hemer, Sarah A1 - Konrad, Christian A1 - Spiliotis, Markus A1 - Koziol, Uriel A1 - Schaack, Dominik A1 - Förster, Sabine A1 - Gelmedin, Verena A1 - Stadelmann, Britta A1 - Dandekar, Thomas A1 - Hemphill, Andrew T1 - Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development N2 - Background The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. Results Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. Conclusions Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs. KW - Cestode KW - Tapeworm KW - Echinococcus KW - Echinococcosis KW - Insulin KW - Receptor kinase KW - Kinase inhibitor KW - Host-parasite interaction Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110357 ER - TY - THES A1 - Herrmann, Alexander Michael T1 - CD8+ Lymphozyten mediierter Angriff auf Neuronen des ZNS: Relevanz von Granzym B und Perforin für akute elektrophysiologische Veränderungen T1 - CD8+ lymphocyte-mediated attack on neurons of the CNS: Relevance on granzyme B and perforin for acute electrophysiological alterations N2 - Zytotoxische CD8+ T-Lymphozyten spielen in vielen inflammatorischen, aber auch primär neurodegenerativen Erkrankungen eine wichtige Rolle. Daher besitzt die Fragestellung inwiefern CD8+ ZTL Neurone direkt schädigen und ggf. welche mechanistischen Aspekte dieser Schädigung zugrunde liegen, eine hohe Relevanz. Um diese Fragestellung eingehender zu beleuchten, wurde mit dem OT-I-System gearbeitet. Dieses gut vorcharakterisierte CD8+ T-Zell-Modell besitzt den Vorteil, dass diese transgenen Zellen nur eine Peptidsequenz des Ovalbumin (OVA) Protein als spezifisches Antigen erkennen. Zunächst wurden in der vorliegenden Arbeit Co-Kultivierungs-Experimente durchgeführt. Hierzu wurden akut isolierte murine Hippokampus-Neurone unter verschiedenen Bedingungen mit OT-I Lymphozyten co-kultiviert. Hierbei konnte gezeigt werden, dass unter Antigenpräsentation der Neurone signifikant mehr Neurone in die Apoptose/Nekrose geführt werden, als unter Kontroll-Bedingungen, in denen entweder kein Antigen oder ein Antigen, das nicht von OT-I Lymphozyten erkannt wird, präsentiert wird. Nachdem die Antigen-abhängigen zytotoxischen Effekte auf Neurone gezeigt werden konnten, wurde mithilfe elektrophysiologischer Techniken die mechanistischen und funktionellen Konsequenzen des direkten neuronalen/OT-I-vermittelten Zellkontakts untersucht. Bei diesem experimentellen Ansatz wurde durch elektrisches Auslenken eines Neurons nach Kontakt mit einem OT-I Lymphozyt die passiven elektrischen Parameter der Neuronenmembran gemessen. In diesen Messungen konnte gezeigt werden, dass nach unmittelbarem Kontakt eines Neurons mit einem OT-I Lymphozyt der neuronale Membranwiderstand reduziert wird bzw. die Leitfähigkeit der Zellmembran erhöht wird. Diese Änderung der neuronalen Membran-Leitfähigkeit findet in einem Zeitraum von 10 min nach dem Zell-Zell-Kontakt statt. Auch hier konnte gezeigt werden, dass dieser Einfluss von OT-I Lymphozyten auf Neurone strikt Antigen-abhängig ist. Zur Untersuchung des Mechanismus der OT-I T-Lymphozyten auf Neurone wurde das Augenmerk auf verschiedene T-Zell-induzierte Apoptosewegegelegt. Es konnte gezeigt werden, dass durch Blockieren der Fas/FasL-Interaktion mittels eines Antikörpers kein Unterschied, weder in der neuronalen Apoptoserate nach Co-Kultivierung, noch eine Änderung der passiven neuronalen Membran-Leitfähigkeit auftritt. Weiterhin wurde die Rolle der von T-Zellen sezernierten Granula Perforin und Granzym B untersucht. Um den Einfluss dieser Granula aufzuklären, wurden OT-I Lymphozyten verwendet, die entweder defizient für Perforin oder Granzym B waren. In diesem experimentellen Ansatz wurde gezeigt, dass ausschließlich Perforin für die Erniedrigung des passiven neuronalen Membran-Widerstandes verantwortlich ist. Diese Erhöhung der neuronalen Membranleitfähigkeit führte aber nicht direkt zum neuronalen Zelltod. Vielmehr wurde durch