Refine
Has Fulltext
- yes (218) (remove)
Is part of the Bibliography
- yes (218)
Year of publication
- 2023 (218) (remove)
Document Type
- Doctoral Thesis (218) (remove)
Language
- English (218) (remove)
Keywords
- Thrombozyt (6)
- Biene (5)
- Tissue Engineering (5)
- Topologischer Isolator (5)
- Angststörung (4)
- Funktionelle Kernspintomografie (4)
- Maschinelles Lernen (4)
- Mikroskopie (4)
- Zellzyklus (4)
- ARPES (3)
Institute
- Graduate School of Life Sciences (102)
- Theodor-Boveri-Institut für Biowissenschaften (21)
- Institut für Pharmazie und Lebensmittelchemie (14)
- Physikalisches Institut (11)
- Institut für Psychologie (10)
- Institut für Organische Chemie (9)
- Julius-von-Sachs-Institut für Biowissenschaften (9)
- Rudolf-Virchow-Zentrum (9)
- Institut für Theoretische Physik und Astrophysik (8)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (8)
Sonstige beteiligte Institutionen
- Helmholtz Institute for RNA-based Infection Research (HIRI) (2)
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg (2)
- Anthropology Department University of Tennessee, Knoxville (1)
- Carl-Ludwig-Institut für Physiologie, Universität Leipzig (1)
- Chair of Experimental Biomedicine I (1)
- Deutsches Zentrum für Luft- und Raumfahrt (DLR) (1)
- EMBL Heidelberg (1)
- Evangelisches Studienwerk e.V. (1)
- Fachgebiet für Populationsgenomik bei Nutztieren, Universität Hohenheim (1)
- Forschungsverbund ForChange des Bayrischen Kultusministeriums (1)
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.
Respiratory infections are a significant health concern worldwide, and the airway epithelium plays a crucial role in regulating airway function and modulating inflammatory processes. However, most studies on respiratory infections have used cell lines or animal models, which may not accurately reflect native physiological conditions, especially regarding human pathogens. We generated human nasal mucosa (hNM) and tracheobronchial mucosa (hTM) models to address this issue using primary human airway epithelial cells and fibroblasts. We characterised these human airway tissue models (hAM) using high speed video microscopy, single cell RNA sequencing, immunofluorescence staining,
and ultrastructural analyses that revealed their complexity and cellular heterogeneity. We demonstrated that Bordetella pertussis virulence factor adenylate cyclase toxin (CyaA) elevated the intracellular production of cyclic adenosine monophosphate (cAMP) and secretion of interleukin (IL) 6, IL 8, and human beta defensin 2 (HBD2). In addition, we compared the responses of the tissue models from two different anatomical sites (the upper and lower respiratory mucosa) and are the first to report such differential susceptibility towards CyaA using 3D primary airway cell derivedmodels. The effect of toxin treatment on the epithelial barrier integrity of the tissue models was assessed by measuring the flux of fluorescein isothiocyanate (FITC)-conjugated dextran across the models. Though we observed a cell type specific response with respect to intracellular cAMP production and IL 6, IL 8, and HBD2 secretion in the models treated with CyaA on the apical side, the epithelial membrane barrier integrity was not compromised. In addition to toxin studies, using these characterised models, we established viral infection studies for Influenza A (IAV), Respiratory Syncytial Virus subtype B (RSV), and severe acute respiratory syndrome coronavirus 2. We visualised the morphological consequences of the viral infection using ultrastructural analysis
and immunofluorescence. We verified the effective infection in hAM by measuring the viral RNA using RTqPCR and detected elevated cytokine levels in response to infection using biochemical assays. In contrast to cell lines, studies on viral infection using hAM demonstrated that infected areas were localized to specific regions. This led to the formation of infection hotspots, which were more likely to occur when models derived from different donors were infected separately with all three viruses. IAV infected tissue models replicate the clinical findings of H1N1 infection, such as mucus
hypersecretion, cytokine release, and infection-associated epithelial cell damage.Finally, we paved the steps towards understanding the impact of IAV infection on disease models. We generated hTM from biopsies obtained from chronic obstructive pulmonary disease (COPD) patients. As a model to study the impact of COPD on respiratory infections, considering the increase in COPD cases in the past decade and the continued predicted increase in the future. We established the IAV infection
protocol to capture the early infection signatures in non-COPD and COPD conditions using scRNA-seq. We investigated the infection kinetics of IAV (H1N1-clinical isolate) in hTM and found that viruses were actively released approximately 24 hours post infection. The scRNA-seq data from the hTM derived from non-COPD and COPD patients, revealed lower levels of SCGB1A1 (club cell marker) gene expression in the COPD-control group compared to the non-COPD control group, consistent with previous clinical studies. Furthermore, we observed that IAV infection elevated SCGB1A1 gene expression especially in secretory cells of both the COPD and non COPD groups. This may imply the role of club cells as early responders during IAV infection providing epithelial repair, regeneration, and resistance to spread of infection. This is the first study to address the molecular diversity in COPD and non-COPD disease models infected with IAV investigating the early response (6 h) of specific cell types in the human lower airways towards infection using scRNA-seq. These findings
highlight the potential interplay between COPD, IAV infection, and altered vulnerability to other viral infections and respiratory illnesses making the hAM applicable for addressing more specific research questions and validating potential targets, such as SCGB1A1 targeted therapy for chronic lung diseases. Our findings demonstrate the potential of the hNM and hTM for investigating respiratory infections, innate immune responses, and trained immunity in non-immune cells. Our experiments show that hAM may represent a more accurate representation of the native physiological condition and improve our understanding of the disease mechanisms. Furthermore, these models promote non-animal research as they replicate clinical findings. We can further increase their complexity by incorporating dynamic flow systems and immune cells catered to the research question.
Motivated by the perceived great potential of chiral polymers, the presented work aimed at the investigation of synthesis, solubility and optical activity of chiral poly(2,4-disubstituted-2-oxazoline)s. A novel polymeric carrier based on ABA-type triblock copolymers poly(2-oxazoline)s with chiral and racemic hydrophobic blocks was developed for the formulation of chiral and achiral drugs (Fig. 5.1). Poly(2-methyl-2-oxazoline) (pMeOx) was used as hydrophilic A block, and poly(2-ethyl-4-ethyl-2-oxazoline) (pEtEtOx) and poly(2-propyl-4-methyl-2-oxazoline) (pPrMeOx) were used as hydrophobic B blocks. Curcumin (CUR), paclitaxel (PTX) and chiral/racemic ibuprofen (R/S/RS-IBU) were applied as model drugs. Nanoformulations were prepared consisting of these triblock copolymers and model drugs. ...
This dissertation focuses on Mip (macrophage infectivity potentiator protein) inhibitors in response to increasing antibiotic resistance. The study follows an antivirulence approach, which aims to inhibit the non-essential Mip protein without exerting too much selective pressure. Three focus areas were (1) development and synthesis of a fluorescent probe for screening Mip inhibitors via fluorescence polarization; (2) design and synthesis of broad spectrum Mip inhibitors bearing a side chain; and (3) understanding the metabolism of Mip inhibitors and identification of active metabolites.
A sub-study addressed the biotinylation of anti-leishmanial compounds from Valeriana wallichii rhizomes, with three tracer molecules synthesized for future pull-down experiments.
RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease “Neurodevelopmental Disorder with severe Motor Impairment and Absent Language” (NEDMIAL;OMIM#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3’ → 5’ RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome’s mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5’TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.
Additive manufacturing processes such as 3D printing are booming in the industry due to their high degree of freedom in terms of geometric shapes and available materials. Focusing on patient-specific medicine, 3D printing has also proven useful in the Life Sciences, where it exploits the shape fidelity for individualized tissues in the field of bioprinting. In parallel, the current systems of bioreactor technology have adapted to the new manufacturing technology as well and 3D-printed bioreactors are increasingly being developed. For the first time, this work combines the manufacturing of the tissue and a tailored bioreactor, significantly streamlining the overall process and optimally merging the two processes. This way the production of the tissues can be individualized by customizing the reactor to the tissue and the patient-specific wound geometry. For this reason, a common basis and guideline for the cross-device and cross-material use of 3D printers was created initially. Their applicability was demonstrated by the iterative development of a perfusable bioreactor system, made from polydimethylsiloxane (PDMS) and a lignin-based filament, into which a biological tissue of flexible shape can be bioprinted. Cost-effective bioink-replacements and in silico computational fluid dynamics simulations were used for material sustainability and shape development. Also, nutrient distribution and shear stress could be predicted in this way pre-experimentally.
As a proof of functionality and adaptability of the reactor, tissues made from a nanocellulose-based Cellink® Bioink, as well as an alginate-based ink mixed with Me-PMeOx100-b-PnPrOzi100-EIP (POx) (Alginate-POx bioink) were successfully cultured dynamically in the bioreactor together with C2C12 cell line. Tissue maturation was further demonstrated using hMSC which were successfully induced to adipocyte differentiation. For further standardization, a mobile electrical device for automated media exchange was developed, improving handling in the laboratory and thus reduces the probability of contamination.
In all the projects presented, it is evident that the selection of suitable separation conditions is only one side of the coin. Equally crucial in the development of methods for the quality assessment of APIs/drugs is the right detection system.
The application of CAD as an alternative to UV detection at low wavelength of the two weak chromophore main degradation products of the very polar, zwitterionic API carbocisteine requires the volatility of the mobile phase. Therefore, as a substitute for the non-volatile ion pairing reagent tetrabutylammonium hydroxide (TBAOH), six different volatile alkylamines as well as a RP/SAX mixed-mode column were evaluated. The best selectivity and separation performance comparable to TBAOH was achieved with the RP/SAX column and a mixture of formic acid and trifluoroacetic acid. For the simultaneous optimisation of the evaporation temperature of the CAD as a function of two chromatographic parameters, a central composite design was chosen and the “desirability function” was subsequently applied for modelling. In addition, column bleeding was investigated with a second RP/SAX column (different batch) with the result that the acetonitrile percentage had to be adjusted and preconditioning by injection of concentrated samples is essential. The final mixed-mode method was finally validated with both columns according to the ICH Q2 (R1) guideline.
Based on this, an MS-compatible method was developed with little effort using an identical RP/SAX column in UPLC dimension for the untargeted analysis by HRMS of two carbocisteine-containing prototype syrup formulations. For a comprehensive characterisation, HRMS and MS/HRMS data were recorded simultaneously by information dependent acquisition mode. Based on the exact masses, isotope patterns and an in silico plausibility check of the fragment spectra, the prediction of the structures of the unknown impurities was possible. In both syrup samples, which had been stored for nine months at 40 °C and 75 % r.h., two additional impurities of carbocisteine (i.e. lactam of the sulfoxides and disulphide between cysteine and thioglycolic acid) were identified by comparison with the corresponding prototype placebo samples using general unknown comparative screening. In addition, the formation of Maillard products by binary mixtures with 13C-labelled sugars was revealed in the sucrose-containing formulation.
For the promising hyphenation of the UV detector with the CAD for the simultaneous detection of all UV-active impurities of the cholesterol-lowering drug simvastatin and the only weak chromophore dihydrosimvastatin, the Ph. Eur. method had to be adapted. Besides replacing phosphoric acid with trifluoroacetic acid, the gradient also had to be adjusted and a third critical peak pair was observed. Based on validation experiments (according to the ICH Q2 (R1) guideline), the suitability of the CAD for sensitive detection (LOQ = 0.0175 % m/m) was proven.
To further investigate the robustness of the adapted method and CAD, a Plackett-Burman design was chosen. None of the factors had a statistically significant effect on the S/N of the CAD in the ranges tested. Regarding the three critical peak pairs, on the other hand, the factors to be controlled were statistically established, so that a targeted correction is possible if the system suitability test is not passed. The idea of employing a hyphenated UV-CAD system was finally applied to the structurally closely related lovastatin and its specified impurity dihydrolovastatin. Here, the CAD showed a significantly better S/N compared to the compendial UV detection at 200 nm.
The suitability of CAD for the analysis of non-volatile fatty acids in polysorbate 80 (PS80) as favourable alternative to the Ph. Eur. GC method (no time-consuming, error-prone and toxic derivatisation) has already been demonstrated. The aim of this project was therefore to develop a robust method with a focus on the AQbD principles, which can be used for the analysis of other excipients with similar fatty acid composition. After the definition of the analytical target profile and a risk assessment by means of an Ishikawa diagram, a suitable C18 column and the chromatographic framework conditions (formic acid concentration and initial/final gradient conditions) were selected after only few preliminary runs. The remaining critical method parameters were then investigated with the help of DoE and RSM. Using the obtained model equations, Monte Carlo simulations were performed to create the method operable design region as a region of theoretical robustness. After validation according to ICH Q2 (R1), the fatty acid composition of a magnesium stearate batch was successfully analysed as a further application example in addition to PS80.
The CAD was able to prove its potential in all the issues investigated in the context of this doctoral thesis. As a cost-effective alternative compared to MS instruments, it thus closes a gap in the quality assessment of APIs or excipients without a suitable chromophore. The easy method transfer to (HR)MS instruments also allows for a unique degree of sample characterisation through untargeted approaches in case of new impurities. For resource- and time-efficient work, the possibilities and limitations of software tools for method development and data evaluation as well as the application of risk-based approaches such as AQbD should also be considered.
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).
This PhD thesis addresses the photophysics of selected small organic molecules with the purpose of using them for efficient and even novel light sources. In particular, the studies presented focused on revealing the underlying exciton dynamics and determining the transition rates between different molecular states. It was shown how the specific properties and mechanisms of light emission in fluorescent molecules, molecules with phosphorescence or thermally activated delayed fluorescence (TADF), biradicals, and multichromophores can be utilized to build novel light-emitting devices. The main tool employed here was the analysis of the emitters’ photon statistics, i.e. the analysis of the temporal distribution of emitted photons, during electrical or optical excitation. In the introduction of this work, the working principle of an organic light-emitting diode (OLED) was introduced, while Chapter 2 provided the physical background of the relevant properties of organic molecules and their interaction with light. In particular, the occurrence of discrete energy levels in organic semiconductors and the process of spontaneous light emission were discussed. Furthermore, in this chapter a mathematical formalism was elaborated with the goal to find out what kind of information about the studied molecule can be obtained by analyzing its photon statistics. It was deduced that the intensity correlation function g (2)(t) contains information about the first two factorial moments of the photon statistics and that higher order factorial moments do not contain any additional information about the system under study if the system is always in the same state after the emission of a photon. To conclude the introductory part, Chapter 3 introduced the utilized characterization methods including confocal microscopy of single molecules, time correlated single photon counting and temperature dependent photoluminescence measurements. To provide the background necessary for an understanding of for the following result chapters, in Section 4.1 a closer look was taken at the phenomenon of blinking and photobleaching of individual molecules. For a squaraine-based fluorescent emitter rapid switching between a bright and dark state was observed during photoexcitation. Using literature transition rates between the molecular states, a consistent model was developed that is able to explain the distribution of the residence times of the molecule in the bright and dark states. In particular, an exponential and a power-law probability distribution was measured for the time the molecule resides in tis bright and dark state, respectively. This behavior as well as the change in photoluminescence intensity between the two states was conclusively explained by diffusion of residual oxygen within the sample, which had been prepared in a nitrogen-filled glovebox. For subsequent samples of this work, thin strips of atomic aluminum were deposited on the matrices to serve as oxygen getter material. This not only suppressed the efficiency of photobleaching, but also noticeably prolonged the time prior to photobleaching, which made many of the following investigations possible in the first place. For emitters used in displays, emission properties such as narrow-band luminescence and short fluorescence lifetimes are desired. These properties can be influenced not only by the emitter molecule itself, but also by the interaction with the chosen environment. Therefore, before focusing on the photophysics of individual small organic molecules, Section 4.2 highlighted the interaction of a perylene bisimide-based molecular species with its local environment in a disordered polymethyl methacrylate matrix. In a statistical approach, individual photophysical properties were measured for 32 single molecules and correlations in the variation of the properties were analyzed. This revealed how the local polarity of the molecules’ environment influences their photophysics. In particular, it was shown how an increase in local polarity leads to a red-shifted emission, narrower emission lines, broader vibronic splitting between different emission lines in combination with a smaller Huang-Rhys parameter, and a longer fluorescence lifetime. In the future, these results may help to embed individual chromophores into larger macromolecules to provide the chromophore with the optimal local environment to exhibit the desired emission properties. The next two sections focused on a novel and promising class of chromophores, namely linear coordinated copper complexes, synthesized in the group of Dr. Andreas Steffen at the Institute of Inorganic Chemistry at the University of Würzburg. In copper atoms, the d-orbitals are fully occupied, which prevents undesirable metal-centered d-d⋆ states, which tend to lie low in energy and recombine non-radiatively. Simultaneously, the copper atom provides a flexible coordination geometry, while complexes in their linear form are expected to exhibit the least amount of excited state distortions. Depending on the chosen ligands, these copper complexes can exhibit phosphorescence as well as temperature activated delayed fluorescence. In Section 4.3, a phosphorescent copper complex with a chlorine atom and a 1-(2,6-diisopropylphenyl)-3,3,5,5-tetramethyl-2-pyrrolidine-ylidene- ligand was tested for its suitability as an optically active material in an OLED. For this purpose, an OLED with a polyspirobifluorene-based copolymer matrix and the dopant at a concentration of 20 wt% was electrically excited. Deconvolution of the emission spectrum in contributions from the matrix and the dopant revealed that 60 % of the OLEDs emission was due to the copper complex. It was also shown that the shape of the emission spectrum of the copper complex remains unchanged upon incorporation into the OLED, but is red-shifted by about 233 meV. In Section 4.4, a second copper complex exhibiting thermally activated delayed fluorescence was analyzed. This complex comprised a carbazolate as well as a 2-(2,6- diisopropyl)-phenyl-1,1-diphenyl-isoindol-2-ium-3-ide ligand and was examined in the solid state and at the single-molecule level, where single photon emission was recorded up to an intensity of 78’000 counts per second. The evaluation of the second-order autocorrelation function of the emitted light proved an efficient transition between singlet and triplet excited states on the picosecond time scale. In the solid state, the temperature- dependent fluorescence decay of the complex was analyzed after pulsed photoexcitation in the temperature range between 300 K and 5 K. From these measurements, a small singlet-triplet energy gap of only 65 meV and a triplet sublevel splitting of 3.0 meV were derived. The transition rates between molecular states could also be determined. Here, the fast singlet decay time of τS1 = 9.8ns proved the efficient thermally activated delayed fluorescence process, which was demonstrated for the first time for this new class of copper(I) complexes thus. While the use of thermally activated delayed fluorescence is a potential way to harness otherwise long-living dark triplet states, radicals completely avoid dark triplet states. However, this usually comes with the huge drawback of the molecules being chemically unstable. Therefore, two chemically stable biradical species were synthesized in the framework of the DFG research training school GRK 2112 on Molecular biradicals: structure, properties and reactivity, by Yohei Hattori in the group of Prof. Dr. Christoph Lambert and Rodger Rausch in the group of Prof. Dr. Frank Würthner at the Institute of Organic Chemistry at the University of Würzburg, respectively. In Section 4.5, it was investigated how these molecules can be used in OLEDs. In the first isoindigo based biradical (6,6’-bis(3,5-di-tert-butyl-4-phenoxyl)-1,1’-bis(2- ethylhexyl)-[3,3’-biindolinyl-idene]-2,2’-dione) two tert-butyl moieties kinetically block chemical reactions at the place of the lone electrons and an electron-withdrawing core shifts the electron density into the center of the chromophore. With these properties, it was possible to realize a poly(p-phenylene vinylene) copolymer based OLED doped with the biradical and to observe luminescence during optical as well as electrical excitation. Analyzing shapes of the photo- and electroluminescence spectra at different doping concentrations, Förster resonance energy transfer was determined to be the dominant transition mechanism for excitons from the matrix to the biradical dopants. Likewise, OLEDs could be realized with the second diphenylmethylpyridine based birad- ical (4-(5-(bis(2,4,6-trichlorophenyl)methyl)-4,6-dichloropyridin-2-yl)-N-(4-(5-(bis(2,4,6- -trichlorophenyl)methyl)-4,6-dichloropyridin-2-yl)phenyl)-N-(4-methoxyphenyl)aniline) as dopant. In this biradical, chlorinated diphenylmethyl groups protect the two unpaired electrons. Photo- and electroluminescence spectra showed an emission in the near in- frared spectral range between 750 nm and 1000 nm. Also, Förster resonance energy trans- fer was the dominant energy transfer mechanism with an transfer efficiency close to 100 % even at doping concentrations of only 5 wt%. In addition to demonstrating the working OLEDs based in biradicals, the detection of luminescence of the two biradical species in devices also constitutes an important step toward making use of experimental techniques such as optically detected electron spin resonance, which could provide information about the electronic states of the emitter and their spin manifold during OLED operation. Another class of emitters studied are molecules in which several chromophores are co- valently linked to form a macrocyclic system. The properties of these multichromophores were highlighted in Section 4.6. Here, it was analyzed how the photophysical behavior of the molecules is affected by the covalent linking, which determines the interaction be- tween the chromophores. The first multichromophore, 2,2’-ditetracene, was synthesized by Lena Ross in the group of Prof. Dr. Anke Krüger at the Institute of Organic Chemistry at the University of Würzburg and was analyzed in this work both at the single-molecule level and in its aggregated crystalline form. While the single crystals were purified and grown in a vertical sublimation oven, the samples for the single molecule studies were prepared in matrices of amorphous polymethyl methacrylate and crystalline anthracene. Tetracene was analyzed concurrently to evaluate the effects of covalent linking. In samples where the distance between two molecules is sufficiently large, tetracene and 2,2’-ditracene show matching emission profiles with the only difference in the Franck-Condon factors and a de- creased photoluminescence decay time constant from 14 ns for tetracene to 5 ns for 2,2’- ditracene, which can be attributed to the increased density of the vibrational modes in 2,2’-ditracene. Evaluation of the photon statistics of individual 2,2’-ditracene molecules however showed that the system does not behave as two individual chromophores but as a collective state, preserving the spectral properties of the two tetracene chromophores. Complementary calculations performed by Marian Deutsch in the group of Prof. Dr. Bernd Engels at the Institute of Physical and Theoretical Chemistry at the University of Würzburg helped to understand the processes in the materials and could show that the electronic and vibronic modes of 2,2’-ditracene are superpositions of the modes occurring in tetracene. In contrast, single-crystalline 2,2’-ditetracene behaves significantly different than tetracene, namely exhibiting a red shift in photoluminescence of 150 meV, caused by an altered crys- talline packing that lowers the S1-state energy level. Temperature-dependent photolu- minescence measurements revealed a rich emission pattern from 2,2’-ditetracene single crystals. The mechanisms behind this were unraveled using photoluminescence lifetime density analysis in different spectral regions of the emission spectrum and at different tem- peratures. An excimer state was identified that is located about 5 meV below the S1-state, separated by a 1 meV barrier, and which can decay to the ground state with a time constant of 9 ns. Also, as the S1-state energy level is lowered below the E(S1) ≥ 2 ×E(T1) threshold, singlet fission is suppressed in 2,2’-ditetracene in contrast to tetracene. Therefore, at low temperatures, photoluminescence is enhanced by a factor of 46, which could make 2,2’- ditetracene a useful material for future applications in devices such as OLEDs or lasers. The second multichromophore species, para-xylylene bridged perylene bisimide macrocycles, were synthesized by Peter Spenst in the group of Prof. Dr. Frank Würthner at the Institute of Organic Chemistry at the University of Würzburg, by linking three and four perylene bisimides, respectively. To reveal the exciton dynamics in these macrocycles, highly diluted monomers as well as trimers and tetramers were doped into matrices of polymethyl methacrylate to create thin films in which individual macrocycles could be analyzed. The emission spectra of the macrocycles remained identical to those of the monomers, indicating weak coupling between the chromophores. Single photon emission could be verified for monomers as well as macrocycles, as exciton-exciton annihilation processes suppress the simultaneous emission of two photons from one macrocycle. Nevertheless, the proof of the occurrence of a doubly excited state was obtained by excitation power dependent photon statistics measurements. The formalism developed in the theory part of this thesis for calculating the photon statistics of multichromophore systems was used here to find a theoretical model that matches the experimental results. The main features of this model are a doubly excited state, fast singlet-singlet annihilation, and an efficient transition from the doubly excited state to a dark triplet state. The occurrence of triplet-triplet annihilation was demonstrated in a subsequent experiment in which the macrocycles were excited at a laser intensity well above the saturation intensity of the monomer species. In contrast to the monomers, the trimers and tetramers exhibited neither a complete dark state nor saturation of photoluminescence. Both processes, efficient singlet-singlet and triplet-triplet annihilation make perylene bisimide macrocycles exceptionally bright single photon emitters. These advantages were utilized to realize a room temperature electrically driven fluorescent single photon source. For this purpose, OLEDs were fabricated using polyvinylcarbazole and 2-tert-butylphenyl-5-biphenyl-1,3,4-oxadiazol blends as a host material for perylene bisimide trimers. Photon antibunching could be observed in both optically and electrically driven devices, representing the first demonstration of electrically driven single photon sources using fluorescent emitters at room temperature. As expected from the previous optical experiments, the electroluminescence of the molecules was exceptionally bright, emitting about 105 photons per second, which could be seen even by eye under the microscope. Finally, in the last section 4.7 of this thesis, two additional measurement schemes were proposed as an alternative to the measurement of the second-order correlation function g (2)(t) of single molecules, which only provides information about the first two factorial moments of the molecules’ photon statistics. In the first scheme, the g (3)(t) function was measured with three photodiodes, which is a consequential extension of the Hanbury Brown and Twiss measurement with two photodiodes. It was demonstrated how measuring the g (3)(t) function is able to identify interfering emitters with non-Poisson statistics in the experiment. The second setup was designed with an electro-optic modulator that repeatedly gen- erates photoexcitation in the form of a step function. The recording of luminescence transients for different excitation intensities yields the same results as the correspond- ing g (2)-functions measured on single emitters, both in their shape and in their depen- dence on excitation power. To demonstrate this concept, the TADF emitter TXO-TPA (2- [4-(diphenylamino)phenyl]-10,10-dioxide-9H-thioxanthen-9-one) was doped at a concen- tration of 10−4 wt% in a mCP (1,3-Bis(N-carbazolyl)benzene) matrix. This concentration was low enough that TXO-TPA molecules did not interact with each other, but an ensem- ble of molecules was still present in the detection volume. The intramolecular transition rates between singlet and triplet states of TXO-TPA could be derived with an error of at most 5 %. Other experimental techniques designed to obtain this information require ei- ther lengthy measurements on single molecules, where sample preparation is also often a challenge, or temperature-dependent fluorescence lifetime measurements, which require a cryostat, which in turn places constraints on the sample design used. In future, this ap- proach could establish a powerful method to study external factors influencing molecular transition rates. Overall, this thesis has introduced new molecular materials, revealed their photophys- ical properties, and demonstrated how they can be used to fabricate efficient and even novel light sources.
In contrast to the well described molecular basis for S-type anion currents, the genes underlying R-type anion currents were unknown until 2010. Meyer S. and colleagues (2010) showed that, localized in the guard cell plasma membrane, AtALMT12 is an R-type anion channel involved in stomatal closure. However, knocking out AtALMT12 did not fully shut down R-type currents; the almt12 loss-of-function mutant has residual R-type-like currents indicating that ALMT12 is not the only gene encoding Arabidopsis thaliana R-type channels (Meyer S. et al., 2010). This PhD thesis is focussed on understanding the properties, regulation and molecular nature of the R-type channels in Arabidopsis thaliana plants. To fulfil these aims, the patch clamp technique was used to characterize electrical features of R-type currents in various conditions such as the presence/absence of ATP, variation in cytosolic calcium concentration or the presence of cytosolic chloride. Electrophysiological study revealed many similarities between the features of Arabidopsis thaliana R-type currents (Col0) and residual R-type currents (the almt12 loss-of-function mutant). Strong voltage dependency, channel activity in the same voltage range, position of maximal recorded current and blockage by cytosolic ATP all pointed to a shared phylogenetic origin of the channels underlying these R-type currents. Expression patterns of the ALMT family members for Col0 and the almt12 mutant revealed ALMT13 and AMT14 as potential candidates of the R-type channels. Electrical characterization of Col0, almt12 and the two double loss-of-function mutants (almt12/almt13 and almt12/almt14) strongly suggest that ALMT13 mediates the calcium-dependent R-type current component that is directly regulated by cytosolic calcium. Additionally, similarly to ALMT12, ALMT14 could participate as a calcium-independent R-type anion channel. Differences in response to the cytosolic calcium concentration between ALMT12, ALMT13 and ALMT14 suggest their possible involvement in different signalling pathways leading to stomatal closure. Moreover, a study performed for the two Arabidopsis thaliana ecotypes Col0 and WS showed drastically increased ALMT13 expression for WS, which is related to R-type current properties. The WS ecotype has calcium-dependent R-type current behaviour, while it is calcium-independent in Col0. Furthermore, this plant line showed lower peak current densities compared to Col0 and almt mutants. These facts strongly suggest interaction between ALMT12 and ALMT13, with ALMT13 as a repressor of the ALMT12. Acquired patch clamp data revealed sulphate-dependent increases in ALMT13 current. This could be caused by changes in absolute open probability and/or permeability for sulphate and possibly chloride and links ALMT13 with sulphate-mediated stomatal closure under drought stress. It was then confirmed that ATP affects R-type currents. In contrast to Vicia faba, ATP was identified as a negative regulator of the Arabidopsis thaliana R-type anion channels. The effect of ATP is ambiguous but there is a high probability that it is a result of direct block and phosphorylation. However, the phosphorylation site and place of ATP binding needs further investigation.
