@article{FlorenMupepeleMuelleretal.2014, author = {Floren, Andreas and Mupepele, Anne-Christine and M{\"u}ller, Tobias and Dittrich, Marcus}, title = {Are Temperate Canopy Spiders Tree-Species Specific?}, doi = {10.1371/journal.pone.0086571}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111413}, year = {2014}, abstract = {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.}, language = {en} } @article{AlsheimerLinkLeubneretal.2014, author = {Alsheimer, Manfred and Link, Jana and Leubner, Monika and Schmitt, Johannes and G{\"o}b, Eva and Benavente, Ricardo and Jeang, Kuan-Teh and Xu, Rener}, title = {Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function}, doi = {10.1371/journal.pgen.1004099}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111355}, year = {2014}, abstract = {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.}, language = {en} } @phdthesis{Kaiser2014, author = {Kaiser, Dorkas}, title = {Termites and ants in BURKINA FASO (WEST AFRICA): taxonomic and functional diversity along land-use gradients; ecosystem services of termites in the traditional ZA{\"I} SYSTEM}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107001}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {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 Za{\"i} 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 Za{\"i} 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 Za{\"i} 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 Za{\"i} system: crusted bare soil (initial stage), millet field, young and old forest. In each of the four Za{\"i} 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 Za{\"i} 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 Za{\"i} 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 Za{\"i} 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 Za{\"i} 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.}, subject = {Termiten}, language = {en} } @phdthesis{KubischgebWiegand2014, author = {Kubisch (geb. Wiegand), Franziska}, title = {Learning in botanical gardens: Investigating educational methods during an instruction about plants and water}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111620}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {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.}, subject = {Konstruktive Didaktik}, language = {en} } @article{DjuzenovaMemmelSukhorukovetal.2014, author = {Djuzenova, Cholpon S. and Memmel, Simon and Sukhorukov, Vladimir L. and H{\"o}ring, Marcus and Westerling, Katherine and Fiedler, Vanessa and Katzer, Astrid and Krohne, Georg and Flentje, Michael}, title = {Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status}, doi = {10.1371/journal.pone.0087052}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111322}, year = {2014}, abstract = {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.}, language = {en} } @article{RocesPielstroem2014, author = {Roces, Flavio and Pielstr{\"o}m, Steffen}, title = {Soil Moisture and Excavation Behaviour in the Chaco Leaf-Cutting Ant (Atta vollenweideri): Digging Performance and Prevention of Water Inflow into the Nest}, doi = {10.1371/journal.pone.0095658}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111298}, year = {2014}, abstract = {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.}, language = {en} } @article{WiegeringKorbThalheimeretal.2014, author = {Wiegering, Armin and Korb, Doreen and Thalheimer, Andreas and K{\"a}mmerer, Ulrike and Allmanritter, Jan and Matthes, Niels and Linnebacher, Michael and Schlegel, Nicolas and Klein, Ingo and Erg{\"u}n, S{\"u}leyman and Germer, Christoph-Thomas and Otto, Christoph}, title = {E7080 (Lenvatinib), a Multi-Targeted Tyrosine Kinase Inhibitor, Demonstrates Antitumor Activities Against Colorectal Cancer Xenografts}, doi = {10.1016/j.neo.2014.09.008}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111165}, year = {2014}, abstract = {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.}, language = {en} } @article{RudelPrustySiegletal.2014, author = {Rudel, Thomas and Prusty, Bhupesh K. and Siegl, Christine and Gulve, Nitish and Mori, Yasuko}, title = {GP96 Interacts with HHV-6 during Viral Entry and Directs It for Cellular Degradation}, doi = {10. 