die einhergehende Depolarisation des Neurons die elektrische Aktivität der Zelle vermindert, sodass es zu einem sogenannten „electrical silencing“ kommt. Dieser Umstand konnte auch in der Betrachtung der spontanen Netzwerkaktivität von Neuronenkulturen gezeigt werden. Hierfür wurden hoch dichte Neuronenkulturen auf MEA-Chips kultiviert. Mit Hilfe dieser MEA konnten die Summenfeldpotentiale der Neuronenkulturen detektiert werden. Hierbei wurde beobachtet, dass nach Beladung der Neuronen mit dem spezifischen OT-I-Antigen und OT-I Zellen eine Verringerung der spontanen Netzwerkaktivität einhergeht. Auch in diesem Effekt konnte eine Antigen-Spezifität nachgewiesen werden. Da der Prozess der zellulären Apoptose mit einem Anstieg der intrazellulären Ca2+-Konzentration einhergeht, und Perforin als Ca2+-durchlässiger unselektiver Porenbildner fungiert, wurden zur Überprüfung der Hypothese calcium imaging-Experimente durchgeführt. Analog zu den elektrophysiologischen Messungen wurde gezeigt, dass nach direktem Zell-Zell-Kontakt zwischen Neuron und OT-I Lymphozyt eine Erhöhung der intrazellulären Ca2+-Konzentration zu messen ist. Dass diese Änderung des neuronalen Ca2+-Einstroms durch Perforin-abhängige Membranporen hervorgerufen wird, konnte durch die Verwendung von Perforin-defizienten OT-I Lymphozyten bewiesen werden. Unter Verwendung von Perforin-defizienten OT-I Lymphozyten wurde keine Änderung der neuronalen Ca2+-Konzentration ermittelt. Weiterhin wurde in diesem experimentellen Ansatz gezeigt, dass auch der OT-I-vermittelte neuronale Ca2+-Anstieg strikt Antigen-abhängig ist.Zusammengefasst konnte in dieser Arbeit gezeigt werden, dass MHC-I/Antigen-vermittelte CD8+ Lymphozyten-Interaktion mit einem Neuron zu „electrical silencing“ des Neurons führt. Dieser Prozess ist klar Perforin-abhängig, führt jedoch nicht zum unmittelbaren Zelltod des Neurons. N2 - Cytotoxic CD8+ T cells are considered as important effector cells contributing to neuronal damage in inflammatory and primary degenerative disorders in the CNS. Hence, it is highly relevant to know to what extent CD8+ T-lymphocytes can contribute to neuronal damage in these disorders. To challenge this question, we used the murine OT-I system. The advantage of this well-characterized transgenic model is that OT-I CD8+ T-lymphocytes are restricted to one single antigen – one peptide sequence of Ovalbumin (Ova). In a first set of experiments, OT-I lymphocytes were co-cultured with neurons that presented Ova in a MHC-I specific context on their surface. As control, neurons without any antigen or neurons that presented a scrambled peptide form (SIY) were used. These co-culture experiments indicates that neuronal killing by OT-I lymphocytes is a MHC-I and antigen-dependent mechanism. To clarify the underlying mechanism and the functionally consequences in this OT-I/neuron interaction, we performed electrophysiological patch-clamp analysis to measure the influence from one single OT-I T-cell on a single neuron. For this purpose, we established a special protocol to stimulate the neuronal membrane to measure the passive electrical parameters after a direct OT-I contact. These measurements revealed a significant antigen restricted reduction in neuronal membrane resistance. This effect could be detected within 10 min after the direct cell-cell contact. To challenge the underlying cellular mechanisms we analyzed several known apoptosis pathways. In a first set of experiments, we investigated the Fas/FasL interaction. To answer this question, we used a blocking FasL antibody, to interrupt this pathway. These experiments showed no changes in neuronal apoptosis, neither in co-cultivation experiments nor in the electrophysiological situation. As next step we investigate the role of CD8+ lymphocyte derived granula perforin and granzyme B. Therefore we used OT-I T-cells that are either deficient for perforin or