The story of the ALMT family, as examined in this thesis, sheds light on the complexity of the stomatal closure process.
SUMOylation, as a post-translational modification, plays a crucial role in several biological processes. Small ubiquitin-like modifier (SUMO) proteins can be reversibly linked to the lysine residues located within specific motifs on numerous target proteins, leading to the change of stability, localization, activity of target proteins, mostly by promoting or interfering with the interaction with other molecules. Consequently, it can regulate gene transcription, migration, cell cycle progression, cellular responses to stress, and tumorigenesis.
NFATc1 belongs to the Nuclear Factor of Activated T-cells (NFAT) transcription factor family, which is dephosphorylated and translocates to the nucleus upon cell stimulation, which provokes Ca2+ signalling. NFAT plays a crucial role in the development and function of the immune system. NFATc1 has three SUMOylation sites at the position of aa 349, 702, and 914. In our previous study, we demonstrated that point mutations performed on the SUMOylation sites on all three or only at the lysine residues K702 and K914 lead to enhanced expression of IL-2 in vitro. To evaluate the function of SUMOylation of NFATc1 on T cell-mediated immunity in vivo, we not only generated a transgenic mouse strain (NFATc1/ΔS+ mouse) by point mutations from Lysine to Arginine on the two SUMOylation sites within exon 10 of Nfatc1 to prevent their SUMOylation, but in combination created another mouse strain (NFATc1/ΔBC+ mouse) that is completely Nfatc1 exon 10-ablated by using the LoxP/Cre system. In NFATc1/ΔS+ T cells, we observed enhanced IL-2 production and less IL-17A and IFN-γ expression. In line with exon 10 bearing the relevant SUMO sites, NFATc1/ΔBC+ CD4+ T cells behaved similarly as NFATc1/ΔS+ ones. The mechanism is that elevated IL-2 secretion can counteract the expression of IL-17A and IFN-γ via STAT5 and Blimp-1 induction. Afterwards, Blimp-1 suppressed IL-2 itself as well as Bcl2A1. Next, we performed two disease models with our NFATc1/ΔS+ mice. In a major mismatch model for acute graft-versus-host disease, we found that the mice transplanted with NFATc1/ΔS+ CD3+ T cells developed less severe disease, and T cells proliferated less due to increased Tregs. Moreover, when transferring 2D2.NFATc1/ΔS+ Th1 plus Th17 cells to Rag1-/- mice to induce experimental autoimmune encephalitis, we also observed ameliorated disease compared to animals with transferred WT T cells as well as increased Tregs.
Taking all data together, the deficiency in SUMOylation of NFATc1 leads to an elevated IL-2 secretion in T cells and subsequent activation of STAT5, which competes with STAT3 to inhibit IL-17A production and promotes Treg expansion, as well as to an enforcement of Blimp-1 expression, which suppresses IFN-γ and IL-2 expression. Consequently and despite a short phase of enhanced IL-2 secretion, the deficiency of SUMOylation on NFATc1 can protect from autoreactive and alloreactive diseases.
Moreover, to further understand the function of SUMOylation of NFATc1 in humans, we started by establishing an in vitro 3D culture system for tonsil organoids, which was successful in the presence of feeder cells, along with IL-4 and IL-7 cytokines. To confirm that our 3D tonsil organoids can respond to real antigens, we used CMV peptides and peptides of spike proteins from Covid-19 as real antigens, and co-cultured with tonsil organoids, which indeed can generate memory cells and plasmablasts. In the end, we also compared 3D to 2D cultures. Although the total numbers of all B cell subsets were much less in 3D culture than that in 2D culture, still, it indicates that this in-vitro culture system has its limitation, while being usable to produce the similar results as 2D did. Therefore, this 3D culture system can be used as a platform to investigate NFATc1/ΔS+ or NFATc1/ΔBC+ TFH and TFR cells in the dynamic of human GC responses.
Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets.
Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin.
In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear.
Short functional peptidic probes can maximize the potential of high-end microscopy techniques and multiplex imaging assays and provide new insights into normal and aberrant molecular, cellular and tissue function. Particularly, the visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques and relies either on genetic manipulation or on antibodies that underperform in tissue immunofluorescence. Starting from an endogenous activity-related ligand of gephyrin, a universal marker of the inhibitory post-synapse, I developed a short peptidic multivalent binder with exceptional affinity and selectivity to gephyrin. By tailoring fluorophores to the binder, I have obtained Sylite, a probe for the visualization of inhibitory synapses, with an outstanding signal-to-background ratio, that bests the “gold standard” gephyrin antibodies both in selectivity and in tissue immunofluorescence. In tissue Sylite benefits from simplified handling, provides robust synaptic labeling in record-short time and, unlike antibodies, is not affected by staining artefacts. In super-resolution microscopy Sylite precisely localizes the post-synapse and enables accurate pre- to post-synapse measurements. Combined with complimentary tracing techniques Sylite reveals inhibitory connectivity and profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the periaqueductal gray brain region. Lastly, upon probe optimization for live cell application and with the help of novel thiol-reactive cell penetrating peptide I have visualized inhibitory synapses in living neurons. Taken together, my work provided a versatile probe for conventional and super-resolution microscopy and a workflow for the development and application of similar compact functional synthetic probes.
Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research.
Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition.
Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 %, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 %. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments.
Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation.
As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets.
N-heterocyclic carbenes (NHC) are utilized for the stabilization of reactive compounds, for the activation of strong bonds, and as ligands in transition metal chemistry. In contrast to neutral NHCs, few examples of anionic or even dianionic NHCs are known. One approach for the synthesis of anionic carbenes is the deprotonation of neutral or anionic precursors, bearing Lewis acids instead of alkyl or aryl substituents. Following this strategy, novel anionic and dianionic NHCs, featuring weakly coordinating fluorinated borane and phosphorane substituents or coordinating tricyanoborane substituents were synthesized within the scope of this thesis. These carbenes possess unprecedented stabilities compared to related species. Furthermore, their electronic and steric properties can be directly adjusted by the type of Lewis acid attached. Their potential as ligands with highly shielding weakly coordinating substituents next to the carbene coordination center was demonstrated by the syntheses of the respective NHC selenium adducts and NHC gold(I) complexes. In contrast anionic NHCs with coordinating tricyanoborane moieties have an outstanding potential as ditopic ligands with coordination being possible at the carbene center and via the cyano groups. Their beneficial ligand properties were demonstrated by the syntheses of the respective NHC selenium adducts and NHC nickeltricarbonyl complexes. The combination of electronic properties, the large buried volume, the negative charge, the possibility to act as ditopic or ligands with weakly coordinating groups, and the ease of accessibility render borane- and phosphorane functionalized NHCs unique novel ligands. A further project of this PhD thesis deals with the steric properties of Lewis acids. Therefore, an easy-to-apply model was designed to quantify the steric demand of Lewis acids. Using the results of this evaluation, a second model was developed which judges the steric repulsion in Lewis acid/base adduct formation for arbitrary sets of acids and bases.
Abstract
Neuropathic pain affects 6.9 to 10% of the general population, arises from lesion or disease of the somatosensory nervous system and is still challenging to treat. Indeed, current treatments efficacy are relatively low and present strong side effects. To that extent, identifying new targets and developing new treatment strategies constitute a priority. The blood nerve barrier consists of the endoneurial micro-blood vessels and the perineurium sealed by tight junctions constituted of tight junction proteins such claudin-5 and claudin-1. As the functional blood nerve barrier allows nerve tissue protection from external elements and maintains homeostasis, a destabilization or a disruption leads to infiltration of immunocytes promoting neuroinflammation and increased inflammatory mediators that can sensitize nociceptors and enhance pain. Thus resealing the blood nerve barrier in case of neuropathic pain could be a possible treatment strategy.
Specialised proresolving mediators such lipoxin A4 and resolvin D1 are small lipids that bind to receptors such the formylpeptide recptor 2 (FPR2) and resolve inflammation. Specially resolvin D1 as anti-inflammatory and analgesic properties. Thus using resolvin D1 or eventually other specialized proresolving mediators in neuropathic pain could reseal the blood nerve barrier and resolve neuropathic pain. The present work aimed to characterize the blood nerve barrier in a preclinical model of diabetic polyneuropathy and nerve injury (chronic constriction injury) and to identify specialized proresolving mediators that seal the blood nerve barrier and thereby alleviate neuropathic pain.
In diabetic polyneuropathy, the blood nerve barrier is permeable only to small molecules, which is due to the loss of claudin-1 in the perineurium and a reduced number of blood vessel- associated macrophages. Interestingly, blood nerve barrier permeability did not occur until four to eight weeks after diabetes induction, whereas mechanical hyperalgesia was measurable as early as two weeks. This suggests a pain-maintaining rather than a pain-triggering role of the blood nerve barrier.
In case of chronic constriction injury, a resolution process of both mechanical and thermal hyperalgesia occurs between three to six weeks after injury. Here, the blood nerve barrier is permeable to both small and large molecules from the beginning. The pain recovery process occurs primarily in parallel with the sealing of the endoneurial barrier to large molecules such as fibrinogen from the plasma and its degradation. Perineurium is still permeable nine weeks after injury. Metabolomic analyses show that especially precursors of Resolvin D1 as well as its receptor FPR2, are upregulated at the beginning of pain resolution. Application of resolvin
D1 loaded nanoparticles or agonists of FPR2 at the injury site before the onset of pain resolution accelerates the process and fibrinogen is no longer detectable in the endoneurium. Depending on the nerve damage, the blood nerve barrier is affected to varying degrees. Direct mechanical trauma and the accompanying inflammation lead to a more pronounced and long-lasting permeability - independent hyperalgesia. Possibly permeability, at least for small molecules, is important for prolonged reparative processes. In the nerve, permeability of capillaries in particular depends not only on tight junctions but also on other cells: in addition to macrophages, pericytes could also have a sealing effect. Endoneurial fibrinogen triggers pain; the exact mechanism remains to be investigated. Resolvin-containing nanoparticles were particularly effective and could be used locally as they contain endogenous substances in non- toxic particles.
Magnetic systems underlie the physics of quantum mechanics when reaching the limit of few or even single atoms. This behavior limits the minimum size of magnetic bits in data storage devices as spontaneous switching of the magnetization leads to the loss of information. On the other hand, exactly these quantum mechanic properties allow to use such systems in quantum computers. Proposals to realize qubits involve the spin states of single atoms as well as topologically protected Majorana zero modes, that emerge in coupled systems of magnetic atoms in proximity to a superconductor. In order to implement and control the proposed applications, a detailed understanding of atomic spins and their interaction with the environment is required.
In this thesis, two different systems of magnetic adatoms coupled to metallic and superconducting surfaces are studied by means of scanning tunneling microscopy (STM) and spectroscopy: Co atoms on the clean Cu(111) were among the first systems exhibiting signatures of the Kondo effect in an individual atom. Yet, a recent theoretical work proposed an alternative interpretation of these early experimental results, involving a newly described many-body state. Spin-averaged and -polarized experiments in high magnetic fields presented in this thesis confirm effects beyond the Kondo effect that determine the physics in these Co atoms and suggest a potentially even richer phenomenology than proposed by theory.
The second studied system are single and coupled Fe atoms on the superconducting Nb(110) surface. Magnetic impurities on superconducting surfaces locally induce Yu-Shiba-Rusinov (YSR) states inside the superconducting gap due to their pair breaking potential. Coupled systems of such impurities exhibit YSR bands and, if the bands cross the Fermi level such that the band structure is inverted, host Majorana zero modes. Using the example of Fe atoms on Nb(110), the YSR states’ dependence on the adatom–substrate interaction as well as the interatomic YSR state coupling is investigated. In the presence of oxygen on the Nb surface, the adatom–substrate interaction is shown to be heavily modified and the YSR states are found to undergo a quantum phase transition, which can be directly linked to a modified Kondo screening.
STM tips functionalized with CO molecules allow to resolve self-assembled one-dimensional chains of Fe atoms on the clean Nb(110) surface to study the YSR states’ coupling. Mapping out the states’ wave functions reveals their symmetry, which is shown to alter as a function of the states’ energy and number of atoms in the chain. These experimental results are reproduced in a simple tight-binding model, demonstrating a straightforward possibility to describe also more complex YSR systems toward engineered, potentially topologically non-trivial states.
One of the features that defines humans as extraordinarily social beings is their striking susceptibility to the gaze of others. The research reported in this dissertation was undertaken to advance our understanding of the role of gaze cues in low-level attentional and higher-order cognitive processes. In particular, effects of gaze were examined with regard to three aspects of human cognition: (1) social attention, (2) social interaction and (3) social understanding. Chapter 1 consists of three manuscripts that investigate the boundary conditions of attention capture by direct gaze and how gaze direction is integrated with facial context information. Manuscript 1 and 2 suggest two necessary requirements for attention capture by direct gaze: a meaningful holistic facial context and sharp foveal vision, respectively. Manuscript 3 shows approach/avoidance-congruency effects between gaze direction and emotion expression on attention. Chapter 2 of this dissertation explores the role of gaze in more naturalistic social scenarios. Manuscript 4 demonstrates that gaze behavior during a conversation shapes our perception of another person. Manuscript 5 builds on these findings by showing that these perceptions define our willingness to act in a prosocial way towards our interaction partner. Finally, chapter 3 adopts a broader perspective on social cognition research with a special focus on methodological aspects. Manuscript 6 is a review highlighting the significance of methodological aspects in social cognition research and stressing the importance of sophisticated decisions on task and stimulus materials. Manuscript 7 introduces a new instrument for the assessment of social understanding in adolescents. Initial application in a young sample group indicates that an understanding of another person’s mental states is a capacity that is still developing throughout adolescence. Both manuscripts of this final chapter include eye tracking data that suggest a relationship between gaze behavior and social understanding, a finding that further emphasizes the complex and multifaceted nature of social cognition. I conclude from the findings of this dissertation that research can benefit from adopting a broad view in terms of methodological as well as temporal aspects in order to capture human social cognition in its entirety.
Explaining the baryon asymmetry of the Universe has been a long-standing problem of particle physics, with the consensus being that new physics is required as the Standard Model (SM) cannot resolve this issue. Beyond the Standard Model (BSM) scenarios would need to incorporate new sources of \(CP\) violation and either introduce new departures from thermal equilibrium or modify the existing electroweak phase transition. In this thesis, we explore two approaches to baryogenesis, i.e. the generation of this asymmetry.
In the first approach, we study the two-particle irreducible (2PI) formalism as a means to investigate non-equilibrium phenomena. After arriving at the renormalised equations of motions (EOMs) to describe the dynamics of a phase transition, we discuss the techniques required to obtain the various counterterms in an on-shell scheme. To this end, we consider three truncations up to two-loop order of the 2PI effective action: the Hartree approximation, the scalar sunset approximation and the fermionic sunset approximation. We then reconsider the renormalisation procedure in an \(\overline{\text{MS}}\) scheme to evaluate the 2PI effective potential for the aforementioned truncations. In the Hartree and the scalar sunset approximations, we obtain analytic expressions for the various counterterms and subsequently calculate the effective potential by piecing together the finite contributions. For the fermionic sunset approximation, we obtain similar equations for the counterterms in terms of divergent parts of loop integrals. However, these integrals cannot be expressed in an analytic form, making it impossible to evaluate the 2PI effective potential with the fermionic contribution. Our main results are thus related to the renormalisation programme in the 2PI formalism: \( (i) \)the procedure to obtain the renormalised EOMs, now including fermions, which serve as the starting point for the transport equations for electroweak baryogenesis and \( (ii) \) the method to obtain the 2PI effective potential in a transparent manner.
In the second approach, we study baryogenesis via leptogenesis. Here, an asymmetry in the lepton sector is generated, which is then converted into the baryon asymmetry via the sphaleron process in the SM. We proceed to consider an extension of the SM along the lines of a scotogenic framework. The newly introduced particles are charged odd under a \(\mathbb{Z}_2\) symmetry, and masses for the SM neutrinos are generated radiatively. The \(\mathbb{Z}_2\) symmetry results in the lightest BSM particle being stable, allowing for a suitable dark matter (DM) candidate. Furthermore, the newly introduced heavy Majorana fermionic singlets provide the necessary sources of \(CP\) violation through their Yukawa interactions and their out-of-equilibrium decays produce a lepton asymmetry. This model is constrained from a wide range of observables, such as consistency with neutrino oscillation data, limits on branching ratios of charged lepton flavour violating decays, electroweak observables and obtaining the observed DM relic density. We study leptogenesis in this model in light of the results of a Markov chain Monte Carlo scan, implemented in consideration of the aforementioned constraints. Successful leptogenesis in this model, to account for the baryon asymmetry, then severely constrains the available parameter space.
Influence of Carbon Additives on the Electrochemical Performance of Modern Lead-Acid Batteries
(2023)
In the first part of this thesis, a validation of both short-term and long-term DCA tests on 2 V laboratory cells is focussed. The aim is to improve the laboratory cell level measurement technology for dynamic charge acceptance regarding the investigation of carbon additives. To address this issue, it is crucial to apply carbon additives generating a remarkable difference in charge acceptance. For this purpose, five different carbon additives providing a variation in the specific external surface were included as additives in the negative plates of 2 V lead-acid cells. Both short-term (charge acceptance test 2 from SBA and DCA from EN) and long-term (Run-in DCA from Ford) DCA tests were executed on the lead-acid cells. Further understanding of the mechanism was studied by applying electrochemical methods like cyclic voltammetry and electrochemical impedance spectroscopy.
The second part of this thesis aims to understand the impact of carbon surface functional groups on the electrochemical activity of the negative electrodes as well as the DCA of 2 V lead-acid cells. In order to address this topic, commercially available activated carbon was modified by different chemical treatments to incorporate specific surface functional groups in the carbon structure. A series of activated carbons having a broad range of pH was prepared, which were used as additives in the negative electrodes. The corresponding lead-acid cells were subjected to cyclic voltammetry and DCA test according to EN. Further, the physical and chemical properties of the functionalized carbon additives were intensively analyzed to establish a structure-property relationship with a focus on DCA.
A plethora of novel material concepts are currently being investigated in the condensed matter research community. Some of them hold promise to shape our everyday world in a way that silicon-based semiconductor materials and the related development of semiconductor devices have done in the past. In this regard, the last decades have witnessed an explosion of studies concerned with so called ‘’quantum materials’’ with emerging novel functionalities. These could eventually lead to new generations of electronic and/or spintronic devices. One particular material class, the so called topological materials, play a central role. As far as their technological applicability is concerned, however, they are still facing outstanding challenges to date.
Predicted for the first time in 2005 and experimentally verified in 2007, two-dimensional topological insulators (2D TIs) (a.k.a. quantum spin Hall insulators) exhibit the outstanding property of hosting spin-polarized metallic states along the boundaries of the insulating 2D bulk material, which are protected from elastic single-particle backscattering and give rise to the quantum spin Hall effect (QSHE). Owing to these peculiar properties the QSHE holds promise for dissipationless charge and/or spin transport. However, also in today’s best 2D TIs the observation of the QSHE is still limited to cryogenic temperatures of maximum 100 K. Here, the discovery of bismuthene on SiC(0001) has marked a milestone towards a possible realization of the QSHE at or beyond room-temperature owing to the massively increased electronic bulk energy gap on the order of 1 eV. This thesis is devoted to and motivated by the goal of advancing its synthesis and to build a deeper understanding of its one-particle and two-particle electronic properties that goes beyond prior work.
Regarding the aspect of material synthesis, an improved growth procedure for bismuthene is elaborated that increases the domain size of the material considerably (by a factor of ≈ 3.2 - 6.5 compared to prior work). The improved film quality is an important step towards any future device application of bismuthene, but also facilitates all further basic studies of this material.
Moreover, the deposition of magnetic transition metals (Mn and Co) on bismuthene is investigated. Thereby, the formation of ordered magnetic Bi-Mn/Co alloys is realized, their structure is resolved with scanning tunneling microscopy (STM), and their pristine electronic properties are resolved with scanning tunneling spectroscopy (STS) and photoemission spectroscopy (PES). It is proposed that these ordered magnetic Bi-Mn/Co-alloys offer the potential to study the interplay between magnetism and topology in bismuthene in the future.
In this thesis, a wide variety of spectroscopic techniques are employed that aim to build an understanding of the single-particle, as well as two-particle level of description of bismuthene's electronic structure. The techniques involve STS and angle-resolved PES (ARPES) on the one hand, but also optical spectroscopy and time-resolved ARPES (trARPES), on the other hand. Moreover, these experiments are accompanied by advanced numerical modelling in form of GW and Bethe-Salpeter equation calculations provided by our theoretical colleagues. Notably, by merging many experimental and theoretical techniques, this work sets a benchmark for electronic structure investigations of 2D materials in general.
Based on the STS studies, electronic quasi-particle interferences in quasi-1D line defects in bismuthene that are reminiscent of Fabry-Pérot states are discovered. It is shown that they point to a hybridization of two pairs of helical boundary modes across the line defect, which is accompanied by a (partial) lifting of their topological protection against elastic single-particle backscattering.
Optical spectroscopy is used to reveal bismuthene's two-particle elecronic structure. Despite its monolayer thickness, a strong optical (two-particle) response due to enhanced electron-hole Coulomb interactions is observed. The presented combined experimental and theoretical approach (including GW and Bethe-Salpeter equation calculations) allows to conclude that two prominent optical transitions can be associated with excitonic transitions derived from the Rashba-split valence bands of bismuthene. On a broader scope this discovery might promote further experiments to elucidate links of excitonic and topological physics.
Finally, the excited conduction band states of bismuthene are mapped in energy and momentum space employing trARPES on bismuthene for the first time. The direct and indirect band gaps are succesfully extracted and the effect of excited charge carrier induced gap-renormalization is observed. In addition, an exceptionally fast excited charge carrier relaxation is identified which is explained by the presence of a quasi-metallic density of states from coupled topological boundary states of domain boundaries.
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.
This work in the field of digital literary stylistics and computational literary studies is concerned with theoretical concerns of literary genre, with the design of a corpus of nineteenth-century Spanish-American novels, and with its empirical analysis in terms of subgenres of the novel. The digital text corpus consists of 256 Argentine, Cuban, and Mexican novels from the period between 1830 and 1910. It has been created with the goal to analyze thematic subgenres and literary currents that were represented in numerous novels in the nineteenth century by means of computational text categorization methods. The texts have been gathered from different sources, encoded in the standard of the Text Encoding Initiative (TEI), and enriched with detailed bibliographic and subgenre-related metadata, as well as with structural information.
To categorize the texts, statistical classification and a family resemblance analysis relying on network analysis are used with the aim to examine how the subgenres, which are understood as communicative, conventional phenomena, can be captured on the stylistic, textual level of the novels that participate in them. The result is that both thematic subgenres and literary currents are textually coherent to degrees of 70–90 %, depending on the individual subgenre constellation, meaning that the communicatively established subgenre classifications can be accurately captured to this extent in terms of textually defined classes.
Besides the empirical focus, the dissertation also aims to relate literary theoretical genre concepts to the ones used in digital genre stylistics and computational literary studies as subfields of digital humanities. It is argued that literary text types, conventional literary genres, and textual literary genres should be distinguished on a theoretical level to improve the conceptualization of genre for digital text analysis.
Depressive disorders represent one of the main sources for the loss of healthy years of life. One of the reasons for this circumstance is the recurrent course of these disorders, which can be interrupted by current therapeutic approaches, especially in the shortterm, but seem to be maintained at least in part in the long-term. Subsequently, on one hand, this thesis deals with methodological measurement issues in the longitudinal prediction of depressive courses. On the other hand, it addresses two currently discussed neuroscience-based treatment approaches, which are investigated experimentally in a basic-psychological manner and reviewed in the light of their potential to translate results to the application in patient care. These two approaches each address potential mechanisms that may negatively impact long-term disease trajectories: First, stable endophenotypes for vulnerability factors that could regain control over the organism and reactivate maladaptive experiences, or behaviors with increasing temporal distance from therapeutic methods are focused on. In the studies presented, these were influenced by a recently rediscovered method of neuromodulation (transcranial low-intensity focused ultrasound) which is discussed in light of its unique capability to address even deepest, subcortical regions at a high spatial resolution. Lastly, as a second approach, an experimental design for the use of reconsolidation interference is presented, which could provide a first insight into the applicability of corresponding protocols in the field of depressive disorders and thus contribute to the modification, instead of inhibition, of already mentioned endophenotypes. In sum, methodological considerations for monitoring and predicting long-term courses of depression are deducted before two approaches are discussed that could potentially exert positive influences on the recurrent nature of depressive symptoms on their own, in combination with each other, or as augmentation for existing therapeutic procedures.
Ubiquitylation is a protein post translational modification, in which ubiquitin is covalently attached to target protein substrates resulting in diverse cellular outcomes. Besides ubiquitin, various ubiquitin-like proteins including FAT10 exist, which are also conjugated to target proteins. The underlying modification mechanisms are conserved. In the initial step, ubiquitin or a ubiquitin-like protein is thioester-linked to a catalytic cysteine in the E1activating enzyme in an ATP-dependent manner. The respective protein modifier is then transferred to an E2 conjugating enzyme in a transthioesterification reaction. Finally, an E3 ubiquitin ligase E3 catalyzes the covalent attachment of the protein modifier to a substrate. In the case of ubiquitin, multiple ubiquitin molecules can be attached to a substrate in the form of either linear or branched polyubiquitin chains but also as single ubiquitin modifications. Depending on the nature of the ubiquitin chain, the substrates are destined to various cellular processes such as their targeted destruction by the proteasome but also non-degradative outcomes may occur.
As stated above FAT10 is a ubiquitin-like protein modifier which typically targets proteins for proteasomal degradation. It consists of two ubiquitin-like domains and is mainly expressed in cells of the human immune system. The reported involvement of FAT10 modifications in cancers and other diseases has caught the attention of the scientific community as an inhibition of the FAT10ylation process may provide avenues for novel therapeutic approaches. UBA6 is the E1 activating enzyme that resides at the apex of the FAT10 proteasomal degradation pathway. UBA6 not only recognizes FAT10 but can also activate ubiquitin as efficiently as the ubiquitin specific E1 UBA1. The dual specificity of UBA6 may complicate the inhibition FAT10ylation since targeting the active site of UBA6 will also inhibit the UBA6-catalyzed ubiquitin activation. Therefore, it is important to understand the underlying principles for the dual specificity of UBA6 prior to the development of compounds interfering with FAT10ylation.
In this thesis important novel insights into the structure and function of UBA6 were derived by X-ray crystallography and biochemical methods. The first crystal structure of UBA6 reveals the multidomain architecture of this enzyme in atomic detail. The enzyme is composed of a rigid core including its active and inactive adenylation domains as well as a 4 helix bundle. Overall, the molecule adopts a “Y” shape architecture with the core at the base and the first and second catalytic half domains forming one arm of the “Y” and the ubiquitin fold domain constituting the other arm. While UBA6 shares the same domain architecture as UBA1, substantial differences were revealed by the crystal structure. In particular, the first catalytic half domain undergoes a significant shift to a position more distal from the core. This rigid body movement is assumed to generate room to accommodate the second ubiquitin-like domain of FAT10. Differences are also observed in a hydrophobic platform between the core and the first catalytic half domain and the adenylation active site in the core, which together from the binding sites for ubiquitin and FAT10. Site directed mutagenesis of key residues in these areas altered the UBA6-catalyzed activation of ubiquitin and FAT10. UBA6 variants were generated with the goal of trying to block the activation of FAT10 while still maintaining that of ubiquitin activation, in order to fully explain the dual specificity of UBA6. However, none of these mutations could block the activation of FAT10, while some of these UBA6 variants blocked ubiquitin activation. Preliminary inhibition assays with a group of E1 inhibitors belonging to the adenosyl sulfamate family demonstrated potent inhibition of FAT10ylation for two compounds. The dual specificity of UBA6 hence needs to be further examined by biochemical and structural methods. In particular, the structure of a complex between UBA6 and ubiquitin or FAT10 would provide key insights for further biochemical studies, ultimately allowing the targeted inhibition of the FAT10ylation machinery.
Deep learning enables enormous progress in many computer vision-related tasks. Artificial Intel- ligence (AI) steadily yields new state-of-the-art results in the field of detection and classification. Thereby AI performance equals or exceeds human performance. Those achievements impacted many domains, including medical applications.
One particular field of medical applications is gastroenterology. In gastroenterology, machine learning algorithms are used to assist examiners during interventions. One of the most critical concerns for gastroenterologists is the development of Colorectal Cancer (CRC), which is one of the leading causes of cancer-related deaths worldwide. Detecting polyps in screening colonoscopies is the essential procedure to prevent CRC. Thereby, the gastroenterologist uses an endoscope to screen the whole colon to find polyps during a colonoscopy. Polyps are mucosal growths that can vary in severity.
This thesis supports gastroenterologists in their examinations with automated detection and clas- sification systems for polyps. The main contribution is a real-time polyp detection system. This system is ready to be installed in any gastroenterology practice worldwide using open-source soft- ware. The system achieves state-of-the-art detection results and is currently evaluated in a clinical trial in four different centers in Germany.