1371/journal.pone.0113962}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111068}, year = {2014}, abstract = {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.}, language = {en} } @article{WiegeringIsbertDietzetal.2014, author = {Wiegering, Armin and Isbert, Christoph and Dietz, Ulrich A. and Kunzmann, Volker and Ackermann, Sabine and Kerscher, Alexander and Maeder, Uwe and Flentje, Michael and Schlegel, Nicolas and Reibetanz, Joachim and Germer, Christoph-Thomas and Klein, Ingo}, title = {Multimodal therapy in treatment of rectal cancer is associated with improved survival and reduced local recurrence - a retrospective analysis over two decades}, doi = {10.1186/1471-2407-14-816}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110606}, year = {2014}, abstract = {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.}, language = {en} } @article{BrehmHemerKonradetal.2014, author = {Brehm, Klaus and Hemer, Sarah and Konrad, Christian and Spiliotis, Markus and Koziol, Uriel and Schaack, Dominik and F{\"o}rster, Sabine and Gelmedin, Verena and Stadelmann, Britta and Dandekar, Thomas and Hemphill, Andrew}, title = {Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development}, doi = {10.1186/1741-7007-12-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110357}, year = {2014}, abstract = {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.}, language = {en} } @article{BrehmKoziolRauschendorferetal.2014, author = {Brehm, Klaus and Koziol, Uriel and Rauschendorfer, Theresa and Rodr{\´i}guez, Luis Zanon and Krohne, Georg}, title = {The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis}, doi = {10.1186/2041-9139-5-10}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110315}, year = {2014}, abstract = {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.}, language = {en} } @article{SchultzBaier2014, author = {Schultz, J{\"o}rg and Baier, Herbert}, title = {ISAAC - InterSpecies Analysing Application using Containers}, doi = {10.1186/1471-2105-15-18}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110124}, year = {2014}, abstract = {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.}, language = {en} } @article{RostMuellerKelleretal.2014, author = {Rost, Simone and M{\"u}ller, Elisabeth and Keller, Alexander and Fregin, Andreas and M{\"u}ller, Clemens R.}, title = {Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling}, doi = {10.1186/1471-2156-15-17}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110095}, year = {2014}, abstract = {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.}, language = {en} } @article{VerghoKneitzRosenwaldetal.2014, author = {Vergho, Daniel and Kneitz, Susanne and Rosenwald, Andreas and Scherer, Charlotte and Spahn, Martin and Burger, Maximilian and Riedmiller, Hubertus and Kneitz, Burkhard}, title = {Combination of expression levels of miR-21 and miR-126 is associated with cancer-specific survival in clear-cell renal cell carcinoma}, doi = {10.1186/1471-2407-14-25}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110061}, year = {2014}, abstract = {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.}, language = {en} } @phdthesis{Huber2014, author = {Huber, Annette}, title = {Chlamydial deubiquitinase ChlaDUB1 as regulator of host cell apoptosis and new target for anti-chlamydial therapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110013}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {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.}, subject = {Chlamydia trachomatis}, language = {en} } @phdthesis{Hondke2014, author = {Hondke, Sylvia}, title = {Elucidation of WISP3 function in human mesenchymal stem cells and chondrocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109641}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {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.