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Sonstige beteiligte Institutionen
- DNA Analytics Core Facility, Biocenter, University of Wuerzburg, Wuerzburg, Germany (1)
- EMBL, Structural and Computational Biology Unit, Heidelberg, Germany (1)
- IZKF Laboratory for Microarray Applications, University Hospital of Wuerzburg, Wuerzburg, Germany (1)
- Microarray Core Unit, Interdisciplinary Center for Clinical Science, University of Würzburg, Versbacher Straße, Würzburg 97080, Germany (1)
Bees have had an intimate relationship with humans for millennia, as pollinators of fruit, vegetable and other crops and suppliers of honey, wax and other products. This relationship has led to an extensive understanding of their ecology and behavior. One of the most comprehensively understood species is the Western honeybee, Apis mellifera. Our understanding of sex-specific investment in other bees, however, has remained superficial. Signals and cues employed in bee foraging and mating behavior are reasonably well understood in only a handful of species and functional adaptations are described in some species. I explored the variety of sensory adaptations in three model systems within the bees. Females share a similar ecology and similar functional morphologies are to be expected. Males, engage mainly in mating behavior. A variety of male mating strategies has been described which differ in their spatiotemporal features and in the signals and cues involved, and thus selection pressures. As a consequence, males’ sensory systems are more diverse than those of females. In the first part I studied adaptations of the visual system in honeybees. I compared sex and caste-specific eye morphology among 5 species (Apis andreniformis, A. cerana, A. dorsata, A. florea, A. mellifera). I found a strong correlation between body size and eye size in both female castes. Queens have a relatively reduced visual system which is in line with the reduced role of visual perception in their life history. Workers differed in eye size and functional morphology, which corresponds to known foraging differences among species. In males, the eyes are conspicuously enlarged in all species, but a disproportionate enlargement was found in two species (A. dorsata, A. florea). I further demonstrate a correlation between male visual parameters and mating flight time, and propose that light intensities play an important role in the species-specific timing of mating flights. In the second study I investigated eye morphology differences among two phenotypes of drones in the Western honeybee. Besides normal-sized drones, smaller drones are reared in the colony, and suffer from reduced reproductive success. My results suggest that the smaller phenotype does not differ in spatial resolution of its visual system, but suffers from reduced light and contrast sensitivity which may exacerbate the reduction in reproductive success caused by other factors. In the third study I investigated the morphology of the visual system in bumblebees. I explored the association between male eye size and mating behavior and investigated the diversity of compound eye morphology among workers, queens and males in 11 species. I identified adaptations of workers that correlate with distinct foraging differences among species. Bumblebee queens must, in contrast to honeybees, fulfill similar tasks as workers in the first part of their life, and correspondingly visual parameters are similar among both female castes. Enlarged male eyes are found in several subgenera and have evolved several times independently within the genus, which I demonstrate using phylogenetic informed statistics. Males of these species engage in visually guided mating behavior. I find similarities in the functional eye morphology among large-eyed males in four subgenera, suggesting convergent evolution as adaptation to similar visual tasks. In the remaining species, males do not differ significantly from workers in their eye morphology. In the fourth study I investigated the sexual dimorphism of the visual system in a solitary bee species. Males of Eucera berlandi patrol nesting sites and compete for first access to virgin females. Males have enlarged eyes and better spatial resolution in their frontal eye region. In a behavioral study, I tested the effect of target size and speed on male mate catching success. 3-D reconstructions of the chasing flights revealed that angular target size is an important parameter in male chasing behavior. I discuss similarities to other insects that face similar problems in visual target detection. In the fifth study I examined the olfactory system of E. berlandi. Males have extremely long antennae. To investigate the anatomical grounds of this elongation I studied antennal morphology in detail in the periphery and follow the sexual dimorphism into the brain. Functional adaptations were found in males (e.g. longer antennae, a multiplication of olfactory sensilla and receptor neurons, hypertrophied macroglomeruli, a numerical reduction of glomeruli in males and sexually dimorphic investment in higher order processing regions in the brain), which were similar to those observed in honeybee drones. The similarities and differences are discussed in the context of solitary vs. eusocial lifestyle and the corresponding consequences for selection acting on males.