The thesis presents two additional key contributions: One is a polyp detection system with ex- tended vision tested in an animal trial. Polyps often hide behind folds or in uninvestigated areas. Therefore, the polyp detection system with extended vision uses an endoscope assisted by two additional cameras to see behind those folds. If a polyp is detected, the endoscopist receives a vi- sual signal. While the detection system handles the additional two camera inputs, the endoscopist focuses on the main camera as usual.
The second one are two polyp classification models, one for the classification based on shape (Paris) and the other on surface and texture (NBI International Colorectal Endoscopic (NICE) classification). Both classifications help the endoscopist with the treatment of and the decisions about the detected polyp.
The key algorithms of the thesis achieve state-of-the-art performance. Outstandingly, the polyp detection system tested on a highly demanding video data set shows an F1 score of 90.25 % while working in real-time. The results exceed all real-time systems in the literature. Furthermore, the first preliminary results of the clinical trial of the polyp detection system suggest a high Adenoma Detection Rate (ADR). In the preliminary study, all polyps were detected by the polyp detection system, and the system achieved a high usability score of 96.3 (max 100). The Paris classification model achieved an F1 score of 89.35 % which is state-of-the-art. The NICE classification model achieved an F1 score of 81.13 %.
Furthermore, a large data set for polyp detection and classification was created during this thesis. Therefore a fast and robust annotation system called Fast Colonoscopy Annotation Tool (FastCAT) was developed. The system simplifies the annotation process for gastroenterologists. Thereby the
i
gastroenterologists only annotate key parts of the endoscopic video. Afterward, those video parts are pre-labeled by a polyp detection AI to speed up the process. After the AI has pre-labeled the frames, non-experts correct and finish the annotation. This annotation process is fast and ensures high quality. FastCAT reduces the overall workload of the gastroenterologist on average by a factor of 20 compared to an open-source state-of-art annotation tool.
MDSCs are suppressive immune cells with a high relevance in various pathologies including cancer, autoimmunity, and chronic infections. Surface marker expression of MDSCs resembles monocytes and neutrophils which have immunostimulatory functions instead of suppressing T cells. Therefore, finding specific surface markers for MDSCs is important for MDSC research and therapeutic MDSC manipulation. In this study, we analyzed if the integrin VLA-1 has the potential as a novel MDSC marker. VLA-1 was expressed by M-MDSCs but not by G-MDSCs as well as by Teff cells. VLA-1 deficiency did not impact iNOS expression, the distribution of M-MDSC and G-MDSC subsets, and the suppressive capacity of MDSCs towards naïve and Teff cells in vitro. In mice, VLA-1 had no effect on the homing capability of MDSCs to the spleen, which is a major reservoir for MDSCs. Since the splenic red pulp contains collagen IV and VLA-1 binds collagen IV with a high affinity, we found MDSCs and Teff cells in this area as expected. We showed that T cell suppression in the spleen, indicated by reduced T cell recovery and proliferation as well as increased apoptosis and cell death, partially depended on VLA-1 expression by the MDSCs. In a mouse model of multiple sclerosis, MDSC injection prior to disease onset led to a decrease of the disease score, and this effect was significantly reduced when MDSCs were VLA-1 deficient. The expression of Sema7A by Teff cells, a ligand for VLA-1 which is implicated in negative T cell regulation, resulted in a slightly stronger Teff cell suppression by MDSCs compared to Sema7A deficient T cells. Live cell imaging and intravital 2-photon microscopy showed that the interaction time of MDSCs and Teff cells was shorter when MDSCs lacked VLA 1 expression, however VLA-1 expression had no impact on MDSC mobility. Therefore, the VLA-1-dependent interaction of MDSC and Teff cells on collagen IV in the splenic red pulp is implicated MDSC-mediated Teff cell suppression.
Relativistic effects crucially influence the fundamental properties of many quantum materials. In the accelerated reference frame of an electron, the electric field of the nuclei is transformed into a magnetic field that couples to the electron spin. The resulting interaction between an electron spin and its orbital angular momentum, known as spin-orbit coupling (SOC), is hence fundamental to the physics of many condensed matter phenomena. It is particularly important quantitatively in low-dimensional quantum systems, where its coexistence with inversion symmetry breaking can lead to a splitting of spin degeneracy and spin momentum locking. Using the paradigm of Landau Fermi liquid theory, the physics of SOC can be adequately incorporated in an effective single particle picture. In a weak coupling approach, electronic correlation effects beyond single particle propagator renormalization can trigger Fermi surface instabilities such as itinerant magnetism, electron nematic phases, superconductivity, or other symmetry broken states of matter.
In this thesis, we use a weak coupling-based approach to study the effect of SOC on Fermi surface instabilities and, in particular, superconductivity. This encompasses a weak coupling renormalization group formulation of unconventional superconductivity as well as the random phase approximation. We propose a unified formulation for both of these two-particle Green’s function approaches based on the notion of a generalized susceptibility.
In the half-Heusler semimetal and superconductor LuPtBi, both SOC and electronic correlation
effects are prominent, and thus indispensable for any concise theoretical description. The metallic and weakly dispersive surface states of this material feature spin momentum locked Fermi surfaces, which we propose as a possible domain for the onset of unconventional surface superconductivity. Using our framework for the analysis of Fermi surface instability and combining it with ab-initio density functional theory calculations, we analyse the surface band structure of LuPtBi, and particularly its propensity towards Cooper pair formation. We study how the presence of strong SOC modifies the classification of two-electron wave functions as well as the screening of electron-electron interactions. Assuming an electronic mechanism, we identify a chiral superconducting condensate featuring Majorana edge modes to be energetically favoured over a wide range of model parameters.
Mineral biocements are brittle materials, which usually results in catastrophic failure during mechanical loading. Here, previous works demonstrated the feasibility of reducing brittleness by a dual-setting approach, in which a silica sol was simultaneously gelled during the setting of a brushite forming cement. The current thesis aimed at further improving this concept by both using a novel silicate based cement matrix for an enhanced bonding between cement and silica matrix as well as multifunctional silica precursors to increase the network density of the gel. Due to its well-known biocompatibility and osteogenic regeneration capacity, baghdadite was chosen as mineral component of such composites. This required in a first approach the conversion of baghdadite ceramics into self-setting cement formulations. This was investigated initially by using baghdadite as reactive filler in a brushite forming cement (Chapter 4). Here, the ß-TCP component in a equimolar mixture of ß-TCP and acidic monocalcium phosphate anhydrous was subsequently replaced by baghdadite at various concentrations (0, 5, 10, 20, 30, 50, and 100 wt%) to study the influence on physicochemical cement properties such as mechanical performance, radiopacity, phase composition and microstructure. X-ray diffraction profiles demonstrated the dissolution of baghdadite during the cement reaction without affecting the crystal structure of the precipitated brushite phase. In addition, EDX analysis showed that calcium is homogeneously distributed in the cement matrix, while zirconium and silicon form cluster-like aggregates ranging in size from a few micrometers to more than 50 µm. X-ray images and µ-CT analyses indicate improved X-ray visibility with increased incorporation of baghdadite in brushite cement, with an aluminum equivalent thickness nearly doubling at a baghdadite content of 50 wt%. At the same time, the compressive strength of brushite cement increased from 12.9 ± 3.1 MPa to 21.1 ± 4.1 MPa at a baghdadite content of 10 wt%. Cell culture medium conditioned with powdered brushite cement approached physiological pH values when increasing amounts of baghdadite were added to the cement (pH = 6.47 for pure brushite, pH = 7.02 for brushite with 20 wt% baghdadite substitution). Baghdadite substitution also affected the ion content in the culture medium and thus the proliferation activity of primary human osteoblasts in vitro. The results demonstrated for the first time the suitability of baghdadite as a reactive cement additive for improving the radiopacity, mechanical performance, and cytocompatibility of brushite cements.
A second approach (Chapter 5) aimed to produce single component baghdadite cements by an increase of baghdadite solubility to initiate a self-setting cement reaction. For this, the material was mechanically activated by longer grinding times of up to 24h leading to both a decrease in particle and crystallite size as well as a partial amorphization of baghdadite. Baghdadite cements were formed by adding water at a powder to liquid ratio of 2.0 g/ml. Maximum compressive strengths were determined to be ~2 MPa after 3 days of setting for a 24-hour ground material. Inductively coupled plasma mass spectrometry (ICP-MS) measurements showed an incongruent dissolution profile of the set cements, with preferential dissolution of calcium and only minor release of zirconium ions. Cement formation occurs under alkaline conditions, with the unground raw powder resulting in a pH of 11.9 during setting, while prolonged grinding increases the pH to about 12.3.
Finally, mechanically activated baghdadite cements were combined with inorganic silica networks (Chapter 6) to create dual-setting cements with a further improvement of mechanical performance. While a modification of the cement pastes with a TEOS derived sol was already thought to improve strength, it was hypothesized that using multi-arm silica precursors can further enhance their mechanical performance due to a higher network density. In addition, this should also reduce pore size of both gels and cement and hence will be able to adjust the release kinetics of incorporated drugs. For this, multi-armed silica precursors were synthesized by the reaction of various multivalent alcohols (ethylene glycol, glycerine, pentaerythrit) with an isocyanate modified silica precursor. After hydrolysis under acidic conditions, the sols were mixed with baghdadite cement powders in order to allow a simultaneous gel formation and cement setting. Since the silica monomers have a high degree of linkage sites, this resulted in a branched network that interpenetrated with the growing cement crystals. In addition to minor changes in the crystalline phase composition as determined by X-ray diffraction, the novel composites exhibited improved mechanical properties with up to 20 times higher compressive strength and further benefit from an about 50% lower overall porosity than the reference pure baghdadite cement. In addition, the initial burst release of the model drug vancomycin was completely inhibited by the added silica matrix. This observation was verified by testing for the antimicrobial activity with Staphylococcus aureus by measuring the inhibition zones of selected samples after 24 h and 48 h, whereas the antimicrobial effectiveness of a constant vancomycin release could be demonstrated.
The current thesis clearly demonstrated the high potential of baghdadite as a cement formulation for medical application. The initially poor mechanical properties of such cements can be overcome by special processing techniques or by combination with silica networks. The achieved mechanical performance is > 10 MPa and hence suitable for bone replacement under non-load bearing conditions. The high intrinsic radiopacity as well as the alkaline pH during setting may open the way ahead to further dental applications, e.g. as root canal sealers or filler in dental composites. Here, the high pH is thought to lead to antimicrobial properties of such materials similar to commonly applied calcium hydroxide or calcium silicates, however combined with an intrinsic radiopacity for X-ray imaging. This would simplify such formulations to single component materials which are less susceptible to demixing processes during transport, storage or processing.
Onchocerciasis, the world's second-leading infectious cause of blindness in humans
–prevalent in Sub-Saharan Africa – is caused by Onchocerca volvulus (O. volvulus), an
obligatory human parasitic filarial worm. Commonly known as river blindness,
onchocerciasis is being targeted for elimination through ivermectin-based mass
drug administration programs. However, ivermectin does not kill adult parasites,
which can live and reproduce for more than 15 years within the human host. These
impediments heighten the need for a deeper understanding of parasite biology and
parasite-human host interactions, coupled with research into the development of
new tools – macrofilaricidal drugs, diagnostics, and vaccines. Humans are the only
definitive host for O. volvulus. Hence, no small-animal models exist for propagating
the full life cycle of O. volvulus, so the adult parasites must be obtained surgically
from subcutaneous nodules. A two-dimensional (2D) culture system allows that
O. volvulus larvae develop from the vector-derived infective stage larvae (L3) in vitro
to the early pre-adult L5 stages. As problematic, the in vitro development of
O. volvulus to adult worms has so far proved infeasible. We hypothesized that an
increased biological complexity of a three-dimensional (3D) culture system will
support the development of O. volvulus larvae in vitro. Thus, we aimed to translate
crucial factors of the in vivo environment of the developing worms into a culture
system based on human skin. The proposed tissue model should contain 1. skinspecific
extracellular matrix, 2. skin-specific cells, and 3. enable a direct contact of
larvae and tissue components. For the achievement, a novel adipose tissue model
was developed and integrated to a multilayered skin tissue comprised of epidermis,
dermis and subcutis. Challenges of the direct culture within a 3D tissue model
hindered the application of the three-layered skin tissue. However, the indirect coculture
of larvae and skin models supported the growth of fourth stage (L4) larvae in
vitro. The direct culture of L4 and adipose tissue strongly improved the larvae
survival. Furthermore, the results revealed important cues that might represent the
initial encapsulation of the developing worm within nodular tissue. These results
demonstrate that tissue engineered 3D tissues represent an appropriate in vitro
environment for the maintenance and examination of O. volvulus larvae.
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.
Social contact is an integral part of daily life. Its health-enhancing effects include reduced negative affective experiences of fear and anxiety, a phenomenon called social buffering. This dissertation studied different forms of social contact and their anxiety-buffering effects with diverse methodologies.
The laboratory-based first study investigated minimal social contact in the context of pain relief learning. Results showed that the observed decreased autonomic and increased subjective fear responses following pain relief learning were independent of social influence. The minimalistic and controlled social setting may have prevented social buffering. Study 2 targeted social buffering in daily life using Ecological Momentary Assessment. We repeatedly assessed individuals’ state anxiety, related cardiovascular responses, and aspects of social interactions with smartphones and portable sensors on five days. Analyses of over 1,500 social contacts revealed gender-specific effects, e.g., heart rate-reducing effects of familiarity in women, but not men. Study 3 examined anxiety, loneliness, and related social factors in the absence of social contact due to social distancing. We constructed and validated a scale measuring state and trait loneliness and isolation, and analysed its link to mental health. Results include a social buffering-like relation of lower anxiety with more trait sociability and sense of belonging.
In sum, the studies showed no fear reduction by minimal social contact, but buffering effects relating to social and personal factors in more complex social situations. Anxiety responses during daily social contacts were lower with more familiar or opposite-gender interaction partners. During limited social contact, lower anxiety related to inter-individual differences in sociability, social belonging, and loneliness. By taking research from lab to life, this dissertation underlined the diverse nature of social contact and its relevance to mental health.
The mammalian central clock, located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, controls circadian rhythms in behaviour such as the sleep-wake cycle. It is made up of approximately 20,000 heterogeneous neurons that can be classified by their expression of neuropeptides. There are three major populations: AVP neurons (arginine vasopressin), VIP neurons (vasoactive intestinal peptide), and GRP neurons (gastrin releasing peptide). How these neuronal clusters form functional units to govern various aspects of rhythmic behavior is poorly understood. At a molecular level, biological clocks are represented by transcriptional-posttranslational feedback loops that induce circadian oscillations in the electrical activity of the SCN and hence correlate with behavioral circadian rhythms. In mammals, the sleep wake cycle can be accurately predicted by measuring electrical muscle and brain activity. To investigate the link between the electrical activity of heterogeneous neurons of the SCN and the sleep wake cycle, we optogenetically manipulated AVP neurons in vivo with SSFO (stabilized step function opsin) and simultaneously recorded an electroencephalogram (EEG) and electromyogram (EMG) in freely moving mice. SSFO-mediated stimulation of AVP positive neurons in the anterior hypothalamus increased the total amount of wakefulness during the hour of stimulation. Interestingly, this effect led to a rebound in sleep in the hour after stimulation. Markov chain sleep-stage transition analysis showed that the depolarization of AVP neurons through SSFO promotes the transition from all states to wakefulness. After the end of stimulation, a compensatory increase in transitions to NREM sleep was observed. Ex vivo, SSFO activation in AVP neurons causes depolarization and modifies the activity of AVP neurons. Therefore, the results of this thesis project suggest an essential role of AVP neurons as mediators between circadian rhythmicity and sleep-wake behaviour.
The last years have witnessed an exciting scientific quest for intriguing topological phenomena in time-dependent quantum systems. A key to many manifestations of topology in dynamical systems relies on the effective dimensional extension by time-periodic drives. An archetypal example is provided by the Thouless pump in one spatial dimension, where a robust and quantized charge transport can be described in terms of an integer quantum Hall effect upon interpreting time as an extra dimension. Generalizing this fundamental concept to multifrequency driving, a variety of higher-dimensional topological models can be engineered in dynamical synthetic dimensions, where the underlying topological classification leads to quantized pumping effects in the associated lower-dimensional time-dependent systems.
In this Thesis, we explore how correlations profoundly impact the topological features of dynamical synthetic quantum materials. More precisely, we demonstrate that the interplay of interaction and dynamical synthetic dimension gives rise to striking topological phenomena that go beyond noninteracting implementations. As a starting point, we exploit the Floquet counterpart of an integer quantum Hall scenario, namely a two-level system driven by two incommensurate frequencies. In this model, the topologically quantized response translates into a process in which photons of different frequencies are exchanged between the external modes, referred to as topological frequency conversion. We extend this prototypical setup to an interacting version, focusing on the minimal case of two correlated spins equally exposed to the external drives. We show that the topological invariant determining the frequency conversion can be changed by odd integers, something explicitly forbidden in the noninteracting limit of two identical spins. This correlated topological feature may, in turn, result in an enhancement of the quantized response.
Robust response signals, such as those predicted for the topological frequency converter, are of fundamental interest for potential technological applications of topological quantum matter. Based on an open quantum system implementation of the frequency converter, we propose a novel mechanism of topological quantization coined ''topological burning glass effect''. Remarkably, this mechanism amplifies the local response of the driven two-level system by an integer that is proportional to the number of environmental degrees of freedom to which the system is strongly coupled. Specifically, our findings are illustrated by the extension of the frequency converter to a central spin model. There, the local energy transfer mediated exclusively by the central spin is significantly enhanced by the collective motion of the surrounding spins. In this sense, the central spin adopts the topological nature of the total system in its non-unitary dynamics, taking into account the correlations with the environment.
The life of an exciton: From ultrafast nonradiative relaxation to high quantum yield fluorescence
(2023)
This thesis focuses on understanding and predicting processes in chromophores after electronic state excitation, particularly the impact on luminescence - the spontaneous emission of light. It considers the effect of processes preceding luminescence on emission properties, which are challenging to predict, especially in complex aggregates.
For example, excitation energy transfer is a crucial process in understanding luminescence, as it allows the emission to occur from different molecular units than where the absorption occurs. This can lead to significant shifts in emission wavelength and fluorescence quantum yields. The thesis offers solutions to model this process effectively, understanding the impact of excitation energy and exciton coupling disorder on energy transfer rates and linking simulated energy transfer to experimental measurements.
The work further explores excimer formation - an undesired luminescence loss channel due to its significant stabilization of the electronic state. Usually, the molecules obey a stacked conformation with parallel orientation to maximize the orbital overlap. This energetic lowering of the excited state can often result in trapping of the dimer in this state due to a deep minimum on the potential energy surface. The excimer formation dynamics, structural rearrangement, and its influence on singlet-correlated triplet pair states formation, critical for the singlet-fission process, have been extensively studied.
The thesis also focuses on another luminescence loss channel triggered by conical intersections between the electronic ground and the first excited states. A new model is introduced to overcome limitations in current simulation methods, considering the solvent's electrostatic and frictional effects on the barriers. The model accurately describes merocyanine dyes' solvent-dependent photoluminescence quantum yields and characterizes all relaxation channels in different BODIPY oligomer series.
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.
Mass spectrometry-based quantification of steroids for the diagnostic workup of adrenal tumors
(2023)
Tumors of the adrenal gland belong to the most frequent neoplasms in humans with a prevalence of 3–10 % in adults. The aim of the diagnostic workup is the identification of potentially hormone-secreting and / or malignant tumors, because most of these tumors will require surgical resection. Malignant adrenocortical carcinomas (ACC) are very rare and associated with a poor prognosis in advanced stages, therefore, an early and accurate diagnosis is crucial.
Within this thesis, two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the quantification of steroids in different biomaterials were developed to improve the diagnostic workup of adrenal tumors.
First, an LC-MS/MS method for the simultaneous quantification of cortisol and dexamethasone in serum samples after dexamethasone suppression test (DST) was developed, validated, and applied to 400 clinical samples. Newly established method-specific threshold concentrations for cortisol and dexamethasone increased DST specificity from 67.5 % to 92.4 % while preserving 100 % sensitivity.
Second, an LC-MS/MS method for the quantification of eleven urinary steroids was developed and validated to improve the differentiation between ACC and adrenocortical adenomas (ACA). A decision tree requiring only two steroids was trained for classification and tested based on 24 h urine samples from 268 patients with adrenal tumor. Malignancy was excluded with a negative predictive value of 100 % in an independent validation cohort of 84 samples of 24-h urine. A newly proposed simplified diagnostic workflow with urinary steroid profiling as first tier test could obviate additional adrenal-specific imaging in 42 of 64 patients with ACA.
The new DST method is already in clinical use at the University Hospital Würzburg, whereas the classification model based on urinary steroid profiling will require prospective validation in a larger cohort.
Glioblastoma (GB) is the most aggressive malignant adult brain tumour with a median survival rate of only 15 months. GB tumours are characterized by necrotic and hypoxic core, which leads to nutrient deficient areas contributing to invasive, diffuseinfiltrative and angiogenic nature of these tumours. Cells exposed to nutrient deficient conditions and are known to reprogram their metabolism to produce or procure macro molecules from their environment. This makes cancer cells uniquely dependent on transcriptional regulators and a window of opportunity to target them. Sterol regulatory element binding protein 1 (SREBP1) is a transcriptional regulator of de-novo fatty acid synthesis in cells. The aim of this thesis was to investigate if SREBP1 was involved in restructuring the transcriptional regulation of genes involved in fatty acid biosynthesis upon low serum condition, in mediating interaction with other cell types in the tumour bulk such as endothelial cells, in regulating cancer stem like cells and finally to study its upstream regulation in GB. Global transcriptional analysis on GB cells exposed to low serum conditions revealed that SREBP1 regulated several fatty acid biosynthesis and phospholipid metabolic processes. PLA2G3 was identified as a novel target of SREBP1 in GB that was uniquely regulated in low serum condition. Analysis of total fatty acid and lipid species revealed that loss of SREBP1 in low serum condition changes the proportion of saturated, MUFAs and PUFAs. These changes were not specific to loss of PLA2G3 but as a result of downregulation of many genes regulated by SREBP1 in the fatty acid biosynthetic pathway. Next, treatment of HUVEC’s (endothelial cells) with condition medium from SREBP1-silenced U87 cells inhibited sprouting and tube formation capacity compared to the control condition, emphasizing the role of SREBP1 in angiogenesis and release of signalling mediators. Further, SREBP1 was shown to be important for proliferation of patient derived stem like cells and becomes indispensable for forming neurospheres in long term cultures, indicating its role in maintaining stemness. Also, inhibition of SREBP function by blocking the esterification of cholesterol using inhibitors targeting SOAT1 showed impairment in the viability of GB cells exposed to serum-depleted condition. Overall, SREBP1 plays an important role in maintaining tumour growth in nutrient deficient conditions and help in interaction with tumour microenvironment contributing to the aggressiveness of this tumour and poses itself as an attractive and unique target for GB treatment
Diabetes mellitus is an incurable, metabolic disease, which is associated with severe long-term complications. The in vitro generation of pancreatic β-cells from human induced pluripotent stem cells (hiPSCs) represent a promising strategy for a curative therapy of diabetes mellitus. However, current differentiation strategies largely fail to produce functional β-cells in vitro and require an additional in vivo transplantation to achieve terminal maturation. Previous studies demonstrated a beneficial effect of the extracellular matrix (ECM) on the survival and sustained function of adult, isolated islets of Langerhans. This raises the question whether organ-specific cell-ECM interactions might represent the missing link driving the final stage of β-cell development. In order to address this issue, this study investigated the impact of the pancreas ECM on in vitro β-cell differentiation and its use for the establishment of a pancreatic endocrine organ model.
To this purpose, a pancreas-specific ECM scaffolds (PanMa) was derived from porcine pancreata using whole organ decellularization with Sodium Deoxycholate. In a first step, the generated PanMa was thoroughly characterized using (immuno-) histological stainings, scanning electron microscopy and DNA quantification as well as perfusion and recellularization experiments with endothelial cells. Based on these data, a scoring system (PancScore) for a standardized PanMa generation was developed. Next, the generated PanMa was tested for the presence of tissue-specific ECM features. Therefore, the biophysical and physico-structural characteristics, such as rigidity, porosity and hygroscopy were analyzed using rheological measurements, particle diffusion analyses as well as a water evaporation assay and compared to the properties of ECM scaffolds derived from porcine small intestine (SISser) and lung (LungMa) to examine organ-specific scaffold cues. Following the thorough scaffold characterization, the impact of the PanMa on pluripotency and early development of hiPSC was studied. To this purpose, gene and protein expression of hiPSCs during maintenance culture and spontaneous differentiation on the PanMa were assessed. In a next step, the impact of the PanMa on the pancreatic endocrine differentiation of hiPSCs was tested. Therefore, the PanMa was used as a liquid media supplement or as a solid scaffold during the directed differentiation of hiPSC towards either pancreatic hormone-expressing cells (Rezania et al. 2012; Rezania et al. 2014) or maturing β-cells (Rezania et al. 2014). The impact of the PanMa on the generated cells was examined by gene expression analysis, immunohistochemical staining of important stage markers, as well as glucose stimulated insulin secretion assays. In a last part of this study, the potential of the PanMa for the prolonged culture of hiPSC derived endocrine cells for the establishment of an in vitro organ model of the endocrine pancreas was examined. Therefore, a PanMa-derived hydrogel was generated and used for the encapsulation and culture of hiPSC-derived hormone-expressing cells (HECs). The influence of the PanMa-hydrogel culture was analyzed on gene, protein and functional level by gene expression analysis, immunohistochemical stainings and glucose stimulated insulin secretion.
Whole organ decellularization resulted in the generation of an acellular PanMa scaffold, with low amounts of residual DNA and a preserved ECM micro- and ultrastructure, including important ECM components, such as collagen I, III and IV. Furthermore, the PanMa maintained an intact vessel system and was verified as cytocompatible as demonstrated by the successful recellularization of the arterial system with human endothelial cells. In comparison to SISser and LungMa, the PanMa was characterized as a relative soft, hygroscopic scaffold with a collagen-fiber based structure. Furthermore, the findings indicate that the ECM-specific properties have a relevant effect on the stem cell character and early multi-lineage decisions of hiPSCs. In this regard, maintenance of hiPSCs on the PanMa resulted in a slightly changed expression of pluripotency genes (OCT4, SOX2 and NANOG) and a weak immunohistochemical signal for NANOG protein, indicating a PanMa-dependent impact on hiPSC pluripotency. Strikingly, this presumption was corroborated by the finding that culture on the PanMa promoted an endodermal development of hiPSCs during spontaneous differentiation. In line with that, pancreatic differentiation of hiPSC on both the PanMa and SISser resulted in a significant decrease of glucagon and somatostatin gene expression as well as an unaltered insulin expression, suggesting an ECM-driven suppression of the development of non β-cell endocrine cells. However, this change did not result in an improved glucose stimulated insulin secretion of the generated HECs. Moreover, use of the PanMa as a hydrogel allowed prolonged culture of these cells in a defined culture system. HECs were viable after 21 days of culture, however already showed an altered islet morphology as well as a slightly decreased glucose stimulated insulin secretion.
Altogether, this study demonstrates a relevant biological effect of tissue specific ECM cues on the in vitro differentiation of hiPSCs. More specifically, the data indicate an involvement of the ECM in the endocrine commitment of hiPSC-derived pancreatic cells during directed differentiation highlighting the ECM as an important regulator of pancreatic development. Collectively, these findings emphasize the relevance of the ECM for the fabrication of functional hiPSC-derived cell types and suggest a much stronger consideration of organ specific ECM cues for tissue engineering approaches as well as clinical translation in regenerative medicine.
To reach their target site, systemic pesticides must enter the plant from a spray droplet applied in the field. The uptake of an active ingredient (AI) takes place via the barrier-forming cuticular membrane, which is the outermost layer of the plant, separating it from the surrounding environment. Formulations are usually used which, in addition to the AI, also contain stabilizers and adjuvants. Adjuvants can either have surface-active properties or they act directly as barrier-modifying agents. The latter are grouped in the class of accelerating adjuvants, whereby individual variants may also have surface-active properties. The uptake of a pesticide from a spray droplet depends essentially on its permeability through the cuticular barrier. Permeability defines a combined parameter, which is the product of AI mobility and AI solubility within the cuticle. In recent decades, several tools have been developed that allowed the determination of individual parameters of organic compound penetration across the cuticular membrane. Nevertheless, earlier studies showed that mainly cuticular waxes are the barrier-determining component of the cuticular membrane and additionally, it was shown that mainly the very-long-chain aliphatic compounds (VLCAs) are responsible for establishing an effective barrier. However, the barrier-determining role of the individual VLCAs, being classified according to their respective functional groups, is still unknown.
Therefore, the following objectives were pursued and achieved in this work: (1) A new ATR-FTIR-based approach was developed to measure the temperature-dependent real-time diffusion kinetics of organic models for active ingredients (AIs) in paraffin wax, exclusively consisting of very-long chain alkanes. (2) The developed ATR-FTIR approach was applied to determine the diffusion kinetics of self-accelerating adjuvants in cuticular model waxes of different VLCA composition. At the same time, wax-specific changes were recorded in the respective IR spectra, which provided information about the respective wax modification. (3) The ATR-FTIR method was used to characterize the diffusion kinetics, as well as to determine the wax-specific sorption capacities for an AI-modeling organic compound and water in cuticular model waxes after adjuvant treatment. Regarding the individual chemical compositions and structures, conclusions were drawn about the adjuvant-specific modes of action (MoA).