granzyme B. Using these experimental conditions, we could show that only perforin is responsible for changing passive electrical parameters. However, these reductions in neuronal membrane resistance did not lead immediately in neuronal cell death, but rather led to a depolarization and therefore to an electrical silencing of the neuron. This electrical silencing was also shown to occur in the spontaneous network activity in a neuronal network. The network activity was measured on a high density neuron network cultivated on a MEA. These MEA measurements revealed a decrease in the total spike activity after loading of OT-I lymphocytes on an antigen presenting neuronal network. Due to the increase of the intracellular Ca2+ level in the process of cell death and the Ca2+ selectivity of perforin membrane pores, we hypothesized that neuronal silencing and neuronal cell death elicited by perforin pores might lead to an intracellular Ca2+ increase. To proof this hypothesis we established a calcium imaging experiment in an OT-I/neuron contact situation. These measurements were done in the same manner as the electrophysiological measurements. Ca2+ imaging indicated increasing Ca2+ levels in neurons after application of perforin releasing OT-I lymphocytes. Furthermore, these experiments revealed a strictly antigen dependence for Ca2+ increase in target cells. In conclusion, we could show that MHC-I/antigen-mediated CD8+ lymphocyte interactions with neurons led to their electrical silencing. This process was perforin dependent. However this process was not causally linked to neuronal cell death. KW - Antigen CD8 KW - T-Lymphozyt KW - Lymphozyten mediierter Angriff auf Neurone KW - Nervendegeneration KW - Lymphozyten KW - neurone Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-109124 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 - Peter, Stefanie T1 - Hemmung der Myc-Funktion durch niedermolekulare Inhibitoren der E3-Ubiquitin-Ligase Huwe1 T1 - Inhibition of Myc function using small molecule inhibitors of the E3 ubiquitin ligase Huwe1 N2 - Die Deregulation des Transkriptionsfaktors Myc ist ein zentraler Mechanismus in der kolorektalen Karzinogenese. Die Myc-Deletion in Tumormodellen hemmt das Wachstum von Kolonkarzinomen, somit stellt die Inaktivierung von Myc einen Ansatzpunkt in der Behandlung von kolorektalen Tumoren dar. Die direkte Inhibition von Myc ist schwierig, da Myc keine katalytische Aktivität besitzt und stattdessen für die Myc-Funktion nötige Protein-Protein- oder Protein-DNA-Interaktionen angegriffen werden müssen. Die E3-Ubiquitin-Ligase Huwe1 interagiert sowohl mit Myc als auch mit dem Myc-interagierenden Protein Miz1 und ist im Kolonkarzinom überexprimiert. Huwe1 ubiquitiniert Myc und induziert darüber dessen Transaktivierungsfunktion. Die Inaktivierung von Huwe1 ist somit eine vielversprechende Möglichkeit für die Inhibition der Myc-Funktion und die Therapie des Kolonkarzinoms. In dieser Arbeit wird mittels shRNA-vermittelter Depletion von Huwe1 in Zellkulturexperimenten gezeigt, dass Huwe1 für die Proliferation von Kolonkarzinomzelllinien und für die Transaktivierung von Myc-Zielgenen benötigt wird. Mit zwei von Boehringer Ingelheim identifizierten niedermolekularen Huwe1-Inhibitoren (BI8622 und BI8626) ist es möglich, die Huwe1-Funktion spezifisch in Zellen zu blockieren. Die Huwe1-Inhibitoren induzieren einen Proliferationsarrest in kolorektalen Karzinomzelllinien, wohingegen die Substanzen auf embryonale Stammzellen keine Auswirkungen haben. Die Inaktivierung von Huwe1 führt zu einer Akkumulation von Miz1 an Promotoren Myc-aktivierter Zielgene und darüber zu einer vermehrten Bildung repressiver Myc/Miz1-Komplexe, was mit einer Deacetylierung von Histon H3 und einer transkriptionellen Repression Myc-gebundener Gene assoziiert ist. Miz1 akkumuliert nach Huwe1-Inhibition ebenso an direkten Miz1-Zielgenen, deren Expression