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- Chemical Biology Laboratory, National Cancer Institue, Frederick (USA) (1)
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To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca\(^{2+}\). In our search for species-dependent functional TPC1 channel variants with different luminal Ca\(^{2+}\) sensitivity, we found in total three acidic residues present in Ca\(^{2+}\) sensor sites 2 and 3 of the Ca\(^{2+}\)-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca\(^{2+}\). When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca\(^{2+}\) sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca\(^{2+}\) sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.
Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.
Recent studies link increased ozone (O\(_3\)) and carbon dioxide (CO\(_2\)) levels to alteration of plant performance and plant-herbivore interactions, but their interactive effects on plant-pollinator interactions are little understood. Extra floral nectaries (EFNs) are essential organs used by some plants for stimulating defense against herbivory and for the attraction of insect pollinators, e.g., bees. The factors driving the interactions between bees and plants regarding the visitation of bees to EFNs are poorly understood, especially in the face of global change driven by greenhouse gases. Here, we experimentally tested whether elevated levels of O\(_3\) and CO\(_2\) individually and interactively alter the emission of Volatile Organic Compound (VOC) profiles in the field bean plant (Vicia faba, L., Fabaceae), EFN nectar production and EFN visitation by the European orchard bee (Osmia cornuta, Latreille, Megachilidae). Our results showed that O\(_3\) alone had significant negative effects on the blends of VOCs emitted while the treatment with elevated CO\(_2\) alone did not differ from the control. Furthermore, as with O\(_3\) alone, the mixture of O\(_3\) and CO\(_2\) also had a significant difference in the VOCs’ profile. O\(_3\) exposure was also linked to reduced nectar volume and had a negative impact on EFN visitation by bees. Increased CO\(_2\) level, on the other hand, had a positive impact on bee visits. Our results add to the knowledge of the interactive effects of O\(_3\) and CO\(_2\) on plant volatiles emitted by Vicia faba and bee responses. As greenhouse gas levels continue to rise globally, it is important to take these findings into consideration to better prepare for changes in plant-insect interactions.
Natural DNA storage allows cellular differentiation, evolution, the growth of our children and controls all our ecosystems. Here, we discuss the fundamental aspects of DNA storage and recent advances in this field, with special emphasis on natural processes and solutions that can be exploited. We point out new ways of efficient DNA and nucleotide storage that are inspired by nature. Within a few years DNA-based information storage may become an attractive and natural complementation to current electronic data storage systems. We discuss rapid and directed access (e.g. DNA elements such as promotors, enhancers), regulatory signals and modulation (e.g. lncRNA) as well as integrated high-density storage and processing modules (e.g. chromosomal territories). There is pragmatic DNA storage for use in biotechnology and human genetics. We examine DNA storage as an approach for synthetic biology (e.g. light-controlled nucleotide processing enzymes). The natural polymers of DNA and RNA offer much for direct storage operations (read-in, read-out, access control). The inbuilt parallelism (many molecules at many places working at the same time) is important for fast processing of information. Using biology concepts from chromosomal storage, nucleic acid processing as well as polymer material sciences such as electronical effects in enzymes, graphene, nanocellulose up to DNA macramé , DNA wires and DNA-based aptamer field effect transistors will open up new applications gradually replacing classical information storage methods in ever more areas over time (decades).
Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.
In the fast-evolving landscape of biomedical research, the emergence of big data has presented researchers with extraordinary opportunities to explore biological complexities. In biomedical research, big data imply also a big responsibility. This is not only due to genomics data being sensitive information but also due to genomics data being shared and re-analysed among the scientific community. This saves valuable resources and can even help to find new insights in silico. To fully use these opportunities, detailed and correct metadata are imperative. This includes not only the availability of metadata but also their correctness. Metadata integrity serves as a fundamental determinant of research credibility, supporting the reliability and reproducibility of data-driven findings. Ensuring metadata availability, curation, and accuracy are therefore essential for bioinformatic research. Not only must metadata be readily available, but they must also be meticulously curated and ideally error-free. Motivated by an accidental discovery of a critical metadata error in patient data published in two high-impact journals, we aim to raise awareness for the need of correct, complete, and curated metadata. We describe how the metadata error was found, addressed, and present examples for metadata-related challenges in omics research, along with supporting measures, including tools for checking metadata and software to facilitate various steps from data analysis to published research.
Highlights
• Data awareness and data integrity underpins the trustworthiness of results and subsequent further analysis.
• Big data and bioinformatics enable efficient resource use by repurposing publicly available RNA-Sequencing data.
• Manual checks of data quality and integrity are insufficient due to the overwhelming volume and rapidly growing data.
• Automation and artificial intelligence provide cost-effective and efficient solutions for data integrity and quality checks.
• FAIR data management, various software solutions and analysis tools assist metadata maintenance.
Machine learning techniques are excellent to analyze expression data from single cells. These techniques impact all fields ranging from cell annotation and clustering to signature identification. The presented framework evaluates gene selection sets how far they optimally separate defined phenotypes or cell groups. This innovation overcomes the present limitation to objectively and correctly identify a small gene set of high information content regarding separating phenotypes for which corresponding code scripts are provided. The small but meaningful subset of the original genes (or feature space) facilitates human interpretability of the differences of the phenotypes including those found by machine learning results and may even turn correlations between genes and phenotypes into a causal explanation. For the feature selection task, the principal feature analysis is utilized which reduces redundant information while selecting genes that carry the information for separating the phenotypes. In this context, the presented framework shows explainability of unsupervised learning as it reveals cell-type specific signatures. Apart from a Seurat preprocessing tool and the PFA script, the pipeline uses mutual information to balance accuracy and size of the gene set if desired. A validation part to evaluate the gene selection for their information content regarding the separation of the phenotypes is provided as well, binary and multiclass classification of 3 or 4 groups are studied. Results from different single-cell data are presented. In each, only about ten out of more than 30000 genes are identified as carrying the relevant information. The code is provided in a GitHub repository at https://github.com/AC-PHD/Seurat_PFA_pipeline.
CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.
PRO-Simat is a simulation tool for analysing protein interaction networks, their dynamic change and pathway engineering. It provides GO enrichment, KEGG pathway analyses, and network visualisation from an integrated database of more than 8 million protein-protein interactions across 32 model organisms and the human proteome. We integrated dynamical network simulation using the Jimena framework, which quickly and efficiently simulates Boolean genetic regulatory networks. It enables simulation outputs with in-depth analysis of the type, strength, duration and pathway of the protein interactions on the website. Furthermore, the user can efficiently edit and analyse the effect of network modifications and engineering experiments. In case studies, applications of PRO-Simat are demonstrated: (i) understanding mutually exclusive differentiation pathways in Bacillus subtilis, (ii) making Vaccinia virus oncolytic by switching on its viral replication mainly in cancer cells and triggering cancer cell apoptosis and (iii) optogenetic control of nucleotide processing protein networks to operate DNA storage. Multilevel communication between components is critical for efficient network switching, as demonstrated by a general census on prokaryotic and eukaryotic networks and comparing design with synthetic networks using PRO-Simat. The tool is available at https://prosimat.heinzelab.de/ as a web-based query server.
Grading, immunohistochemistry and c-kit mutation status are criteria for assessing the prognosis and therapeutic options of canine cutaneous mast cell tumours (MCTs). As a subset, canine digital MCTs have rarely been explored in this context. Therefore, in this retrospective study, 68 paraffin-embedded canine digital MCTs were analysed, and histological grading was assessed according to Patnaik and Kiupel. The immunohistochemical markers KIT and Ki67 were used, as well as polymerase chain reaction (PCR) for mutational screening in c-kit exons 8, 9, 11 and 14. Patnaik grading resulted in 22.1% grade I, 67.6% grade II and 10.3% grade III tumours. Some 86.8% of the digital MCTs were Kiupel low-grade. Aberrant KIT staining patterns II and III were found in 58.8%, and a count of more than 23 Ki67-positive cells in 52.3% of the cases. Both parameters were significantly associated with an internal tandem duplication (ITD) in c-kit exon 11 (12.7%). French Bulldogs, which tend to form well-differentiated cutaneous MCTs, had a higher proportion of digital high-grade MCTs and ITD in c-kit exon 11 compared with mongrels. Due to its retrospective nature, this study did not allow for an analysis of survival data. Nevertheless, it may contribute to the targeted characterisation of digital MCTs.
Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.
The increasing loss of pollinators over the last decades has become more and more evident. Intensive use of plant protection products is one key factor contributing to this decline. Especially the mixture of different plant protection products can pose an increased risk for pollinators as synergistic effects may occur. In this study we investigated the effect of the fungicide Cantus® Gold (boscalid/dimoxystrobin), the neonicotinoid insecticide Mospilan® (acetamiprid) and their mixture on honeybees. Since both plant protection products are frequently applied sequentially to the same plants (e.g. oilseed rape), their combination is a realistic scenario for honeybees. We investigated the mortality, the sucrose responsiveness and the differential olfactory learning performance of honeybees under controlled conditions in the laboratory to reduce environmental noise. Intact sucrose responsiveness and learning performance are of pivotal importance for the survival of individual honeybees as well as for the functioning of the entire colony. Treatment with two sublethal and field relevant concentrations of each plant protection product did not lead to any significant effects on these behaviors but affected the mortality rate. However, our study cannot exclude possible negative sublethal effects of these substances in higher concentrations. In addition, the honeybee seems to be quite robust when it comes to effects of plant protection products, while wild bees might be more sensitive.
Highlights
• Mix of SBI fungicides and neonicotinoids can lead to synergistic effects for bees.
• Combination of non-SBI fungicide and neonicotinoid in field-realistic doses tested.
• Synergistic effect on mortality of honeybees.
• No effects on sucrose responsiveness and learning performance of honeybees.
• Synergistic effects by other pesticide mixtures or on wild bees cannot be excluded.