In the first chapter, the ATR-FTIR based approach to determine organic compound diffusion kinetics in paraffin wax was successfully established. The diffusion kinetics of the AI modelling organic compounds heptyl parabene (HPB) and 4-cyanophenol (CNP) were recorded, comprising different lipophilicities and molecular volumes typical for AIs used in pesticide formulations. Derived diffusion coefficients ranged within 10-15 m2 s-1, thus being thoroughly higher than those obtained from previous experiments using an approach solely investigating desorption kinetics in reconstituted cuticular waxes. An ln-linear dependence between the diffusion coefficients and the applied diffusion temperature was demonstrated for the first time in cuticular model wax, from which activation energies were derived. The determined activation energies were 66.2 ± 7.4 kJ mol-1 and 56.4 ± 9.8 kJ mol-1, being in the expected range of already well-founded activation energies required for organic compound diffusion across cuticular membranes, which again confirmed the significant contribution of waxes to the cuticular barrier. Deviations from the assumed Fickian diffusion were attributed to co-occurring water diffusion and apparatus-specific properties.
In the second and third chapter, mainly the diffusion kinetics of accelerating adjuvants in the cuticular model waxes candelilla wax and carnauba wax were investigated, and simultaneously recorded changes in the wax-specific portion of the IR spectrum were interpreted as indications of plasticization. For this purpose, the oil derivative methyl oleate, as well as the organophosphate ester TEHP and three non-ionic monodisperse alcohol ethoxylates (AEs) C12E2, C12E4 and C12E6 were selected. Strong dependence of diffusion on the respective principal components of the mainly aliphatic waxes was demonstrated. The diffusion kinetics of the investigated adjuvants were faster in the n-alkane dominated candelilla wax than in the alkyl ester dominated carnauba wax. Furthermore, the equilibrium absorptions, indicating equilibrium concentrations, were also higher in candelilla wax than in carnauba wax. It was concluded that alkyl ester dominated waxes feature higher resistance to diffusion of accelerating adjuvants than alkane dominated waxes with shorter average chain lengths due to their structural integrity. This was also found either concerning candelilla/policosanol (n-alcohol) or candelilla/rice bran wax (alkyl-esters) blends: with increasing alcohol concentration, the barrier function was decreased, whereas it was increased with increasing alkyl ester concentration. However, due to the high variability of the individual diffusion curves, only a trend could be assumed here, but significant differences were not shown. The variability itself was described in terms of fluctuating crystalline arrangements and partial phase separation of the respective wax mixtures, which had inevitable effects on the adjuvant diffusion. However, diffusion kinetics also strongly depended on the studied adjuvants. Significantly slower methyl oleate diffusion accompanied by a less pronounced reduction in orthorhombic crystallinity was found in carnauba wax than in candelilla wax, whereas TEHP diffusion was significantly less dependent on the respective wax structure and therefore induced considerable plasticization in both waxes. Of particular interest was the AE diffusion into both waxes. Differences in diffusion kinetics were also found here between candelilla blends and carnauba wax. However, these depended equally on the degree of ethoxylation of the respective AEs. The lipophilic C12E2 showed approximately Fickian diffusion kinetics in both waxes, accompanied by a drastic reduction in orthorhombic crystallinity, especially in candelilla wax, whereas the more hydrophilic C12E6 showed significantly retarded diffusion kinetics associated with a smaller effect on orthorhombic crystallinity. The individual diffusion kinetics of the investigated adjuvants sometimes showed drastic deviations from the Fickian diffusion model, indicating a self-accelerating effect. Hence, adjuvant diffusion kinetics were accompanied by a distinct initial lag phase, indicating a critical concentration in the wax necessary for effective penetration, leading to sigmoidal rather than to exponential diffusion kinetics.
The last chapter dealt with the adjuvant-affected diffusion of the AI modelling CNP in candelilla and carnauba wax. Using ATR-FTIR, diffusion kinetics were recorded after adjuvant treatment, all of which were fully explicable based on the Fickian model, with high diffusion coefficients ranging from 10-14 to 10-13 m2 s-1. It is obvious that the diffusion coefficients presented in this work consistently demonstrated plasticization induced accelerated CNP mobilities. Furthermore, CNP equilibrium concentrations were derived, from which partition- and permeability coefficients could be determined. Significant differences between diffusion coefficients (mobility) and partition coefficients (solubility) were found on the one hand depending on the respective waxes, and on the other hand depending on treatment with respective adjuvants. Mobility was higher in candelilla wax than in carnauba wax only after methyl oleate treatment. Treatment with TEHP and AEs resulted in higher CNP mobility in the more polar alkyl ester dominated carnauba wax. The partition coefficients, on the other hand, were significantly lower after methyl oleate treatment in both candelilla and carnauba wax as followed by TEHP or AE treatment. Models were designed for the CNP penetration mode considering the respective adjuvants in both investigated waxes. Co-penetrating water, which is the main ingredient of spray formulations applied in the field, was likely the reason for the drastic differences in adjuvant efficacy. Especially the investigated AEs favored an enormous water uptake in both waxes with increasing ethoxylation level. Surprisingly, this effect was also found for the lipophilic TEHP in both waxes. This led to the assumption that the AI permeability is not exclusively determined by adjuvant induced plasticization, but also depends on a “secondary plasticization”, induced by adjuvant-attracted co-penetrating water, consequently leading to swelling and drastic destabilization of the crystalline wax structure.
The successful establishment of the presented ATR-FTIR method represents a milestone for the study of adjuvant and AI diffusion kinetics in cuticular waxes. In particular, the simultaneously detectable wax modification and, moreover, the determinable water uptake form a perfect basis to establish the ATR-FTIR system as a universal screening tool for wax-adjuvants-AI-water interaction in crop protection science.
Magnetism is a phenomenon ubiquitously found in everyday life. Yet, together with superconductivity and superfluidity, it is among the few macroscopically realized quantum states. Although well-understood on a quasi-classical level, its microscopic description is still far from being solved. The interplay of strong interactions present in magnetic condensed-matter systems and the non-trivial commutator structure governing the underlying spin algebra prevents most conventional approaches in solid-state theory to be applied.
On the other hand, the quantum limit of magnetic systems is fertile land for the development of exotic phases of matter called spin-liquids. In these states, quantum fluctuations inhibit the formation of magnetic long-range order down to the lowest temperatures. From a theoretical point of view, spin-liquids open up the possibility to study their exotic properties, such as fractionalized excitations and emergent gauge fields. However, despite huge theoretical and experimental efforts, no material realizing spin-liquid properties has been unambiguously identified with a three-dimensional crystal structure. The search for such a realization is hindered by the inherent difficulty even for model calculations. As most numerical techniques are not applicable due to the interaction structure and dimensionality of these systems, a methodological gap has to be filled.
In this thesis, to fill this void, we employ the pseudo-fermion functional renormalization group (PFFRG), which provides a scheme to investigate ground state properties of quantum magnetic systems even in three spatial dimensions.
We report the status quo of this established method and extend it by alleviating some of its inherent approximations. To this end, we develop a multi-loop formulation of PFFRG, including hitherto neglected terms in the underlying flow equations consistently, rendering the outcome equivalent to a parquet approximation. As a necessary prerequisite, we also significantly improve the numerical accuracy of our implementation of the method by switching to a formulation respecting the asymptotic behavior of the vertex functions as well as employing state-of-the-art numerical algorithms tailored towards PFFRG. The resulting codebase was made publicly accessible in the open-source code PFFRGSolver.jl.
We subsequently apply the technique to both model systems and real materials. Augmented by a classical analysis of the respective models, we scan the phase diagram of the three-dimensional body-centered cubic lattice up to third-nearest neighbor coupling and the Pyrochlore lattice up to second-nearest neighbor. In both systems, we uncover in addition to the classically ordered phases, an extended parameter regime, where a quantum paramagnetic phase appears, giving rise to the possibility of a quantum spin liquid.
Additionally, we also use the nearest-neighbor antiferromagnet on the Pyrochlore lattice as well as the simple cubic lattice with first- and third-nearest neighbor couplings as a testbed for multi-loop PFFRG, demonstrating, that the inclusion of higher loop orders has quantitative effects in paramagnetic regimes and that the onset of order can be signaled by a lack of loop convergence.
Turning towards material realizations, we investigate the diamond lattice compound MnSc\(_2\)S\(_4\), explaining on grounds of ab initio couplings the emergence of a spiral spin liquid at low temperatures, but above the ordering transition.
In the Pyrochlore compound Lu\(_2\)Mo\(_2\)O\(_5\)N\(_2\), which is known to not magnetically order down to lowest temperatures, we predict a spin liquid state displaying a characteristic gearwheel pattern in the spin structure factor.
In this thesis, we are interested in numerically preserving stationary solutions of balance laws. We start by developing finite volume well-balanced schemes for the system of Euler equations and the system of MHD equations with gravitational source term. Since fluid models and kinetic models are related, this leads us to investigate AP schemes for kinetic equations and their ability to preserve stationary solutions. Kinetic models typically have a stiff term, thus AP schemes are needed to capture good solutions of the model. For such kinetic models, equilibrium solutions are reached after large time. Thus we need a new technique to numerically preserve stationary solutions for AP schemes. We find a criterion for SP schemes for kinetic equations which states, that AP schemes under a particular discretization are also SP. In an attempt to mimic our result for kinetic equations in the context of fluid models, for the isentropic Euler equations we developed an AP scheme in the limit of the Mach number going to zero. Our AP scheme is proven to have a SP property under the condition that the pressure is a function of the density and the latter is obtained as a solution of an elliptic equation. The properties of the schemes we developed and its criteria are validated numerically by various test cases from the literature.
Current therapeutic strategies efficiently improve survival in patients after myocardial infarction (MI). Nevertheless, long-term consequences such as heart failure development, are still one of the leading causes of death worldwide. Inflammation is critically involved in the cardiac healing process after MI and has a dual role, contributing to both tissue healing and tissue damage. In the last decade, a lot of attention was given to targeting inflammation as a potential therapeutic approach in MI, but the poor understanding of inflammatory cell heterogeneity and function is a limit to the development of immune modulatory strategies. The recent development of tools to profile immune cells with high resolution has provided a unique opportunity to better understand immune cell heterogeneity and dynamics in the ischemic heart.
In this thesis, we employed single-cell RNA-sequencing combined with detection of epitopes by sequencing (CITE-seq) to refine our understanding of neutrophils and monocytes/macrophages heterogeneity and dynamic after experimental myocardial infarction.
Neutrophils rapidly invade the infarcted heart shortly after ischemic damage and have previously been proposed to display time-dependent functional heterogeneity. At the single-cell level, we observed dynamic transcriptional heterogeneity in neutrophil populations during the acute post-MI phase and defined previously unknown cardiac neutrophil states. In particular, we identified a locally acquired SiglecFhi neutrophil state that displayed higher ROS production and phagocytic ability compared to newly recruited neutrophils, suggesting the acquisition of specific function in the infarcted heart. These findings highlight the importance of the tissue microenvironment in shaping neutrophil response.
From the macrophage perspective, we characterized MI-associated monocyte-derived macrophage subsets, two with a pro-inflammatory gene signature (MHCIIhiIl1βhi) and three Trem2hi macrophage populations with a lipid associated macrophage (LAM) signature, also expressing pro-fibrotic and tissue repair genes. Combined analysis of blood monocytes and cardiac monocyte/macrophages indicated that the Trem2hi LAM signature is acquired in the infarcted heart.
We furthermore characterized the role of TREM2, a surface protein expressed mainly in macrophages and involved in macrophage survival and function, in the post-MI macrophage response and cardiac repair. Using TREM2 deficient mice, we demonstrate that acquisition of the LAM signature in cardiac macrophages after MI is partially dependent on TREM2. While their cardiac function was not affected, TREM2 deficient mice showed reduced collagen deposition in the heart after MI. Thus, our data in Trem2-deficient mice highlight the role of TREM2 in promoting a macrophage pro-fibrotic phenotype, in line with the pro-fibrotic/tissue repair gene signature of the Trem2hi LAM-signature genes.
Overall, our data provide a high-resolution characterization of neutrophils and macrophage heterogeneity and dynamics in the ischemic heart and can be used as a valuable resource to investigate how these cells modulate the healing processes after MI. Furthermore, our work identified TREM2 as a regulator of macrophage phenotype in the infarcted heart
The universal two-child policy was introduced by the central government of China in 2016 to respond to the country’s deteriorating population problems, but it was soon replaced by a three-child policy in 2021 given that it failed to continuously boost fertility in Chinese society. This dissertation empirically investigates the implementation of universal two-child policy in three Chinese major cities. Based on the data collected through semi-structured interviews with leaders of local family planning agencies, it finds that local officials are primarily devoted to coping with the discontent of the bereaved single-child parents (shidu families), which is an unexpected consequence of the historical one-child policy, rather than working on the tasks regarding birth encouragement. The dissertation suggests understanding the implementation of China’s population policy within the framework of both historical and rational choice institutionalism. The target responsibility system as an effective tool of the central authority drives local agents to fix their attention at tasks that have larger impact on their career. The shifted focus in the implementation of the universal two-child policy is a result of local officials’ emphasis on the task of maintaining social stability. Shidu families are deemed as a salient threat to social order because their discontent with the state support has incurred continuous petitions at both the national and local level, which would severely undermine local officials’ career advancement. However, in the meantime, stability maintenance is found to have become alienated as reflected by the rising costs and that it replaced birth support to be the focus of local family planning agents in the universal two-child policy era. Since the conflict between the shidu group and the state is unlikely to be resolved, the future population policy design and enforcement will continue to be constrained by the shidu problem.
The nervous system relies on an orchestrated assembly of complex cellular entities called neurons, which are specifically committed to information management and transmission. Inter-neuronal communication takes place via synapses, membrane-membrane junctions which ensure efficient signal transfer. Synaptic neurotransmission involves release of presynaptic neurotransmitters and their reception by cognate receptors at postsynaptic terminals. Inhibitory neurotransmission is primarily mediated by the release of neurotransmitters GABA (γ-Aminobutyric acid) and glycine, which are precisely sensed by GABA type-A receptors (GABAARs) and glycine receptors (GlyRs), respectively. GABAAR assembly and maintenance is coordinated by various postsynaptic neuronal factors including the scaffolding protein gephyrin, the neuronal adaptor collybistin (CB) and cell adhesion proteins of the neuroligin (NL) family, specifically NL2 and NL4.
At inhibitory postsynaptic specializations, gephyrin has been hypothesized to form extended structures underneath the plasma membrane, where its interaction with the receptors leads to their stabilization and impedes their lateral movement. Gephyrin mutations have been associated with various brain disorders, including autism, schizophrenia, Alzheimer’s disease, and epilepsy. Furthermore, gephyrin loss is lethal and causes mice to die within the first post-natal day. Gephyrin recruitment from intracellular deposits to postsynaptic membranes primarily relies on the adaptor protein CB.
As a moonlighting protein, CB, a guanine nucleotide exchange factor (GEF), also catalyzes a nucleotide exchange reaction, thereby regenerating the GTP-bound state of the small GTPase Cdc42 from its GDP-bound form. The CB gene undergoes alternative splicing with the majority of CB splice variants featuring an N-terminal SH3 domain followed by tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains. Previous studies demonstrated that the most widely expressed, SH3-domain containing splice variant (CB2SH3+) preferentially adopts a closed conformation, in which the N-terminally located SH3 domain forms intra-molecular interaction with the DH-PH domain tandem. Previous cell-based studies indicated that SH3 domain-encoding CB variants remain untargeted and colocalize with intracellular gephyrin deposits and hence require additional factors which interact with the SH3 domain, thus inducing an open or active conformation. The SH3 domain-deficient CB isoform (CB2SH3-), on the contrary, adopts an open conformation, which possess enhanced postsynaptic gephyrin-clustering and also effectively replenishes the GTP-bound small GTPase-Cdc42 from its GDP-bound state.
Despite the fundamental role of CB as a neuronal adaptor protein maintaining the proper function of inhibitory GABAergic synapses, its interactions with the neuronal scaffolding protein gephyrin and other post synaptic neuronal factors remain poorly understood. Moreover, CB interaction studies with the small GTPase Cdc42 and TC10, a closely related member of Cdc42 subfamily, remains poorly characterized. Most importantly, the roles of the neuronal factors and small GTPases in CB conformational activation have not been elucidated.
This PhD dissertation primarily focuses on delineating the molecular basis of the interactions between CB and postsynaptic neuronal factors. During the course of my PhD dissertation, I engineered a series of CB FRET (Förster Resonance Energy Transfer) sensors to characterize the CB interaction with its binding partners along with outlining their role in CB conformational activation. Through the aid of these CB FRET sensors, I analyzed the gephyrin-CB interaction, which, due to technical limitations remained unaddressed for more than two decades (refer Chapter 2 for more details). Subsequently, I also unraveled the molecular basis of the interactions between CB and the neuronal cell adhesion factor neuroligin 2 (refer chapter 2) and the small GTPases Cdc42 and TC10 (refer chapter 3) and describe how these binding partners induce a conformational activation of CB.
In summary, this PhD dissertation provides strong evidence of a closely knit CB communication network with gephyrin, neuroligin and the small GTPase TC10, wherein CB activation from closed/inactive to open/active states is effectively triggered by these ligands.
As part of the parasympathetic nervous system, muscarinic receptors are involved in the regulation of numerous functions in the human body. However, targeting a specific subtype of muscarinic receptors is challenging due to the high degree of similarity within the binding site of the endogenous neurotransmitter acetylcholine. Therefore, this study focused on the investigation of dualsteric ligands. Such hybrid ligands target the orthosteric acetylcholine binding site and, simultaneously, a distinct allosteric binding site. Since allosteric binding regions show significant structural differences throughout muscarinic receptor subtypes, it was aimed to produce selective ligands by means of combination of two pharmacophores in one molecule. Herein, the thienopyridine derivatives LY2033298 and LY2119620 were chosen as allosteric moieties. Based on literature studies, the investigated allosteric modulators were analyzed in terms of adequate attachment points for the combination with an orthosteric agonist. As orthosteric units, muscarinic superagonist iperoxo, xanomeline, and TMA were applied in this work. Since the distance between orthosteric and allosteric moieties plays a crucial role for dualsteric ligand binding, the linker chain length was also varied. Pharmacological investigations of the synthesized hybrid ligands were perfomed via FRET- and BRET-assay measurements.
Environmental issues have emerged especially since humans burned fossil fuels, which led to air pollution and climate change that harm the environment. These issues’ substantial consequences evoked strong efforts towards assessing the state of our environment.
Various environmental machine learning (ML) tasks aid these efforts. These tasks concern environmental data but are common ML tasks otherwise, i.e., datasets are split (training, validatition, test), hyperparameters are optimized on validation data, and test set metrics measure a model’s generalizability. This work focuses on the following environmental ML tasks: Regarding air pollution, land use regression (LUR) estimates air pollutant concentrations at locations where no measurements are available based on measured locations and each location’s land use (e.g., industry, streets). For LUR, this work uses data from London (modeled) and Zurich (measured). Concerning climate change, a common ML task is model output statistics (MOS), where a climate model’s output for a study area is altered to better fit Earth observations and provide more accurate climate data. This work uses the regional climate model (RCM) REMO and Earth observations from the E-OBS dataset for MOS. Another task regarding climate is grain size distribution interpolation where soil properties at locations without measurements are estimated based on the few measured locations. This can provide climate models with soil information, that is important for hydrology. For this task, data from Lower Franconia is used.
Such environmental ML tasks commonly have a number of properties: (i) geospatiality, i.e., their data refers to locations relative to the Earth’s surface. (ii) The environmental variables to estimate or predict are usually continuous. (iii) Data can be imbalanced due to relatively rare extreme events (e.g., extreme precipitation). (iv) Multiple related potential target variables can be available per location, since measurement devices often contain different sensors. (v) Labels are spatially often only sparsely available since conducting measurements at all locations of interest is usually infeasible. These properties present challenges but also opportunities when designing ML methods for such tasks.
In the past, environmental ML tasks have been tackled with conventional ML methods, such as linear regression or random forests (RFs). However, the field of ML has made tremendous leaps beyond these classic models through deep learning (DL). In DL, models use multiple layers of neurons, producing increasingly higher-level feature representations with growing layer depth. DL has made previously infeasible ML tasks feasible, improved the performance for many tasks in comparison to existing ML models significantly, and eliminated the need for manual feature engineering in some domains due to its ability to learn features from raw data. To harness these advantages for environmental domains it is promising to develop novel DL methods for environmental ML tasks.
This thesis presents methods for dealing with special challenges and exploiting opportunities inherent to environmental ML tasks in conjunction with DL. To this end, the proposed methods explore the following techniques: (i) Convolutions as in convolutional neural networks (CNNs) to exploit reoccurring spatial patterns in geospatial data. (ii) Posing the problems as regression tasks to estimate the continuous variables. (iii) Density-based weighting to improve estimation performance for rare and extreme events. (iv) Multi-task learning to make use of multiple related target variables. (v) Semi–supervised learning to cope with label sparsity. Using these techniques, this thesis considers four research questions: (i) Can air pollution be estimated without manual feature engineering? This is answered positively by the introduction of the CNN-based LUR model MapLUR as well as the off-the-shelf LUR solution OpenLUR. (ii) Can colocated pollution data improve spatial air pollution models? Multi-task learning for LUR is developed for this, showing potential for improvements with colocated data. (iii) Can DL models improve the quality of climate model outputs? The proposed DL climate MOS architecture ConvMOS demonstrates this. Additionally, semi-supervised training of multilayer perceptrons (MLPs) for grain size distribution interpolation is presented, which can provide improved input data. (iv) Can DL models be taught to better estimate climate extremes? To this end, density-based weighting for imbalanced regression (DenseLoss) is proposed and applied to the DL architecture ConvMOS, improving climate extremes estimation. These methods show how especially DL techniques can be developed for environmental ML tasks with their special characteristics in mind. This allows for better models than previously possible with conventional ML, leading to more accurate assessment and better understanding of the state of our environment.
The use of digital media by children and young people offers opportunities for communication, collaboration, and participation. However, to prepare them for the risks and challenges of media usage, promoting digital competencies of students and teachers is an indispensable goal for educational institutions. To meet this requirement, teacher education must be opened to innovative pedagogical concepts for initial teacher education that considers new technologies in a reflective, action-oriented way to promote competencies. Therefore, this work aims to promote the technological pedagogical content knowledge (TPACK) of prospective teachers that enables the purposeful integration of social virtual reality (social VR) into the classroom. Consequently, a pedagogical concept is developed and evaluated in an iterative research and development process following the design- based research approach (DBR) through four consecutive studies. The first study involved an analysis of the requirements of teachers and students for the effective use of social VR in the classroom. The second study examined how prospective teachers perceive teaching and learning activities within two theory-driven scenarios in social VR. The third study investigated the development of Technological Pedagogical Content Knowledge (TPACK) among students in social VR compared to video-based communication. Finally, the fourth study measured the development of TPACK in social VR using epistemic network analysis, finding that social VR can be an effective tool for teacher education, emphasizing the importance of authentic contexts and practical experiences for effective teaching in social VR. In the concluding chapter, appropriate implications for teacher education research and practice are derived from findings. For example, that a deeper understanding of TPACK as metacognitive awareness could enhance teacher education for media integration. It also highlights the need for digital literacy in seminars that address new technologies, emphasizing the importance of considering moral values and sustainability when using VR.
The holy grail of structural biology is to study a protein in situ, and this goal has been fast approaching since the resolution revolution and the achievement of atomic resolution. A cell's interior is not a dilute environment, and proteins have evolved to fold and function as needed in that environment; as such, an investigation of a cellular component should ideally include the full complexity of the cellular environment. Imaging whole cells in three dimensions using electron cryotomography is the best method to accomplish this goal, but it comes with a limitation on sample thickness and produces noisy data unamenable to direct analysis. This thesis establishes a novel workflow to systematically analyse whole-cell electron cryotomography data in three dimensions and to find and identify instances of protein complexes in the data to set up a determination of their structure and identity for success. Mycoplasma pneumoniae is a very small parasitic bacterium with fewer than 700 protein-coding genes, is thin enough and small enough to be imaged in large quantities by electron cryotomography, and can grow directly on the grids used for imaging, making it ideal for exploratory studies in structural proteomics. As part of the workflow, a methodology for training deep-learning-based particle-picking models is established.
As a proof of principle, a dataset of whole-cell Mycoplasma pneumoniae tomograms is used with this workflow to characterize a novel membrane-associated complex observed in the data. Ultimately, 25431 such particles are picked from 353 tomograms and refined to a density map with a resolution of 11 Å. Making good use of orthogonal datasets to filter search space and verify results, structures were predicted for candidate proteins and checked for suitable fit in the density map. In the end, with this approach, nine proteins were found to be part of the complex, which appears to be associated with chaperone activity and interact with translocon machinery.
Visual proteomics refers to the ultimate potential of in situ electron cryotomography: the comprehensive interpretation of tomograms. The workflow presented here is demonstrated to help in reaching that potential.
The present cumulative dissertation summarizes three clinical studies, which examine
subgroups of patients within the fibromyalgia syndrome (FMS). FMS entails chronic pain and
associated symptoms, and its pathophysiology is incompletely understood (1). Previous studies
show that there is a subgroup of patients with FMS with objective histological pathology of the
small nerve fibers of the peripheral nervous system (PNS). Another subgroup of FMS patients
does not show any signs of pathological changes of the small nerve fibers. The aim of this
dissertation was to compare FMS patients with healthy controls, and these two FMS subgroups
for differences in the central nervous system (CNS) in order to explore possible interactions
between PNS and the CNS. Regarding the CNS, differences of FMS patients with healthy
controls have already been found in studies with small sample sizes, but no subgroups have yet
been identified. Another aim of this thesis was to test whether the subgroups show a different
response to different classes of pain medication. The methods used in this thesis are structural
and functional magnetic resonance imaging (MRI), magnetic resonance diffusion imaging and
magnetic resonance spectroscopy. For the evaluation of clinical symptoms, we used
standardized questionnaires. The subgroups with and without pathologies of the PNS were
determined by skin biopsies of the right thigh and lower leg based on the intraepidermal nerve
fiber density (IENFD) of the small nerve fibers.
1) In the first MRI study, 43 female patients with the diagnosis of FMS and 40 healthy
control subjects, matched in age and body mass index, were examined with different MRI
sequences. Cortical thickness was investigated by structural T1 imaging, white matter integrity
by diffusion tensor imaging and functional connectivity within neuronal networks by functional
resting state MRI. Compared to the controls, FMS patients had a lower cortical volume in
bilateral frontotemporoparietal regions and the left insula, but a higher cortical volume in the
left pericalcarine cortex. Compared to the subgroup without PNS pathology, the subgroup with
PNS pathology had lower cortical volume in both pericalcarine cortices. Diffusion tensor
imaging revealed an increased fractional anisotropy (FA) of FMS patients in corticospinal
pathways such as the corona radiata, but also in regions of the limbic systems such as the fornix
and cingulum. Subgroup comparison again revealed lower mean FA values of the posterior
thalamic radiation and the posterior limb of the left internal capsule in the subgroup with PNS
pathology. In the functional connectivity analysis FMS patients, compared to controls, showed
a hypoconnectivity between the right median frontal gyrus and the posterior cerebellum and
the right crus cerebellum, respectively. In the subgroup comparisons, the subgroup with PNS
pathology showed a hyperconnectivity between both inferior frontal gyri, the right posterior
parietal cortex and the right angular gyrus. In summary, these results show that differences in
brain morphology and functional connectivity exist between FMS patients with and without
PNS pathology. These differences were not associated with symptom duration or severity and,
in some cases, have not yet been described in the context of FMS. The differences in brain
morphology and connectivity between subgroups could also lead to a differential response to
treatment with centrally acting drugs. Further imaging studies with FMS patients should take
into account this heterogeneity of FMS patient cohorts.
2) Following the results from the first MRI study, drug therapies of FMS patients and
their treatment response were compared between PNS subgroups. As there is no licensed drug
for FMS in Europe, the German S3 guideline recommends amitriptyline, duloxetine and
pregabalin for temporary use. In order to examine the current drug use in FMS patients in
Germany on a cross-sectional basis, 156 patients with FMS were systematically interviewed.
The drugs most frequently used to treat pain in FMS were non-steroidal anti-inflammatory
drugs (NSAIDs) (28.9%), metamizole (15.4%) and amitriptyline (8.8%). Pain relief assessed by
patients on a numerical rating scale from 0-10 averaged 2.2 points for NSAIDs, 2.0 for
metamizole and 1.5 for amitriptyline. Drugs that were discontinued for lack of efficacy and not
for side effects were acetaminophen (100%), flupirtine (91.7%), selective serotonin reuptake
inhibitors (81.8%), NSAIDs (83.7%) and weak opioids (74.1%). Patients were divided into
subgroups with and without PNS pathology as determined by skin biopsies. We found no
differences in drug use and effect between the subgroups. Taken together, these results show
that many FMS patients take medication that is not in accordance with the guidelines. The
reduction of symptoms was best achieved with metamizole and NSAIDs. Further longitudinal
studies on medication in FMS are necessary to obtain clearer treatment recommendations.
3) Derived from previous pharmacological and imaging studies (with smaller case
numbers), there is a hypothesis in the FMS literature that hyperreactivity of the insular cortex
may have an impact on FMS. The hyperreactivity seems to be due to an increased concentration
of the excitatory neurotransmitter glutamate in the insular cortex of FMS patients. The
hypothesis is supported by magnetic resonance spectroscopy studies with small number of
cases, as well as results from pharmacological studies with glutamate-inhibiting medication.