Within the last decades, land use intensification reduced the heterogeneity of habitats and landscapes. The resulting pauperization led to habitats and landscapes that are spatially or temporally limited in food and nesting resources for solitary bees and wasps. Hence, biodiversity and ecosystem processes are seriously threatened. The impacts of changing resource conditions for valuable pollinators and (pest) predators remain poorly studied as well as their top-down regulation by natural enemies. Further, the reproductive success of solitary bees as response to changed resource distribution within foraging ranges is rarely examined. We considered trap-nesting bees, wasps and their antagonists as suitable model organisms to fill these gaps of knowledge, since trap nests provide insight into otherwise hidden trophic interactions, like parasitism and predation, as well as ecological processes, like pollination and reproduction. Moreover, trap-nesting species are established as essential biodiversity indicator taxa. Thus, we first asked in Chapter II how the reproduction of cavity-nesting bees and wasps in grasslands depends on local management Moreover, we tested land use effects on the effectiveness of two groups of antagonists in regulating bee and wasp populations by excluding ground-dwelling antagonists. We characterized nest closure type to determine their protective function against antagonist attacks. In a highly replicated, large-scaled study, we provided 95 grassland sites in three geographic regions in Germany with 760 trap-nests. The full factorial design comprised mown and unmown plots as well as plots with and without access of ground-dwelling predators to the trap nests. The colonization of bees and wasps was unaffected by ground-dwelling antagonists. However, excluding ground-dwellers enhanced the attack rate of flying antagonists. Experimental mowing marginally affected the colonization of wasps but not attack rates. Nevertheless, both treatments – mowing and predator exclusion – significantly interacted. The exclusion of ground-dwellers on mown plots resulted in higher attack rates of flying antagonists, whereas on unmown plots this effect of ground-dweller-exclusion on the attack rate of flying antagonists was not visible. Further, attack rates were determined by nest closure material, local abundance of different nest closure types as well as closure-associated antagonist species. In Chapter III, we studied the relative impact of local land use intensity, landscape composition and configuration on the species richness and abundance of bees, wasps and their antagonists. We analysed abundances and species numbers of hosts and their antagonists as well as parasitism rate and conducted a comprehensive landscape mapping. The digitized landscape data were the basis for further calculations of landscape metrics, like landscape composition and configuration within eight spatial scales ranging from 250 to 2,000 m radii. We used a compound, additive index of local land use intensity. Host abundance was only marginally negatively affected by local land use intensity. However, landscape composition at small spatial scales enhanced the species richness and abundance of hosts, while species richness and abundance of antagonists was positively related to landscape configuration at larger spatial scales. In the last study, presented in Chapter IV, we observed nesting bees on a selection of 18 grassland sites in two of the three research regions. We estimated the importance of resource distribution for pollen-nectar trips and consequences for the reproductive success of the solitary Red Mason Bee (Osmia bicornis). Local land use intensity, local flower cover as well as landscape composition and configuration were considered as critical factors of influence. We equipped each grassland site with eight trap nests and 50 female bees. Different nest building activities, like foraging trips for pollen and nectar, were measured. After the nesting season, we calculated measures of reproductive success. Foraging trips for pollen and nectar were significantly shorter in spatially complex landscapes but were neither affected by local metrics nor landscape composition. We found no evidence that the duration of pollen-nectar trips determines the reproductive success. Thus, to maintain trophic interactions and biodiversity, local land use as well as landscape diversity and spatial complexity should be accounted for to create spatial and temporal stability of food and nesting resources within small spatial scales. Concrete steps to support pollinator populations include hedges, sown field margins or other linear elements. These measures that enhance the connectivity of landscapes can also support flying antagonists.
This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.