bleibt aber unbeeinflusst. Diese Daten weisen darauf hin, dass eine kontinuierliche Degradierung von Miz1 durch Huwe1 zur Transaktivierung von Myc-Zielgenen in Kolonkarzinomzellen nötig ist. Damit wurde ein neuer Mechanismus identifiziert, über den Huwe1 die Myc-Transaktivierung reguliert und der eine tumorzellspezifische Repression der Myc-Funktion mit Hilfe von Huwe1-Inhibitoren ermöglicht. N2 - Deregulation of the transcription factor Myc is a central driver in colorectal carcinogenesis. Deletion of Myc in murine tumor models inhibits the growth of colon carcinoma, therefore inactivation of Myc is an attractive possibility for treating these types of malignancies. Since Myc does not contain any catalytic activity, protein-protein or protein-DNA interactions necessary for Myc function need to be modified, making direct targeting of Myc difficult. The E3 ubiquitin ligase Huwe1 interacts both with Myc and the Myc-interacting protein Miz1 and is overexpressed in colorectal cancer. Huwe1 ubiquitinates Myc thereby inducing its transactivation function. Hence, inactivation of Huwe1 is a reasonable approach for inhibition of Myc function and treatment of colon cancer. In this work depletion of Huwe1 via shRNA shows that Huwe1 is required for the proliferation of colon carcinoma cell lines and for transactivation of Myc target genes. Two novel small molecule inhibitors of Huwe1 (BI8622 and BI8626), which were identified by Boehringer Ingelheim, block Huwe1 function in cells specifically. The Huwe1 inhibitors induce a proliferation arrest in colorectal cancer cell lines, but not in embryonic stem cells. Inactivation of Huwe1 leads to an accumulation of Miz1 at promoters of Myc-activated target genes and to an increased formation of repressive Myc/Miz1 complexes at these sites. This is associated with deacetylation of histone H3 and transcriptional repression of Myc-bound genes. Additionally, Miz1 accumulates at direct Miz1 target genes upon Huwe1 inhibition but this does not influence expression of these genes. These data suggest that a continuous degradation of Miz1 by Huwe1 is required for transcriptional activation of Myc target genes in colon cancer cells. This identified a new principle how Huwe1 regulates Myc transactivation allowing a tumor cell-specific repression of Myc function via small molecule inhibitors of Huwe1. KW - Myc KW - Ubiquitin KW - Huwe1 KW - Kolonkarzinom KW - Colonkrebs Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-104449 ER - TY - THES A1 - Ok [geb. Hofmann], Claudia Barbara T1 - Isoform-spezifische Analyse der PI3-Kinase (Klasse I) im Multiplen Myelom T1 - Isoform-specific analysis of the PI3-kinase (class I) in multiple myeloma N2 - Das Multiple Myelom (MM) ist eine unheilbare Erkrankung, die aus einer klonalen Proliferation maligner Plasmazellen im Knochenmark hervorgeht. Dabei liegt ein komplexes Signalnetzwerk vor, das zum Überleben und Wachstum der MM-Zellen führt. Das MM ist durch eine enorme genetische und phänotypische Heterogenität gekennzeichnet. Die konstitutive Aktivierung des PI3K/Akt-Signalwegs spielt bei ungefähr der Hälfte der Patienten mit MM eine wichtige Rolle für das Überleben der MM-Zellen und ist daher ein potentieller therapeutischer Ansatzpunkt. Isoform-spezifische Untersuchungen der katalytischen Untereinheiten der Klasse I-PI3K (p110α, p110β, p110γ, p110δ) sollten zur Erkenntnis führen, welche dieser Isoformen für das MM Zellüberleben wichtig sind, um spezifischere Behandlungen mit möglichst geringen Nebenwirkungen zu erlauben. Dafür wurden zunächst Isoform-spezifische Knockdown-Experimente mit MM Zelllinien durchgeführt und sowohl deren Überleben als auch die Aktivierung der nachgeschalteten Komponenten im PI3K Signalweg untersucht. Zur Verifizierung der Ergebnisse wurden sowohl MM Zelllinien als auch Primärzellen mit Isoform-spezifischen PI3K-Inhibitoren behandelt (BYL 719 für p110α, TGX 