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The evolution of cell-free protein synthesis (CFPS) over recent decades has made it a widely used system for expressing membrane proteins (MPs). Unlike traditional methods, CFPS allows direct and translocon-independent expression of MPs within lipid membranes, such as liposomes or nanodiscs (NDs), without the need for detergent solubilization. This open nature of CF systems enables customization of the experimental environment, including expression conditions, choice of nanoparticles (NPs), lipid composition, and addition of stabilizing molecules.
Membrane scaffold protein (MSP)-based NDs emerged as a gold standard for cotranslational solubilization of MPs using the CF-system. This approach allowed not only biochemical characterization, but also structural studies of MPs and even GPCRs. However, to solubilize MPs inside nanoparticles via the traditional reconstitution route, apart from MSPs other scaffolds were successfully implemented, e.g. the saposin A (commercially known as Salipro) scaffold system or the synthetic styrene maleic acid lipid particles (SMALPs). In this study the potential of saposin A-based nanoparticles (SapNPs) was explored for cotranslational MP solubilization.
Three strategies for applying SapNPs in CF systems were investigated: preassembly, (i) coassembly (ii), and coexpression (iii). (i) Preassembly involved forming SapNPs before CF expression and adding them to the CF reaction. In coassembly mode SapA and lipids were mixed in the CF reaction for spontaneous assembly with the synthesized MP. In coexpression mode lipids were added to the CF reaction while coexpressing SapA with the MP target. Proteorhodopsin (PR) served as a model protein to evaluate these strategies due to its ability to oligomerize and straightforward quantification using the cofactor retinal. Preassembled SapNPs provided homogeneous, aggregate-free particles yielding up to 200 µM solubilized PR inside in the CF reaction. Coassembly was also successfully applied to produce PR/SapNP complexes at slightly lower yields, however the system was prone to produce soluble aggregates at too high PR template concentrations and overall needed more adjustments. Coexpression resulted in PR yields below 20 µM and was not considered viable for MP production. Finally, the preassembled SapNPs were used to produce functional G-protein coupled receptor probes. Despite lower overall performance compared to MSP-based systems, SapNPs showed potential as an alternative in CF systems for specific MPs.
The second optimization approach was directed at the CF lysate itself. CF synthesis for NMR analysis benefits from selective labeling schemes enabled by truncated amino acid (AA) metabolic pathways in lysates, reducing spectral ambiguity. However, residual enzymatic AA conversions persist, leading to label dilution and ambiguous NMR spectra. This study aimed to eliminate these residual activities in the E. coli A19 strain, generating optimized CF lysates for NMR applications.
The approach involved cumulative gene deletions of the most problematic scrambling enzymes. The new strain, “Stablelabel,” included deletions and modifications in genes asnA, ansA, ansB, glnA, aspC, and ilvE, effectively eliminating background activities of L-Asn, L-Asp, and conversions of L-Glu to L-Asp and L-Gln. However, residual conversion of L-Gln to L-Glu persisted due to glutaminase activity of several glutaminases using the inhibitor 6 diazo-5-oxo-L-norleucine (DON). Stablelabel showed a slightly slower growth than A19, and an overall good performance with 2.7 mg/mL GFP expressed in the reaction mixture (RM) compared to the parental A19 strain with 3.5 mg/mL. Furthermore, the strain was successfully applied to demonstrate methyl group labeling of MPs using preconverted L-val and L-leu from their respective precursors 2-ketoisovalerate and 4-methyl-2-oxovalerate.
In this study, lipid nanoparticle particle-and strain engineering vividly demonstrated the potential of CFPS systems and their versatility. While the SapNP system requires further engineering to potentially reach the efficiency of the well-studied MSP NDs, this study provides an example of nanoparticle characterization allowing new insights into NP behavior in CF systems. Furthermore, it was shown that strain engineering is a straightforward solution to tailor CF lysates to the individual requirements. After this thesis was submitted, Stablelabel in fact was successfully applied for backbone assignment of casein kinase 1, thereby demonstrating its suitability to express complex targets for NMR studies.
This thesis is concerned with the investigation of static and dynamic properties of quantum Heisenberg paramagnets in the absence of a magnetic field and therefore for vanishing magnetization. For this purpose a new formulation of the spin functional renormalization group (SFRG) is employed. The first manifestations of the SFRG were developed by Krieg and Kopietz, motivated by the FRG approach to ordinary field theories and the older works of Vaks, Larkin and Pikin on diagrammatic methods for spin operators.
The main idea is to study quantum spin systems by considering the evolution of correlation functions under a continuous deformation of the interaction between magnetic moments, starting from a solvable limit. This leads to nonperturbative results for quantities like the spin-spin correlation function. After a basic introduction to the phenomena and concomitant problems discussed in this thesis, a detailed description of the SFRG method in its initial formulation is given in the second chapter. We start with the generating functional of connected imaginary-time spin-correlation functions GΛ [h], for which an exact flow equation is derived. A particular issue, already pointed out by Krieg and Kopietz, arises here, namely the singular non-interacting limit of its subtracted Legendre transform ΓΛ [m]. As a consequence the initial condition of that functional does not have a proper series expansion in powers of m. This prevents us from working directly within a pure one-particle irreducible (1-PI) parametrization of the correlation functions, as is often done in the context of field theories. Thus motivated, we develop a workaround explicitly tailored to paramagnets, which provides us with a functional that has a well-behaved Legendre transform. The new approach is based on a different treatment of fluctuations at zero and finite frequencies, analogous to a previous hybrid formulation for the symmetry-broken phase. Certain properties, considered to be highly relevant for isotropic paramagnets, as well as previous observations, already made in the study of simpler spin systems like the Ising model, serve as additional justifications for choosing this construction.
In the third chapter our new method is assessed by calculating the dynamic susceptibility G(k, iω) and thus the dynamic structure factor S(k, ω) in the symmetric phase. For this purpose an approximate integral equation for the dynamic polarization function Π̃(k, iω) was derived. This equation results from a truncation of the hierarchy of flow equations and contains static quantities, that are assumed to be known from another source. Our first application is the high-temperature limit T → ∞ in d ≤ 3 dimensions. Salient features, believed to be part of the spin dynamics in isotropic Heisenberg magnets are also exhibited by our solution, like (anomalous) diffusion in a suitable hydrodynamic limit. Moreover we obtain the same order of magnitude for the diffusion coefficient D as in experiments and other theoretical calculations. Other aspects do not entirely agree with previous approaches.
Afterwards we continue by investigating systems close to the critical point Tc. Dynamic scaling forms for Π̃(k, iω) and S(k, ω), which, like spin diffusion, are postulated on the basis of quite general physical arguments, are reproduced. Agreement of the line-shapes 2with neutron scattering experiments at T = Tc is found to be satisfying, with deviations for ω → 0, that may be attributed to the simplicity of the approximation, like at infinite temperature.
Finally, we focus our attention on the thermodynamic properties of isotropic Heisenberg paramagnets by calculating the static susceptibility G(k). For this purpose we employ simple truncation schemes of the flow equations for the static self-energy ΣΛ (k) and four-spin vertex ΓΛ , together with a basic ansatz for the dynamic polarization Π̃(k, iω) in quantum systems. As a result we obtain transition temperatures Tc of three-dimensional nonfrustrated magnets within an accuracy of 5 percent compared to established benchmark values from Quantum Monte Carlo and high temperature expansion series. We conclude this chapter by giving an outlook on the application of our method to frustrated systems, which may require a combined non-trivial calculation of static and dynamic properties.
The attention on the protein PURA has increased recently following the discovery of the rare PURA Syndrome. This neurodevelopmental disorder is caused by de novo mutations in the PURA gene. Notably, our collaborators could show that the protein PURA can bind DNA and RNA in vitro. As a result, I was motivated to explore PURA's cellular RNAbinding activity. Furthermore, I inquired on the connection of PURA-RNA binding to the cellular effect of a reduction of functional PURA as present in PURA Syndrome patients.
To investigate the binding of PURA and the impact of PURA de ciency on cellular RNA and protein expression, I performed an integrative computational analysis of multimodal data from complementary high-throughput experiments. An essential component was the examination of UV Crosslinking and immunoprecipitation (CLIP) experiments, which can query the global RNA-binding behaviour of a given protein in a cellular context. As the processing and analysis of CLIP data are rather complex, I introduce an automated command line tool for the processing of CLIP data named racoon_clip as part of this dissertation. Therefore, this dissertation comprises two major segments. Firstly, I describe the implementation and usage of racoon clip for CLIP data analysis. Secondly, I discuss my research on the protein PURA, demonstrating its global RNA-binding properties, the effects of PURA depletion and its association with neuronal functions and P-bodies, among others.
racoon_clip is a command line application that I have developed for processing of individualnucleotide resolution CLIP (iCLIP) and enhanced CLIP (eCLIP) experiments - two of the most commonly used types of CLIP experiments - in a comparable and user-friendly way.
For this, I built racoon_clip as an automated work how that encompasses all CLIP processing steps from raw data to single-nucleotide resolution crosslink events. racoon_clip is available as a command line tool that users can run with a single command. The work how is implemented with Snakemake work how management providing computational advantage tages including parallelisation, scalability and portability of the work how. The main task of racoon_clip is to extract single-nucleotide crosslink events from iCLIP, iCLIP2, eCLIP and similar data types. To strike a balance between being highly customisable and easy to use, racoon_clip supplies pre-set options for the most common types of experiments.
Additionally, it is possible for users to create a custom setup of barcode and adapter architectures, which allows them to use the software for other types of CLIP data. While accounting for the different architectures in the reads, the performed central processing steps remain the same. This leads to a high degree of comparability between the different experiment types, which I demonstrate in the exemplary processing of U2AF2 iCLIP and eCLIP data. Taken together, I am confident that racoon_clip will be beneficial to numerous researchers interested in RNA-Protein interactions as it offers easily accessible processing for CLIP data and enhances the comparability of multiple CLIP datasets across di erent experiment types.
In the second part of this dissertation, I focus on the cellular function of the RNAbinding protein PURA. Through in-depth computational analysis of one iCLIP data set of endogenous PURA and two iCLIP data sets of overexpressed PURA in HeLa cells, I establish that PURA is a global RNA-binding protein. It preferentially binds RNAs in either the coding sequence (CDS) or the 3' untranslated region (3'UTR) of mature protein-coding transcripts by recognising a Purine-rich degenerated sequence motif. Even though overexpression of PURA results in less specific binding behaviour, the same overall binding patterns as from endogenous PURA persist. Overall characteristics of PURA binding remain similar in three distinct PURA iCLIP data sets with and without PURA overexpression.
To learn about the molecular consequences of a depletion of functional PURA in a cellular context, I used a 50% reduction of PURA in HeLa cells as a model for the heterozygous loss of PURA in PURA Syndrome and evaluated its impact on global RNA and protein expression. The results demonstrate that PURA depletion globally a ects RNA and protein expression. Additionally, I integrate PURA RNA binding with the changes in expression of RNAs and proteins in the context of PURA depletion. This reveals 234 targets of PURA that are bound by PURA and are impacted at both RNA and protein levels by the PURA protein. RNAs that are bound by PURA or change in abundance upon PURA depletion are enriched in neuronal development factors, RNA lifecycle regulators, and mitochondrial factors, among others. Consistent with a possible role of PURA in neuronal transport, there is considerable overlap between PURA bound transcripts and transcripts, that are transported to the dendritic end of neurons.
Notably, there is a link between PURA and P-bodies, as documented by the enrichment of PURA-bound RNAs in both the P-body and stress granule transcriptome. Further, PURA was found by our collaborators to be localised within P-bodies and P-body numbers were strongly reduced in cells that are depleted of PURA. This absence might be attributed to the downregulation of the proteins encoded by the PURA targets LSM14A and DDX6 as both of them were previously identified as essential for P-body formation.
Overall, the reduction of P-body numbers in PURA depletion, the neuronal function of PURA, and its association with mitochondria and RNA lifecycle regulation may indicate the cellular foundation of both PURA Syndrome and related neuronal diseases.
In summary, I present a versatile and user-friendly computational tool for the analysis of CLIP data. Subsequently, I conduct a thorough computational analysis of CLIP and other high-throughput data in the context of the RNA-binding protein PURA, which offers valuable insights into the cellular functions of PURA. These insights advance our understanding of the impact of PURA loss in PURA Syndrome and other disease contexts.
Mitochondrial RNA granules (MRGs) are membraneless, highly specialized compartments that play an essential role in the post-transcriptional regulation of mitochondrial gene expression. This regulation is crucial for maintaining energy production, controlling metabolic functions and ensuring homeostasis in cells. Dysregulation of mitochondrial genes has been linked to various human diseases, including neurodegenerative and metabolic disorders as well as certain types of cancer.
MRGs are composed of different RNA species, including mitochondrial precursor RNA (pre-RNA), mature tRNAs, rRNAs and mRNAs complexed with multiple proteins involved in RNA processing and mitoribosome assembly. However, despite the significance of MRGs, their protein composition, structural organization, stability and dynamics during stress conditions remain elusive. In the study reported here, I adopted a three-step approach to address the aforementioned fundamental issues.
First and foremost, I identified the protein composition of MRGs and unveiled their architectural complexity. To characterize the MRG proteome, I applied the cutting-edge TurboID-based proximity labeling approach combined with quantitative mass spectrometry. Proximity labeling was conducted on 20 distinct MRG-associated human proteins, resulting in the identification of more than 1,700 protein-protein interactions. This expansive dataset enabled me to create a comprehensive network, providing valuable insights into both the (sub)architecture as well as the core structure of MRGs in-depth.
Secondly, I investigated the spatio-temporal dynamics of MRGs under various mitochondrial stress conditions. To monitor the morphological alterations and compositional changes of MRGs, I utilized time-resolved confocal fluorescence microscopy and proteomics, respectively. In this analysis, I applied IMT1, the first specific inhibitor that selectively targets mitochondrial transcription. Using this methodology, I pinpointed precise conditions that triggered MRGs’ disassembly during stress, followed by their reassembly when nascent RNA production was restored. The results of this examination elucidate that MRGs are highly dynamic and stress adaptive structures, capable of rapid dissolution and reassembly, a process closely connected to mitochondrial transcription.
Thirdly, I aimed to explore the impact of RNA turnover on MRGs’ integrity during stress, employing confocal fluorescence microscopy and quantitative real-time PCR. I observed that depletion of MRG proteins associated with RNA degradation counteracts MRGs’ disassembly under stress conditions, a phenomenon attributed to the accumulation of double-stranded RNA (dsRNA). These results emphasize the critical role of pre-RNA turnover in maintaining MRG integrity and reveal that MRGs can be stabilized by dsRNA.
Taken together, the comprehensive investigation reported in this thesis has substantially broadened and deepened our understanding of MRGs’ complexity. By identifying their molecular structure and dynamics, I have gained significant insights into the fundamental characteristics and biological functions of MRGs in cellular processes. This knowledge contributes to the identification of disease-related pathways linked to mitochondrial gene expression and may inspire future studies to develop novel therapeutic approaches.
In almost all parts of the world the industrialisation grows continuously and thus, the chemical pollution of natural waters has become a major public concern. A major consequence and one of the key environmental problems we are facing today is the increasing contamination of freshwater systems with chemicals. The chemicals are detected in wastewater, surface (river) water, ground water and drinking water ubiquitously in natural waters and not only in industrialised areas. The main point sources for water pollution and the release of these synthetic organic substances of human origin, so called micropollutants (MPs), are wastewater treatment plants (WWTPs). These MPs such as pharmaceuticals, personal care products, disinfectant chemicals, chemicals used in the industry and in households, contraceptives, hormones, food additives, artificial sweeteners, pesticides, biocides, and many emerging contaminants are only incompletely removed by the existing conventional wastewater treatment technologies. The MPs end up in the water cycle and have adverse effects on wildlife aquatic ecosystems and human health even at very low concentrations. Therefore, advanced wastewater treatment (AWWT) technologies, such as ozonation, treatment with activated carbon, biofiltration, membrane bioreactors (MBRs) or exposure to ultraviolet light are investigated as options to upgrade conventional WWTPs. However, several studies show that especially the ozonation of wastewater generates diverse transformation products (TPs) with unknown properties. These TPs could be more toxic than the mother compound. Thus, a post-treatment after the ozonation process is required.
The present thesis was part of the BMBF-funded TransRisk project dealing with “the characterisation, communication, and minimisation of risks of emerging pollutants and pathogens in the water cycle”. One main objective was the investigation of conventional treated wastewater after a full-scale ozonation with four post-treatments (each non-aerated and aerated granular activated carbon (GAC) filtration and biofiltration) in comparison to a MBR treatment of raw (untreated) wastewater separately and in combination with an additional ozonation on a pilot WWTP. For this purpose, the wastewater samples were characterised with a comprehensive battery of in vitro and in vivo bioassays. The in vitro bioassays were performed to detect endocrine activities (such as (anti)estrogenic and (anti)androgenic activities), genotoxicity, and mutagenicity. The results showed a decreased estrogenic activity due to the conventional wastewater treatment as well as the ozonation, but a distinct increase of the anti-estrogenic activity and the mutagenicity in the ozonated wastewater, possibly caused by new formed TPs, that were reduced after the post-treatments whereas the GAC filtration performed better than the biofiltration. The in vivo bioassays included for example the impact of the wastewater on mortality, reproduction, development, and energy reserves of the test organisms. The in vivo on-site tests with the mudsnail Potamopyrgus antipodarum and with the amphipod Gammarus fossarum indicated a major impact of conventional treated wastewater, ozonated wastewater, and MBR treated wastewater. The flow channel experiments in the laboratory with Gammarus pulex pointed to a serious impact of an estrogenic effluent on life-history traits of the amphipod. Finally, an ozonation of the wastewater with subsequent GAC filtration represented the most promising option. In addition, chemical analyses of 40 selected MPs, so called tracer substances, performed in parallel to the in vitro and in vivo bioassays underlined this assumption.
A second main objective was the optimisation of the preparation of water and wastewater samples for ecotoxicological in vitro bioassays because common sample preparation techniques are predominantly adapted for chemical analyses. Therefore, the impact of sample filtration, long-term acidification with following neutralisation as well as the enrichment with solid phase extraction (SPE) in combination with short-term acidification were investigated using amongst others raw (untreated) wastewater, hospital wastewater, conventional treated and ozonated wastewater, surface water, and ground water. Overall, eleven in vitro bioassays were performed for the detection of endocrine activities, genotoxicity, and mutagenicity. The results show that sample filtration and acidification/neutralisation significantly affected the outcome of the bioassays especially the anti-estrogenic activity and the mutagenicity whereas the sample filtration had a minor impact than the acidification. Thus, the testing of untreated (waste)water samples is advisable because the sample is minimally processed. Furthermore, the SPE extracts showed in parts high cytotoxic effects whereby no conclusions on the results of the bioassays were possible. However, the enrichment of endocrine activity and mutagenicity was predominantly effective but depended on the used SPE cartridge and the pH value of the (waste)water samples. Based on the results the use of a Telos C18/ENV cartridge and an acidified sample is recommendable. In the end, there is a need to optimise the sample preparation for in vitro bioassays to reach their maximum outcome for the best possible assessment of the water quality.
The main focus of this thesis is the application of the nonperturbative Functional Renormalization Group (FRG) to the study of low-energies effective models for Quantum Chromodynamics (QCD). The study of effective field theories and models is crucial for our understanding of physics, especially when we deal with fundamental interaction theories like QCD. In particular, the ultimate goal is the understanding of the critical properties of these models in such a way that we can have an insight on the actual critical phenomena of QCD, with a special focus on its chiral phase transition. The choice of the FRG method derives from the fact that it belongs to the class of functional non-perturbative methods and has also the advantage of linking physics at different energy scales. These features make FRG perfectly compatible with the task of studying non-perturbative phenomena and in particular phase transitions, like the ones expected for strongly interacting matter. However, the functional nature of the FRG approach and of the Wetterich equation has a consequence that its exact resolution is hardly possible, and an ansatz for the effective action is generally needed. In this work we choose to adopt the local-potential approximation (LPA), which prescribes to stop at zeroth order in the expansion in derivative operators of the quantum effective action, including only the quantum effective potential. In this work we exploited the key observation that the FRG flow equation can be cast, for specific models and truncation schemes, in the form of an advection-diffusion, possibly with a source term. This type of equation belongs to the class of problems faced in the context of viscous hydrodynamics. Therefore, an innovative approach to the solution of the FRG flow equation consists in the choice of a method developed specifically for the resolution of this class of hydrodynamic equations. In particular, the Kurganov-Tadmor finite-volume scheme is adopted. Throughout this work we apply this scheme to the study of different physical systems, showing the reliability and the flexibility of this approach.
In the first part of the thesis, we discuss the well-known O(N) model, using the hydrodynamic formulation to solve the FRG flow equation in the LPA truncation. We focus on the study of the critical behaviour of the system and calculate the corresponding critical exponents. Particular attention is given to the error estimation in the extraction of critical exponents, which is a needed and not widely explored aspect. The results are well compatible with others in the literature, obtained with different perturbative and nonperturbative methods, which validates the procedure. In the second part of the thesis, we introduce the quark-meson model as a low-energy effective model for QCD, with a specific focus on its chiral symmetry-breaking pattern and the subsequent dynamical quark-mass generation. The LPA flow equation is of the advection-diffusion type, with an extra source contribution which is due to the inclusion of fermionic degrees of freedom. We thus adopt the developed numerical techniques to derive the phase diagram of the model, which is in agreement with the one obtained with other techniques in the literature.
We also follow another possible way for the study of the critical properties of the quark-meson model: the so-called thermodynamic geometry. This approach is based on the interpretation of the parameter space of the system as a differential manifold. One can then obtain relevant information about the phase transitions from the Ricci scalar. We studied the chiral crossover investigating the behavior of the Ricci scalar up to the critical point, featuring a peaking behavior in the presence of the crossover. We then repeated this analysis in the chiral limit, where the phase transition is expected to be of second order. Via this geometric technique it is possible to have a different view on the chiral phase transition of QCD. This is the case since this approach is based on the calculation of quantities which are influenced by higher-order momenta of the thermodynamic potential, thus allowing for a more comprehensive analysis of the phase transition.
Finally, we exploit the numerical advancement to face the issue of the regulator choice in the FRG calculations. This is one of the most delicate issues which arise when using approximations to solve the FRG flow equation and deserves extensive investigation. In particular, we performed a vacuum parameter study and used the RG consistency requirement to determine the impact of the choice of the regulator on the physical observables and on the phase diagram of the model. Via this study we develop a systematic method to comparison the results obtained via different regulators. We show the importance of the choice of an appropriate UV cutoff in the determination of UV-independent IR observables and, consequently, the impact on the latter that the truncation of the effective average action and the choice of the regulator have.
Autism Spectrum Disorder (ASD) is a neurodevelopmental condition with an onset in early development. ASD has varying degrees of severity and thus affects people differently throughout their lives. Early diagnosis of ASD is essential to provide children with individually-tailored support.8 Eye-tracking may contribute to an earlier diagnosis: Several studies showed differences in eye movements between people with autism spectrum disorder (ASD) and typically developing controls (TD). Different eye movements may contribute to different visual perception that perpetuates to problems in attention, communication and social interaction.
Eye movements are divided into: (1) Fixations (2) Saccades (fast and short eye movements) and (3) Smooth Pursuit Eye Movements (SPEM). SPEM follow the target in a continuous manner. The latter are the subject of the present thesis. SPEM consist of two phases: the open loop phase (= phase of initiation, first 50- 100ms) and the closed loop phase (= phase of maintenance, after about 100ms). SPEM are usually measured by a gain index. It is defined as the ratio of smooth pursuit velocity and visual target velocity and ideally equals to 1.2
In young children, corneal-reflection (CR) eye-tracking is usually applied to quantify eye movement. It allows precise measurements without the use of potentially intrusive devices.
Studies in ASD reported deficits in open loop and closed loop pursuit in children and adults with a mean age of 19.32 (TD) and 20.04 (ASD) years. However, SPEM in preschoolers with ASD remain understudied, although this developmental phase is crucial to the development of non-social and social attentional abilities.
In the present study 66 toddlers and preschoolers (18 to 72 months; ASD: n = 33, TD: n = 33) with matched cognitive abilities and sex were assessed. The main objective was to compare the gain index (Smooth Pursuit Gain = SPG). SPEM were compared between groups with gain index as a dependent measure. We hypothesized that participants with ASD show lower average gain compared to the control group.
We could show a significant group influence on the gain when considering interactions between target velocity and group (p = 0.041). The TD group showed a greater dependence on the increasing object speed than the ASD group with a trend of -0.30 ± 0.11 in the TD group and a trend of -0.13 ± 0.12 in the ASD group. Across groups, the gain decreased with increasing target velocity and dropped faster in vertical than in horizontal trials. Additionally, participants showed a lower SPG in vertical sequences than in horizontal sequences. This supports the general validity of the measure.
Toddlers and preschoolers represent a group that has been subject of little research to date. In addition, there has been only a limited number of studies analyzing SPEM in ASD. To check for a possible group difference without interactions a study with a larger sample size at fixed target velocity and target direction should follow.
Die Arbeit behandelt die Messung von Photonen mit Teilchendetektoren, die auf digitalen Silizium-Pixelsensoren basieren. Diskutiert werden zwei wesentliche Schritte in den Upgrade-Programmen des ALICE-Experiments am CERN-LHC:
1. FOCAL-Detektor-Upgrade (2027): Untersuchung der Detektorantwort des elektromagnetischen Pixel-Kalorimeters EPICAL-2 und der Form elektromagnetischer Schauer durch Teststrahl-Messungen und Monte Carlo Simulationen.
2. ALICE 3-Upgrade (2035): Simulationsstudien zum Untergrund in der Messung von Photonen mit sehr kleinem Transversalimpuls.
Teil 1: Performance des elektromagnetischen Pixel-Kalorimeters EPICAL-2
Detektordesign und Testmessungen: EPICAL-2, ein SiW-Sandwich-Design-Kalorimeter mit ALPIDE Sensoren, besitzt eine Tiefe von ca. 20 Strahlungslängen und etwa 25 Millionen Pixel. Testmessungen wurden an der Universität Utrecht (kosmische Myonen) sowie am DESY und CERN-SPS (Elektronen) durchgeführt.
Simulation und Validierung: Das EPICAL-2 wird im Simulationspaket Allpix2 implementiert, um die Testmessungen zu validieren und das Detektorverhalten zu untersuchen. Systematische Variationen bestätigen die Stabilität und Reproduzierbarkeit der Simulation.
Datenaufbereitung und Schauerprofile: Im Rahmen der Datenanalyse werden fehlerhafte Pixel ausgeschlossen, Pixel-Treffer zu Clustern gruppiert, Chips kalibriert und der Strahlwinkel korrigiert. Das longitudinale Profil elektromagnetischer Schauer zeigt, dass das Schauermaximum in der Simulation etwas tiefer liegt als in den Testdaten, was auf zusätzliches Material oder eine unvollständige Beschreibung der Schauerentwicklung in der Simulation zurückzuführen sein könnte. Das laterale Profil zeigt, dass eine Schauertrennung im Millimeter-Bereich möglich ist.
Energieantwort und -auflösung: Die nicht-lineare Energieantwort wird sowohl in Testdaten als auch in Simulationen beobachtet. Die Energieauflösung des EPICAL-2 für Cluster ist besser als für Pixeltreffer und vergleichbar mit dem analogen CALICE-Prototypen. Simulationen ohne Strahlenergie-Fluktuationen zeigen eine bessere Energieauflösung als in den Testdaten.
Teil 2: Untergrund in der Messung von Photonen in ALICE 3
Simulationssetup: Die ALICE 3-Detektorgeometrie wird in GEANT4 implementiert, um den Untergrund in der Messung weicher Photonen zu untersuchen. Simulationen mit PYTHIA und GEANT4 zeigen, dass der Untergrund hauptsächlich aus Zerfallsphotonen und Photonen aus externer Bremsstrahlung besteht.
Ergebnisse der Untergrundstudien: Der Untergrund durch Photonen aus externer Bremsstrahlung dominiert und liegt im Akzeptanzbereich des FCT um einen Faktor von 5 bis 10 über dem theoretischen Signal weicher Photonen. In der Simulation wird das Material zu 8%—14% X0 in ALICE 3 bestimmt, wobei bereits bei 5% X0 der Untergrund genauso stark ist wie das erwartete Signal.
Möglichkeiten zur Untergrundreduzierung: Untersuchungen zeigen, dass ein Elektron-Veto das Signal-zu-Untergrund-Verhältnis um den Faktor 30 verbessern und eine Materialreduktion durch ein optimiertes Strahlrohr um den Faktor 7.
Die Ergebnisse des ersten Teils dieser Arbeit demonstrieren insgesamt die gute Performance des EPICAL-2 in Bezug auf die Energiemessung und die Bestimmung der Schauerform. Darüber hinaus unterstützen sie den Einsatz digitaler Kalorimeter im FOCAL-Upgrade des ALICE-Experiments und zeigen das Potenzial der digitalen Kalorimetertechnologie für zukünftige Hochenergiephysik-Experimente.
Die Ergebnisse des zweiten Teils dieser Arbeit liefern einen wesentliche Beitrag zum geplanten ALICE 3-Upgrade. Weiterhin veranschaulichen sie, wie ein Elektron-Veto und die Reduzierung des Materials zusammen eine vielversprechende Messstrategie bilden können.
Inflammation is a crucial host defense mechanism activated in response to injury or infection. Its primary goal is to eliminate the source of the disturbance, repair the damaged tissue, and restore homeostasis. Inflammatory processes can be recognized through increased blood flow, higher vascular permeability, and the recruitment of leukocytes and plasma proteins to the tissue. A pathogen-induced inflammation triggers various pro- and anti-inflammatory processes. Local tissue cells and Toll-like receptors call upon innate immune cells like neutrophils, dendritic cells (DCs), and monocytes to respond to the intruder. They move across the endothelium and respond to local signals by releasing mediators or cytotoxic compounds, phagocytosing, or polarizing. To study local pathogen-induced inflammation, a zymosan-induced inflammation model was used in the hind paws of mice, which caused a Toll-like receptor 2 mediated inflammation. Multi-Epitope-Ligand-Cartography (MELC) was used for multiple sequential immunohistochemistry with 40 different antibodies on the same tissue. Bioinformatic analysis and graphical representation revealed a specific inflammatory architecture consisting of three major areas based on macrophage polarization and their cellular neighborhoods: a core region containing the pathogen, a pro-inflammatory region containing M1-like macrophages, and a region containing anti-inflammatory cells. This discovery highlights the coexistence of pro- and antiinflammatory processes during an ongoing inflammation and challenges the concept of a gradual temporal transition from pro- to anti-inflammation. Flow cytometry of the whole paw was performed to support and refine the MELC results. Eosinophils were used as a specific immune cell population to investigate their role in the inflammatory structure. They were found to be present in all three inflammatory regions, adapting their cytokine profile according to their localization. Depleting eosinophils reduced Interleukin 4 (IL-4)- levels, increased edema formation, and mechanical and thermal hypersensitivities during inflammation resolution. In the absence of eosinophils, pro- and anti-inflammatory region could not be determined in the inflammatory architecture, neutrophil numbers increased, and efferocytosis and M2-macrophage polarization were reduced. IL-4 administration restored these regions, normalized neutrophil numbers, efferocytosis, M2-macrophage polarization, and resolution of zymosan-induced hypersensitivity. The results show that eosinophils expressing IL-4 support the resolution of inflammation by enabling the development of an anti-inflammatory framework that encloses pro-inflammatory regions.