Efficient spatial orientation in the natural environment is crucial for the survival of most animal species. Cataglyphis desert ants possess excellent navigational skills. After far-ranging foraging excursions, the ants return to their inconspicuous nest entrance using celestial and panoramic cues. This review focuses on the question about how naïve ants acquire the necessary spatial information and adjust their visual compass systems. Naïve ants perform structured learning walks during their transition from the dark nest interior to foraging under bright sunlight. During initial learning walks, the ants perform rotational movements with nest-directed views using the earth’s magnetic field as an earthbound compass reference. Experimental manipulations demonstrate that specific sky compass cues trigger structural neuronal plasticity in visual circuits to integration centers in the central complex and mushroom bodies. During learning walks, rotation of the sky-polarization pattern is required for an increase in volume and synaptic complexes in both integration centers. In contrast, passive light exposure triggers light-spectrum (especially UV light) dependent changes in synaptic complexes upstream of the central complex. We discuss a multisensory circuit model in the ant brain for pathways mediating structural neuroplasticity at different levels following passive light exposure and multisensory experience during the performance of learning walks.
Land-use intensification and climate change threaten ecosystem functions. A fundamental, yet often overlooked, function is decomposition of necromass. The direct and indirect anthropogenic effects on decomposition, however, are poorly understood. We measured decomposition of two contrasting types of necromass, rat carrion and bison dung, on 179 study sites in Central Europe across an elevational climate gradient of 168–1122 m a.s.l. and within both local and regional land uses. Local land-use types included forest, grassland, arable fields, and settlements and were embedded in three regional land-use types (near-natural, agricultural, and urban). The effects of insects on decomposition were quantified by experimental exclusion, while controlling for removal by vertebrates. We used generalized additive mixed models to evaluate dung weight loss and carrion decay rate along elevation and across regional and local land-use types. We observed a unimodal relationship of dung decomposition with elevation, where greatest weight loss occurred between 600 and 700 m, but no effects of local temperature, land use, or insects. In contrast to dung, carrion decomposition was continuously faster with both increasing elevation and local temperature. Carrion reached the final decomposition stage six days earlier when insect access was allowed, and this did not depend on land-use effect. Our experiment identified different major drivers of decomposition on each necromass form. The results show that dung and carrion decomposition are rather robust to local and regional land use, but future climate change and decline of insects could alter decomposition processes and the self-regulation of ecosystems.
Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.
Background: The cabbage moth, Mamestra brassicae, is a polyphagous pest that attacks several crops. Here, the sublethal and lethal effects of chlorantraniliprole and indoxacarb were investigated on the developmental stages, detoxification enzymes, reproductive activity, calling behavior, peripheral physiology, and pheromone titer of M. brasssicae. Methods: To assess pesticide effects, the second instar larvae were maintained for 24 h on a semi-artificial diet containing insecticides at their LC\(_{10}\), LC\(_{30}\), and LC\(_{50}\) concentrations. Results: M. brassicae was more susceptible to chlorantraniliprole (LC\(_{50}\) = 0.35 mg/L) than indoxacarb (LC\(_{50}\) = 1.71 mg/L). A significantly increased developmental time was observed with both insecticides at all tested concentrations but decreases in pupation rate, pupal weight, and emergence were limited to the LC50 concentration. Reductions in both the total number of eggs laid per female and the egg viability were observed with both insecticides at their LC\(_{30}\) and LC\(_{50}\) concentrations. Both female calling activity and the sex pheromone (Z11-hexadecenyl acetate and hexadecenyl acetate) titer were significantly reduced by chlorantraniliprole in LC\(_{50}\) concentration. Antennal responses of female antennae to benzaldehyde and 3-octanone were significantly weaker than controls after exposure to the indoxocarb LC\(_{50}\) concentration. Significant reductions in the enzymatic activity of glutathione S-transferases, mixed-function oxidases, and carboxylesterases were observed in response to both insecticides.
Outdoor dust covers a shattered range of microbial agents from land over transportation, human microbial flora, which includes pathogen and commensals, and airborne from the environment. Dust aerosols are rich in bacterial communities that have a major impact on human health and living environments. In this study, outdoor samples from roadside barricades, safety walls, and fences (18 samples) were collected from Abu Dhabi, UAE and bacterial diversity was assessed through a 16S rRNA amplicon next generation sequencing approach. Clean data from HiSeq produced 1,099,892 total reads pairs for 18 samples. For all samples, taxonomic classifications were assigned to the OTUs (operational taxonomic units) representative sequence using the Ribosomal Database Project database. Analysis such as alpha diversity, beta diversity, differential species analysis, and species relative abundance were performed in the clustering of samples and a functional profile heat map was obtained from the OTUs by using bioinformatics tools. A total of 2814 OTUs were identified from those samples with a coverage of more than 99%. In the phylum, all 18 samples had most of the bacterial groups such as Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Twelve samples had Propionibacteria acnes and were mainly found in RD16 and RD3. Major bacteria species such as Propionibacteria acnes, Bacillus persicus, and Staphylococcus captis were found in all samples. Most of the samples had Streptococcus mitis, Staphylococcus capitis. and Nafulsella turpanensis and Enhydrobacter aerosaccus was part of the normal microbes of the skin. Salinimicrobium sp., Bacillus alkalisediminis, and Bacillus persicus are halophilic bacteria found in sediments. The heat map clustered the samples and species in vertical and horizontal classification, which represents the relationship between the samples and bacterial diversity. The heat map for the functional profile had high properties of amino acids, carbohydrate, and cofactor and vitamin metabolisms of all bacterial species from all samples. Taken together, our analyses are very relevant from the perspective of out-door air quality, airborne diseases, and epidemics, with broader implications for health safety and monitoring.
The increasing availability and variety of global satellite products and the rapid development of new algorithms has provided great potential to generate a new level of data with different spatial, temporal, and spectral resolutions. However, the ability of these synthetic spatiotemporal datasets to accurately map and monitor our planet on a field or regional scale remains underexplored. This study aimed to support future research efforts in estimating crop yields by identifying the optimal spatial (10 m, 30 m, or 250 m) and temporal (8 or 16 days) resolutions on a regional scale. The current study explored and discussed the suitability of four different synthetic (Landsat (L)-MOD13Q1 (30 m, 8 and 16 days) and Sentinel-2 (S)-MOD13Q1 (10 m, 8 and 16 days)) and two real (MOD13Q1 (250 m, 8 and 16 days)) NDVI products combined separately to two widely used crop growth models (CGMs) (World Food Studies (WOFOST), and the semi-empiric Light Use Efficiency approach (LUE)) for winter wheat (WW) and oil seed rape (OSR) yield forecasts in Bavaria (70,550 km\(^2\)) for the year 2019. For WW and OSR, the synthetic products’ high spatial and temporal resolution resulted in higher yield accuracies using LUE and WOFOST. The observations of high temporal resolution (8-day) products of both S-MOD13Q1 and L-MOD13Q1 played a significant role in accurately measuring the yield of WW and OSR. For example, L- and S-MOD13Q1 resulted in an R\(^2\) = 0.82 and 0.85, RMSE = 5.46 and 5.01 dt/ha for WW, R\(^2\) = 0.89 and 0.82, and RMSE = 2.23 and 2.11 dt/ha for OSR using the LUE model, respectively. Similarly, for the 8- and 16-day products, the simple LUE model (R\(^2\) = 0.77 and relative RMSE (RRMSE) = 8.17%) required fewer input parameters to simulate crop yield and was highly accurate, reliable, and more precise than the complex WOFOST model (R\(^2\) = 0.66 and RRMSE = 11.35%) with higher input parameters. Conclusively, both S-MOD13Q1 and L-MOD13Q1, in combination with LUE, were more prominent for predicting crop yields on a regional scale than the 16-day products; however, L-MOD13Q1 was advantageous for generating and exploring the long-term yield time series due to the availability of Landsat data since 1982, with a maximum resolution of 30 m. In addition, this study recommended the further use of its findings for implementing and validating the long-term crop yield time series in different regions of the world.
Rapid and accurate yield estimates at both field and regional levels remain the goal of sustainable agriculture and food security. Hereby, the identification of consistent and reliable methodologies providing accurate yield predictions is one of the hot topics in agricultural research. This study investigated the relationship of spatiotemporal fusion modelling using STRAFM on crop yield prediction for winter wheat (WW) and oil-seed rape (OSR) using a semi-empirical light use efficiency (LUE) model for the Free State of Bavaria (70,550 km\(^2\)), Germany, from 2001 to 2019. A synthetic normalised difference vegetation index (NDVI) time series was generated and validated by fusing the high spatial resolution (30 m, 16 days) Landsat 5 Thematic Mapper (TM) (2001 to 2012), Landsat 7 Enhanced Thematic Mapper Plus (ETM+) (2012), and Landsat 8 Operational Land Imager (OLI) (2013 to 2019) with the coarse resolution of MOD13Q1 (250 m, 16 days) from 2001 to 2019. Except for some temporal periods (i.e., 2001, 2002, and 2012), the study obtained an R\(^2\) of more than 0.65 and a RMSE of less than 0.11, which proves that the Landsat 8 OLI fused products are of higher accuracy than the Landsat 5 TM products. Moreover, the accuracies of the NDVI fusion data have been found to correlate with the total number of available Landsat scenes every year (N), with a correlation coefficient (R) of +0.83 (between R\(^2\) of yearly synthetic NDVIs and N) and −0.84 (between RMSEs and N). For crop yield prediction, the synthetic NDVI time series and climate elements (such as minimum temperature, maximum temperature, relative humidity, evaporation, transpiration, and solar radiation) are inputted to the LUE model, resulting in an average R\(^2\) of 0.75 (WW) and 0.73 (OSR), and RMSEs of 4.33 dt/ha and 2.19 dt/ha. The yield prediction results prove the consistency and stability of the LUE model for yield estimation. Using the LUE model, accurate crop yield predictions were obtained for WW (R\(^2\) = 0.88) and OSR (R\(^2\) = 0.74). Lastly, the study observed a high positive correlation of R = 0.81 and R = 0.77 between the yearly R\(^2\) of synthetic accuracy and modelled yield accuracy for WW and OSR, respectively.