Background
Angiogenesis represents a highly multi-factorial and multi-cellular complex (patho-) physiologic event involving endothelial cells, tumor cells in malignant conditions, as well as bone marrow derived cells and stromal cells. One main driver is vascular endothelial growth factor (VEGFA), which is known to interact with endothelial cells as a survival and mitogenic signal. The role of VEGFA on tumor cells and /or tumor stromal cell interaction is less clear. Condition specific (e.g. hypoxia) or tumor specific expression of VEGFA, VEGF receptors and co-receptors on tumor cells has been reported, in addition to the expression on the endothelium. This suggests a potential paracrine/autocrine loop that could affect changes specific to tumor cells.
Methods
We used the monoclonal antibody against VEGFA, bevacizumab, in various in vitro experiments using cell lines derived from different tumor entities (non small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC) and renal cell carcinoma (RCC)) in order to determine if potential VEGFA signaling could be blocked in tumor cells. The experiments were done under hypoxia, a major inducer of VEGFA and angiogenesis, in an attempt to mimic the physiological tumor condition. Known VEGFA induced endothelial biological responses such as proliferation, migration, survival and gene expression changes were evaluated.
Results
Our study was able to demonstrate expression of VEGF receptors on tumor cells as well as hypoxia regulated angiogenic gene expression. In addition, there was a cell line specific effect in tumor cells by VEGFA blockade with bevacizumab in terms of proliferation; however overall, there was a limited measurable consequence of bevacizumab therapy detected by migration and survival.
Conclusion
The present study showed in a variety of in vitro experiments with several tumor cell lines from different tumor origins, that by blocking VEGFA with bevacizumab, there was a limited autocrine or cell-autonomous function of VEGFA signaling in tumor cells, when evaluating VEGFA induced downstream outputs known in endothelial cells.
Background
Phytoplankton communities are often used as a marker for the determination of fresh water quality. The routine analysis, however, is very time consuming and expensive as it is carried out manually by trained personnel. The goal of this work is to develop a system for an automated analysis.
Results
A novel open source system for the automated recognition of phytoplankton by the use of microscopy and image analysis was developed. It integrates the segmentation of the organisms from the background, the calculation of a large range of features, and a neural network for the classification of imaged organisms into different groups of plankton taxa. 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 presented system has a flexible framework which easily allows expanding it to include additional taxa in the future.
Conclusions
The implemented automated microscopy and the new open source image analysis system - PlanktoVision - showed classification results that were comparable or better than existing systems and the exclusion of non-plankton particles could be greatly improved. The software package is published as free software and is available to anyone to help make the analysis of water quality more reproducible and cost effective.
Background
The actin cytoskeleton is essential for many physiological processes of eukaryotic cells. The emergence of new actin fibers is initiated by actin nucleators. Whereas most of them are evolutionary old, the cordon-bleu actin nucleator is classified as vertebrate specific.
Findings
Using sensitive methods for sequence similarity detection, we identified homologs of cordon-bleu not only in non-vertebrate chordates but also in arthropods, molluscs, annelids and platyhelminthes. These genes contain only a single WH2 domain and therefore resemble more the vertebrate cordon-bleu related 1 protein than the three WH2 domain containing cordon-bleu. Furthermore, we identified a homolog of the N-terminal, ubiquitin like, cobl domain of cordon-bleu in the cnidarian Nematostella vectensis.
Conclusion
Our results suggest that the ur-form of the cordon-bleu protein family evolved already with the emergence of the bilateria by the combination of existing cobl and WH2 domains. Following a vertebrate specific gene-duplication, one copy gained two additional WH2 domains leading to the actin nucleating cordon-bleu. The function of the ur-form of the cordon-bleu protein family is so far unknown. The identification of a homolog in the model organism Drosophila melanogaster could facilitate its experimental characterization.
Morphemes are the smallest meaningful parts of words and therefore represent a natural unit to study the evolution of words. To analyze the influence of language change on morphemes, we performed a large scale analysis of German and English vocabulary covering the last 200 years. Using a network approach from bioinformatics, we examined the historical dynamics of morphemes, the fixation of new morphemes and the emergence of words containing existing morphemes. We found that these processes are driven mainly by the number of different direct neighbors of a morpheme in words (connectivity, an equivalent to family size or type frequency) and not its frequency of usage (equivalent to token frequency). This contrasts words, whose survival is determined by their frequency of usage. We therefore identified features of morphemes which are not dictated by the statistical properties of words. As morphemes are also relevant for the mental representation of words, this result might enable establishing a link between an individual’s perception of language and historical language change.