Studies from animal models have also shown that an artificially induced increase in glutamate
in the insular cortex can lead to reduced skin innervation. Therefore, the aim of this study was
to compare glutamate and GABA concentrations in the insular cortex of FMS patients with
those of healthy controls using magnetic resonance imaging. There was no significant
difference of both neurotransmitters between the groups. In addition, there was no correlation
between the neurotransmitter concentrations and the severity of clinical symptoms. There
were also no differences in neurotransmitter concentrations between the subgroups with and
without PNS pathology. In conclusion, our study could not show any evidence of a correlation
of glutamate and GABA concentrations with the symptoms of FMS or the pathogenesis of
subgroups with PNS pathologies.
The monarch butterfly (Danaus plexippus) performs one of the most astonishing behaviors in the animal kingdom: every fall millions of these butterflies leave their breeding grounds in North Amerika and migrate more than 4.000 km southwards until they reach their overwintering habitat in Central Mexico. To maintain their migratory direction over this enormous distance, the butterflies use a time-compensated sun compass. Beside this, skylight polarization, the Earth’s magnetic field and specific mountain ranges seem to guide the butterflies as well the south. In contrast to this fascinating orientation ability, the behavior of the butterflies in their non-migratory state received less attention. Although they do not travel long distances, they still need to orient themselves to find food, mating partners or get away from competitors. The aim of the present doctoral thesis was to investigate use of visual cues for orientation in migrating as well as non-migrating monarch butterflies. For this, field experiments investigating the migration of the butterflies in Texas (USA) were combined with experiments testing the orientation performance of non-migratory butterflies in Germany.
In the first project, I recorded the heading directions of tethered butterflies during their annual fall migration. In an outdoor flight simulator, the butterflies maintained a southwards direction as long as they had a view of the sun’s position. Relocating the position of the sun by 180° using a mirror, revealed that the sun is the animals’ main orientation reference. Furthermore, I demonstrated that when the sun is blocked and a green light stimulus (simulated sun) is introduced, the animals interpreted this stimulus as the ‘real’ sun. However, this cue was not sufficient to set the migratory direction when simulated as the only visual cue in indoor experiments. When I presented the butterflies a linear polarization pattern additionally to the simulated sun, the animals headed in the correct southerly direction showing that multiple skylight cues are required to guide the butterflies during their migration.
In the second project, I, furthermore, demonstrated that non-migrating butterflies are able to maintain a constant direction with respect to a simulated sun. Interestingly, they ignored the spectral component of the stimulus and relied on the intensity instead. When a panoramic skyline was presented as the only orientation reference, the butterflies maintained their direction only for short time windows probably trying to stabilize their flight based on optic-flow information. Next, I investigated whether the butterflies combine celestial with local cues by simulating a sun stimulus together with a panoramic skyline. Under this conditions, the animals’ directedness was increased demonstrating that they combine multiple visual cues for spatial orientation.
Following up on the observation that a sun stimulus resulted in a different behavior than the panoramic skyline, I investigated in my third project which orientation strategies the butterflies use by presenting different simulated cues to them. While a bright stripe on a dark background elicited a strong attraction of the butterflies steering in the direction of the stimulus, the inverted version of the stimulus was used for flight stabilization. In contrast to this, the butterflies maintained arbitrary directions with a high directedness with respect to a simulated sun. In an ambiguous scenery with two identical stimuli (two bright stripes, two dark stripes, or two sun stimuli) set 180° apart, a constant flight course was only achieved when two sun stimuli were displayed suggesting an involvement of the animals’ internal compass. In contrast, the butterflies used two dark stripes for flight stabilization and were alternatingly attracted by two bright stripes. This shows that monarch butterflies use stimulus-dependent orientation strategies and gives the first evidence for different neuronal pathways controlling the output behavior.
Beyond the four canonical nucleosides as primary building blocks of RNA, posttranscriptional modifications give rise to the epitranscriptome as a second layer of genetic information. In eukaryotic mRNA, the most abundant posttranscriptional modification is N6-methyladenosine (m6A), which is involved in the regulation of cellular processes. Throughout this thesis, the concept of atomic mutagenesis was employed to gain novel mechanistic insights into the substrate recognition by human m6A reader proteins as well as in the oxidative m6A demethylation by human demethylase enzymes. Non-natural m6A atomic mutants featuring distinct steric and electronic properties were synthesized and incorporated into RNA oligonucleotides. Fluorescence anisotropy measurements using these modified oligonucleotides revealed the impact of the atomic mutagenesis on the molecular recognition by the human m6A readers YTHDF2, YTHDC1 and YTHDC2 and allowed to draw conclusions about structural prerequisites for substrate recognition. Furthermore, substrate recognition and demethylation mechanism of the human m6A demethylase enzymes FTO and ALKBH5 were analyzed by HPLC-MS and PAGE-based assays using the modified oligonucleotides synthesized in this work.
Modified nucleosides not only expand the genetic alphabet, but are also extensively researched as drug candidates. In this thesis, the antiviral mechanism of the anti-SARS-CoV-2 drug remdesivir was investigated, which causes delayed stalling of the viral RNA-dependent RNA polymerase (RdRp). Novel remdesivir phosphoramidite building blocks were synthesized and used to construct defined RNA-RdRp complexes for subsequent studies by cryogenic electron microscopy (cryo-EM). It was found that the 1'-cyano substituent causes Rem to act as a steric barrier of RdRp translocation. Since this translocation barrier can eventually be overcome by the polymerase, novel derivatives of Rem with potentially improved antiviral properties were designed.
Pulmonary artery embolism (PE) is a common condition and an even more common clinical suspect. The computed tomography pulmonary angiogram (CTPA) is the main medical imaging tool used to diagnose a suspected case of PE. To gain a better impression of the effects of a PE on the perfusion and hence the gas exchange, a functional imaging method is beneficial. One approach for functional imaging using radiation exposure is the generation of color-coded iodine perfusion maps acquired by Dual-Energy Computed Tomography (DECT), which enable the detection of perfusion defects in the pulmonary parenchyma. In contrast to the existing approach of DECT with iodine color-coded maps, the SElf-gated Non-Contrast-Enhanced FUnctional Lung (SENCEFUL) MRI technique offers the possibility to interpret perfusion maps without any radiation exposure or application of contrast agents. The measurement in SENCEFUL MRI can be performed during conditions of free breathing and without electrocardiogram triggering.
The purpose of this study was to determine whether PE can be diagnosed on the basis of visible perfusion defects in the perfusion maps of SENCEFUL MRI and in the iodine-coded maps of DECT and to compare the diagnostic performance of these methods. Both SENCEFUL-MRI and iodine distribution maps from DECT have been compared with the CTPA of ten patients with PE. Additionally, the functional images were compared with each other on a per-patient basis.
The iodine perfusion maps of DECT had a sensitivity of 84.2 % and specificity of 65.2 % for the diagnosis of PE. The SENCEFUL technique in MRI showed a sensitivity of 78.9 % and a specificity of 26.1 %. When comparing the whole lung depicted in both series of functional images, the main perfusion defect location matched in four of ten patients (40 %).
In conclusion, this work found that DECT iodine maps have higher sensitivity and specificity in the diagnosis of pulmonary embolism compared with SENCEFUL MRI.
Coxiella burnetii, a Gram negative obligate intracellular bacterium, is the causative
agent of Q fever. It has a world wide distribution and has been documented to
be capable of causing infections in several domestic animals, livestock species,
and human beings. Outbreaks of Q fever are still being observed in livestock
across animal farms in Europe, and primary transmission to humans still oc-
curs especially in animal handlers. Public health authorities in some countries
like Germany are required by law to report human acute cases denoting the
significance of the challenge posed by C. burnetii to public health.
In this thesis, I have developed a platform alongside methods to address the
challenges of genomic analyses of C. burnetii for typing purposes. Identification
of C. burnetii isolates is an important task in the laboratory as well as in the
clinics and genotyping is a reliable method to identify and characterize known
and novel isolates. Therefore, I designed and implemented several methods
to facilitate the genotyping analyses of C. burnetii genomes in silico via a web
platform. As genotyping is a data intensive process, I also included additional
features such as visualization methods and databases for interpretation and
storage of obtained results. I also developed a method to profile the resistome
of C. burnetii isolates using a machine learning approach. Data about antibiotic
resistance in C. burnetii are scarce majorly due to its lifestyle and the difficulty
of cultivation in laboratory media. Alternative methods that rely on homology
identification of resistance genes are also inefficient in C. burnetii, hence, I
opted for a novel approach that has been shown to be promising in other
bacteria species. The applied method relied on an artificial neural network as
well as amino acid composition of position specific scoring matrix profile for
feature extraction. The resulting model achieved an accuracy of ≈ 0.96 on test
data and the overall performance was significantly higher in comparison to
existing models. Finally, I analyzed two new C. burnetii isolates obtained from
an outbreak in Germany, I compared the genome to the RSA 493 reference
isolate and found extensive deletions across the genome landscape.
This work has provided a new digital infrastructure to analyze and character-
ize C. burnetii genomes that was not in existence before and it has also made a
significant contribution to the existing information about antibiotic resistance
genes in C. burnetii.
The discovery, heterologous expression, and characterization of channelrhodopsin-2 (ChR2) – a light-sensitive cation channel found in the green alga Chlamydomonas reinhardtii – led to the success of optogenetics as a powerful technology, first in neuroscience. ChR2 was employed to induce action potentials by blue light in genetically modified nerve cells. In optogenetics, exogenous photoreceptors are expressed in cells to manipulate cellular activity. These photoreceptors were in the beginning mainly microbial opsins. During nearly two decades, many microbial opsins and their mutants were explored for their application in neuroscience. Until now, however, the application of optogenetics to plant studies is limited to very few reports. Several optogenetic strategies for plant research were demonstrated, in which most attempts are based on non-opsin optogenetic tools. Opsins need retinal (vitamin A) as a cofactor to generate the functional protein, the rhodopsin. As most animals have eyes that contain animal rhodopsins, they also have the enzyme - a 15, 15'-Dioxygenase - for retinal production from food-supplied provitamin A (beta-carotene). However, higher plants lack a similar enzyme, making it difficult to express functional rhodopsins successfully in plants. But plant chloroplasts contain plenty of beta-carotene. I introduced a gene, coding for a 15, 15'-Dioxygenase with a chloroplast target peptide, to tobacco plants. This enzyme converts a molecule of β-carotene into two of all-trans-retinal. After expressing this enzyme in plants, the concentration of all-trans-retinal was increased greatly. The increased retinal concentration led to increased expression of several microbial opsins, tested in model higher plants. Unfortunately, most opsins were observed intracellularly and not in the plasma membrane. To improve their localization in the plasma membrane, some reported signal peptides were fused to the N- or C-terminal end of opsins. Finally, I helped to identify three microbial opsins -- GtACR1 (a light-gated anion channel), ChR2 (a light-gated cation channel), PPR (a light-gated proton pump) which express and work well in the plasma membrane of plants. The transgene plants were grown under red light to prevent activation of the expressed opsins. Upon illumination with blue or green light, the activation of these opsins then induced the expected change of the membrane potential, dramatically changing the phenotype of plants with activated rhodopsins.
This study is the first which shows the potential of microbial opsins for optogenetic research in higher plants, using the ubq10 promoter for ubiquitous expression. I expect this to be just the beginning, as many different opsins and tissue-specific promoters for selective expression now can be tested for their usefulness. It is further to be expected that the here established method will help investigators to exploit more optogenetic tools and explore the secrets, kept in the plant kingdom.
This thesis examines the electronic properties of two materials that promise the realization and observation of novel exotic quantum phenomena. For this purpose, angle-resolved photoemission forms the experimental basis for the investigation of the electronic properties. Furthermore, the magnetic order is investigated utilizing X-ray dichroism measurements.
First, the bulk and surface electronic structure of epitaxially grown HgTe in its three-dimensional topological insulator phase is investigated. In this study, synchrotron radiation is used to address the three-dimensional band structure and orbital composition of the bulk states by employing photon-energy-dependent and polarization-dependent measurements, respectively. In addition, the topological surface state is examined on in situ grown samples using a laboratory photon source. The resulting data provide a means to experimentally localize the bulk band inversion in momentum space and to evidence the momentum-dependent change in the orbital character of the inverted bulk states.
Furthermore, a rather new series of van der Waals compounds, (MnBi\(_2\)Te\(_4\))(Bi\(_2\)Te\(_3\))\(_n\), is investigated. First, the magnetic properties of the first two members of the series, MnBi\(_2\)Te\(_4\) and MnBi\(_4\)Te\(_7\), are studied via X-ray absorption-based techniques. The topological surface state on the two terminations of MnBi\(_4\)Te\(_7\) is analyzed using circular dichroic, photon-energy-dependent, and spin-resolved photoemission. The topological state on the (MnBi\(_2\)Te\(_4\))-layer termination shows a free-standing Dirac cone with its Dirac point located in the bulk band gap. In contrast, on the (Bi\(_2\)Te\(_3\))-layer termination the surface state hybridizes with the bulk valences states, forming a spectral weight gap, and exhibits a Dirac point that is buried within the bulk continuum. Lastly, the lack of unambiguous evidence in the literature showing a temperature-dependent mass gap opening in these magnetic topological insulators is discussed through MnBi\(_2\)Te\(_4\).
Monarch butterflies are famous for their annual long-distance migration. Decreasing temperatures and reduced daylight induce the migratory state in the autumn generation of monarch butterflies. Not only are they in a reproductive diapause, they also produce fat deposits to be prepared for the upcoming journey: Driven by their instinct to migrate, they depart from their eclosion grounds in the northern regions of the North American continent and start their southern journey to their hibernation spots in Central Mexico. The butterflies cover a distance of up to 4000 km across the United States. In the next spring, the same butterflies invert their preferred heading direction due to seasonal changes and start their northward spring migration. The spring migration is continued by three consecutive butterfly generations, until the animals repopulate the northern regions in North America as non-migratory monarch butterflies. The monarch butterflies’ migratory state is genetically and epigenetically regulated, including the directed flight behavior. Therefore, the insect’s internal compass system does not only have to encode the butterflies preferred, but also its current heading direction. However, the butterfly’s internal heading representation has to be matched to external cues, to avoid departing from its initial flight path and increasing its risk of missing its desired destination. During the migratory flight, visual cues provide the butterflies with reliable orientation information. The butterflies refer to the sun as their main orientation cue. In addition to the sun, the butterflies likely use the polarization pattern of the sky for orientation. The sky compass signals are processed within a region in the brain, termed the central complex (CX). Previous research on the CX neural circuitry of the monarch butterflies demonstrated that tangential central complex neurons (TL) carry the visual input information into the CX and respond to a simulated sun and polarized light. However, whether these cells process additional visual cues like the panoramic skyline is still unknown. Furthermore, little is known about how the migratory state affects visual cue processing. In addition to this, most experiments studying the monarch butterfly CX focused on how neurons process single visual cues. However, how combined visual stimuli are processed in the CX is still unknown.
This thesis is investigating the following questions:
1) How does the migratory state affect visual cue processing in the TL cells within the monarch butterfly brain?
2) How are multiple visual cues integrated in the TL cells?
3) How is compass information modulated in the CX?
To study these questions, TL neurons from both animal groups (migratory and non-migratory) were electrophysiologically characterized using intracellular recordings while presenting different simulated celestial cues and visual sceneries. I showed that the TL neurons of migratory butterflies are more narrowly tuned to the sun, possibly helping them in keeping a directed flight course during migration. Furthermore, I found that TL cells encode a panoramic skyline, suggesting that the CX network combines celestial and terrestrial information. Experiments with combined celestial stimuli revealed that the TL cells combine both cue information linearly. However, if exposing the animals to a simulated visual scenery containing a panoramic skyline and a simulated sun, the single visual cues are weighted differently. These results indicate that the CX’s input region can flexibly adapt to different visual cue conditions. Furthermore, I characterize a previously unknown neuron in the monarch butterfly CX which responds to celestial stimuli and connects the CX with other brain neuropiles. How this cell type affects heading direction encoding has yet to be determined.
Regional climate models (RCMs) are tools used to project future climate change at a regional scale. Despite their high horizontal resolution, RCMs are characterized by systematic biases relative to observations, which can result in unrealistic interpretations of future climate change signals. On the other hand, bias correction (BC) is a popular statistical post-processing technique applied to improve the usability of output from climate models. Like every other statistical technique, BC has its strengths and weaknesses. Hence, within the regional context of Germany, and for temperature and precipitation, this study is dedicated to the assessment of the impact of different BC techniques on the RCM output. The focuses are on the impact of BC on the RCM’s statistical characterization, and physical consistency defined as the spatiotemporal consistency between the bias-corrected variable and the simulated physical mechanisms governing the variable, as well as the correlations between the bias-corrected variable and other (simulated) climate variables. Five BC techniques were applied in adjusting the systematic biases in temperature and precipitation RCM outputs. The BC techniques are linear scaling, empirical quantile mapping, univariate quantile delta mapping, multivariate quantile delta mapping that considers inter-site dependencies, and multivariate quantile delta mapping that considers inter-variable dependencies (MBCn). The results show that each BC technique adds value in reducing the biases in the statistics of the RCM output, though the added value depends on several factors such as the temporal resolution of the data, choice of RCM, climate variable, region, and the metric used in evaluating the BC technique. Further, the raw RCMs reproduced portions of the observed modes of atmospheric circulation in Western Europe, and the observed temperature, and precipitation meteorological patterns in Germany. After the BC, generally, the spatiotemporal configurations of the simulated meteorological patterns as well as the governing large-scale mechanisms were reproduced.
However, at a more localized spatial scale for the individual meteorological patterns, the BC changed the simulated co-variability of some grids, especially for precipitation. Concerning the co-variability among the variables, a physically interpretable positive correlation was found between temperature and precipitation during boreal winter in both models and observations. For most grid boxes in the study domain and on average, the BC techniques that do not adjust inter-variable dependency did not notably change the simulated correlations between the climate variables. However, depending on the grid box, the (univariate) BC techniques tend to degrade the simulated temporal correlations between temperature and precipitation. Further, MBCn which adjusts biases in inter-variable dependency has the skill to improve the correlations between the simulated variables towards observations.
Cancer is one of the leading causes of death worldwide, with currently assessed chances to develop at least one cancer in a lifetime for about 20%. High cases rates and mortality require the development of new anticancer therapies and treatment strategies. Another important concern is toxicity normally associated with conventional therapy methods, such as chemo- and radiotherapy. Among many proposed antitumoral agents, oncolytic viruses are still one of the promising and fast-developing fields of research with almost a hundred studies published data on over 3000 patients since the beginning of the new millennia.
Among all oncolytic viruses, the Vaccinia virus is arguably one of the safest, with an extremely long and prominent history of use, since it was the one and only vaccine used in the Smallpox Eradication Program in the 1970s. Interestingly enough, it was the first oncolytic virus proven to have tumor tropism in vitro and in vivo in laboratory settings, and this year we can celebrate an unofficial 100th anniversary since the publication of the fact. While being highly immunogenic, Vaccinia virus DNA replication takes place in the cytoplasm of the infected cell, and virus genes never integrate into the host genome. Another advantage of using Vaccinia as an oncolytic agent is its high genome capacity, which allows inserting up to 25 kbps of exogenous genes, thus allowing to additionally arm the virus against the tumor.
Oncolytic virus action consists of two major parts: direct oncolysis and immune activation against the tumor, with the latter being the key to successful treatment. To this moment, preclinical research data are mostly generated in immunocompromised xenograft models, which have hurdles to be properly translated for clinical use. In the first part of the current study, fourteen different recombinant Vaccinia virus strains were tested in two different murine tumor cell lines and corresponding immunocompetent animal models. We found, that Copenhagen backbone Vaccinia viruses while being extremely effective in cell culture, do not show significant oncolytic efficacy in animals. In contrast, several of the LIVP backbone viruses tested (specifically, IL-2 expressing ones) have little replication ability when compared to the Copenhagen strain, but are able to significantly delay tumor growth and prolong survival of the treated animals. We have also noted cytokine related toxicity of the animals to be mouse strain specific.
We have also tested the virus with the highest therapeutic benefit in combination with romidepsin and cyclophosphamide. While the combination with histone deacetylase inhibitor romidepsin did not result in therapeutic benefit in our settings, the addition of cyclophosphamide significantly improved the efficacy of the treatment, at the same time reducing cytokine-associated toxicity of the IL-2 expressing virus.
In the second part of the work, we analyzed the ability of adipose-derived mesenchymal stem cells to serve as a carrier for the oncolytic Vaccinia virus. We showed for the first time that the cells can be infected with the virus and can generate virus progeny. They are also able to survive for a substantially long time and, when injected into the bloodstream of tumor-bearing animals, produce the virus that is colonizing the tumor. Analysis of the systemic distribution of the cells after injection revealed that infected and uninfected cells are not distributed in the same manner, possibly suggesting that infected cells are getting recognized and cleared by an impaired immune system of athymic mice faster than non-infected cells. Despite this, injection of virus-loaded adipose-derived mesenchymal stem cells to human A549 tumor-bearing xenograft mice resulted in rapid tumor regression and reduced virus-related side effects of the treatment when compared to injection of the naked virus.
In conclusion, we have tested two different approaches to augmenting oncolytic Vaccinia virus therapy. First, the combination of recombinant Vaccinia virus expressing IL-2 and cyclophosphamide showed promising results in a syngeneic mouse model, despite the low permissivity of murine cells to the virus. Second, we loaded the oncolytic Vaccinia virus into mesenchymal stem cells and have proven that they can potentially serve as a vehicle for the virus.
Two-dimensional (2D) topological insulators are a new class of materials with properties that are
promising for potential future applications in quantum computers. For example, stanene represents
a possible candidate for a topological insulator made of Sn atoms arranged in a hexagonal
lattice. However, it has a relatively fragile low-energy spectrum and sensitive topology. Therefore,
to experimentally realize stanene in the topologically non-trivial phase, a suitable substrate
that accommodates stanene without compromising these topological properties must be found.
A heterostructure consisting of a SiC substrate with a buffer layer of adsorbed group-III elements
constitutes a possible solution for this problem. In this work, 2D adatom systems of Al and In
were grown epitaxially on SiC(0001) and then investigated structurally and spectroscopically by
scanning tunneling microscopy (STM) and photoelectron spectroscopy.
Al films in the high coverage regime \( (\Theta_{ML}\approx2\) ML\( ) \) exhibit unusually large, triangular- and
rectangular-shaped surface unit cells. Here, the low-energy electron diffraction (LEED)
pattern is brought into accordance with the surface topography derived from STM. Another Al
reconstruction, the quasi-one-dimensional (1D) Al phase, exhibits a striped surface corrugation,
which could be the result of the strain imprinted by the overlayer-substrate lattice mismatch.
It is suggested that Al atoms in different surface areas can occupy hexagonal close-packed and
face-centered cubic lattice sites, respectively, which in turn lead to close-packed transition regions
forming the stripe-like corrugations. On the basis of the well-known herringbone reconstruction
from Au(111), a first structural model is proposed, which fits well to the structural data from
STM. Ultimately, however, thermal treatments of the sample could not generate lower coverage
phases, i.e. in particular, a buffer layer structure.
Strong metallic signatures are found for In high coverage films \( (\Theta_{ML}\approx3\) to \(2\) ML\() \) by
scanning tunneling spectroscopy (STS) and angle-resolved photoelectron spectroscopy (ARPES),
which form a \( (7\times7) \), \( (6\times4\sqrt{3}) \), and \( (4\sqrt{3}\times4\sqrt{3}) \) surface reconstruction. In all these In phases
electrons follow the nearly-free electron model. Similar to the Al films, thermal treatments could
not obtain the buffer layer system.
Surprisingly, in the course of this investigation a triangular In lattice featuring a \( (1\times1) \)
periodicity is observed to host massive Dirac-like bands at \( K/K^{\prime} \) in ARPES. Based on this
strong electronic similarity with graphene at the Brillouin zone boundary, this new structure is
referred to as \textit{indenene}. An extensive theoretical analysis uncovers the emergence of an electronic
honeycomb network based on triangularly arranged In \textit{p} orbitals. Due to strong atomic spin-orbit
coupling and a comparably small substrate-induced in-plane inversion symmetry breaking this
material system is rendered topologically non-trivial. In indenene, the topology is intimately
linked to a bulk observable, i.e., the energy-dependent charge accumulation sequence within the
surface unit cell, which is experimentally exploited in STS to confirm the non-trivial topological
character. The band gap at \( K/K^{\prime} \), a signature of massive Dirac fermions, is estimated by
ARPES to approximately 125 meV. Further investigations by X-ray standing wave, STM, and
LEED confirm the structural properties of indenene. Thus, this thesis presents the growth and
characterization of the novel quantum spin Hall insulator material indenene.
In this in-vitro study, teeth were imaged using photoacoustic tomography (PAT), cone-beam computed tomography (CBCT), and micro-computed tomography (µ-CT). The study had aim: to identify the best wavelength for PAT images to determine the accuracy of the three imaging methods, and to determine whether PAT images of teeth can achieve acceptable reconstruction quality.
Aging is known to be a risk factor for structural abnormalities and functional decline in the nervous system. Characterizing age-related changes is important to identify putative pathways to overcome deleterious effects and improve life quality for the elderly. In this study, the peripheral nervous system of 24-month-old aged C57BL/6 mice has been investigated and compared to 12-month-old adult mice. Aged mice showed pathological alterations in their peripheral nerves similar to nerve biopsies from elderly human individuals, with nerve fibers showing demyelination and axonal damage. Such changes were lacking in nerves of adult 12-month-old mice and adult, non-aged humans. Moreover, neuromuscular junctions of 24-month-old mice showed increased denervation compared to adult mice. These alterations were accompanied by elevated numbers of macrophages in the peripheral nerves of aged mice. The neuroinflammatory conditions were associated with impaired myelin integrity and with a decline of nerve conduction properties and muscle strength in aged mice.
To determine the pathological impact of macrophages in the aging mice, macrophage depletion was performed in mice by oral administration of CSF-1R specific kinase (c-FMS) inhibitor PLX5622 (300 mg/kg body weight), which reduced the number of macrophages in the peripheral nerves by 70%. The treated mice showed attenuated demyelination, less muscle denervation and preserved muscle strength. This indicates that macrophage-driven inflammation in the peripheral nerves is partially responsible for the age-related neuropathy in mice.
Based on previous observations that systemic inflammation can accelerate disease progression in mouse models of neurodegenerative diseases, it was hypothesized that systemic inflammation can exacerbate the peripheral neuropathy found in aged mice. To investigate this hypothesis, aged C57BL/6 mice were intraperitoneally injected with a single dose of lipopolysaccharide (LPS; 500 μg/kg body weight) to induce systemic inflammation by mimicking bacterial infection, mostly via activation of Toll-like receptors (TLRs). Altered endoneurial macrophage activation, highlighted by Trem2 downregulation, was found in LPS injected aged mice one month after injection. This was accompanied by a so far rarely observed form of axonal perturbation, i.e., the occurrence of “dark axons” characterized by a damaged cytoskeleton and an increased overall electron density of the axoplasm. At the same time, however, LPS injection reduced demyelination and muscle denervation in aged mice. Interestingly, TREM2 deficiency in aged mice led to similar changes to LPS injection. This suggests that LPS injection likely mitigates aging-related demyelination and muscle denervation via Trem2 downregulation.
Taken together, this study reveals the role of macrophage-driven inflammation as a pathogenic mediator in age-related peripheral neuropathy, and that targeting macrophages might be an option to mitigate peripheral neuropathies in aging individuals. Furthermore, this study shows that systemic inflammation may be an ambivalent modifier of age-related nerve damage, leading to a distinct type of axonal perturbation, but in addition to functionally counteracting, dampened demyelination and muscle denervation. Translationally, it is plausible to assume that tipping the balance of macrophage polarization to one direction or the other may determine the functional outcome in the aging peripheral nervous system of the elderly.
As central components of life, DNA and RNA encode the genetic information. However, RNA performs several functions that exceed the competences stated in the ‘central dogma of life‘. RNAs undergo extensive post-transcriptional processing like chemical modifications. Among all classes of RNA, tRNAs are the most extensively modified. Their modifications are chemically diverse and vary from simple methylations (e.g. m3C, m6A) to more complex residues, like isopentenyl group (e.g. i6A, hypermodifications: e.g. ms2i6A) or even amino acids (e.g. t6A). Depending on their location within the overall structure, modifications can have an impact on tRNA stability and structure, as well as affinity for the ribosome and translation efficiency and fidelity. Given the importance of tRNA modifications new tools are needed for their detection and to study their recognition by proteins and enzymatic transformations.