Efficient modeling and mitigation of quadrupole errors in synchrotrons and their beam transfer lines
(2023)
This thesis investigates the problem of estimating quadrupole errors on synchrotrons as well as how to minimize the influence of quadrupole errors for beam transfer lines (beamlines). It emphasizes the importance to treat possible error sources in all parts of an accelerator in order to provide constantly high beam quality to the experimental stations. While the presented methods have been investigated by using the example of the SIS18 synchrotron and the HEST beamlines at GSI Helmholtz Centre for Heavy Ion Research, they are equally relevant for the future synchrotrons and beamlines of the Facility for Antiproton and Ion Research in Europe (FAIR).
Part 1 discusses the problem of estimating quadrupole errors via orbit response measurements at synchrotrons. An emphasis is put on investigating the influence of the availability of steerer magnets and beam position monitors (BPMs) on the solvability of the inverse problem as well as on the propagation of measurement uncertainty for the estimation of quadrupole errors. The problem is approached via analytical considerations as well as via dedicated simulation studies. By developing an analytical expression for the Jacobian matrix, the theoretical boundaries for the solvability of the inverse problem are derived. Moreover, it is shown that the analytical expressions for the Jacobian matrix can be used during the fitting procedure to achieve a significant improvement in the computational efficiency by a factor $N_{steerers} \times N_{quadrupoles}$, where $N$ denotes the number of lattice elements of the respective type. The presented results are tested via dedicated measurements at the SIS18 synchrotron.
Part 2 discusses – complementary to part 1 – the influence of quadrupole errors in beam transfer lines with respect to the beam quality requirements given by the experimental stations. A preventive approach is presented which allows to minimize the influence of possible quadrupole errors on the degradation of beam quality. By identifying and selecting robust quadrupole configurations, a stable operation of the beamline can be enabled and the time needed by operators to readjust the beamline parameters can be reduced. The concept of beamline robustness is developed and is studied with the help of dedicated simulations. The simulation results are used to identify certain properties that distinguish robust from nonrobust quadrupole configurations. Also, various methods for improving the computational process of identifying robust quadrupole configurations are presented. The methods and results are tested via dedicated measurements at two different beamlines at GSI Helmholtz Centre for Heavy Ion Research and at Forschungszentrum Jülich.
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
Die vorliegende Dissertation mit dem Titel “Structural dynamics of eukaryotic H/ACA RNPs from Saccharomyces cerevisiae & Structural dynamics of the Guanidine-II riboswitch from Escherichia coli” besteht aus zwei Projekten. Das erste Projekt befasst sich mit den eukaryotischen H/ACA Ribonukleoproteinen (RNP) aus der Hefe. Diese können sequenzspezifisch in der RNA ein Uridin Nukleotid in das Rotationsisomer Pseudouridin (Ψ) umwandeln. Die H/ACA RNPs bestehen aus einer Leit-RNA und vier Proteinen, der katalytisch aktiven Pseudouridylase Cbf5, Nhp2, Gar1 und Nop10. Die Leit-RNA besteht in Eukaryoten konserviert aus zwei Haarnadelstrukturen, die von einem H-Box oder ACA-Box Sequenzmotiv gefolgt sind. In jeder dieser Haarnadeln befindet sich ein ungepaarter Bereich, die sogenannte Pseudouridylierungstasche, wo durch komplementäre Basenpaarung die Ziel-RNA gebunden wird. Fehlerhafte H/ACA RNPs können beim Menschen zu schweren Krankheiten wie verschiedenen Krebsarten oder dem Knochenmarksversagen Dyskeratosis congenita führen, aber sie bieten auch Möglichkeiten zum Einsatz als Therapiemethode. In dieser Arbeit wurde hauptsächlich der zweiteilige Aufbau der H/ACA RNPs untersucht.
Dafür wurden zunächst die einzelnen Komponenten hergestellt werden. Cbf5, Nop10 und Gar1 wurden zusammen heterolog in E. coli exprimiert und gereinigt. Außerdem wurden mehrere Deletionsvarianten von Gar1 hergestellt. Zusätzlich wurde die Leit-RNA unmarkiert über T7 Transkription synthetisiert, sowie sechs verschiedene FRET-Konstrukte mit verschiedenen Markierungschemas der Fluorophore Cy3 und Cy5 über DNA-geschiente Ligation. Anschließend wurde über Größenausschlusschromatographie und radioaktiven Aktivitätsassays geprüft, dass sich die aktiven H/ACA RNPs in vitro aus den einzelnen Komponenten rekonstituieren lassen.
In smFRET Experimenten wurden einzelne Haarnadelstrukturen mit dem zweiteiligen Komplexen verglichen. Dabei konnte gezeigt werden, dass die H3 Haarnadel durch die Anwesenheit von H5 dynamischer und heterogener wurde, während H5 überwiegend unbeeinflusst war. Außerdem konnte die dreidimensionale Orientierung der Haarnadelstrukturen in verschiedenen Assemblierungsschritten mittels smFRET untersucht werden. Hier deutete sich an, dass in Abwesenheit von Proteinen beide Haarnadeln eher entgegengesetzt stehen als in einer parallelen Konformation. Cbf5 scheint den Linker zwischen den Beiden auszustrecken bzw. zu orientieren und die Haarnadelstrukturen etwas gegeneinander zu neigen. Ein Zusammenspiel von Nhp2 und Gar1 war nötig um die oberen Bereiche der Haarnadeln zusammenzuziehen. Es konnte auch ein Modell für den vollen H/ACA RNP vorgeschlagen werden. Im kompletten Komplex könnte das Zusammenziehen der Haarnadelstrukturen durch Nhp2 und Gar1 mit dem Effekt von Cbf5 konkurrieren und könnte hauptsächlich den oberen Bereich von H3 betreffen. Zum Schluss wurde das Zusammenspiel von Gar1 und Nhp2 auf eine Abhängigkeit von den RGG Domänen von Gar1 hin untersucht. Hier besteht möglicherweise eine Hierarchie, die eine Kooperativität von den N- und C-terminalen Domänen benötigt.
Das zweite Projekt befasst sich mit dem Guanidin-II Riboschalter aus E. coli. Der Riboschalter kann das toxische Molekül Guanidinium (Gdm+) spezifisch in seiner Aptamerdomäne binden und dadurch die Genexpression von Proteinen zur Detoxifizierung von Gdm+ aktivieren. Der Riboschalter besteht aus zwei Haarnadelstrukturen, mit einer Schleife, die aus der Sequenz ACGR besteht, wobei R ein Purin ist. In einem vorgeschlagenen Modell soll die Ribosomenbindestelle (Shine-Dalgarno Sequenz) in Abwesenheit von Ligand mit dem Linker komplementär Basenpaaren und so die Translation verhindern. Mit Ligand würde sich dann eine Schleifen-Schleifen Interaktion mit den beiden CG Basen ausbilden, wodurch die Anti-Shine-Dalgarno Sequenz nicht mehr zugänglich wäre. Bisherige Studien arbeiteten zumeist nur mit der Aptamerdomäne, den einzelnen Haarnadeln oder noch kleineren Elementen. In dieser Arbeit wurden die Strukturdynamiken von verschiedenen Längen, auch mit der Expressionsplatform, untersucht. Außerdem wurden verschiedene Mutationen analysiert und die Effekte auf den Riboschalter in seiner natürlichen Umgebung in E. coli.
Zunächst mussten insgesamt 24 FRET-Konstrukte hergestellt werden, die sich in Länge, Markierungsschema und Mutationen unterschieden. Hierfür wurde DNA-geschiente Ligation verwendet. Dank der verschiedenen Fluorophorpositionen konnte ein konformationelles Modell für die Aptamerdomäne vorgeschlagen werden. In diesem Modell könnte in Abwesenheit von Ionen das Aptamer offen vorliegen. Durch Mg2+ würde sich bereits eine lockere Schleifen-Schleifen Interaktion ausbilden. Zusätzlich deuten die Ergebnisse auf eine neue Konformation hin, der stabilisierten Schleifen-Schleifen Interaktion, bei der der Linker zusätzlich mit den Haarnadelstrukturen interagiert, beispielswese mit den Purinen an der vierten Schleifenposition...
Caspase-2 is the evolutionary most conserved member of the caspase family and was shown to be involved in genotoxic stress induced apoptosis, control of aneuploidy, and ageing related metabolic changes. However, its role in apoptosis seems redundant due to the observation, that knockout does not inhibit apoptotic signalling exclusively. Instead, knockout of caspase-2 leads to tumor susceptibility in vivo, which led to the assumption, that caspase-2 has non-apoptotic functions and can act as a tumor suppressor. The underlying mechanism of the tumor suppressor activity of caspase-2 has not been clarified so far. Furthermore, caspase-2, has a prominent, and as pro-enzyme exclusive localisation in the nucleus and other subcellular compartments, implicating a distinct and location specific role.
In this study, a novel caspase-2 specific substrate, termed p54nrb, was identified. P54nrb is harbouring a caspase-2 specific cleavage site at the aspartate residue D422, and cleavage of p54nrb leads apparently to disruption of its putative DNA binding domain at the C-terminus.
P54nrb is a nuclear multifunctional RNA and DNA binding protein, known for roles in transcriptional regulation, DNA unwinding and repair, RNA splicing, and retention of defective RNA. Overexpression of p54nrb has been observed in several human cancers, such as cervix carcinoma, melanoma, and colon carcinoma.
Data from this study revealed, that depletion of p54nrb in tumor cell lines results in a loss of resistance to drug induced cell death and to reduced capability of anchorage independent growth, which is functionally equivalent to a reduced tumorigenic potential. Meanwhile, p54nrb depletion alone is not cytotoxic.
The investigation of p54nrb dependent gene regulations by high resolution quantitative proteomics uncovered an altering expression of multiple tumorigenic genes. For two of these candidates, the tumorigenic protease cathepsin-Z and the anti-apoptotic gelsolin, p54nrb dependent expression was detected universally in all three investigated tumor cell lines, cervix carcinoma, melanoma, and colon carcinoma. Additionally, a direct interaction of p54nrb with the cathepsin Z and gelsolin encoding DNA, but not with their corresponding mRNA, could be demonstrated.
Conjointly, this study unveils a novel mechanistic feature of caspase-2 as a tumor suppressor. The caspase-2—p54nrb axis can orchestrate the levels of several tumorigenic proteins and thereby determine the cell death susceptibility and long-term tumor survival. These findings might be of great value for future therapeutic interventions and for overcoming drug resistance of tumors.
Investigation of the kinematics involved in compton scattering and hard X-ray photoabsorption
(2023)
The present work investigates the kinematics of Compton scattering at gaseous, internally-cool helium and molecular nitrogen targets in the high- and the low-energy regime. Additionally, photoionization at molecular nitrogen with high-energy photons is investigated. These exeprimental regimes were previously inaccessible due to the extremely small cross sections involved. Nowadays, the third- and fourth-generation synchrotron machines produce sufficient photon flux, enabling the investiagtion of the above processes. The utilized cold-target recoil-ion momentum spectroscopy (COLTRIMS) technique further increases the detection efficiency of the observed processes, since it enables full-solid-angle detection by exploiting momentum conservation.
Compton scattering is investigated at both high (helium and N2) and low (helium) photon energies. In the high-energy regime, the impulse approximation is mostly valid, which is not the case for the low-energy regime. The impulse approximation assumes that the Compton-scattering process takes place at a free electron with a momentum distribution as if it was bound, thus ignoring the binding energy of the system. In the low-energy regime, the impulse approximation is not valid.
Photoionization is investigated at high photon energies, where the linear momentum of the photon cannot be neglected, as is the fashion of the commonly used dipole approximation.
Anthropogenic activities have a major impact on our planet and rapidly drive biodiversity loss in ecosystems at a global scale. Particularly over the last century, rising CO2 emissions significantly raised global temperatures and increased the intensity and frequency of droughts and heatwaves. Additionally, agricultural land use and fossil fuel combustion contribute to the continuous release of nitrogen (N) and phosphorus (P) into ecosystems worldwide through extensive fertilization and deposition from the atmosphere. It is important to understand how these rapid changes affect the evolution of plant populations and their adaptive potential. Adaptation by natural selection (i.e., adaptive evolution) within a few generations is an essential process as a response to rapid environmental changes. Rapid evolution of plant populations can be detected by using the so-called resurrection approach. Here, diaspores (i.e., seeds) from a population are collected before (ancestors) and after (descendants) a potential selection pressure (e.g., consecutive years of drought or changes in nutrient supply). Comparing phenotypes of ancestors and descendants in a common environment such as an outside garden, greenhouse, or climate chamber, may then reveal evolutionary changes. Ideally, plants are first grown in a common environment for an intermediate refresher generation to reduce parental and storage effects.
The aim of this thesis was to investigate the occurrence of adaptive evolution in natural plant populations in response to rapidly changing environments over the past three decades. I conducted three experiments using the resurrection approach to generate comprehensive data on the adaptive processes that acted on three plant populations from three different species over the last three decades. Furthermore, I filled knowledge gaps in plant evolutionary ecology and conceptually developed the resurrection approach further.
In Chapter I, I performed a novel approach by testing for adaptive evolution in natural plant populations using the resurrection approach in combination with in-situ transplantations. I cultivated seedlings from ancestors (23 – 26 years old) and contemporary descendants of three perennial species (Melica ciliata, Leontodon hispidus and Clinopodium vulgare) from calcareous grasslands in the greenhouse and In Chapter III, I assessed the reproducibility of phenotypic differences between genotypes among three different growth facilities (climate chamber, greenhouse, and outdoor garden). I also evaluated differences in phenotypic expression between plants grown after one vs. two intermediate generations (i.e., refresher generations). I performed this experiment within the framework of the resurrection approach and compared ancestors and descendants of the same population of Leontodon hispidus.
I observed very strong differences among plants growing in the different growth facilities. I found a significant interaction between the growth facility and the temporal origin (ancestors vs. descendants): descendants had significantly larger rosettes than ancestors only in the greenhouse and they flowered significantly later than ancestors exclusively in the climate chamber. I did not find significant differences between intermediate generations within the growth facilities. Overall, Chapter III shows that the use of a particular experimental system can dictate the presence and magnitude of phenotypic differences. This implies that absence of evidence is not evidence of absence when it comes to investigating genetically based trait differentiation among plant origins (in space or time). Experimental systems should be carefully designed to provide meaningful conditions, ideally mimicking the environmental conditions of the population’s origins. Finally, growing a second intermediate generation did not impact the genetic differences of ancestors and descendants within the environments, supporting the idea that only one intermediate generation may be sufficient to reduce detectable parental and storage effects.
The resurrection approach allows a better understanding of rapid plant adaptation, but some limitations deserve to be highlighted. I only studied one population per species, and Chapters II and III only focus on one population of L. hispidus, which is also hampering generalizations, as adaptive potential can vary greatly among populations of the same species. I only compared the ancestral genotypes to one descendant sample with a long time span in between (26 – 28 years), which makes it hard to pinpoint the selection agents that caused the genetic differentiation among the sampling years. Hence, closely monitoring biotic and abiotic factors of the studied populations between the ancestral and descendant sampling in future studies, would make identifying the responsible selection pressures more precise. I also recommend sampling multiple populations over consecutive years to improve the robustness of results and make generalizations more approachable.Furthermore, combining the resurrection approach with other methods such as in-situ transplantations will be valuable to offset the limitation that adaptations cannot be proven under artificial conditions (e.g., in the greenhouse).
The nucleus reuniens drives hippocampal goal‑directed trajectory sequences for route planning
(2023)
Goal-directed spatial navigation requires accurate estimates of one’s position and destination, as well as careful planning of a route between them to avoid known obstacles in the environment. Despite its general importance across species, the neural circuitry supporting the ability for route planning remains largely unclear. Previous studies described that place cells in the hippocampal CA1 encode the animal's next movement direction (Wood et al., 2000; Ito et al., 2015) and upcoming navigational routes (Pfeiffer & Foster, 2013). However, it has been shown that part of the CA1 activity representing the animal’s future behaviors is not necessarily generated in the hippocampus, but is derived from the medial prefrontal cortex (PFC) via the nucleus reuniens of the thalamus (RE) (Ito et al., 2015). Notably, the importance of the PFC in navigation has been demonstrated in several studies, including the recent finding of a goal map in the orbitofrontal cortex (Basu et al., 2021). Therefore, I hypothesized that information flow from the PFC to CA1 via the RE plays a key role in route planning.
To assess the animals' route planning ability, I designed a new navigation task in which a rat has to navigate to a fixed target location from various starting positions in an arena. Furthermore, by adding an L-shaped wall in the maze and removing all light sources in the experimental room, this task forced the animals to plan a wall-avoiding route without relying on direct sensory perceptions. I confirmed that rats could learn this task successfully, memorizing the wall location and taking a smooth wall-avoidance route. To test the role of the RE, I inactivated RE neurons by expressing the inhibitory opsin SwiChR++, which resulted in a significant deficit in the animal’s route planning ability, taking a longer non-smooth path to the destination. By contrast, this manipulation did not affect navigation performance when a straight goal-directed route was available, suggesting a specific role of the RE in route planning. I further found that DREADDs-mediated inactivation of neurons in the bilateral hippocampi resulted in a similar deficit in route planning ability, implying cooperation between the RE and the hippocampus.
I finally examined the activity of hippocampal CA1 neurons with and without RE inactivation. While neurons in the hippocampus exhibited brief trajectory sequences corresponding to the animal’s subsequent goal-directed journey, I found that this goal-directed bias of trajectory events was significantly reduced by RE inactivation, likely associated with route-planning deficits in these animals.
Altogether, this dissertation demonstrates the role of the RE from both behavioral and neural coding perspectives, identifying a pivotal circuit element supporting the animal’s route-planning ability.
The equation of state (EoS) of matter at extremely high temperatures and densities is currently not fully understood, and remains a major challenge in the field of nuclear physics. Neutron stars harbor such extreme conditions and therefore serve as celestial laboratories for constraining the dense matter EoS. In this thesis, we present a novel algorithm that utilizes the idea of Bayesian analysis and the computational efficiency of neural networks to reconstruct the dense matter equation of state from mass-radius observations of neutron stars. We show that the results are compatible with those from earlier works based on conventional methods, and are in agreement with the limits on tidal deformabilities obtained from the gravitational wave event, GW170817. We also observe that the resulting squared speed of sound from the reconstructed EoS features a peak, indicating a likely convergence to the conformal limit at asymptotic densities, as expected from quantum chromodynamics. The novel algorithm can also be applied across various fields faced with computational challenges in solving inverse problems. We further examine the efficiency of deep learning methods for analyzing gravitational waves from compact binary coalescences in this thesis. In particular, we develop a deep learning classifier to segregate simulated gravitational wave data into three classes: signals from binary black hole mergers, signals from binary neutron star mergers, or white noise without any signals. A second deep learning algorithm allows for the regression of chirp mass and combined tidal deformability from simulated binary neutron star mergers. An accurate estimation of these parameters is crucial to constrain the underlying EoS. Lastly, we explore the effects of finite temperatures on the binary neutron star merger remnant from GW170817. Isentropic EoSs are used to infer the frequencies of the rigidly rotating remnant and are noted to be significantly lower compared to previous estimates from zero temperature EoSs. Overall, this thesis presents novel deep learning methods to constrain the neutron star EoS, which will prove useful in future, as more observational data is expected in the upcoming years.
Pericytes are capillary-associated mural cells involved in the maintenance and the stability of the vascular network. This thesis aims to investigate the role of pericytes in the heart in the context of ageing and disease. We highlight the malignant effects of the remodelling in the heart and stress the focus on the role of cardiac pericytes in this context. We show that ageing reduces pericyte coverage and that myocardial infarction (MI) causes an activation of these cells. Single-nuclei and single-cell RNA sequencing analysis of murine hearts further revealed that the expression of the Regulator of G-protein signalling 5 (Rgs5) is reduced in cardiac pericytes both in ageing and transiently at day 1 and day 3 after MI. The loss of RGS5 in pericytes drives an entropic state of these mural cells characterized by morphological changes, excessive extracellular deposition and enhanced Gaq mediated GPCR signalling. The deletion of RGS5 in pericytes causes cardiac systolic dysfunction, induces myocardial fibrosis, and drives the activation of cardiac fibroblasts in a TGFb-dependent manner. In conclusion, our results describe the importance of pericytes maintaining cardiac homeostasis, identify RGS5 as a key regulator of this process and propose pericytes as crucial mediators of cardiac fibrosis and possible therapeutic targets to prevent cardiovascular disease.
The prefrontal cortex (PFC) is considered the cognitive center of the mammalian brain. It is involved in a variety of cognitive functions such as decision making, working memory, goal-directed behavior, processing of emotions, flexible action selection, attention, and others (Fuster, 2015). In rodents, these functions are associated with the medial prefrontal cortex (mPFC). Experiments in mice and rats have shown that neurons in the mPFC are necessary for successful performance of many cognitive tasks. Moreover, measurements of neural activity in the mPFC show excitation or inhibition in different cells in relation to specific aspects of the tasks to be solved. To date, however, it is largely unknown whether prefrontal neurons are stably activated during the same behaviors within a task and whether similar aspects are represented by the same neurons in different tasks. In addition, it is unclear how specifically neurons are activated, for example, whether cells that are activated in response to reward are activated in a different task without reward in a different situation or remain inactive. To address these questions, we recorded the same neurons in the mPFC of mice over the course of several weeks while the animals performed various behaviors.
To do this, we expressed GCaMP6 in pyramidal neurons in the mPFC of mice. A small lens was implanted in the same location and a miniature microscope ("miniscope") was used to record neural activity. Later the extracted neurons got aligned based on their shape and position across multiple days and sessions. The mice performed five different behavioral tests while neural activity was measured: A spatial working memory test in a T-maze, exploration of the elevated plus maze (EPM), a novel object recognition (NO) test including free open field (OF) exploration, a social interaction (SI) test and discriminatory auditory fear conditioning (FC). Each task was repeated at least twice to check for stable task encoding across sessions. Behavioral performance and neural correlates to specific task events were similar to earlier studies across all tasks. We utilized generalized linear models (GLM) to determine which behavioral variables most strongly influence neural activity in the mPFC. The position of the mouse in the environment was found to explain most of the variance in neural activity, together with movement speed they were the strongest predictors of neural activity across all tasks. Reward time points in the working memory test, the conditioned stimulus after fear conditioning, or head direction in general were also strongly encoded in the mPFC.
Many of the recorded neurons showed a stable spatial activity profile across multiple sessions of the same task. Similarly, cells that coded for position in one task tended to code for position in other tasks. Not only did the same cells code for position across multiple tasks, but cells also coded for movement speed and head direction. This indicates that at least these general behavioral variables are each represented by the same neurons in the mPFC. Interestingly, the stability of position or speed coding did not depend on the time between two sessions, but only on whether it was within the same or across different tasks. Within the same task, stability was slightly higher than across different tasks.
To find out whether task-specific behavioral aspects were also stably encoded in the mPFC, difference scores as the difference in neural activity between two task aspects like left- and right-choice trials or exposed and enclosed locations were calculated. Many cells encoded these aspects stably across different sessions of each task. Both the left-right differences in the different phases of the working memory test, the open-closed-arm differences in the elevated plus maze, the different activity between center and corners in the open field, the social target-object differences in the social interaction test, and the differences between the two tones during fear conditioning were all stably encoded across the population of mPFC cells. Only the distinction between the novel and the familiar object during object recognition was not stably encoded, but also the preference for the novel object was not present in the second session of novel object exploration.
There was also an overlap in coding for different aspects within a task across multiple sessions. For example, cells stably encoded left-right differences in the T-maze between different sessions as a function of walking direction across different phases of working memory, an aspect that we could already show within one session (Vogel, Hahn et al., 2022). During fear conditioning, the same cells showed a discrimination between CS+ and CS- that also responded to the start of CS+.
Consistency in the neurons activity across different tasks was also found, but only between tasks with similar demands, the elevated plus-maze and free exploration of the open field. Cells that were more active in the open arms also showed more activity in the center of the open field and vice versa. This could be an indicator that the cells were coding for anxiety or exposure across those tasks, indicating that neurons in the mPFC also stably encode general task aspects independent of the specific environment. However, it remains unclear what exactly these neurons encode; in the case of a general fear signal, one would also expect activation during fear conditioning which could not be found.
Overall, we found that neurons in the mPFC of mice encoded multiple general behavioral variables across multiple tasks and task-specific variables were encoded stably within each of the tested tasks. However, we found little task-specific variables that were systematically encoded by the same neurons with the exception being the elevated plus-maze and open field exploration, two tasks with similar features.
Impact of pectin dietary supplementation on experimental food allergy via gut microbiota modulation
(2023)
In recent years, dietary fibers gained focus in regard of their immune-modulatory effects and the potentially beneficial effect on allergies. The dietary fiber and prebiotic pectin is able to promote growth and activity of beneficial bacteria and thereby induce modulation of different immune responses. However, structurally different types of pectin might promote different immune-modulatory responses and to date the optimal pectin type for induction of beneficial health effects is not identified. Furthermore, it is still unclear, whether pectins provide a beneficial effect on certain allergies, such as food allergy.
Having this in consideration, this study examined the immune-modulatory effects of structurally different pectins on naive as well as peach allergic mice. Furhtermore, the impact of dietary pectin supplementation on composition and diversity of the murine gut microbiota was determined.
This study showed that dietary pectin intervention was able to suppress allergy-related Th2 responses considering humoral and cellular immune responses. Only apple-derived high-methoxyl pectin revealed an impact on total IgA levels and affected the microbial richness. Furthermore, it is not known whether the effects observed with the two pectins are caused by modulations of the bacterial composition or induced at least partly by direct interaction with the immune cells. Further studies are required to fully understand the mechanisms underlying the immune-modulatory capacities of different pectins.
Finally, the obtained results generated evidence that dietary pectin intervention can beneficially modulate the immune response in healthy mice and – at least partially – suppress allergy-related immune responses in a model of food allergy, depending on the structural characteristics of the used pectin.
Mitochondria perform essential energetic, metabolic and signalling functions within the cell. To fulfil these, the integrity of the mitochondrial proteome has to be preserved. Therefore, each mitochondrial subcompartment harbours its own system for protein quality control. However, if the capacity of mitochondrial chaperones and proteases is overloaded, mitochondrial misfolding stress (MMS) occurs. Upon this stress condition, mitochondria communicate with the nucleus to increase the transcription of nuclear encoded mitochondrial chaperones and proteases. This proteotoxic stress pathway was termed the mitochondrial unfolded protein response (UPRmt) aiming at restoring protein homeostasis. Despite being discovered over 25 years ago, the signalling molecules released by stressed mitochondria as well as the corresponding receptor and transcription factor remain poorly understood. With this study, we aimed at characterising the underlying signalling events and mechanisms of how mitochondria react to misfolded proteins. First, we aimed to establish different methods to induce MMS that triggers the transcriptional induction of mitochondrial chaperones and proteases detected by quantitative polymerase chain reaction. We were able to induce UPRmt signalling by overexpression of an aggregation-prone protein and by knock-down or inhibition of mitochondrial protein quality control components. To study the signalling in a time-resolved manner, we focused on the usage of the mitochondrial HSP90 inhibitor GTPP and the mitochondrial LONP1 protease inhibitor CDDO.
Early time point RNA sequencing analysis of cells stressed with GTPP or CDDO revealed upregulated genes in response to oxidative stress. Indeed, measurements of mitochondrial superoxide with the fluorescent dye MitoSOX showed increased levels of reactive oxygen species (ROS) upon MMS induction. In contrast, there was no induction of mitochondrial chaperones and proteases when combining MMS with antioxidants. Compartment-specific targeting of the hydrogen peroxide sensor HyPer7 revealed increased ROS levels in the intermembrane space and matrix of mitochondria, followed by elevated ROS levels in the cytosol at later time points. The importance of cytosolic ROS for the signalling was supported by preventing UPRmt induction with an inhibitor blocking the outer mitochondrial membrane pore. Thus, ROS were identified as an essential UPRmt signal.
To understand which cytosolic factor is modified by ROS, redox proteomics was performed. Here, reversible changes on cysteine residues of the HSP40 co-chaperone DNAJA1 were observed upon MMS. Consequently, transcriptional induction of UPRmt genes was abolished by DNAJA1 knock-down. To understand the function of DNAJA1 during UPRmt signalling, quantitative interaction proteomics upon MMS revealed an increased binding to mitochondrial proteins and its interaction partner HSP70. Immunoprecipitation confirmed a ROS-dependent interaction between HSP40 and HSP70. Increased binding to mitochondrial proteins represented a cytosolic interaction of DNAJA1 with mitochondrial precursor proteins, whose accumulation was confirmed by western blot. Moreover, a fluorescent protein targeted to mitochondria accumulated in the cytosol during GTPP treatment, confirming a reduced import efficiency upon MMS. Preventing the accumulation of precursors by a translation inhibitor or depletion of a general mitochondrial transcription factor resulted in reduced UPRmt activation. Thus, DNAJA1 is essential for UPRmt signalling, since its oxidation by mitochondrial ROS and its enhanced recruitment to mitochondrial precursors allows the integration of both MMS-induced signals.
To link these findings to an increased transcription of mitochondrial chaperones and proteases, we screened for transcription factors accumulating in the nucleus upon MMS by cellular fractionation mass spectrometry. We demonstrated that specifically HSF1 accumulates in nuclei of cells stressed with GTPP or CDDO. Depletion of HSF1 by knock-down or knock-out resulted in the abrogation of the UPRmt-specific transcriptional response. HSF1 activation was visualised by nuclear accumulation on western blot, a process inhibited by ROS and precursor suppression. Moreover, DNAJA1 depletion prevented HSF1 activation. Ultimately, we proved by immunoprecipitation that the inhibitory interaction between HSF1 and HSP70 is reduced upon MMS.
Thus, we conclude that MMS increases mitochondrial ROS that are released into the cytosol. In addition, the import efficiency is reduced upon MMS, resulting in the accumulation of non-imported mitochondrial precursor proteins in the cytosol. Both signals are recognised via DNAJA1 oxidation and substrate binding. The concurrent recruitment of HSP70 to DNAJA1 results in the loss of the inhibitory HSP70-HSF1 interaction. Thus, active HSF1 can migrate to the nucleus to initiate transcription of mitochondrial chaperones and proteases. These findings are in accordance with observations in yeast, where mistargeted mitochondrial proteins activate cellular stress responses. Our results highlight a surprising interconnection and dependence of the mitochondrial and the cytosolic proteostasis network, in which the UPRmt is activated by a combination of two mitochondria-specific proteotoxic stress signals.
This dissertation constitutes a series of successive research papers, starting with the characterization of various optogenetic tools up to the establishment of purely optical electrophysiology in living animals.
Optogenetics has revolutionized neurobiology as it allows stimulation of excitable cells with exceptionally high spatiotemporal resolution. To cope with the increasing complexity of research issues and accompanying demands on experimental design, the broadening of the optogenetic toolbox is indispensable. Therefore, one goal was to establish a wide variety of novel rhodopsin-based actuators and characterize them, among others, with respect to their spectral properties, kinetics, and efficacy using behavioral experiments in Caenorhabditis elegans. During these studies, the applicability of highly potent de- and hyperpolarizers with adapted spectral properties, altered ion specificity, strongly slowed off-kinetics, and inverted functionality was successfully demonstrated. Inhibitory anion channelrhodopsins (ACRs) stood out, filling the gap of long-sought equivalent hyperpolarizing tools, and could be convincingly applied in a tandem configuration combined with the red-shifted depolarizer Chrimson for bidirectional stimulation (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES). A parallel study aimed to compare various rhodopsin-based genetically encoded voltage indicators (GEVIs) in the worm: In addition to electrochromic FRET-based GEVIs that use lower excitation intensity, QuasAr2 was particularly convincing in terms of voltage sensitivity and photostability in C. elegans. However, classical optogenetic approaches are quite static and only allow perturbation of neural activity. Therefore, QuasAr2 and BiPOLES were combined in a closed-loop feedback control system to implement the first proof-of-concept all-optical voltage clamp to date, termed the optogenetic voltage clamp (OVC). Here, an I-controller generates feedback of light wavelengths to bidirectionally stimulate BiPOLES and keep QuasAr’s fluorescence at a desired level. The OVC was established in body wall muscles and various types of neurons in C. elegans and transferred to rat hippocampal slice culture. In the worm, it allowed to assess altered cellular physiology of mutants and Ca2+-channel characteristics as well as dynamical clamping of distinct action potentials and associated behavior.