Copy number variations (CNVs) of the KITLG gene seem to be involved in the oncogenesis of digital squamous cell carcinoma (dSCC). The aims of this study were (1) to investigate KITLG CNV in giant (GS), standard (SS), and miniature (MS) schnauzers and (2) to compare KITLG CNV between black GS with and without dSCC. Blood samples from black GS (22 with and 17 without dSCC), black SS (18 with and 4 without dSSC; 5 unknown), and 50 MS (unknown dSSC status and coat colour) were analysed by digital droplet PCR. The results are that (1) most dogs had a copy number (CN) value > 4 (range 2.5–7.6) with no significant differences between GS, SS, and MS, and (2) the CN value in black GS with dSCC was significantly higher than in those without dSCC (p = 0.02). CN values > 5.8 indicate a significantly increased risk for dSCC, while CN values < 4.7 suggest a reduced risk for dSCC (grey area: 4.7–5.8). Diagnostic testing for KITLG CNV may sensitise owners to the individual risk of their black GS for dSCC. Further studies should investigate the relevance of KITLG CNV in SS and the protective effects in MS, who rarely suffer from dSCC.
Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC). This may potentially suggest an underlying genetic predisposition not yet completely elucidated. Some authors have suggested a potential correlation between the number of copies KIT Ligand (KITLG) and the predisposition of dogs to DSCC, containing a higher number of copies in those affected by the neoplasm. In this study, the aim was to evaluate a potential correlation between the number of copies of the KITLG and the histological grade of malignancy in dogs with DSCC. For this, 72 paraffin-embedded DSCCs with paired whole blood samples of 70 different dogs were included and grouped according to their haircoat color as follow: Group 0/unknown haircoat color (n = 11); Group 1.a/black non-Schnauzers (n = 15); group 1.b/black Schnauzers (n = 33); group 1.c/black and tan dogs (n = 7); group 2/tan animals (n = 4). The DSCCs were histologically graded. Additionally, KITLG Copy Number Variation (CNV) was determined by ddPCR. A significant correlation was observed between KITLG copy number and the histological grade and score value. This finding may suggest a possible factor for the development of canine DSCC, thus potentially having an impact on personalized veterinary oncological strategies and breeding programs.
Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.
Agricultural abandonment is one of the main land-use changes in Europe, and its consequences on biodiversity are context- and taxa-dependent. While several studies have worked on this topic, few have focused on traditional orchards, especially in different landscapes and under a Mediterranean climate. In this context, we aimed to determine the effects of almond orchard abandonment on the communities of three groups of beneficial arthropods and the role of the landscape context in modulating these effects. Between February and September 2019, four samplings were carried out in twelve almond orchards (three abandoned and three traditional (active orchards under traditional agricultural management) located in simple landscapes as well as three abandoned and three traditional in complex landscapes). Abandoned and traditional almond orchards harbor different arthropod communities and diversity metrics that are strongly conditioned by seasonality. Abandoned orchards can favor pollinators and natural enemies, providing alternative resources in simple landscapes. However, the role that abandoned orchards play in simple landscapes disappears as the percentage of semi-natural habitats in the landscape increases. Our results show that landscape simplification, through the loss of semi-natural habitats, has negative consequences on arthropod biodiversity, even in traditional farming landscapes with small fields and high crop diversity.
Orthopteran diversity in steep slope vineyards: the role of vineyard type and vegetation management
(2023)
The abandonment of traditional agricultural practices and subsequent succession are major threats to many open-adapted species and species-rich ecosystems. Viticulture on steep slopes has recently suffered from strong declines due to insufficient profitability, thus increasing the area of fallow land considerably. Changing cultivation systems from vertically oriented to modern vineyard terraces offers an opportunity to maintain management economically viable and thus reduces further abandonment. Hillside parallel terraces favor mechanization, and their embankments offer large undisturbed areas that could provide valuable habitats. We investigated the effects of vineyard abandonment, different vineyard management types (vertically oriented vs. terraced), and local parameters on Orthoptera diversity in 45 study sites along the Upper Middle Rhine Valley in Germany. Our results show that woody structures and vineyard abandonment reduced Orthoptera diversity at the local and landscape scale due to decreased habitat quality, especially for open-adapted species. In contrast, open inter-rows of actively managed vineyard types supported heat-adapted Caelifera species. On terrace embankments, extensive management and taller vegetation benefited Ensifera species, while short and mulched vegetation in vertically oriented vineyards favored the dominance of one single Caelifera species. Our results highlight the significance of maintaining viticultural management on steep slopes for the preservation of both open-adapted Orthoptera species and the cultural landscape.
(1) Background: Clear cell renal cell carcinoma extending into the inferior vena cava (ccRCC\(^{IVC}\)) represents a clinical high-risk setting. However, there is substantial heterogeneity within this patient subgroup regarding survival outcomes. Previously, members of our group developed a microRNA(miR)-based risk classifier — containing miR-21-5p, miR-126-3p and miR-221-3p expression — which significantly predicted the cancer-specific survival (CSS) of ccRCC\(^{IVC}\) patients. (2) Methods: Examining a single-center cohort of tumor tissue from n = 56 patients with ccRCC\(^{IVC}\), we measured the expression levels of miR-21, miR-126, and miR-221 using qRT-PCR. The prognostic impact of clinicopathological parameters and miR expression were investigated via single-variable and multivariable Cox regression. Referring to the previously established risk classifier, we performed Kaplan–Meier analyses for single miR expression levels and the combined risk classifier. Cut-off values and weights within the risk classifier were taken from the previous study. (3) Results: miR-21 and miR-126 expression were significantly associated with lymphonodal status at the time of surgery, the development of metastasis during follow-up, and cancer-related death. In Kaplan–Meier analyses, miR-21 and miR-126 significantly impacted CSS in our cohort. Moreover, applying the miR-based risk classifier significantly stratified ccRCC\(^{IVC}\) according to CSS. (4) Conclusions: In our retrospective analysis, we successfully validated the miR-based risk classifier within an independent ccRCC\(^{IVC}\) cohort.
Protein folding achieves a clear solution structure in a huge parameter space (the so-called protein folding problem). Proteins fold in water, and get by this a highly ordered structure. Finally, inside a protein crystal for structure resolution, you have everywhere the same symmetries as there is everywhere the same unit cell. We apply this to qubit interactions to do fundamental physics:
in a modified cosmology, we replace the big bang by a condensation event in an eternal all-encompassing ocean of free qubits. Interactions of qubits in the qubit ocean are quite rare but provide a nucleus or seed for a new universe (domain) as the qubits become decoherent and freeze-out into defined bit ensembles. Second, we replace inflation by a crystallization event triggered by the nucleus of interacting qubits to which rapidly more and more qubits attach (like in everyday crystal growth). The crystal unit cell guarantees same symmetries everywhere inside the crystal. The textbook inflation scenario to explain the same laws of nature in our domain is replaced by the unit cell of the crystal formed.
Interacting qubits solidify, quantum entropy decreases (but increases in the ocean around). In a modified inflation scenario, the interacting qubits form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. Then standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements. We explain by cosmological crystallization instead of inflation: early creation of large-scale structure of voids and filaments, supercluster formation, galaxy formation, and the dominance of matter: the unit cell of our crystal universe has a matter handedness avoiding anti-matter.
We prove initiation of qubit interactions can only be 1,2,4 or 8-dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. Crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness.
The phase space of the crystal agrees with the standard model of the basic four forces for n quanta. It includes all possible ensemble combinations of their quantum states m, a total of n**m states. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients. However, in our four dimensions there is only one bit overlap to neighbor states left (almost solid, only below Planck quantum there is liquidity left). The E8 symmetry of heterotic string theory has six curled-up, small dimensions which help to keep the qubit crystal together and will never expand.
Mathematics focusses on the Hurwitz proof applied to qubit interaction, a toy model of qubit interaction and repulsive forces of qubits. Vacuum energy gets appropriate low inside the crystal. We give first energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit decoherence / crystal formation. Scalar fields for color interaction/confinement and gravity are derived from the qubit-interaction field.
Single-molecule dynamics at a bottleneck: a systematic study of the narrow escape problem in a disc
(2023)
Diffusion facilitates numerous reactions within the biological context of a cell. It is remarkable how the cost-efficient random process of Brownian motion promotes fast reactions. From the narrow escape theory, it is possible to determine the mean first passage time of such processes based on their reaction space and diffusion coefficient. The narrow escape theory of Brownian particles is characterized by a confining domain with reflective boundaries and a small reaction site. In this thesis, the mean first passage time was systematically tested in a disc as a function of the escape opening size in vitro and in silico. For the in vitro experiments, a model system of patterned supported-lipid bilayers (SLB) was established. Such a model is prepared by a combined colloid metalization approach, where a gold scaffold on glass facilitates assembly of SLB patches of distinct sizes through vesicle fusion. The model setup was evaluated and found to match all necessary requirements to test the nar- row escape problem in vitro. In particular, the reflectivity of the boundaries, the unhindered, free diffusion of the tracer lipids, and the distinct area were assessed. Observed results of the mean first passage time agreed with the theory of the narrow escape problem. There was excellent agreement in both absolute values and across a range of small escape opening sizes. Additionally, I developed a straightforward method, a correction factor, to calculate the mean first passage time from incomplete experimental traces. By re-scaling the mean first passage time to the fraction of particles that escaped, I was able to overcome the lifetime limitations of fluorescent probes. Previously inaccessible measurements of the mean first passage time relying on fluorescent probes will be made possible through this approach. The in vitro experiments were complemented with various in silico experiments. The latter were based on random walk simulations in discs, mimicking the in vitro situation with its uncertainties. The lifetime of single particles was either set sufficiently long to allow all particles to escape, or was adjusted to meet the lifetime limitations observed in the in vitro experiments. A comparison of the mean first passage time from lifetime-unlimited particles to the corrected, lifetime-limited particles did support the use of the correction factor. In agreement with the narrow escape theory, it was experimentally found that the mean first passage time is independent of the start point of the particle within the domain. This is when the particle adheres to a minimum distance to the escape site. In general, the presented random walk simulations do accurately represent the in vitro experiments in this study. The required hardware for the establishment of an astigmatism-based 3D system was installed in the existing microscope. The first attempts to analyze the obtained 3D imaging data gave insight into the potential of the method to investigate molecule dynamics in living trypanosome cells. The full functionality will be realized with the ongoing improvement of image analysis outside of this thesis.
Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host.
Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression.
A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts.
The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes.
In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection.
After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host.
In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.
The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration.
Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.
The western honeybee (Apis mellifera) is widely known as the honey producer and pollinator managed by beekeepers but neglected as a wild bee species. Central European honeybee populations have been anthropogenically disturbed since about 1850 through introgression and moderate artificial selection but have never been truly domesticated due to a lack of mating control. While their decline in the wild was historically attributed to the scarcity of nesting cavities, a contemporary view considers the invasion of the parasitic mite Varroa destructor in the 1970s as the major driver. However, there are no longitudinal population data available that could substantiate either claim. Based on the insight that introduced European honeybees form viable wild populations in eastern North America and reports on the occurrence of wild-living colonies from various European countries, we systematically studied the ecology of wild-living honeybees in Germany. First, we investigated whether wild-living honeybees colonising German forests form a self-sustaining population. Second, we asked how the parasite burden of wild-living colonies relates to that of managed colonies. And third, we explored whether the winter mortality of wild-living colonies is associated with parasite burden, nest depredation, or the lack of resources on the landscape scale.
Between 2017 and 2021, we monitored listed trees with black woodpecker cavities for honeybees in the managed forests of three study regions (Swabian Alb, counties Coburg and Lichtenfels, county Weilheim-Schongau). Continuity of occupation was determined using microsatellite genetic markers. Wild-living colonies predictably colonised forests in summer, when about 10% of all cavities were occupied. The annual colony survival rate and colony lifespan (based on N=112 colonies) were 10.6% and 0.6 years, with 90% of colonies surviving summer (July–September), 16% surviving winter (September–April), and 72% surviving spring (April–July). The average maximum and minimum colony densities were 0.23 (July) and 0.02 (April) colonies per km^2. During the (re-)colonisation of forests in spring, swarms preferred cavities that had already been occupied by other honeybee colonies. We estimate the net reproductive rate of the population to be R0= 0.318, meaning that it is currently not self-sustaining but maintained by the annual immigration of swarms from managed hives. The wild-living colonies are feral in a behavioural sense.
We compared the occurrence of 18 microparasites among feral colonies (N=64) and managed colonies (N=74) using qPCR. Samples were collected in four regions (the three regions mentioned above and the city of Munich) in July 2020; they consisted of 20 workers per colony captured at flight entrances. We distinguished five colony types representing differences in colony age and management histories. Besides strong regional variation, feral colonies consistently hosted fewer microparasite taxa (median: 5, range 1–8) than managed colonies (median: 6, range 4–9) and had different parasite communities. Microparasites that were notably less prevalent among feral colonies were Trypanosomatidae, Chronic bee paralysis virus, and Deformed wing viruses A and B. In the comparison of five colony types, parasite burden was lowest in newly founded feral colonies, intermediate in overwintered feral colonies and managed nucleus colonies, and highest in overwintered managed colonies and hived swarms. This suggests that the natural mode of colony reproduction by swarming, which creates pauses in brood production, and well-dispersed nests, which reduce horizontal transmission, explain the reduced parasite burden in feral compared to managed colonies.
To explore the roles of three potential drivers of feral colony winter mortality, we combined colony observations gathered during the monitoring study with data on colony-level parasite burden, observations and experiments on nest depredation, and landscape analyses. There was no evidence for an effect of summertime parasite burden on subsequent winter mortality: colonies that died (N=57) did not have a higher parasite burden than colonies that survived (N=10). Camera traps (N=15) installed on cavity trees revealed that honeybee nests are visited by a range of vertebrate species throughout the winter at rates of up to 10 visits per week. Four woodpecker species, great tits, and pine martens acted as true nest depredators. The winter survival rate of colonies whose nest entrances were protected by screens of wire mesh (N=32) was 50% higher than that of colonies with unmanipulated entrances (N=40). Analyses of land cover maps revealed that the landscapes surrounding surviving colonies (N=19) contained on average 6.4 percentage points more resource-rich cropland than landscapes surrounding dying colonies (N=94).
We estimate that tens of thousands of swarms escape from apiaries each year to occupy black woodpecker cavities and other hollow spaces in Germany and that feral colonies make up about 5% of the regional honeybee populations. They are unlikely to contribute disproportionately to the spread of bee diseases. Instead, by spatially complementing managed colonies, they contribute to the pollination of wild plants in forests. Honeybees occupying tree cavities likely have various effects on forest communities by acting as nest site competitors or prey, and by accumulating biomass in tree holes. Nest depredation (a consequence of a lack of well-protected nest sites) and food resource limitation seem to be more important than parasites in hampering feral colony survival. The outstanding question is how environmental and intrinsic factors interact in preventing population establishment. Nest boxes with movable frames could be used to better study the environmental drivers of feral colonies’ mortality. Pairs of wild (self-sustaining) and managed populations known to exist outside Europe could provide answers to whether modern apiculture creates honeybee populations maladapted to life in the wild. In Europe, large continuous forests might represent evolutionary refuges for wild honeybees.
Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain.
The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis.
Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group’s macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera.
Honeybees are among the few animals that rely on eusociality to survive. While the
task of queen and drones is only reproduction, all other tasks are accomplished by sterile
female worker bees. Different tasks are mostly divided by worker bees of different ages
(temporal polyethism). Young honeybees perform tasks inside the hive like cleaning and
nursing. Older honeybees work at the periphery of the nest and fulfill tasks like guarding
the hive entrance. The oldest honeybees eventually leave the hive to forage for resources
until they die. However, uncontrollable circumstances might force the colony to adapt or
perish. For example, the introduced Varroa destructor mite or the deformed wing virus
might erase a lot of in-hive bees. On the other hand, environmental events might kill a
lot of foragers, leaving the colony with no new food intake. Therefore, adaptability of
task allocation must be a priority for a honeybee colony.
In my dissertation, I employed a wide range of behavioral, molecular biological and analytical techniques to unravel the underlying molecular and physiological mechanisms of
the honeybee division of labor, especially in conjunction with honeybee malnourishment.
The genes AmOARα1, AmTAR1, Amfor and vitellogenin have long been implied to
be important for the transition from in-hive tasks to foraging. I have studied in detail
expression of all of these genes during the transition from nursing to foraging to understand how their expression patterns change during this important phase of life. My focus
lay on gene expression in the honeybee brain and fat body. I found an increase in the
AmOARα1 and the Amforα mRNA expression with the transition from in-hive tasks to
foraging and a decrease in expression of the other genes in both tissues. Interestingly,
I found the opposite pattern of the AmOARα1 and AmTAR1 mRNA expression in the
honeybee fat body during orientation flights. Furthermore, I closely observed juvenile
hormone titers and triglyceride levels during this crucial time. Juvenile hormone titers
increased with the transition from in-hive tasks to foraging and triglyceride levels decreased.
Furthermore, in-hive bees and foragers also differ on a behavioral and physiological level.
For example, foragers are more responsive towards light and sucrose. I proposed that
modulation via biogenic amines, especially via octopamine and tyramine, can increase
or decrease the responsiveness of honeybees. For that purpose, in-hive bees and foragers were injected with both biogenic amines and the receptor response was quantified
1
using electroretinography. In addition, I studied the behavioral response of the bees to
light using a phototaxis assay. Injecting octopamine increased the receptor response and
tyramine decreased it. Also, both groups of honeybees showed an increased phototactic
response when injected with octopamine and a decreased response when injected with
tyramine, independent of locomotion.
Additionally, nutrition has long been implied to be a driver for division of labor. Undernourished honeybees are known to speed up their transition to foragers, possibly to
cope with the missing resources. Furthermore, larval undernourishment has also been
implied to speed up the transition from in-hive bees to foragers, due to increasing levels
of juvenile hormone titers in adult honeybees after larval starvation. Therefore, I reared
honeybees in-vitro to compare the hatched adult bees of starved and overfed larvae to
bees reared under the standard in-vitro rearing diet. However, first I had to investigate
whether the in-vitro rearing method affects adult honeybees.
I showed effects of in-vitro rearing on behavior, with in-vitro reared honeybees foraging
earlier and for a shorter time than hive reared honeybees. Yet, nursing behavior was
unaffected.
Afterwards, I investigated the effects of different larval diets on adult honeybee workers.
I found no effects of malnourishment on behavioral or physiological factors besides a
difference in weight. Honeybee weight increased with increasing amounts of larval food,
but the effect seemed to vanish after a week.
These results show the complexity and adaptability of the honeybee division of labor.
They show the importance of the biogenic amines octopamine and tyramine and of the
corresponding receptors AmOARα1 and AmTAR1 in modulating the transition from inhive bees to foragers. Furthermore, they show that in-vitro rearing has no effects on
nursing behavior, but that it speeds up the transition from nursing to foraging, showing
strong similarities to effects of larval pollen undernourishment. However, larval malnourishment showed almost no effects on honeybee task allocation or physiology. It seems
that larval malnourishment can be easily compensated during the early lifetime of adult
honeybees.
Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus
(2023)
Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.
DNA methylation acts as a major epigenetic modification in mammals, characterized by the transfer of a methyl group to a cytosine. DNA methylation plays a pivotal role in regulating normal development, and misregulation in cells leads to an abnormal phenotype as is seen in several cancers. Any mutations or expression anomalies of genes encoding regulators of DNA methylation may lead to abnormal expression of critical molecules. A comprehensive genomic study encompassing all the genes related to DNA methylation regulation in relation to breast cancer is lacking. We used genomic and transcriptomic datasets from the Cancer Genome Atlas (TGCA) Pan-Cancer Atlas, Genotype-Tissue Expression (GTEx) and microarray platforms and conducted in silico analysis of all the genes related to DNA methylation with respect to writing, reading and erasing this epigenetic mark. Analysis of mutations was conducted using cBioportal, while Xena and KMPlot were utilized for expression changes and patient survival, respectively. Our study identified multiple mutations in the genes encoding regulators of DNA methylation. The expression profiling of these showed significant differences between normal and disease tissues. Moreover, deregulated expression of some of the genes, namely DNMT3B, MBD1, MBD6, BAZ2B, ZBTB38, KLF4, TET2 and TDG, was correlated with patient prognosis. The current study, to our best knowledge, is the first to provide a comprehensive molecular and genetic profile of DNA methylation machinery genes in breast cancer and identifies DNA methylation machinery as an important determinant of the disease progression. The findings of this study will advance our understanding of the etiology of the disease and may serve to identify alternative targets for novel therapeutic strategies in cancer.
Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.
Nebennierenrindenkarzinome (NNR-Ca; engl. adrenocortical carcinoma (ACC)) zählen zu den sehr seltenen Tumorentitäten. Die Prognose für die Patient*innen ist insgesamt eher schlecht, kann aber, im Einzelnen betrachtet, sehr heterogen sein. Eine zuverlässige Prognose anhand klinischer und histopathologischer Marker – wie dem Tumorstadium bei Diagnose, dem Resektionsstatus und dem Proliferationsindex Ki-67 –, die routinemäßig erhoben werden, ist nicht für alle Erkrankten möglich. Außerdem wird deren Behandlung dadurch erschwert, dass Therapeutika fehlen, von denen ein Großteil der Patient*innen profitiert. Umfassende Multi-Omics-Studien aus den letzten Jahren halfen nicht nur das Wissen über Pathomechanismen in NNR-Cas zu erweitern, es konnte auch gezeigt werden, dass sich Patient*innen anhand molekularer Marker in Subgruppen mit jeweils unterschiedlicher Prognose einteilen lassen. Mit molekulargenetischen Untersuchungen wurden außerdem potentielle neue Therapieziele gefunden. Diese Erkenntnisse finden bisher jedoch keine oder kaum Anwendung, da die Analysen den zeitlichen und finanziellen Rahmen, der für den routinemäßigen Einsatz im Klinikalltag zu erfüllen wäre, deutlich überschreiten. Ziel dieser Arbeit war es, eine Strategie zur verbesserten Patientenversorgung der NNR-CaPatient*innen zu etablieren. Dafür sollte geklärt werden, ob ausgewählte molekulare prognostische Marker mit Methoden, die theoretisch einfach in den Klinikalltag zu implementieren wären, gefunden werden können. Außerdem sollte nach prädiktiven Markern gesucht werden, die helfen, NNR-Ca-Patient*innen zielgerichtet zu therapieren. Statt exom- oder genomweite Analysen durchzuführen wurden gezielt krebs- beziehungsweise NNR-Ca-assoziierte Gene mittels NGS (Next-Generation Sequencing) oder SangerSequenzierung (zusammen 161 Gene) und Pyrosequenzierung (4 Gene) auf somatische Veränderungen hin untersucht. Die Analysen wurden an DNA (Desoxyribonukleinsäure) durchgeführt, die aus FFPE (mit Formalin fixiert und in Paraffin eingebettet)-Gewebe isoliert worden war, welches standardmäßig nach Tumoroperationen in Pathologien für Untersuchungen zur Verfügung steht. Durch Analyse der Sequenzierergebnisse von insgesamt 157 Patient*innen aus einem retrospektiven (107 Patient*innen) und einem prospektiven Studienteil (50 Patient*innen) konnten in NNR-Cas bereits beschriebene Veränderungen von Genen und Signalwegen sowie Methylierungsunterschiede gefunden werden. Anhand der Sequenzierdaten der retrospektiven Studie wurden molekulare prognostische Marker (Anzahl an proteinverändernden Varianten pro Tumorprobe, Veränderungen im P53/Rb- und/oder dem Wnt/ß-Catenin-Signalweg und dem Methylierungsstatus von CpG-Inseln von vier 2 Tumorsuppressorgenen (GSTP1, PAX5, PAX6 und PYCARD)) definiert und für jeden einzelnen Marker ein signifikanter Zusammenhang zur Länge des progressionsfreien Überlebens (PFS) der Patient*innen gefunden. Durch die Kombination der molekularen Marker mit den klinischen und histopathologischen Markern war es zudem möglich, einen COMBI-Score zu bilden, der, verglichen mit den klinischen und histopathologischen Markern, eine spezifischere und sensitivere Aussage darüber erlaubt, ob Patient*innen innerhalb von 2 Jahren ein Fortschreiten der Tumorerkrankung erfahren. Mit Hilfe der Sequenzierdaten wurden in beiden Kohorten außerdem Veränderungen gefunden, die als prädiktive Marker zum Einsatz von zielgerichteten Therapien vewendet werden könnten. Als vielversprechendstes Therapieziel wurde – bei 46 Tumoren in der retrospektiven und 7 Tumoren in der prospektiven Studie – CDK4 identifiziert. CDK4/CDK6-Inhibitoren sind für die Behandlung von fortgeschrittenem und metastasiertem Brustkrebs von der Lebensmittel- überwachungs- und Arzneimittelbehörde (FDA; engl. Food and Drug Administration) zugelassene Therapeutika und bei anderen soliden Tumoren Gegenstand von Studien. Im Rahmen der Arbeit konnten außerdem von 12 Patient*innen jeweils zwei Tumoren molekulargenetisch untersucht und die Ergebnisse verglichen werden. Die Analyse zeigte, dass der Methylierungsstatus – im Vergleich zu Veränderungen in der DNA-Sequenz – der stabilere prognostische Marker ist. Mit dieser Arbeit wurde gezeigt, dass molekulare prognostische und prädiktive Marker für den Einsatz zielgerichteter Therapien mit Methoden identifiziert werden können, die sich im klinischen Alltag bei der Behandlung von NNR-Ca-Patient*innen implementieren lassen. Um einen allgemein anerkannten Leitfaden zu etablieren, fehlen allerdings noch die Ergebnisse weiterer – vor allem prospektiver – Studien zur Validierung der hier präsentierten Ergebnisse. Die gewonnenen Erkenntnisse sind jedoch als wichtiger Schritt in Richtung personalisierter Medizin bei Nebennierenrindenkarzinomen anzusehen.
The behavior of honeybees and bumblebees relies on a constant sensory integration of abiotic or biotic stimuli. As eusocial insects, a sophisticated intraspecific communication as well as the processing of multisensory cues during foraging is of utter importance. To tackle the arising challenges, both honeybees and bumblebees have evolved a sophisticated olfactory and visual processing system.
In both organisms, olfactory reception starts at the antennae, where olfactory sensilla cover the antennal surface in a sex-specific manner. These sensilla house olfactory receptor neurons (ORN) that express olfactory receptors. ORNs send their axons via four tracts to the antennal lobe (AL), the prime olfactory processing center in the bee brain. Here, ORNs specifically innervate spheroidal structures, so-called glomeruli, in which they form synapses with local interneurons and projection neurons (PN). PNs subsequently project the olfactory information via two distinct tracts, the medial and the lateral antennal-lobe tract, to the mushroom body (MB), the main center of sensory integration and memory formation. In the honeybee calyx, the sensory input region of the MB, PNs synapse on Kenyon cells (KC), the principal neuron type of the MB. Olfactory PNs mainly innervate the lip and basal ring layer of the calyx. In addition, the basal ring receives input from visual PNs, making it the first site of integration of visual and olfactory information. Visual PNs, carrying sensory information from the optic lobes, send their terminals not only to the to the basal ring compartment but also to the collar of the calyx. Receiving olfactory or visual input, KCs send their axons along the MB peduncle and terminate in the main output regions of the MB, the medial and the vertical lobe (VL) in a layer-specific manner. In the MB lobes, KCs synapse onto mushroom body output neurons (MBON). In so far barely understood processes, multimodal information is integrated by the MBONs and then relayed further into the protocerebral lobes, the contralateral brain hemisphere, or the central brain among others.
This dissertation comprises a dichotomous structure that (i) aims to gain more insight into the olfactory processing in bumblebees and (ii) sets out to broaden our understanding of visual processing in honeybee MBONs.
The first manuscript examines the olfactory processing of Bombus terrestris and specifically investigates sex-specific differences. We used behavioral (absolute conditioning) and electrophysiological approaches to elaborate the processing of ecologically relevant odors (components of plant odors and pheromones) at three distinct levels, in the periphery, in the AL and during olfactory conditioning. We found both sexes to form robust memories after absolute conditioning and to generalize towards the carbon chain length of the presented odors. On the contrary, electroantennographic (EAG) activity showed distinct stimulus and sex-specific activity, e.g. reduced activity towards citronellol in drones. Interestingly, extracellular multi-unit recordings in the AL confirmed stimulus and sex-specific differences in olfactory processing, but did not reflect the differences previously found in the EAG. Here, farnesol and 2,3-dihydrofarnesol, components of sex-specific pheromones, show a distinct representation, especially in workers, corroborating the results of a previous study. This explicitly different representation suggests that the peripheral stimulus representation is an imperfect indication for neuronal representation in high-order neuropils and ecological importance of a specific odor.
The second manuscript investigates MBONs in honeybees to gain more insights into visual processing in the VL. Honeybee MBONs can be categorized into visually responsive, olfactory responsive and multimodal. To clarify which visual features are represented at this high-order integration center, we used extracellular multi-unit recordings in combination with visual and olfactory stimulation. We show for the first time that information about brightness and wavelength is preserved in the VL. Furthermore, we defined three specific classes of visual MBONs that distinctly encode the intensity, identity or simply the onset of a stimulus. The identity-subgroup exhibits a specific tuning towards UV light. These results support the view of the MB as the center of multimodal integration that categorizes sensory input and subsequently channels this information into specific MBON populations.
Finally, I discuss differences between the peripheral representations of stimuli and their distinct processing in high-order neuropils. The unique activity of farnesol in manuscript 1 or the representation of UV light in manuscript 2 suggest that the peripheral representation of a stimulus is insufficient as a sole indicator for its neural activity in subsequent neuropils or its putative behavioral importance. In addition, I discuss the influence of hard-wired concepts or plasticity induced changes in the sensory pathways on the processing of such key stimuli in the peripheral reception as well as in high-order centers like the AL or the MB. The MB as the center of multisensory integration has been broadly examined for its olfactory processing capabilities and receives increasing interest about its visual coding properties. To further unravel its role of sensory integration and to include neglected modalities, future studies need to combine additional approaches and gain more insights on the multimodal aspects in both the input and output region.
