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Rafts: Rafts sind spezialisierte Domänen biologischer Membranen, die sich durch ihre spezifische Lipid- und Proteinzusammensetzung auszeichnen (zur Übersicht siehe Simons und Toomre, 2000). Die am besten beschriebenen Rafts sind die Caveolae, doch es gibt noch weitere weniger gut charakterisierte Rafttypen. Rafts werden verschiedene zelluläre Funktionen zugeschrieben wie z.B. gerichteter Transport von Membranproteinen, Endozytose und Signaltransduktion. Diese Funktionen erfüllen sie vornehmlich, indem sie verschiedene Proteine und Lipide bedingt durch ihre biophysikalischen Eigenschaften selektiv aufnehmen oder ausschließen. Viele Raftproteine sind über gesättigte Acylketten, wie Myristat oder Palmitat, oder einen GPIAnker mit der Membran assoziiert. Transmembranproteine, wie z.B. der EGFRezeptor, können jedoch auch in Rafts angereichert sein. Besonders an der Plasmamembran dienen Rafts als Signaltransduktionszentren, indem sie beteiligte Rezeptoren und Signalmoleküle konzentrieren.
Reggie-Proteine: Bei der Suche nach Proteinen, die bei der Regeneration von verletzten Sehnerven von Fischen hochreguliert werden, wurden Reggie-1 und Reggie-2 entdeckt (Schulte et al., 1997). Gleichzeitig wurden diese Proteine bei der Suche nach neuen Raftproteinen gefunden und als Flotillin-1 (=Reggie-2) und Flotillin-2 (=Reggie-1) bezeichnet (Bickel et al., 1997). Reggie-1 und -2 haben ein Molekulargewicht von 47 kDa und sind auf Aminosäuren-Basis zu 44% identisch. Homologe zu Reggie-1 wurden bislang in Mensch, Maus, Ratte und Fisch, wie auch in D. melanogaster gefunden. Die evolutionäre Konservierung der Reggies ist, mit beispielsweise 80% zwischen Ratte und Goldfisch, sehr hoch und weist auf eine wichtige Funktion hin, die Sequenzkonservierung verlangt. Reggie-1 wird ubiquitär exprimiert, wogegen Reggie-2 ein weniger verbreitetes Expressionsmuster aufweist. Reggie-1 ist vornehmlich an der Plasmamembran und an Endosomen lokalisiert. Die subzelluläre Lokalisation von Reggie-2 hängt vom Zelltyp ab...
Bioactive small molecules are used in many research areas as important tools to uncover biological pathways, interpret phenotypic changes, deconvolute protein functions and explore new therapeutic strategies in disease relevant cellular model systems. To unlock the full potential of these small molecules and to ensure reliability of results obtained in cellular assays, it is crucial to understand the properties of these small molecules. These properties encompass their activity and potency on their designated target(s), their selectivity towards unintended off-targets and their phenotypic effects in a cellular system. Approved drugs often engage with multiple targets, which can be beneficial for some applications such as treatment of cancer where several pathways need to be inhibited for treatment efficacy. However, targeting multiple key proteins in diverse pathways also increases the possibility for unspecific or unwanted side effects. For many drugs the entire target space that they modulate is not known. This makes it difficult to use these drugs for target deconvolution or functional assays with the aim to understand the underlying biological processes. In contrast to drugs, for mechanistic studies, a good alternative are chemical tool compounds so called chemical probes that are usually exclusively selective as well as chemogenomic compounds, that inhibit several targets but have narrow selectivity profiles. Because they are mechanistic tools, chemical tool compounds must meet stringent quality criteria and they are therefore well characterized in terms of their potency, selectivity and cellular on-target activity. To ensure that an observed phenotypic effect caused by a compound can be attributed to the described target(s), it is essential to study also properties of chemical tools leading to unspecific cellular effects. There are a variety of unspecific effects that can be caused by physiochemical compound properties that can interfere with phenotypic assays as well as functional compound evaluations. One of these effects is low solubility causing toxicity or intrinsic fluorescence potentially interfering with assay readouts. But unanticipated cellular responses can also arise from unspecific binding, accumulation in cellular compartments or damage caused to organelles such as mitochondria or the cytoskeleton that can result in the induction of diverse forms of cell death.
In this study, we investigated the influence of a variety of small molecules on distinct cell states, by establishing and validating high-content imaging assays, which we called Multiplex assay. This assay portfolio enabled us to detect different cellular responses using diverse fluorescent reporters, such as the influence of a compound on cell viability, induction of cell death programs and modulation of the cell cycle. Additionally, general compound properties such as precipitation and intrinsic fluorescence were simultaneously detected. The assay is adaptable to assess other cellular properties of interest, such as mitochondrial health, changes in cytoskeletal morphology or phospholipidosis. A significant advantage of the assay is that we are using live cells, so we can capture dynamic cellular changes and fluctuations that can be crucial for the understanding of cellular responses.
Der Hirntumor Glioblastom (GBM) ist aufgrund seines infiltrativen Wachstums, der hohen intra- und intertumoralen Heterogenität, der hohen Therapieresistenz als auch aufgrund der sogenannten gliomartigen Stammzellen sehr schwer zu behandeln und führt fast immer zu Rezidiven. Da es in den letzten Jahrzehnten kaum Fortschritte in der Behandlung des GBMs gab, bis auf die Therapie mit Tumortherapiefeldern, wird weiterhin nach alternativen Zelltodtherapien geforscht, wie zum Beispiel dem Autophagie-abhängigen Zelltod. Der Autophagie-abhängige Zelltod ist durch einen erhöhten autophagischen Flux gekennzeichnet und obwohl die Autophagie, als auch selektive Formen wie die Lysophagie und Mitophagie, normalerweise als überlebensfördernde Mechanismen gelten, konnten viele Studien eine duale Rolle in der Tumorentstehung, -progression und -behandlung aufzeigen, die vor allem vom Tumortyp und stadium abhängt. Um die zugrunde liegenden Mechanismen des durch Medikamente induzierten Autophagie-abhängigen Zelltods im GBM weiter zu entschlüsseln, habe ich in meiner Dissertation verschiedene Substanzen untersucht, die einen Autophagie-abhängigen Zelltod induzieren.
In einer zuvor in unserem Labor durchgeführten Studie konnte gezeigt werden, dass das Antipsychotikum Pimozid (PIMO) und der Opioidrezeptor-Antagonist Loperamid (LOP) einen Autophagie-abhängigen Zelltod in GBM Zellen induzieren können. Darauf aufbauend habe ich die Fähigkeit zur Induktion des Autophagie-abhängigen Zelltods in weiteren Zellmodellen validiert. Dies bestätigte einen erhöhten autophagischen Flux nach PIMO und LOP Behandlung, während der Zelltod als auch der autophagische Flux in Autophagie-defizienten Zellen reduziert war. In weiteren Versuchen konnte ich die Involvierung der LC3-assoziierten Phagozytose (LAP), ein Signalweg der auf die Funktion einiger autophagischer Proteine angewiesen ist, ausschließen. Weiterhin konnte ich eine massive Störung des Cholesterin- und Lipidstoffwechsels beobachten. Unter anderem akkumulierte Cholesterin in den Lysosomen gefolgt von massiven Schäden des lysosomalen Kompartiments und der Permeabiliserung der lysosomalen Membran. Dies trug einerseits zur Aktivierung überlebensfördernder Lysophagie als auch der Zell-schädigenden „Bulk“-Autophagie bei. Letztendlich konnte aber die erhöhte Lysophagie die Zellen nicht vor dem Zelltod retten und die Zellen starben einen Autophagie-abhängigen lysosomalen Zelltod. Da die Eignung von LOP als Therapie für das GBM aufgrund der fehlenden Blut-Hirn-Schranken Permeabilität und von dem Antipsychotikum PIMO aufgrund teils schwerer Nebenwirkungen eingeschränkt ist, habe ich mich im weiteren Verlauf meiner Dissertation mit einer Substanz mit einem anderen Wirkmechanismus beschäftigt.
Der Eisenchelator und oxidative Phosphorylierungs (OXPHOS) Inhibitor VLX600 wurde zuvor berichtet mitochondriale Dysfunktion und Zelltod in Kolonkarzinomzellen zu induzieren. Allerdings hat meines Wissens nach bisher noch keine Studie die therapeutische Eignung von VLX600 für das GBM untersucht. Hier zeige ich eine neuartige Autophagie-abhängige Zelltod-induzierende Fähigkeit von VLX600 für GBM Zellen, da der Zelltod signifikant in Autophagie-defizienten Zellen aber nicht durch Caspase-Inhibitoren gehemmt wurde und der autophagische Flux erhöht war. Darüber hinaus konnte ich die Hemmung der OXPHOS und die Induktion von mitochondrialem Stress in GBM Zellen bestätigen und weiterhin aufzeigen, dass VLX600 nicht nur die mitochondriale Homöostase stört, sondern auch zu einer BNIP3-BNIP3L-abhängigen Mitophagie führt, die wahrscheinlich durch HIF1A reguliert wird aber keinen erkennbaren Nettoeffekt auf den von VLX600 induzierten Zelltod hat. Demnach induziert VLX600 letale „Bulk“-Autophagie in den hier verwendeten Zellmodellen. Darüber hinaus konnte ich zeigen, dass die Eisenchelatierung durch VLX600 eine große Rolle für den von VLX600-induzierten Zelltod spielt aber auch für die Mitophagie Induktion, Histon Lysin Methylierung und den ribosomalen Stress. Letztendlich ist es wahrscheinlich ein Zusammenspiel all dieser Faktoren, die zur Zelltodinduktion durch VLX600 führen und interessanterweise werden Eisenchelatoren bereits in präklinischen und klinischen Studien für Krebstherapien untersucht. Dabei könnten gewisse metabolische Eigenschaften verschiedener Tumorzellen die Sensitivität von Wirkstoffen, die auf den Metabolismus wirken wie VLX600, beeinflussen was in zukünftigen Studien beachtet werden sollte um den bestmöglichsten Therapieerfolg zu erzielen. Zusammenfassend unterstützt meine Dissertation die duale Rolle der Autophagie, die stark vom jeweiligen Kontext abhängt und befürwortet die weitere Forschung von Substanzen, die einen Autophagie-abhängigen Zelltod induzieren, für das GBM.