Ultimately, the optogenetic actuators and sensors implemented in the course of this cumulative work enabled to synergistically combine the advantages of imaging- and electrode-based techniques, thus providing the basis for noninvasive, optical electrophysiology in behaving animals.
This thesis investigates the structure of the translocase of the outer membrane (TOM) complex in mitochondria, focusing on the TOM holo complex through single-particle electron cryo-microscopy (cryoEM) complemented by mass spectrometry and computational structure prediction. Mitochondria, crucial for energy production in eukaryotic cells, import most of their proteins from the cytoplasm. These proteins enter through the TOM complex, which in its core form consists of a membrane-embedded homodimer of Tom40 pores, two Tom22 cytoplasmic receptors, and six small TOM stabilizing subunits (Tom7, Tom6, and Tom5). The holo complex includes two additional subunits, Tom70 and Tom20, whose stoichiometry and positioning are less understood due to their easy dissociation during isolation of the complex. CryoEM analysis revealed the high-resolution structure of the Neurospora crassa TOM core complex at 3.3 Å, containing all core subunits, and the presence of a central phospholipid causing the Tom40 dimer to tilt to 20°. Furthermore, a 4 Å resolution map indicated the binding of a precursor protein as it transitions through the translocation barrel. Finally, at 6-7 Å resolution, the structure of the TOM holo complex highlighted Tom20's flexibility as it interacts with the core complex, emphasizing its role in protein translocation. This work provides significant insights into the architecture and functioning of the TOM complex, contributing to the understanding of mitochondrial protein import mechanisms.
Life and biological resilience rely on the execution of precise gene expression profiles. A key mechanism to ensure cellular homeostasis is the regulation of protein synthesis. Recent studies have unveiled an intrinsic regulatory capacity of ribosomes, previously considered mere executors of mRNA translation. Neurons in particular finely regulate protein synthesis, at both global and local levels. This sustains their complex morphology and allows them to rapidly transmit, integrate, and respond to external stimuli. In this thesis, I investigated the neuronal ribosome and how subcellular environments and physiological perturbations shape it, by profiling its molecular composition, functional interconnections, and cellular distribution.
First, I used genetic engineering, biochemical purification, and mass spectrometry, to characterize in an unbiased manner the translation machinery specifically from excitatory and inhibitory neurons of the mouse cortex. I found that neuronal ribosomes commonly interact with RNA-binding proteins, components of the cytoskeleton, and proteins associated with the endoplasmic reticulum and vesicles. In line with the requirement for local protein synthesis in the distal parts of neurons, we observed that neuronal ribosomes preferentially interact with proteins involved in cellular transport. Remarkably, I observed a strong association between ribosomes and pre-synaptic vesicles, which suggests a potential regulatory interaction between local translation and neuronal activity.
Intriguingly, I and others have observed mRNAs encoding for core ribosomal proteins (RPs) among the genes most enriched in neuronal processes. This observation challenges two historical assumptions of ribosome biology: (1) new RPs are incorporated only into newly forming ribosomes, and (2) this incorporation occurs only in the nucleus and perinuclear region. In my PhD, I aimed to directly test these two assumptions and if proven wrong ask whether and why neurons would localize RP mRNAs far from their known assembly site.
Employing a combination of metabolic labeling and highly sensitive mass spectrometry techniques, I discovered that a subset of RPs rapidly and dynamically binds on and off mature ribosomes. Strikingly, this incorporation does not depend on the supply of new ribosomes from the nucleus. Therefore, my data refuted the assumption that ribosomes are built and degraded as a unit and revealed a more dynamic view of these machines, which can actively exchange core components. In particular, I found that the association of certain exchanging RPs is influenced by location (e.g., cell body versus neurites) and cellular state (e.g., post-oxidative stress). Neurons may use this mechanism to repair and/or specialize their protein synthesis machinery in a rapid and context-dependent manner.
Finally, I asked whether some steps of ribosome biogenesis could also take place in distal processes. Although most steps of ribosome assembly occur within the nucleus, the final stages of maturation are known to occur in the cytosol. By combining several imaging and biochemical approaches, I found that cytosolic (but not nuclear) pre-ribosomal particles are present in neuronal processes. Through the incorporation of new RPs into these immature particles, neurons may be able to locally “turn on” previously incompetent ribosomes. This may enable regions near synapses to enhance and customize their translational capacity, independently of the central pool of ribosomes from the cell body. Indeed, I observed that synaptic plasticity induces a maturation of cytosolic pre-ribosomes.
In summary, this thesis shows how neuronal ribosomes can sense cellular states, respond by adjusting their core composition, and in doing so influence the local capacity for protein synthesis. By overturning long-held assumptions in ribosome biology, this work highlights new molecular mechanisms of gene expression and enriches our understanding of the rapid and dynamic strategies cells employ to operate, thrive, and adaptively respond to environmental changes.
Precise regulation of gene expression networks is required to develop and maintain a healthy organism before and after birth and throughout adulthood. Such networks are mostly comprised of regulatory proteins, but meanwhile many long non-coding transcripts (lncRNAs) are shown to participate in these regulatory processes. The functions and mechanisms of these lncRNAs vary greatly, however they are often associated with transcriptional regulation. Three lncRNAs, namely Sweetheart RNA (Swhtr), Fetal-lethal noncoding developmental regulatory RNA / Foxf1 adjacent non-Coding developmental regulatory RNA (Fendrr) and lncFsd2, were studied in this work to demonstrate the variety of cellular and biological processes that require lncRNA-mediated fine-tuning, in regard to the cardiopulmonary system.
Swhtr was found to be expressed exclusively in cardiomyocytes and became critical for regeneration after myocardial injury. Mice lacking Swhtr did not show issues under normal conditions, but failed to undergo compensatory hypertrophic remodeling after injury, leading to increased mortality. This effect was rescued by re-expressing Swhtr, demonstrating importance of the RNA. Genes dependent on Swhtr during cardiac stress were found to likely be regulated by NKX2-5 through physical interaction with Swhtr. Fendrr was found to be expressed in lung and interacted with target promoters through its RNA:dsDNA binding domain, the FendrrBox, which was partially required for Fendrr function. Fendrr, together with activated WNT signaling, regulated fibrosis related target genes via the FendrrBox in fibroblasts. LncFsd2, an ubiquitously expressed lncRNA, showed possible interaction with the striated muscle specific Fsd2, but its exact function and regulatory role remain unclear in muscle physiology. Immunoprecipitation and subcellular fractionation experiments suggest that lncFsd2 might be involved in nuclear retention of Fsd2 mRNA, thus fine-tuning FSD2 protein expression. These investigations have shed light on the roles of these lncRNAs in stress responses, fibrosis-related gene regulation, and localization processes, advancing our understanding of cardiovascular and pulmonary maintenance, reaction to injury, and diseases. The diverse and intricate roles of these three lncRNAs highlight how they influence various cellular processes and disease states, offering avenues for exploring lncRNA functions in different biological contexts.
The theoretical and experimental investigation of exotic hadrons like tetraquarks is an important branch of modern elementary particle physics. In this thesis I investigate different four-quark systems using lattice QCD and search for evidence of stable tetraquark states or resonances.
Lattice QCD as a non-perturbative approach to QCD allows an accurate and reliable determination of the masses of strongly bound hadrons.
However, most tetraquarks appear as weakly bound states or resonances, which makes a theoretical investigation using lattice QCD difficult due to the finite spatial volume. A rigorous treatment of such systems is feasible using the so-called Lüscher method. This allows to calculate the scattering amplitude based on the finite-volume energy spectrum determined in a lattice QCD calculation. Similarly to the analysis of experimental data, this scattering amplitude can be used to determine the binding energies of bound states or the masses and decay widths of resonances in the infinite volume.
In my work I calculate the low-energy energy spectra of different four-quark systems and use - if necessary - the Lüscher method to determine the masses of potential tetraquark states.
I focus on systems consisting of two heavy antiquarks and two light quarks, where at least one of the heavy antiquarks is a bottom quark.
Even though such tetraquarks have not yet been experimentally detected, they are considered promising candidates for particles that are stable with respect to the strong interaction.
A decisive step for successfully calculating low-lying energy levels for such four-quark systems is a carefully chosen set of creation operators, which represent the physical states most accurately. In addition to operators that generate a local structure where all four quarks are located at the same space-time point, I also use so-called scattering operators that resemble two spatially separated mesons. These scattering operators turned out to be relevant for successfully determining the lowest energy levels and are therefore essential, especially if a Lüscher analysis is carried out.
In my work, I considered two different lattice setups to study the four-quark systems $\bar{b}\bar{b}ud$ with $I(J^P)=0(1^+) $, $\bar{b}\bar{b}us$ with $J^P=1^+ $ and $\bar{b}\bar{c}ud$ with $I(J^P)=0(0^+) $ and $I(J^P)=0(1^+) $ and to predict potential tetraquark states. In both setups, I considered scattering operators. While in the first setup I used them only as annihilation operators, in the second setup they were included both as creation and annihilation operators. Additionally, in the second lattice setup, I performed a simplified investigation of the $\bar{b}\bar{b}ud$ system with $I(J^P)=0(1^-) $, which is a potential candidate for a tetraquark resonance. The results of the investigation of the mentioned four-quark systems can be summarized as follows:
For the $ \bar{b}\bar{b}ud $ four-quark system with $ I(J^P)=0(1^+) $ I found a deeply bound ground state slightly more than $ 100\,\textrm{MeV} $ below the lowest meson-meson threshold. The existence of a corresponding $\bar{b}\bar{b}ud$ tetraquark in the infinite volume was confirmed using a Lüscher analysis and possible systematic errors due to the use of lattice QCD were taken into account.
Similar results were obtained for the $ \bar{b}\bar{b}us $ four-quark system with $ J^P=1^+ $. Again, I found a ground state well below the lowest meson-meson threshold, but slightly weaker bound than for the $ \bar{b}\bar{b}ud $ system. Effects due to the finite volume turned out to be negligible for this system, as already predicted for the $ \bar{b}\bar{b}ud $ system. \item For the $ \bar{b}\bar{c}ud $ four-quark systems with $ (J^P)=0(0^+) $ and $ (J^P)=0(1^+) $ I was able to rule out the existence of a deeply bound tetraquark states based on the energy spectrum in the finite volume. However, by means of a scattering analysis using the Lüscher method, I found evidence a broad resonance for both channels.
In the case of the $ \bar{b}\bar{b}ud $ four-quark system with $ I(J^P)=0(1^-) $, I could neither confirm the existence of a resonance, nor rule out its existence with certainty.
In particular, my investigations showed that the results of the two different lattice simulations are consistent. The theoretical prediction of the bound tetraquark states $\bar{b}\bar{b}ud$ and $\bar{b}\bar{b}us$ as well as the tetraquark resonances in the $\bar{b}\bar{c}ud$ system in this work represent an important contribution to the future experimental search for exotic hadrons and can support the discovery of previously unobserved particles.
This work aimed to investigate the regulation and activity of 5-lipoxygenase (5-LO), the central enzyme in leukotriene biosynthesis, in two colorectal cancer cell lines. The leukotriene pathway is positively correlated with the progression of several solid malignancies; however, factors regulating 5-LO expression and activity in tumors are poorly understood.
Cancer development, as well as cancer progression, are strongly dependent on the tumor microenvironment. In the conventional monolayer culture of cancer cell lines, cell-matrix and cell-cell interactions present in native tumors are absent. Furthermore, it is already known that various colon cancer cell lines dysregulate several important signaling pathways due to 3D growth. Therefore, the expression of the leukotriene cascade in HT-29 and HCT-116 colorectal cancer cells was investigated within a three-dimensional context using multicellular tumor spheroids to mimic a more physiological environment compared to conventional cell culture. Especially the expression of 5-LO, cPLA2α, and LTA4 hydrolase was altered due to threedimensional (3D) cell growth, which was investigated by qPCR and Western blot analysis. High cellular density in monolayer cultures led to similar results. The observed 5-LO upregulation was found inversely correlated with cell proliferation, determined by cell cycle analysis, and activation of PI3K/mTORC-2- and MEK-1/ERK-dependent pathways, determined using pharmacological pathway inhibition, stable shRNA knockdown cell lines, and analysis via qPCR and Western blot analysis. Following, the transcription factor E2F1 and its target gene MYBL2 were identified to play a role in the repression of 5-LO during cell proliferation. For this purpose, several stable MYBL2 over-expression and ALOX5 reporter cell lines were prepared and analyzed. Since 5-LO was already identified as a direct p53 target gene, the influence of p53, which is variably expressed in the cell lines (HT-29, p53 R273H mut; HCT-116 p53 wt; HCT-116 p53 KO), was investigated as well. Furthermore, HCT-116 cells carrying a p53 knockout were investigated. The PI3K/mTORC-2- and MEK-1/ERK-dependent suppression of 5-LO was also found in tumor cells from other origins (Capan-2, Caco-2, MCF-7), which was determined using pharmacological pathway inhibition and following analysis via qPCR. This suggests that the identified mechanism might apply to other tumor entities as well.
5-LO activity was previously described as attenuated in HT-29 and HCT-116 cells compared to polymorphonuclear leukocytes, which express a highly active 5-LO. However, the present study showed that the enzyme activity is indeed low but inducible in HT-29 and HCT-116 cells. Of note, the general lipid mediator profile and the mediator concentrations were comparable to those of M2 macrophages. Finally, the analysis of substrate availability in HT-29 and HCT-116 cells revealed a vast difference between formed metabolite concentrations and supplemented fatty acid concentrations, indicating that the substrates are either transformed into lipoxygenase-independent metabolites or are esterified into the cellular membrane.
In summary, the data presented in this work demonstrate that 5-LO expression and activity are tightly regulated in HT-29 and HCT-116 cells and fine-tuned due to environmental conditions. The cells suppress 5-LO during proliferation but upregulate the expression and activity of the enzyme under cellular stress-triggering conditions. This implies a possible role of 5-LO in manipulating the tumor stroma to support a tumor-promoting microenvironment.
Xylose, an abundant sugar fraction of lignocellulosic biomass, is a five-carbon skeleton molecule. Since decades, utilization of this sugar has gained much attention and has been in particular focus as a substrate for production of biofuels like ethanol by microbial hosts, including Saccharomyces cerevisiae. In this yeast, xylose is naturally not used as a carbon source, but its utilization could be achieved by metabolic engineering either via the oxidoreductive route or through the isomerase pathway. Both pathways share xylulose as a common intermediate that must be phosphorylated before entering the endogenous metabolism via the non-oxidative pentose phosphate pathway (noxPPP). Besides this, in some bacteria a non-phosphorylating oxidative pathway for xylose degradation exists, known as Weimberg pathway, where a molecule of xylose is converted by a series of enzymes - xylose dehydrogenase (XylB), xylonate dehydratase (XylD), 3-keto-2-deoxy-xylonate dehydratase (XylX) and α-ketoglutarate semialdehyde dehydrogenase (KsaD) - to form α-ketoglutarate (AKG). Besides having several useful properties as a product, AKG could also be used for cell growth as an intermediate of the tricarboxylic acid (TCA) cycle. One target of the present study is to establish a functional Weimberg pathway in S. cerevisiae. Previous studies have shown that this task is not trivial, for instance due to the toxicity of xylonate (the first metabolite of the pathway) and the involvement of an iron-sulfur cluster dependent enzyme, the D-xylonate dehydratase. The assembly of iron-sulfur clusters on a heterologous protein in yeast is known to be challenging.
To establish the Weimberg pathway in yeast, the genes xylB, xylD, and xylX were obtained from Caulobacter cresentus and ksaD was from Corynebacterium glutamicum. In a variant, the dehydratase xylD was replaced with orf41 from Arthrobacter nicotinovorans, which is believed to be independent of iron-sulfur clusters. Growth of yeast cells on xylose as a sole carbon source was expected as an indicator of a functional Weimberg pathway. However, the heterologous expression of the codon optimized genes was not sufficient to reach this goal. Due to the complexity of the interactions of the heterologous pathway with the endogenous cellular processes, it was assumed that potential limitations could be overcome by adaptive laboratory evolution, using xylose as a sole source of carbon. Increasing selection pressure was applied on a strain with Weimberg pathway genes integrated into the genome over several generations. As a variant of the evolutionary engineering approach, mutator strains were generated. For this, RAD27 and MSH2 genes were deleted, which are involved in nucleotide excision and mismatch repair mechanisms, respectively. Some of the resulting strains PRY24, PRY25, PRY27 and PRY28 were able grow in xylose as a sole carbon source after evolutionary engineering. As a control, a non-mutator strain PRY19 was also included. Strikingly, only the mutator strains were able to consume xylose as a sole carbon source, which shows the feasibility of the approach.
In addition to the mutator strain strategy, a further approach employed in the present study was the simultaneous expression of the Weimberg pathway in the cytosol and mitochondria. This was based on the reasoning that the iron-sulfur cluster biogenesis on XylD may be improved in the organelle and that the AKG is an intermediate of the TCA cycle. In the strain AHY02, all enzymes of the pathway were tagged with mitochondrial targeting signals in addition to a full cytosolically localized pathway. The localization of the mitochondrial variants was confirmed by fluorescence microscopy. Together with AHY02, CEN.PK2-1C wild type strain was also included as a control for evolution. When a selection pressure on xylose was applied, both strains - AHY02 and CEN.PK2-1C - were able to grow in the course of evolution. Deletion of the xylulokinase (XKS1) gene was found to be detrimental for both evolved strains in xylose-containing media. This suggests that the evolution of the endogenous oxidoreductive and noxPPP genes is responsible for growth of the evolved cells. For the evolved strain AHY02, it could also be possible that the Weimberg pathway genes supported to growth in addition to the oxidoreductive route. To elucidate the underlying molecular mechanisms, genome sequencing and reverse engineering approaches would be necessary in future.
In addition to screening for growth on xylose as a sole carbon source, a less stringent screening system was created to examine even a minor flux of xylose towards AKG. For this, all genes necessary for conversion of isocitrate to AKG where deleted, yielding a glutamate auxotrophic strain. In this system, the cells can grow on other carbon sources, whereas xylose is only provided as a source of AKG for the synthesis of glutamate...
Precise intensity monitoring at CRYRING@ESR: on designing a Cryogenic Current Comparator for FAIR
(2023)
In the field of today’s beam intensity diagnostic there is a significant gap in the non-interceptive, calibrated measurement of the absolute intensity of continuous (unbunched) dc beams with current amplitudes below 1 μA. At the Facility for Antiproton and Ion Research (FAIR) low-intensity DC beams will occur during slow extraction from the synchrotrons as well as for coasting beams of highly-charged or exotic nuclei in the storage rings. The lack of adequate beam instrumentation limits the experimental program as well as the accuracy of experimental results.
The Cryogenic Current Comparator (CCC) can close the diagnostic gap with a high-precision dc current reading independent of ion-species and of beam parameters. However, the established detector design based on a core with high magnetic permeability and on a radial shield geometry has well-known weaknesses concerning magnetic shielding efficiency and intrinsic current noise. To eliminate these weaknesses, a novel coreless CCC with a co-axial shield was constructed and combined with a high-performance SQUID contributed by the Leibniz-Institute of Photonic Technology (Leibniz-IPHT Jena). The new axial CCC model was compared to a radial CCC with the established design provided by the Friedrich-Schiller-University Jena. According to numerical simulations prepared at TU Darmstadt and test measurements of the detectors in the laboratory, the new design offered a significant improvement of the shielding factor – from 75dB to 207dB at the required dimensions – and eliminated all noise contributions from the core material, promising an improved current resolution. Although the lower inductance of the pickup coil reduced the coupling to the beam significantly, the noise properties of the new CCC type were comparable to the classical version with a high-permeability core. However, the expected decrease of the low-frequency noise and thus an increase of the current resolution could not be observed at this stage of development.
Consequently, the classical CCC based on the radial shielding and high-permeability core had to be installed in CRYRING@ESR to provide best possible intensity measurements for the upcoming experimental campaign. In CRYRING the CCC was operated with beam currents between 1nA and 20μA and with different ion species (H, Ne, O, Pb, U). It was shown that the CCC provides a noise-limited current resolution of better than 3.2 nArms at a bandwidth of 200 kHz as well as a noise level below 40 pA/√Hz above 1 kHz. During the operation, the main noise sources of the accelerator environment had to be identified and suitable mitigation strategies were developed. Temperature and pressure fluctuations were suppressed with a newly-designed cryogenic support system based on a 70 l helium bath cryostat, developed and built in collaboration with the Institut für Luft- und Kältetechnik Dresden, in combination with a helium re-liquefier. The cryogenic operating time was restricted to around 7 days, which must be expanded significantly in the future. Digital filters were developed to remove the perturbations of the helium liquefier and of the neighboring dipole magnets. Given the promising results the CCC system can be considered as a prototype for future CCCs at FAIR.
The Compressed Baryonic Matter (CBM) is one of the core experiments at the future Facility for Anti-proton and Ion Research (FAIR), Darmstadt, Germany. Its goal is to investigate nuclear matter characteristics at high net-baryon densities and moderate temperatures. The Silicon Tracking System (STS) is a central detector system of CBM.
It is placed inside a 1Tm magnet and operated at a temperature of about −10 °C to keep radiation-induced bulk current in the 300μm double-sided microstrip silicon sensors low. The design of the STS aims to minimize the material budget in the detector acceptance (2.5° < θ < 25°). In order to do so, the readout electronics is placed outside the active area, and the analog signals are transported via ultra-thin micro-cables. The STS comprises eight tracking stations with 876 modules. Each module is assembled on a carbon fiber ladder, which is subsequently mounted in the C-shaped aluminum frame.
The scope of the thesis focused on developing a modular control system framework that can be implemented for different sizes of experimental setups. The developed framework was used for setups that required a remote operation, like the irradiation of the powering modules for the front-end electronics (FEE), but also in laboratory-based setups where the automation and archiving were needed (thermal cycling of the STS electronics).
The low voltage powering modules will be placed in the vicinity of the experiment, therefore they will experience a total dose of up to 40mGy over the 10 years of STS lifetime.
To estimate the effects of the radiation on the low-voltage module performance, a dedicated irradiation campaign took place. It aimed at estimating the rate of radiation induced soft errors, that lead to the switch off of the FEE.
Regular power cycles of multiple front-end boards (FEBs) pose a risk to the experiment operation. Firstly, such behavior could negatively influence the physics performance but also have deteriorating effects on the hardware. It was further assessed what are the limitations of the FEBs with respect to the thermal cycling and the mechanical stress. The results served as an indication of possible failure modes of the FEB at the end of STS lifetime. Failure modes after repeated cycles and potential reasons were determined (e.g., Coefficient of Thermal Expansion (CTE) difference between the materials).
Due to the conditions inside the STS efficient temperature and humidity monitoring and control are required to avoid icing or water condensation on the electronics or silicon sensors. The most important properties of a suitable sensor candidate are resilience to the magnetic field, ionizing radiation tolerance, and fairly small size.
A general strategy for ambient parameters monitoring inside the STS was developed, and potential sensor candidates were chosen. To characterize the chosen relative humidity sensors the developed control framework was introduced. A sampling system with a ceramic sensor and Fiber Optic Sensors (FOS) were identified as reliable solutions for the distributed sensing system. Additionally, the industrial capacitive sensors will be used as a reference during the commissioning.
Two different designs of FOS were tested: a hygrometer and 5 sensors multiplexed in an array. The FOS hygrometer turned out to be a more reliable solution. One of the possible reasons for a worse performance is a relatively low distance between the subsequent sensors (15 cm) and a thicker coating. The results obtained from the time response study pointed out that the thinner coating of about 15μm should be a good compromise between the humidity sensitivity and the time response.
The implementation of the containerized-based control system framework for the mSTS is described in detail. The deployed EPICS-based framework proved to be a reliable solution and ensured the safety of the detector for almost 1.5 years. Moreover, the data related to the performance of the detector modules were analyzed and significant progress in the quality of modules was noted. Obtained data was also used to estimate the total fluence, which was based on the leakage current changes.
The developed framework provided a unique opportunity to automate and control different experimental setups which provided crucial data for the STS. Furthermore, the work underlines the importance of such a system and outlines the next steps toward the realization of a reliable Detector Control System for STS.
This thesis deals with several aspects of non-perturbative calculations in low-dimensional quantum field theories. It is split into two main parts:
The first part focuses on method development and testing. Using exactly integrable QFTs in zero spacetime dimensions as toy models, the need for non-perturbative methods in QFT is demonstrated. In particular, we focus on the functional renormalization group (FRG) as a non-perturbative exact method and present a novel fluid-dynamic reformulation of certain FRG flow equations. This framework and the application of numerical schemes from the field of computational fluid dynamics (CFD) to the FRG is tested and benchmarked against exact results for correlation functions. We also draw several conclusions for the qualitative understanding and interpretation of renormalization group (RG) flows from this fluid-dynamic reformulation and discuss the generalization of our findings to realistic higher-dimensional QFTs.
The topics discussed in the second part are also manifold. In general, the second part of this thesis deals with the Gross-Neveu (GN) model, which is a prototype of a relativistic QFT. Even though being a model in two spacetime dimensions, it shares many features of realistic models and theories for high-energy particle physics, but also emerges as a limiting case from systems in solid state physics. Especially, it is interesting to study the model at non-vanishing temperatures and densities, thus, its thermodynamic properties and phase structure.
First, we use this model to test and apply our findings of the first part of this thesis in a realistic environment. We analyze how the fluid-dynamic aspects of the FRG realize themselves in the RG flow of a full-fledged QFT and how we profit from this numeric framework in actual calculations. Thereby, however, we also aim at answering a long-standing question: Is there still symmetry breaking and condensation at non-zero temperatures in the GN model, if one relaxes the commonly used approximation of an infinite number of fermion species and works with a finite number of fermions? In short: Is matter (in the GN model) in a single spatial dimension at non-zero temperature always gas-like?
In general, we also use the GN model to learn about the correct description of QFTs at non-zero temperatures and densities. This is of utmost relevance for model calculations in low-energy quan- tum chromodynamics (QCD) or other QFTs in medium and we draw several conclusions for the requirements for stable calculations at non-zero chemical potential.
Large international airports were identified as sources of ultrafine particles (UFPs) (Hu et al., 2009; Yu et al., 2012; Hsu et al., 2013; Keuken et al., 2015; Hudda and Fruin, 2016). Since September 2017 UFP emissions originating from the Frankfurt International Airport, Germany are monitored by the Hessian Agency for Nature Conservation, Environment and Geology (HLNUG) showing elevated UFP concentrations during airport operating hours (05:00–23:00 CET) (Ditas et al., 2022). Referring to that, the organic chemical composition of aviation-related UFPs emerging from the Frankfurt Airport was analysed by performing a comprehensive non-target screening of UFP filter samples.
Aluminium-filter samples were collected at an air quality monitoring station 4 km north of the Frankfurt Airport, using a 13-stage impactor system (Nano-MOUDI). The chemical
characterization of UFPs in the size range of 10-18 nm, 18-32 nm and 32-56 nm was accomplished by ultra-high-performance liquid chromatography, heated electrospray ionisation and mass analysis using an Orbitrap high-resolution mass spectrometer. Non-target screening revealed that the majority of detected compounds belong to homologous series of two different types of organic esters, which are base stocks of aircraft lubrication oils.
In reference to five different jet engine lubrication oils of various manufacturers, the corresponding lubricant base stocks and their additives, two amines and one organophosphate, were identified in the UFPs by the use of matching retention time, exact mass and MS/MS fragmentation pattern of single organic molecules. The quantitative analysis of the jet engine oil constituents in the aviation-related UFPs with diameters < 56 nm was accomplished by standard addition. By characterizing the Nano-MOUDI, loss factors for each size stage were determined and used for correction accordingly. Particle-number size distribution measurements, conducted parallel to the filter sampling, enabled the determination of the jet engine oil contribution to the UFP mass.
Furthermore, the nucleation and particle formation potential of a commonly used synthetic jet engine lubrication oil was investigated in the laboratory. Thermodenuder experiments at 20 °C and 300 °C were carried out to monitor the gas-to-particle partitioning behaviour of jet engine oils. At 300 °C a significantly higher number of particles with a mean diameter of ~10 nm are formed, leading to a more than fivefold increase in total particle numbers compared to 20 °C. Particle diameters of the newly formed oil particles in the laboratory experiment appeared in the same size region as UFPs emerging from Frankfurt Airport. Particles originating from the Frankfurt city centre direction showed larger diameters.
Results indicate that aircraft emissions strongly influence the total mass of 10-18 nm particles. The jet oil fraction decreases for bigger particles (e.g., 18-56 nm), implying that these oils form new particles in the cooling exhaust gases of aircraft engines. In addition, non-target screening and in vitro bioassays on aviation-related PM2.5 filter samples were combined to provide indications for potential toxicologically relevant compounds in dependence of different wind directions and airport operations. Most recently, the applied non-target screening method was also used to identify seasonal variations in the organic aerosol composition in Beijing.
Regulatory required, classical toxicity studies for environmental hazard assessment are costly, time consuming, and often lack mechanistic insights about the toxic mode of action induced through a compound. In addition, classical toxicological non-human animal tests raise serious ethical concerns and are not well suited for high throughput screening approaches. Molecular biomarker-based screenings could be a suitable alternative for identifying particular hazardous effects (e.g. endocrine disruption, developmental neurotoxicity) in non-target organisms at the molecular level. This, however, requires a better mechanistic understanding of different toxic modes of action (MoA) to describe characteristic molecular key events and respective markers.
Ecotoxicgenomics, which uses modern day omic technologies and systems biology approaches to study toxicological responses at the molecular level, are a promising new way for elucidating
the processes through which chemicals cause adverse effects in environmental organisms. In this context, this PhD study was designated to investigate and describe MoA-characteristic
ecotoxicogenomic signatures in three ecotoxicologically important aquatic model organisms of different trophic levels (Danio rerio, Daphnia magna and Lemna minor).
Applying non-target transcriptomic and proteomic methodologies post chemical exposure, the aim was to identify robust functional profiles and reliable biomarker candidates with potential
predictive properties to allow for a differentiation among different MoA in these organisms. For the sublethal exposure studies in the zebrafish embryo model (96 hpf), the acute fish embryo toxicity test guideline (OECD 236) was used as conceptual framework. As different test compounds with known MoA, the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyrostatic 6-propyl-2-thiouracil (6-PTU), as well as six nerve- and muscle-targeting insecticides (abamectin, carbaryl, chlorpyrifos, fipronil, imidacloprid and methoxychlor) were evaluated. Furthermore, a novel sublethal immune challenge assay in early zebrafish embryos (48 hpf) was evaluated for its potential to assess immuno-suppressive effects at the gene expression level. Therefore, toxicogenomic profiles after an immune response inducing stimulus with and without prior clobetasol propionate (CP) treatment were compared. For the aquatic invertebrate D. magna, the study was performed with previously determined low effect concentrations (EC5 & EC20) of fipronil and imidacloprid according to the acute immobilization test in water flea (OECD 202). The aim was to compare toxicogenomic signatures of the GABA-gated chloride channel blocker (fipronil) and the nAChR agonist (imidacloprid). With similar low effect concentrations, a shortened 3 day version of the growth inhibition test with L. minor (OECD 221) was conducted to find molecular profiles differentiating between photosynthesis and HMG-CoA reductase inhibitory effects. Here, the biological interpretation of the molecular stress response profiles in L. minor due to the lack of functional annotation of the reference genome was particularly challenging. Therefore, an annotation workflow was developed based on protein sequence homology predicted from the genomic reference sequences.