221 für p110β, CAY10505 für p110γ und CAL 101 für p110δ) und in gleicher Weise untersucht. In beiden Versuchsansätzen stellte sich die katalytische Untereinheit p110α als wichtigste Isoform für das Überleben von MM Zellen mit konstitutiv phosphoryliertem Akt Signal heraus. Weder der Knockdown noch die pharmakologische Inhibition der anderen drei Isoformen (p110β, p110γ, p110δ) führten in MM-Zelllinien zur Beeinträchtigung des Zellüberlebens. Auch reagierten die Primärzellen von MM Patienten größtenteils nicht mit Apoptose auf eine Behandlung mit TGX 221, CAY10505 oder CAL 101. Aufbauend auf der postulierten Bedeutung von p110α, wurde der dafür spezifische Inhibitor BYL 719 mit bereits klinisch etablierten Therapeutika in Kombination verwendet, woraus eine im Vergleich zur Einzelbehandlung verstärkte Apoptose resultierte. Insgesamt deuten diese Daten darauf hin, dass PI3K/p110α eine therapeutisch nutzbare Zielstruktur zur Behandlung des Multiplen Myeloms darstellt. Daher scheinen weitergehende prä-klinische Studien mit p110α Inhibitoren erfolgversprechend. N2 - Multiple myeloma (MM) is an incurable disease, which results from clonal proliferation of malignant plasma cells in the bone marrow. Thereby, a complex signaling network regulates the survival and growth of MM cells. This malignant hematological disease is characterized by profound genetic and phenotypical heterogeneity. The PI3K/Akt signaling pathway is constitutively activated in about 50% of patients with MM and therefore plays an important role for the survival of MM cells. Accordingly, treatment of MM patients with the most isoform-specific drugs may be a desirable goal to achieve therapeutic utility with a minimum of undesired side effects. Therefore, an isoform-specific analysis of the catalytic subunits of the PI3K class I (p110α, p110β, p110γ, p110δ) was undertaken to reveal their individual role(s) for MM cell survival. Initially, isoform-specific knockdown experiments in MM cell lines were performed to assess their survival and the activation states of down-stream components of the PI3K pathway. These experiments were then complemented using isoform-specific pharmacological inhibitors (BYL 719 for p110α, TGX 221 for p110β, CAY10505 for p110γ and CAL 101 for p110δ) in MM cells and primary MM cells. Cell lines with constitutively phosphorylated Akt reduced this signal after p110α knockdown or pharmacologic inhibition and these treatments also affected their survival. Conversely, neither knockdown nor drug-mediated inhibition of any of the other three p110 isoforms influenced MM cell survival. In addition, whereas most primary MM samples were sensitive against BYL-719 only a few samples displayed apoptotic effects when treated with TGX 221, CAY10505 or CAL-101. These results showed that p110α is the major contributor of PI3K-mediated cell survival, and therefore the inhibitor BYL 719 was tested in combination with clinically relevant therapeutics for MM. Such treatment led to increased rates of apoptosis in MM cell lines in comparison to the respective single drug treatments. Taken together, we assume that PI3K/p110α is a therapeutically valuable target structure for the treatment of MM that would warrant more extensive pre-clinical studies. KW - Phosphatidylinositolkinase KW - Isomer KW - Signaltransduktion KW - Plasmozytom KW - p110alpha KW - BYL-719 KW - Carfilzomib KW - Melphalan KW - Lenalidomid KW - Pomalidomid KW - Bortezomib KW - synergistische Effekte KW - Phospho-Akt KW - Zellüberleben KW - pi3kinase KW - Multiples Myelom KW - Knochenmark KW - Isoform Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-108466 ER - TY - THES A1 - Krüger, Alice T1 - Die Entwicklung regenerativer Implantatmatrices auf der Basis von Kollagen Typ I zur Anwendung bei degenerativen Bandscheibenerkrankungen T1 - The development of regenerative implantatmatrices based on Collagen type 1 for