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 strong force is one of the four fundamental interactions, and the theory of it is called Quantum Chromodynamics (QCD). A many-body system of strongly interacting particles (QCD matter) can exist in different phases depending on temperature (T) and baryonic chemical potential (µB). The phases and transitions between them can be visualized as µB−T phase diagram. Extraction of the properties of the QCD matter, such as compressibility, viscosity and various susceptibilities, and its Equation of State (EoS) is an important aspect of the QCD matter study. In the region of near-zero baryonic chemical potential and low temperatures the QCD matter degrees of freedom are hadrons, in which quarks and gluons are confined, while at higher temperatures partonic (quarks and gluons) degrees of freedom dominate. This partonic (deconfined) state is called quark-gluon plasma (QGP) and is intensively studied at CERN and BNL. According to lattice QCD calculations at µB=0 the transition to QGP is smooth (cross-over) and takes place at T≈156 MeV. The region of the QCD phase diagram, where matter is compressed to densities of a few times normal nuclear density (µB of several hundreds MeV), is not accessible for the current lattice QCD calculations, and is a subject of intensive research. Some phenomenological models predict a first order phase transition between hadronic and partonic phases in the region of T≲100 MeV and µB≳500 MeV. Search for signs of a possible phase transition and a critical point or clarifying whether the smooth cross-over is continuing in this region are the main goals of the near future explorations of the QCD phase diagram.
In the laboratory a scan of the QCD phase diagram can be performed via heavy-ion collisions. The region of the QCD phase diagram at T≳150 MeV and µB≈0 is accessible in collisions at LHC energies (√sNN of several TeV), while the region of T≲100 MeV and µB≳500 MeV can be studied with collisions at √sNN of a few GeV. The QCD matter created in the overlap region of colliding nuclei (fireball) is rapidly expanding during the collision evolution. In the fireball there are strong temperature and pressure gradients, extreme electromagnetic fields and an exchange of angular momentum and spin between the system constituents. These effects result in various collective phenomena. Pressure gradients and the scattering of particles, together with the initial spatial anisotropy of the density distribution in the fireball, form an anisotropic flow - a momentum (azimuthal) anisotropy in the emission of produced particles. The correlation of particle spin with the angular momentum of colliding nuclei leads to a global polarization of particles. A strong initial magnetic field in the fireball results in a charge dependence and particle-antiparticle difference of flow and polarization.
Anisotropic flow is quantified by the coefficients vₙ from a Fourier decomposition of the azimuthal angle distribution of emitted particles relative to the reaction plane spanned by beam axis and impact parameter direction. The first harmonic coefficient v₁ quantifies the directed flow - preferential particle emission either along or opposite to the impact parameter direction. The v₁ is driven by pressure gradients in the fireball and thus probes the compressibility of the QCD matter. The change of the sign of v₁ at √sNN of several GeV is attributed to a softening of the EoS during the expansion, and thus can be an evidence of the first order phase transition. The global polarization coefficient PH is an average value of the hyperon’s spin projection on the direction of the angular momentum of the colliding system. It probes the dynamics of the QCD matter, such as vorticity, and can shed light on the mechanism of orbital momentum transfer into the spin of produced particles.
In collisions at √sNN of several GeV, which probe the region of the QCD phase diagram at T≲100 MeV and µB≳500 MeV, hadron production is dominated by u and d quarks. Hadrons with strange quarks are produced near the threshold, what makes their yields and dynamics sensitive to the density of the fireball. Thus measurement of flow and polarization, in particular of (multi-)strange particles, provides experimental constraints on the EoS, that allows to extract transport coefficients of the QCD matter from comparison of data with theoretical model calculations of heavy-ion collisions.
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This thesis aims to investigate the properties of hadronic matter by analyzing fluctuations of conserved charges. A transport model (SMASH) is used for these studies to achieve this. The first part of this thesis focuses on examining transport coefficients, specifically the diffusion coefficients of conserved charges and the shear viscosity. The second part investigates equal-time correlations of particle numbers in the form of cumulants. The last chapter studies different aspects of the isobar collision systems Ru and Zr.
As a first step, the hadronic medium and interactions between its constituents are introduced, and simultaneously, their impact on transport coefficients is investigated. The methodology is verified by comparing the results of SMASH with Chapman-Enskog calculations, followed by examining 3-to-1 multi-particle reactions, revealing their influence on shear viscosity and electrical diffusion. The analysis of the full hadron gas considers angle-dependent cross-sections and additional elastic cross-sections via the AQM description, showing significant impacts on transport coefficients. The dependency on the number of degrees of freedom is explored, with noticeable effects on diffusion coefficients but a smaller influence on the shear viscosity. At non-zero baryon chemical potential, the diffusion coefficients are strongly influenced, while the shear viscosity remains unaffected. Overall, the study underscores the importance of individual cross-sections and the modeling of interactions on transport coefficients.
The following chapter explores fluctuations of conserved charges, crucial for understanding phase transitions in heavy-ion collision from the quark-gluon plasma to the hadronic phase. Using SMASH, the impact of global charge conservation on particle number cumulants in subvolumes of boxes simulating infinite matter is studied. Comparisons with simpler systems highlights the influence of hadronic interactions on cumulants, especially via charge annihilation processes and the results from SMASH shows agreement with analytical calculations. Calculations at finite baryon chemical potential reveals a transition from a Poisson to Skellam distribution within the net proton cumulants. It is shown that an unfolding procedure to obtain the net baryon fluctuations from the net proton ones deviates from the actual net baryon result, particularly in larger volumes. Finally, net proton correlations at vanishing baryon chemical potential align with ALICE measurements and the net proton cumulants are unaffected by deuteron formation.
In the next step, the goal is to investigate critical fluctuations in the hadronic medium. Therefore, the hadronic system is initialized with critical equilibrium fluctuations by coupling the hadron resonance gas with the 3D Ising model. The single-particle probability distributions are derived from the principle of maximum entropy. Evolving these distributions in SMASH, their development in an expanding sphere adjusted to experimental conditions can be analyzed. It reveals resonance decay and formations as the primary source that affects the particle cumulants. Because of isospin randomization processes, critical fluctuations are better preserved in net nucleon numbers. However, for the strongest coupling investigated in this work, correlations of the critical field are still present in the final state of the evolution in the net proton fluctuations. Examining cumulant dependence on rapidity windows shows a non-monotonic trend.
In the third part, collisions involving the isobars Ru and Zr are studied at a center-of-mass energy of 200 GeV. Initially, SMASH is used to study the initial conditions to hydrodynamical simulations, emphasizing the importance of the nuclear structure of isobars on the geometry of the collision area. It is found that the deformation parameters notably influence the initial state. Correlations between nucleon-nucleon pairs on eccentricity fluctuations yield no significant effect. Subsequently, the hydrodynamic model vHLLE evolves the previously explored initial conditions and for the transition between the hydrodynamic and kinetic descriptions, the Cooper-Frye formula is used. Usage of the canonical ensemble ensures the exact conservation of the conserved charges B, Q, and S. The neutron skin effect, which changes the charge distribution within Ru nuclei, is additionally considered. Fluctuations are assessed, revealing suppression in large rapidity windows due to global charge conservation. The hadronic phase modifies fluctuations of net pions, net kaons, and net protons via annihilation processes, yet fluctuations remain unaffected by the neutron skin effect.
Partial melting of crustal and mantle rocks under pressure from impedance spectroscopy measurements
(2004)
The purpose of this work is to achieve a better understanding of the physical properties of rocks during partial melting processes. The electrical conductivity of some crustal and upper mantle rocks was measured prior and above the melting under pressure. The variations of the electrical conductivity were compared with the distribution of melt in partially molten rock samples. The electrical conductivity was estimated from the impedance spectroscopy at temperatures between 800 and 1450˚C and at pressures between 0.3 and 2 GPa. These measurements were performed in a piston cylinder apparatus. At temperatures above the melting, samples were equilibrated during a long time and subsequently quenched. Thin sections were prepared and topology, volume fraction and chemical composition of melt was analyzed by using a microprobe. Above the solidus temperature, the electrical conductivity increases for about 1 to 2 orders of magnitude in comparison with non-melted rocks. The "melt effect" seems to reflect the formation of an interconnected network of melt. When a complete melt connectivity is established, the charge transport follows the network of the formed melt films at grain boundaries. Usually, it takes a long time in order to reach a steady state of the electrical resistance in partially molten rocks. Only when a steady state of the electrical resistance is achieved, the bulk conductivity of a sample can be measured properly. The time-independent electrical conductivity were found only after 200 h of annealing time at a desired temperature.
Usually, the measurements of a dihedral angle on grain-liquid interfaces in rocks show that the wetting of grain faces start to develop at temperatures slightly above the solidus temperature. The development of these faces should lead to a continuous melt network even at small melt fractions of few wt.%. This result is not confirmed by our electrical conductivity measurements. The complete interconnection of the melt phase, which was mark by an increase of the electrical conductivity, corresponds to a temperature significantly above the solidus temperature, for at least 30-50˚C. The development of these faces stimulate a significant increase of the electrical conductivity, and corresponds to the occurence of at least 5 wt.% of a melt fraction. This result could be explained by deviations from the textural equilibrium of a melt phase topology in partially molten samples due to heterogeneous grain size distribution, misorientation of grains and anisotropy of the superficial energy of adjacent grain boundaries.
Some mixing models that allow to calculate the electrical conductivity of a composite as a function of a melt fraction were examined and the results of these calculations are discussed.
The experimental results were compared to the conductivity data obtained from magnetotelluric and electromagnetic measurements in the Northern part of mid-Atlantic ridge where a series of magma chambers are presumably located. There is a good agreement between our conductivity values for a melt fraction of 10-13 the conductivity estimated in the Reykjanes ridge zone.