With this PhD work, it was shown how transcriptomic, proteomic and computational systems biology approaches can be coupled with aquatic toxicological tests, to gain important mechanistic insights into adverse effects at the molecular level. In general, for the different investigated adverse effects for the different organisms, biomarker candidates were identified, which describe a potential functional link between impaired gene expressions and previously reported apical effects. For the assessed chemicals in the zebrafish embryo model, biomarker candidates for thyroid disruption as well as developmental toxicity targeting the heart and central nervous system were described. The biomarkers derived from nerve- and muscletargeting insecticides were associated with three major affected processes: (1) cardiac muscle cell development and functioning, (2) oxygen transport and hypoxic stress and (3) neuronal development and plasticity. To our knowledge, this is the first study linking neurotoxic insecticide exposure and affected expression of important regulatory genes for heart muscle (tcap, actc2) and forebrain (npas4a) development in a vertebrate model. The proposed immunosuppression assay found CP to affect innate immune induction by attenuating the response of genes involved in antigen processing, TLR signalling, NF-КB signalling, and complement activation ...
As one of the most widespread infectious diseases in the world, it is currently estimated that approximately 296 million people globally are chronically infected with Hepatitis B virus (HBV), the consequences of HBV infection cause more than 620,000 deaths each year. Although safe and effective HBV vaccines have reduced the incidence of new HBV infections in most countries, there are still around 1.5 million new infections each year. HBV remains a major health problem because there is no large-scale effective vaccination strategy in many countries with a high burden of disease, many people with chronic HBV infection are not receiving effective and timely treatment, and a complete cure for chronic infection is still far from being achieved.
Since its discovery, HBV has been identified as an enveloped DNA virus with a diameter of 42 nm. For efficient egress from host cells, HBV is thought to acquire the viral envelope by budding into multivesicular bodies (MVBs) and escape from infected cells via the exosome release pathway. It is clear that HBV hijacks the host vesicle system to complete self-assembly and propagation by interacting with factors that mediate exosome formation. Consequently, the overlap with exosome biogenesis, using MVBs as the release platform, raises the possibility for the release of exosomal HBV particles. Currently, virus containing exosomal vesicles have been described for several viruses. In light of this, this study explored whether intact HBV-virions wrapped in exosomes are released by HBV-producing cells.
First, this study established a robust method for efficient separation of exosomes from HBV virions by a combination of differential ultracentrifugation and iodixanol density gradient centrifugation. Fractionation of the density gradient revealed that two populations of infectious viral particles can be separated from the culture fluids of HBV-producing cells. The population present in the low-density peak co-migrates with the exosome markers. Whereas the population that appeared in the high-density fractions was the classical HBV virions, which are rcDNA-containing nucleocapsids encapsulated by the HBV envelope.
Subsequently, the characterization of this low-density population was performed, namely the highly purified exosome fraction was systematically investigated. Relying on the detergent sensitivity of the exosome membrane and the outer envelope of the HBV virus, disruption of the exosome structure by treatment with limited detergent revealed the presence of HBsAg in the exosomes. At the same time, mild and limited NP-40 treatment of highly purified exosomes and a further combination of density gradient centrifugation resulted in the stepwise release of intact HBV virions and naked capsids from the exosomes generated by HBV-producing cells. This implies the presence of intact HBV particles encapsulated by the host membrane.
The presence of exosome-encapsulated HBV particles was consequently also verified by suppressing the morphogenesis of MVBs or exosomes. Impairment of MVB- or exosome-generation with small molecule inhibitors has significantly inhibited the release of host membrane-encapsulated HBV particles as well. Likewise, silencing of exosome-related proteins caused a diminution of exosome output, which compromised the budding efficiency of wrapped HBV.
Moreover, electron microscopy images of ultra-thin sections combined with immunogold staining visualized the hidden virus in the exosomal structure. Additionally, the presence of LHBs on the surface of exosomes derived from HBV-expressing cells was also observed.
As expected, these exosomal membrane-wrapped HBV particles can spread productive infection in differentiated HepaRG cells. In HBV-susceptible cells, as LHBs on the membrane surface, this type of exosomal HBV appeared to be uptaken in an NTCP receptor-dependent manner.
Taken together these data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV. Exosomes hijacked by HBV act as a transporter impacting the dissemination of the virus.
Ischemic heart disease caused by occlusion of coronary vessels leads to the death of downstream tissues, resulting in a fibrotic scar that cannot be resolved. In contrast to the adult mammalian heart, the adult zebrafish heart can regenerate following injury, enabling the study of the underlying cellular and molecular mechanisms. One of the earliest responses that take place after cardiac injury in adult zebrafish is coronary revascularization. Previous transcriptomic data from our lab show that vegfc, a well-known regulator of lymphatic development, is upregulated early after injury and peaks at 96 hours post cryoinjury, coinciding with the peak of coronary endothelial cell proliferation. To test the hypothesis that vegfc is involved in coronary revascularization, I examined its expression pattern and found that it is expressed by coronary endothelial cells after cardiac damage. Using a loss-of-function approach to block Vegfc signaling, I found that it is required for coronary revascularization during cardiac regeneration. Notably, blocking Vegfc signaling resulted in a significant reduction in cardiomyocyte regeneration. Using transcriptomic analysis, I identified the extracellular matrix component gene emilin2a and the chemokine gene cxcl8a as effectors of Vegfc signaling. During cardiac regeneration, cxcl8a is expressed in epicardium-derived cells, while the gene encoding its receptor cxcr1 is expressed on coronary endothelial cells. I found that overexpressing emilin2a increases coronary revascularization, and induces cxcl8a expression. Using loss-of-function approaches, I observed that both cxcl8a and cxcr1 are required for coronary revascularization after cardiac injury.
Altogether, my findings indicate that Vegfc acts as an angiocrine factor that plays an important role in regulating cardiac regeneration in zebrafish. Mechanistically, Vegfc promotes the expression of emilin2a, which promotes coronary proliferation, at least in part by enhancing Cxcl8a-Cxcr1 signaling. This study helps in understanding the mechanisms underlying coronary revascularization during cardiac regeneration, with promising therapeutic applications for human heart regeneration.
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
Sensors for high rate charge particle tracking have to withstand the harsh radiation doses deposited by the particles to be sensed. This holds particularly for the novel CMOS Monolithic Active Pixel Sensors, which are considered a promising sensor technology for future vertex detectors due to their very light material budget and excellent spatial resolution. To resist the radiation doses expected close to the interaction regions of heavy-ion experiments, the sensors have to be hardened against radiation doses, which exceed the native tolerance of CMOS technology significantly. In this thesis, the results of non-ionizing radiation hardness studies at the IKF on sensor prototypes developed at the IPHC in Strasbourg are presented. Our results demonstrate that the CMOS sensors evaluated in the context of this thesis can withstand non-ionizing radiation of up to 5×10^14 neq/cm^2. This hardness qualifies them as promising candidates for use in future vertex detectors.
The stellar nucleosynthesis of elements heavier than iron can primarily be attributed to neutron capture reactions in the s and r process. While the s process is considered to be well understood with regards to the stellar sites, phases and conditions where it occurs, nucleosynthesis networks still need accurate neutron capture cross sections
with low uncertainties as input parameters. Their quantitative outputs for the isotopic abundances produced in the s process, coupled with the observable solar abundances, can be used to indirectly infer the expected r process abundances. The two stable gallium isotopes, 69Ga and 71Ga, have been shown in sensitivity studies to have considerable impact on the weak s process in massive stars. The available experimental data, mostly derived from neutron activation measurements for quasi-stellar neutron spectra at kBT = 25 keV, show disagreements up to a factor of three.
Determining the differential neutron capture cross section can provide input data for the whole range of astrophysically relevant energies. To that end, a neutron time of flight experimental campaign at the n_TOF facility at CERN was performed for three months, using isotopically enriched samples of both isotopes. The data taken at the EAR1 experimental area covered a wide neutron energy range from thermal to several hundred keV. The respective differential and spectrum averaged neutron capture cross sections for 69Ga and 71Ga were determined in this thesis. They show good agreement with the evaluated cross sections for 71Ga, but reproduce the deviations from the evaluated data that other, more recent activation measurements showed for 69Ga.
The main task of modern large experiments with heavy ions, such as CBM (FAIR), STAR (BNL) and ALICE (CERN) is a detailed study of the phase diagram of quantum chromodynamics (QCD) in the quark-gluon plasma (QGP), the equation of state of matter at extremely high baryonic densities, and the transition from the hadronic phase of matter to the quark-gluon phase.
In the thesis, the missing mass method is developed for the reconstruction of short-lived particles with neutral particles in their decay products, as well as its implementation in the form of fast algorithms and a set of software for prac- tical application in heavy ion physics experiments. Mathematical procedures implementing the method were developed and implemented within the KF Par- ticle Finder package for the future CBM (FAIR) experiment and subsequently adapted and applied for processing and analysis of real data in the STAR (BNL) experiment.
The KF Particle Finder package is designed to reconstruct most signal particles from the physics program of the CBM experiment, including strange particles, strange resonances, hypernuclei, light vector mesons, charm particles and char- monium. The package includes searches for over a hundred decays of short-lived particles. This makes the KF Particle Finder a universal platform for short-lived particle reconstruction and physics analysis both online and offline.
The missing mass method has been proposed to reconstruct decays of short-lived charged particles when one of the daughter particles is neutral and is not regis- tered in the detector system. The implementation of the missing mass method was integrated into the KF Particle Finder package to search for 18 decays with a neutral daughter particle.
Like all other algorithms of the KF Particle Finder package, the missing mass method is implemented with extensive use of vector (SIMD) instructions and is optimized for parallel operation on modern many-core high performance com- puter clusters, which can include both processors and coprocessors. A set of algorithms implementing the method was tested on computers with tens of cores and showed high speed and practically linear scalability with respect to the num- ber of cores involved.
It is extremely important, especially for the initial stage of the CBM experiment, which is planned for 2025, to demonstrate already now on real data the reliability of the developed approach, as well as the high efficiency of the current implemen- tation of both the entire KF Particle Finder package, and its integral part, the missing mass method. Such an opportunity was provided by the FAIR Phase-0 program, motivating the use in the STAR experiment of software packages orig- inally developed for the CBM experiment.
Application of the method to real data of the STAR experiment shows very good results with a high signal-to-background ratio and a large significance value. The results demonstrate the reliability and high efficiency of the missing mass method in the reconstruction of both charged mother particles and their neutral daughter particles. Being an integral part of the KF Particle Finder package, now the main approach for reconstruction and analysis of short-lived particles in the STAR experiment, the missing mass method will continue to be used for the physics analysis in online and offline modes.
The high quality of the results of the express data analysis has led to their status as preliminary physics results with the right to present them at international physics conferences and meetings on behalf of the STAR Collaboration.
In Europe, the sugar refinery is largely based on sugar beets. This route for obtaining household sugar results in a large amount of biomass waste, consisting mainly of the insoluble beet resi-dues, e.g., cell wall fragments. To a vast moiety this debris consists of the polymer pectin (up to 20% in the dry total solids). The structure of pectin is based on a backbone of D-galacturonic acid units (GalA), but also contains various other sugar monomers, predominantly L-arabinose, D-galactose, L-rhamnose and D-xylose. The amount of GalA adds up to a moiety of up to 70% with-in this sugar cocktail. So far, this debris is only fed to cattle or simply burnt. In nature, pectin is a common substrate for various organisms. The degradation of pectin-rich biomass is often per-formed by filamentous fungi like Hypocrea jecorina (also known as Trichoderma reesei) and As-pergillus niger, which evolved pectinases to degrade the pectin backbone and pathways to con-sume the monomer GalA as a sole carbon source. The fungal catabolism of pectin residues starts with the reduction of GalA to L-galactonate (GalOA) by a GalA-reductase. Even though filamen-tous fungi are native hosts of the GalA-catabolism and certain engineering approaches have al-ready been demonstrated, this class of organisms remains challenging with regard to bioreactor cultivation and tedious genetic accessibility. In contrast, the yeast S. cerevisiae is well known in fermentation processes and easily modified by a versatile set of genetic tools. So far, first ap-proaches have already been conducted to transfer the GalA utilization pathways into S. cerevisiae, but these approaches indicated limitations regarding GalA-uptake and redox cofac-tor replenishment due to the relatively high oxidative state of GalA compared to other sugars like glucose and galactose. Furthermore, the generally strongly increased demand for redox co-factors must be met by GalA reduction by finding new cofactor sources or redirecting reactions of the core metabolism.
This work aimed at the production of GalOA, which is the first intermediate of the fungal GalA catabolism. This compound shows an interesting range of potential applications, for instance as a food and cosmetic additive. To overcome the oxidized character of GalA, the presence of a more reduced co-substrate as a redox donor and as a carbon and energy source was required. To further enhance the reduction of GalA, modulation of the redox-cofactor supply and enzyme engineering were performed.
This work is about resumptive and non-resumptive relative clauses (RCs) in the three big Ibero-Romance languages: Spanish, Portuguese, and Catalan. In (1), the examined structures are exemplified for Spanish: (1a.) No conozco el hombre que viste _ ayer. “I don’t know the man that you saw yesterday.” (1b.) Es este el hombre que le enviaron el libro. “This is the man to whom they sent the book.” (1c.) Es este el hombre a quien le enviaron el libro. “This is the man to whom they sent the book.”
(1a.) displays a non-resumptive, or canonical, RC, which is characterized by the canonical use of a relativizing operator and a gap in the subordinate’s object position, a piece of evidence which has induced most of the generative literature to assume wh-movement of the relative operator in the sense of Chomsky (1977). The last two decades, however, have seen a big debate regarding the exact derivational analysis, starting with Kayne’s (1994) antisymmetry theory and the following focus on reconstruction and anti-reconstruction effects in RCs. This search for the correct starting site of the RC’s head noun has dismissed the original Head External Analysis (HEA) (Chomsky 1965, 1977) and led to the development of a Head Raising Analysis (RA) (Kayne 1994, Bianchi 1999, a.o.) and a Matching Analysis (MA) (Munn 1994, Sauerland 1998, a.o.). The discussion in this work argues that the data on reconstruction and anti-reconstruction effects are not sufficiently clear and reliable in order to adopt one of the head-internal analyses, i.e. a HEA or a MA. Instead, the work follows a variant of the HEA proposed for Portuguese by Rinke & Aßmann (2017), which adheres to standard assumptions about Romance syntax, and avoids the empirical problems that the other proposals have to face. Arguing that the HEA holds for all Ibero-Romance languages, this work also takes a stance in the debate around the categorical status of the relativizing element que and argues that it is always a D-element, and never of category C, i.e. there is no such thing as a relativizing complementizer (cf. also Kayne 2010, Kato & Nunes 2008, Poletto & Sanfelici 2018).
The work argues that wh-movement in a HEA fashion is the correct analysis also for resumptive relative clauses as in (1b., c.), which crucially lack a gap in argument position but show a resumptive pronominal element instead. Furthermore, it takes advantage of the fact that the choice of such genetically closely related languages like Spanish, Portuguese, and Catalan enables research to address the phenomenon under consideration from a microcomparative perspective, which is “the closest we can come … to a controlled experiment in comparative syntax” (Kayne 2005: 281-282). The descriptive literature suggests that, at least for Spanish and Catalan, there are two types of a resumptive RC structure available: a simple resumption as in (1b.), including mere que, and a complex resumption structure which displays a more complex relativizer like a quien in combination with a resumptive pronoun (1c.). However, a corpus study carried out for this work reveals that speakers of the three languages behave alike insofar as the only resumptive RC used in spontaneous speech is a simple-resumption structure, while complex resumption never occurs. Additionally, a multivariate analysis shows that in all three languages, grammatical case is the most important factor when it comes to the possibility of a resumptive structure in RCs: with a dative argument, simple resumption is obligatory, while for accusative and nominative arguments, resumption is optional. The discussion concludes that simple and complex resumption constitute different phenomena also on a structural level: the latter one is argued to be a subcase of clitic doubling, and therefore, receives an analysis along the lines of Pineda (2016), who argues against a dative alternation in Romance languages and locates the (non-)realisation of the dative clitic in a transitive clitic-doubling structure outside of syntax, it being a case of silent variation along the lines of Sigurðsson (2004) and Kayne (2005). From this perspective, it follows naturally that in Portuguese, complex resumption structures are ungrammatical. Simple resumption, on the other hand, which is a possible structure in all three languages, is argued to represent the phonological counterpart of “scattered deletion”, i.e. the preferred interpretation for an A’-chain according to Chomsky (2003): in the operator position SpecCP, every feature except for the operator feature is deleted, resulting in the phonological outcome que, while in the variable position, everything but the operator is interpreted, resulting in a pronominal element according to the argument’s phi-features.
This work takes a stance in the latest topics on generative analyses for relative clauses. Using not only theoretical considerations but conclusions drawn from empirical data on three languages, it offers a new perspective on pending questions and proposes to take a fresh look on supposedly outdated analyses.
Gait analysis as a clinical examination method has been increasingly used in recent years. In particular, the external knee adduction moment was often used as a surrogate measure for internal medial knee joint loading, e.g., in elderly individuals with medial knee osteoarthritis. Therefore, the knee adduction moment is also associated with the progression of knee osteoarthritis. Children and adolescents with valgus malalignment have been found to experience a reduced external knee adduction moment, but internal knee joint contact forces, particularly in the lateral compartment, were not previously studied.
First, medial and lateral knee joint contact forces were studied using muskulosceletal modeling in young individuals with and without valgus malalignment treated by guided growth. In addition, a systematic literature review was conducted to explore the relationship between external joint moments and internal joint contact forces. Finally, this relationship was investigated in children and adolescents with and without valgus malalignment. Furthermore, we examined whether statistical models could be determined to accurately predict internal knee joint contact forces by commonly used parameters from three-dimensional gait analysis, such as external knee joint moments.
It was found that guided growth normalized knee joint contact forces after treatment. In addition, the static radiographic mechanical axis angle correlated better after the treatment when the patients showed a typical limb alignment compared to the correlation before guided growth with the valgus malalignment due to compensating strategies during gait. Furthermore, the systematic review showed that the peak medial knee joint contact force was best predicted by the knee adduction moment and even better together with the knee flexion moment in the first half of stance. However, for the second half of stance of the medial knee joint contact force and the entire stance of the lateral knee joint contact force, only low correlations with knee adduction and/or flexion moment were found. Finally, statistical models could be determined with high accuracy for both medial and lateral knee joint contact force, for both peaks in the first and second half of stance, and for both study groups of children and adolescents with and without valgus malalignment by including knee adduction and flexion moment as predictors.
These results demonstrate the importance of examining not only the external knee adduction moment but also the knee flexion moment and, even better, the medial and lateral knee joint contact forces when evaluating knee joint loading. With these statistical models, clinicians can predict the medial and lateral knee joint contact forces without the need to perform musculoskeletal simulations and can therefore use standard three-dimensional gait analysis parameters such as knee adduction and flexion moment. This can improve guided growth treatment in children and adolescents with valgus malalignment with regard to implantation or explantation of the growth restricting plates or to rebound. Instrumented gait analysis could be particularly helpful in borderline cases, as kinematic compensation mechanisms during gait may play a role and the static radiograph alone does not provide information about dynamic joint loads.
Single-electron transport in focused electron beam induced deposition (FEBID)-based nanostructures
(2022)
Mit steigender Komplexität von integrierten Schaltungen im Nanometer-Maÿstab werden immer innovativere Techniken nötig, um diese zu fabrizieren. Dies erfordert einen starken Fokus auf die Kontrolle der Fabrikation akkurater Strukturen und der Materialreinheit, und dies im Zusammenhang mit einer skalierbaren Produktion. In diesem Kontext hat Elektronenstrahlinduzierte Abscheidung (engl. Focused Electron Beam Induced Deposition, FEBID) eine wachsende Aufmerksamkeit im Bereich der Nanostrukturierung gewonnen. Der FEBID-Prozess basiert auf der lokalen Abscheidung von Material auf einem Substrat. Das Deponat entsteht durch die Spaltung von Präkursor-Molekülen durch die Interaktion mit einem Elektronenstrahl entsteht. Als Beispiel sei hier der Präkursor Me3PtCpMe angeführt. Das auf dem Substrat abgelagerte Material besteht aus wenigen Nanometer großen Kristalliten aus Platin, welche in einer Matrix aus amorphem Kohlenstoff eingebettet sind. Die Pt-C FEBID Ablagerungen sind nano-granulare Metalle, deren elektrische Transporteigenschaften die Folge des Zusammenspiels von diffusivem Transport von Ladungen innerhalb der Pt-Kristalliten und temperaturabhängigen Tunneleffekten sind. Das größte Interesse an diesen Materialien liegt an der Möglichkeit, Strukturen für technische Anwendungen im Nanometerbereich herstellen zu können.
In dieser Arbeit wurden Anwendungen, die auf Einzelelektroneneffekten beruhen, ausgewählt, um die FEBID basierte Probenpräparation zu testen. Um Einzelelektronentransport zu ermöglichen, der auf dem Tunneln einzelner Elektronen basiert, müssen alle Parameter wie Grösse und Abstände der Strukturen genauestens definiert sein. Im Rahmen dieser Arbeit wurden Einzelelektronenbausteine entwickelt, die auf zwei unterscheidlichen Anwendungen des Pt-C FEBID-Prozesses basieren. Die beiden Anwendungen sind: 1) Arrays von Gold-Nanopartikeln (Au-NP), welche mittels Pt-Strukturen kontaktiert wurden, die mit FEBID präpariert und anschlieÿend aufgereinigt wurden; 2) Einzelelektronentransistoren (engl. Single-Electron Transistors, SET), deren Inseln aus elektronennachbestrahlten Pt-C FEBID Deponate bestehen. Die elektrischen Eigenschaften der präparierten Nanostrukturen wurden charakterisiert und mit der erzielten Auflösung und Materialqualität in Relation gesetzt. Es wurden Optimierungen an der Präparationsmethode durchgeführt, welche direkt die Leitfähigkeit des Pt-C FEBID-Materials erhöhen. Dies kann durch die Änderung der
Karbonmatrix oder die Erhöhung des metallischen Gehalts der Struktur geschehen. In dieser Arbeit wurde eine katalytische Aufreinigungsmethode von Pt-C FEBID Strukturen für zwei Anwendungen genutzt: zum Einen wurden die aufgereinigten Strukturen als Keimschichten für die nachfolgende ortsgenaue Atomlagenabscheidung (engl. Area-Selective Atomic Layer
Deposition, AS-ALD) von Pt-Dünnschichten genutzt. Zum Anderen wurde diese Technik dafür genutzt, Metallbrücken zwischen den bereits durch Auftropfen zufällig auf dem Substrat aufgebrachten NP-Gruppen und den zuvor aufgebrachten UV-Lithographie (UVL) präparierten Cr-Au Kontakten zu erzeugen. Eine NP-Gruppe ist ein periodisches, granulares Array von Partikeln, welche uniform in Größe und Form sind und einen unterschiedlichen Grad von Ordnung inne haben. Durch die Art des Aufbringens kann die Anordnung der Nanopartikel durch Lösen und Erzeugen der Verbindungen beeinflusst werden. Diese Systeme zeigen ein Verhalten wie Tunnelkontakte mit Coulombblockade und eine Verteilung der Schwellspannung. Die Ergebnisse der elektrischen Messungen bestätigen den Einzelelektronentransport durch die Nanopartikel in einem typischen Elektronentransportregime mit schwacher Kopplung. Trotz dieser Ergebnisse war die Anwendung dieser Technik für die SET Nanostrukturierung nicht erfolgreich. Die Ursache
konnte zurückgeführt werden auf das Vorhandensein von Pt-Partikeln in der Nähe der Kontakte zu den Au-NP-Arrays. Die Pt-Partikel sind durch den FEBID Fertigungsprozess in
der Nähe der vorgegebenen Struktur entstanden. Aus diesem Grund wurde das FEBID Co-Deponat in der folgenden SET-Nanofabrikation entfernt.
Ein SET basiert auf einer Nano-Insel, welche durch Tunnelkontakte mit Source- und Drain-Elektroden verbunden ist. Darüber hinaus besteht eine kapazitive Verbindung zu einer
oder mehreren Gate-Elektrode(n). Innerhalb der Insel gibt es eine feste Anzahl von Elektronen.
In dieser Arbeit wurden die Source-, Drain- und Gate-Kontakte durch Ätzen mittels eines fokussierten Gallium-Strahls erzeugt, was Abstände von 50nm ermöglichte, wohingegen die SET Insel mit Pt-C FEBID-Material erzeugt wurde. Die Leitfähigkeit der Insel aus Pt-C wurde mit anschließender Elektronenbestrahlung erhöht. Als letzter Präparationsmethode wurde ein neueartiges Argon-Ätzverfahren genutzt, um die durch FEBID erzeugten Co-Ablagerungen in der direkten Umgebung der Insel zu entfernen. Durch die Elektronennachbestrhalung kann die Kopplung der einzelnen metallischen Kristalliten angepasst werden. Die Auswirkungen unterschiedlicher starker Tunnelkontakte auf die elektronischen Eigenschaften der Insel und die daraus resultierende Performanz des SETs wurden in dieser Arbeit beobachtet ...
Folgend auf den ersten Realisierungen von Bose-Einstein Kondensaten erschienen weitere innovative Experimente, die sich in den optischen Gittern gefangenen Quantengasen widmeten. In diesen zahlreichen, wissenschaftlichen Untersuchungen konnten die Eigenschaften von Bose-Einstein Kondensaten besser verstanden werden. Das Prinzip von Vielteilchensystemen, gefangen in einem periodischen Potential, bot eine Plattform zur Untersuchung weiterer Quantenphasen.
Eine konzeptionell einfache Modifikation von solchen Systemen erhält man durch die Kopplung der Grundzustände der gefangenen Teilchen an hoch angeregten Zuständen mithilfe einer externen Lichtquelle. Im Falle dessen, dass diese Zustände nahe der Ionisationsgrenze des Atoms liegen, spricht man von Rydberg-Zuständen und Atome, welche zu diesen Zuständen angeregt werden, bezeichnet man als Rydberg-Atome. Eines der vielen charakteristischen Eigenschaften von Rydberg-Atomen ist die Fähigkeit über große Entfernungen jenseits der atomaren Längenskalen zu wechselwirken. Im Rahmen von Vielteilchensystemen wurden dementsprechend Kristallstrukturen aus gefangenen Rydberg-Atomen experimentell beobachtet.
Nun stellt sich die Frage, was mit einem gefangenen Bose-Einstein Kondensat passiert, dessen Teilchen an langreichweitig wechselwirkenden Zuständen gekoppelt sind. Gibt es ein Parameterregime, in dem sowohl Kristallstruktur als auch Suprafluidität in solchen Systemen koexistieren können? Dies ist die zentrale Frage dieser Arbeit, die sich mit der Theorie von gefangenen Quantengasen gekoppelt an Rydberg-Zuständen auseinandersetzt.
This thesis is concerned with the study of symmetry breaking phenomena for several different semilinear partial differential equations. Roughly speaking, this encompasses equations whose symmetries are not necessarily inherited by their solutions, which is particularly interesting for ground state solutions.
Die vorliegende Dissertation stellt eine Methode zur Löslichkeitsbestimmung vor, die für die Anwendung im Rahmen von BCS-Biowaiver Monografien entwickelt wurde. Der Methode und dem dafür konzipierten Studienprotokoll liegt das Prinzip der „Minimallöslichkeit“ zugrunde. Damit lässt sich einfach, kosteneffizient und wissenschaftlich verlässlich feststellen, ob ein Arzneistoff „hochlöslich“ gemäß den BCS-Biowaiver Richtlinien der Gesundheitsbehörden FDA, EMA und WHO ist und sich dementsprechend generische Produkte des Arzneistoffs grundsätzlich für das BCS-Biowaiver Zulassungsverfahren eignen.
Dieses Verfahren für die Zulassung von Generika erlaubt die Beurteilung der Bioäquivalenz eines festen generischen Arzneimittels zur peroralen Anwendung auf Basis von in vitro-Freisetzungsuntersuchungen anstatt von in vivo-Studien wie z.B. pharmakokinetischen Studien am Menschen und erleichtert dadurch eine Marktzulassung sowohl durch Zeit- als auch Kosteneinsparung. Die Anwendung des Verfahrens ist von Vorteil, um die Verfügbarkeit von qualitativ hochwertigen, generischen (und damit kostengünstigen) Arzneimitteln zu erhöhen. Dies ist besonders wünschenswert für die Verfügbarkeit von gemäß der Weltgesundheitsorganisation essenziellen Arzneistoffen und unter denen gerade von solchen, die zur Bekämpfung von Krankheiten mit nur wenigen und/oder teuren therapeutischen Alternativen benötigt werden.
Entstanden ist die Löslichkeitsbestimmungsmethode im Rahmen von zwei Projekten, die beide zu diesem Ziel einer guten globalen Gesundheitsversorgung beitragen: die Erstellung der Biowaiver Monografien von Proguanilhydrochlorid (ein Malaria-Prophylaktikum) und Cefalexinmonohydrat (ein Antibiotikum aus der Gruppe der Cephalosporine) setzt die Publikationsreihe „Biowaiver Monograph Series“ der FIP Focus Group „Bioclassification/Biowaiver“ fort. Jede Monografie gibt eine umfassende wissenschaftliche Empfehlung zur Eignung eines Wirkstoffs der WHO „Model List of Essential Medicines“ und seiner generischen Produkte für das BCS-Biowaiver Verfahren hinsichtlich aller regulatorisch geforderten Aspekte ab. Proguanilhydrochlorid (BCS Klasse III – „hochlöslich“ und nicht „hoch permeabel“) und Cefalexinmonohydrat (BCS Klasse I – „hochlöslich“ und „hoch permeabel“) sind beide für dieses Zulassungsverfahren geeignet.
Im Zuge des anderen Projektes wurde die Löslichkeit und anschließend die BCS Klasse von Wirkstoffen bestimmt, die der 16. und 17. Version der WHO „Model List of Essential Medicines“ neu hinzugefügt wurden. Neun von 16 untersuchten Wirkstoffen, die in feste, perorale Arzneimittel formuliert werden können, sind im Hinblick auf ihre BCS Klasse für das eine Zulassung per BCS-Biowaiver geeignet. Eine umfangreichere Empfehlung könnte im Rahmen einer Biowaiver Monografie gegeben werden.
Die experimentelle Bestimmung der Löslichkeit über einen pH-Wert-Bereich von 1-6,8 war essenzieller Bestandteil beider Projekte, da Literaturdaten zur Löslichkeit der Wirkstoffe nicht oder nur unvollständig vorlagen. Die entwickelte Methode basiert auf einer im Kleinmaßstab angesetzten „Shake-Flask“-Methode zur Bestimmung der thermodynamischen Löslichkeit, wird jedoch in einem Zeitrahmen von 24 Stunden durchgeführt. Sie nutzt die höchste Dosis der Wirkstoffe als Substanzmenge, um zu bestimmen, ob dieser „hochlöslich“ gemäß den BCS-Biowaiver Richtlinien ist oder nicht. Die Methode bzw. das dazugehörige Studienprotokoll beinhalten Empfehlungen zu den einzelnen Schritten der Durchführung, der Auswahl der Medien und Herausforderungen wie Präzipitation (Fallbeispiel: Proguanilhydrochlorid) und Zersetzungsreaktionen (Fallbeispiel: Cefalexinmonohydrat). Löslichkeitsdaten, die mit dieser Methode erhoben werden, können für eine Zulassung per BCS-Biowaiver bei den Gesundheitsbehörden eingereicht werden, aber auch für ein Vorab-Screening genutzt werden, dass „hochlösliche“ Arzneistoffe aus einer Vielzahl von Substanzen herauszufiltern soll, um nähere Untersuchungen im Rahmen einer Biowaiver Monografie anzuschließen.