retreatment of degenerative disc deseases N2 - Degenerative Bandscheibenerkrankungen wie Protrusionen oder vorgefallenes Nukle-usgewebe führen häufig zu chronischen Schmerzen und schränken die Bewegungsmo-bilität sehr ein. Operative Behandlungsmöglichkeiten wie die Nukleotomie oder die Fusion von Wirbelkörpern stellen traumatische Eingriffe in das komplexe System der Wirbelsäule dar. Biologische Verfahren, durch die eine Regeneration des geschädigten Gewebes erzielt werden kann, sind klinisch bisher nicht etabliert. Ziel dieser Arbeit ist die Entwicklung, Herstellung und Testung regenerativer azellulä-rer Implantatmatrices auf der Basis von Kollagen Typ I, die den degenerierten Nukleus pulposus ersetzen sollen. Insbesondere eine Höhenminderung der Bandscheibe kann zu Anschlussdegenerationen benachbarter Segmente führen. Dies soll durch die Implan-tatmatrix ausgeglichen werden. Nach der Konstruktion und dem Bau eines Reaktors aus dem Hochleistungskunststoff Polytetrafluorethylen (PTFE), der allen Anforderungen eines CE-Konformitätsbewertungsverfahrens entspricht, wird eine hoch verdichtete Kollagen Typ I Matrix mit einer Stärke von 1 mm hergestellt. Diese kann über den Pro-zess der Lyophilisation auf 0,6 mm weiter reduziert werden. Es gelingt, die Matrix in einer Edelstahlhülse zu platzieren, über die mit Hilfe eines passgenauen Führungssta-bes die endoskopische Implantation in die Nukleuskavität erfolgen soll. Im Rahmen der Interkorporellen Fusionstage des Diakonie Klinikums Stuttgart wird das operative Handling an einem humanem Präparat simuliert. Die Implantation erfolgt offen über einen transforaminalen Zugang in zwei nukleotomierte Segmente der lumbalen Wir-belsäule. Die anwesenden Wirbelsäulenchirurgen beurteilen die Möglichkeit der endo-skopischen Applikation als positiv und machbar. Durch den Zusatz des Polysaccharids Hyaluronsäure gelingt es, die Quelleigenschaften der hoch verdichteten Matrix zu steigern, so dass diese wie natives Nukleusgewebe in der Lage ist, Flüssigkeit in Ruhe wieder aufzunehmen. Das Quellpotential und die da-mit einhergehende Volumenzunahme nach Kompression sind für ein Nukleusersatzma-terial essentiell. Die hier verwendete Hyaluronsäure geht jedoch im offenen System der in vitro Inkubation innerhalb von 11 Tagen verloren. Dennoch zeigen sich weitere Vorteile gegenüber der Matrix ohne Hyaluronsäure-Zusatz innerhalb der Testungen heraus. Diese sind neben dem erhöhten Quellpotential z. B. eine gesteigerte Rate der Zellproliferation der verwendeten bovinen und humanen Bandscheibenzellen (bBSZ und hBSZ) sowie humanen mesenchymalen Stammzellen (hMSC), die über die Be-stimmung der Zellzahl und Viabilität ermittelt wird. Zudem zeigt sich eine gesteigerte mechanische Stabilität, die über die Spannungs-Kompressions-Messungen evaluiert wird. Über Lebend-/ Totfärbungen und Zytotoxizitätstests an Monolayerkulturen kann zudem nachgewiesen werden, dass die notwendige Endsterilisation durch γ-Bestrahlung zu keinen zytotoxischen Veränderungen der Matrix führt. Da die verdich-tete Implantatmatrix azellulär als Medizinprodukt der Klasse III eingesetzt werden soll, wird als ergänzende Matrix zur Füllung kleinster Hohlräume die zunächst flüssige ChondroFillerliquid Matrix (ein Knorpelersatzmaterial der Firma Amedrix GmbH, Esslin-gen) durch den Zusatz von Hyaluronsäure modifiziert und in der Zellkultur getestet. Da es sich hierbei um ein Zweikammerspritzensystem handelt, ist die Verwendung von Additiva wie z. B. Stammzellen technisch möglich. Die Ermittlung der maximalen Inku-bationszeit von Zellen in verschieden konzentrierten hyperosmotischen Neutralisations-lösungen ergibt eine Dauer von 5 min, bis irreversible Zellschäden auftreten. In Migra-tionsversuchen kann gezeigt werden, dass die ChondroFillerliquid Matrix als Konektiv zwischen nativem Nukleusgewebe und verdichteter Implantatmatrix fungiert. Des Wei-teren synthetisieren