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.
Das westphälische Modell für Staatsinstitutionen, einschließlich nationaler Exekutive, Legislative und Judikative, hat sich aus den Ereignissen europäischer Geschichte heraus entwickelt. Seit dem Ende des Kalten Krieges dient es als grundlegendes Paradigma für Internationale Interventionen zum Wiederaufbau von gescheiterten - oder zum Aufbau von neuen - Staaten. Für die internationale Gemeinschaft fungiert das westphälische Modell als Maß zur Beurteilung ihrer Interventionen, wie zum Beispiel in Somalia, Kambodscha oder den Balkanstaaten. In den meisten Fällen gilt eine durch sie beaufsichtigte oder gar durchgeführte ‚freie und faire’ Wahl als hauptsächliche Massnahme zur Bildung eines ‚westphälischen’ und demokratischen Staates. Die Erfolgsrate solcher internationalen Friedenseinsätze und ‚state-building operations’ ist jedoch enttäuschend. Bei näherer Betrachtung der Misserfolge des letzten Jahrzehnts wird deutlich, daß sich die lokalen Gesellschaftssysteme der betroffenen Bevölkerungen oft beträchtlich von liberaler Demokratie unterscheiden. Dies ist insbesondere der Fall in Gesellschaften deren Ordnung nicht auf Staatsinstiutionen basiert. Ihnen liegen sozio-politische Systeme zugrunde die sich oft mit dem Paradigma des westlichen Staatssystems nur schwer vereinen lassen. Um im Rahmen internationaler Friedenseinsätze erfolgreich Staatstrukturen zu etablieren, ist es daher notwendig lokale Sozialstrukturen und lokale Konzepte politischer Legitimität und Autorität zu addressieren. Erst mit solchem Verständnis ist es möglich einen Staatsapparat in den Augen der Bevölkerung zu legitimieren. Ist Letzteres nicht der Fall, so kann sich eine Regierung zwar in Übereinstimmung mit internationalen Menschenrechten befinden, oder alle wichtigen demokratischen Einrichtungen vorweisen, jedoch dennoch dem Prinzip der Partizipation durch die Bevölkerung widersprechen. Ist dies das Endresultat eines internationalen Friedenseinsatzes, so hat die internationale Gemeinschaft ihre eigenen Werte bestaetigt. Jedoch herrscht kein Vertrauen zwischen der Bevölkerung und Regierung, da letztere nicht kompatibel mit dem Versaendnis der Bürger ist. Der ‚demokratische’ Staat ist nur schwerlich funktionsfähig.Der internationale Einsatz in Osttimor illustriert dieses Problem. Hier wurden die Vereinten Nationen (VN) mit dem Wiederaufbau und der Verwaltung eines Staates betraut (UNTAET ‚Übergangsregierung der Vereinten Nationen in Osttimor’). Zum ersten mal in der Geschichte übernahm die international Gemeinschaft damit die Souveränität über ein territoriales Gebiet...
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.
Fungi belonging to the Rhytismatales (Ascomycota) are parasites or endophytes of plants, some are saprophytes. Their fruiting bodies are localized in different organs of the host plants belonging to many different families of gymnosperms and angiosperms. Many species of Rhytismatales are known on species of Pinaceae, Ericaceae, and Poaceae. These fungi usually have ascomata that are more or less embedded in host tissue and open by longitudinal or radial splits. They have a more or less carbonized covering stroma, thin-walled, iodine negative asci, and ascospores usually covered by gelatinous sheaths.
In the present study, two lists of species of Rhytismatales in China are presented. One is based on literature and includes 103 species in 15 genera. The second one contains the names of the species in the present study, 57 species in 20 genera based on 90 specimens I collected in the Yunnan and Anhui province in China during July to August in 2001. 31 species in the second list are new species or new records for China, so we presently know 134 species in 22 genera of Rhytismatales for China. 28 new species of Rhytismatales are proposed, 21 species from the Yunnan province and seven from the Anhui province. Among them, three new species are proposed in three new genera, Nematococcomyces, New Genus 1, and New Genus 2, respectively. The 28 new species are Cerion sp., Coccomyces spp. 1-2, Colpoma spp. 1-2, Hypoderma spp. 1-6, Lirula sp., Lophodermella sp., Lophodermium spp. 1-5, Nematococcomyces rhododendri C.-L. Hou, M. Piepenbr. & Oberw., Neococcomyces sp., New Genus 1 sp., New Genus 2 sp., Rhytisma spp. 1-2, Soleella sp., Terriera spp. 1-2, and Therrya sp. The genus Davisomycella is proposed as a synonym of Lophodermella based on observations of the morphology, ecology, and the infected organ. The four genera Cerion, Naemacyclus, Terriera, and Therrya, and three species, Hypoderma rubi, Lophodermium uncinatum, and Naemacyclus pinastri, are reported for the first time for China. All the new taxa, the newly recorded ones, as well as six species which had not been illustrated in detail before, are carefully described and illustrated by line drawings in the present study.
The results show that species of Rhytismatales are highly diverse especially in the natural vegetation in high mountainous areas in China. Most species of Rhytismatales are conspicuously host specific. The diversity of Rhytismatales is closely related to that of the preferred hosts, which are members of Pinaceae, Ericaceae, and Cupressaceae. Based on the detailed morphological observations, the significance of different morphological characteristics for a natural classification of Rhytismatales is discussed. Genera are traditionally defined by character states of a few characteristics, namely the opening patterns of ascomata, the depth of ascomata in the host tissue, and asci and ascospore shape. Data from collections in the field, detailed morphological investigation, and molecular data show, however, that the ecology, the infected organ, the host relationship, and many other characteristics have to be combined to circumscribe natural groups.
The discussion of the systematic significance of morphological characteristics is complemented by molecular data. In the present study, partial nuclear large subunit rDNA sequences of 52 specimens representing 38 species are used to analyse phylogenetic relationships for members of Rhytismatales.
Most species of Rhytismatales are placed in a monophyletic group corresponding to the Rhytismatales in the Maximum Parsimony analysis. The delimitation of the Rhytismatales from the Helotiales is, however, difficult. Cyclaneusma minus should be transferred from the Rhytismatales to the Helotiales, and Cudonia circinans and Spathularia flavida from the Helotiales to the Rhytismatales. These tranfers have previously been proposed based on SSU rDNA analysis by other authors. New Genus 1 sp. has morphological characteristics typical for species of Rhytismatales. In the LSU rDNA analysis, however, it is more closely related to Helotiales rather than toRhytismatales. Therefore New Genus 1 sp. is placed in the Helotiales.
Tryblidiopsis pinastri is morphologically intermediate between members of Rhytismataceae and Cudoniaceae. LSU rDNA sequences in the present study show that T. pinastri is more closely related to species of Cudoniaceae. Therefore, this species is removed from the Rhytismataceae to the Cudoniaceae. The delimitation of further families could not be resolved in the present analysis.
Though many new morphological, ecological, and molecular phylogenetic findings are contributed for the first time, the systematic conclusions at generic, family, and order level can only be fragmentary in the present study. With more collections and more molecular data of the worldwide 450 known and many more unknown species of Rhytismatales at hand, a natural system combining morphological and molecular analysis can be elaborated.
Cytochrome P450 (CYP) enzymes oxidize, peroxidize and/or reduce cholesterol, vitamins, steroids, xenobiotics and numerous pharmacological substances in an oxygen- and NADPHdependent manner. Since many CYP isozymes are also capable of metabolizing arachidonic acid to biologically active products, CYP enzymes are often described as the third pathway of arachidonic acid metabolism i.e., in addition to cyclooxygenases and lipoxygenases. CYP enzymes are predominantly expressed in the liver while others, such as members of the CYP 2J, CYP 2C and CYP 4A subfamilies, can be detected in extrahepatic tissues, particularly in the cardiovascular system. Recent data suggest that a CYP 2C enzyme(s) expressed in coronary artery endothelial cells generate epoxyeicosatrienoic acids (5,6-; 8,9-; 11,12- and 14,15-EET) which contribute to the acute control of vascular tone and the longterm regulation of vascular homeostasis.
The expression of CYP 2C in coronary artery endothelial cells is regulated by a number of stimuli, such as cyclic stretch and fluid shear stress as well as by the corticosteroid cortisol and a number of CYP substrates (nifedipine, cerivastatin and -naphthoflavone). However, the signalling pathways and the transcription factors involved in regulating the expression of the gene are unknown.
Since most of the CYP 2C enzymes are transcriptionally regulated, we were interested in identifying the CYP 2C isoform(s) expressed in porcine coronary artery endothelial cells (PCAEC) as well as determining its/their promoter sequence(s). The overall goal was to study the involvement of different transcription factor binding elements in the regulation of the CYP 2C gene(s). Porcine coronary arteries were used given the possibility of analysing the results obtained at the cellular level with alterations in vascular function. Comparison of the porcine CYP 2C and the human CYP 2C8 and 2C9 promoters was also a major goal of this study.
To identify the relevant porcine CYP 2C isoform nested RT-PCR was performed using total RNA from porcine coronary artery endothelial cells. Comparison of the sequence of the product of this reaction with the NCBI database suggested that the CYP 2C expressed in PCAEC was approximately 85% homologous with the human CYP 2C9 enzyme. To obtain the full length CYP 2C isoform 5´ rapid amplification of cDNA end (5´ RACE) was performed using a downstream reverse gene specific primer which is conserved in all of the porcine CYP 2C isoforms. The intention behind using such a primer was to amplify all the possible CYP cDNAs expressed in PCAEC. With the 5´ RACE technology it was possible not only to identify the exact isoform (CYP 2C34) expressed in PCAEC, but it was also possible to amplify 550 bp of the 5´ upstream region. This result was authenticated by comparing the protein/nucleotide sequence with other human CYP 2C genes such as CYP 2C8 and CYP 2C9 as well as different porcine CYP 2C genes (CYP 2C34, CYP 2C49). Multiple protein/nucleotide sequence alignment revealed approximately 85-90% sequence identity. An exon1-2 specific radio-labelled probe of the CYP 2C34 gene was then used to screen a porcine genomic library for positive genomic clones containing the promoter region of the CYP 2C34 gene.