The chemical synthesis of these naturally occurring tRNA modifications as phosphoramidite building blocks is a prerequisite to incorporate the desired modification via solid-phase synthesis into oligonucleotides. With the help of the m3C, (ms2)i6A, and t6A oligonucleotides, the importance and impact of tRNA modifications was investigated in this thesis. To this end, the role of METTL8 as the methyltransferase responsible for the installation of the methyl group at C32 for mt-tRNAThr and mt-tRNASer(UCN) was resolved. Thereby, the respective adenosine modification on position 37 is essential for the effectiveness of the enzyme. Besides, by means of NMR analysis, CD spectroscopy, thermal denaturation experiments, and native page separation, the impact of m3C32 on the structure of the tRNA ASLs was shown. The modification appeared to fine-tune the tRNA structure to optimize mitochondrial translation. To investigate the regulation of the dynamic modification pathway of m3C, demethylation assays were performed with the modified tRNA-ASLs and the (α-KG)- and Fe(II)-dependent dioxygenase ALKBH1 and ALKHB3. A demethylation activity of ALKBH3 on the mt-tRNAs was observed, even though it has so far only been described as a cytoplasmic enzyme. Whether this is physiologically relevant and ALKBH3 present a mitochondrial localization needs further validation. In addition, ALKBH1 was confirmed to not be able to demethylate m3C on mt-tRNAs, but indications for a deprenylation and exonuclease activity were found. Furthermore, the aforementioned naturally occurring modifications were utilized to find analytical tools that can determine the modification levels by DNAzymes, which cleave RNA in the presence of a specific modification. Selective DNA enzymes for i6A, as well as the three cytidine isomers m3C, m4C, and m5C have been identified and characterized.
Besides the naturally occurring tRNA modifications, the investigation on artificially modified nucleosides is also part of this thesis. Nucleosides with specific properties for desired applications can be created by modifying the scaffold of native nucleosides.
During the pandemic, the potential of antiviral nucleoside analogues was highlighted for the treatment of the SARS-CoV-2 infection. For examinations of the potential drug-candidate Molnupiravir, the N4-hydroxycytidine phosphoramidite building block was synthesized and incorporated into several RNA oligonucleotides. A two-step model for the NHC-induced mutagenesis of SARS-CoV-2 was proposed based on RNA elongation, thermal denaturation, and cryo-EM experiments using the modified RNA strands with the recombinant SARS-CoV-2 RNA-dependent RNA polymerase. Two tautomeric forms of NHC enable base pairing with guanosine in the amino and with adenosine in the imino form, leading to error catastrophe after the incorporation into viral RNA. These findings were further corroborated by thermal melting curve analysis and NMR spectroscopy of the NHC-containing Dickerson Drew sequence. In conclusion, the anti-amino form in the NHC-G base pair was assigned by NMR analysis using a 15N-labeld NHC building block incorporated into the Dickerson Drew sequence.
This thesis also addressed the synthesis of a 7-deazaguanosine crosslinker with a masked aldehyde as a diol linker for investigations of DNA-protein interactions. The diol functional group can be unmasked to release the reactive aldehyde, which can specifically form a covalent bond with amino acids Lys or Arg within the protein complex condensin. The incorporation of the synthesized phosphoramidite and triphosphate building blocks were shown and the functionality of the PCR product containing the crosslinker was demonstrated by oxidation and the formation of a covalent bond with a fluorescein label.
The development of assays that detect changes in this methylation pattern of m6A could provide new insights into important biological processes. In the last project of this thesis, the influence of RNA methylation states on the structural properties of RNA was analyzed and a fluorescent nucleoside analog (8-vinyladenosine) as molecular tools for such assays was developed. Initial experiments with the fluorescent nucleoside analog N6-methyl-8-vinyladenosine (m6v8A) were performed and revealed a strong fluorescence enhancement of the free m6v8A nucleoside by the installation of the vinyl moiety at position 8.
Overall, this thesis contributes to various research topics regarding the application of naturally occurring and artificial nucleoside analogues. Starting with the chemical synthesis of RNA and DNA modifications, this thesis has unveiled several open questions regarding the dynamic (de-)methylation pathway of m3C and the mechanism of action of molnupiravir through in-depth analysis and provided the basis for further investigations of the protein complex condensin, and a new fluorescent nucleoside analog m6v8A.
Ibrutinib serves as an efficient second-line therapy in relapsed/refractory mantle cell lymphoma. However, resistance to the BTK inhibitor results in a poor prognosis for patients. Since the mechanisms leading to resistance in initially responding tumor cells are poorly understood, this work aimed to decipher acquired features in ibrutinib-surviving cells of a sensitive mantle cell lymphoma cell line and evaluate these potential therapeutic targets in ibrutinib-treated mantle cell lymphoma.
Time-resolved single-cell RNA sequencing was performed to track the transcriptomic evolution of REC-1 cells across 6 and 48 hours of treatment. Single-cell analysis uncovered a subpopulation of REC-1 with potentially greater aggressiveness and survival advantage by benefiting from interaction with the tumor microenvironment. Upregulation of B-cell receptor genes, elevated surface antigen expression of CD52 and metabolic rewiring to higher dependence on oxidative phosphorylation were identified as further potential resistance features of ibrutinib-surviving cells. RNA sequencing after prolonged incubation corroborated the increase in CD52 and oxidative phosphorylation as dominant characteristics of the cells surviving the 4-day treatment, highlighting their potential as therapeutic targets in combination with ibrutinib treatment. Concomitant use of ibrutinib and the oxidative phosphorylation inhibitor IACS-010759 increased toxicity compared to ibrutinib monotherapy due to higher apoptosis and greater inhibition of proliferation. For anti-CD52 therapy, a consecutive approach with ibrutinib pretreatment followed by incubation of surviving cells with a CD52 monoclonal antibody and human serum yielded a synergistic effect, as ibrutinib-surviving mantle cell lymphoma cells were rapidly depleted by complement-dependent cytotoxicity. Regarding the effects on primary tumor cells from mantle cell lymphoma patients, ibrutinib induced upregulation of CD52 in some cases, and increased toxicity of anti-CD52 therapy was observed in ibrutinib-sensitive patient samples after pretreatment with the BTK inhibitor. The likely favorable in vivo efficacy of an anti-CD52 therapy might therefore be restricted to a subgroup of mantle cell lymphoma patients, also in view of the associated side effects.
Given the need for new therapeutic options in mantle cell lymphoma to overcome resistance to ibrutinib, this work highlights the potentially beneficial use of an oxidative phosphorylation inhibitor as add-on therapy. In addition, the findings suggest to further assess the value of anti-CD52 therapy as consolidation to ibrutinib in ibrutinib-sensitive patients with elevated CD52 surface levels on tumor cells to target resistant clones and minimize risk of minimal residual disease and relapse.
The present thesis introduce different synthetic strategies towards a variety of polycyclic aromatic dicarboximides (PADIs) with highly interesting and diverse properties. This included tetrachlorinated, tetraaryloxy- and tetraaryl-substituted dicarboximides, fused acceptor‒donor(‒acceptor) structures as well as sterically shielded rylene and nanographene dicarboximides. The properties and thus the disclosure of structure‒property relationships of the resulting dyes were investigated in detail among others with UV‒vis absorption spectroscopy, fluorescence spectroscopy, cyclic voltammetry and single crystal X-ray analysis. For instance, some of the fused and substituted PADIs offer strong absorption of visible and near infrared (NIR) light, NIR emission and low-lying LUMO levels. On the contrary, intriguing optical features in the solid-state characterize the rylene dicarboximides with their bulky N-substituents, while the devised sterically enwrapped nanographene host offered remarkable complexation capabilities in solution.
The light-gated cation channel Channelrhodopsin-2 was discovered and characterized in 2003. Already in 2005/2006 five independent groups demonstrated that heterologous expression of Channelrhodopsin-2 is a highly useful and simply applicable method for depolarizing and thereby activating nerve cells. The application of Channelrhodopsin-2 revolutionized neuroscience research and the method was then called optogenetics. In recent years more and more light-sensitive proteins were successfully introduced as “optogenetic tools”, not only in neuroscience. Optogenetic tools for neuronal excitation are well developed with many different cation-conducting wildtype and mutated channelrhodopsins, whereas for inhibition of neurons in the beginning (2007) only hyperpolarizing ion pumps were available. The later discovered light-activated anion channels (anion channelrhodopsins) can be useful hyperpolarizers, but only at low cytoplasmic anion concentration. For this thesis, I optimized CsR, a proton-pumping rhodopsin from Coccomyxa subellipsoidea, which naturally shows a robust expression in Xenopus laevis oocytes and plant leaves. I improved the expression and therefore the photocurrent of CsR about two-fold by N-terminal modification to the improved version CsR2.0, without altering the proton pump function and the action spectrum. A light pulse hyperpolarised the mesophyll cells of CsR2.0-expressing transgenic tobacco plants (N. tabacum) by up to 20 mV from the resting membrane potential of -150 to -200 mV. The robust heterologous expression makes CsR2.0 a promising optogenetic tool for hyperpolarization in other organisms as well. A single R83H point-mutation converted CsR2.0 into a light-activated (passive) proton channel with a reversal potential close to the Nernst potential for intra-/extra-cellular H+ concentration. This light-gated proton channel is expected to become a further useful optogenetic tool, e.g. for analysis of pH-regulation in cells or the intercellular space. Ion pumps as optogenetic tools require high expression levels and high light intensity for efficient pump currents, whereas long-term illumination may cause unwanted heating effects. Although anion channelrhodopsins are effective hyperpolarizing tools in some cases, their effect on neuronal activity is dependent on the cytoplasmic chloride concentration which can vary among neurons. In nerve cells, increased conductance for potassium terminates the action potential and K+ conductance underlies the resting membrane potential in excitable cells. Therefore, several groups attempted to synthesize artificial light-gated potassium channels but 2 all of these published innovations showed serious drawbacks, ranging from poor expression over lacking reversibility to poor temporal precision. A highly potassium selective light-sensitive silencer of action potentials is needed. To achieve this, I engineered a light-activated potassium channel by the genetic fusion of a photoactivated adenylyl cyclase, bPAC, and a cAMP-gated potassium channel, SthK. Illumination activates bPAC to produce cAMP and the elevated cAMP level opens SthK. The slow diffusion and degradation of cAMP makes this construct a very light-sensitive, long-lasting inhibitor. I have successfully developed four variants with EC50 to cAMP ranging from 7 over 10, 21, to 29 μM. Together with the original fusion construct (EC50 to cAMP is 3 μm), there are five different light- (or cAMP-) sensitive potassium channels for researchersto choose, depending on their cell type and light intensity needs.
Alzheimer´s disease (AD) is a neurodegenerative disease and the most common form of dementia with still no preventive or curative treatment. Besides several risk factors, age is one of the major risks for AD and with an aging society, there is an urgent need for disease modifying agents. The strategy to address only one target within the intertwined network of AD failed so far.
Natural products especially the phytochemical flavonoids, which are poly-phenolic natural products, have shown great potential as disease modifying agents against neurodegenerative disorders like Alzheimer´s disease (AD) with activities even in vivo. Flavonoids are produced by many plants and the native Californian plant Eriodictyon californicum is particularly rich in flavonoids. One of the major flavonoids of E. californicum is sterubin, a very potent agent against oxidative stress and inflammation, two hallmarks and drivers of AD and neurodegeneration. Herein, racemic sterubin was synthesized and separated into its pure (R)- and (S)-enantiomer by chiral HPLC. The pure enantiomers showed comparable neuroprotection in vitro with no significant differences. The stereoisomers were configurationally stable in methanol, but fast racemization was observed in culture medium. Moreover, the activity of sterubin was investigated in vivo, in an AD mouse model. Sterubin showed a significant positive impact on short- and long-term memory at low dosages.
A promising concept for the increase of activity of single flavonoids is hybridization with aromatic acids like cinnamic or ferulic acids. Hybridization of the natural products taxifolin and silibinin with cinnamic acid led to an overadditive effect of these compounds in phenotypic screening assays related to neurodegeneration and AD. Because there are more potent agents as taxifolin or silibinin, the hybrids were further developed, and different flavonoid cinnamic acid hybrids were synthesized. The connection between flavonoids and cinnamic acid was achieved by an amide instead of a labile ester to improve the stability towards hydrolysis to gain better “druggability” of the compounds. To investigate the oxidation state of the C-ring of the flavonoid part, the dehydro analogues of the respective hybrids were also synthesized. The compounds show neuroprotection against oxytosis, ferroptosis and ATP-depletion in the murine hippocampal cell line HT22. While no overall trend within the flavanones compared to the flavones could be assigned, the taxifolin and the quercetin derivative were the most active compounds in course of all assays. The quercetin derivate even shows greater activity than the taxifolin derivate in every assay. As desired no hydrolysis product was found in cellular uptake experiments after 4h, whereas different metabolites were found. The last part of this work focused on synthetic bioisoteres of the natural product curcumin. Due to the drawbacks of curcumin and flavonoids arising from poor pharmacokinetics, rapid metabolism and sometimes instability in aqueous medium, we have examined the biological activity of azobenzene compounds designed as bioisoteres of curcumin, carrying the pharmacophoric catechol group of flavonoids. These bioisosteres exceeded their parent compounds in counteracting intracellular oxidative stress, neuroinflammation and amyloid-beta aggregation. By incorporating an azobenzene moiety and the isosteric behaviour to the natural parent compounds, these compounds may act as molecular tools for further investigation towards the molecular mode of action of natural products.
Due to the global aging society and the enormous global incidence and prevalence rates that will result in the coming years, Alzheimer's Dementia (AD) represents a growing challenge for the health care system. The pathogenesis, which is unclear in parts, the chronic progression of AD, which often lasts for years, as well as insufficient diagnostic and therapeutic options complicate an adequate psychotherapeutic and medical approach to the disease. To date, AD is also considered an incurable disease.
Therefore, it is essential to gain deeper insights into the early detection or even prevention of AD. Consideration of prodromal syndromes such as Mild Cognitive Impairment (MCI) can provide significant evidence about high-risk groups for AD progression and differentiate cognitively "normal" aging individuals from those with pathological cognitive decline. Thus, for example, functional Near-Infrared Spectroscopy (fNIRS) imaging helps identify early neurodegenerative processes. In contrast, potential risk factors and predictors of later-onset clinical symptoms of MCI and AD can most often be revealed and quantified via the use of neuropsychiatric test batteries.
The present thesis consists of four studies and aimed to assess and describe the pathological cognitive decline in a sample of elderly study participants (age: ≥ 70 years; N = 604 at baseline) of the longitudinal, observational, and prospective "Vogel Study" from Würzburg, Germany, who were primarily healthy at baseline, over two measurement time points approximately 3 years apart, to differentiate between healthy and diseased study participants and to define predictors of MCI/AD and longitudinal study dropout.
Studies 1 and 2 differentiated healthy study participants from MCI patients based on the baseline hemodynamic response of the parietal cortex recorded by fNIRS during the processing of a paradigm (here: Angle Discrimination Task [ADT]) for visual-spatial processing performance. Neuronal hypoactivity was found in the MCI patients, with both healthy study participants and MCI patients showing higher superior and right hemispheric activation. MCI patients had more difficulty resolving the paradigm. Thus, no evidence of possible compensatory mechanisms was uncovered in the MCI patients.
Study 3 first defined the four latent factors declarative memory, working memory, attention, and visual-spatial processing based on structural equation model (SEM) calculations of the sample using adequate measurement (in-)variant confirmatory factor models from the baseline assessment to the first of a total of two follow-up assessments after approximately 3 years. This allowed a dimensional assessment of pathological cognitive decline versus classificatory-categorical assignment (healthy/diseased) of the sample. In addition, the superiority of the latent factor approach over a composite approach was demonstrated. Next, using a mixed-model approach, predictive analyses were calculated for the prediction of latent factors at first follow-up by baseline risk factors. The sex of study participants proved to be the best predictor of cognitive change in all the cognitive domains, with females performing better than men in the memory domains. Specifically, for declarative memory, older age predicted lower performance regardless of sex. Additional predictive evidence emerged for low serum levels of Brain-Derived Neurotrophic Factor (BDNF) on lower attention performance and higher depression symptoms on lower visual-spatial processing performance.
Study 4 further reported baseline predictors of study dropout at first follow-up. Cognitive performance, as defined in Study 3 using the four latent cognitive factors, was a predictor of study dropout for cognitive decline in the domains of declarative memory, attention, and visual-spatial processing. Conspicuous dementia screening on the Mini-Mental Status Examination (MMSE) also predicted dropout.
Overall, both the use of fNIRS imaging to detect visual-spatial processing performance in the parietal cortex during applying ADT and the dimensional perspective of the neuropsychiatric test battery in the context of prediction and dropout analyses were found to be suitable for early detection research of MCI and AD. Finally, the results will be interpreted in the overall context and implications, limitations, and perspectives will be discussed.
PRC1 serves as a microtubule-bundling protein and is a potential therapeutic target for lung cancer
(2023)
Protein regulator of cytokinesis 1 (PRC1) is a microtubule-associated protein with essential roles in mitosis and cytokinesis. Furthermore, the protein is highly expressed in several cancer types which is correlated with aneuploidy and worse patient outcome. In this study it was investigated, whether PRC1 is a potential target for lung cancer as well as its possible nuclear role.
Elevated PRC1 expression was cell cycle-dependent with increasing levels from S-phase to G2/M-phase of the cell cycle. Thereby, PRC1 localized at the nucleus during interphase and at the central spindle and midbody during mitosis and cytokinesis. Genome-wide expression profiling by RNA sequencing of ectopically expressed PRC1 resulted in activation of the p53 pathway. A mutant version of PRC1, that is unable to enter the nucleus, induced the same gene sets as wildtype PRC1, suggesting that PRC1 has no nuclear-specific functions in lung cancer cells. Finally, PRC1 overexpression leads to proliferation defects, multi-nucleation, and enlargement of cells which was directly linked to microtubule-bundling within the cytoplasm.
For analysis of the requirement of PRC1 in lung cancer, different inducible cell lines were generated to deplete the protein by RNA interference (RNAi) in vitro. PRC1 depletion caused proliferation defects and cytokinesis failures with increased numbers of bi- and multi-nucleated cells compared to non-induced lung cancer cells. Importantly, effects in control cells were less severe as in lung cancer cells. Finally, p53 wildtype lung cancer cells became senescent, whereas p53 mutant cells became apoptotic upon PRC1 depletion. PRC1 is also required for tumorigenesis in vivo, which was shown by using a mouse model for non-small cell lung cancer driven by oncogenic K-RAS and loss of p53. Here, lung tumor area, tumor number, and high-grade tumors were significantly reduced in PRC1 depleted conditions by RNAi.
In this study, it is shown that PRC1 serves as a microtubule-bundling protein with essential roles in mitosis and cytokinesis. Expression of the protein needs to be tightly regulated to allow unperturbed proliferation of lung cancer cells. It is suggested that besides phosphorylation of PRC1, the nuclear localization might be a protective mechanism for the cells to prevent perinuclear microtubule-bundling. In conclusion, PRC1 could be a potential target of lung cancer as mono therapy or in combination with a chemotherapeutic agent, like cisplatin, which enhanced the negative effects on proliferation of lung cancer cells in vitro.
This thesis identifies how the printing conditions for a high-resolution additive manufacturing technique, melt electrowriting (MEW), needs to be adjusted to process electroactive polymers (EAPs) into microfibers. Using EAPs based on poly(vinylidene difluoride) (PVDF), their ability to be MEW-processed is studied and expands the list of processable materials for this technology.
The anaerobe Fusobacterium nucleatum (F. nucleatum) is an important member of the oral microbiome but can also colonize different tissues of the human body. In particular, its association with multiple human cancers has drawn much attention.
This association has prompted growing interest into the interaction of F. nucleatum with cancer, with studies focusing primarily on the host cells. At the same time, F. nucleatum itself remains poorly understood, which includes its transcriptomic architecture but also gene regulation such as global stress responses that typically enable survival of bacteria in new environments. An important aspect of such regulatory networks is the post-transcriptional regulation, which is entirely unknown in F. nucleatum. This paucity extents to any knowledge on small regulatory RNAs (sRNAs), despite their important role as post-transcriptional regulators of the bacterial physiology.
Investigating the above stated aspects is further complicated by the fact that F. nucleatum is phylogenetically distant from all other bacteria, displays very limited genetic tractability and lacks genetic tools for dissecting gene function.
This leaves many open questions on basic gene regulation in F. nucleatum, such as if the bacterium combines transcriptional and post-transcriptional regulation in its adaptation to a changing environment.
To begin answering this question, this works elucidated the transcriptomic landscape of F. nucleatum by performing differential RNA-seq (dRNA-seq). Conducted for five representative strains of all F. nucleatum subspecies and the closely related F. periodonticum, the analysis globally uncovered transcriptional start sites (TSS), 5'untranslated regions (UTRs) and improved the existing annotation. Importantly, the dRNA-seq analysis also identified a conserved suite of sRNAs specific to Fusobacterium.
The development of five genetic tools enabled further investigations of gene functions in F. nucleatum. These include vectors that enable the expression of different fluorescent proteins, inducible gene expression and scarless gene deletion in addition to transcriptional and translational reporter systems.
These tools enabled the dissection of a Sigma E response and uncovered several commonalities with its counterpart in the phylogenetically distant Proteobacteria. The similarities include the upregulation of genes involved in membrane homeostasis but also a Simga E-dependent regulatory sRNA. Surprisingly, oxygen was found to activated Sigma E in F. nucleatum contrasting the typical role of the factor in envelope stress.
The non-coding Sigma E-dependent sRNA, named FoxI, was shown to repress the translation of several envelope proteins which represented yet another parallel to the envelope stress response in Proteobacteria.
Overall, this work sheds light on the RNA landscape of the cancer-associated bacterium leading to the discovery of a conserved global stress response consisting of a coding and a non-coding arm. The development of new genetic tools not only aided the latter discovery but also provides the means for further dissecting the molecular and infection biology of this enigmatic bacterium.
Platelets play an important role in the body, since they are part of the hemostasis
system, preventing and stopping blood loss. Nevertheless, when platelet or
coagulation system function are impaired, uncontrolled bleedings but also irreversible
vessel occlusion followed by ischemic tissue damage can occur. Therefore,
understanding platelet function and activation, mechanisms which are controlled by a
variety of platelet membrane receptors and other factors is important to advance out
knowledge of hemostasis and platelet malfunction. For a complete picture of platelet
function and their modulating behavior it is desired to be able to quantify receptor
distributions and interactions of these densely packed molecular ensembles in the
membrane. This challenges scientists for several reasons. Most importantly, platelets
are microscopically small objects, challenging the spatial resolution of conventional
light microscopy. Moreover, platelet receptors are highly abundant on the membrane
so even super-resolution microscopy struggles with quantitative receptor imaging on
platelets.
With Expansion microscopy (ExM), a new super-resolution technique was introduced,
allowing resolutions to achieve super-resolution without using a super-resolution
microscope, but by combining a conventional confocal microscopy with a highly
processed sample that has been expanded physically. In this doctoral thesis, I
evaluated the potential of this technique for super-resolution platelet imaging by
optimizing the sample preparation process and establishing an imaging and image
processing pipeline for dual-color 3D images of different membrane receptors. The
analysis of receptor colocalization using ExM demonstrated a clear superiority
compared to conventional microscopy. Furthermore, I identified a library of
fluorescently labeled antibodies against different platelet receptors compatible with
ExM and showed the possibility of staining membrane receptors and parts of the
cytoskeleton at the same time.
This work introduced the reader to all relevant fields to tap into an ultrasound-based state of charge estimation and provides a blueprint for the procedure to achieve and test the fundamentals of such an approach. It spanned from an in-depth electrochemical characterization of the studied battery cells over establishing the measurement technique, digital processing of ultrasonic transmission signals, and characterization of the SoC dependent property changes of those signals to a proof of concept of an ultrasound-based state of charge estimation.
The State of the art & theoretical background chapter focused on the battery section on the mechanical property changes of lithium-ion batteries during operation. The components and the processes involved to manufacture a battery cell were described to establish the fundamentals for later interrogation. A comprehensive summary of methods for state estimation was given and an emphasis was laid on mechanical methods, including a critical review of the most recent research on ultrasound-based state estimation. Afterward, the fundamentals of ultrasonic non-destructive evaluation were introduced, starting with the sound propagation modes in isotropic boundary-free media, followed by the introduction of boundaries and non-isotropic structure to finally approach the class of fluid-saturated porous media, which batteries can be counted to. As the processing of the ultrasonic signals transmitted through lithium-ion battery cells with the aim of feature extraction was one of the main goals of this work, the fundamentals of digital signal processing and methods for the time of flight estimation were reviewed and compared in a separate section.
All available information on the interrogated battery cell and the instrumentation was collected in the Experimental methods & instrumentation chapter, including a detailed step-by-step manual of the process developed in this work to create and attach a sensor stack for ultrasonic interrogation based on low-cost off-the-shelf piezo elements.
The Results & discussion chapter opened with an in-depth electrochemical and post-mortem interrogation to reverse engineer the battery cell design and its internal structure. The combination of inductively coupled plasma-optical emission spectrometry and incremental capacity analysis applied to three-electrode lab cells, constructed from the studied battery cell’s materials, allowed to identify the SoC ranges in which phase transitions and staging occur and thereby directly links changes in the ultrasonic signal properties with the state of the active materials, which makes this work stand out among other studies on ultrasound-based state estimation. Additional dilatometer experiments were able to prove that the measured effect in ultrasonic time of flight cannot originate from the thickness increase of the battery cells alone, as this thickness increase is smaller and in opposite direction to the change in time of flight. Therefore, changes in elastic modulus and density have to be responsible for the observed effect.
The construction of the sensor stack from off-the-shelf piezo elements, its electromagnetic shielding, and attachment to both sides of the battery cells was treated in a subsequent section. Experiments verified the necessity of shielding and its negligible influence on the ultrasonic signals. A hypothesis describing the metal layer in the pouch foil to be the transport medium of an electrical coupling/distortion between sending and receiving sensor was formulated and tested. Impedance spectroscopy was shown to be a useful tool to characterize the resonant behavior of piezo elements and ensure the mechanical coupling of such to the surface of the battery cells. The excitation of the piezo elements by a raised cosine (RCn) waveform with varied center frequency in the range of 50 kHz to 250 kHz was studied in the frequency domain and the influence of the resonant behavior, as identified prior by impedance spectroscopy, on waveform and frequency content was evaluated to be uncritical. Therefore, the forced oscillation produced by this excitation was assumed to be mechanically coupled as ultrasonic waves into the battery cells.
The ultrasonic waves transmitted through the battery cell were recorded by piezo elements on the opposing side. A first inspection of the raw, unprocessed signals identified the transmission of two main wave packages and allowed the identification of two major trends: the time of flight of ultrasonic wave packages decreases with the center frequency of the RCn waveform, and with state of charge. These trends were to be assessed further in the subsequent sections. Therefore, methods for the extraction of features (properties) from the ultrasonic signals were established, compared, and tested in a dedicated section. Several simple and advanced thresholding methods were compared with envelope-based and cross-correlation methods to estimate the time of flight (ToF). It was demonstrated that the envelope-based method yields the most robust estimate for the first and second wave package. This finding is in accordance with the literature stating that an envelope-based method is best suited for dispersive, absorptive media [204], to which lithium-ion batteries are counted. Respective trends were already suggested by the heatmap plots of the raw signals vs. RCn frequency and SoC. To enable such a robust estimate, an FIR filter had to be designed to preprocess the transmitted signals and thereby attenuate frequency components that verifiably lead to a distorted shape of the envelope.
With a robust ToF estimation method selected, the characterization of the signal properties ToF and transmitted energy content (EC) was performed in-depth. A study of cycle-to-cycle variations unveiled that the signal properties are affected by a long rest period and the associated relaxation of the multi-particle system “battery cell” to equilibrium. In detail, during cycling, the signal properties don’t reach the same value at a given SoC in two subsequent cycles if the first of the two cycles follows a long rest period. In accordance with the literature, a break-in period, making up for more than ten cycles post-formation, was observed. During this break-in period, the mechanical properties of the system are said to change until a steady state is reached [25]. Experiments at different C-rate showed that ultrasonic signal properties can sense the non-equilibrium state of a battery cell, characterized by an increasing area between charge and discharge curve of the respective signal property vs. SoC plot. This non-equilibrium state relaxes in the rest period following the discharge after the cut-off voltage is reached. The relaxation in the rest period following the charge is much smaller and shows little C-rate dependency as the state is prepared by constant voltage charging at the end of charge voltage. For a purely statistical SoC estimation approach, as employed in this work, where only instantaneous measurements are taken into account and the historic course of the measurement is not utilized as a source of information, the presence of hysteresis and relaxation leads to a reduced estimation accuracy. Future research should address this issue or even utilize the relaxation to improve the estimation accuracy, by incorporating historic information, e.g., by using the derivative of a signal property as an additional feature. The signal properties were then tested for their correlation with SoC as a function of RCn frequency. This allowed identifying trends in the behavior of the signal properties as a function of RCn frequency and C-rate in a condensed fashion and thereby enabled to predict the frequency range, about 50 kHz to 125 kHz, in which the course of the signal properties is best suited for SoC estimation.