bBSZ, hBSZ und hMSC sulfatierte Glykosaminoglykane und behal-ten dabei ihr charakteristisches Genexpressionsprofil. Die chondrogene Differenzie-rung durch die Verwendung eines chondrogenen Differenzierungsmediums gelingt bei den hMSC bereits nach einer Kultivierungsdauer von 14 d. Die Zellverteilung in den Implantatmatrices und deren Morphologie entspricht dem nativen Nukleusgewebe. Die biomechanische Testung an einem international anerkannten Modellsystem für humane Wirbelsäulen – der Kalbswirbelsäule – ergibt, dass die Nukleotomie zu einer Erhöhung des Range of Motion (RoM) in alle Richtungen nach Flexion/Extension, Seit-neigung rechts/links und axiale Rotation rechts/links sowie zu einer Höhenreduktion des Segments im Vergleich zum Intaktzustand führt. Nach der Implantation der ver-dichteten Implantatmatrix wird der RoM deutlich reduziert. Das Segment weist dadurch eine hohe Steifigkeit ähnlich dem Intaktzustand auf. Die Höhenreduktion kann durch die Implantation beinahe vollständig wieder ausgeglichen werden. Im Rahmen der zyklischen Dauerbelastungen treten jedoch Implantatextrusionen auf. Zudem nimmt die Steifigkeit deutlich ab, der RoM hingegen wieder zu. Da das bovine Modell jedoch nicht der in vivo Situation entspricht und beispielsweise eine zunehmende In-tegration des Implantats durch Einwachsen nicht ermöglicht, ist die hohe Extrusionsra-te als nicht realistisch zu werten. Klinische Studien am Tier und Mensch müssen zeigen, inwieweit derartige Extrusionen ohne die Verwendung eines Anulusverschlußsystems auftreten. Im Rahmen der vorliegenden Arbeit ist es gelungen, einen geeigneten Reaktor zu ent-wickeln und mit diesem eine biokompatible, stabile und quellfähige Matrix herzustel-len, die den Höhenverlust nach einer Nukleotomie auszugleichen vermag. Die modifi-zierte ChondroFillerliquid Matrix stellt eine ideale Ergänzung dar, da über diese Zellen oder andere Additiva verabreicht werden können und deren konektive Wirkung die Zellbesiedlung der azellulären Matrix begünstigt. N2 - Degenerative disc deseases like the protrusion or slipped disc tissue of the nucleus fre-quently cause chronic pain and restrict the freedom of movement. Surgical therapies like the nucleotomy or spinal fusion are traumatic procedures for the complex system of the spine. Biologic therapies which can lead to a regeneration of the damaged tis-sue were not clinically established until now. The aim of this work is the development, fabrication and testing of regenerative acel-lular implant matrices based on collagen type I, which should replace the degenerated nucleus pulposus. Especially a reduction of the disc height can cause degenerations of the adjacent segments. This should be balanced by the implant matrix. After the reac-tor construction and its fabrication made of a high-performance plastic polytetrafluo-rethylene (PTFE), which corresponds to the requirements of a CE-conformity assess-ment procedure, a highly condensed collagen type I matrix with a thickness of 1 mm is fabricated. Through lyophilisation this thickness can be reduced to 0.6 mm. It succeeds to place the matrix inside a stainless steel sleeve which should permit the endoscopic implantation into the nucleus cavity with the help of custom-fit leading-bar. During the intercorporelle spinal fusion days of the Diakonie Klinikum Stuttgart the surgical han-dling is simulated on a human speciment. The implantation occurs in an open trans-foraminal access into two nucleotomised segments of the lumbar spine. The participat-ing spinal surgeons evaluate the possibility of the endoscopic application as positive and feasible. The addition of polysaccharide hyaluronic acid leads to an increasement of the swell-ing capacities of the highly condensed matrix. At rest, the matrix was subsequently able to absorb fluids the same way as native nucleus tissue. The increased swelling potential and the accompanied