For the isolation of 5´ flanking region of CYP 2C34 gene a PCR-based directional genome walking strategy was used in which the positive porcine genomic BAC clones were taken as a DNA template. Four arbitrarily designed universal walking primers and a gene-specific primer derived from the CYP 2C34 gene sequence were employed and led to the identification and isolation of 1.4 kb of the 5´ flanking region.
The 1.4 kb 5´ flanking region of CYP 2C34 gene contains multiple transcription factor binding sites including glucocorticoid-responsive element (GRE), hypoxia-responsive element (HRE), CAAT-enhancer binding protein (C/EBP), stress responsive element (STRE) consensus sequences. CYP 2C34 promoter constructs were generated and reporter gene activity (luciferase) activity was compared with that of a promoterless vector (pGL3-Basic) at first in HEK cells and then in PCAEC. After using cortisol as a positive control to demonstrate that the promoter constructs generated were functional we determined the effects of physiologically relevant stimuli i.e., hypoxia and cyclic stretch. Additional experiments with zinc sulphate were performed in a preliminary analysis of the role of Zn2+ inducible transcription factors and might be cooperative heterodimerization formation with these transcription factor with C/EBP in the regulation of CYP 2C34 expression. With all these stimuli, reporter gene activity of CYP 2C34 promoter was significantly (3-8 fold) increased over values obtained in unstimulated cells.
Analysis of the regions that are essential for the induction of promoter activity in response to the different stimuli of interest have to be performed in combination with gel shift assays, siRNA experiments as well as site-directed mutagenesis experiments. Comparison of the regulation of the CYP 2C34 gene and correlation with changes in vascular function (in isolated porcine coronary arteries) should deliver information relevant to the regulation of the CYP 2C enzyme expressed in human coronary artery endothelial cells. The recent demonstration of a clinically relevant role for CYP 2C9 in coronary heart disease underlines the importance of such a study.
Determination of the structure of complex I of Yarrowia lipolytica by single particle analysis
(2004)
Komplex I enthält ein Flavinmononukleotid sowie mindestens acht Eisen- Schwefel Zentren als redoxaktive Cofaktoren. Da ein wesentlicher Teil des mitochondrialen Genoms für Untereinheiten von Komplex I codiert, betrifft eine Vielzahl von mitochondrialen Erkrankungen diesen Enzymkomplex.
Komplex I wurde bisher aus Mitochondrien, Chloroplasten und Bakterien isoliert. Die Minimalform von Komplex I wird in Bakterien gefunden, wo er aus 14 (bzw 13 im Falle einer Genfusion) Untereinheiten besteht und eine Masse von etwa 550 kDa aufweist. Generell werden sieben hydrophile und sieben hydrophobe Untereinheiten mit über 50 vorhergesagten Transmembranhelices gefunden. Im Komplex I aus Eukaryoten wurde eine grössere Anzahl zusätzlicher, akzessorischer Untereinheiten nachgewiesen. Hier werden die sieben hydrophoben Untereinheiten vom mitochondrialen Genom codiert, während alle anderen Untereinheiten kerncodiert sind und in das Mitochondrium importiert werden müssen.
Die obligat aerobe Hefe Yarrowia lipolytica wurde als Modellsystem zur Untersuchung von eukaryotischem Komplex I etabliert. Die bisher am besten untersuchte Hefe Saccharomyces cerevisiae enthält keinen Komplex I. Hier wird die Oxidation von NADH durch eine andere Klasse von sogenannten alternativen NADH Dehydrogenasen durchgeführt. Auch Y. lipolytica enthält ein solches alternatives Enzym, das allerdings mit seiner Substratbindungsstelle zur Aussenseite der inneren Mitochondrienmembran orientiert ist. Durch molekularbiologische Manipulation konnte eine interne Version dieses Enzymes exprimiert werden, wodurch es möglich ist, letale Defekte in Komplex I Deletionsmutanten zu kompensieren. Mittlerweile wurden alle Voraussetzungen geschaffen, um kerncodierte Untereinheiten von Komplex I aus Y. lipolytica gezielt genetisch zu verändern. Die Proteinreinigung wird durch die Verwendung einer auf einem His-tag basierenden Affinitätsreinigung erheblich erleichtert...
The transcriptional regulator RcsB controls the expression of a minimum of 20 different genes having diverse functionalities and biosynthetic operons in the family of Enterobacteriaceae. While in the heterodimeric complex with the co activator RcsA, the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various co activators, resulting in recognition of different DNA targets. In this study the interaction of RcsB with some of these DNA targets have been analysed by a diverse array of techniques including gel shift assay and SPR. The solution structure of the C-terminal DNA-binding domain of RcsB from Erwinia amylovora spanning amino acid residues 129-215 has been solved in this study by heteronuclear NMR spectroscopy. The C-terminal domain is composed of four α-helices where the two central helices of the H-T-H motif are similar to the structures of the regulatory proteins GerE, NarL and TraR. The DNA-binding activity of the C-terminal domain alone is established for the first time in this study and was specified by fluorescence spectroscopy, SPR and NMR titration experiments. The molecular interaction between the individual RcsB domains was analysed by cross-linking experiments and heteronuclear NMR spectroscopy and the amino acid residues of the C-terminal domain involved in this interaction were identified precisely. Another important part of this project was the cell-free production of different Trp analogue labelled RcsB protein. RcsB protein was produced in quite a good yield with different Trp analogue having spectrally enhanced properties. The isolated RcsB alloproteins proved to be ideal for protein interaction studies by fluorescence spectroscopy and the very first evidence of an oligomerization of RcsB due to molecular association has been put forth from these studies. The phosphorylated state of the RcsB protein was mimicked by a beryllofluoride complex in order to study its role in transcriptional regulation. It was found that RcsB alone could bind to DNA targets upon this modification by the beryllofluoride complex. Thus the phosphorylation of the protein that involves the Asp 56 residue induces a structural change of the protein followed probably by a domain movement also, so that the C-terminal domain having the H-T-H DNA binding motif that was previously eclipsed by the N-terminal domain is relieved of this constraint.
Taphonomy and palaeoecology of Laetoli as well as Makuyuni, Arusha region in northern Tanzania
(2004)
This thesis is the result of the Hominid Corridor research Project in Tanzania since 1993 to 1995 that include Pliocene and Pleistocene localities. The localities under study include Laetoli and Manyara area in Arusha Region, northern Tanzania. The thesis has the following specific objectives: firstly, to identify taxa recovered from the studied assemblages; secondly, to underpin taphonomic history of the assemblages under study; thirdly, to elucidate further palaeoecological reconstruction of the assemblages; and finally, to examine surface fossil fauna modifications including agents of modifications either hominids or carnivores.
The Upper Laetolil Beds are dated at 3.5 million years ago (Ma) and the Ndolanya Beds are bracketed in age between 3.5 and 2.41 Ma. The Naibadad Beds, also from Laetoli area, are date to be between 2.2 to 2.1 Ma. The Naibadad Beds are correlated with the base of Bed I at Olduvai Gorge. There are so far no absolute dates for Manyara assemblages. Based on biostratigraphic correlation, the younger overlying unit, the Upper Manyara Beds are estimated to belong to Later Pleistocene and the Lower Manyara Beds are estimated to belong to Early Pleistocene. The Upper Manyara Beds are correlated to the age of Bed III at Olduvai Gorge, while the Lower Manyara Beds are interpreted to span the same contemporaneity with the upper part of Bed II at Olduvai Gorge.
At Laetoli localities, terrestrial mammals while localities from Manyara besides terrestrial mammals dominate fauna; they include aquatic species such as fish, crocodiles and hippopotamus. The main families recovered from Upper Laetolil Beds complement those already recovered from former research works by other workers. This is also true for the younger overlying stratigraphic horizon, the Upper Ndolanya Beds. Thus, mammalian families recovered from Upper Laetolil Beds include Bovidae, Carnivora, Elephantidae, Equidae, Lagomorpha, Suidae, Rodentia, Hominoidea and Rhenocerotidae. Remains of an invertebrate, Gastropoda were also recovered. For Upper Ndolanya Beds include almost the same families recovered from Upper Laetolil Beds, but based on former recovery of fossil fauna, these Beds outnumber greatly the Upper Laetolil Beds in bovid composition by 20 per cent. Such a change in species composition is noticed also from South African localities and East African localities such as the East Turkana. This is interpreted to be due to climatic change drier environments that included species adapted to such palaeoclimates.
For the first time, our team has been able to retrieve specimens identifiable to taxa, a pattern that not possible from previous workers who claimed to have recovered too sparse specimens to be identifiable to any taxon.
The Upper Manyara Beds as well as Lower Manyara taxonomic composition include aquatic species besides the large terrestrial mammalian fauna retrieved from there. In due regard, the former horizon is attributed to have affinity with Olduvai Bed III components and the latter, older horizon, is attributed to have affinity with upper parts of Bed II times at Olduvai Gorge. The Lower Manyara Beds can be said to have, in relative terms, affinity to species recovered from site RC 11 of the Chiwondo Beds, Malema region in northern Malawi, although the former site may be equable to the terminal age of the latter locality.
Fossil hominid remains; attributable to genus Homo and possibly species Homo erectus have been recovered from two localities, Mk 2 and Mk, along Lower Manyara Beds. On the other hand, stone tools, identified to belong to the Acheulian industrial technocomplex, were recovered from site Mk 4.
All of fossil fauna from Laetoli sites were mostly exfoliated and there shows to be little effect in terms of hydrodynamic sorting of the fossil bones. However, intense carnivore activity is witnessed due to the almost one to one ratio of proximal to distal ends. This is also true for the Lower Manyara Beds locality. Through examination of surface modifications of the fossil fauna, it has been established that there was carnivore consumption of ungulates. There is no evidence of hominid involvement that has to be testified by stone tools.