Machine learning (ML) techniques have evolved rapidly in recent years and have shown impressive capabilities in feature extraction, pattern recognition, and causal inference. There has been an increasing attention to applying ML to medical applications, such as medical diagnosis, drug discovery, personalized medicine, and numerous other medical problems. ML-based methods have the advantage of processing vast amounts of data.
With an ever increasing amount of medical data collection and large, inter-subject variability in the medical data, automated data processing pipelines are very much desirable since it is laborious, expensive, and error-prone to rely solely on human processing. ML methods have the potential to uncover interesting patterns, unravel correlations between complex features, learn patient-specific representations, and make accurate predictions. Motivated by these promising aspects, in this thesis, I present studies where I have implemented deep neural networks for the early diagnosis of epilepsy based on electroencephalography (EEG) data and brain tumor detection based on magnetic resonance spectroscopy (MRS) data.
In the project for early diagnosis of epilepsy, we are dealing with one of the most common neurological disorders, epilepsy, which is characterized by recurrent unprovoked seizures. It can be triggered by a variety of initial brain injuries and manifests itself after a time window which is called the latent period. During this period, a cascade of structural and functional brain alterations takes place leading to an increased seizure susceptibility.
The development and extension of brain tissue capable of generating spontaneous seizures is defined as epileptogenesis (EPG).
Detecting the presence of EPG provides a precious opportunity for targeted early medical interventions and, thus, can slow down or even halt the disease progression. In order to study brain signals in this latent window, animal epilepsy models are used to provide valuable data as it is extremely difficult to obtain this data from human patients. The aim of this study is to discover biomarkers of EPG using animal models and then to find the equivalent and counterparts in human patients' data. However, the EEG features for EPG are not well-understood and there is not a sufficiently large amount of annotated data for ML-based algorithms. To approach this problem, firstly, I utilized the timestamp information of the recorded EEG from an animal epilepsy model where epilepsy is induced by an electrical stimulation. The timestamp serves as a form of weak supervision, i.e., before and after the stimulation. Secondly, I implemented a deep residual neural network and trained it with a binary classification task to distinguish the EEG signals from these two phases. After obtaining a high discriminative ability on the binary classification task, I proposed to divide further the time span after the stimulation for a three-class classification, aiming to detect possible stages of the progression of the latent EPG phase. I have shown that the model can distinguish EEG signals at different stages of EPG with high accuracy and generalization ability. I have also demonstrated that some of the learned features from the network are clinically relevant.
In the task of detecting brain tumors based on MRS data, I first proposed to apply a deep neural network on the MRS data collected from over 400 patients for a binary classification task. To combat the challenge of noisy labeling, I developed a distillation step to filter out relatively ``cleanly'' labeled samples. A mixing-based data augmentation method was also implemented to expand the size of the training set. All the experiments were designed to be conducted with a leave-patient-out scheme to ensure the generalization ability of the model. Averaged across all leave-patient-out cross-validation sets, the proposed method performed on par with human neuroradiologists, while outperforming other baseline methods. I have demonstrated the distillation effect on the MNIST data set with manually-introduced label noise as well as providing visualization of the input influences on the final classification through a class activation map method.
Moreover, I have proposed to aggregate information at the subject level, which could provide more information and insights. This is inspired by the concept of multiple instance learning, where instance-level labels are not required and which is more tolerant to noisy labeling. I have proposed to generate data bags consisting of instances from each patient and also proposed two modules to ensure permutation invariance, i.e., an attention module and a pooling module. I have compared the performance of the network in different cases, i.e., with and without permutation-invariant modules, with and without data augmentation, single-instance-based and multiple-instance-based learning and have shown that neural networks equipped with the proposed attention or pooling modules can outperform human experts.
To this day, stroke is the leading cause of death and disability worldwide. Due to increasing age of the world population and poor lifestyle, the incidence is further rising. Besides mechanical thrombectomy as a surgical option, there is a lack of therapeutic options with recombinant tissue plasminogen activator (rt-PA) being the only approved drug for treatment for ischemic stroke. However, there are various problems that make the administration of rt-PA difficult. In particular, it can only be given for ischemic (not hemorrhagic) stroke, and there is a narrow time frame of 4.5 hours after onset of stroke, in which it can be successfully applied. While the success rates of combined thrombectomy with rt-PA are around 60%, less than 5% of patients receive this therapy.
ß-Hydroxybutyrate (BHB) is a ketone body that is formed in high amounts during fasting and lipolysis. Ketone bodes and the ketogenic diet have been shown to have neuroprotective properties in neurodegenerative diseases. In prior work of our group, the ketogenic diet was shown to have beneficial effects in mice after transient ischemia. In the present work, a single dose of BHB was tested for beneficial effects. For this purpose, microdialysis was used to demonstrate that BHB can cross the blood-brain barrier. For the next series of experiments, transient cerebral ischemia was induced in mice for 90 minutes by unilaterally occluding the middle cerebral artery (MCAO) with a silicone-covered filament. Behavioral tests one day after BHB administration showed that the moderate dose of 30 mg/kg, given immediately after reperfusion, improved the neurological score significantly whereas a lower (10 mg/kg) and a higher dose (100 mg/kg) had no effects The main part of the experiments focused on mitochondrial respiration as a potential mechanism of action for BHB. In isolated mitochondria from mouse brain, BHB (1-10 mM) was able to stimulate mitochondrial respiration stronger than pyruvate, but not as strong as succinate.. In the following experiments, MCAO was induced in vivo, and mitochondria were isolated and investigated ex vivo. Experiments were conducted 60 minutes, 24 hours, 72 hours, and 7 days after cerebral ischemia and reperfusion. Besides mitochondrial respiration (normalized to mitochondrial protein content or citrate synthase activity), several other parameters were monitored: the development of bodyweight throughout the experiment, citrate synthase activity, plasma metabolites and behavior to assess motor functions. Three behavioral tests were conducted: first, the Corner test, an experiment for measuring the extent of unilateral movement. Here, if a stroked mouse is put into a narrow corner (30°), it is most likely to turn unilaterally to the right, whereas an unimpaired mouse will turn to both sides randomly. From a total of 10 turns, a laterality index was calculated. Second, in the Chimney test, the mouse walks heads first into a tube. Once it reaches the end, the tube is tipped 90 degrees to stand on the table vertically. Motorically impaired animals have difficulties crawling backwards up to the top of the tube. The experiment was stopped if an animal did not reach the top of the tube within 60 seconds. Third, in the Rotarod test, the mouse is placed on a rotating beam on which it is supposed to walk for at least 60 seconds, and the time when the animal falls off the rotating tube is measured.
All animals that had undergone ischemia showed massive weight loss until 72 hours after reperfusion. Weight loss then stagnated and there was a trend of increasing weight 7 days after reperfusion. The behavioral analysis showed that 24 hours after reperfusion, BHB-treated animals performed significantly better in the Corner test, meaning their moving patterns were more heterogeneous than those of saline-treated animals and in the Chimney test. 72 hours after reperfusion, BHB-treated animals still performed significantly better in the Chimney test, but 7 days after reperfusion, the performances of BHB- and saline-treated animals were no longer different from each other in any of the behavioral tests. In separate experiments, the plasma metabolites glucose, lactate, and pyruvate were changed in the animals that had undergone ischemia but were not affected by BHB administration.
Mitochondrial respiration was tested at four time points after the administration of BHB after reperfusion – 60 minutes, 24 hours, 72 hours, and 7 days after transient cerebral ischemia. 60 minutes later, data showed an increase of oxygen consumption of the complexes I and II. OxPhos was also increased but the effect at this point, did not reach statistical significance. 24 hours after reperfusion, this effect was consolidated: complex I, complex II and OxPhos respiration were significantly improved in the BHB-treated group compared to saline...
This thesis examines the referential properties of prenominal possessive modifiers in Serbian. The focus of the investigation is on the configurations that have been claimed to violate Binding Principles B and C: lexical or pronominal possessives modifying a noun in subject position binding a pronoun or an R-expression in object position. Such constructions have been claimed to be ungrammatical in Serbian due to the alleged adjectival status of the possessive and its respective syntactic position as NP-adjoined (Despić 2013).
The present thesis takes up the ongoing debate about the categorial status of Serbian possessives as adjectives or determiners. Based on several arguments, such as word order, binding of anaphora, coordination, and the fact that they are typically represented by either nouns or pronouns, it is concluded that possessives rather behave like full noun phrases than adjectives. Therefore, I analyse possessives as DPs from a categorial point of view.
In a second step, the syntactic position of the possessives within the Serbian noun phrase has been investigated. Based on theoretical arguments (cf. Bašić 2004) and empirical evidence, I propose a structural position that would accommodate the binding facts and the referential possibilities in these configurations. In line with Kayne (1994), Bernstein and Tortora (2005) and Alexiadou et al. (2007), I assume that possessives occupy SpecAgrP in Serbian, where they move from their base position (SpecPossP). Thirdly, I question the (im)possibility of coreference with possessives in comparison to ‘typical’ binding constructions without possessives by providing empirical evidence from three experimental studies, showing that coreference between possessive modifiers and objects is indeed available in Serbian.
The results from Experiment 1 (a picture selection task) have shown that coreference between a lexical possessive and a (clitic or strong) pronoun is allowed in Serbian. Further, there is a tendency that the coreferential reading is preferred with clitics, while the disjoint reference is preferred with strong pronouns. The fact that coreference is possible, does not necessarily mean that it is always available as the only interpretation, but can be influenced by other (pragmatic) factors. The same is observed in Experiments 2 and 3 as coreference was chosen between pronominal possessives modifying a noun in subject position and R-expressions but rejected between pronouns and R-expressions in a forced-choice task, suggesting a structural difference – no c-command – in the former case. The results from the self-paced reading task corroborate this finding.
Importantly, the experimental results provide evidence that possessive configurations are not violating Binding Principles B and C. This implies that Serbian possessive constructions do not c-command out of the noun phrase, as predicted by the proposed syntactic analysis.
The findings from all experiments contribute to the bigger picture concerning the nature and behaviour of Serbian possessives and cast doubt on the cross-linguistic DG/AG parameter. Instead, the theoretical arguments and the empirical results from the experiments rather speak for a parallel structure of possessive noun phrases in Serbian and English and ultimately in favour of the Universal DP Hypothesis.
The present work deals with photoionization in the realm of the absorption of one single photon. The formal treatment of one-photon ionization usually employs a semi-classical approach, where the electron’s initial and final states are described as quantum-mechanical wave functions but the photon is treated as a classical electromagnetic wave. In the calculation of photoionization cross sections with this semi-classical method, there is an often used approximation which is called the electric dipole approximation. Mathematically, the application of the dipole approximation corresponds to truncating the series expansion of an exponential after the leading term. Physically, this means neglecting the linear photon momentum and the spatial dependence of the light field. The dipole approximation is valid if the wavelength of the light is much larger than the spatial extent of the target and if the photon momentum is small compared to the momenta of the reaction products, which is generally the case for photon energies short above the electron binding energy.
For the present work, we experimentally investigated nondipolar photoionization, i.e., one-photon ionization at high photon energies where the dipole approximation breaks down. In our experiments, we irradiated single atoms and molecules with such high-energetic photons and measured the three-dimensional momentum distributions of the reaction fragments to uncover the effects of the linear photon momentum and the spatially-dependent light field on photoionization. Our observations allow the first profound insight into photoionization that reveals all photon properties, i.e., photon energy, spin, linear momentum, and the speed of light. Hopefully, our efforts make a constructive contribution to the understanding and the further exploration of light-matter interaction.
Since Vietnamese is an isolating language, word order plays an important role in identifying the function of a particular word. Yet in some contexts word order may be flexible especially in the case of special information-structural settings. Discontinuous noun phrases constitute a specific case of non-canonical word order in Vietnamese.
I have conducted two read-speech experiments in order to find out whether there are prosodic or intonational effects in a comparison between continuous and discontinuous noun phrases in Vietnamese. In the first experiment, speakers from the Northern dialect were recorded and in the second experiment speakers from the Southern dialect. The results showed prosodic differences in the two word order conditions in both dialects. The duration of the classifier is significantly longer (p<0.001, ANOVA calculation) in the case of discontinuous noun phrases and the rising tone (sắc) is clearly articulated as rising. In the case of continuous noun phrases, the duration of the classifier is significantly shorter (p<0.001, ANOVA calculation) and a classifier with rising tone may lose its rising property. These prosodic effects are related to prosodic boundaries. In the case of discontinuous noun phrases, the classifier constitutes the prosodic boundary, whereas with continuous noun phrases, the (right) prosodic boundary occurs further to the right.
I assume that in Vietnamese there is generally a correspondence between syntactic and prosodic structure as in Selkirk (2011) and Féry (2017).
This means that for example the DP hai trái cam ‘two oranges’ (two CLF orange) is matched by a prosodic phrase, thus (hai trái cam)Φ. However, when the noun cam ‘orange’ is separated from the numeral-classifier complex, the noun and the classifier form a prosodic phrase on their own: (hai trái)Φ. It can thus be concluded that intonation effects in Vietnamese are not only present when expressing sentence modality and when changing the role of function words (Đỗ et al. 1998 and Hạ & Grice 2010), but they also play a role in word order change, as in discontinuous nominal phrases.
When it comes to syntactic aspects of discontinuous noun phrases, I discuss whether split constructions in Vietnamese involve movement as proposed by Trịnh (2011) or base-generation as put forward by Fanselow & Féry (2006). I argue for base-generation analysis since the second part of a discontinuous NP (remnant) may also occur outside of discontinuous noun phrases without its head noun and some discontinuous noun phrases do not have a continuous counterpart. My study confirms the connection between syntax and prosody.
The two parts of the discontinuous noun phrase form their own phrases syntactically as well as prosodically.
Acute myeloid leukemia (AML) is one of the most frequently occurring and fatal types of leukemia. Initiated by genetic alterations in hematopoietic stem and progenitor cells, rapidly proliferating cancer cells (leukemic blasts) infiltrate the bone marrow and damage healthy hematopoiesis. Subgroups of AML are defined by underlying molecular and cytogenetic abnormalities, which are decisive for treatment and prognosis. For AML patients that can be intensively treated, the first line treatment remains a combination of cytarabine and anthracycline, which was developed in the 1970s. While this treatment regimen clears the disease and reinstates normal hematopoiesis (complete remission, CR) in 60% to 80% of patients below the age of 60, CR rates in patients above the age of 60 are only 40% to 50%. Relapse and refractory disease are the major cause of death of AML patients, despite large efforts to improve risk-adjusted post-remission therapy with further chemotherapy cycles and, if possible, allogeneic bone marrow transplantation. Elderly patients are particularly difficult to treat because of age-related comorbidities and because their disease tends to relapse more often than the disease of younger patients. Thus, the cure rates of AML vary with age, with 5-year survival rates of about 50% in young patients, and less than 20% in patients above the age of 65 years. With the median age of AML patients being 68 years, the need for novel therapeutic options is immense. The recent approval of eight new agents (venetoclax, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, gemtuzumab ozogamicin and CPX-351 (liposomal cytarabine and daunorubicin)) has added considerably to the therapeutic armamentarium of AML and has increased cure rates in specific subgroups of AML. However, the high heterogeneity among patients, clonal evolution and commonly occurring drug resistance, which cause the high relapse rates, remain a substantial problem in the treatment of AML. Therefore, a better understanding of currently used therapeutics and further development of novel therapeutics is urgently needed.
In recent years, attention has increasingly focused on therapeutic strategies to interfere with the metabolic requirements of cancer cells. The last three decades have provided extensive insights into the diversity and flexibility of AML metabolism. AML cells use different sources of nutrients compared to normal hematopoietic progenitor cells and reprogram their metabolic pathways to fulfill their exquisite anabolic and energetic needs. As a result, they develop high metabolic plasticity that enables them to thrive in the bone marrow microenvironment, where oxygen and nutrient availability are subject to constant change.
Cancer cells, specifically AML cells, have a strong dependency for the amino acid glutamine. Glutamine serves in energy production, redox control, cell signaling as well as an important nitrogen source. The only enzyme capable of de novo glutamine synthesis is glutamine synthetase (GS). GS catalyzes glutamine production from glutamate and ammonium. In AML, the metabolic role and dependency of GS is poorly understood. Here, we investigated the effects of GS deletion on AML growth, and its functional relevance in AML metabolism. Genetic deletion of GS resulted in a significant decrease of cell growth in vitro, and impaired leukemia progression in vivo in a xenotransplantation mouse model. Interestingly, the dependency of AML cell growth on GS was shown to be independent of its functional role in glutamine synthesis. Glutamine starvation did not increase the dependency of the AML cells on GS, nor did increased glutamine availability rescue the GS-knockout-associated growth disadvantage. Instead, functional studies revealed the role of GS in the detoxification of ammonium. GS-deficient cells showed elevated ammonium secretion as well as a higher sensitivity towards the toxic metabolite. Exogenous provision of 15N-labeled ammonium was detoxified by GS-driven incorporation into glutamine. Studies on cells that had gained resistance to GS-knockout-mediated growth inhibition indicated enzymes involved in the urea cycle and the arginine biogenesis pathway to compensate for a loss of GS. Together, these findings unveiled GS as an important ammonium scavenger in AML.
Clinical studies on AML patients revealed increased ammonium concentrations in the blast-infiltrated bone marrow compared to peripheral blood. In line with this finding, proteome and transcriptome analysis of AML blasts showed a significant upregulation of GS in AML compared to healthy progenitors, further indicating its importance in ammonium detoxification.
Analyzing pathways that contribute to ammonium production revealed protein uptake followed by amino acid catabolism as a yet not identified mechanism supporting AML growth. Protein endocytosis and subsequent proteolytic degradation were shown to rescue AML cells from otherwise growth-inhibiting glucose or amino acid depletion. Furthermore, protein metabolization led to the reactivation of the mammalian target of rapamycin (mTOR) signaling pathway, which was deactivated upon leucine and glutamine depletion, revealing protein consumption as an important alternative source of amino acids in AML.
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The book deals with a comprehensive constellation of narrative and visual, often counterposed representations of the causes, course, and results of the assault on the Palace of Justice of Colombia by a guerrilla commando and the immediate counterattack launched by state security forces on November 6, 1985, as well as with the local memorial traditions in which the production, circulation and reproduction of these representations have taken place between 1985 and 2020. The research on which it is based was grounded in the method and perspective of classical anthropology, in as much as qualitative fieldwork and the search for the perspective of the actors involved have played a central role. Within that context, memory entrepreneurs belonging to diverse sectors, from the far-right to the human rights movement, were followed through multisited fieldwork in various locations of Colombia, as well as in various countries of America and Europe. The analyses of fieldwork data, documental sources, and visual representations that constitute the core of the argument are framed in the field of memory studies and mainly based on theoretical and methodological resources from Pierre Bourdieu’s Field Theory, Jeffrey Alexander’s theory of social trauma, and Ernst Gombrich’s characterization of iconological analysis.
The book is composed of four chapters preceded by an introduction and followed by the conclusions and documental appendices, and substantiates three main theses. The first is that the Palace of Justice events were a radio- and television-broadcasted dispersed tragedy that affected the lives of actors from different social sectors and regions of Colombia, who have launched since 1985 multiple memorial initiatives in different fields of culture, thereby contributing to the formation and intergenerational transmission of a widespread cultural trauma. The second is that the narrative and visual representations at the core of that trauma express a vast universe of local representational traditions that can be traced at least until the early 20th century, and therefore preexists the so-called Colombian “memory boom”, dated to the mid-1990s. As an example of the preexistence and longstanding impact of these traditions, the local usage of the figure of “holocaust” for representing the effects of politically motivated violence is analyzed regarding the Palace of Justice events, but also traced to other representations emerged in the decade of 1920. The third thesis is that analyzing the diverse, frequently counterposed accounts of political violence elaborated within these traditions provides an opportunity to explore a wide variety of understandings of the causes and characteristics of the longstanding Colombian social and armed conflict.
Keywords: Political violence, Cultural trauma, Collective Memory, Iconology, Holocaust, Colombia.
Characteristics of critical incident reporting systems in primary care: an international survey
(2022)
Aim: The aim of the study was to support the development of future critical incident reporting systems (CIRS) in primary care by collecting information on existing systems. Our focus was on processes used to report and analyse incidents, as well as strategies used to overcome difficulties.
Methods: Based on literature from throughout the world, we identified existing CIRS in primary care. We developed a questionnaire and sent it to operators of a purposeful sample of 17 CIRS in primary care. We used cross-case analysis to compare the answers and pinpoint important similarities and differences in the CIRS in our sample.
Results: Ten CIRS operators filled out the questionnaire, and 9 systems met the inclusion criteria. The sample of CIRS came from 8 different countries and was rather heterogeneous. The reporting systems invited a broad range of professions to report, with some also including reports by patients. In most cases, reporting was voluntary and conducted via an online reporting form. Reports were analysed locally, centrally, or both. The various CIRS used interesting ideas to deal with barriers. Some, for example, used confidential reporting modes as a compromise between anonymity and the need for follow-up investigations, whereas others used smartphone applications and call centres to speed up the reporting process.
Conclusion: We found multiple CIRS that have operated in primary care for many years, have received a high number of reports and were largely developed in accordance with recommendations found in literature. Although primary care in Germany differs from other countries, these CIRS could serve as an inspiration for CIRS in German primary care.
We present new results on nonlocal Dirichlet problems established by means of suitable spectral theoretic and variational methods, taking care of the nonlocal feature of the operators. We mainly address: First, we estimate the Morse index of radially symmetric sign changing bounded weak solutions to a semilinear Dirichlet problem involving the fractional Laplacian. In particular, we derive a conjecture due to Bañuelos and Kulczycki on the geometric structure of the second Dirichlet eigenfunctions. Secondly, we study a small order asymptotics with respect to the parameter s of the Dirichlet eigenvalues problem for the fractional Laplacian. Thirdly, we deal with the logarithmic Schrödinger operator. In particular, we provide an alternative to derive the singular integral representation corresponding to the associated Fourier symbol and introduce tools and functional analytic framework for variational studies. Finaly, we study nonlocal operators of order strictly below one. In particular, we investigate interior regularity properties of weak solutions to the associated Poisson problem depending on the regularity of the right-hand side.
The thesis is composed of four Chapters.
In the first Chapter, the boundary expression of the one-sided shape derivative of nonlocal Sobolev best constants is derived. As a simple consequence, we obtain the fractional version of the so-called Hadamard formula for the torsional rigidity and the first Dirichlet eigenvalue. An application to the optimal obstacle placement problem for the torsional rigidity and the first eigenvalue of the fractional Laplacian is given.
In the second Chapter, we introduce and prove a new maximum principle for doubly antisymmetric functions. The latter can be seen as the first step towards studying the optimal obstacle placement problem for the second fractional eigenvalue. Using the new maximum principle we derive new symmetry results for odd solutions to semilinear Dirichlet boundary value problems with Lipschitz nonlinearity.
In the third Chapter, we derive new integration by parts formula for the fractional Laplace operator with a general globally Lipschitz vector field and in particular, we obtain a new Pohozaev type identity generalizing the one obtained by X. Ros-Oton and J. Serra. As an application we obtain nonexistence results for semilinear Dirichlet boundary problems in bounded domains that are not necessarly starshaped.
In the last Chapter, we study symmetry properties of second eigenfunctions of annuli. Using results from the first Chapter and the maximum principle in Chpater 2, we extend the result on the optimal obstacle placement problem from the first eigenvalue to the second eigenvalue.
Acute myeloid leukemia (AML) is a neoplastic disease of an early myeloid precursor cell in hematopoiesis. It leads to the accumulation of monoclonal cells in the bone marrow and the peripheral blood, showing a differentiation block and deregulated self-renewal. Frequently, the leukemic cells exhibit genetic aberrations with reciprocal chromosomal translocations. These translocations induce the formation of a fusion protein, that can lead to new cellular functions and a transformation into a leukemic cell. Common chromosomal translocation in AML are t(8;21) or t(15;17), which cause the formation of the fusion proteins AML1/ETO and PML/RARα and determine the leukemic phenotype of the AML.
The translocation t(6;9) leads to the formation of the fusion protein DEK/CAN and is of special interest, because of its association with mostly young patients and a very aggressive course of the disease. The fusion product induces leukemia in a small subset of hematopoietic stem cells, but its mechanism of leukemogenesis is greatly unknown.
The intention of this work was to characterize the DEK/CAN-induced AML on a molecular genetic level to gain a deeper understanding of the disease pathogenesis. Therefore, gene expression analysis with polymerase chain reaction (PCR) and microarray analysis was performed.
To detect DEK/CAN in different cell lines by PCR and real-time quantitative PCR (qPCR), specific primers and probes were designed, and a standardized workflow was established. Emphasis was placed on the optimization of RNA isolation, DNase treatment, cDNA synthesis with following PCR and qPCR, which enabled the detection of the fusion product DEK/CAN in the cell lines 32B, Phoenix and FKH-1. To quantify the fusion product DEK/CAN, the method of qPCR with absolute and relative quantification was used. Absolute quantification enabled the calculation of an exact copy number of the fusion transcript DEK/CAN with a detection limit of 50 copies/µl at a sensitivity of 10-6, which is of importance in determining the minimal residual disease (MRD) of patients with DEK/CAN-positive AML. MRD detection by qPCR is a highly sensitive diagnostic method to identify leukemic cells, even in low cell counts. This enables a thorough evaluation of the treatment response and allows an early detection of changes in the MRD level as part of the remission control.
Additionally, a microarray gene expression analysis was performed to identify alterations in relevant target genes and associated signaling pathways in DEK/CAN-positive cells.
Because of DEK/CAN’s potential to induce leukemia in a subset of hematopoietic stem cells, Sca+/Lin- cells of the bone marrow of C57Bl/6 mice were used and transfected with the gene products DEK/CAN and PML/RARα. Microarray analysis led to the identification of 16 different genes of interest, which demonstrated significant alterations of gene expression in DEK/CAN-positive cells. They were validated and quantified with TaqMan assay assisted qPCR. The elevated expression of the transcription factors TRIM25, HIF1α and ATF2, in DEK/CAN-positive cells, indicated an altered transcription factor activity and interaction with DNA in the nucleus. The localization of DEK/CAN in the nucleus emphasizes this assumption. Also, the upregulated expression of the nuclear export receptor XPO1 suggested changes in nuclear transport processes and impaired export activity in DEK/CAN-positive cells.
Furthermore, the results demonstrated changes of gene expression in genes that are involved in the JAK/STAT signaling pathway. PTPRC, the Protein Tyrosine Phosphatase Receptor Type C, functions as a direct inhibitor of JAKs (Janus Kinases) and STATs (Signal Transducers and Activators of Transcription) and their associated signaling pathway.
It was shown that the gene expression of PTPRC was significantly reduced in DEK/CAN-positive cells. This allowed the assumption, that the reduced expression of PTPRC led to a loss of inhibition and thus a consecutive hyperactivation of the JAK/STAT signaling pathway. This hypothesis was supported by an independent activation of PIM1, a target gene of STAT5 and the activation of LMO2, a direct target gene of JAK2. In addition, the transmembrane receptor CSF1R, which is directly involved in STAT activation, also showed an upregulation in gene expression.
The results of this work show an activation of the JAK/STAT signaling pathway in DEK/CAN-positive cells, which may be a key mechanism in DEK/CAN-induced leukemogenesis.
Considering treatment options in the future, the addition of targeted therapy, such as pan-JAK inhibitors, to the standard therapy, could be a chance to improve the overall survival rate and the prognosis of t(6;9)-positive AML.
This work describes the development and characterization of two instruments and their data evaluation, which contributes to a better understanding of new particle formation and growth, as well as their interactions with clouds. Both instruments were characterized at the Cosmics Leaving Outdoor Droplets (CLOUD) experiment at the European Center for Nuclear Research (CERN).
The expansion of actors and instruments in sovereign debt markets through bond financing generated a coordination problem among bondholders during the debt restructuring process. There is a risk that an individual bondholder will be passive or act against the restructuring slowing down or even precluding the process of restructuring even though it is in the general interest of bondholders as a group, not to mention the population of the country experiencing the shortage of funds for public welfare. In particular, the disruptions to sovereign debt restructuring by frivolous litigation is considered as one of the main threats.
This dissertation is the first major study devoted to sovereign bonds structured through a trust arrangement and the promising features that such a legal structure possesses for an effective and efficient sovereign debt restructuring. It provides a comprehensive inquiry into the evolution of the mechanisms to coordinate creditors, with a focus on bondholders and institutional frameworks which facilitated this coordination. It examines intriguing primary sources from League of Nations archives and provides in-depth case studies on the functionality of the trustees in sovereign bond restructurings performed by Argentina in 2016 and Ecuador in 2008.
Assessing the utility of trust arrangements to address coordination problems, this thesis is driven by the puzzle: How to better balance (i) the need for smooth sovereign debt restructurings, which by definition entails some losses for creditors, with (ii) bondholders’ legitimate interests? What approach can be used in constructing a legal and institutional framework for trustees to promote the best interest of the bondholders in sovereign debt restructuring? As a solution, it seems that incentives for bond trustees to pursue debt sustainability will achieve both goals.
In this regard, recognition of the concept of debt sustainability, being in substance the IMF and WB debt sustainability assessment, as the best interest of bondholders in sovereign debt restructuring is beneficial from multiple aspects. It enables a bond trustee to excel in its role as a guardian of bondholders by following the best interest of bondholders in exercising its discretion. Moreover, it fosters an equilibrium between the interests of private creditors and a state taking into account its socio-political aspects.
Facial expression recognition is linked to clinical and neurofunctional differences in autism
(2022)
Background: Difficulties in social communication are a defining clinical feature of autism. However, the underlying neurobiological heterogeneity has impeded targeted therapies, and requires new approaches to identifying clinically relevant bio-behavioural subgroups. In the largest autism cohort to date, we comprehensively examined difficulties in facial expression recognition, a key process in social communication, as a bio-behavioural stratification biomarker, and validated them against clinical features and neurofunctional responses.
Methods: Between 255 and 488 participants aged 6-30 years with autism, typical development and/or mild intellectual disability completed the Karolinska Directed Emotional Faces task, the Reading the Mind in the Eyes Task and/or the Films Expression Task. We first examined mean-group differences on each test. Then we used a novel intersection approach that compares two centroid and connectivity-based clustering methods to derive subgroups based on the combined performance across the three tasks. Measures and subgroups were then related to clinical features and neurofunctional differences measured using fMRI during a fearful face-matching task.
Results: We found significant mean-group differences on each expression recognition test. However, cluster analyses showed that these were driven by a low-performing autistic subgroup (~30% of autistic individuals who performed below 2SDs of the neurotypical mean on at least one test), while a larger subgroup (~70%) performed within 1SD on at least 2 tests. The low-performing subgroup also had on average significantly more social-communication difficulties and lower activation in the amygdala and fusiform gyrus than the high-performing subgroup.
Limitations: Findings of autism expression recognition subgroups and their characteristics require independent replication. This is currently not possible, as there is no other existing data set that includes all relevant measures. However, we demonstrated high internal robustness (91.6%) of findings between two clustering methods with fundamentally different assumptions, which is a critical pre-condition for independent replication.
Conclusions: We identified a subgroup of autistic individuals with expression recognition difficulties and showed that this related to clinical and neurobiological characteristics. If replicated, expression recognition may serve as bio-behavioural stratification biomarker and aid in the development of targeted interventions for a subgroup of autistic individuals.