Bone Morphogenetic Proteins (BMPs) are important growth factors that regulate many cellular processes. During embryogenesis they act as morphogens and play a critical role during organ development. They influence cell fates via concentration-gradients in the embryos where cells transduce this extracellular information into gene expression profiles and cell fate decisions. How receiving cells decode and quantify BMP2/4 signals is hardly understood. There is little data on the quantitative relationships between signal input, transducing molecules, their states and location, and ultimately their ability to integrate graded systemic inputs and generate qualitative responses. Understanding this signaling network on a quantitative level should be considered a prerequisite for efficient pathway modulation, as the BMP pathway is a prime target for therapeutic invention. Hence, we quantified the spatial distribution of the main signal transducer of the BMP2/4 pathway in response to different types and levels of stimuli in c2c12 cells. We found that the subcellular localization of Smad1 is independent of ligand concentration. In contrast, Smad1 phosphorylation levels relate proportionally to BMP2 ligand concentrations and they are entirely located in the nucleus. Interestingly, we found that BMP2 stimulates target gene expression in non-linear, wave-like forms. Amplitudes showed a clear concentration-dependency, for sustained and transient stimulation. We found that even burst-stimulation triggers gene-expression wave-like modulations that are detectable for at least 30 h. Finally, we show here that target gene expression oscillations depend on receptor kinase activity, as the kinase drives further expression pulses without receptor reactivation and the target gene expression breaks off after inhibitor treatment in c2c12 cells.
Bone Morphogenetic Proteins (BMPs) are key regulators for a lot of diverse cellular processes. During embryonic development these proteins act as morphogens and play a crucial role particularly in organogenesis. BMPs have a direct impact on distinct cellular fates by means of concentration-gradients in the developing embryos. Using the diverse signaling input information within the embryo due to the gradient, the cells transduce the varying extracellular information into distinct gene expression profiles and cell fate decisions. Furthermore, BMP proteins bear important functions in adult organisms like tissue homeostasis or regeneration. In contrast to TGF-ß signaling, currently only little is known about how cells decode and quantify incoming BMP signals. There is poor knowledge about the quantitative relationships between signal input, transducing molecules, their states and location, and finally their ability to incorporate graded systemic inputs and produce qualitative responses. A key requirement for efficient pathway modulation is the complete comprehension of this signaling network on a quantitative level as the BMP signaling pathway, just like many other signaling pathways, is a major target for medicative interference. I therefore at first studied the subcellular distribution of Smad1, which is the main signal transducing protein of the BMP signaling pathway, in a quantitative manner and in response to various types and levels of stimuli in murine c2c12 cells. Results indicate that the subcellular localization of Smad1 is not dependent on the initial BMP input. Surprisingly, only the phospho-Smad1 level is proportionally associated to ligand concentration. Furthermore, the activated transducer proteins were entirely located in the nucleus. Besides the subcellular localization of Smad1, I have analyzed the gene expression profile induced by BMP signaling. Therefore, I examined two endogenous immediate early BMP targets as well as the expression of the stably transgenic Gaussia Luciferase. Interestingly, the results of these independent experimental setups and read-outs suggest oscillating target gene expression. The amplitudes of the oscillations showed a precise concentration-dependence for continuous and transient stimulation. Additionally, even short-time stimulation of 15’ activates oscillating gene-expression pulses that are detectable for at least 30h post-stimulation. Only treatment with a BMP type I receptor kinase inhibitor leads to the complete abolishment of the target gene expression. This indicated that target gene expression oscillations depend directly on BMP type I receptor kinase activity.
Background
The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact.
Methodology and Principal Findings
We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples.
Conclusion
Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.