Nitric oxide (NO) is a potent mediator with pleiotropic functions such as inhibition of platelet aggregation, smooth muscle relaxation and regulation of neuronal transmission. These effects are mostly mediated by intracellular NO-sensitive guanylyl cyclases (GCs) which convert GTP into the second messenger, cGMP. This messenger in turn activates multiple downstream effectors such as cGMP-dependent protein kinases, cGMP-regulated ion channels and cGMPdependent phosphodiesterases. Mammalian NO-sensitive GCs are obligate heterodimers of an α and β subunit each. Given that these enzymes play a key role in cGMP-mediated pathways, one may anticipate that mechanisms other than allosteric activation via NO may exist to regulate the production and turnover of cGMP. In this thesis, novel aspects of the regulation of the most abundantly expressed GC heterodimer α1β1 are presented.
A possible mechanism of regulation that was tested here, is tyrosine phosphorylation. Using anti-phosphotyrosine antibodies, the phosphorylation of the β1 subunit was detected after incubation of β1-overexpressing COS-1 cells with protein tyrosine phosphatase (PTP) inhibitors such as pervanadate and bpV(phen). β1 phosphorylation on tyrosines was also observed in PC-12 cells which endogenously express GC and in rat aorta after inhibition of PTPs. Furthermore, hydrogen peroxide was found to be a physiological stimulus for the induction of reversible β1 tyrosine phosphorylation in intact cells. Using phenylalanine mutants of different tyrosines, residue 192 (Y192) of β1 was identified as the major phosphorylation site. Consistent with this finding, sequence analyses showed that Y192 forms part of a motif that resembles a preferential target site for Src-like kinases. When tyrosine-phosphorylated, this motif exposes a typical SH2 docking site for members of the Src kinase family.
Experiments with inhibitors of Src kinases, PP1 and PP2, clearly showed that phosphorylation of Y192 is Src-dependent. Preincubation of β1-expressing cells with these inhibitors significantly reduced the level of phosphorylated β1 after bpV(phen) treatment. Furthermore, co-expression of β1 with Src led to a strong phosphorylation of this subunit. Co-precipitation experiments showed that Src interacts with GC. Interestingly, kinases of the Src family are recruited to β1 via the SH2 domain upon phosphorylation of Y192. Together, these results indicate that Src kinases phosphorylate tyrosine 192 thereby creating a docking site for their own SH2 domains. Kinase bound to GC may then catalyze phosphorylation of GC or other downstream effectors. Inhibition of PTPs altered GC activity in two ways: it increased both the basal activity and the YC-1- and BAY 41-2272-stimulated activity two-fold, and it reduced the sensitivity of the enzyme towards NO. The detailed mechanism of action is still unknown, but experiments using the mutant β1[Y192F] demonstrated that residue 192 is not responsible for these effects.
Another major focus of this thesis was the identification of novel GC binding proteins. Using the yeast two-hybrid approach, the carboxy-terminal portion of a protein named AGAP1 (amino acid (aa) 399-804) was found to interact with the catalytic domain of α1 (aa 466-690) and with the regulatory domain of β1 (aa 1-348). Human AGAP1 is a multidomain protein of 804 amino acids with a calculated molecular mass of 89,1 kDa comprising an Arf-GAP (GAP:GTPase activating protein), a putative GTPase domain, two Ankyrin repeats and a PHdomain. Co-precipitation experiments using lysates from mammalian cells overexpressing both binding partners confirmed the interaction of AGAP1 with the GC subunits. Immunofluorescence analyses demonstrated that AGAP1 co-localizes with GC in the cytoplasm of COS-1 cells.
In Northern blots, AGAP1 mRNA was detected in various human and murine tissues showing a comparable expression pattern described for the mRNA of α1 and β1. Using an AGAP1-specific antibody, endogenous protein was precipitated from lysates of HEK-293 cells derived from human embryonic kidney. The same antibody efficiently cross-reacted with the rat homologue (rAGAP1) and immunoprecipitated endogenous rAGAP1 from lysates of PC-12 cells, aorta and heart. The molecular mass of rAGAP1 is larger than that of the human protein, possibly due to an additional exon present in the rat genome. Like β1, AGAP1 is a substrate for tyrosine kinases. Phosphorylation of AGAP1 was detected after inhibition of PTPs or by coexpression of Src. Furthermore, the kinase inhibitor PP2 strongly impaired phosphorylation of AGAP1 after pervanadate treatment suggesting that tyrosine kinases of the Src family are involved. Measurements of cGMP production showed that AGAP1 has no influence on the activity of NO-sensitive GC. Interestingly, inhibition of PTPs potently increased the complex formation between AGAP1 and GC indicating that the interaction between these two proteins is modulated by reversible tyrosine phosphorylation. Whether this effect is due to the phosphorylation of AGAP1 or GC is still unknown. AGAP1 associates with endosomes and exposes Arf-GAP activity towards Arf1 and Arf5 which are involved in vesicular transport. Thus, one may hypothesize that binding of α1β1 to AGAP1 targets GC to distinct subcellular compartments in close proximity to cGMP-dependent effectors, thereby optimizing cGMP generation and fostering cGMP-driven actions.
Taken together, these results demonstrate that beside the modulation of GC by NO the enzyme is regulated by tyrosine phosphorylation and interaction with AGAP1.
Aim of the present study was the characterization of the RORa receptor (Retinoidrelated Orphan Receptor a). RORa is a member of the nuclear receptor family and is involved into the differentiation of Purkinje cells, inflammation, arteriosclerosis, and bone mineralization. Nuclear receptors are transcription factors and mediate biological responses within target cells to outer signals such as lipophilic hormones. They are involved in development, growth, differentiation, proliferation, apoptosis, and maintenance of homeostasis. Ligand binding, posttranslational modifications, and cofactor recruitment control their activity. Nearly all nuclear receptors share a common modular structure with an Nterminal A/B region, a DNA-binding domain (DBD) that is composed of two zinc finger motifs, a hinge region, and a C-terminal ligand-binding domain (LBD). The RORs comprise the subtypes RORa, RORb, and RORg, which are encoded by different genes. All isoforms of the respective subtypes only differ in their A/B domain. This study focused mainly on the exploration of the gene structure, expression, and subcellular distribution of RORa...
Metastatic rhabdomyosarcoma (RMS) is one of the most challenging tumor entities in pediatric oncology caused by treatment resistances and immune escape. Novel chimeric antigen receptor (CAR) immunotherapies as specific, effective and safe treatment provide antitumor cytotoxicity by soluble factors and ligands/receptor signals. Besides its intrinsic potential as innate immune cell the ErbB2-sprecific CAR-engineered natural killer (NK)-92 cell line NK-92/5.28.z also provides CAR-mediated cytotoxicity, resulting in a high lytic capacity against 2D and 3D RMS cell structures in vitro. Also in a xenograft model using immune deficient NOD/Scid/IL2Rγ-/- (NSG) mice inhibited NK-92/5.28.z the tumor growth as long as the cells were administered and therefore prolonged the survival of the animals. The NK-92/5.28.z were distributed by the blood circulation and subsequently infiltrated the tumor tissue. Due to the malignant origin of the NK-92 cell line the cells must be irradiated prior to the use in patients. While the irradiation hampered the proliferation of NK-92/5.28.z cells, the cytotoxicity against RMS cells in vitro is retained for at least 24 hours. In the xenograft model irradiated NK-92/5.28.z cells inhibited the tumor growth but to a lower extent than untreated cells, as irradiated cells have only a limited life span in vivo no durable persistence and remission was achieved. Therefore, combinatorial approaches were focused and while blocking of the PD-1/PD-L1 axis did not resulted in a significantly enhanced tumor cell lysis, the combinatorial treatment with proteasome inhibitor bortezomib exhibited a significant enhanced cytotoxicity against RMS cells at least in vitro. Bortezomib itself induces caspase mediated apoptosis and also the upregulates the expression of TRAIL receptor DR5. The corresponding ligand TRAIL is expressed on the surface of the NK-92/5.28.z and pursuing experiments with purified TRAIL and bortezomib revealed a synergism. NK-92/5.28.z as an off-the-shelf product is therefore feasible for the therapy of metastatic RMS, but it might be necessary to support the cytotoxicity by additive agents like proteasome inhibitor bortezomib to archive durable remission.
Another cell population suitable for RMS CAR-immunotherapy are cytokine induced killer (CIK) cells, a heterogenous cell population generated from autologous PBMCs consisting of T, NK and T-NK cells. Lentivirally transduced ErbB2-specific CAR-CIK cells were previously shown to inhibit the tumor engraftment in a RMS xenograft model. However, lentiviral transduced adoptive immunotherapies bear risks for the transfer in patients, therefore the Sleeping Beauty Transposon System (SBTS) as a non-viral method, which integrates the CAR coding DNA by a cut-and-paste mechanism from a minicircle (MC) into the CIK cells genome is more feasible for the generation of CAR-CIK cells. The Sleeping beauty transposase mRNA and the MC were transferred in the cell by nucleofection, different factors influence the transfection efficiency and viability of the CIK cells in this harsh procedure. In preliminary experiments with MC Venus, a MC encoding eGFP, the highest transfection efficiency with the best proliferative capacity was achieved with cells on day 3 of CIK culture and without the addition of autologous monocytes as feeder cells. For the CAR construct the protocol was further improved by adjusting crucial factors, for this construct the best results were achieved on day 0, without irradiated PBMCs as feeder cells and cultivation in X-Vivo10 medium supplemented with human fresh frozen plasma. The X-Vivo10 medium enhanced the percentage of NK- and T-NK cells significantly compared to CAR-CIK cells cultured in RPMI. Since the gene transfer by SBTS resulted in CAR-CIK cells stably expressing a CAR in all subpopulations, resulting in a significantly enhanced cytotoxicity against RMS cells in vitro, these cells were compared to lentiviral transduced CAR-CIK cells in vitro and in vivo. While the SBTS CAR-CIK cells were superior to viral CAR-CIK cells in 2D short-term assays, the viral cells showed higher lytic capacity in 3D spheroid long-term assays. In a RMS xenograft model lentiviral CAR-CIK cells significantly prolonged the survival of mice and persisted, whereas SBTS CAR-CIKs did not favor the overall survival compared to untreated controls and also did not persist. Phenotypic analysis revealed a highly cytotoxic CD8+ and late effector memory dominant phenotype for SBTS CAR-CIK cells supporting short-term cytotoxicity but also more prone for exhaustion, while viral CAR-CIK cells showed a more balanced phenotype for memory and cytotoxicity. Therefore, the SBTS is feasible for the ErbB2-CAR gene transfer in CAR-CIK resulting in a stable CAR-expression with high short-term cytotoxicity, but these cells are also more prone to exhaustion and the protocol might be adapted further to prevent this limitation for in vivo application.