Protein biosynthesis is a fundamental process across all domains of life. Polypeptides are produced by translating the genetic information of the messenger RNA (mRNA) into amino acids. This elaborate procedure is divided into the four distinct phases: initiation, elongation, termination, and ribosome recycling. The phases are controlled and regulated by a multitude of translation factors. During initiation, the ribosome assembles on the mRNA. Initiation factors (IFs) bind to the small ribosomal subunit (SSU) and assist the recruitment of mRNA and initiator transfer RNA (tRNA), which delivers the first amino acid methionine. After positioning the SSU at the start codon of the mRNA, additional IFs support the joining of the large ribosomal subunit (LSU). Next, elongation factors (EFs) deliver amino-acylated tRNAs (aa-tRNAs) to the translating ribosome and assist kinetic proofreading and ribosome subunit translocation after the catalytic transfer of the polypeptide onto the aa-tRNA. When a stop codon is reached, translation is terminated by release factors (RFs) that hydrolyze the peptidyl-tRNA to release the nascent protein chain. Afterwards, the ribosome is recycled in Eukaryotes and Archaea by the conserved and essential factor ABCE1, which splits the ribosome into the LSU and SSU. ABCE1 remains bound to the SSU forming the post-splitting complex (post-SC). mRNA translation closes into a cycle by recruitment of IFs to the post-SC and the start of a new round of initiation. The post-SC presents the platform for translation initiation. However, the role of ABCE1 in initiation remains elusive. Therefore, the main goal of my thesis was to unravel the molecular mechanism of ABCE1 on the post-SC and during initiation complex (IC) assembly.
Using a reconstituted system, the high-resolution structure of the archaeal post-SC was solved by cryogenic electron microscopy (cryo-EM) following the native splitting route. It was the first complete model of an archaeal SSU at atomic resolution and revealed a previously undescribed ribosomal protein, which we termed eS21. The hinge 2 region of ABCE1 was identified to be the major interaction interface that anchors to the SSU. Functional characterization of single residue mutations in hinge 2 unraveled essential interactions with the ribosomal RNA backbone of the SSU. Sensing of SSU-binding was found to be allosterically transmitted to the nucleotide-binding sites (NBSs) for integration into the ATPase cycle of ABCE1.
Reconstitution of the archaeal translation apparatus allowed for dissection of IC assembly in the presence of ABCE1. Three different ICs were resolved by cryo-EM. The results were in accordance with recent structural findings of eukaryotic translation initiation and highlighted that the involvement of ABCE1 is conserved.
In a semi-native approach, recombinant ABCE1 was pulled-down from crenarchaeal cell lysates. Mass spectrometric analysis of co-immunoprecipitated ribosomal complexes identified the association of numerous translation factors to the post-SC in a cellular context. The establishment of the genetic toolbox of the acidothermophilic Sulfolobus acidocaldarius allowed the homologous expression of ABCE1. Pull-down of native ABCE1 revealed similar ribosomal complexes as the semi-native and reconstituted approaches. Together, my results gave first physiological relevance of ABCE1 involvement in mRNA translation initiation in Archaea. Native archaeal ABCE1-ICs were vitrified for structural analysis by cryo-EM. Thereby, future structural analysis will allow to analyze the interactions of ABCE1 on native ICs and identify its role in IC assembly.
To address the molecular process of IC assembly, the binding affinity of aIF1 to the SSU was determined by fluorescence polarization. Similar studies will allow for a detailed functional analysis on IF recruitment to the SSU in presence of ABCE1.
mRNA surveillance and ribosome-associated quality control (RQC) mechanisms evolved to ensure cell viability. The pathways overcome ribosome stalling and defective translation components. Stalled ribosomes are terminated by special RFs, which do not hydrolyze the peptidyl-tRNA, but allow dissociation of the ribosome by ABCE1. Faulty messages are degraded via mRNA decay pathways and the LSU is rescued by RQC factors. Recently, the bacterial RQC factor MutS2 was identified to specifically target collided di- and polysomes but its molecular mechanism remains unknown. In this thesis, initial functional analyses showed tri-phosphate specific nucleotide binding of MutS2. While the dissociation of collided disomes by MutS2 could not be observed, the results pave the way for future in vitro studies of bacterial RQC factors acting on specific ribosome populations.
In the future, mRNA translation research must focus on complex quality control processes to comprehensively understand this fundamental cellular process in a holistic context.
As part of two drilling campaigns of the International Continental Scientific Drilling Program (ICDP), several geophysical borehole measurements were carried out by the Leibniz Institute for Applied Geophysics (LIAG) in two lakes. The acquired data was used to answer stratigraphic and paleoclimatic research questions, including the establishment of robust age-depth models and the construction of continuous lithological profiles.
Lake Towuti is located on Sulawesi (Indonesia), within the "Indo-Pacific Warm Pool" (IPWP), a globally important region for atmospheric heat and moisture budgets. The lake exists for approximately one million years, but its exact age is uncertain. We present the first agedepth model for the approximately 100 m continuous sediment sequence from the central part of the lake. The basis for this model is the magnetic susceptibility measured in the borehole and a tephra layer with an age of about 797 ka at 72 m depth. Our age-depth model is inferred from cyclostratigraphic analysis of borehole data and covers a period from 903 ± 11 to 131 ± 67 ka. We suggest that orbital eccentricity and/or changes between global cold and warm periods are responsible for hydroclimatic changes in the IPWP, that these changes affect sedimentation processes in Lake Towuti, and that we can measure and observe this effect in the sediment properties today. Additionally, we created a continuous artificial lithological profile from a series of different borehole data using cluster analysis. This provides information from parts of the borehole where no sediment is available due to core loss.
Lake Ohrid is 1.36 million years old and is located on the Balkan Peninsula on the border between Albania and North Macedonia. The primary hole 'DEEP' in the central part of the lake has been the subject of several investigations, but information about sediments of the marginal locations 'Pestani' and 'Cerava' have not been published yet. In our study, we use natural gamma radiation (GR) measured in the borehole to generate an age-depth model for DEEP. This is performed using the correlation of GR to the global LR04 reference record of Lisiecki and Raymo (2005).
The age information is then transferred via prominent seismic marker horizons to the other two sites, Pestani and Cerava, where it provides the first age-control points for the construction of age-depth models from correlation of GR to LR04. The generated age-depth models are tested using cyclostratigraphic methods, but the limits of this approach are revealed. At DEEP, sedimentation rates (SR) from the cyclostratigraphic method and the correlative approach differ by 2.8 %, at Pestani this difference is 16.7 %, and at Cerava the quality of the data does not allow a reliable evaluation of SR using the cyclostratigraphic approach. We used cluster analysis to construct artificial lithological profiles at all three sites and integrated them into the respective age-depth models. This enables us to determine which sediment types were deposited at what time, and we recognize the change between warm and cold periods in the sediment properties at all three locations. The analyses in this study were all performed on borehole and seismic data and thus do not involve sediment core data. Especially at Pestani and Cerava, new insights into the sedimentological history of Lake Ohrid could be obtained.
In the last part we discuss the occurrence of the half-precession (HP) signal in the European region during the last one million years. The focus is on Lake Ohrid, but a range of other proxies, from the eastern Mediterranean, across the European continent, up to Greenland are analyzed in regards to HP. Applying filters, we focus on the frequency range with a period of 13-8.5 ka and only HP remains in the records. We use correlative methods to determine the clarity of the HP signal in proxies distributed across the European realm. Additionally, we determined the development of HP over time. The HP signal is clearest in the southeast and decreases toward the north. It is further more pronounced in interglacial periods and in the younger part (<621 ka) of most proxies. We suggest that there are mechanisms that transmit the HP signal from its origin near the equator to higher latitudes via different processes. In this context, for instance, the African monsoon, the Nile River and the Mediterranean outflow via the Strait of Gibraltar can be important factors.
People can describe spatial scenes with language and, vice versa, create images based on linguistic descriptions. However, current systems do not even come close to matching the complexity of humans when it comes to reconstructing a scene from a given text. Even the ever-advancing development of better and better Transformer-based models has not been able to achieve this so far. This task, the automatic generation of a 3D scene based on an input text, is called text-to-3D scene generation. The key challenge, and focus of this dissertation, now relate to the following topics:
(a) Analyses of how well current language models understand spatial information, how static embeddings compare, and whether they can be improved by anaphora resolution.
(b) Automated resource generation for context expansion and grounding that can help in the creation of realistic scenes.
(c) Creation of a VR-based text-to-3D scene system that can be used as an annotation and active-learning environment, but can also be easily extended in a modular way with additional features to solve more contexts in the future.
(d) Analyze existing practices and tools for digital and virtual teaching, learning, and collaboration, as well as the conditions and strategies in the context of VR.
In the first part of this work, we could show that static word embeddings do not benefit significantly from pronoun substitution. We explain this result by the loss of contextual information, the reduction in the relative occurrence of rare words, and the absence of pronouns to be substituted. But we were able to we have shown that both static and contextualizing language models appear to encode object knowledge, but require a sophisticated apparatus to retrieve it. The models themselves in combination with the measures differ greatly in terms of the amount of knowledge they allow to extract.
Classifier-based variants perform significantly better than the unsupervised methods from bias research, but this is also due to overfitting. The resources generated for this evaluation are later also an important component of point three.
In the second part, we present AffordanceUPT, a modularization of UPT trained on the HICO-DET dataset, which we have extended with Gibsonien/telic annotations. We then show that AffordanceUPT can effectively make the Gibsonian/telic distinction and that the model learns other correlations in the data to make such distinctions (e.g., the presence of hands in the image) that have important implications for grounding images to language.
The third part first presents a VR project to support spatial annotation respectively IsoSpace. The direct spatial visualization and the immediate interaction with the 3D objects should make the labeling more intuitive and thus easier. The project will later be incorporated as part of the Semantic Scene Builder (SeSB). The project itself in turn relies on the Text2SceneVR presented here for generating spatial hypertext, which in turn is based on the VAnnotatoR. Finally, we introduce Semantic Scene Builder (SeSB), a VR-based text-to-3D scene framework using Semantic Annotation Framework (SemAF) as a scheme for annotating semantic relations. It integrates a wide range of tools and resources by utilizing SemAF and UIMA as a unified data structure to generate 3D scenes from textual descriptions and also supports annotations. When evaluating SeSB against another state-of-the-art tool, it was found that our approach not only performed better, but also allowed us to model a wider variety of scenes. The final part reviews existing practices and tools for digital and virtual teaching, learning, and collaboration, as well as the conditions and strategies needed to make the most of technological opportunities in the future.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
This work ties in with the investigation of the intermediate valent states and valence fluctuations in certain europium based intermetallic systems. Valence fluctuations are a property of the electronic system of a compound that is possibly accompanied by structural effects, which, in some cases, are quite noticable. By assuming how the changes in the electronic system and in the crystal lattice are connected, valence _uctuations of europium are believed to be a possible probe for the theory of quantum critical elasticity, which is investigated on by the SFB TRR 288 (Frankfurt, Mainz, Karlsruhe, Bochum, Dresden).
Here, the proceedings in growing single crystals of di_erent compounds related to this _eld of research are reported. This includes the ThCr2Si2 (122) type compounds EuPd2Si2 as well as the doping series EuPd2(Si1-xGex)2, the Europium based ternary Phosphides EuFe2P2, EuCo2P2, EuNi2P2 and EuRu2P2, and attempts to grow compounds of a derived 1144 structure by ordered substitution of half the Europium, EuKRu4P4.
The largest part of this work focusses on the EuPd2Si2 system, which exhibits intermediate valent europium and a temperature dependent transition between two di_erent intermediate valent states of europium. Crystals of this system were grown using the Czochralski method with a levitating melt and an europium excess flux after a two step prereaction process. Also, explorations of a PdSi-rich flux and external flux methods are reported. Ten Czochralski grown experiments, in six generations iteratevely seeded by the previous generation, were prepared.
Thermodynamical and structural analyses of the crystals located the transition between the di_erent intermediate valent states of europium between 140K and 165 K, transitioning from a high temperature Eu2.3+ state to a low temperature Eu2.7+ state, and classified it as a second order transition. To this transition a lattice anomaly of the a-parameter collapsing about 2% is connected, while the c-parameter remains largely unaffected. Large differences between individual samples can be explained by combining thermodynamical and structural analyses with compositional analysis, revealing the valence transition temperature as strongly dependent on the sample composition and Pd-Si site interchanges.
Searching to change the character of the valence transition to first order, silicon was substituted by germanium to introduce negative pressure. Germanium substituted samples of EuPd2(Si1-xGex)2 were grown using the Czochralski method with the optimized parameters from the growth experiments for the undoped compound. Samples were prepared with a nominal substitution of x = 0.05, x = 0.10, x = 0.15, x = 0.20 (twice) and x = 0.30. For the EuPd2(Si1-xGex)2 system, a phase diagram for the europium valence states is derived from chemical and thermodynamical characterizations.
n ternary europium phosphides EuT2P2, the position of the compounds in the generalized phase diagram and the question of long range magnetic order or valence transition appear connected to an isostructural transition of the tetragonal crystal structure, drastically decreasing the length of the c-parameter while establishing covalent bonds between phosphorus atoms of different interlayers of the structure, the so called ‚collapse‘. While EuFe2P2, EuT2P2 and EuCo2P2 display both long range magnetic order and a non-collapsed crystal structure, EuNi2P2 shows both a valence transition between two intermediate valent states at a characteristic temperature of 36K - accompanied by a small lattice anomaly of the a-parameter shrinking about 0.2% - and a collapsed crystal structure. Samples of EuFe2P2, EuCo2P2 and EuNi2P2 were grown in tin flux and using solid-solid sintering approaches.
Single crystals of EuFe2P2, EuCo2P2 and EuRu2P2 were investigated at ESRF in Grenoble with single crystal X-ray di_ractometry on a pressure range up to 15GPa and at temperatures down to 15K to investigate the nature of the structural transitions in the compounds. While in EuCo2P2 the structural transition occurs as a transition of first order at all temperatures (e.g. at 2GPa for 15 K), in EuFe2P2 and EuRu2P2 the structural collapse evolves over a broad pressure range up to 8GPa and as a transition of second order troughout the temperature ranges, albeit seeming to sharpen at lower temperatures. From the crystallographic data, elastic constants of the compounds could be derived, revealing EuFe2P2 and EuRu2P2 as unexpectedly elastic materials.
In order to probe the structural collapse at more accessible pressures, crystals with a sturcture derived from the 122 structure, but with ordered 50% substitution of europium and hence altering the symmetry from I4/mmm to P4/mmm in a 1144 structure, were exploratively pursued. Different experiments to obtain EuAT4P4 (with A = K, Rb, Cs and T = Fe, Ru) from binary or ternary prereactants or directly from the elements remained largely unsuccessful.
Locomotion, the way animals independently move through space by active muscle contractions, is one of the most apparent animal behaviors. However, in many situations it is more beneficial for animals to actively prevent locomotion, for instance to briefly stop before reorienting with the aim of avoiding predators, or to save energy and recuperate from stress during sleep. The molecular and cellular mechanisms underlying such locomotion inhibition still remain elusive. So, the aim of this study was to utilize the practical genetic model organism Caenorhabditis elegans to efficiently tackle relevant questions on how animals are capable of suppressing locomotion.
Nerve cells, mostly called neurons, are known to control locomotion patterns by activating some and inhibiting other muscle groups in a spatiotemporal manner via local secretion of molecules known as neurotransmitters. This study particularly focuses on whether neuropeptides modulate such neurotransmission to prevent locomotion. Neuropeptides are small protein-like molecules that are secreted by specific neurons and that act in the brain by activating G protein-coupled receptors (GPCRs) expressed in other target neurons. They can act as hormones, neuromodulators or neurotransmitters. DNA sequences coding for neuropeptides and their cognate receptors are similar across diverse species and thus indicate evolutionary conservation of their molecular signaling pathways. This could potentially also imply that regulatory functions of specific neuropeptides are also similar across species and are thus meaningful to unravel more general mechanisms for instance underlying locomotion inhibition.
Specifically, we find that the modulatory interneuron RIS constitutes a dedicated stop neuron of which the activity is sufficient to initiate rapid locomotion arrest in C. elegans while maintaining its body posture. Similar to its known function in larval sleep, RIS requires RFamide neuropeptides encoded by the flp 11 gene for this activity, in addition to GABA. Furthermore, we find that spontaneous calcium activity transients in RIS are compartmentalized and correlated with locomotion stop. These findings illustrate that a single neuron can regulate both stopping and sleeping phenotypes.
Secondly, we show that C. elegans RPamide neuropeptides encoded by nlp-22 and nlp-2 regulate sleep and wakefulness, respectively. We unexpectedly find that these peptides activate gonadotropin-releasing hormone (GnRH)-like receptors dose dependently and we highlight their sequence resemblance to other bilaterian GnRH-like neuropeptides. In addition, we show that these receptors are expressed in distinct subsets of neurons that are associated with motor behavior. Finally, we show that nlp 22 encoded peptides signal through GNNR 6 receptors to regulate larval sleep and that nlp 2 encoded peptides require both GNRR 3 and GNRR 6 receptors to promote wakefulness.
In sum, we find that locomotion inhibition in C. elegans is regulated by multiple, but evolutionary conserved RFamide and GnRH-like RPamide neuropeptidergic signaling pathways.
We study the polarization of relativistic fluids using the relativistic density operator at global and local equilibrium. In global equilibrium, a new technique to compute exact expectation values is introduced, which is used to obtain the exact polarization vector for fields of any spin. The same result has been extended to the case of massless fields. Furthermore, it is demonstrated that at local equilibrium not only the thermal vorticity but also the thermal shear contribute to the polarization vector. It is shown that assuming an isothermal local equilibrium, the new term can solve the polarization sign puzzle in heavy ion collisions.
Heart development is a dynamic process modulated by various extracellular and intracellular cues. Cardiac progenitors in vertebrates such as the zebrafish, migrate over to the midline after differentiation from the epiblast (Bakkers, 2011; Rosenthal & Harvey, 2010; Stainier et al., 1996; Trinh & Stainier, 2004). These progenitors form a cardiac disc at the midline which elongates into the linear heart tube. The differentiation and migration of cardiac precursors is modulated by signaling interactions between cardiac precursor cells and their extracellular environment known as the Extracellular Matrix (ECM). Studies have shown that Cell-ECM interactions play a crucial role in sculpting the heart during early morphogenic events (Davis CL, 1924; Männer & Yelbuz, 2019; Rosenthal & Harvey, 2010). One key factor to these processes is the presence of a specialized ECM known as the Basement Membrane (BM). Extracellular basement membrane proteins such as Fibronectin have been shown to modulate these very early migration processes of the cardiomyocyte progenitors (Trinh & Stainier, 2004). As the heart develops further, the linear heart tube is composed of myocardial cells with an inner endothelial cell lining separated by a layer of thick jelly like substance called the cardiac jelly (Barry A, 1948; Davis CL, 1924; Little et al., 1989). The cardiac jelly also called the cardiac basement membrane, has been shown to regulate distinct developmental events during cardiogenesis. This early CJ contains components of the basal lamina such as laminins, fibronectin, hyaluronan as well as non-fibrillar collagens such as Collagen IV (Little et al., 1989). In this study, I aimed to identify ECM molecules of the Basement Membrane in the heart and identify their role in the modulation of cardiac development and regeneration using the zebrafish as my model organism.
I identified genes belonging to the Zebrafish Matrisome expressed during cardiac developmental and regeneration and performed CRISPR/Cas9 sgRNA mediated mutagenesis. I also developed overexpression tools for these genes.
Agrinp168 mutants exhibited no obvious gross morphology defects during cardiac development and were adult viable. Adult mutants exhibited reduced cardiomyocyte proliferation, but no significant difference in cardiomyocyte dedifferentiation post cardiac cryoinjury.
Decorin overexpression through mRNA injections led to increased myocardial wall thickness and DN dcn overexpression through mRNA injections led to loss of cardiac looping during early development.
Mutants for Small Leucine Rich Proteoglycan (SLRP) prelp generated using CRISPR/Cas9 mutagenesis exhibited cardiovascular defects. Close observation of prelp mutant hearts revealed a reduced heart rate and impaired fractional shortening of the ventricle. prelp mutants exhibited an enlarged atrium at 48 hpf and 72 hpf as well as a reduced ventricle size at 72 hpf. Chamber size in the mutant hearts were enlarged irrespective of contractility of the heart. Mutants showed an increased number of Atrial cardiomyocytes, but no change in cell size. On the molecular level, extracellular Laminin localization was disrupted in prelp mutants along with an increase in thickness and volume of the cardiac HA in the CJ suggesting a potential compensatory role, or retention of immaturity of the cardiac jelly in the prelp mutants. Transcriptomics analysis on the prelp mutant hearts revealed downregulation of ECM organization and ECM-Receptor interaction processes in the mutants. Gene Ontology analysis on prelp mutants hearts transcriptome revealed increased MAPK signaling. Interestingly, genes related to degradation of cardiac HA and maturation of cardiac jelly were downregulated, and genes related to epithelial identity of cardiomyocytes were upregulated. Analysis of the mutant hearts at single cell resolution revealed increased number of mutants exhibiting rounded up cardiomyocytes and loss of apical Podocalyxin. Truncated forms of prelp were generated to identify domain specific roles for Prelp, and reintroduction of N-terminal truncated Prelp into the mutants rescued the basal lamina localization and cardiac jelly volume phenotypes. Myocardium specific re-establishment of prelp expression revealed a marked rescue of the mutant cardiovascular phenotype suggesting that tissue specific expression of prelp is not required so long as Prelp is secreted into the CJ. With these data, I’ve elucidated the role of ECM SLRPs in modulation of cardiac chamber morphogenesis process and regeneration of the heart.
In order to understand the origin of the elements in the universe, one must understand the nuclear reactions by which atomic nuclei are transformed. There are many different astrophysical environments that fulfill the conditions of different nucleosynthesis processes. Even though great progress has been made in recent decades in understanding the origin of the elements in the universe, some questions remain unanswered. In order to understand the processes, it is necessary to measure cross sections of the involved reactions and constrain theoretical model predictions. A variety of methods have been developed to measure nuclear reaction cross sections relevant for nuclear astrophysics. In this thesis, two different experiments and their results, both using the well-established activation method, are presented.
A measurement of the proton capture cross section on the p-nuclide 96Ru was performed at the Institute of Structure and Nuclear Astrophysics ISNAP - Notre Dame, USA. The main goal of this experiment was to compare the results with those obtained by Mei et al. in a pioneering experiment using the method of inverse kinematics at the GSI Helmholtzzentrum für Schwerionenforschung GmbH - Darmstadt, Germany. Therefore, the activations were taken out at the same center of mass energies of 9 MeV, 10 MeV and 11 MeV. Another activation was taken out at an energy of 3.2 MeV to compare the result to a measurement of Bork et al. who also used the activation method. While the results at 3.2 MeV agree quite well with those of Bork et al., the results at higher energies show significantly smaller cross sections than those measured by Mei et al.. Experimental details, the data analysis and sources of uncertainties are discussed.
The second part of this thesis describes a neutron capture cross section experiment. At the Institut für Kernphysik - Goethe Universtität Frankfurt an experimental setup allows to produce quasi maxwell-distributed neutron fields to measure maxwell-averaged cross sections (MACS) relevant for s-process nucleosynthesis. The setup was upgraded by a fast electric linear guide to transport samples from the activation to the detection site. The cyclic activation of the sample allows to increase the signal-to-noise ratio and to measure neutron captures that lead to nuclei with
half-lives on the order of seconds. In a first campaign, MACS of the reactions 51V(n,γ), 107,109Ag(n,γ) and 103Rh(n,γ) were measured. The new components of the setup aswell as the data analysis framework are described and the results of the measurements are discussed.
Tinnitus is a symptom experienced by most people at least once in their lifetime. In most documented cases, a new onset of chronic tinnitus can be chronologically correlated with hearing loss. However, tinnitus can also occur in people with (apparently) normal hearing and remains without a traceable preceding cause. Despite the frequency of occurrence of tinnitus, the pathophysiological mechanisms are still not fully understood. A currently proposed hypothesis focuses on a "hidden" hearing loss called synaptopathy as a pathomechanism of tinnitus in normal hearing subjects. In the present study, the objective was to test whether finestructure audiometry or measurement of otoacoustic emissions can reveal possibly overlooked hearing impairment in presumed normalhearing individuals with chronic tinnitus. Thus, a hearing loss not audiologically detectable by the usual methods would supplement or replace the presumed synaptopathic pathomechanism. Another objective was to attempt to replicate the existing findings of another research group on synaptopathy as cause for tinnitus in normal hearing people. Schaette and McAlpine (2011) were able to demonstrate a significant difference in wave I amplitudes between groups of normal hearing subjects with and without chronic tinnitus by deriving clickevoked auditory brainstem potentials, thus supporting the hypothesis of synaptopathy18.
For the present study, a cohort of normal-hearing subjects consisting of a group of tinnitus subjects (N = 15) and a control group (N = 14) was tested. Manual puretone audiometry with 11 test frequencies was conducted to determine hearing performance. Inclusion criteria were defined as air conducted hearing thresholds of 10 dB HL or lower. A deviation at a test frequency of 15 dB HL or less was tolerated. Data of tinnitus characteristics, such as pitch and intensity, were collected by presentation and matching of comparative tones, quality and subjective disturbance by questionnaire. Furthermore, data was obtained from both test groups by Békésy gliding frequency audiometry (794 test frequencies), as well as DPOAE measurement (36 test frequencies) and auditory brainstem response (ABR) audiometry (derivation of early auditory evoked potentials). The results showed a correlation of the determined tinnitus comparison pitch with the frequency location of the largest deviation (impairment) from the normal hearing curve in the Békésy gliding frequency audiometry (p = 0.032). All further analyses of the finestructure hearing curve (steepness of hearing loss, slope, number of hearing loss dips) showed no statistically significant relationship between the morphology of the fine-structure hearing curve and tinnitus characteristics. Finestructure measurement revealed areas of hearing loss that were not mapped in manual puretone audiometry. These "undetected" hearing losses would have led to the exclusion of 12 of 29 subjects (41.4 %) if the finestructure hearing curve had been used as an inclusion criterion. A direct comparison of the mean finestructure hearing curves of both test groups showed a statistically significant better mean hearing performance of the tinnitus group (p < 0.05) in 3 different test frequency ranges (1.5 kHz, 3 kHz, 7 kHz) with a maximum of 4 dB HL. Analy-sis of the mean amplitudes of wave I of the ABRs showed, contrary to expectation, a weak trend toward higher amplitudes in the tinnitus group (p = 0.06). According to Schaette and McAlpine (2011), synaptopathy pathogenesis should have resulted in an opposite trend, i.e., a decrease in wave I amplitude in the tinnitus group. As a secondary finding, a weak trend between wave I amplitude and subjectively perceived disturbance of tinnitus was demonstrated (p = 0.06). Statistical analysis of the parameters determined from the DPOAE measurements did not reveal any significant differences between the tinnitus group and control group. Direct comparison of the DPOAE and finestructure hearing curves, revealed a significant difference in the differences of the frequencyspecific measurements around 2.4 kHz (p = 0.007).
The results of the study suggest that in previous studies with supposedly normal hearing tinnitus subjects there were unrecognized hearing losses that either went unrecognized by the screening by manual puretone audiometry, or subjects with previously aboveaverage hearing experienced a subtle spontaneous decrease in their hearing as tinnitus pathogenesis. This assumption is also supported by the fact that there is a significant correlation between the frequency range of the greatest hearing loss in the finestructure hearing curves and the tinnitus frequency.
The suspected pathomechanism of synaptopathy in "normal hearing" subjects with tinnitus could not be confirmed. The correlation between wave I amplitudes and subjectively perceived disturbance by tinnitus, indicated by the data of this study, should be investigated in more detail in future studies. Further research with more accurate measurement methods and larger subject groups is needed to clarify the hypothesis "Genesis of chronic subjective tinnitus without hearing loss".
Chapter I of this work addressed the piggyBac (PB) transposon system, a non-viral genome engineering tool that is capable of efficiently performing stable integration of DNA sequences into a target cells genome and has already been used in clinical trials. However, the PB transposase has the problematic property of preferentially integrating transposons near transcriptional start sites (TSSs). This increases the likelihood of causing genotoxic effects, limiting its potential use as a tool in clinical applications. It has been shown in the past that the PB transposase shows physical interactions with BET proteins (e.g. BRD4) through Co-IP experiments. Representatives of these proteins are part of the transcriptional activation complex and are abundant at TSSs. Accordingly, it was previously proposed that this interaction is the underlying cause for the biased integration preference. For the first chapter of this thesis, the goal was to disrupt this interaction potentially modifying said integration preference. A secondary structure hypothesized to be mainly responsible for said interaction was extensively mutated resulting in several PB variants that were analyzed for their interaction capacity through a series of Co-IP experiments with BRD4. In total, seven substitutions were identified (E380F, V390K, T392Y, M394R, K407C, K407Q, and K407V) which exhibited reduced interaction capacity with BRD4. Each of the aforementioned mutants were used to generate integration libraries and, through NGS, it was determined if the integration preferences of the respective mutants had changed. In the immediate range 200 base pairs up- and downstream from known TSSs all mutants used exhibited a reduced integration bias. At a wider observation window 3 kbp up- and downstream from TSSs, further mutants with the substitutions M394R, T392Y and V390K showed a reduction in integration frequency of 17.3%, 1.5% and 5.4%, respectively, compared to the wildtype. Of particular note was the M394R mutant, which showed a reduction in all window sizes analyzed with a maximum of 65% less integration preference in the immediate vicinity of TSSs, theoretically generating a safety advantage over the wildtype transposase.
Chapter II was dedicated to the overall safety improvement for transposon-based gene modification and addresses the time point after the transgene has already been integrated and serious side effects may not be preventable. With this in mind, the aim was to develop a novel suicide-switch that can be stably introduced into cells via transposition, and reliably leads to cell death of the modified cells once activated. A system based on CRISPR/Cas9 was developed, where single guide RNAs were used to guide the Cas9 nuclease to Alu elements. These are short, repetitive sequences, which are distributed over the human genome in more than one million copies. Inducing double strand breaks within these elements would lead to genomic fragmentation and cell death. To be inducible, a transcriptional as well as post- translational control mechanism was added. Transcription of the Cas9 nuclease was regulated using a tet-on system, making expression dependent on doxycycline (DOX) supplementation. Furthermore, a version of the Cas9 nuclease called arC9 was used that allows double strand break generation only in the presence of 4-Hydroxytamoxifen (4-HT). Together with an expression cassette for the Alu-specific guide RNA and an expression cassette for the reverse tetracycline controlled transactivator all components were arranged between transposase-specific recognition sequences on a plasmid to allow transposon-system based gene transfer. The system was tested in HeLa cells. First, conditional expression of the arC9 nuclease was confirmed by addition of 1 μg/ml DOX. Second, the suicide-switch was further induced by adding 200 nM 4-HT and protein extracts were assayed for the KAP1 phosphorylation. Only upon induction with DOX and 4-HT phosphorylated KAP1 was detected, indicating DNA damage. Further, extensive growth and survival experiments were conducted to determine the effect of suicide-switch induction on cell proliferation and survival. Between 24 and 48 hours after induction, a halt in cell division was detected, after which extensive cell death was observed. Within 5 days post induction, >99% of all cells were eliminated. In the absence of both inducers, no significant differences in survival were observed compared to control cells line lacking Alu-specific guide RNAs. Microscopic examinations of the <1% surviving cell fraction revealed a senescence-associated phenotype and showed no signs of resumption of the cell division process. Accordingly, the second chapter of this thesis also achieved its goal in developing a functional suicide-switch that can be inserted into human cells via transposition, is highly dependent on the necessary induction signals, and exhibits excellent elimination capabilities in the context tested.
Many countries have restricted public life during the SARS-CoV2 pandemic. As related measures limited the access to sports facilities, this dissertation aimed (1) to examine changes in physical activity (PA) and well-being in affected countries, and (2) to determine the effectiveness of a digital home exercise program in this context.