increase of volume after compression are essential for a nucleus replacement material. However within 11 days the used hyaluronic acid gets lost in the open system of the in vitro incubation. Nevertheless there are further ad-vantages of this matrix shown in the scope of testing compared to the matrix without the addition of hyaluronic acid. Besides the swelling potential these advantages are e.g. an increased rate of cell proliferation of the used bovine and human disc cells (bBSZ and hBSZ) and human mesenchymal stem cells (hMSC), identified by the num-ber and viability of these cells as well as an increased mechanical stability of the ma-trix, which is shown by tension-compression-measurements. Live/dead staining and cytotoxicity tests on monolayer cultures showed that the required final sterilization through γ-irradiation does not lead to cytotoxic changes of the matrix. Because the condensed matrix should be used as an acellular medical device of classification III the preliminary liquid ChondroFillerliquid matrix (a cartilage replacement material of the company Amedrix GmbH, Esslingen), which is able to fill smallest cavities, is modified by the addition of hyaluronic acid and tested in cell culture experiments. As this matrix comes in a two chamber syringe system the substitution of additives e.g. stem cells is technically feasible. The determination of the maximum cell incubation time in differ-ent concentrated hyperosmotic neutralization solutions showed a maximum incubation time of five min before irreversible cell damages occur. Migration experiments can show that the ChondroFillerliquid matrix acts as a connection between the native nucleus tissue and the condensed implant matrix. Furthermore bBSZ, hBSZ and hMSC synthe-size sulfated glycosaminoglycanes and maintain their characteristic gene expression profile. Also a chondrogenic differentiation of the hMSC takes place followed by the use of a chondrogenic differentiation medium after a cultivation period of 14 d. The cell distribution and their morphology were similar to native nucleus tissue. The bio-mechanical testing which was done in an international accepted model system of the human spine – the calf spine – showed that the nucleotomy leads to an increased range of motion (RoM) of the segment in all directions to flexion/extension, lateral bending right/left and axial rotation left/right as well as a reduction of the height of the segment compared to the intact condition. Subsequently to the implantation of the condensed implant matrix the RoM is clearly reduced. The segment exhibits a high stiffness similar to the intact condition. The height reduction can be corrected almost completely by the implant matrix. During the cyclic compression however implant ex-trusions appear. Moreover the stiffness of the segment decreases clearly while the RoM increases. As the bovine model might not be applicable completely to the in vivo situation e.g it does not facilitate an increasing integration through the ingrowth of the implant, the high extrusion rate is assessed as unrealistic. Clinical studies on animals and humans have to show in which extent extrusions appear without the use of an anu-lar closure device system. Within the scope of this work it succeeds to develop an eligible reactor with which the fabrication of a biocompatible, dimensionally stable and swellable matrix which in turn is able to restore the disc height after a nucleotomy. The modified ChondroFillerliquid matrix represents an ideal supplement to the condensed matrix as it offers the possi-bility to apply cells and other additives and furthermore favours the cell seeding of the acellular condensed matrix by its connective effect. KW - Bandscheibenkrankheit KW - Bandscheibenerkrankung KW - Implantat KW - disc deseases KW - Kollagen KW - Implantatmatrices Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-106504 ER -