This work underlines the hard-to-treat characteristics of metastatic RMS, but also shows some approaches for further evaluation like the combination of NK-92/5.28.z cells with bortezomib and the feasibility of the generation of CAR-CIK cells via SBTS.
In narratology, a widely recognized method involves exploring the connection between implied authors and implied readers. It entails correlating abstract narrative components within a text to understand the conveyed message and the multitude of interpretations it can offer. The present study adopts an implied reader-oriented approach to analyze three selected novels from the twentieth and twenty-first centuries—one Nigerian, one Caribbean, and one Kurdish. The aim is to explore the potential readings within these texts, considering the hermeneutic process of critical reading. The selected texts include Chinua Achebe’s Things Fall Apart, (1958), Same Selvon’s The Lonely Londoners, (1956), and Karwan Kakesur’s The Channels of the Armed Monkeys, (2011). This approach closely examines the communication between the author and reader of the text, with a special focus on the varying levels of communication between the components of the narration, including fictional and implied fictional communication.
The implied fictional communication occurs between a narrative agent known as ‘the implied author’ and its fictional counterpart ‘the implied reader’ rather than between the real, flesh and blood authors and readers. I argue that this level of communication is coded, and the act of decoding it is part of the reading process performed by the reader. Certain texts can propose different and sometimes opposing readings which are initially and purposefully designed by the implied author and addressed to different implied readers. These readings are not necessarily the results of different real readers but rather incorporated ones predetermined by the implied author only to be acknowledged and uncovered by the readers. In other words, the latent meaning is and always was an integral part of the text and is not something created by the imaginative reader or critic. The core interest of my thesis lies in identifying prompts and suggestions within the narrative of the selected texts and ultimately understanding the readerships prestructured in them. Identifying the different readers within those texts will provide new reinterpretations that can add undetected values to the reading process and sometimes suggests opposing readings to how those texts have so far been read. Additionally, it is the objective of this thesis to propose new ways that readers can interact with reading literature that would result in a more aesthetic and entertaining reading experience besides providing ways to be more informed and aware of the cues certain narrative texts contain.
There have been numerous critical studies on both narratology and postcolonial or minority literatures; however, there has been little scholarly work that attempts to utilize narratology as a theoretical foundation for understanding postcolonial and minority fiction.
This study examines fictional texts from Nigerian, Caribbean, and Kurdish literature, employing the narratological concept known as ‘Multiple Implied Readers’. By incorporating concepts from Brian Richardson’s ‘Singular Text, Multiple Implied Readers’, and Peter J. Rabinowitz’s ‘authorial audiences’, I explore the various readerships that the texts could encompass. This exploitation may lead to the discovery of new readings, interpretations, and meanings that would otherwise remain undetected. These structures introduce provocative indeterminacies that challenge the reader’s synthesis of information into coherent configurations of meaning. Consequently, this approach not only enhances the reading experience but also opens doors to new interpretations of the text. In some cases, these interpretations could even dismantle prior understandings and propose entirely new readings.
The concepts of the implied author and implied reader have been studied before in relation to various disciplines of narratology. However, by applying them in conjunction with the relatively less researched subject of multiple implied readers, I aim to shed light on important aspects of these readings. This exploration could prove beneficial for literature students as well as critical readers of literary texts, revealing the potential of these texts to accommodate more than one implied reader within their narratives.
While high-quality climate reconstructions of some past warm periods in the Cenozoic era now exist, the geological processes responsible for driving the observed longterm changes in atmospheric CO2 are not sufficiently well understood. The long-term change in atmospheric CO2 across the Cenozoic has been proposed to be driven by processes such as terrestrial weathering, organic carbon production and burial, reverse weathering, and volcanic degassing. One way of constraining the relative importance of the various driving forces proposed so far is to better understand the degree to which ocean chemistry has changed because the chemistry of seawater responds to geologic processes that drive atmospheric CO2. In addition, knowledge of the concentration of the major elements in seawater is crucial for accurately applying proxies such as those based on the boron isotopic composition and Mg/Ca of marine carbonates (a proxy for palaeo pH/CO2 and palaeotemperature, respectively). Previously reported records of seawater composition are primarily derived from fluid inclusions in marine evaporites; however, the results are sparse due to the limited availability of such deposits. In this thesis, changes in the Eocene seawater chemistry were reconstructed using trace element (elements/Ca) and isotopic (δ26Mg) proxies in a Larger Benthic Foraminifera (LBFs), i.e., Nummulites sp., to constrain the driving processes of long-term changes in seawater chemistry.
To achieve the objective of this thesis, first, a measurement protocol was established using LA-ICPMS to measure the K/Ca ratio simultaneously with other element/calcium ratios, which is challenging due to the interference of ArH+ on K+. Utilising this newly established measurement protocol, laboratory-cultured Operculina ammonoides grown at different seawater calcium concentrations ([Ca2+]), repeated at different temperatures, as well as modern O. ammonoides collected from different regions exhibiting a range of seawater parameters, were investigated. A significant correlation was observed between K/Casw and K/CaLBF, allowing K/CaLBF to potentially be used as a proxy for seawater major ion reconstructions. In addition, modern O. ammonoides demonstrated no significant influence of most seawater parameters (temperature, salinity, pH, or [CO32-]) on K/CaLBF. Modern O.
ammonoides were also assessed for their Mg isotopic composition (δ26Mg), revealing no significant effect of temperature or salinity on δ26MgLBF. Furthermore, the Mg isotopic fractionation in O. ammonoides was found to be close to that of inorganic calcite, indicating minimal vital effects in these large benthic foraminifera.
Operculina ammonoides is the nearest living relative of the abundant Eocene genus Nummulites, enabling the reconstruction of seawater chemistry using the calibration based on O. ammonoides. The trace elemental/calcium proxies, including Na/Ca, K/Ca, and Mg/Ca, as well as the δ26Mg proxy, were investigated in Eocene Nummulites. The result showed that during the Eocene, [Ca2+]sw was 1.6-2 times higher, while [K+]sw was ~2 times lower than the modern seawater composition. Furthermore, [Mg2+]sw decreased from the early Eocene (54.3− +9 7..69 mmol kg-1 at ~55 Ma) to Late Eocene (37.8− +4 4..3 4 mmol kg-1 at ~31 Ma), followed by
an increase toward modern seawater [Mg]. In contrast, the variability in δ26Mgsw values remained within a narrow range of ~0.3 ‰ throughout the Cenozoic. The reconstructed [Ca2+]sw agrees with the suggestion that Cenozoic seawater chemistry changes can be explained via a change in the seafloor spreading rate. When combined with existing records, the observed minimal change in δ26Mgsw with an increase in [Mg2+]sw suggests an additional possible role of a decrease in the formation of authigenic clay minerals coincident with the Cenozoic decline in deep ocean temperature, which is also supported by the increase in the [K+]sw reconstructed here for the first time. This finding highlights that the reduction in seafloor-spreading rate and decline in reverse weathering during the Cenozoic era has played a significant role in the evolution of seawater chemistry, emphasizing the importance of these processes in driving long-term changes in the carbon cycle.
The core of this work is represented by the investigation of the chiral phase transition, using Monte Carlo simulations and unimproved staggered fermions, both in the weak and strong coupling regimes of Quantum Chromodynamics. Based on recent results from Monte Carlo simulations, both using unimproved staggered fermions and Wilson fermions, the chiral phase transition in the continuum and chiral limit shows compatibility with a second-order phase transition for Nf (number of flavours) in range [2:7], at zero baryon chemical potential. This achievement relies on the analytic continuation of Nf to non-integer values on the lattice, which allows to make use of extrapolation techniques to the chiral limit, where simulations are not possible. Furthermore, these results provide a resolution to the ambiguous scenario for Nf = 2 in the chiral limt. The first part of this thesis is devoted to the investigation of the chiral phase transition when a non-zero imaginary baryon chemical potential is involved, whose value corresponds to the 81% of the Roberge-Weiss one. Using the same extrapolation techniques aforementioned, the order of the chiral phase transition in the continuum and chiral limit shows compatibility with a second-order phase transition for Nf in range [2:6], highlighting a lack of dependence of the order of the chiral phase transition on the imaginary baryon chemical potential value. The second part of this thesis is about the study of the extension of the first-order chiral region in the strong coupling regime, at zero baryon chemical potential. Using Monte Carlo techniques, this can be done by investigating the Z2 boundary on a coarse lattice, whose temporal extent reads Nt = 2, and simulations are realised for Nf = 4, 8. The results in the weak coupling regime show, for $Nt = 8, 6, 4 and fixed Nf value, an inflating first-order chiral region. As in the strong coupling limit a second-order chiral phase transition is expected, the first-order chiral region has to shrink as the strong coupling regime is approached, resulting in a non-monotonic behaviour of the Z2 boundary. For Nf = 8, a critical mass on the Z2 boundary has been obtained, confirming the expected non-monotonic behaviour. For Nf = 4 the results do not provide a unique conclusion: Either a Z2 boundary at extremely low bare quark mass or a second-order chiral phase transition in the O(2) universality class in the chiral limit can take place. In addition to the two main topics, the performances of the second-order minimum norm integrator (2MN) and the fourth-order minimum norm integrator (4MN) have been compared, after implementing the 4MN one in the CL2QCD code used to realise our simulations. The 2MN integrator had already been implemented in the code since the first version was released. The two integrators belong to the class of symplectic integrators and represent an essential component of the RHMC algorithm, involved in our investigation. This step is extremely important, in order to guarantee the best quality when collecting data from simulations, and the results of the comparison suggested to favor the 2MN integrator, for both the topics.