Part 1 (PA/well-being) of the dissertation was a digital survey administered in 14 countries. Participants reported a 41 - 42% reduction of PA (NPAQ-SF) during restrictions (n=13,503 valid responses). Compliance with international PA guidelines decreased by nearly 19%. Mental well-being declined substantially (n=14,975 responses; 68.1 to 51.9 points on the WHO5 index) and the proportion of individuals at risk of depression tripled (14.2% to 45.2%). Physical well-being (SF-36 Pain) decreased slightly (85.8% to 81.3%). About two thirds (68.1%) of the respondents reported being interested in digital home exercise.
For Part 2 (digital home exercise) of the dissertation, an international multicenter randomized, controlled trial was performed allocating healthy adults (n=763; 33±12 years) to an intervention (IG) or control (CG) group. In contrast to the CG, the IG was offered live-streamed home exercise for four weeks. Subsequently, both groups had access to pre-recorded workouts for another four weeks. Outcomes were measured weekly using validated questionnaires. Mixed-models data analyses revealed an up to 1.65-fold (95% CI: 1.4-1.94; week 1) increase of PA relative to the CG. Moreover, small improvements in exercise motivation (SKK scale), psychological well-being (WHO-5 index), sleep quality (MOS Sleep Scale), and anxiety symptoms (GAD-7 Scale) were observed for IG.
The results of this dissertation suggest that public life restrictions associated with the pandemic had significant adverse effects on movement behavior and well-being. Digital home exercise can help to maintain and/or increase health- beneficial PA and well-being and may hence represent a supportive element of viral containment efforts.
Mechanism of the MHC I chaperone TAPBPR and its role in promoting UGGT1-mediated quality control
(2022)
Information about the health status of most nucleated cells is provided through peptides presented on major histocompatibility complex I (pMHC I) on the cell surface. T cell receptors of CD8+ T cells constantly monitor these complexes and allow the immune system to detect and eliminate infected or cancerous cells. Antigenic peptides displayed on MHC I are typically derived from the cellular proteome and are translocated into the lumen of the endoplasmic reticulum (ER) by the ATP-binding cassette (ABC) transporter associated with antigen processing (TAP), which is part of the peptide-loading complex (PLC). In a process called peptide editing, the MHC I-dedicated chaperone tapasin (Tsn) selects peptides for their ability to form stable complexes with MHC I. While initial peptide loading is catalyzed in the confines of the PLC, the second quality control is mediated by TAPBPR, operating in the peptide-depleted cis-Golgi network. TAPBPR was shown to have a more fine-tuning effect on the presented peptide repertoire rather than initial peptide selection. The fundamental mechanism of peptide editing was illuminated by two crystal structures of TAPBPR in complex with peptide-receptive MHC I. Notably, one of these structures reported a structural element that inserted into the peptidebinding pocket. The so-called scoop loop was assumed to be involved in mediating peptide exchange but the underlying mechanism remained undefined. Additionally, latest results suggested that TAPBPR mediates the interaction of the glucosyltransferase UGGT1 with peptide-receptive MHC. To expand the current knowledge of quality control processes in the antigen presentation pathway, the contribution of the scoop loop in peptide editing and the role of TAPBPR in UGGT1-mediated quality control needs to be elucidated. In the first part of this study, TAPBPR proteins with various loop lengths were designed to scrutinize the contribution of the scoop loop in chaperoning peptidereceptive MHC I. In a light-driven approach, the ability of TAPBPR variants to form stable complexes with peptide-free MHC I was tested. These results demonstrated that in a peptide-depleted environment, the scoop loop is of critical importance for TAPBPR to chaperone intrinsically unstable, peptidereceptive MHC I clients. Moreover, fluorescence polarization-based assays allowed the pursuit of peptide exchange in different, native-like environments. Peptide displacement activities of TAPBPR variants illustrated that catalyzed peptide editing is primarily induced by structural elements outside the scoop loop. In a peptide-depleted environment, the scoop loop occupies the position of the peptide C-terminus and acts as an internal peptide surrogate. By combining complex formation and fluorescence polarization experiments, the scoop loop of TAPBPR was shown to be critically important in stabilizing empty MHC I and functions as an internal peptide selector. In the second part of this study, a novel in-vitro glucosylation assay was established to examine the role of TAPBPR in UGGT1-catalyzed re-glucosylation of TAPBPR-bound MHC I clients. Therefore, a peptide-free MHC I-TAPBPR complex with defined glycan species was designed which served as physiological substrate for UGGT1. By subjecting the recombinantly expressed HLA-A*68:02- TAPBPR complex and UGGT1 proteins to the new in-vitro system, UGGT1 was shown to catalyze the transfer of a glucose residue to the N-linked glycan of TAPBPR-bound Man9GlcNAc2-HLA-A*68:02. Moreover, a high-affinity, photocleavable peptide was applied to dissociate the MHC I-chaperone complex. However, in the absence of TAPBPR, no glucosyltransferase activity was observed. Generation of peptide-free MHC I through UV illumination also showed no activity, and only the addition of TAPBPR could restore UGGT1-mediated reglucosylation of the empty MHC I. Independent of the peptide status of HLAA*68:02, the combination of protein glycoengineering and LC-MS analysis implicated that UGGT1 exclusively acts on TAPBPR-chaperoned HLA-A*68:02. The newly established system provided insights into the function of TAPBPR during UGGT1-catalyzed re-glucosylation activity and quality control of MHC I. Taken together, the scoop loop allows TAPBPR to function as MHC I chaperone through stabilizing peptide-receptive MHC I. In a peptide-depleted environment, the loop structure serves as an internal peptide surrogate and can only be dislodged by a high-affinity peptide. Based on these findings, TAPBPR fulfills a dual function in the second level of quality control. On the one hand, TAPBPR functions as peptide editor, shaping the repertoire of presented peptides. On the other hand, TAPBPR mediates peptide-receptive MHC I clients to the folding sensor UGGT1. Here, TAPBPR is essential to promote UGGT1-catalyzed reglucosylation of the N-linked glycan, giving MHC I a second chance to be loaded with an optimal peptide cargo in the peptide loading complex.
KMT2A-rearrangements are causative for 70-80% all infant acute lymphoblastic leukemias (Pieters et al., 2019, 2007). Among these, the translocation t(4;11)(q21;23) generating the oncogenic fusion genes KMT2A::AFF1 and AFF1::KMT2A is the most frequent one, accounting for almost every second case of KMT2A-r infant ALL (Meyer et al., 2018). Despite passing a multimodal chemotherapy, 64% of patients achieve an event including relapse or death within four years from diagnosis, and overall survival three years from relapse remains poor with only 17% (Driessen et al., 2016; Pieters et al., 2019, 2007). Vari-ous studies have shown that relapse and therapy resistance were not mediated by chemotherapy-induced mutagenesis as there was no accumulation of secondary mutations in the dominant leukemic clone between diagnosis and relapse (Agraz-Doblas et al., 2019; Andersson et al., 2015; Bardini et al., 2011; Dobbins et al., 2013; Driessen et al., 2013; Mullighan et al., 2007).
Intriguingly, exclusively infant t(4;11) ALL patients were reported to subdivide in two groups depending on the level of HOXA gene cluster expression (Trentin et al., 2009). The HOXAlo group displayed a high expression of IRX1 and the HOXAhi group a low expression of IRX1 (Symeonidou and Ottersbach, 2021; Trentin et al., 2009). Importantly, the HOXAlo/IRX1hi group was characterized to possess a strongly ele-vated relapse incidence compared to the HOXAhi/IRX1lo group (Kang et al., 2012; Stam et al., 2010). IRX1 was identified to upregulate the Early growth response genes EGR1, EGR2 and EGR3 (Kühn et al., 2016).
The doctoral project “EGR-mediated relapse mechanisms in infant t(4;11) acute lymphoblastic leuke-mia” aimed to investigate a potential correlation between the HOXAlo-IRX1-EGR axis and relapse development in infant t(4;11) ALL. The primary objective was to clarify through which molecular mechanism(s) relapse development despite continuous chemotherapy could be achieved. In this context, the role of the EGR genes has been investigated. In addition, this project aimed to disclose molecular targets which could offer novel therapeutic interventions to interfere with therapy resistance and relapse formation.
This thesis has two main parts.
The first part is based on our publication [1], where we use perturbation theory to calculate decay rates of magnons in the Kitaev-Heisenberg-Γ (KHΓ) model. This model describes the magnetic properties of the material α-RuCl 3 , which is a candidate for a Kitaev spin liquid. Our motivation is to validate a previous calculation from Ref. [2]. In this thesis, we map out the classical phase diagram of the KHΓ model. We use the Holstein-Primakoff
transformation and the 1/S expansion to describe the low temperature dynamics of the Kitaev-Heisenberg-Γ model in the experimentally relevant zigzag phase by spin waves. By parametrizing the spin waves in terms of hermitian fields, we find a special parameter region within the KHΓ model where the analytical expressions simplify. This enables us to construct the Bogoliubov transformation analytically. For a representative point in the special parameter region, we use these results to numerically calculate the magnon damping, which is to leading order caused by the decay of single magnons into two. We also calculate the dynamical structure factor of the magnons.
The second part of this thesis is based on our publication [3], where we use the functional renormalization group to analyze a discontinuous quantum phase transition towards a non-Fermi liquid phase in the Sachdev-Ye-Kitaev (SYK) model. In this thesis, we perform a disorder average over the random interactions in the SYK model. We argue that in the thermodynamic limit, the average renormalization group (RG) flow of the SYK model is identical to the RG flow of an effective disorder averaged model. Using the functional RG, we find a fixed point describing the discontinuous phase transition to the non-Fermi liquid phase at zero temperature. Surprisingly, we find a finite anomalous dimension of the fermions, which indicates critical fluctuations and is unusual for a discontinuous transition. We also determine the RG flow at zero temperature, and relate it to the phase diagram known from the literature.
Oceanic islands only comprise a small amount of the Earth’s land area but harbour a disproportionate amount of global biodiversity. This vast diversity is not only reflected in the taxonomic uniqueness of island biota but also in the remarkable evolution of functional traits. Functional traits, i.e. measurable characteristics that strongly influence the fitness of species, determine how a species responds to its environment and can help to gain more insights into the biogeographical, ecological and evolutionary processes that have shaped island biodiversity. However, research in island biogeography has primarily focused on species richness, and knowledge of functional trait patterns on oceanic islands is scarce. Hence, in this dissertation, I have explored how trait-based approaches can increase our understanding of how biodiversity on oceanic islands assembles and how it is driven by the environment. The Canary Islands (Spain) are a particularly suitable model system to investigate patterns and drivers of biodiversity. The archipelago is characterised by a high variation in environmental heterogeneity and inhabits a unique and well-described native flora. Therefore, I have investigated five principal research questions using the flora (Spermatophytes) of the Canary Islands as a study object. First, I have analysed how climate and biogeography shape the assembly of the Canary Islands flora using a novel trait-based approach. Second, the question of whether rare climates link to functional trait distinctiveness in the native Canary Islands flora was addressed. Third, I have examined how intraspecific trait variation is represented in the native flora of oceanic islands focusing on the succulent scrub of La Palma (Canary Islands). Fourth, this dissertation investigated whether scientific floras can be reliable sources for trait data of plants native to oceanic islands. Finally, I have explored how climate change may impact the native Canary Islands flora by analysing possible climate change-induced shifts in plant species distribution and plant traits.
The results of my dissertation expand the understanding of the importance of biogeography and the environment in determining the functional composition of island floras. I have assessed that traits of endemic plant species did not expand the functional trait space of the Canary Islands but were packed with the ones of non-endemic species. This result hints at a trait convergence in endemic species, possibly driven by non-adaptive speciation processes. Moreover, I have evidenced that humidity is a critical driver of functional diversity in native plant assemblages and particularly leads to a high trait convergence in arid environments via environmental filtering. In contrast, alien species have expanded the Canary Islands flora’s functional trait space. I further have shown that in contrast to native species assemblages, alien species assemblages are characterised by an increasing functional diversity with increasing aridity. This contrasting pattern of functional diversity could pose a potential risk to the native flora of the Canary Islands as a low functional diversity is expected to reduce the resilience of species assemblages to the establishment of more functionally diverse alien plant species. However, in this dissertation, I also have revealed that endemic plant species on the Canary Islands show a high intraspecific variation in arid environments, possibly as an adaptation to environmental stress. Intraspecific variation could help endemic plant species have a competitive advantage over alien species and be more resilient to environmental changes. Furthermore, in this dissertation, I have shown that scientific floras and taxonomic monographs could be used to gain information on quantitative functional traits of plants native to oceanic islands. This finding is particularly relevant for advances in trait-based research, as coverage of trait data for oceanic island floras is extremely poor in global trait databases. Hence, for some of the studies included in this dissertation, trait data were retrieved from scientific floras and taxonomic monographs and used to answer novel scientific research questions. Thus, I have used trait data from the literature to analyse the effect of climate change on the range size of plants native to the Canary Islands. Identifying plant species of particular conservation concern is critical on oceanic islands as many island species have limited distributions and small population sizes, and their niche tracking is impeded by insularity. I have revealed that single-island endemic plants gain less and lose more climatically suitable areas than archipelago endemic and non-endemic native plants due to a climate change-induced decrease in precipitation until 2100...
The relevant field of interest in High Energy Physics experiments is shifting to searching and studying extremely rare particles and phenomena. The search for rare probes requires an increase in the number of available statistics by increasing the particle interaction rate. The structure of the events also becomes more complicated, the multiplicity of particles in each event increases, and a pileup appears. Due to technical limitations, such data flow becomes impossible to store fully on available storage devices. The solution to the problem is the correct triggering of events and real-time data processing.
In this work, the issue of accelerating and improving the algorithms for reconstruction of the charged particles' trajectories based on the Cellular Automaton in the STAR experiment is considered to implement them for track reconstruction in real-time within the High-Level Trigger. This is an important step in the preparation of the CBM experiment as part of the FAIR Phase-0 program. The study of online data processing methods in real conditions at similar interaction energies allows us to study this process and determine the possible weaknesses of the approach.
Two versions of the Cellular Automaton based track reconstruction are discussed, which are used, depending on the detecting systems' features. HFT~CA Track Finder, similar to the tracking algorithm of the CBM experiment, has been accelerated by several hundred times, using both algorithm optimization and data-level parallelism. TPC~CA Track Finder has been upgraded to improve the reconstruction quality while maintaining high calculation speed. The algorithm was tuned to work with the new iTPC geometry and provided an additional module for very low momentum track reconstruction.
The improved track reconstruction algorithm for the TPC detector in the STAR experiment was included in the HLT reconstruction chain and successfully tested in the express production for the online real data analysis. This made it possible to obtain important physical results during the experiment runtime without the full offline data processing. The tracker is also being prepared for integration into a standard offline data processing chain, after which it will become the basic track search algorithm in the STAR experiment.
In this thesis we discuss the group Out(Gal_K) of outer automorphism of the absolute Galois group Gal_K of a p-adic number field K. Using results about the mapping class group of a surface S, as well as a result by Jannsen--Wingberg on the structure of the absolute Galois group Gal_K, we construct a large subgroup of Out(Gal_K) arising as images of certain Dehn twists on S.
Background: Increasing numbers of patients surviving malignant bone tumors around the knee joint have led to an increasing importance to investigate long-term results. This study assessed the long-term results of rotationplasty after resection of malignant bone tumors regarding functional outcome and quality of life to allow better comparison with other treatment options in bone cancer treatment.
Procedure: 60 participants who underwent rotationplasty due to bone cancer took part in this multicentric questionnaire- based study. The long-term functional outcome was measured by the Musculoskeletal tumor society score (MSTS) and the Tegner activity level scale. The health-related quality of life (HRQL) was assessed by using the Short Form Health Survey (SF-36).
Results: Patients treated with rotationplasty (median follow- up of 22 years, range 10–47 years) regained a high level of activity (median MSTS score of 24). Even a return to high level sports was possible (mean Tegner activity level scale of 4). Duration of follow-up did not influence the functional outcome. HRQL scores were comparable to the general German popula tion. Concerns of psychological problems due to the unusual appearance of the rotated foot have not been confirmed.
Conclusion: Rotationplasty can be a good alternative to en- doprosthetic replacement or amputation, either as primary surgery or as a salvage procedure. Especially for growing children and very active patients rotationplasty should be considered.
Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with a multifarious clinical presentation. Even though many genetic risk factors have been identified and studied in mouse models, the neurophysiological mechanisms underlying the autistic phenotype are still unclear. Based on the high rates of comorbidity with epilepsy, it was hypothesized that the balance between excitation and inhibition in neural circuits may be disrupted in autistic individuals.
In this dissertation, synaptic and network activity was measured in three different genetically modified mouse models that exhibit the characteristic behavioral abnormalities of the disorder: the Neurobeachin (Nbea) haploinsufficient mouse, the Neuroligin-3 (Nlgn3) knockout (KO) mouse, and the Neuroligin-4 (Nlgn4) KO mouse. Each of the affected proteins is involved in the formation and/or function of synapses in the central nervous system. Therefore, it was posited that the reduction or deletion of these proteins might alter the balance of excitatory to inhibitory synaptic transmission in individual neurons and in neural circuits. Extracellular recordings in the hippocampal dentate gyrus of anesthetized mice revealed that the excitation-inhibition (E-I) balance was reduced in Nbea haploinsufficient and Nlgn4 KO mice, but unchanged in Nlgn3 KO mice despite a reduction in excitatory synaptic transmission to dentate granule cells. Unexpectedly, the intrinsic excitability of dentate granule cells was altered in all three mouse models. These results imply that a homeostatic increase in the intrinsic excitability is able to compensate for the decreased excitatory transmission in Nlgn3 KO mice, whereas the decreased intrinsic excitability in the Nbea haploinsufficient and Nlgn4 KO mice leads to a reduction in the E-I balance. Taken together, these findings suggest that the influence of genetic factors on the E-I balance might be a potential common mechanism underlying the development of ASD.
Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
A promising strategy to reduce the dependency from fossil fuels is to use the yeast Saccharomyces cerevisiae to bioconvert renewable non-food feedstocks or waste streams, like lignocellulosic biomass, into bioethanol and other valuable molecule blocks. Lignocellulosic feedstocks contain glucose and significant fractions of the pentoses xylose and arabinose in varying proportions depending on the biomass type. S. cerevisiae is an efficient glucose consumer, but it cannot metabolize xylose and arabinose naturally. Therefore, extensive research using recombinant DNA techniques has been conducted to introduce and improve the biochemical pathways necessary to utilize these non-physiological substrates. However, any functional pathway capable of metabolizing D xylose and L arabinose in S. cerevisiae requires the transport of these sugars across the plasma membrane. The endogenous sugar transport system of S. cerevisiae can conduct a limited uptake of D-xylose and L-arabinose; this uptake enables only basal growth when the enzymatic pathways are provided. For this reason, the uptake of D xylose and L-arabinose has been recognized as a limiting step for the efficient utilization of these non-physiological substrates.
Gal2, a member of the major facilitator superfamily, is one of the most studied hexose transporters in S. cerevisiae. Although its expression is repressed in the presence of glucose, it also transports this sugar with high affinity when constitutively expressed. Recent efforts to engineer yeast strains for the utilization of plant biomass have unraveled the ability of Gal2 to transport non-physiological substrates like xylose and arabinose, among others. Improving Gal2 kinetic and substrate specificity, particularly for pentoses, has become a crucial target in strain engineering. The main goal of this study is to improve the utilization of xylose and arabinose by increasing the cell permeability of these non physiological substrates through the engineering of the galactose permease Gal2.
GAL2 gene expression depends on galactose, which acts as an inducer; nevertheless, even in the presence of galactose, glucose act as a strict repressor; consequently, GAL2 gene is usually placed under the control of a constitutive promoter. However, the presence of glucose additionally triggers the Gal2 degradation, which is mediated by the covalent attachment of the small 76 amino acid protein ubiquitin (Ub) to the targeted transporter; in a multi-step process called ubiquitination.
Ubiquitination of hexose permeases involves the activation of the Ub molecule by the E1 Ub-activating enzyme using ATP; then, the activated Ub is transferred to a specific Ub-conjugating enzyme E2, which donates the Ub indirectly through a specific HECT E3 enzyme (Rsp5) to a lysine residue of the substrate, with the aid of an adaptor protein which recognizes the target (Rsp5-adaptor). Ubiquitinated permeases are sent by membrane invagination to early endosomes, where they encounter ESCRTs (endosomal sorting complex required for transport). The targeted permeases are sorted in intralumenal vesicles (ILV) inside of the endosome, which after several cycles, turns into a multivesicular body (MVB) that subsequently fuses with the vacuole to expose the protein content of the ILVs to lumenal hydrolases for degradation.
Gal2 contains 30 lysine residues that may accept the ubiquitin molecule, which targets its degradation. It is known that mono-ubiquitination by Rsp5 on multiple lysine residues is necessary to internalize Gal2 (Horak & Wolf, 2001). However, the authors did not identify the specific lysine residues involved in the ubiquitination processes. This study screened several Gal2 variants where lysine residues were mutated or removed from the protein sequence to discover which lysine residues are likely involved in ubiquitination and consequent turnover of the transporter. The results of the screening showed that mutation of the N terminal lysine residues 27, 37, and 44 to arginine (Gal23KR) produced a functional transporter that, when fused with GFP (Gal23KR_GFP), showed an exclusive localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b).
This study furthermore evaluated upstream signals caused by phosphorylation which triggers ubiquitination and consequent turnover of the targeted protein; using similar screening approaches to assess the stabilization of Gal2 by lysine residue modifications, it was possible to identify that N terminal serine residues 32, 35, 39, 48, 53, and 55 are likely involved in the internalization of Gal2, since a Gal2 construct where all these serines were mutated to alanine residues and tagged with GFP (Gal26SA_GFP) exhibited practically complete localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b)...
Mutational analysis of ribosomal DNA and maturation-scheme analysis of ribosomal RNA in A. thaliana
(2022)
Ribosome biogenesis is a fundamental cellular process beginning with long precursor rRNA transcription from multi-copies of repetitive 45S ribosomal DNAs. At the subunit level, the primary pre-rRNA transcript encapsuled in 90S protein-RNA complex undergoes decisive splitting in two chief ways for further maturation into large (LSU) and small (SSU) ribosomal subunit. The usage of specific rDNA copies from defined chromosomes and their selective role during growth and development have been a topic of interest owing to its contribution to specialized ribosome theory which proposes non-monolithic functions for ribosomes and thereby their mRNA translation potential. Dual-guide CRISPR/Cas9 mediated disruption of rDNA regions resulted in stable disruption of up to 2.5% and 5% of all rDNA copies in hetero- and homozygous (ploop KD) conditions, respectively. At the RNA level, the mutation excised a critical structural element, P-loop on the LSU 25S rRNA. Mutation caused a dosage dependent defect with homozygosity leading to severe developmental defects through vegetative and reproductive growth phases which is manifested in their proteome by means of disregulation through both increase and decrease of several gene ontological categories of proteins in mutants. Interestingly, the mutation on chromosome 4 triggered dosage compensation through rRNA expression from chromosome 2 further compounded by ectopic rRNA biogenesis defects. The mutated copies however are not incorporated in the translating ribosomes and as a direct or indirect consequence led to elevated basal autophagic levels in the mutants.
The primary 35S transcript is known to undergo two modes of initial cleavages at the pre-rRNA level that aid in their subsequent maturation. Root cell culture (RCC) studies shows that these cells contain a novel ITS2-first cleaved precursor even under control growth conditions, P-C2 adding a third maturation means for the 35S pre-rRNA. This maturation path is further known to be triggered under elevated growth temperature forming a novel adaptive response in Arabidopsis and two other crop plants, tomato, and rice. Taken together, the pulse-chase labeling analysis of control and stressed tissues uncovers the fine-tuned pre-rRNA schematics with crossovers between multiple maturation paths.
Lipopolysaccharide (LPS) is a major glycolipid component in the outer leaflet of the outer membrane of Gram-negative bacteria and known as endotoxin exhibited by the lipid A moiety, which serves as a membrane anchor. The effective permeability barrier properties of the outer membrane contributed by the presence of LPS in the extracellular layer of the outer membrane confer Gram-negative bacteria a high resistance against hydrophobic compounds such as antibiotics, bile salts and detergents to survive in harsh environments. The biogenesis of LPS is well studied in Escherichia coli (herewith E. coli) and the LPS transport (Lpt) is carried out by a transenvelope complex composed of seven essential proteins (LptABCDEFG), which are located in the three compartments of the cell such as the outer membrane, the inner membrane and the periplasm. The Lpt system also exists in Anabaena sp. PCC 7120 (herewith Anabaena sp.), however, homologues of LptC and LptE are still missing. BLAST search failed to identify a homologue of LptC, in contrast, the secondary structure analysis using the Pfam database based on the existing ecLptC secondary structure identified one open reading frame All0231 as the putative Anabaena sp. homologue of LptC, which is designated anaLptC. Despite the low sequence similarity, the secondary structure alignment between anaLptC and ecLptC using the HHpred server showed that both proteins share high secondary structural similarities. The genotypic analysis of the insertion mutant anaLptC did not identify a fully segregated genome and its phenotypic analysis revealed that it was sensitive against chemicals, suggesting that the analptC gene is essential for the growth of Anabaena sp. and involved in the outer membrane biogenesis. This is further supported by the observation of the small cell phenotype in the anaLptC mutant via transmission electron microscopy. Moreover, physical interactions between the anaLptC periplasmic domain with anaLptA as well as with anaLptF were established, indicating that the anaLptC periplasmic domain is correctly folded and alone functional and that the transmembrane helix is not required for the interaction with anaLptA and anaLptF. Furthermore, the reduction of the O-antigen containing LPS was observed in the insertion mutant anaLptC and the dissociation constant Kd of the anaLptC periplasmic domain for ecLPS was determined.The three-dimensional structure of the periplasmic domain of anaLptC was solved by X-ray crystallography with a resolution of 2.8 Å. The structural superposition between the ecLptC crystal structure (PDB number 3my2) and the crystal structure of anaLptC periplasmic domain obtained by this study showed the similarity in the folding of the two proteins with a Cα r.m.s.d value of about 1 Å and confirmed that the length of anaLptC is more than two times longer than that of ecLptC. The structural comparison also revealed that both structures share the typical β-jellyroll fold and conserved amino acids, which were shown in ecLptC to bind to LPS in vivo and found in anaLptC. Overall, these data strongly suggest that anaLptC is involved in the transport of LPS and support the model whereby the bridge spanning the inner membrane and the outer membrane would be assembled via interactions of the structurally conserved β-jellyroll domains shared by five (LptACDFG) out of seven Lpt proteins.
The majority of B-cell precursor acute leukemias in infants are associated with the chromosomal translocation t(4;11)(q21;q23), resulting in the fusion of the mixed-lineage leukemia (MLL) and ALL1-fused gene of chromosome 4 (AF4) genes. While the fusion protein MLL-AF4 is expressed in all t(4;11) patients and essential for leukemia progression, the distinct role of the reciprocal fusion protein AF4-MLL, that is expressed in only 50-80% of t(4;11) leukemia patients (Meyer et al., 2018), remains unclear. In addition, t(4;11) leukemia could so far exclusively be generated in vivo in the presence of AF4-MLL and independent of the co-expression of MLL-AF4 (Bursen et al., 2010).
In a multifactorial approach inhibiting histone deacetylases (HDACs) and expressing the dominant negative mutation of Taspase1 (dnTASP1), both MLL fusion proteins were targeted simultaneously to evaluate a possible cooperative effect between MLL-AF4 and AF4-MLL during the progression of leukemia. Of note, neither HDACi nor dnTASP1 expression negatively affect endogenous MLL, but rather endorse its function hampered by the MLL fusion proteins (Ahmad et al., 2014; Bursen et al., 2004; Zhao et al., 2019). The mere expression of dnTASP1 failed to induce apoptosis, whereas dnTASP1 could elevate apoptosis levels significantly in HDACi-treated t(4;11) cells underlining the therapeutic potential of co-inhibiting both MLL fusion proteins.
Next, the impact of inhibiting either MLL-AF4 or AF4-MLL in vivo was resolved using whole transcriptome analysis. In PDX cells obtained by the Jeremias Laboratory (Völse, 2020) that co-expressed both t(4;11) fusion proteins, the knock-down of MLL-AF4 revealed the down-regulation of pivotal hemato-malignant factors. The expression of dnTASP1 led to massive deregulation of cell-cycle genes in vivo. Considering that the inhibition of particularly MLL-AF4 but not AF4-MLL impaired leukemic cell growth in vivo (Völse, 2020), the results of this work suggest a cooperative effect between both fusion proteins, while the loss of AF4-MLL during leukemia progression appears not essential.
Thereafter, a possible short-term role of AF4-MLL during the establishment of t(4;11) leukemia was analyzed. For this purpose, an in vitro t(4;11) model was constructed to investigate the transforming potential of transiently expressed AF4-MLL in cells constitutively expressing MLL-AF4, putatively reflecting the situation in vivo. Due to the lack of a leukemic background of the applied cell line, the aim was to investigate the long-term potential of AF4-MLL to significantly alter the epigenome rather than mimicking the development of leukemia. Strikingly, short-term-expressed AF4-MLL in cooperation with MLL-AF4 exerted durable epigenetic effects on gene transcription and chromatin accessibility. The here obtained in vitro data suggest a clonal evolutionary process initiated by AF4-MLL in a cooperative manner with MLL-AF4. Importantly, no long-term changes in chromatin accessibility could be observed by the transient expression of either MLL-AF4 or AF4-MLL alone.
All in all, considering endogenous MLL, MLL-AF4 and AF4-MLL in a targeted treatment is a promising approach for a more tailored therapy against t(4;11) leukemia, and AF4-MLL is suggested to act in a cooperative manner with MLL-AF4 especially during the development of a t(4;11) leukemia.
High-resolution, compactness, scalability, efficiency – these are the critical requirements which imaging radar systems have to fulfil in applications such as environmental monitoring, cloud mapping, body sensing or autonomous driving. This thesis presents a modular millimetre-wave frequency modulated continuous-wave (FMCW) radar front-end solution intended for such applications. High-resolution is achieved by enlarging the operating frequency band of the radar system. This can be realized at millimetre-wave frequencies due to the large spectrum availability. Furthermore, the size of components decreasing with increasing frequency makes millimetre-wave systems a good candidate for compactness. However, the full integration of radar front-ends is a challenge at millimetre-wave frequencies due to poor signal integrity and spectral purity, which are essential for imaging applications. The proposed radar uses an alternative technique and tackles this limitation by featuring highly-integrable architectures, specifically the Hartley architecture for signal conversion and enhanced push-pull amplifier for harmonic suppression. The resolution of imaging radars can be further improved by increasing the number of transmitters and receivers. This has spurred the investigation of spectrum, time and energy-efficient multiplexing techniques for multi-input multi-output (MIMO) radar systems. The FMCW radar architecture proposed in this thesis is based on code-division technique using intra-pulse, also called intra-chirp modulation. This advanced scalable and non-complex solution, made possible by the latest achievements on direct digital synthesis for signal generation, guarantees signal integrity and compact size implementation. The proposed architecture is investigated by a thorough system analysis. A transmitter module and a receiver module for a 35 GHz imaging radar prototype are designed, fabricated and fully characterized to validate the feasibility of our novel approach for high-resolution highly-integrated MIMO front-ends.