The final section provided a proof of concept of the ultrasound-based SoC estimation, by applying a support vector regression (SVR) to before thoroughly studied ultrasonic signal properties, as well as current and battery cell voltage. The included case study was split into different parts that assessed the ability of an SVR to estimate the SoC in a variety of scenarios. Seven battery cells, prepared with sensor stacks attached to both faces, were used to generate 14 datasets. First, a comparison of self-tests, where a portion of a dataset is used for training and another for testing, and cross-tests, which use the dataset of one cell for training and the dataset of another for testing, was performed. A root mean square error (RMSE) of 3.9% to 4.8% SoC and 3.6% to 10.0% SoC was achieved, respectively. In general, it was observed that the SVR is prone to overestimation at low SoCs and underestimation at high SoCs, which was attributed to the pronounced hysteresis and relaxation of the ultrasonic signal properties in this SoC ranges. The fact that higher accuracy is achieved, if the exact cell is known to the model, indicates that a variation between cells exists. This variation between cells can originate from differences in mechanical properties as a result of production variations or from differences in manual sensor placement, mechanical coupling, or resonant behavior of the ultrasonic sensors. To mitigate the effect of the cell-to-cell variations, a test was performed, where the datasets of six out of the seven cells were combined as training data, and the dataset of the seventh cell was used for testing. This reduced the spread of the RMSE from (3.6 - 10.0)% SoC to (5.9 – 8.5)% SoC, respectively, once again stating that a databased approach for state estimation becomes more reliable with a large data basis. Utilizing self-tests on seven datasets, the effect of additional features on the state estimation result was tested. The involvement of an additional feature did not necessarily improve the estimation accuracy, but it was shown that a combination of ultrasonic and electrical features is superior to the training with these features alone. To test the ability of the model to estimate the SoC in unknown cycling conditions, a test was performed where the C-rate of the test dataset was not included in the training data. The result suggests that for practical applications it might be sufficient to perform training with the boundary of the use cases in a controlled laboratory environment to handle the estimation in a broad spectrum of use cases.
In comparison with literature, this study stands out by utilizing and modifying off-the-shelf piezo elements to equip state-of-the-art lithium-ion battery cells with ultrasonic sensors, employing a range of center frequencies for the waveform, transmitted through the battery cell, instead of a fixed frequency and by allowing the SVR to choose the frequency that yields the best result. The characterization of the ultrasonic signal properties as a function of RCn frequency and SoC and the assignment of characteristic changes in the signal properties to electrochemical processes, such as phase transitions and staging, makes this work unique. By studying a range of use cases, it was demonstrated that an improved SoC estimation accuracy can be achieved with the aid of ultrasonic measurements – thanks to the correlation of the mechanical properties of the battery cells with the SoC.
The focus of this thesis was to investigate how PCL and PLGA react to the heat exposure that comes with the MEW process over a defined timespan.
To assess the thermal stability of PCL during MEW over 25 d, an automated collection of fibers has been used to determine the CTS on each day of heating for three different temperatures. PCL is exceptionally stable over 25 d at 75 °C, whereas for 85 °C and 95 °C a slight upward trend during the last 10 d could be observed, which is an indication for thermal degradation. Same trend could be observed for diameter of fibers produced at a fixed collector speed. For all temperatures, CTS during the first 5 d decreased due to inhomogeneities of the melt. Physical analysis of the fibers by XRD and mechanical testing showed no significant changes.
To investigate the chemical details of the thermal durability, PCL was artificially aged over 25 d at 75 °C, 85 °C and 95 °C. Data from GPC analysis and rheology revealed that PCL is degrading steadily at all three temperatures. Combined with GC-MS analysis, two different mechanisms for degradation could be observed: random chain scission and unzipping. Additional GPC experiment using a mixture of PCL and a fluorescence labelled PCL showed that PCL was undergoing ester interchange reactions, which could explain its thermal stability.
PLGA was established successfully as material for MEW. GPC results revealed that PLGA degraded heavily in the one-hour preheating period. To reduce the processing temperature, ATEC was blended with PLGA in three mixtures. This slowed down degradation and a processing window of 6 h could be established. Mechanical testing with fibers produced with PLGA and all three blends was performed. PLGA was very brittle, whereas the blends showed an elastic behavior. This could be explained by ester interchange reactions that formed a loosely crosslinked network with ATEC.
The original habitat of native European honey bees (\(Apis\) \(mellifera\)) is forest, but currently there is a lack of data about the occurrence of wild honey bee populations in Europe. Prior to being kept by humans in hives, honey bees nested as wild species in hollow trees in temperate forests. However, in the 20th century, intensification of silviculture and agriculture with accompanying losses of nesting sites and depletion of food resources caused population declines in Europe. When the varroa mite (Varroa destructor), an invasive ectoparasite from Asia, was introduced in the late 1970s, wild honey bees were thought to be eradicated in Europe. Nevertheless, sporadic, mostly anecdotal, reports from ornithologists or forest ecologists indicated that honey bee colonies still occupy European forest areas. In my thesis I hypothesize that near-natural deciduous forests may provide sufficient large networks of nesting sites representing refugia for wild-living honey bees. Using two special search techniques, i.e. the tracking of flight routes of honey bee foragers (the “beelining” method) and the inspection of known cavity trees, I collected for the first time data on the occurrence and density of wild-living honey bees in forest areas in Germany (CHAPTER 3). I found wild-living honey bee colonies in the Hainich national park at low densities in two succeeding years. In another forest region, I checked known habitat trees containing black woodpecker cavities for occupation by wild-living honey bee colonies. It turned out that honey bees regularly use these cavities and occur in similar densities in both studied forest regions, independent of the applied detection method. Extrapolating these densities to all German forest areas, I estimate several thousand wild-living colonies in Germany that potentially interact in different ways with the forest environment. I conclude that honey bees regularly colonize forest areas in Germany and that networks of mapped woodpecker cavities offer unique possibilities to study the ecology of wild-living honey bees over several years.
While their population status is ambiguous and the density of colonies low, the fact that honey bees can still be found in forests poses questions about food supply in forest environments. Consequently, I investigated the suitability of woodlands as a honey bee foraging habitat (CHAPTER 4). As their native habitat, forests are assumed to provide important pollen and nectar sources for honey bee colonies. However, resource supply might be spatially and temporally restricted and landscape-scale studies in European forest regions are lacking. Therefore, I set up twelve honey bee colonies in observation hives at locations with varying degree of forest cover. Capitalizing on the unique communication behaviour, the waggle dance, I examined the foraging distances and habitat preferences of honey bees over almost an entire foraging season. Moreover, by connecting this decoded dance information with colony weight recordings, I could draw conclusions about the contribution of the different habitat types to honey yield. Foraging distances generally increased with the amount of forest in the surrounding landscape. Yet, forest cover did not have an effect on colony weight. Compared to expectations based on the proportions of different habitats in the surroundings, colonies foraged more frequently in cropland and grasslands than in deciduous and coniferous forests, especially in late summer when pollen foraging in the forest is most difficult. In contrast, colonies used forests for nectar/honeydew foraging in early summer during times of colony weight gain emphasizing forests as a temporarily significant source of carbohydrates. Importantly, my study shows that the ecological and economic value of managed forest as habitat for honey bees and other wild pollinators can be significantly increased by the continuous provision of floral resources, especially for pollen foraging.
The density of these wild-living honey bee colonies and their survival is driven by several factors that vary locally, making it crucial to compare results in different regions. Therefore, I investigated a wild-living honey bee population in Galicia in north-western Spain, where colonies were observed to reside in hollow electric poles (CHAPTER 5). The observed colony density only in these poles was almost twice as high as in German forest areas, suggesting generally more suitable resource conditions for the bees in Galicia. Based on morphometric analyses of their wing venation patterns, I assigned the colonies to the native evolutionary lineage (M-lineage) where the particularly threatened subspecies \(Apis\) \(mellifera\) \(iberiensis\) also belongs to. Averaged over two consecutive years, almost half of the colonies survived winter (23 out of 52). Interestingly, semi-natural areas both increased abundance and subsequent colony survival. Colonies surrounded by more semi-natural habitat (and therefore less intensive cropland) had an elevated overwintering probability, indicating that colonies need a certain amount of semi-natural habitat in the landscape to survive. Due to their ease of access these power poles in Galicia are, ideally suited to assess the population demography of wild-living Galician honey bee colonies through a long-term monitoring.
In a nutshell, my thesis indicates that honey bees in Europe always existed in the wild. I performed the first survey of wild-living bee density yet done in Germany and Spain. My thesis identifies the landscape as a major factor that compromises winter survival and reports the first data on overwintering rates of wild-living honey bees in Europe. Besides, I established methods to efficiently detect wild-living honey bees in different habitat. While colonies can be found all over Europe, their survival and viability depend on unpolluted, flower rich habitats. The protection of near-natural habitat and of nesting sites is of paramount importance for the conservation of wild-living honey bees in Europe.
The Seville Strategy spurred a signifi cant paradigm shift in UNESCO’s MAB Programme, re-conceptualising the research programme as a modern tool for the dual mandate of nature conservation and sustainable development. However, many biosphere reserves failed to comply with the new regulations and in 2013 the ‘Exit Strategy’ was announced to improve the quality of the global network.
This study presents a global assessment of the implementation of the quality enhancement strategies, highlighting signifi cant differences worldwide through 20 country-specifi c case studies. It concludes that the strategies have been fundamental in improving the credibility and coherence of the MAB Programme. Challenges in the implementation were not unique to individual countries but were common to all Member States with pre-Seville sites, and in many states the process has led to a rejuvenation of national biosphere reserve networks.
Cancer is one of the leading causes of death worldwide. The underlying tumorigenesis is driven by the accumulation of alterations in the genome, eventually disabling tumor suppressors and activating proto-oncogenes.
The MYC family of proto-oncogenes shows a strong deregulation in the majority of tumor entities. However, the exact mechanisms that contribute to MYC-driven oncogenesis remain largely unknown. Over the past decades, the influence of the MYC protein on transcription became increasingly apparent and was thoroughly investigated. Additionally, in recent years several publications provided evidence for so far unreported functions of MYC that are independent of a mere regulation of target genes. These findings suggest an additional role of MYC in the maintenance of genomic stability and this role is strengthened by key findings presented in this thesis.
In the first part, I present data revealing a pathway that allows MYC to couple transcription elongation and DNA double-strand break repair, preventing genomic instability of MYC-driven tumor cells. This pathway is driven by a rapid transfer of the PAF1 complex from MYC onto RNAPII, a process that is mediated by HUWE1. The transfer controls MYC-dependent transcription elongation and, simultaneously, the remodeling of chromatin structure by ubiquitylation of histone H2B. These regions of open chromatin favor not only elongation but also DNA double-strand break repair.
In the second part, I analyze the ability of MYC proteins to form multimeric structures in response to perturbation of transcription and replication. The process of multimerization is also referred to as phase transition. The observed multimeric structures are located proximal to stalled replication forks and recruit factors of the DNA-damage response and transcription termination machinery. Further, I identified the HUWE1-dependent ubiquitylation of MYC as an essential step in this phase transition. Cells lacking the ability to form multimers display genomic instability and ultimately undergo apoptosis in response to replication stress.
Both mechanisms present MYC as a stress resilience factor under conditions that are characterized by a high level of transcriptional and replicational stress. This increased resilience ensures oncogenic proliferation.
Therefore, targeting MYC’s ability to limit genomic instability by uncoupling transcription elongation and DNA repair or disrupting its ability to multimerize presents a therapeutic window in MYC-dependent tumors.
For formative evaluations of user experience (UX) a variety of methods have been developed over the years. However, most techniques require the users to interact with the study as a secondary task. This active involvement in the evaluation is not inclusive of all users and potentially biases the experience currently being studied. Yet there is a lack of methods for situations in which the user has no spare cognitive resources. This condition occurs when 1) users' cognitive abilities are impaired (e.g., people with dementia) or 2) users are confronted with very demanding tasks (e.g., air traffic controllers). In this work we focus on emotions as a key component of UX and propose the new structured observation method Proxemo for formative UX evaluations. Proxemo allows qualified observers to document users' emotions by proxy in real time and then directly link them to triggers. Technically this is achieved by synchronising the timestamps of emotions documented by observers with a video recording of the interaction.
In order to facilitate the documentation of observed emotions in highly diverse contexts we conceptualise and implement two separate versions of a documentation aid named Proxemo App. For formative UX evaluations of technology-supported reminiscence sessions with people with dementia, we create a smartwatch app to discreetly document emotions from the categories anger, general alertness, pleasure, wistfulness and pride. For formative UX evaluations of prototypical user interfaces with air traffic controllers we create a smartphone app to efficiently document emotions from the categories anger, boredom, surprise, stress and pride. Descriptive case studies in both application domains indicate the feasibility and utility of the method Proxemo and the appropriateness of the respectively adapted design of the Proxemo App.
The third part of this work is a series of meta-evaluation studies to determine quality criteria of Proxemo. We evaluate Proxemo regarding its reliability, validity, thoroughness and effectiveness, and compare Proxemo's efficiency and the observers' experience to documentation with pen and paper. Proxemo is reliable, as well as more efficient, thorough and effective than handwritten notes and provides a better UX to observers. Proxemo compares well with existing methods where benchmarks are available.
With Proxemo we contribute a validated structured observation method that has shown to meet requirements formative UX evaluations in the extreme contexts of users with cognitive impairments or high task demands. Proxemo is agnostic regarding researchers' theoretical approaches and unites reductionist and holistic perspectives within one method.
Future work should explore the applicability of Proxemo for further domains and extend the list of audited quality criteria to include, for instance, downstream utility. With respect to basic research we strive to better understand the sources leading observers to empathic judgments and propose reminisce and older adults as model environment for investigating mixed emotions.
Summary
Platelet activation and aggregation at sites of vascular injury is critical to prevent excessive blood loss, but may also lead to life-threatening ischemic disease states, such as myocardial infarction and stroke. Glycoprotein (GP) VI and C type lectin-like receptor 2 (CLEC-2) are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter protein (SLAP) and SLAP2 are involved in the regulation of immune cell receptor surface expression and signaling, but their function in platelets is unknown. As revealed in this thesis, single deficiency of SLAP or SLAP2 in mice had only moderate effects on platelet function, while SLAP/SLAP2 double deficiency resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity and thrombin generation following (hem)ITAM-coupled, but not G protein-coupled receptor activation. Slap-/-/Slap2-/- mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. These results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.
GPVI has emerged as a promising novel pharmacological target for treatment of thrombotic and inflammatory disease states, but the exact mechanisms of its immunodepletion in vivo are incompletely understood. It was hypothesized that SLAP and SLAP2 may be involved in the control of GPVI down-regulation because of their role in the internalization of immune cell receptors. As demonstrated in the second part of the thesis, SLAP and SLAP2 were dispensable for antibody-induced GPVI down-regulation, but anti-GPVI treatment resulted in prolonged strong thrombocytopenia in Slap-/-/Slap2-/- mice. The profound thrombocytopenia likely resulted from the powerful platelet activation which the anti-GPVI antibody induced in Slap-/-/Slap2-/- platelets, but importantly, not in wild-type platelets. These data indicate that the expression and activation state of key modulators of the GPVI signaling cascade may have important implications for the safety profile and efficacy of anti-GPVI agents.
Small GTPases of the Rho family, such as RhoA and Cdc42, are critically involved in the regulation of cytoskeletal rearrangements during platelet activation, but little is known about the specific roles and functional redundancy of both proteins in platelet biogenesis. As shown in the final part of the thesis, combined deficiency of RhoA and Cdc42 led to marked alterations in megakaryocyte morphology and the generation of platelets of heterogeneous size and granule content. Despite severe hemostatic defects and profound thrombo¬cytopenia, circulating RhoA-/-/Cdc42-/- platelets were still capable of granule secretion and the formation of occlusive thrombi. These results implicate the existence of both distinct and overlapping roles of RhoA and Cdc42 in platelet production and function.
Empathy, the act of sharing another person’s affective state, is a ubiquitous driver for helping others and feeling close to them. These experiences are integral parts of human behavior and society. The studies presented in this dissertation aimed to investigate the sustainability and stability of social closeness and prosocial decision-making driven by empathy and other social motives. In this vein, four studies were conducted in which behavioral and neural indicators of empathy sustainability were identified using model-based functional magnetic resonance imaging (fMRI).
Applying reinforcement learning, drift-diffusion modelling (DDM), and fMRI, the first two studies were designed to investigate the formation and sustainability of empathy-related social closeness (study 1) and examined how sustainably empathy led to prosocial behavior (study 2). Using DDM and fMRI, the last two studies investigated how empathy combined with reciprocity, the social norm to return a favor, on the one hand and empathy combined with the motive of outcome maximization on the other hand altered the behavioral and neural social decision process.
The results showed that empathy-related social closeness and prosocial decision tendencies persisted even if empathy was rarely reinforced. The sustainability of these empathy effects was related to recalibration of the empathy-related social closeness learning signal (study 1) and the maintenance of a prosocial decision bias (study 2). The findings of study 3 showed that empathy boosted the processing of reciprocity-based social decisions, but not vice versa. Study 4 revealed that empathy-related decisions were modulated by the motive of outcome maximization, depending on individual differences in state empathy.
Together, the studies strongly support the concept of empathy as a sustainable driver of social closeness and prosocial behavior.
After the discovery of three-dimensional topological insulators (TIs), such as tetradymite chalcogenides Bi$_2$Se$_3$, Bi$_2$Te$_3$ and Sb$_2$Te$_3$ – a new class of quantum materials characterized by their unique surface electronic properties – the solid state community got focused on topological states that are driven by strong electronic correlations and magnetism. An important material class is the magnetic TI (MTI) exhibiting the quantum anomalous Hall (QAH) effect, i.e. a dissipationless quantized edge-state transport in the absence of external magnetic field, originating from the interplay between ferromagnetism and a topologically non-trivial band structure. The unprecedented opportunities offered by these new exotic materials open a new avenue for the development of low-dissipation electronics, spintronics, and quantum computation. However, the major concern with QAH effect is its extremely low onset temperature, limiting its practical application. To resolve this problem, a comprehensive understanding of the microscopic origin of the underlying ferromagnetism is necessary.
V- and Cr-doped (Bi,Sb)$_2$Te$_3$ are the two prototypical systems that have been widely studied as realizations of the QAH state. Finding microscopic differences between the strongly correlated V and Cr impurities would help finding a relevant model of ferromagnetic coupling and eventually provide better control of the QAH effect in these systems. Therefore, this thesis first focuses on the V- and Cr-doped (Bi,Sb)$_2$Te$_3$ systems, to better understand these differences. Exploiting the unique capabilities of x-ray absorption spectroscopy and magnetic circular dichroism (XAS/XMCD), combined with advanced modeling based on multiplet ligand-field theory (MLFT), we provide a detailed microscopic insight into the local electronic and magnetic properties of these systems and determine microscopic parameters crucial for the comparison with theoretical models, which include the $d$-shell filling, spin and orbital magnetic moments. We find a strongly covalent ground state, dominated by the superposition of one and two Te-ligand-hole configurations, with a negligible contribution from a purely ionic 3+ configuration. Our findings indicate the importance of the Te $5p$ states for the ferromagnetism in (Bi, Sb)$_2$Te$_3$ and favor magnetic coupling mechanisms involving $pd$-exchange. Using state-of-the-art density functional theory (DFT) calculations in combination with XMCD and resonant photoelectron spectroscopy (resPES), we reveal the important role of the $3d$ impurity states in mediating magnetic exchange coupling. Our calculations illustrate that the kind and strength of the exchange coupling varies with the impurity $3d$-shell occupation. We find a weakening of ferromagnetic properties upon the increase of doping concentration, as well as with the substitution of Bi at the Sb site. Finally, we qualitatively describe the origin of the induced magnetic moments at the Te and Sb sites in the host lattice and discuss their role in mediating a robust ferromagnetism based on a $pd$-exchange interaction scenario. Our findings reveal important clues to designing higher $T_{\text{C}}$ MTIs.
Rare-earth ions typically exhibit larger magnetic moments than transition-metal ions and thus promise the opening of a wider exchange gap in the Dirac surface states of TIs, which is favorable for the realization of the high-temperature QAH effect. Therefore, we have further focused on Eu-doped Bi$_2$Te$_3$ and scrutinized whether the conditions for formation of a substantial gap in this system are present by combining spectroscopic and bulk characterization methods with theoretical calculations. For all studied Eu doping concentrations, our atomic multiplet analysis of the $M_{4,5}$ x-ray absorption and magnetic circular dichroism spectra reveals a Eu$^{2+}$ valence, unlike most other rare earth elements, and confirms a large magnetic moment. At temperatures below 10 K, bulk magnetometry indicates the onset of antiferromagnetic ordering. This is in good agreement with DFT results, which predict AFM interactions between the Eu impurities due to the direct overlap of the impurity wave functions. Our results support the notion of antiferromagnetism coexisting with topological surface states in rare-earth doped Bi$_2$Te$_3$ and corroborate the potential of such doping to result in an antiferromagnetic TI with exotic quantum properties.
The doping with impurities introduces disorder detrimental for the QAH effect, which may be avoided in stoichiometric, well-ordered magnetic compounds. In the last part of the thesis we have investigated the recently discovered intrinsic magnetic TI (IMTI) MnBi$_6$Te$_{10}$, where we have uncovered robust ferromagnetism with $T_{\text{C}} \approx 12$ K and connected its origin to the Mn/Bi intermixing. Our measurements reveal a magnetically intact surface with a large moment, and with FM properties similar to the bulk, which makes MnBi$_6$Te$_{10}$ a promising candidate for the QAH effect at elevated temperatures. Moreover, using an advanced ab initio MLFT approach we have determined the ground-state properties of Mn and revealed a predominant contribution of the $d^5$ configuration to the ground state, resulting in a $d$-shell electron occupation $n_d = 5.31$ and a large magnetic moment, in excellent agreement with our DFT calculations and the bulk magnetometry data. Our results together with first principle calculations based on the DFT-GGA$+U$, performed by our collaborators, suggest that carefully engineered intermixing plays a crucial role in achieving a robust long-range FM order and therefore could be the key for achieving enhanced QAH effect properties.
We expect our findings to aid better understanding of MTIs, which is essential to help increasing the temperature of the QAH effect, thus facilitating the realization of low-power electronics in the future.
About 2.4 billion years ago, nature has fundamentally revolutionized life on earth by inventing the multi-subunit protein complex photosystem II, the only molecular machine in nature that catalyzes the thermodynamically demanding photosynthetic splitting of water into oxygen and reducing equivalents. Nature chose a distorted Mn4CaO5 cluster as catalyst, better known as oxygen-evolving complex (OEC), thus recognizing the need for transition metals to achieve high-performance catalysts. The curiosity has always driven mankind to mimic nature’s achievements, but the performance of natural enzymes such as the oxygen-evolving complex in photosystem II remain commonly unmatched. An important role in fine-tuning and regulating the activity of natural enzymes is attributed to the surrounding protein domain, which facilitates substrate preorganization within well-defined nanoenvironments.
In light of growing energy demands and the depletion of fossil fuels, the unparalleled efficiency of natural photosynthesis inspires chemists to artificially mimic its natural counterpart to generate hydrogen as a ‘solar fuel’ through the light-driven splitting of water. As a result, significant efforts have been devoted in recent decades to develop molecular water oxidation catalysts based on earth-abundant transition metals and the discovery of the Ru(bda) (bda: 2,2’ bipyridine-6,6’-dicarboxylate) catalyst family enabled activities comparable to the natural OEC. Similar to the natural archetypes, the design of homogeneous catalysts that interplay judiciously with the second coordination sphere of the outer ligand framework proved to be a promising concept for catalyst design. In this present thesis, novel supramolecular design approaches for enzyme like activation of substrate water molecules for the challenging oxidative water splitting reaction were established via tailor-made engineering of the secondary ligand environment of macrocyclic Ru(bda) catalysts.
The 1st chapter provides a detailed review of the development of synthetic approaches to triarylboranes from their first report nearly 135 years ago to the present. In the 2nd chapter, a novel and convenient methodology is reported for the one-pot synthesis of sterically-congested triarylboranes, using bench-stable aryltrifluoroborates as the boron source. The new procedure gives access to symmetrically- and unsymmetrically-substituted triarylboranes. The borylated triarylboranes are suggested as building blocks for the design of functional materials. In the 3rd chapter, four luminescent tetracationic bis-triarylborane DNA and RNA sensors that show high binding affinities, in several cases even in the nM range, are investigated. The molecular structures of two of the neutral precursors reveal some structural flexibility for these compounds in the solid state. The compounds were found to be highly emissive even in water and DNA and RNA binding affinities were found to be dependent on linker length and flexibility. Strong SERS responses for three of the four compounds demonstrate the importance of triple bonds for strong Raman activity in molecules of this compound class. In chapter 4, the compound class of water-soluble tetracationic bis-triarylborane chromophores is extended by EDOT-linked compounds and those are compared to their thiophene-containing analogs. Absorption and emission are significantly red-shifted in these compounds, compared to their thiophene-containing analogs and, due to a large Stokes shift, one of the reported compounds exhibits the most bathochromically shifted emission, observable well into the near infrared region, of all tetracationic water-soluble bis-triarylborane chromophores reported to date. Long-lived excited states, completely quenched by oxygen, were observed for the water-stable compounds of this study via transient absorption spectroscopy and a quantum yield for singlet oxygen formation of 0.6 was determined for one of them.
Stomata are pores in the leaf surface, formed by pairs of guard cells. The guard cells modulate the aperture of stomata, to balance uptake of CO2 and loss of water vapor to the atmosphere. During drought, the phytohormone abscisic acid (ABA) provokes stomatal closure, via a signaling chain with both Ca2+-dependent and Ca2+-independent branches. Both branches are likely to activate SLAC1-type (Slow Anion Channel Associated 1) anion channels that are essential for initiating the closure of stomata. However, the importance of the Ca2+-dependent signaling branch is still debated, as the core ABA signaling pathway only possesses Ca2+-independent components. Therefore, the aim of this thesis was to address the role of the Ca2+-dependent branch in the ABA signaling pathway of guard cells.
In the first part of the thesis, the relation between ABA-induced Ca2+ signals and stomatal closure was studied, with guard cells that express the genetically encoded Ca2+-indicator R-GECO1-mTurquoise. Ejection of ABA into the guard cell wall rapidly induced stomatal closure, however, only in ¾ of the guard cells ABA evoked a cytosolic Ca2+ signal. A small subset of stomata (¼ of the experiments) closed without Ca2+ signals, showing that the Ca2+ signals are not essential for ABA-induced stomatal closure. However, stomata in which ABA evoked Ca2+ signals closed faster as those in which no Ca2+ signals were detected. Apparently, ABA-induced Ca2+ signals enhance the velocity of stomatal closure. In addition to ABA, hyperpolarizing voltage pulses could also trigger Ca2+ signals in wild type guard cells, which in turn activated S-type anion channels. However, these voltage pulses failed to elicit S-type anion currents in the slac1/slah3 guard cells, suggesting that SLAC1 and SLAH3 contribute to Ca2+-activated conductance. Taken together, our data indicate that ABA-induced Ca2+ signals enhance the activity of S-type anion channels, which accelerates stomatal closure.
The second part of the thesis deals with the signaling pathway downstream of the Ca2+ signals. Two types of Ca2+-dependent protein kinase modules (CPKs and CBL/CIPKs) have been implicated in guard cells. We focused on the protein kinase CIPK23 (CBL-Interacting Protein Kinase 23), which is activated by the Ca2+-dependent protein CBL1 or 9 (Calcineurin B-Like protein 1 or 9) via interacting with the NAF domain of CIPK23. The CBL1/9-CIPK23 complex has been shown to affect stomatal movements, but the underlying molecular mechanisms remain largely unknown. We addressed this topic by using an estrogen-induced expression system, which specifically enhances the expression of wild type CIPK23, a phosphomimic CIPK23T190D and a kinase dead CIPK23K60N in guard cells. Our data show that guard cells expressing CIPK23T190D promoted stomatal opening, while CIPK23K60N enhanced ABA-induced stomatal closure, suggesting that CIPK23 is a negative regulator of stomatal closure. Electrophysiological measurements revealed that the inward K+ channel currents were similar in guard cells that expressed CIPK23, CIPK23T190D or CIPK23K60N, indicating that CIPK23-mediated inward K+ channel AKT1 does not contribute to stomatal movements. Expression of CIPK23K60N, or loss of CIPK23 in guard cells enhanced S-type anion activity, while the active CIPK23T190D inhibited the activity of these anion channels. These results are in line with the detected changes in stomatal movements and thus indicate that CIPK23 regulates stomatal movements by inhibiting S-type anion channels. CIPK23 thus serves as a brake to control anion channel activity. Overall, our findings demonstrate that CIPK23-mediated stomatal movements do not depend on CIPK23-AKT1 module, instead, it is achieved by regulating S-type anion channels SLAC1 and SLAH3.
In sum, the data presented in this thesis give new insights into the Ca2+-dependent branch of ABA signaling, which may help to put forward new strategies to breed plants with enhanced drought stress tolerance, and in turn boost agricultural productivity in the future.
Serverless computing is an emerging cloud computing paradigm that offers a highlevel
application programming model with utilization-based billing. It enables the
deployment of cloud applications without managing the underlying resources or
worrying about other operational aspects. Function-as-a-Service (FaaS) platforms
implement serverless computing by allowing developers to execute code on-demand
in response to events with continuous scaling while having to pay only for the
time used with sub-second metering. Cloud providers have further introduced
many fully managed services for databases, messaging buses, and storage that also
implement a serverless computing model. Applications composed of these fully
managed services and FaaS functions are quickly gaining popularity in both industry
and in academia.
However, due to this rapid adoption, much information surrounding serverless
computing is inconsistent and often outdated as the serverless paradigm evolves.
This makes the performance engineering of serverless applications and platforms
challenging, as there are many open questions, such as: What types of applications
is serverless computing well suited for, and what are its limitations? How should
serverless applications be designed, configured, and implemented? Which design
decisions impact the performance properties of serverless platforms and how can
they be optimized? These and many other open questions can be traced back to an
inconsistent understanding of serverless applications and platforms, which could
present a major roadblock in the adoption of serverless computing.