}, subject = {Knorpelzelle}, language = {en} } @phdthesis{Herrmann2014, author = {Herrmann, Alexander Michael}, title = {CD8+ Lymphozyten mediierter Angriff auf Neuronen des ZNS: Relevanz von Granzym B und Perforin f{\"u}r akute elektrophysiologische Ver{\"a}nderungen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109124}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Zytotoxische CD8+ T-Lymphozyten spielen in vielen inflammatorischen, aber auch prim{\"a}r neurodegenerativen Erkrankungen eine wichtige Rolle. Daher besitzt die Fragestellung inwiefern CD8+ ZTL Neurone direkt sch{\"a}digen und ggf. welche mechanistischen Aspekte dieser Sch{\"a}digung 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. Zun{\"a}chst wurden in der vorliegenden Arbeit Co-Kultivierungs-Experimente durchgef{\"u}hrt. Hierzu wurden akut isolierte murine Hippokampus-Neurone unter verschiedenen Bedingungen mit OT-I Lymphozyten co-kultiviert. Hierbei konnte gezeigt werden, dass unter Antigenpr{\"a}sentation der Neurone signifikant mehr Neurone in die Apoptose/Nekrose gef{\"u}hrt werden, als unter Kontroll-Bedingungen, in denen entweder kein Antigen oder ein Antigen, das nicht von OT-I Lymphozyten erkannt wird, pr{\"a}sentiert wird. Nachdem die Antigen-abh{\"a}ngigen 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 Leitf{\"a}higkeit der Zellmembran erh{\"o}ht wird. Diese {\"A}nderung der neuronalen Membran-Leitf{\"a}higkeit 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-abh{\"a}ngig 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 Antik{\"o}rpers kein Unterschied, weder in der neuronalen Apoptoserate nach Co-Kultivierung, noch eine {\"A}nderung der passiven neuronalen Membran-Leitf{\"a}higkeit auftritt. Weiterhin wurde die Rolle der von T-Zellen sezernierten Granula Perforin und Granzym B untersucht. Um den Einfluss dieser Granula aufzukl{\"a}ren, wurden OT-I Lymphozyten verwendet, die entweder defizient f{\"u}r Perforin oder Granzym B waren. In diesem experimentellen Ansatz wurde gezeigt, dass ausschließlich Perforin f{\"u}r die Erniedrigung des passiven neuronalen Membran-Widerstandes verantwortlich ist. Diese Erh{\"o}hung der neuronalen Membranleitf{\"a}higkeit f{\"u}hrte aber nicht direkt zum neuronalen Zelltod. Vielmehr wurde durch die einhergehende Depolarisation des Neurons die elektrische Aktivit{\"a}t der Zelle vermindert, sodass es zu einem sogenannten „electrical silencing" kommt. Dieser Umstand konnte auch in der Betrachtung der spontanen Netzwerkaktivit{\"a}t von Neuronenkulturen gezeigt werden. Hierf{\"u}r 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 Netzwerkaktivit{\"a}t einhergeht. Auch in diesem Effekt konnte eine Antigen-Spezifit{\"a}t nachgewiesen werden. Da der Prozess der zellul{\"a}ren Apoptose mit einem Anstieg der intrazellul{\"a}ren Ca2+-Konzentration einhergeht, und Perforin als Ca2+-durchl{\"a}ssiger unselektiver Porenbildner fungiert, wurden zur {\"U}berpr{\"u}fung der Hypothese calcium imaging-Experimente durchgef{\"u}hrt. Analog zu den elektrophysiologischen Messungen wurde gezeigt, dass nach direktem Zell-Zell-Kontakt zwischen Neuron und OT-I Lymphozyt eine Erh{\"o}hung der intrazellul{\"a}ren Ca2+-Konzentration zu messen ist. Dass diese {\"A}nderung des neuronalen Ca2+-Einstroms durch Perforin-abh{\"a}ngige 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 {\"A}nderung der neuronalen Ca2+-Konzentration ermittelt. Weiterhin wurde in diesem experimentellen Ansatz gezeigt, dass auch der OT-I-vermittelte neuronale Ca2+-Anstieg strikt Antigen-abh{\"a}ngig ist.Zusammengefasst konnte in dieser Arbeit gezeigt werden, dass MHC-I/Antigen-vermittelte CD8+ Lymphozyten-Interaktion mit einem Neuron zu „electrical silencing" des Neurons f{\"u}hrt. Dieser Prozess ist klar Perforin-abh{\"a}ngig, f{\"u}hrt jedoch nicht zum unmittelbaren Zelltod des Neurons.