The health status of every nucleated cell in the human body is monitored through peptides presented by major histocompatibility complex class I (MHC I) to T-cell receptors of CD8+ T-cells. Thereby, the adaptive immune system ensures the recognition and elimination of infected or cancerous cells. MHC I molecules comprise the polymorphic heavy chain (hc) and the light chain β2-microglobulin (β2m). More than 13,000 allomorphs of the MHC I hc have been identified. All MHC I hcs associate with β2m but differ in their binding preferences for peptides, ensuring the presentation of a large peptide pool. After maturation of MHC I hc/β2m heterodimers in the endoplasmic reticulum (ER), most of the peptide-deficient MHC I molecules are recruited to the peptide-loading complex (PLC). There, they go through peptide loading and editing before they are released as stable peptide-MHC I (pMHC I) complexes and traffic to the cell surface for antigen presentation.
During the stringent quality control of MHC I peptide loading and editing within the PLC, the chaperone tapasin in conjunction with the oxidoreductase ERp57 stabilizes peptide-receptive MHC I molecules and alters the peptide cargo for high immunogenicity by catalyzing peptide-exchange. The tapasin-homologue TAP-binding protein related (TAPBPR) is involved in downstream quality control, editing the peptide repertoire of MHC I molecules that slipped through peptide proofreading by tapasin. Both chaperones were shown to adopt similar binding-modes for MHC I, suggesting related mechanisms of peptide editing. Nevertheless, the MHC I specific chaperones operate in different subcellular locations with differing assistance. While TAPBPR mediates peptide-exchange solely in the peptide-poor environment of the cis-Golgi and ER-Golgi intermediate compartment (ERGIC), tapasin functions mainly within the PLC together with ERp57 and the lectin-like chaperone calreticulin. Calreticulin with its lectin-, arm- and C-terminal domain contacts the MHC I heterodimer, ERp57 and the C-terminal domain of tapasin, respectively. Notably, the interaction site between calreticulin and tapasin has not yet been elucidated experimentally at molecular detail. The depletion of tapasin leads to a compromised immune response and a change in the pool of peptide cargo. The numerous MHC I allomorphs vary in their plasticity and their dependence on tapasin for the loading of optimal peptides. Moreover, the conformational plasticity of MHC I correlates with their dependence on tapasin. However, the molecular basis on how tapasin edits the various MHC I allomorphs and the structural features that are essential for peptide exchange catalysis at atomic resolution remained elusive.
In the first part of this thesis, the trimeric complex of tapasin–ERp57/calreticulin was analyzed. To this end, laser induced liquid bead ionization mass spectrometry (LILBID-MS) was performed as part of a collaboration and revealed the trimeric assembly for tapasin–ERp57 and calreticulin. Furthermore, additional to a wildtype construct of calreticulin, a second construct, lacking the acidic helix of calreticulin that was found to come to close contact with tapasin, was utilized for isothermal titration calorimetry (ITC). A micromolar affinity of wildtype calreticulin to tapasin–ERp57 was determined. Previous biochemical and NMR studies utilizing the P-domain of calreticulin and solely ERp57 provided a micromolar affinity for the complex of calreticulin and ERp57. In this study, no interaction of calreticulin lacking the acidic helix with tapasin–ERp57 could be measured by ITC. However, these results undergo with findings that calreticulin lacking the acidic helix impairs the function of the PLC. Most likely, the negatively charged acidic helix is located in a groove of tapasin, carrying a more positive charge. Taken together, the functional data demonstrates the importance of the acidic helix of calreticulin for assembly of the trimeric subunit of calreticulin/tapasin–ERp57.
In the main part of this study an MHC I–tapasin–ERp57 complex was structurally analyzed. Therefore, a photo-triggered approach was chosen to assemble the transient complex of MHC I–tapasin–ERp57. Various allomorphs were screened for complex formation with the tapasin–ERp57 heterodimer after photocleavage by size exclusion chromatography (SEC), resulting in mouse MHC I H2-Db as the suited allomorph. Microseed matrix screening was performed. Crystals diffracting X-rays to a resolution of 2.7 Å were obtained showing one tetrameric tapasin–ERp57–MHC I complex per asymmetric unit.
The MHC I-chaperone structure shows molecular rearrangements upon MHC I engagement and unveils structural features of tapasin, involved in peptide-exchange catalysis...
In the last twenty years, there has been splendid progress in energy conversion technologies to have sustainable energy sources. For example, solar cells contribute significantly to energy production as the sun is an enormous source for renewable energy. Currently, the most common commercialized photovoltaic devices are silicon-based. The scientists' main targets are high efficiency, low cost, environmentally friendly, and easy to synthesize new semiconductor materials to replace silicon. Furthermore, understanding the photophysical properties of these materials is very important for designing high efficient photoconversion systems.
This thesis investigates the photophysics of lead-based wide-bandgap perovskites with different dimensionality (2D, 3D) and how they can be optimized for optoelectronic applications. In chapter 1, we present the background and progress in perovskite research. The basic concepts of semiconductor and spectroscopic methods of the applied techniques in this work are discussed in chapter 2.
In the first project (chapter 3.1), we used our time-resolved techniques to study the ultrafast dynamics of energy transfer from the inorganic to the organic layer in a series of three lead-based mixed-halide 2D perovskites containing benzyl ammonium (BA), 1-naphthyl methyl ammonium (NMA), and 1-pyrene methyl ammonium (PMA) thin films.
In the second project (chapter 3.2), we used time-resolved spectroscopic techniques to study the effect of adding 5% of Cs on the dynamics of a mixed-cation wide bandgap bromide-based 3D perovskite.
In another side project (chapter 4), we present the photophysics properties of newly synthesized new Schiff bases containing indole moieties using piperidine as an organic base catalyst and Au@TiO2 as a heterogeneous catalyst. Finally, the results of this work are summarized in Chapter 5 with an outlook and a discussion of open questions for further research.
Ceramide synthase (CerS) is the enzyme responsible for the de novo synthesis of ceramide. In this process, the different CerS isoforms are substrate-specific and produce ceramides of different chain lengths. Ceramides form the backbone for other sphingolipids and are enriched in membrane microdomains called lipid rafts. Lipid rafts are important signaling platforms for many transmembrane proteins, but can also act as bioactive lipids. Depending on the chain length, the effects on signaling pathways can vary. The aim of this work was to further investigate the chain length-specific effects by CerS4 on the progression of inflammatory colon cancer. To understand the tissue-specific effects of CerS4 deficiency on the progression of acute colitis and colitis-associated cancer (CAC), CerS4 knockout models were used. Disease progression of wild-type CerS4 (WT) was compared with that of mice with global CerS4 knockout (CerS4 KO) and mice in which CerS4 deficiency was restricted to T cells (CerS4 LCK/Cre) or intestinal cells (CerS4 Vil/Cre). Acute colitis was induced with sodium dextran sulfate (DSS), whereas azoxymethane (AOM)/DSS combinations were used to induce CAC in mice. The results showed a different disease progression depending on the specific knockout. While CerS4 KO mice were sensitive to DSS. AOM/DSS treatment was lethal for these mice, indicating an important role of CerS4 in other tissues. CerS4 Vil/Cre mice were protected from tumor formation. In contrast, CerS4 LCK/Cre mice experienced increased tumor formation and pan-inflammation. The mechanism behind this is due to the absence of cytotoxic T cells and the increase of regulatory T cells in the CerS4 LCK/Cre mice, demonstrating that CerS4 is critical for T cell function and development. To understand the role of CerS in humans, organoids were prepared from patients and the CerS profile in the different organoids was elucidated. This work provides, for the first time, insights into the CerS profile in human organoids and demonstrates a link between differentiation markers and stem cell markers with CerS. In addition, the role of CerS4 was investigated in vitro using three different colon cell lines-Caco-2 cells, HCT116 cells, and HCT15 cells. Hypoxia induced downregulation of CerS4 in all cell lines. Using the luciferase promoter assay, hypoxia-induced downregulation could already be detected at the promoter. Downregulation of CerS4 and CerS5 in Caco-2 cells and HCT116 cells resulted in different metabolic changes and mitochondrial dynamics after hypoxia. In conclusion, the results show that the role of CerS4 depends on the tissue cell type and stage of colorectal carcinoma, which complicates the consideration of CerS4 as a target in patients.
In the human brain, the incoming light to the retina is transformed into meaningful representations that allow us to interact with the world. In a similar vein, the RGB pixel values are transformed by a deep neural network (DNN) into meaningful representations relevant to solving a computer vision task it was trained for. Therefore, in my research, I aim to reveal insights into the visual representations in the human visual cortex and DNNs solving vision tasks.
In the previous decade, DNNs have emerged as the state-of-the-art models for predicting neural responses in the human and monkey visual cortex. Research has shown that training on a task related to a brain region’s function leads to better predictivity than a randomly initialized network. Based on this observation, we proposed that we can use DNNs trained on different computer vision tasks to identify functional mapping of the human visual cortex.
To validate our proposed idea, we first investigate a brain region occipital place area (OPA) using DNNs trained on scene parsing task and scene classification task. From the previous investigations about OPA’s functions, we knew that it encodes navigational affordances that require spatial information about the scene. Therefore, we hypothesized that OPA’s representation should be closer to a scene parsing model than a scene classification model as the scene parsing task explicitly requires spatial information about the scene. Our results showed that scene parsing models had representation closer to OPA than scene classification models thus validating our approach.
We then selected multiple DNNs performing a wide range of computer vision tasks ranging from low-level tasks such as edge detection, 3D tasks such as surface normals, and semantic tasks such as semantic segmentation. We compared the representations of these DNNs with all the regions in the visual cortex, thus revealing the functional representations of different regions of the visual cortex. Our results highly converged with previous investigations of these brain regions validating the feasibility of the proposed approach in finding functional representations of the human brain. Our results also provided new insights into underinvestigated brain regions that can serve as starting hypotheses and promote further investigation into those brain regions.
We applied the same approach to find representational insights about the DNNs. A DNN usually consists of multiple layers with each layer performing a computation leading to the final layer that performs prediction for a given task. Training on different tasks could lead to very different representations. Therefore, we first investigate at which stage does the representation in DNNs trained on different tasks starts to differ. We further investigate if the DNNs trained on similar tasks lead to similar representations and on dissimilar tasks lead to more dissimilar representations. We selected the same set of DNNs used in the previous work that were trained on the Taskonomy dataset on a diverse range of 2D, 3D and semantic tasks. Then, given a DNN trained on a particular task, we compared the representation of multiple layers to corresponding layers in other DNNs. From this analysis, we aimed to reveal where in the network architecture task-specific representation is prominent. We found that task specificity increases as we go deeper into the DNN architecture and similar tasks start to cluster in groups. We found that the grouping we found using representational similarity was highly correlated with grouping based on transfer learning thus creating an interesting application of the approach to model selection in transfer learning.
During previous works, several new measures were introduced to compare DNN representations. So, we identified the commonalities in different measures and unified different measures into a single framework referred to as duality diagram similarity. This work opens up new possibilities for similarity measures to understand DNN representations. While demonstrating a much higher correlation with transfer learning than previous state-of-the-art measures we extend it to understanding layer-wise representations of models trained on the Imagenet and Places dataset using different tasks and demonstrate its applicability to layer selection for transfer learning.
In all the previous works, we used the task-specific DNN representations to understand the representations in the human visual cortex and other DNNs. We were able to interpret our findings in terms of computer vision tasks such as edge detection, semantic segmentation, depth estimation, etc. however we were not able to map the representations to human interpretable concepts. Therefore in our most recent work, we developed a new method that associates individual artificial neurons with human interpretable concepts.
Overall, the works in this thesis revealed new insights into the representation of the visual cortex and DNNs...
In this thesis, we cover two intimately related objects in combinatorics, namely random constraint satisfaction problems and random matrices. First we solve a classic constraint satisfaction problem, 2-SAT using the graph structure and a message passing algorithm called Belief Propagation. We also explore another message passing algorithm called Warning Propagation and prove a useful result that can be employed to analyze various type of random graphs. In particular, we use this Warning Propagation to study a Bernoulli sparse parity matrix and reveal a unique phase transition regarding replica symmetry. Lastly, we use variational methods and a version of local limit theorem to prove a sufficient condition for a general random matrix to be of full rank.
Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway
(2022)
The human body is permanently exposed to its environment and thus to viruses and other pathogens, which require a flexible response and defense. Alongside to the innate immune system, the adaptive immune system provides highly specialized protection against these threats. The major histocompatibility complex class I (MHC I) antigen presentation system is a cornerstone of the adaptive immune system and a major constituent of cellular immunity. Pathogens such as viruses that invade a cell will leave traces in the form of proteins and peptides which are degraded and loaded onto MHC I molecules. MHC I peptide loading is performed by peptide loading complex (PLC) in the membrane of the endoplasmic reticulum as part of a multifaceted and comprehensive quality control machinery. Monitored by multiple layers of quality assurance, the MHC I molecules consequently display the immune status of the cell on its surface. In this context, the captured fragment of the virus serves as a call for help issued by the cell, alerting the adaptive immune system to the infection to mount an appropriate immune response.
The three-dimensional structure as well as the mechanistic details of parts of this complex machinery were characterized in the context of this dissertation. Among other tools, light-modulable nanotools were developed in this thesis, which permit external regulation of cellular processes in temporal and spatial resolution. Furthermore, methods and model systems for the biochemical characterization of cellular signaling cascades, proteins, as well as entire cell organelles were developed, which are likely to influence the field of cellular immunity and protein biochemistry in the future.
This cumulative work comprises a total of six publications whose scientific key advances will be briefly outlined in this abstract. In the introduction, the scientific background as well as the current state of research and methodological background knowledge are conveyed. The results section condenses the main aspects of the publications and links them to each other. Further details can be retrieved from the attached original publications.
In “Semisynthetic viral inhibitor for light control of the MHC I peptide loading complex, Winter, Domnick et al., Angew Chem Int Ed 2022” a photocleavable viral inhibitor of the peptide loading complex was produced by semi-synthesis. This nanotool was shown to be suitable for both purifying the PLC from human Raji cells as well as reactivating it in a light-controlled manner. Thus, this tool establishes the isolation of a fully intact and functional peptide loading complex for biochemical characterization. In addition, a novel flow cytometric analysis pipeline for microsomes was developed, allowing cellular vesicles to be characterized with single organelle resolution, similar to cells.
In “Molecular basis of MHC I quality control in the peptide loading complex, Domnick, Winter et al., Nat Commun 2022” the peptide loading complex was reconstituted into large nanodiscs, and a cryo-EM structural model of the editing module at 3.7 Å resolution was generated. By combining the structural model with in vitro glycan editing assays, an allosteric coupling between peptide-MHC I assembly and glycan processing was revealed, extending the known model of MHC I loading and dissociation from the PLC. These mechanisms provide a prototypical example for endoplasmic reticulum quality control.
In a related context, in “Structure of an MHC I–tapasin–ERp57 editing complex defines chaperone promiscuity, Müller, Winter et al., Nat Commun 2022” a recombinantly assembled editing module comprised of MHC I-tapasin-ERp57 was crystallized for X-ray structural biology. The resulting crystal structure at a resolution of 2.7 Å permitted the precise identification of characteristic features of the editing module and particularly of the peptide proofreading mechanism of tapasin. This study provided pivotal insights into the tapasin-mediated peptide editing of different MHC I allomorphs as well as similarities to TAPBPR-based MHC I peptide proofreading.
In “TAPBPR is necessary and sufficient for UGGT1-mediated quality control of MHC I, Sagert, Winter et al. (in preparation)” novel insights concerning the peptide proofreader TAPBPR and its close interplay with the folding sensor and glucosyltransferase UGGT1 were obtained. It was shown that TAPBPR is an integral part of the second level of endoplasmic quality control and is indispensable for effective MHC I coordination by UGGT1.
In “Light-guided intrabodies for on-demand in situ target recognition in human cells, Joest, Winter et al., Chem Sci 2021” intracellular nanobodies were equipped with a photocaged target recognition domain by genetic code expansion via amber suppression. These intrabodies, acting as high-affinity binding partners endowed with a fluorophore, could be used in a light-triggered approach to instantaneously visualize their target molecule...
Recent advances in artificial neural networks enabled the quick development of new learning algorithms, which, among other things, pave the way to novel robotic applications. Traditionally, robots are programmed by human experts so as to accomplish pre-defined tasks. Such robots must operate in a controlled environment to guarantee repeatability, are designed to solve one unique task and require costly hours of development. In developmental robotics, researchers try to artificially imitate the way living beings acquire their behavior by learning. Learning algorithms are key to conceive versatile and robust robots that can adapt to their environment and solve multiple tasks efficiently. In particular, Reinforcement Learning (RL) studies the acquisition of skills through teaching via rewards. In this thesis, we will introduce RL and present recent advances in RL applied to robotics. We will review Intrinsically Motivated (IM) learning, a special form of RL, and we will apply in particular the Active Efficient Coding (AEC) principle to the learning of active vision. We also propose an overview of Hierarchical Reinforcement Learning (HRL), an other special form of RL, and apply its principle to a robotic manipulation task.
This work investigated the influence of the CRISPR/Cas9 mediated knockout of 5-lipoxygenase (5-LO) on different adherent tumour cell lines derived from solid tumours. For this, the 5-LO expressing tumour cell lines HCT-116, HT-29, and U-2 OS were transiently transfected using a plasmid carrying the CRISPR/Cas9 complex sequence to the ALOX5 gene. Subsequently, cells were selected using Puromycin and analysed via Western blotting and DNA Sanger sequencing. Cells that were transfected with a control plasmid missing the guide RNA sequence, were used as a control for all experiments.
Differential gene expression analysis, performed after next-generation RNA sequencing, revealed that the expression of various genes was altered after the knockout of 5-LO. In HCT-116 cells, 28 genes were expressed differentially in all 5-LO knockout single-cell clones, while in HT-29 cells the expression of 18 genes and in U-2 OS cells of 234 genes was influenced by the knockout of 5-LO. These findings were validated by real-time qPCR. A lot of the genes that were influenced by the 5-LO knockout are known to be connected to epithelial-mesenchymal-transition (EMT), a process necessary for tumour metastasis. The results from RNA sequencing were the starting point for further investigations. In the following, different aspects of the tumour cell lines were examined. In HT-29, as
well as in U-2 OS cells, it was shown that knockout of the 5-LO resulted in impaired cell proliferation. Also, the formation of three-dimensional tumour spheroids was altered. In HT-29 cells, the knockout of 5-LO increased the number of cells in spheroids. In contrast, in U-2 OS cells, the number of cells per spheroid was decreased, even though the diameter of the spheroids was increased, due to more loosely packed spheroids. The difference between 5-LO positive and negative U-2 OS cells became even more obvious after embedding the spheroids in an artificial extracellular matrix. In that scenario, cells lacking the 5-LO formed smaller spheroids that did not have the same ability to grow into the extracellular matrix as 5-LO positive cells did. Also, directed cell migration was strongly influenced by the knockout of 5-LO. In both, HCT-116 and U-2 OS cells, directed cell migration towards a serum gradient was increased in 5-LO knockout single-cell clones. Pharmacological inhibition of the enzyme was used to investigate, whether canonical or non-canonical functions were responsible for the previously mentioned effects.
Therefore, vector control cells were treated with the 5-LO inhibitors Zileuton and CJ-13610 in different concentrations. Interestingly, only some of the effects mediated by the complete knockout of 5-LO could be reproduced by inhibiting the enzyme, leading to the suggestion, that canonical, as well as non-canonical functions of 5-LO, play a role in these tumour cells.
To conclude, it was shown in this study, that 5-LO affects various cellular functions when expressed in adherent tumour cell lines. These cell line-dependent effects result in altered gene expression, enhanced proliferation, and spheroid formation, as well as impaired cell motility, and can be mediated by enzymatic activity as well as other non-canonical functions.
Oxidative stress is thought to be a driver for several diseases. However, many data to support this concept were obtained by the addition of extracellular H2O2 to cells. This does not reflect the dynamics of intracellular redox modifications. Cells actively control their redox-state, and increased formation of ROS is a response to cellular stress situations such as chronic inflammation.
In this study, it was shown that different types of ROS lead to different metabolic and transcriptomic responses of HUVECs. While 300 μM extracellular H2O2 led to substantial metabolic and transcriptomic changes, the effects of DAO-derived H2O2 and menadione were low to moderate, indicating that the source and the concentration of ROS are important in eliciting changes in metabolism and gene expression.
Specifically, it was identified that acute increases in ROS transiently inactivate the enzyme ω-amidase/NIT2 of the glutaminase II pathway, which supplies cells with anaplerotic α-ketoglutarate. The pathway has not been studied systematically because, as noted above, the major intermediate, KGM, is not commercially available. In the present study, an internal standard for targeted detection of KGM in cells and blood plasma/serum was used. Deletion of NIT2 by CRISPR/Cas9 significantly reduced α-ketoglutarate levels in HUVECs and elevated KGM levels. It appears that in cell culture conditions, hydrolysis of KGM to α-ketoglutarate is very efficient. Knockout of the glutamine transaminases significantly reduced methionine, suggesting that the glutaminase II pathway is an important source of amino acid replenishment.
Similar to genetic silencing of GLS1 [91,92], HUVECs lacking NIT2 showed reduced proliferation and angiogenic sprouting. Furthermore, our results indicate that, at least in HUVECs, the enzyme also locates in the mitochondria where it interacts with key enzymes of glutamine/glutamate/α-ketoglutarate metabolism.
The data of the present work indicate that the glutaminase II pathway is an underappreciated, redox-sensitive pathway for glutamine utilization in HUVECs. Genetic deletion of NIT2 has considerable physiological effects highlighting the importance of glutamine for ECs.
Navigating a complex environment is assumed to require stable cortical representations of environmental stimuli. Previous experimental studies, however, show substantial ongoing remodeling at the level of synaptic connections, even under behaviorally and environmentally stable conditions. It remains unclear, how these changes affect sensory representations on the level of neuronal populations during basal conditions and how learning influences these dynamics.
Our approach is a joint effort between the analysis of experimental data and theory. We analyze chronic neuronal population activity data – acquired by out collaborators in Mainz – to describe population activity dynamics during basal dynamics and during learning (fear conditioning). The data analysis is complemented by the analysis of a circuit model investigating the link between a neural network’s activity and changes in its underlying structure.
Using chronic two-photon imaging data recorded in awake mouse auditory cortex, we reproduce previous findings that responses of neuronal populations to short complex sounds typically cluster into a near discrete set of possible responses. This means that different stimuli evoke basically the same response and are thus grouped together into one of a small set of possible response modes. The near discrete set of response modes can be utilized as a sensitive and robust means to detect and track changes in population activity over time. Doing so we find that sound representations are subject to a significant ongoing remodeling across the time span of days under basal conditions. Auditory cued fear conditioning introduces a bias into these ongoing dynamics, resulting in a differential generalization both on the level of neuronal populations and on the behavioral level. This means that sounds that are perceived similar to the conditioned stimulus (CS+) show an increased co-mapping to the same response mode the CS+ is mapped to. This differential generalization is also observed in animal behavior, where sounds similar to the CS+ result in the same freezing behavior as the CS+, whereas dissimilar sounds do not. These observations could provide a potential mechanism of stimulus generalization, which is one of the most common phenomena associated with post-traumatic stress disorder, on the level of neuronal populations.
To investigate how the aforementioned changes in neuronal population activity are linked to changes in the underlying synaptic connectivity, we devised a circuit model of excitatory and inhibitory neurons. We studied this firing rate model to investigate the effect of gradual changes in the network’s connectivity on its activity. Apart from an input dominated uni-stable regime (one response per stimulus independent of the network) and a network dominated uni-stable regime (one response per network independent of the stimulus), we also find a multi-stable regime for strong recurrent connectivity and a high ratio of inhibition to excitation. In this regime the model reproduces properties of neural population activity in mouse auditory cortex, including sparse activity, a broad distribution of firing rates, and clustering of stimuli into a near discrete set of response modes. This clustering in the multi-stable regime means that, not only can identical stimuli evoke different responses, depending on the network’s initial condition, but different stimuli can also evoke the same response.
Applying gradual drift to the network connectivity we find periods of stable responses, interrupted by abrupt transitions altering the stimulus response mapping. We study the mechanism underlying these transitions by analyzing changes in the fixed points of this network model, employing a method to numerically find all the fixed points of the system. We find that such abrupt transitions typically cannot be explained by the mere displacement of existing fixed points, but involve qualitative changes in the fixed point structure in the vicinity of the response trajectory. We conclude that gradual synaptic drift can lead to abrupt transitions in stimulus responses and that qualitative changes in the network’s fixed point topology underlie such transitions.
In summary we find that cortical networks display ongoing representational drift under basal conditions that is biased towards a differential generalization during fear conditioning. A circuit model is able to reproduce key characteristics of auditory cortex, including a clustering of stimulus responses into a near discrete set of response modes. Implementing synaptic drift into this model leads to periods of stable responses interrupted by abrupt transitions towards new responses.
Die vorgelegte Dissertation behandelt den Einfluss homöostatischer Adaption auf die Informationsverarbeitung und Lenrprozesse in neuronalen Systemen. Der Begriff Homöostase bezeichnet die Fähigkeit eines dynamischen Systems, bestimmte interne Variablen durch Regelmechanismen in einem dynamischen Gleichgewicht zu halten. Ein klassisches Beispiel neuronaler Homöostase ist die dynamische Skalierung synaptischer Gewichte, wodurch die Aktivität bzw. Feuerrate einzelner Neuronen im zeitlichen Mittel konstant bleibt. Bei den von uns betrachteten Modellen handelt es sich um eine duale Form der neuronalen Homöostase. Das bedeutet, dass für jedes Neuron zwei interne Parameter an eine intrinsische Variable wie die bereits erwähnte mittlere Aktivität oder das Membranpotential gekoppelt werden. Eine Besonderheit dieser dualen Adaption ist die Tatsache, dass dadurch nicht nur das zeitliche Mittel einer dynamischen Variable, sondern auch die zeitliche Varianz, also die stärke der Fluktuation um den Mittelwert, kontrolliert werden kann. In dieser Arbeit werden zwei neuronale Systeme betrachtet, in der dieser Aspekt zum Tragen kommt.
Das erste behandelte System ist ein sogennantes Echo State Netzwerk, welches unter die Kategorie der rekurrenten Netzwerke fällt. Rekurrente neuronale Netzwerke haben im Allgemeinen die Eigenschaft, dass eine Population von Neuronen synaptische Verbindungen besitzt, die auf die Population selbst projizieren, also rückkoppeln. Rekurrente Netzwerke können somit als autonome (falls keinerlei zusätzliche externe synaptische Verbindungen existieren) oder nicht-autonome dynamische Systeme betrachtet werden, die durch die genannte Rückkopplung komplexe dynamische Eigenschaften besitzen. Abhängig von der Struktur der rekurrenten synaptischen Verbindungen kann beispielsweise Information aus externem Input über einen längeren Zeitraum gespeichert werden. Ebenso können dynamische Fixpunkte oder auch periodische bzw. chaotische Aktivitätsmuster entstehen. Diese dynamische Vielseitigkeit findet sich auch in den im Gehirn omnipräsenten rekurrenten Netzwerken und dient hier z.B. der Verarbeitung sensorischer Information oder der Ausführung von motorischen Bewegungsmustern. Das von uns betrachtete Echo State Netzwerk zeichnet sich dadurch aus, dass rekurrente synaptische Verbindungen zufällig generiert werden und keiner synaptischen Plastizität unterliegen. Verändert werden im Zuge eines Lernprozesses nur Verbindungen, die von diesem sogenannten dynamischen Reservoir auf Output-Neuronen projizieren. Trotz der Tatsache, dass dies den Lernvorgang stark vereinfacht, ist die Fähigkeit des Reservoirs zur Verarbeitung zeitabhängiger Inputs stark von der statistischen Verteilung abhängig, die für die Generierung der rekurrenten Verbindungen verwendet wird. Insbesondere die Varianz bzw. die Skalierung der Gewichte ist hierbei von großer Bedeutung. Ein Maß für diese Skalierung ist der Spektralradius der rekurrenten Gewichtsmatrix.
In vorangegangenen theoretischen Arbeiten wurde gezeigt, dass für das betrachtete System ein Spektralradius nahe unterhalb des kritischen Wertes von 1 zu einer guten Performance führt. Oberhalb dieses Wertes kommt es im autonomen Fall zu chaotischem dynamischen Verhalten, welches sich negativ auf die Informationsverarbeitung auswirkt. Der von uns eingeführte und als Flow Control bezeichnete duale Adaptionsmechanismus zielt nun darauf ab, über eine Skalierung der synaptischen Gewichte den Spektralradius auf den gewünschten Zielwert zu regulieren. Essentiell ist hierbei, dass die verwendete Adaptionsdynamik im Sinne der biologischen Plausibilität nur auf lokale Größen zurückgreift. Dies geschieht im Falle von Flow Control über eine Regulation der im Membranpotential der Zelle auftretenden Fluktuationen. Bei der Evaluierung der Effektivität von Flow Control zeigte sich, dass der Spektralradius sehr präzise kontrolliert werden kann, falls die Aktivitäten der Neuronen in der rekurrenten Population nur schwach korreliert sind. Korrelationen können beispielsweise durch einen zwischen den Neuronen stark synchronisierten externen Input induziert werden, der sich dementsprechend negativ auf die Präzision des Adaptionsmechanismus auswirkt.
Beim Testen des Netzwerks in einem Lernszenario wirkte sich dieser Effekt aber nicht negativ auf die Performance aus: Die optimale Performance wurde unabhängig von der stärke des korrelierten Inputs für einen Spektralradius erreicht, der leicht unter dem kritischen Wert von 1 lag. Dies führt uns zu der Schlussfolgerung, dass Flow Control unabhängig von der Stärke der externen Stimulation in der Lage ist, rekurrente Netze in einen für die Informationsverarbeitung optimalen Arbeitsbereich einzuregeln.
Bei dem zweiten betrachteten Modell handelt es sich um ein Neuronenmodell mit zwei Kompartimenten, welche der spezifischen Anatomie von Pyramidenneuronen im Kortex nachempfunden ist. Während ein basales Kompartiment synaptischen Input zusammenfasst, der in Dendriten nahe des Zellkerns auftritt, repräsentiert das zweite apikale Kompartiment die im Kortex anzutreffende komplexe dendritische Baumstruktur. In früheren Experimenten konnte gezeigt werden, dass eine zeitlich korrelierte Stimulation sowohl im basalen als auch apikalen Kompartiment eine deutlich höhere neuronale Aktivität hervorrufen kann als durch Stimulation nur einer der beiden Kompartimente möglich ist. In unserem Modell können wir zeigen, dass dieser Effekt der Koinzidenz-Detektion es erlaubt, den Input im apikalen Kompartiment als Lernsignal für synaptische Plastizität im basalen Kompartiment zu nutzen. Duale Homöostase kommt auch hier zum Tragen, da diese in beiden Kompartimenten sicherstellt, dass sich der synaptische Input hinsichtlich des zeitlichen Mittels und der Varianz in einem für den Lernprozess benötigten Bereich befindet. Anhand eines Lernszenarios, das aus einer linearen binären Klassifikation besteht, können wir zeigen, dass sich das beschriebene Framework für biologisch plausibles überwachtes Lernen eignet.
Die beiden betrachteten Modelle zeigen beispielhaft die Relevanz dualer Homöostase im Hinblick auf zwei Aspekte. Das ist zum einen die Regulation rekurrenter neuronaler Netze in einen dynamischen Zustand, der für Informationsverarbeitung optimal ist. Der Effekt der Adaption zeigt sich hier also im Verhalten des Netzwerks als Ganzes. Zum anderen kann duale Homöostase, wie im zweiten Modell gezeigt, auch für Plastizitäts- und Lernprozesse auf der Ebene einzelner Neuronen von Bedeutung sein. Während neuronale Homöostase im klassischen Sinn darauf beschränkt ist, Teile des Systems möglichst präzise auf einen gewünschten Mittelwert zu regulieren, konnten wir Anhand der diskutierten Modelle also darlegen, dass eine Kontrolle des Ausmaßes von Fluktuationen ebenfalls Einfluss auf die Funktionalität neuronaler Systeme haben kann.