T-Zell-aktivierende Formate, wie BiTE (bispecific T-cell engagers) Antikörper und CAR T Zellen haben in den vergangen Jahren die Therapiemöglichkeiten für Tumorpatienten erweitert. Diese Therapeutika verknüpfen T-Zellen mit malignen Zellen über je ein spezifisches Oberflächenmolekül und initiieren, über eine T-Zell-vermittelte Immunantwort, die Lyse der Tumorzelle. Tumorspezifische Antigene sind jedoch selten. Häufig werden Proteine adressiert, die neben den Tumorzellen auch auf gesunden Zellen exprimiert werden. Die Folgen sind toxische Effekte abseits der Tumorzellen auf Antigen-positiven gesunden Zellen (on target/off tumor), welche nicht nur die Dosis des Therapeutikums und dessen Effektivität limitieren, sondern zu geringen bis letalen Begleiterscheinungen führen können. Der Bedarf an effektiven Therapieformen mit geringen Nebenwirkungen ist folglich immer noch sehr hoch. Diese Lücke soll durch ein neues Antikörperformat, sogenannten Hemibodies, geschlossen werden. Hemibodies sind eine neue Klasse von T-Zell-aktivierenden Antikörpern, die sich gegen eine Antigenkombination und nicht einzelne Antigene auf Tumorzellen richten. Sie bestehen aus zwei komplementären Molekülen mit je einer Antigen-bindenden Sequenz, die entweder mit der leichten (VL) oder der schweren (VH) Kette eines T-Zell-aktivierenden anti CD3 Antikörpers fusioniert ist. Nur wenn beide Hemibody-Fragmente gleichzeitig in unmittelbarer Nähe an ihr jeweiliges Antigenepitop auf der Tumorzelle binden, komplementieren die beiden Antikörperkonstrukte über das geteilte anti-CD3 und bilden einen trivalenten T Zell aktivierenden Komplex aus. Diese funktionale Einheit rekrutiert T-Zellen zur Tumorzelle und induzierte die T-Zell-vermittelte Lyse der malignen Zelle.
Im Rahmen der vorliegenden Arbeit wurden geeignete Antigenkombinationen identifiziert und die erste effektive und spezifische Hemibody-basierte Immuntherapie gegen das Multiple Myelom (MM), ohne Nebenwirkungen auf Antigen-einfach-positiven gesunden Zellen, entwickelt. Basierend auf einer umfangreichen Analyse von Kandidaten-Antigenen wurden Kombinationen aus bekannten MM Zielmolekülen, wie BCMA, CD38, CD138, CD229 und SLAMF7, und für das MM unbekannte Oberflächenmolekülen, wie CHRM5 und LAX1, untersucht. Gegen die vielversprechendsten Antigene wurden Hemibodies entwickelt und produziert. Im Zusammenhang mit Analysen zur Produzierbarkeit sowie biochemischen und funktionalen Charakterisierungen, konnte aus 75 initialen Hemibody-Kombinationen drei Kombinationen mit geeigneten Eigenschaften identifiziert werden. Die Bindung von zwei Hemibody-Partnern auf der Oberfläche der MM Zelle führte zur Ausbildung eines trivalenten T-Zell-rekrutierenden Komplexes. Dieser initiierte nachfolgend über eine T-Zell-vermittelte Immunantwort die spezifische Lyse der malignen Zellen, ohne die Viabilität von Antigen-einfach-positiven gesunden Körper- oder Effektor-Zellen zu beeinflussen. Zusätzlich führte eine Hemibody-Therapie in vivo in einem NOD SCID MM-Mausmodel innerhalb von 7 Tagen zur kompletten Remission der MM Zellen. Diese Daten zeigten Hemibodies als ein neues, sehr vielversprechendes Antikörperformat für eine effektive und tumorspezifische Immuntherapie mit potentiell geringen Nebenwirkungen.
Formation of the Aurora-A–MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A–MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A–MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders.
Chapter I – Introduction
Global trade of beans of the cacao tree (Theobroma cacao), of which chocolate is produced, contributes to the livelihoods of millions of smallholder farmers. The understorey tree is native to South America but is nowadays cultivated in many tropical regions. In Peru, a South American country with a particularly high cacao diversity, it is common to find the tree cultivated alongside non-crop trees that provide shade, in so-called agroforestry systems. Because of the small scale and low management intensity of such systems, agroforestry is one of the most wildlife-friendly land-use types, harbouring the potential for species conservation. Studying wildlife-friendly land-use is of special importance for species conservation in biodiversity-rich tropical regions such as Peru, where agricultural expansion and intensification are threatening biodiversity. Moreover, there is a growing body of evidence that shows co-occurrence of high biodiversity levels and high yield in wildlife-friendly cacao farming. Yet studies are restricted to non-native cacao countries, and since patterns might be different among continents, it is important to improve knowledge on wildlife-friendly agroforestry in native countries.
Because studies of wildlife-friendly cultivation processes are still largely lacking for South America, we set out to study multiple aspects of cacao productivity in agroforests in Peru, part of cacao´s region of origin. The natural pollination process of cacao, which is critically understudied, was investigated by trapping flower visitors and studying pollen deposition from macrophotographs (Chapter II). Next, we excluded birds, bats, ants and flying insects and squirrels from cacao trees in a full-factorial field experiment and quantified these animals´ contribution to cacao fruit set, fruit loss and yield (Chapter III). Lastly, we aimed to assess whether fruit quantity and quality of native cacao increases through manually supplementing pollen (Chapter II and IV), and whether microclimatic conditions and the genetic background of the studied varieties limit fruit set (Chapter IV).
Chapter II – Cacao flower visitation: Low pollen deposition, low fruit set and dominance of herbivores
Given the importance of cacao pollination for the global chocolate production, it is remarkable that fruit set limitations are still understudied. Knowledge on flower visitation and the effect of landscape context and local management are lacking, especially in the crop’s region of origin. Moreover, the role of pollen deposition in limiting fruit set as well as the benefits of hand pollination in native cacao are unknown. In this chapter, we aimed to close the current knowledge gaps on cacao pollination biology and sampled flower visitors in 20 Peruvian agroforests with native cacao, along gradients of shade cover and forest distance. We also assessed pollen quantities and compared fruit set between manually and naturally pollinated flowers. We found that herbivores were the most abundant flower visitors in both northern and southern Peru, but we could not conclude which insects are effective cacao pollinators. Fruit set was remarkably low (2%) but improved to 7% due to pollen supplementation. Other factors such as a lack of effective pollinators, genetic pollen incompatibility or resource unavailability could be causing fruit set limitations. We conclude that revealing those causes and the effective pollinators of cacao will be key to improve pollination services in cacao.
Chapter III – Quantifying services and disservices provided by insects and vertebrates in cacao agroforestry landscapes
Pollination and pest control, two ecosystem services that support cacao yield, are provided by insects and vertebrates. However, animals also generate disservices, and their combined contribution is still unclear. Therefore, we excluded flying insects, ants, birds and bats, and as a side effect also squirrels from cacao trees and we assessed fruit set, fruit loss and final yield. Local management and landscape context can influence animal occurrence in cacao agroforestry landscapes; therefore, shade cover and forest distance were included in the analyses. Flying insects benefitted cacao fruit set, with largest gains in agroforests with intermediate shade cover. Birds and bats were also associated with improved fruit set rates and with a 114% increase in yield, potentially due to pest control services provided by these animals. The role of ants was complicated: these insects had a positive effect on yield, but only close to forest. We also evidenced disservices generated by ants and squirrels, causing 7% and 10% of harvest loss, respectively. Even though the benefits provided by animals outweighed the disservices, trade-offs between services and disservices still should be integrated in cacao agroforestry management.
Chapter IV – Cross-pollination improves fruit set and yield quality of Peruvian native cacao
Because yields of the cacao tree are restricted by pollination, hand pollination has been proposed to improve yield quantity and potentially, also quality. However, low self- and cross-compatibility of native cacao, and abiotic conditions could cancel out hand pollination benefits. Yet, the impact of genetic constraints and abiotic conditions on fruit set have not been assessed in native cacao so far. To increase our understanding of the factors that limit fruit set in native cacao, we compared manual self- and cross-pollination with five native genotypes selected for their sensorial quality and simultaneously tested for effects of soil water content, temperature, and relative air humidity. We also compared quality traits between manually and naturally pollinated fruits. Success rates of self-pollination were low (0.5%), but increased three- to eightfold due to cross-pollination, depending on the genotype of the pollen donor. Fruit set was also affected by the interaction between relative air humidity and temperature, and we found heavier and more premium seeds in fruits resulting from manual than natural pollination. Together, these findings show that reproductive traits of native cacao are constrained by genetic compatibility and abiotic conditions. We argue that because of the high costs of hand pollination, natural cross-pollination with native pollen donors should be promoted so that quality improvements can result in optimal economic gains for smallholder farmers.
Chapter V – Discussion
In this thesis, we demonstrated that the presence of flying insects, ants and vertebrates, local and landscape management practices, and pollen supplementation interactively affected cacao yield, at different stages of the development from flower to fruit. First, we showed that fruit set improved by intermediate shade levels and flower visitation by flying insects. Because the effective cacao pollinators remain unknown, we recommend shade cover management to safeguard fruit set rates. The importance of integrating trade-offs in wildlife-friendly management was highlighted by lower harvest losses due to ants and squirrels than the yield benefits provided by birds and bats. The maintenance of forest in the landscape might further promote occurrence of beneficial animals, because in proximity to forest, ants were positively associated with cacao yields. Therefore, an integrated wildlife-friendly farming approach in which shade cover is managed and forest is maintained or restored to optimize ecosystem service provision, while minimizing fruit loss, might benefit yields of native cacao. Finally, manual cross-pollination with native genotypes could be recommended, due to improved yield quantity and quality. However, large costs associated with hand pollination might cancel out these benefits. Instead, we argue that in an integrated management, natural cross-pollination should be promoted by employing compatible genotypes in order to improve yield quantity and quality of native cacao.