Abdominal aortic aneurysm (AAA) is the most common type of aortic aneurysm, which is defined as a dilation of the abdominal aorta over 3.0 cm or more. Surgical repair is the golden standard for the treatment of AAA, in which open surgical repair (OSR) and endovascular aneurysm repair (EVAR) are the main approaches. Technically speaking, the lesion segment of aueurysm is completely replaced by a graft during OSR, while in EVAR, the lesion is insulated by a stentgraft. EVAR is a less invasive treatment than OSR and shows a lower early mortality rate, although the long-term advantages of EVAR over OSR remain inconclusive.
Endoleak, especially the type II endoleak (T2EL), is a common complication after EVAR. According to research, 16-28% of the patients develop a T2EL after EVAR, and it accounts for nearly three in four of all types of endoleaks. Around 30-50% of the T2EL resolved spontaneously during the follow-up, however, it still causes a secondary intervention in many patients. Therefore, it is critical to monitor endoleaks after repair.
Patent aortic branches in the stent-overlapped area and vasa vasorum have been identified as potential sources of blood flow in T2EL. However, the mechanisms of biological changes or remodeling of the aneurysm sac after the repair are still not clear, but they have been considered to play an important role in the development of endoleaks. Unfortunately, it is impossible to obtain a tissue sample of the aortic wall in patients who underwent EVAR.
MicroRNAs (miRNAs) are a class of small single-stranded non-coding RNAs that inhibit the expression of target message RNA (mRNA). miR-29b/29c, miR-155, and miR-15a are miRNAs associated with regulating extracellular matrix (ECM) components, inflammation, and proliferation, respectively. All four miRNAs have been identified as biomarkers of AAA, not only in aneurysm tissue but also extracellular as circulating miRNAs. However, it is still unknown whether they can reflect the biological changes after AAA repair. Thus, we conducted a prospective study to investigate the changes in expression of circulating miR-29b, miR-29c, miR-155, and miR-15a before (T0), 3 days (T1), and 3 months (T2) after AAA repair.
A total of 39 patients were recruited for this study, 17 of whom were repaired by OSR and 22 of whom were repaired by EVAR. Four patients failed the T2 follow-up due to the Covid-19 pandemic. No significant changes were found in the expression of miR-29b, miR-29c, miR-155, and miR-15a. There were also no obvious differences between OSR and EVAR. However, the T1 expression of miR-15a was significantly lower in patients without endoleak after EVAR than in those who developed endoleak after EVAR and those who were repaired by OSR. Unfortunately, these differences did not persist to the T2 follow-up, and no other differences were found among these patients.
In summary, miR-15a is a miRNA that significantly changes in AAA patients. This study demonstrates that the expression of circulating miR-15a is lower in patients without endoleak three days after EVAR, compared to those who had endoleak after EVAR and those who underwent OSR. The results suggest that miR-15a might be involved in the early aortic remodeling after EVAR as an indicator of endoleak.
A powerful technique to distinguish the enantiomers of a chiral molecule is the Coulomb Explosion Imaging (CEI). This technique allows us to determine the handedness of a single molecule. In CEI, the molecule becomes charged by losing many electrons in a very short period of time by interacting with the light. The repulsion forces between the positive charged particles of the molecule leads the molecule to break into parts-fragments. By measuring the three vector momentum of (at least) four fragments, the handedness observable can be determined. In this thesis, CEI is induced by absorption of a single high energy photon, which creates an inner-shell hole (K shell) of the molecule. The subsequent cascade of Auger decays lead to fragmentation. We decided to work with the formic acid molecule in this thesis. Two different experiments were conducted. The first experiment focused on exciting electrons to different energy states, while the second experiment focused on extracting directly a photoelectron to the continuum and measure the angular distribution of the photoelectron in the molecular frame. The primary goal was to search for chiral signal in a pure achiral planar molecule under the previous electron processes. The results of these findings were further implemented to two more molecules.
In the framework of the LHC Injectors Upgrade Project (LIU), the CERN Proton Synchrotron Booster (PSB) went through major upgrades resulting in new effects to study, challenges to overcome and new parameter regimes to explore. To assess the achievable beam brightness limit of the machine, a series of experimental and computational studies in the transverse planes were performed. In particular, the new injection scheme induces optics perturbations that are strongly enhanced near the half-integer resonance. In this thesis, methods for dynamically measuring and correcting these perturbations and their impact on the beam performance will be presented. Additionally, the quality of the transverse beam distributions and strategies for improvement will be addressed. Finally, the space charge effects when dynamically crossing the half-integer resonance will be characterized. The results of these studies and their broader significance beyond the PSB will be discussed.
Methods using environmental DNA to explore and analyze biodiversity from previously unexplored habitats and ecosystems have become increasingly popular in recent years. This is particularly due to the potential reduction in necessary taxonomic expertise, the opportunity to assess microorganismal communities, and decreased time investments required to cover large spatial extents. In forests, the surface of tree bark is an important habitat for epiphytic diversity. Because of the large surface area rich in micro-niches, the seasonal stability of the substrate, and the longevity of trees, tree bark surfaces provide an ideal habitat for many species. Yet, we lack a comprehensive understanding of their communities and the environmental drivers behind the community assembly. These missing links hinder the exploration of the forest microbiome as a whole and limits our understanding of functions of a large forest habitat and its connections to other forest microbiomes. With a holistic eDNA metabarcoding approach, encompassing samples of three major taxonomic groups (e.g. bacteria, fungi, and green algae), as well as simultaneous collections from multiple forest habitats we can contribute to closing these gaps and increase our knowledge of the forest microbiome.
My dissertation is set within the framework of the Biodiversity Exploratories and was conducted in four parts: I. the establishment of an eDNA metabarcoding workflow to reveal the local diversity of the bark surface microbiome; II. the upscaling of the method to large geographic and environmental gradients to uncover the drivers of the microbiome; III. the integration of soil and bark samples to investigate compositional differences in two important forest habitats; IV. the evaluation of eDNA metabarcoding as a tool for biodiversity assessments of lichen diversity in forests.
In the first part, I developed a simple, cost-effective and fast sampling strategy to acquire eDNA samples from the bark of trees in forest ecosystems. Using readily available medical-specimen-collection swabs I sampled bark surfaces of individual trees in Central German forests and used metabarcoding to amplify marker genes of green algae, fungi and bacteria. From the sequencing reads I calculated the first diversity estimates of the major organismal groups of bark surface microbiomes from Central European forests. Overall the methodology produced reliable results, allowing for an expanded sampling in the second part.
In the second part of the dissertation, I expanded the sampling based on the results of part one. I collected bark surface samples from the three regions of the Biodiversity Exploratories covering large spatial and environmental gradients representative for Central European forests. The collection included composite samples from 150 plots and over 750 trees. Utilizing measurements of climatic and forest structure variables provided by the Biodiversity Exploratories, as well as my own community data, I identified the biotic and abiotic drivers behind alpha and beta diversity of the bark surface microbiome.
In the third part, I studied the differences between the bark surface as an unexplored and the soil as an example of a well characterized forest microbiome. Using only the fungal part of the large sampling campaign and soil samples obtained from the same plots at the same time, I assessed the commonalities and differences of the micro-communities of these distinct forest niches. Furthermore, I included two coniferous and one deciduous tree species to examine, if the effect of tree species, previously shown for soil microbiomes, also holds true for the bark surface.
In the last part of my dissertation, I used eDNA in a more applied way as a tool in biodiversity assessments of lichenized fungi. I compared the results from eDNA metabarcoding to an expert floristic mapping conducted in the same plots in 2007/2008. I assigned functional guilds to the fungal taxa obtained in the large sampling campaign and used a subset that was assigned as lichenized fungi.
In conclusion, I showed that eDNA metabarcoding is a valuable tool to reveal the unknown diversity of microorganisms in forest ecosystems. In particular, my results advance our understanding of the bark surface microbiome, an underexplored habitat within forests. The tightly linked interactions of the three major microbial groups underline that studies need to take holistic approaches across multiple taxonomic groups to deepen our understanding of processes governing the assembly of microbiomes. Results from my dissertation may serve as a foundation to inform hypotheses addressing the functions of forest microbiomes. The massive diversity data collected may also contribute to closing the gap in our understanding of macro-organisms and micro-organisms with respect to diversity distributions and patterns of richness, and serve as a baseline for predictions of biodiversity responses under future anthropogenic change.
Biotechnological processes offer better production conditions for a wide variety of goods of industrial interest. The production of aromatic compounds, for example, involves molecules of great value for cosmetic, plastic, agrochemical and pharmaceutic industries. However, the yield of such processes frequently prevents a proper implementtation that would allow the replacement of traditional production processes.
Numerous rational engineering approaches have been attempted to enhance metabolic pathways associated with desired products. Unfortunately, genetic modifications and heterologous pathway expression often lead to a higher metabolic burden on the producing organisms, ultimately leading to reduced production levels and fitness.
This project utilised adaptive laboratory evolution to better understand the development of synthetic cooperative consortia, using S. cerevisiae as a model organism. Specifically, a synthetic cooperative consortium was developed around the exchange of lysine and tyrosine, which was subjected to adaptive laboratory evolution aiming to induce mutations that would improve the system’s fitness either by enhanced production or upgraded stress resistance. Consequently, the mutant strains isolated after the evolution rounds were sequenced to identify relevant variations that could be related to the growth and production phenotypes observed.
The insights derived from this project are expected to contribute to further developing synthetic cooperative consortia with utilitarian purposes.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2023)
Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves.
Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now.
Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide.