In this thesis, we address the lack of performance knowledge surrounding serverless
applications and platforms from multiple angles: we conduct empirical studies
to further the understanding of serverless applications and platforms, we introduce
automated optimization methods that simplify the operation of serverless applications,
and we enable the analysis of design tradeoffs of serverless platforms by
extending white-box performance modeling.
Since the advent of high-throughput sequencing technologies in the mid-2010s, RNA se-
quencing (RNA-seq) has been established as the method of choice for studying gene
expression. In comparison to microarray-based methods, which have mainly been used to
study gene expression before the rise of RNA-seq, RNA-seq is able to profile the entire
transcriptome of an organism without the need to predefine genes of interest. Today,
a wide variety of RNA-seq methods and protocols exist, including dual RNA sequenc-
ing (dual RNA-seq) and multi RNA sequencing (multi RNA-seq). Dual RNA-seq and
multi RNA-seq simultaneously investigate the transcriptomes of two or more species, re-
spectively. Therefore, the total RNA of all interacting species is sequenced together and
only separated in silico. Compared to conventional RNA-seq, which can only investi-
gate one species at a time, dual RNA-seq and multi RNA-seq analyses can connect the
transcriptome changes of the species being investigated and thus give a clearer picture of
the interspecies interactions. Dual RNA-seq and multi RNA-seq have been applied to a
variety of host-pathogen, mutualistic and commensal interaction systems.
We applied dual RNA-seq to a host-pathogen system of human mast cells and Staphylo-
coccus aureus (S. aureus). S. aureus, a commensal gram-positive bacterium, can become
an opportunistic pathogen and infect skin lesions of atopic dermatitis (AD) patients.
Among the first immune cells S. aureus encounters are mast cells, which have previously
been shown to be able to kill the bacteria by discharging antimicrobial products and re-
leasing extracellular traps made of protein and deoxyribonucleic acid (DNA). However,
S. aureus is known to evade the host’s immune response by internalizing within mast
cells. Our dual RNA-seq analysis of different infection settings revealed that mast cells
and S. aureus need physical contact to influence each other’s gene expression. We could
show that S. aureus cells internalizing within mast cells undergo profound transcriptome
changes to adjust their metabolism to survive in the intracellular niche. On the host side,
we found out that infected mast cells elicit a type-I interferon (IFN-I) response in an
autocrine manner and in a paracrine manner to non-infected bystander-cells. Our study
provides the first evidence that mast cells are capable to produce IFN-I upon infection
with a bacterial pathogen.
Neurodegeneration plays an essential role in Parkinson’s disease (PD). Several crucial neuronal pro-and antidegeneration markers were described to be altered in disease models accompanied by neurodegeneration. In the AAV1/2-A53T-aSyn PD rat model progressive time-dependent motor impairment and neurodegeneration in the nigrostriatal tract starting from 2 weeks after PD model induction could be found. Downregulation of Nrf2 in SN and nigrostriatal axon localization, a trend of Tau downregulation in SN and upregulation in axon localization in the AAV1/2-A53T-aSyn PD rat model were observed, indicating potential therapeutic value of these two molecular targets in PD. No alterations of SARM1 and NMNAT2 could be detected, indicating little relevance of these two molecules with our AAV1/2-A53T-aSyn rat model.
Plexus injury often occurs after motor vehicle accidents and results in lifelong disability with severe neuropathic pain. Surgical treatment can partially restore motor functions, but sensory loss and neuropathic pain persist. Regenerative medicine concepts, such as cell replacement therapies for restoring dorsal root ganglia (DRG) function, set high expectations. However, up to now, it is unclear which DRG cell types are affected by nerve injury and can be targeted in regenerative medicine approaches.
This study followed the hypothesis that satellite glial cells (SGCs) might be a suitable endogenous cell source for regenerative medicine concepts in the DRG. SGCs originate from the same neural crest-derived cell lineage as sensory neurons, making them attractive for neural repair strategies in the peripheral nervous system. Our hypothesis was investigated on three levels of experimentation. First, we asked whether adult SGCs have the potential of sensory neuron precursors and can be reprogrammed into sensory neurons in vitro. We found that adult mouse DRG harbor SGC-like cells that can still dedifferentiate into progenitor-like cells. Surprisingly, expression of the early developmental transcription factors Neurog1 and Neurog2 was sufficient to induce neuronal and glial cell phenotypes. In the presence of nerve growth factor, induced neurons developed a nociceptor-like phenotype expressing functional nociceptor markers, such as the ion channels TrpA1, TrpV1 and NaV1.9. In a second set of experiments, we used a rat model for peripheral nerve injury to look for changes in the DRG cell composition. Using an unbiased deep learning-based approach for cell analysis, we found that cellular plasticity responses after nerve injury activate SGCs in the whole DRG. However, neither injury-induced neuronal death nor gliosis was observed. Finally, we asked whether a severe nerve injury changed the cell composition in the human DRG. For this, a cohort of 13 patients with brachial plexus injury was investigated. Surprisingly, in about half of all patients, the injury-affected DRG showed no characteristic DRG tissue. The complete entity of neurons, satellite cells, and axons was lost and fully replaced by mesodermal/connective tissue. In the other half of the patients, the basic cellular entity of the DRG was well preserved. Objective deep learning-based analysis of large-scale bioimages of the “intact” DRG showed no loss of neurons and no signs of gliosis.
This study suggests that concepts for regenerative medicine for restoring DRG function need at least two translational research directions: reafferentation of existing DRG units or full replacement of the entire multicellular DRG structure. For DRG replacement, SGCs of the adult DRG are an attractive endogenous cell source, as the multicellular DRG units could possibly be rebuilt by transdifferentiating neural crest-derived sensory progenitor cells into peripheral sensory neurons and glial cells using Neurog1 and Neurog2.
Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes.
The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration.
Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.
Companies are expected to act as international players and to use their capabilities to provide customized products and services quickly and efficiently. Today, consumers expect their requirements to be met within a short time and at a favorable price. Order-to-delivery lead time has steadily gained in importance for consumers. Furthermore, governments can use various emissions policies to force companies and customers to reduce their greenhouse gas emissions. This thesis investigates the influence of order-to-delivery lead time and different emission policies on the design of a supply chain. Within this work different supply chain design models are developed to examine these different influences. The first model incorporates lead times and total costs, and various emission policies are implemented to illustrate the trade-off between the different measures. The second model reflects the influence of order-to-delivery lead time sensitive consumers, and different emission policies are implemented to study their impacts. The analysis shows that the share of order-to-delivery lead time sensitive consumers has a significant impact on the design of a supply chain. Demand uncertainty and uncertainty in the design of different emission policies are investigated by developing an appropriate robust mathematical optimization model. Results show that especially uncertainties on the design of an emission policy can significantly impact the total cost of a supply chain. The effects of differently designed emission policies in various countries are investigated in the fourth model. The analyses highlight that both lead times and emission policies can strongly influence companies' offshoring and nearshoring strategies.
Fear and anxiety disorders – interaction of AVP and OXT brain systems with the serotonergic system
(2023)
Anxiety disorders pose a great burden onto society and economy and can have devastating consequences for affected individuals. Treatment options are still limited to psychopharmacotherapy originally developed for the treatment of depression and behavioral therapy. A combination of genetic traits together with aversive events is most likely the cause of these diseases. Gene x environment studies are trying to find a link between genetic traits and specific negative circumstances. In a first study, we focused on social anxiety disorder (SAD), which is the second most-common anxiety disorder after specific phobias. We used a social fear conditioning (SFC) paradigm, which is able to mimic the disease in a mouse model. We wanted to investigate protein levels, as well as mRNA expression of immediate early genes (IEGs), to determine brain areas affected by the paradigm. We also included genes of the vasopressin (AVP)-, oxytocin (OXT)-, neuropeptide Y (NPY)-, and the serotonin system, to investigate the effects of SFC on neurotransmitter gene expression levels in brain regions related to social as well as fear-related behavior. AVP and OXT regulate a lot of different social and anxiety-related behaviors, both positive and negative. Finding a link between different neurotransmitter systems in the development of anxiety disorders could help to identify potential targets for new treatment approaches, which are desperately needed, because the rate of patients not responding to available treatment is very high.
We were able to show altered gene expression of the IEGs cFos and Fosl2, as well as a change in number and density of cFOS-positive cells in the dorsal hippocampus, indicating an influence of SFC on neuronal activity. Our results reveal a possible involvement of anterior dentate gyrus (DG), as well as cornu ammonis area 1 (CA1) and CA3 in the dorsal hippocampus during the expression of social fear. Contrary to our hypothesis, we were not able to see changes in neuronal activity through expression changes of IEGs in the amygdala. Significant higher IEG immunoreactivity and gene expression in the dorsal hippocampus of animals without fear conditioning (SFC-), compared to animals with fear conditioning (SFC+), indicate an involvement of different hippocampal regions in two possible scenarios. Either as elevated gene expression in SFC- animals compared to SFC+ animals or as reduction in SFC+ animals compared to SFC- animals. However, this question cannot be answered without an additional control of basal IEG-activity without social interaction. The NPY system in general and the neuropeptide y receptor type 2 in particular seem to be involved in regulating the response to social fear, mostly through the septum region. In addition to that, a possible role for the induction of social fear response could be identified in the serotonergic system and especially the serotonin receptor 2a of the PVN.
In a second study we focused on changes in the serotonergic system. A polymorphism in the human serotonin transporter (5-HTT) gene is associated with higher risks for the development of anxiety disorders. This makes the 5-HTT a widely used target to study possible causes and the development of anxiety disorders. In mice, a genetically induced knockout of the 5-Htt gene is associated with increased anxiety-like behavior. High amounts of stress during pregnancy, also known as prenatal stress, significantly increase the risk to develop psychiatric disorders for the unborn child. We utilized a prenatal stress paradigm in mice heterozygous for the 5-Htt gene. Some of the animals which had been subjected to prenatal stress showed noticeably “unsocial” interaction behavior towards conspecifics. Again, we were searching for links between the serotonergic system and AVP- and OXT systems. Through quantitative gene expression analysis, we were able to show that both AVP and OXT neuromodulator systems are affected through prenatal stress in female mice, but not in male mice. The 5-Htt genotype seems to be only slightly influential to AVP, OXT or any other neurotransmitter system investigated. Gene expression of AVP and OXT brain systems is highly influenced through the estrous cycle stages of female mice. Additionally, we analyzed the AVP and OXT neuropeptide levels of mice with different 5-Htt genotypes and in both sexes, in order to see whether the production of AVP and OXT is influenced by 5-Htt genotype. On neuropeptide level, we were able to identify a sex difference for vasopressin-immunoreactive (ir) cells in the PVN, with male mice harboring significantly more positive cells than female mice.
All presented studies aimed on the improvement of the quality analysis of already monographed drugs. Thereby different LC methods were applied and coupled to i.e., the UV/VIS detector, the CAD or a hyphenation of these detectors, respectively. The choice of the chromatographic system including the detector was largely dependent on the physicochemical properties of the respective analytes.
With the risk-assessment report on the API cetirizine we presented an exemplary tool, that can help to minimize the risk of the occurrence of unexpected impurities. An in- deep analysis of each step within synthesis pathway by means of reaction matrices of all compounds was performed. It is essential to understand the complete impurity profile of all reactants, solvents, and catalysts and to include them in the matrix. Finally, the API of this synthesis was checked if all impurities are identified by this tool. Of note, a shortcoming of such a targeted approach is that impurities can still occur, but they are not captured. This disadvantage can be partially compensated by non-targeted approaches if they are performed in parallel with the other studies that represent most of the impurities. However, this work also shows that even in a supposedly simple synthesis, potentially hundreds of by-products can be formed. For each of them, it must be decided individually whether their formation is probable or how their quantity can be minimized in order to obtain APIs, that are as pure as possible.
In the dapsone project it was aimed to replace the existing old Ph. Eur. TLC method with a modern RP-HPLC method. This was successful and since Ph. Eur. 10.6, the method developed in this work, became a valid monograph. Within the revision process of the monograph, the individual limits for impurities were tightened. However, this new method needs HPLC instrumentation, suitable to perform gradients. As this is not always available in all control laboratories, we also developed an alternative, more simple method using two different isocratic runs for the impurity analysis. The obtained batch results of both, the new pharmacopoeial method and the more simple one, were in a comparable order of magnitude. Furthermore, within the method development stage of the Ph. Eur. method, we could identify one unknown impurity of the impurity reference by high-resolution MS/MS analysis.
Also, in the baclofen project it was aimed to replace the existing Ph. Eur. method with the introduction of an additional impurity to be quantified. A corresponding method was developed and validated. However, due to the harmonization process of the pharmacopoeias, it is currently not used. In addition, we tried to find further, non- 116
SUMMARY
chromophoric impurities by means of the CAD. However, except for one counterion of an impurity, no further impurities were found. Also, the aforementioned new impurity could not be detected above the reporting threshold in the batches analyzed. As the only individually specified impurity A is also present at a low level, it can be concluded that the examined batches of baclofen are very pure.
The use of universal detectors, such as the CAD can be particularly interesting for compounds with no chromophore or those with only a weak chromophore. Therefore, we decided to take a closer look at the impurity profile of acarbose. Currently, acarbose and its impurities are being studied by low wavelength UV detection at 210 nm. Therefore, the question arose whether there are no other impurities in the API that do not show absorption at this wavelength. CAD, which offers consistent detection properties for all non-volatile compounds, is ideally suited for this purpose. However, it was not so easy to use the CAD together with the UV detector, for example, as a hyphenated detection technique, because the Ph. Eur. method uses phosphate buffers. However, this is non-volatile and therefore inappropriate for the CAD. Therefore, an attempt was made to replace the buffer with a volatile one. However, since this did not lead to satisfactory results and rather the self-degradation process of the stationary phase used could be observed by means of the CAD, it was decided to switch to alternative stationary phases. A column screening also revealed further difficulties with acarbose and its impurities: they show an epimerization reaction at the end of the sugar chain. However, since one wanted to have uniform peaks in the corresponding chromatograms, one had to accelerate this reaction significantly to obtain only one peak for each component. This was best achieved by using two stationary phases: PGC and Amide-HILIC. Impurity-profiling methods could be developed on each of the two phases. In addition, as expected, new impurities could be detected, albeit at a low level. Two of them could even be identified by spiking experiments as the sugar fragments maltose and maltotriose.
Taken together, it can be concluded, that this work has contributed significantly to the improvement of the quality analysis of monographed drugs. In addition to the presented general tool for the identification of potential impurities, one of the methods developed, had already been implemented to the Ph. Eur. In an effort to improve the CAD's universal detection capabilities, additional methods have also been developed. Further, new improved methods for the impurity profiling are ready to use.
Metallic nanostructures possess the ability to support resonances in the visible wavelength regime which are related to localized surface plasmons. These create highly enhanced electric fields in the immediate vicinity of metal surfaces. Nanoparticles with dipolar resonance also radiate efficiently into the far-field and hence serve as antennas for light. Such optical antennas have been explored during the last two decades, however, mainly as standalone units illuminated by external laser beams and more recently as electrically driven point sources, yet merely with basic antenna properties. This work advances the state of the art of locally driven optical antenna systems. As a first instance, the electric driving scheme including inelastic electron tunneling over a nanometer gap is merged with Yagi-Uda theory. The resulting antenna system consists of a suitably wired feed antenna, incorporating a tunnel junction, as well as several nearby parasitic elements whose geometry is optimized using analytical and numerical methods. Experimental evidence of unprecedented directionality of light emission from a nanoantenna is provided. Parallels in the performance between radiofrequency and optical Yagi-Uda arrays are drawn. Secondly, a pair of electrically connected antennas with dissimilar resonances is harnessed as electrodes in an organic light emitting nanodiode prototype. The organic material zinc phthalocyanine, exhibiting asymmetric injection barriers for electrons and holes, in conjunction with the electrode resonances, allows switching and controlling the emitted peak wavelength and directionality as the polarity of the applied voltage is inverted. In a final study, the near-field based transmission-line driving of rod antenna systems is thoroughly explored. Perfect impedance matching, corresponding to zero back-reflection, is achieved when the antenna acts as a generalized coherent perfect absorber at a specific frequency. It thus collects all guided, surface-plasmon mediated input power and transduces it to other nonradiative and radiative dissipation channels. The coherent interplay of losses and interference effects turns out to be of paramount importance for this delicate scenario, which is systematically obtained for various antenna resonances. By means of the here developed semi-analytical toolbox, even more complex nanorod chains, supporting topologically nontrivial localized edge states, are studied. The results presented in this work facilitate the design of complex locally driven antenna systems for optical wireless on-chip communication, subwavelength pixels, and loss-compensated integrated plasmonic nanocircuitry which extends to the realm of topological plasmonics.
This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.
This work deals with the acceleration of cardiovascular MRI for the assessment
of functional information in steady-state contrast and for viability assessment
during the inversion recovery of the magnetization. Two approaches
are introduced and discussed in detail. MOCO-MAP uses an exponential
model to recover dynamic image data, IR-CRISPI, with its low-rank plus
sparse reconstruction, is related to compressed sensing.
MOCO-MAP is a successor to model-based acceleration of parametermapping
(MAP) for the application in the myocardial region. To this end, it
was augmented with a motion correction (MOCO) step to allow exponential
fitting the signal of a still object in temporal direction. Iteratively, this
introduction of prior physical knowledge together with the enforcement of
consistency with the measured data can be used to reconstruct an image
series from distinctly shorter sampling time than the standard exam (< 3 s
opposed to about 10 s). Results show feasibility of the method as well as
detectability of delayed enhancement in the myocardium, but also significant
discrepancies when imaging cardiac function and artifacts caused already by
minor inaccuracy of the motion correction.
IR-CRISPI was developed from CRISPI, which is a real-time protocol
specifically designed for functional evaluation of image data in steady-state
contrast. With a reconstruction based on the separate calculation of low-rank
and sparse part, it employs a softer constraint than the strict exponential
model, which was possible due to sufficient temporal sampling density via
spiral acquisition. The low-rank plus sparse reconstruction is fit for the use on
dynamic and on inversion recovery data. Thus, motion correction is rendered
unnecessary with it.
IR-CRISPI was equipped with noise suppression via spatial wavelet filtering.
A study comprising 10 patients with cardiac disease show medical
applicability. A comparison with performed traditional reference exams offer
insight into diagnostic benefits. Especially regarding patients with difficulty
to hold their breath, the real-time manner of the IR-CRISPI acquisition provides
a valuable alternative and an increase in robustness.
In conclusion, especially with IR-CRISPI in free breathing, a major acceleration
of the cardiovascular MR exam could be realized. In an acquisition
of less than 100 s, it not only includes the information of two traditional
protocols (cine and LGE), which take up more than 9.6 min, but also allows
adjustment of TI in retrospect and yields lower artifact level with similar
image quality.
The field of giftedness and gifted education has long been characterized by internal fragmentation and inconsistent definitions of core concepts (e.g., Ambrose et al., 2010; Coleman, 2006; McBee et al., 2012). It was only in recent years that increased efforts have been made to organize available research findings and thereby bring back greater uniformity to the field of giftedness and gifted education. For example, Preckel et al.’s (2020) Talent Development in Achievement Domains (TAD) framework integrates theoretical perspectives and empirical knowledge from different parts of the field. It is general in concept and can be applied to a wide range of achievement domains. By specifically focusing on measurable psychological constructs as well as their relevance at different stages of the talent development process, Preckel et al.’s (2020) TAD framework is well suited as a starting point for generating more domain-specific talent development models.
The present thesis represents one of the first attempts to empirically test the validity of Preckel et al.’s (2020) TAD framework in academic domains using longitudinal data. The longitudinal data came from a sample of former junior students at Julius-Maximilians-Universität (JMU) Würzburg who showed high academic achievement potential.
There were two related research issues: Research Issue 1 first aimed to document in detail how the educational trajectories of former junior students unfold in the years following their Abitur. To this end, a follow-up was conducted among 208 young adults who had participated in the junior study program at JMU Würzburg between the winter semester of 2004/2005 and the summer semester of 2011. The design of the follow-up questionnaire was based on a series of research questions that had emerged from the relevant literature on junior study programs in Germany. The follow-up ran from October 2019 to February 2020. The data were analyzed descriptively and documented as a detailed report. The results of Research Issue 1 revealed that the former junior students continued to be academically (and later professionally) successful long after their school years. For example, at the time of the follow-up, almost all former junior students had earned a bachelor’s and a master’s degree, most often with notable academic successes (e.g., scholarships, awards/prizes). In addition, more than half of those who responded had begun or already completed a doctoral degree, also recording special academic accomplishments (e.g., scientific publications, scholarships). A significant proportion of the former junior students had already entered the workforce at the time of their response. A look at their current professional situation revealed an above-average expression of success indicators (e.g., income, professional status). The clear majority of the former junior students reported that, even in retrospect, they would choose to take part in the junior study program at JMU Würzburg again.
Research Issue 2 aimed to determine the extent to which the structure of Preckel et al.’s (2020) TAD framework could be empirically validated in academic domains. The educational trajectories of 84 former junior students at JMU Würzburg who had chosen a subject from the same subject field in their regular studies as in their junior studies served as the data basis. The educational trajectories were compiled from the former junior students’ follow-up data and from their data on the selection process for the junior study program at JMU Würzburg. Combining the structural assumptions of Preckel et al.’s (2020) TAD framework with relevant insights from individual academic disciplines made it possible to derive hypotheses regarding potential predictors and indicators of the talent development stages aptitude, competence, and expertise in academic domains. Structural equation models were used for data analysis. The results of Research Issue 2 suggested that the talent development stages aptitude, competence, and expertise, while being predictive of each other in their chronological order, could be satisfactorily modeled using framework-compliant indicators in academic domains. In comparison, the talent development stage transformational achievement could not (yet) be modeled based on the longitudinal data. Among the hypothesized predictors, former junior students’ investigative interests and their metacognitive abilities reliably determined the talent development stages competence and expertise, whereas the remaining predictors did not make significant contributions.
Taken together, the results of the present thesis suggest that the validity of Preckel et al.’s (2020) TAD framework can only be partially confirmed in academic domains. Unlike the postulated indicators, the predictors in Preckel et al.’s (2020) TAD framework do not seem to be easily generalizable to academic domains but to be highly specific with regard to the talent domain under consideration. Therefore, a natural progression of the present thesis would be to examine the structure of Preckel et al.’s (2020) TAD framework at the subordinate level of subject fields or even at the level of individual academic disciplines, for example.
Attention-deficit/hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder described in psychiatry today. ADHD arises during early childhood and is characterized by an age-inappropriate level of inattention, hyperactivity, impulsivity, and partially emotional dysregulation. Besides, substantial psychiatric comorbidity further broadens the symptomatic spectrum. Despite advances in ADHD research by genetic- and imaging studies, the etiopathogenesis of ADHD remains largely unclear. Twin studies suggest a heritability of 70-80 % that, based on genome-wide investigations, is assumed to be polygenic and a mixed composite of small and large, common and rare genetic variants. In recent years the number of genetic risk candidates is continuously increased. However, for most, a biological link to neuropathology and symptomatology of the patient is still missing. Uncovering this link is vital for a better understanding of the disorder, the identification of new treatment targets, and therefore the development of a more targeted and possibly personalized therapy.
The present thesis addresses the issue for the ADHD risk candidates GRM8, FOXP2, and GAD1. By establishing loss of function zebrafish models, using CRISPR/Cas9 derived mutagenesis and antisense oligonucleotides, and studying them for morphological, functional, and behavioral alterations, it provides novel insights into the candidate's contribution to neuropathology and ADHD associated phenotypes. Using locomotor activity as behavioral read-out, the present work identified a genetic and functional implication of Grm8a, Grm8b, Foxp2, and Gad1b in ADHD associated hyperactivity. Further, it provides substantial evidence that the function of Grm8a, Grm8b, Foxp2, and Gad1b in activity regulation involves GABAergic signaling. Preliminary indications suggest that the three candidates interfere with GABAergic signaling in the ventral forebrain/striatum. However, according to present and previous data, via different biological mechanisms such as GABA synthesis, transmitter release regulation, synapse formation and/or transcriptional regulation of synaptic components. Intriguingly, this work further demonstrates that the activity regulating circuit, affected upon Foxp2 and Gad1b loss of function, is involved in the therapeutic effect mechanism of methylphenidate. Altogether, the present thesis identified altered GABAergic signaling in activity regulating circuits in, presumably, the ventral forebrain as neuropathological underpinning of ADHD associated hyperactivity. Further, it demonstrates altered GABAergic signaling as mechanistic link between the genetic disruption of Grm8a, Grm8b, Foxp2, and Gad1b and ADHD symptomatology like hyperactivity. Thus, this thesis highlights GABAergic signaling in activity regulating circuits and, in this context, Grm8a, Grm8b, Foxp2, and Gad1b as exciting targets for future investigations on ADHD etiopathogenesis and the development of novel therapeutic interventions for ADHD related hyperactivity. Additionally, thigmotaxis measurements suggest Grm8a, Grm8b, and Gad1b as interesting candidates for prospective studies on comorbid anxiety in ADHD. Furthermore, expression analysis in foxp2 mutants demonstrates Foxp2 as regulator of ADHD associated gene sets and neurodevelopmental disorder (NDD) overarching genetic and functional networks with possible implications for ADHD polygenicity and comorbidity. Finally, with the characterization of gene expression patterns and the generation and validation of genetic zebrafish models for Grm8a, Grm8b, Foxp2, and Gad1b, the present thesis laid the groundwork for future research efforts, for instance, the identification of the functional circuit(s) and biological mechanism(s) by which Grm8a, Grm8b, Foxp2, and Gad1b loss of function interfere with GABAergic signaling and ultimately induce hyperactivity.
In highly polarized neurons, endoplasmic reticulum (ER) forms a dynamic and continuous network in axons that plays important roles in lipid synthesis, Ca2+ homeostasis and the maintenance of synapses. However, the mechanisms underlying the regulation of axonal ER dynamics and its function in regulation of local translation still remain elusive. In the course of my thesis, I investigated the fast dynamic movements of ER and ribosomes in the growth cone of wildtype motoneurons as well as motoneurons from a mouse model of Spinal Muscular Atrophy (SMA), in response to Brain-derived neurotrophic factor (BDNF) stimulation. Live cell imaging data show that ER extends into axonal growth cone filopodia along actin filaments and disruption of actin cytoskeleton by cytochalasin D treatment impairs the dynamic movement of ER in the axonal filopodia. In contrast to filopodia, ER movements in the growth cone core seem to depend on coordinated actions of the actin and microtubule cytoskeleton. Myosin VI is especially required for ER movements into filopodia and drebrin A mediates actin/microtubule coordinated ER dynamics. Furthermore, we found that BDNF/TrkB signaling induces assembly of 80S ribosomes in growth cones on a time scale of seconds. Activated ribosomes relocate to the presynaptic ER and undergo local translation. These findings describe the dynamic interaction between ER and ribosomes during local translation and identify a novel potential function for the presynaptic ER in intra-axonal synthesis of transmembrane proteins such as the α-1β subunit of N-type Ca2+ channels in motoneurons. In addition, we demonstrate that in Smn-deficient motoneurons, ER dynamic movements are impaired in axonal growth cones that seems to be due to impaired actin cytoskeleton. Interestingly, ribosomes fail to undergo rapid structural changes in Smn-deficient growth cones and do not associate to ER in response to BDNF. Thus, aberrant ER dynamics and ribosome response to extracellular stimuli could affect axonal growth and presynaptic function and maintenance, thereby contributing to the pathology of SMA.
Development, Simulation and Evaluation of Mobile Wireless Networks in Industrial Applications
(2023)
Manyindustrialautomationsolutionsusewirelesscommunicationandrelyontheavail-
ability and quality of the wireless channel. At the same time the wireless medium is
highly congested and guaranteeing the availability of wireless channels is becoming
increasingly difficult. In this work we show, that ad-hoc networking solutions can be
used to provide new communication channels and improve the performance of mobile
automation systems. These ad-hoc networking solutions describe different communi-
cation strategies, but avoid relying on network infrastructure by utilizing the Peer-to-
Peer (P2P) channel between communicating entities.
This work is a step towards the effective implementation of low-range communication
technologies(e.g. VisibleLightCommunication(VLC), radarcommunication, mmWave
communication) to the industrial application. Implementing infrastructure networks
with these technologies is unrealistic, since the low communication range would neces-
sitate a high number of Access Points (APs) to yield full coverage. However, ad-hoc
networks do not require any network infrastructure. In this work different ad-hoc net-
working solutions for the industrial use case are presented and tools and models for
their examination are proposed.
The main use case investigated in this work are Automated Guided Vehicles (AGVs)
for industrial applications. These mobile devices drive throughout the factory trans-
porting crates, goods or tools or assisting workers. In most implementations they must
exchange data with a Central Control Unit (CCU) and between one another. Predicting
if a certain communication technology is suitable for an application is very challenging
since the applications and the resulting requirements are very heterogeneous.
The proposed models and simulation tools enable the simulation of the complex inter-
action of mobile robotic clients and a wireless communication network. The goal is to
predict the characteristics of a networked AGV fleet.
Theproposedtoolswereusedtoimplement, testandexaminedifferentad-hocnetwork-
ing solutions for industrial applications using AGVs. These communication solutions
handle time-critical and delay-tolerant communication. Additionally a control method
for the AGVs is proposed, which optimizes the communication and in turn increases the
transport performance of the AGV fleet. Therefore, this work provides not only tools
for the further research of industrial ad-hoc system, but also first implementations of
ad-hoc systems which address many of the most pressing issues in industrial applica-
tions.