In the eusocial insect honeybee (Apis mellifera), many sterile worker bees live together with a reproductive queen in a colony. All tasks of the colony are performed by the workers, undergoing age-dependent division of labor. Beginning as hive bees, they take on tasks inside the hive such as cleaning or the producing of larval food, later developing into foragers. With that, the perception of sweetness plays a crucial role for all honeybees whether they are sitting on the honey stores in the hive or foraging for food. Their ability to sense sweetness is undoubtedly necessary to develop and evaluate food sources. Many of the behavioral decisions in honeybees are based on sugar perception, either on an individual level for ingestion, or for social behavior such as the impulse to collect or process nectar. In this context, honeybees show a complex spectrum of abilities to perceive sweetness on many levels. They are able to perceive at least seven types of sugars and decide to collect them for the colony. Further, they seem to distinguish between these sugars or at least show clear preferences when collecting them. Additionally, the perception of sugar is not rigid in honeybees. For instance, their responsiveness towards sugar changes during the transition from in-hive bees (e.g. nurses) to foraging and is linked to the division of labor. Other direct or immediate factors changing responsiveness to sugars are stress, starvation or underlying factors, such as genotype.
Interestingly, the complexity in their sugar perception is in stark contrast to the fact that honeybees seem to have only three predicted sugar receptors.
In this work, we were able to characterize the three known sugar receptors (AmGr1, AmGr2 and AmGr3) of the honeybee fully and comprehensively in oocytes (Manuscript II, Chapter 3 and Manuscript III, Chapter 4). We could show that AmGr1 is a broad sugar receptor reacting to sucrose, glucose, maltose, melezitose and trehalose (which is the honeybees’ main blood sugar), but not fructose. AmGr2 acts as its co-receptor altering AmGr1’s specificity, AmGr3 is a specific fructose receptor and we proved the heterodimerization of all receptors. With my studies, I was able to reproduce and compare the ligand specificity of the sugar receptors in vivo by generating receptor mutants with CRISPR/Cas9. With this thesis, I was able to define AmGr1 and AmGr3 as the honeybees’ basis receptors already capable to detect all sugars of its known taste spectrum.
In the expression analysis of my doctoral thesis (Manuscript I, Chapter 2) I demonstrated that both basis receptors are expressed in the antennae and the brain of nurse bees and foragers. This thesis assumes that AmGr3 (like the Drosophila homologue) functions as a sensor for fructose, which might be the satiety signal, while AmGr1 can sense trehalose as the main blood sugar in the brain. Both receptors show a reduced expression in the brain of foragers when compared with nurse bees. These results may reflect the higher concentrated diet of nurse bees in the hive. The higher number of receptors in the brain may allow nurse bees to perceive hunger earlier and to consume the food their sitting on. Forager bees have to be more persistent to hunger, when they are foraging, and food is not so accessible. The findings of reduced expression of the fructose receptor AmGr3 in the antennae of nurse bees are congruent with my other result that nurse bees are also less responsive to fructose at the antennae when compared to foragers (Manuscript I, Chapter 2). This is possible, since nurse bees sit more likely on ripe honey which contains not only higher levels of sugars but also monosaccharides (such as fructose), while foragers have to evaluate less-concentrated nectar.
My investigations of the expression of AmGr1 in the antennae of honeybees found no differences between nurse bees and foragers, although foragers are more responsive to the respective sugar sucrose (Manuscript I, Chapter 2). Considering my finding that AmGr2 is the co-receptor of AmGr1, it can be assumed that AmGr1 and the mediated sucrose taste might not be directly controlled by its expression, but indirectly by its co-receptor. My thesis therefore clearly shows that sugar perception is associated with division of labor in honeybees and appears to be directly or indirectly regulated via expression.
The comparison with a characterization study using other bee breeds and thus an alternative protein sequence of AmGr1 shows that co-expression of different AmGr1 versions with AmGr2 alters the sugar response differently. Therefore, this thesis provides first important indications that alternative splicing could also represent an important regulatory mechanism for sugar perception in honeybees.
Further, I found out that the bitter compound quinine lowers the reward quality in learning experiments for honeybees (Manuscript IV, Chapter 5). So far, no bitter receptor has been found in the genome of honeybees and this thesis strongly assumes that bitter substances such as quinine inhibit sugar receptors in honeybees. With this finding, my work includes other molecules as possible regulatory mechanism in the honeybee sugar perception as well. We showed that the inhibitory effect is lower for fructose compared to sucrose. Considering that sugar signals might be processed as differently attractive in honeybees, this thesis concludes that the sugar receptor inhibition via quinine in honeybees might depend on the receptor (or its co-receptor), is concentration-dependent and based on the salience or attractiveness and concentration of the sugar present.
With my thesis, I was able to expand the knowledge on honeybee’s sugar perception and formulate a complex, comprehensive overview. Thereby, I demonstrated the multidimensional mechanism that regulates the sugar receptors and thus the sugar perception of honeybees. With this work, I defined AmGr1 and AmGr3 as the basis of sugar perception and enlarged these components to the co-receptor AmGr2 and the possible splice variants of AmGr1. I further demonstrated how those sugar receptor components function, interact and that they are clearly involved in the division of labor in honeybees. In summary, my thesis describes the mechanisms that enable honeybees to perceive sugar in a complex way, even though they inhere a limited number of sugar receptors. My data strongly suggest that honeybees overall might not only differentiate sugars and their diet by their general sweetness (as expected with only one main sugar receptor). The found sugar receptor mechanisms and their interplay further suggest that honeybees might be able to discriminate directly between monosaccharides and disaccharides or sugar molecules and with that their diet (honey and nectar).
In a modified inflation scenario we replace the “big bang” by a condensation event in an eternal all-compassing big ocean of free qubits in our modified cosmology. Interactions of qubits in the qubit ocean are rare. If they happen, they provide a nucleus for a new universe as the qubits become decoherent and freeze-out into defined bit ensembles. Second, we replace inflation by a crystallization event triggered by the nucleus of interacting qubits to which rapidly more and more qubits attach (like in everyday crystal growth) – the crystal unit cell guarantees same symmetries everywhere. Hence, the textbook inflation scenario to explain the same laws of nature in our domain is replaced by the crystal unit cell of the crystal formed. We give here only the perspective or outline of this modified inflation theory, as the detailed mathematical physics behind this has still to be formulated and described.
Interacting qubits solidify, quantum entropy decreases (but increases in the ocean around). The interacting qubits form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. After that very early events, standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements, but more importantly can explain well by such a type of cosmological crystallization instead of inflation the early creation of large-scale structure of voids and filaments, supercluster formation, galaxy formation, and the dominance of matter: no annihilation of antimatter necessary, rather the unit cell of our crystal universe has a matter handedness avoiding anti-matter.
We prove a triggering of qubit interactions can only be 1,2,4 or 8-dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. Crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness.
The phase space of the crystal agrees with the standard model of the basic four forces for n quanta. It includes all possible ensemble combinations of their quantum states m, a total of n**m states. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients. However, this means that in our four dimensions there is only one bit overlap to neighbor states left (almost solid, only below h dash liquidity left). However, the E8 symmetry of heterotic string theory has six rolled-up, small dimensions which help to keep the qubit crystal together and will never expand.
Finally, we give first energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit decoherence / crystal formation. Scalar fields for color interaction and gravity derive from the permeating qubit-interaction field in the crystal. Hence, vacuum energy gets low inside the qubit crystal. Condensed mathematics may advantageously help to model free (many states denote the same qubit) and bound qubits in phase space.
Salt stress is a major abiotic stress, responsible for declining agricultural productivity. Roots are regarded as hubs for salt detoxification, however, leaf salt concentrations may exceed those of roots. How mature leaves manage acute sodium chloride (NaCl) stress is mostly unknown.
To analyze the mechanisms for NaCl redistribution in leaves, salt was infiltrated into intact tobacco leaves. It initiated pronounced osmotically‐driven leaf movements. Leaf downward movement caused by hydro‐passive turgor loss reached a maximum within 2 h.
Salt‐driven cellular water release was accompanied by a transient change in membrane depolarization but not an increase in cytosolic calcium ion (Ca\(^{2+}\)) level. Nonetheless, only half an hour later, the leaves had completely regained turgor. This recovery phase was characterized by an increase in mesophyll cell plasma membrane hydrogen ion (H\(^{+}\)) pumping, a salt uptake‐dependent cytosolic alkalization, and a return of the apoplast osmolality to pre‐stress levels. Although, transcript numbers of abscisic acid‐ and Salt Overly Sensitive pathway elements remained unchanged, salt adaptation depended on the vacuolar H\(^{+}\)/Na\(^{+}\)‐exchanger NHX1.
Altogether, tobacco leaves can detoxify sodium ions (Na\(^{+}\)) rapidly even under massive salt loads, based on pre‐established posttranslational settings and NHX1 cation/H+ antiport activity. Unlike roots, signaling and processing of salt stress in tobacco leaves does not depend on Ca\(^{2+}\) signaling.
The phylogeny of Euglenophyceae (Euglenozoa, Euglenida) has been discussed for decades with new genera being described in the last few years. In this study, we reconstruct a phylogeny using 18S rDNA sequence and structural data simultaneously. Using homology modeling, individual secondary structures were predicted. Sequence–structure data are encoded and automatically aligned. Here, we present a sequence–structure neighbor‐joining tree of more than 300 taxa classified as Euglenophyceae. Profile neighbor‐joining was used to resolve the basal branching pattern. Neighbor‐joining, maximum parsimony, and maximum likelihood analyses were performed using sequence–structure information for manually chosen subsets. All analyses supported the monophyly of Eutreptiella, Discoplastis, Lepocinclis, Strombomonas, Cryptoglena, Monomorphina, Euglenaria, and Colacium. Well‐supported topologies were generally consistent with previous studies using a combined dataset of genetic markers. Our study supports the simultaneous use of sequence and structural data to reconstruct more accurate and robust trees. The average bootstrap value is significantly higher than the average bootstrap value obtained from sequence‐only analyses, which is promising for resolving relationships between more closely related taxa.