}, subject = {Antigen CD8}, language = {de} } @phdthesis{Roemer2014, author = {R{\"o}mer, Daniela}, title = {Where and how to build? Influence of social and environmental cues on nest building behavior in leaf-cutting ants}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109409}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {This thesis explores the influence of social and environmental cues on the nest building behavior of leaf-cutting ants. Especially, the investigations are aimed at evaluating the mechanisms of nest building and how the nest environment can spatially guide building responses that lead to an adaptive nest architecture. The emergence of nest chambers in the nest of the leaf-cutting ant Acromyrmex lundi were evaluated. Rather than excavating nest chambers in advance, at places where workers encounter suitable environmental conditions for brood and fungus rearing, these items have to be present at a site. When presented in the laboratory with a choice between two otherwise identical digging sites, offering suitable environmental conditions, but one containing brood, the workers displayed a higher excavation activity at the site where they encountered the putative content of a chamber. The shape of the excavated cavity was also more round and chamber-like. It is concluded that leaf-cutting ants respond to social cues during nest building. Excavation is a costly process and colonies have to spend a part of their energy stores on nest building, so that regulatory responses for the control of nest excavation are expected to occur. Worker density at the beginning of the digging process influenced digging activity while the presence of in-nest stores did not. Stored brood and fungus did however influence the architecture of the excavated nest, leading to the excavation of larger chambers and smaller tunnels. While self-organized mechanisms appear to be involved in the nest building process, the social cues of the ants' environment during building clearly influence the nest architecture and lead to an adjustment of the nest size to the current space needs of the colony. Workers secondarily regulated nest size by the opportunistic refilling of unused space with excavated soil pellets. As the ants should provide suitable conditions for brood and fungus rearing, they should show a behavioral response to CO2 concentrations, as the gas is known to hinder fungus respiration. Workers of A. lundi did indeed avoid high CO2-levels for fungus rearing but actually preferred CO2-values in the range encountered close to the soil surface, where this species excavates their nests. However, different CO2-levels did not affect their excavation behavior. While fungus chambers make up part of a leaf-cutting ant nest, most leaf-cutting ants of the genus Atta also spent part of the colony's energy on excavating large, voluminous chambers for waste disposal, rather than scattering the material aboveground. It is expected that leaf-cutting ants also show environmental preferences for waste management. In experiments Atta laevigata workers preferred deposition in a warm and dry environment and showed no preference for specific CO2-levels. The continued accumulation of waste particles in a waste chamber seems to be based on the use of volatiles. These originate from the waste itself, and seem to be used as an orientation cue by workers relocating the material. The ensuing large accumulation of waste at one site should result in the emergence of more voluminous chambers for waste disposal.}, subject = {Nestbau}, language = {en} } @phdthesis{Wolter2014, author = {Wolter, Steve}, title = {Single-molecule localization algorithms in super-resolution microscopy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109370}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Lokalisationsmikroskopie ist eine Methodenklasse der superaufl{\"o}senden Fluoreszenzmikroskopie, deren Methoden sich durch stochastische zeitliche Isolation der Fluoreszenzemission auszeichnen. Das Blinkverhalten von Fluorophoren wird so ver{\"a}ndert, dass gleichzeitige Aktivierung von einander nahen Fluorophoren unwahrscheinlich ist. Bekannte okalisationsmikroskopische Methoden umfassen dSTORM, STORM, PALM, FPALM, oder GSDIM. Lokalisationsmikroskopie ist von hohem biologischem Interesse, weil sie die Aufl{\"o}sung des Fluoreszenzmikroskops bei minimalem technischem Aufwand um eine Gr{\"o}ßenordnung verbessert. Der verbundene Rechenaufwand ist allerdings erheblich, da Millionen von Fluoreszenzemissionen einzeln mit Nanometergenauigkeit lokalisiert werden m{\"u}ssen. Der Rechen- und Implementationsaufwand dieser Auswertung hat die Verbreitung der superaufl{\"o}senden Mikroskopie lange