Earlier flowering of winter oilseed rape compensates for higher pest pressure in warmer climates
(2023)
Global warming can increase insect pest pressure by enhancing reproductive rates. Whether this translates into yield losses depends on phenological synchronisation of pests with their host plants and natural enemies. Simultaneously, landscape composition may mitigate climate effects by shaping the resource availability for pests and their antagonists. Here, we study the combined effects of temperature and landscape composition on pest abundances, larval parasitism, crop damage and yield, while also considering crop phenology, to identify strategies for sustainable management of oilseed rape (OSR) pests under warming climates.
In all, 29 winter OSR crop fields were investigated in different climates (defined by multi‐annual mean temperature, MAT) and landscape contexts in Bavaria, Germany. We measured abundances of adult pollen beetles and stem weevil larvae, pollen beetle larval parasitism, bud loss, stem damage and seed yield, and calculated the flowering date from growth stage observations. Landscape parameters (proportion of non‐crop and OSR area, change in OSR area relative to the previous year) were calculated at six spatial scales (0.6–5 km).
Pollen beetle abundance increased with MAT but to different degrees depending on the landscape context, that is, increased less strongly when OSR proportions were high (1‐km scale), interannually constant (5‐km scale) or both. In contrast, stem weevil abundance and stem damage did not respond to landscape composition nor MAT. Pollen beetle larval parasitism was overall low, but occasionally exceeded 30% under both low and high MAT and with reduced OSR area (0.6‐km scale).
Despite high pollen beetle abundance in warm climates, yields were high when OSR flowered early. Thereby, higher temperatures favoured early flowering. Only among late‐flowering OSR crop fields yield was higher in cooler than warmer climates. Bud loss responded analogously. Landscape composition did not substantially affect bud loss and yield.
Synthesis and applications: Earlier flowering of winter OSR compensates for higher pollen beetle abundance in warmer climates, while interannual continuity of OSR area prevents high pollen beetle abundance in the first place. Thus, regional coordination of crop rotation and crop management promoting early flowering may contribute to sustainable pest management in OSR under current and future climatic conditions.
Cellular growth and proliferation are among the most important processes for cells and
organisms. One of the major determinants of these processes is the amount of proteins
and consequently also the amount of ribosomes. Their synthesis involves several hundred
proteins and four different ribosomal RNA species, is highly coordinated and very
energy-demanding. However, the molecular mechanims of transcriptional regulation of
the protein-coding genes involved, is only poorly understood in mammals.
In this thesis, unbiased genome-wide knockout reporter screens were performed, aiming
to identify previously unknown transcriptional regulators of ribosome biogenesis
factors (RiBis), which are important for the assembly and maturation of ribosomes,
and ribosomal proteins (RPs), which are ribosomal components themself. With that
approach and follow-up (validation) experiments, ALDOA and RBM8A among others,
could be identified as regulators of ribosome biogenesis.
Depletion of the glycolytic enzyme ALDOA led to a downregulation of RiBi- and RPpromoter
driven reporters on protein and transcript level, as well as to a downregulation
of ribosome biogenesis gene transcripts and of mRNAs of other genes important for
proliferation.
Reducing the amount of the exon junction complex protein RBM8A, led to a more prominent
downregulation of one of the fluorescent reporters, but this regulation was independent
of the promoter driving the expression of the reporter. However, acute protein
depletion experiments in combination with nascent RNA sequencing (4sU-Seq)
revealed, that mainly cytosolic ribosomal proteins (CRPs) were downregulated upon
acute RBM8A withdrawal. ChIP experiments showed RBM8A binding to promoters of
RP genes, but also to other chromatin regions. Total POL II or elongating and initiating
POL II levels were not altered upon acute RBM8A depletion.
These data provide a starting point for further research on the mechanisms of transcriptional
regulation of RP and RiBi genes in mammals.
Despite sometimes strong codependencies of insect herbivores and plants, the responses of individual taxa to accelerating climate change are typically studied in isolation. For this reason, biotic interactions that potentially limit species in tracking their preferred climatic niches are ignored. Here, we chose butterflies as a prominent representative of herbivorous insects to investigate the impacts of temperature changes and their larval host plant distributions along a 1.4‐km elevational gradient in the German Alps. Following a sampling protocol of 2009, we revisited 33 grassland plots in 2019 over an entire growing season. We quantified changes in butterfly abundance and richness by repeated transect walks on each plot and disentangled the direct and indirect effects of locally assessed temperature, site management, and larval and adult food resource availability on these patterns. Additionally, we determined elevational range shifts of butterflies and host plants at both the community and species level. Comparing the two sampled years (2009 and 2019), we found a severe decline in butterfly abundance and a clear upward shift of butterflies along the elevational gradient. We detected shifts in the peak of species richness, community composition, and at the species level, whereby mountainous species shifted particularly strongly. In contrast, host plants showed barely any change, neither in connection with species richness nor individual species shifts. Further, temperature and host plant richness were the main drivers of butterfly richness, with change in temperature best explaining the change in richness over time. We concluded that host plants were not yet hindering butterfly species and communities from shifting upwards. However, the mismatch between butterfly and host plant shifts might become a problem for this very close plant–herbivore relationship, especially toward higher elevations, if butterflies fail to adapt to new host plants. Further, our results support the value of conserving traditional extensive pasture use as a promoter of host plant and, hence, butterfly richness.
For all animals the cold represents a dreadful danger. In the event of severe heat loss, animals
fall into a chill coma. If this state persists, it is inevitably followed by death. In poikilotherms
(e.g. insects), the optimal temperature range is narrow compared to homeotherms
(e.g. mammals), resulting in a critical core temperature being reached more quickly. As a
consequence, poikilotherms either had to develop survival strategies, migrate or die. Unlike
the majority of insects, the Western honeybee (Apis mellifera) is able to organize itself into
a superorganism. In this process, worker bees warm and cool the colony by coordinated
use of their flight muscles. This enables precise control of the core temperature in the hive,
analogous to the core body temperature in homeothermic animals. However, to survive the
harsh temperatures in the northern hemisphere, the thermogenic mechanism of honeybees
must be in constant readiness. This mechanism is called shivering thermogenesis, in which
honeybees generate heat using their flight muscles.
My thesis presents the molecular and neurochemical background underlying shivering thermogenesis
in worker honeybees. In this context, I investigated biogenic amine signaling.
I found that the depletion of vesicular monoamines impairs thermogenesis, resulting in
a decrease in thoracic temperature. Subsequent investigations involving various biogenic
amines showed that octopamine can reverse this effect. This clearly indicates the involvement
of the octopaminergic system. Proceeding from these results, the next step was to elucidate
the honeybee thoracic octopaminergic system. This required a multidisciplinary approach to
ultimately provide profound insights into the function and action of octopamine at the flight
muscles. This led to the identification of octopaminergic flight muscle controlling neurons,
which presumably transport octopamine to the flight muscle release sites. These neurons
most likely innervate octopamine β receptors and their activation may stimulate intracellular
glycolytic pathways, which ensure sufficient energy supply to the muscles.
Next, I examined the response of the thoracic octopaminergic system to cold stress conditions.
I found that the thoracic octopaminergic system tends towards an equilibrium,
even though the initial stress response leads to fluctuations of octopamine signaling. My
results indicate the importance of the neuro-muscular octopaminergic system and thus the need for its robustness. Moreover, cold sensitivity was observed for the expression of one
transcript of the octopamine receptor gene AmOARβ2. Furthermore, I found that honeybees
without colony context show a physiological disruption within the octopaminergic system.
This disruption has profound effects on the honeybees protection against the cold.
I could show how important the neuro-muscular octopaminergic system is for thermogenesis
in honeybees. In this context, the previously unknown neurochemical modulation of the
honeybee thorax has now been revealed. I also provide a broad basis to conduct further
experiments regarding honeybee thermogenesis and muscle physiology.
The fusion of methods from several disciplines is a crucial component of scientific development. Artificial Neural Networks, based on the principle of biological neuronal networks, demonstrate how nature provides the best templates for technological advancement. These innovations can then be employed to solve the remaining mysteries of biology, including, in particular, processes that take place on microscopic scales and can only be studied with sophisticated techniques. For instance, direct Stochastic Optical Reconstruction Microscopy combines tools from chemistry, physics, and computer science to visualize biological processes at the molecular level. One of the key components is the computer-aided reconstruction of super-resolved images. Improving the corresponding algorithms increases the quality of the generated data, providing further insights into our biology. It is important, however, to ensure that the heavily processed images are still a reflection of reality and do not originate in random artefacts.
Expansion microscopy is expanding the sample by embedding it in a swellable hydrogel. The method can be combined with other super-resolution techniques to gain additional resolution. We tested this approach on microtubules, a well-known filamentous reference structure, to evaluate the performance of different protocols and labelling techniques.
We developed LineProfiler an objective tool for data collection. Instead of collecting perpendicular profiles in small areas, the software gathers line profiles from filamentous structures of the entire image. This improves data quantity, quality and prevents a biased choice of the evaluated regions. On the basis of the collected data, we deployed theoretical models of the expected intensity distribution across the filaments. This led to the conclusion that post-expansion labelling significantly reduces the labelling error and thus, improves the data quality. The software was further used to determine the expansion factor and arrangement of synaptonemal complex data.
Automated Simple Elastix uses state-of-the-art image alignment to compare pre- and post-expansion images. It corrects linear distortions occurring under isotropic expansion, calculates a structural expansion factor and highlights structural mismatches in a distortion map. We used the software to evaluate expanded fungi and NK cells. We found that the expansion factor differs for the two structures and is lower than the overall expansion of the hydrogel.
Assessing the fluorescence lifetime of emitters used for direct Stochastic Optical Reconstruction Microscopy can reveal additional information about the molecular environment or distinguish dyes emitting with a similar wavelength. The corresponding measurements require a confocal scanning of the sample in combination with the fluorescent switching of the underlying emitters. This leads to non-linear, interrupted Point Spread Functions. The software ReCSAI targets this problem by combining the classical algorithm of compressed sensing with modern methods of artificial intelligence. We evaluated several different approaches to combine these components and found, that unrolling compressed sensing into the network architecture yields the best performance in terms of reconstruction speed and accuracy.
In addition to a deep insight into the functioning and learning of artificial intelligence in combination with classical algorithms, we were able to reconstruct the described non-linearities with significantly improved resolution, in comparison to other state-of-the-art architectures.