verz{\"o}gert. Diese Arbeit beschreibt meine algorithmische Grundstruktur f{\"u}r die Auswertung lokalisationsmikroskopischer Daten. Die Echtzeitf{\"a}higkeit, d.h. eine Auswertegeschwindigkeit oberhalb der Datenaufnahmegeschwindigkeit an normalen Messaufbauten, meines neuartigen und quelloffenen Programms wird demonstriert. Die Geschwindigkeit wird auf verbrauchermarktg{\"a}ngigen Prozessoren erreicht und dadurch spezialisierte Rechenzentren oder der Einsatz von Grafikkarten vermieden. Die Berechnung wird mit dem allgemein anerkannten Gaussschen Punktantwortmodell und einem Rauschmodell auf Basis der gr{\"o}ßten Poissonschen Wahrscheinlichkeit durchgef{\"u}hrt. Die algorithmische Grundstruktur wird erweitert, um robuste und optimale Zweifarbenauswertung zu realisieren und damit korrelative Mikroskopie zwischen verschiedenen Proteinen und Strukturen zu erm{\"o}glichen. Durch den Einsatz von kubischen Basissplines wird die Auswertung von dreidimensionalen Proben vereinfacht und stabilisiert, um pr{\"a}zisem Abbilden von mikrometerdicken Proben n{\"a}her zu kommen. Das Grenzverhalten von Lokalisationsalgorithmen bei hohen Emissionsdichten wird untersucht. Abschließend werden Algorithmen f{\"u}r die Anwendung der Lokalisationsmikroskopie auf verbreitete Probleme der Biologie aufgezeigt. Zellul{\"a}re Bewegung und Motilit{\"a}t werden anhand der in vitro Bewegung von Myosin-Aktin-Filamenten studiert. Lebendzellbildgebung mit hellen und stabilen organischen Fluorophoren wird mittels SNAP-tag-Fusionsproteinen realisiert. Die Analyse des Aufbaus von Proteinklumpen zeigt, wie Lokalisationsmikroskopie neue quantitative Ans{\"a}tze jenseits reiner Bildgebung bietet.}, subject = {Fluoreszenzmikroskopie}, language = {en} } @phdthesis{OkgebHofmann2014, author = {Ok [geb. Hofmann], Claudia Barbara}, title = {Isoform-spezifische Analyse der PI3-Kinase (Klasse I) im Multiplen Myelom}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108466}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {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 {\"U}berleben und Wachstum der MM-Zellen f{\"u}hrt. Das MM ist durch eine enorme genetische und ph{\"a}notypische Heterogenit{\"a}t gekennzeichnet. Die konstitutive Aktivierung des PI3K/Akt-Signalwegs spielt bei ungef{\"a}hr der H{\"a}lfte der Patienten mit MM eine wichtige Rolle f{\"u}r das {\"U}berleben 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 f{\"u}hren, welche dieser Isoformen f{\"u}r das MM Zell{\"u}berleben wichtig sind, um spezifischere Behandlungen mit m{\"o}glichst geringen Nebenwirkungen zu erlauben. Daf{\"u}r wurden zun{\"a}chst Isoform-spezifische Knockdown-Experimente mit MM Zelllinien durchgef{\"u}hrt und sowohl deren {\"U}berleben als auch die Aktivierung der nachgeschalteten Komponenten im PI3K Signalweg untersucht. Zur Verifizierung der Ergebnisse wurden sowohl MM Zelllinien als auch Prim{\"a}rzellen mit Isoform-spezifischen PI3K-Inhibitoren behandelt (BYL 719 f{\"u}r p110α, TGX 221 f{\"u}r p110β, CAY10505 f{\"u}r p110γ und CAL 101 f{\"u}r p110δ) und in gleicher Weise untersucht. In beiden Versuchsans{\"a}tzen stellte sich die katalytische Untereinheit p110α als wichtigste Isoform f{\"u}r das {\"U}berleben von MM Zellen mit konstitutiv phosphoryliertem Akt Signal heraus. Weder der Knockdown noch die pharmakologische Inhibition der anderen drei Isoformen (p110β, p110γ, p110δ) f{\"u}hrten in MM-Zelllinien zur Beeintr{\"a}chtigung des Zell{\"u}berlebens. Auch reagierten die Prim{\"a}rzellen von MM Patienten gr{\"o}ßtenteils nicht mit Apoptose auf eine Behandlung mit TGX 221, CAY10505 oder CAL 101. Aufbauend auf der postulierten Bedeutung von p110α, wurde der daf{\"u}r spezifische Inhibitor BYL 719 mit bereits klinisch etablierten Therapeutika in Kombination verwendet, woraus eine im Vergleich zur Einzelbehandlung verst{\"a}rkte Apoptose resultierte. Insgesamt deuten diese Daten darauf hin, dass PI3K/p110α eine therapeutisch nutzbare Zielstruktur zur Behandlung des Multiplen Myeloms darstellt. Daher scheinen weitergehende pr{\"a}-klinische Studien mit p110α Inhibitoren erfolgversprechend.}, subject = {Phosphatidylinositolkinase }, language = {de} }