For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and morphospecies of hyperparasitic fungi were identified. In addition, 35 historical specimens, including 12 type specimens, were examined for the present work.
DNA of hyperparasitic fungi was isolated directly from conidia, synnemata, apothecia, perithecia or pseudothecia of fresh and dried specimens. The main challenges faced by scientists in doing molecular studies of hyperparasitic fungi are related to the fact that the hyperparasitic fungi are intermingled with tissues of the meliolalean hosts and other organisms present in a given sample. This makes the isolation of DNA exclusively from the hyperparasite difficult. Moreover, hyperparasitic fungi on Meliolales are biotrophs and cannot be grown axenically. The hosts themselves are also biotrophic, further complicating DNA isolation from either partner. These factors have contributed to a lack of reference sequences in public databases. After more than 100 attempts, DNA of 20 specimens of hyperparasitic fungi, representing seven species, has been isolated in the context of the present investigation. Three partial nuclear gene regions were amplified and sequenced: nrLSU, nrSSU and nrITS. The datasets were assembled for phylogenetic analyses applying Maximum Likelihood (ML) and Bayesian inference (BI) methods. DNA sequences of hyperparasitic fungi on Meliolales were generated for the first time in the context of the present investigation.
Hyperparasitic fungi on Meliolales do not represent a single systematic group, but a polyphyletic ecological guild of fungi. Because of this huge diversity, only the systematics of species of perithecioid hyperparasites, as well as of the species of the genera Atractilina and Spiropes known to be hyperparasitic on black mildews was discussed in this thesis, as they represented the most common groups of fungi found in Benin and Panama. The results indicated, for example, the systematic position of Dimerosporiella cephalosporii and Paranectriella minuta in the Sordariomycetes and Dothideomycetes, respectively. In addition, the first record of a hyperparasitic fungus of black mildews in the Lecanoromycetes, namely Calloriopsis herpotricha, is reported here. The systematics of Atractilina parasitica and of some species of Spiropes is also discussed here.
In the context of the present investigation, four species new to science were described. They are presented with detailed descriptions, photos and scientific illustrations. Taxonomic studies of this thesis also generated seven new synonyms, nine new records for Benin, seven for Panama, one for Africa and two for mainland America, as well as the confirmation of one anamorph-teleomorph connection by molecular sequence data.
The ecology of hyperparasitic fungi on Meliolales is complex and far from being completely understood. The hypothesis of host specificity between hyperparasitic fungi, their meliolalean hosts and their plant hosts was tested for the first time, through a tritrophic network analysis. Results indicate that hyperparasites of Meliolales are generalists concerning genera of Meliolales, but apparently specialists at the level of order. In addition, hyperparasitic fungi tend to be found alongside their meliolalean hosts, suggesting a pantropical distribution.
Die Zahl der gramnegativen Bakterien auf der WHO-Liste der Antibiotikaresistenzen hat in den letzten Jahrzehnten erheblich zugenommen. Schätzungen zufolge wird die Antibiotikaresistenz bis 2050 tödlicher sein als Krebs. Die äußere Membran gramnegativer Bakterien ist aufgrund ihres wichtigsten Strukturbestandteils, des Lipopolysaccharids (LPS), sehr anpassungsfähig an Umweltveränderungen. Das LPS macht gramnegative Bakterien von Natur aus resistent gegen viele Antibiotika und führt somit zu Antibiotikaresistenz. Der bakterielle ATP-bindende Kassettentransporter (ABC-Transporter) MsbA spielt eine entscheidende Rolle bei der Regulierung der bakteriellen Außenmembran, indem er das Kern-LPS durch ATP-Hydrolyse über die Innenmembran von gramnegativen Bakterien flockt. Darüber hinaus fungiert diese Floppase als Efflux-Pumpe, indem sie Medikamente durch die innere Membran transportiert, was sie zu einem interessanten Ziel für Medikamente macht. Vor kurzem wurden zwei verschiedene Klassen von MsbA-Inhibitoren entdeckt: (1) Tetrahydrobenzothiophene (TBT), die den LPS-Transport aufheben, und (2) Chinolinderivate, die sowohl die ATP-Hydrolyse als auch die LPS-Translokation blockieren. Darüber hinaus hat die Bestimmung der 3D-Struktur von MsbA durch Rontgen- und Kryo-EM mehrere interessante Zustände der Floppase ergeben. Die Kernspinresonanzspektroskopie ist eine hervorragende biophysikalische Methode zur Ergänzung der vorhandenen 3D-Strukturdaten. Insbesondere ermöglicht die Festkörper-NMR die Untersuchung von Membranproteinen in einer nativen Umgebung (z. B. in einer Lipiddoppelschicht). In der Vergangenheit hat unser Labor mithilfe der Festkörper-NMR einige detaillierte Mechanismen von MsbA aufgedeckt. Trotz der zahlreichen Fortschritte bei der Untersuchung der ABC-Transporterprotein-Superfamilie ist der spezifische Prozess der Substrattranslokation von MsbA noch immer unbekannt. Es wird angenommen, dass dieser Translokationsprozess über die Kopplungshelices (CHs) erfolgt, die sich zwischen der Transmembranregion (TMD) und der Nukleotidbindungsdomäne (NBD) befinden. Nukleotid-Bindungsdomäne (NBD). Zu diesem Zweck wird dem Zusammenspiel zwischen der TMD und der NBD über die CHs besondere Aufmerksamkeit gewidmet, mit dem Ziel, den Prozess der Substrattranslokation mithilfe von funktionellen Assays und Festkörper-NMR zu verstehen. Bei letzterem wurden spezifische Reporter in die CHs eingeführt, um Konformationsänderungen in 2D-spektroskopischen Daten zu verfolgen. Darüber hinaus wurde zeitaufgelöste NMR eingesetzt, um die Auswirkungen verschiedener Substrate in der TMD während der ATP-Hydrolyse in der NBD sichtbar zu machen. Die einzigartigen Reporter in den CHs haben Konformationsänderungen in bestimmten katalytischen Zuständen gezeigt. Darüber hinaus scheinen verschiedene Substrate die Kinetik der ATP-Hydrolyse zu beeinflussen. Die Ergebnisse zeigten, dass einige Substrate einen bevorzugten katalytischen Zustand innerhalb des ATP-Hydrolyse Zyklus aufweisen, der möglicherweise einen gekoppelten oder ungekoppelten Kinasemechanismus hat. Diese Ergebnisse könnten verschiedene Einblicke in die molekulare Struktur potenzieller neuer Antibiotika liefern.
This thesis provides a detailed derivation of dissipative spin hydrodynamics from quantum field theory for systems composed of spin-0, spin-1/2, or spin-1 particles.
The Wigner function formalism is introduced for quantum fields in the respective representations of the Poincaré group, and the conserved currents, i.e., the energy-momentum tensor and the total angular momentum tensor, in various so-called pseudogauges are derived. An expansion around the semiclassical limit in powers of the Planck constant is performed.
Subsequently, kinetic equations are obtained for binary elastic scattering, using both the de Groot-van Leeuwen-van Weert and Kadanoff-Baym method, with the latter retaining the effect of quantum statistics. The resulting collision term features both local and nonlocal contributions, with the latter providing a relaxation mechanism for the spin degrees of freedom of the quasiparticles. The local-equilibrium distribution function is derived from the requirement that the local part of the collision term vanishes.
From quantum kinetic theory, dissipative spin hydrodynamics is then constructed via the method of moments, extended to particles with spin. The system of moment equations is closed via the Inverse-Reynolds Dominance (IReD) approach, resulting in a set of equations of motion describing the evolution of both ideal and dissipative degrees of freedom. The application to polarization phenomena relevant to heavy-ion collisions is discussed.
Upper mantle shear zones are complex systems where deformation is commonly closely interacting with metamorphic (solid-solid) and/or melt/fluid-rock reactions. Here, feedback processes between deformation, reactions, grain size reduction and phase mixing result in strain weakening and the localization of deformation. The expression of these interlinked processes is portrayed by the microfabrics of strained peridotites and pyroxenites. The present thesis is focusing on these processes and their impact on the deformation in three upper mantle shear zones situated in the peridotite massifs of Lanzo (Italian Alps), Erro-Tobbio (Italian Alps) and Ronda (Betic Cordillera, Spain). In all three shear zones, the presence of melt led to phase mixing either by interstitial crystallization of pyroxenes from a Si-saturated and partially also highly evolved melt or by melt-rock reactions of pyroxene porphyroclasts with a Si-undersaturated melt. The effect of melt on the localization of strain is twofold and variable. Enhanced deformation by melt-wetted boundaries is assumed for all shear zones. Additionally, phase mixing by crystallization of interstitial pyroxenes or melt-rock reactions reduce or maintain the grain size by the formation of fine grained neoblasts and secondary phase boundary pinning. In this regard, pre- to early syn-kinematic, map-scale percolation of OH-bearing, evolved melts in the NW Ronda peridotite massif and the associated crystallization of interstitial pyroxenes result in the activation of grain size sensitive deformation mechanisms in the entire melt-effected area. In the rocks collected at Erro-Tobbio, syn-kinematic melt-rock reactions of pyroxene porphyroclasts and Si-undersaturated melt led to the formation of ultramylonitic neoblast tails (grain size ~10 μm). Compared to the adjacent coarser-grained olivine-dominated matrix, the activation of diffusion creep led to an increase in the strain rate by an order of magnitude within interconnected ultramylonitic layers. Strain localization and softening in ultramylonitic layers are also documented in the Lanzo samples. Neoblast tails of pyroxene porphyroclasts were likewise identified as their precursor. The phase assemblage of the tails, including ortho- and clinopyroxene, olivine, plagioclase, and spinel (± amphibole), and their geochemical trends suggest, unlike in Erro-Tobbio, a formation by continuous net-transfer reactions enhanced by the spinel lherzolite to plagioclase lherzolite transition.
The new results obtained from the three studied shear zones underscore the importance of reactions for the interlinked processes of grain size reduction, phase mixing, strain localization and strain softening in upper mantle shear zones. Concerning strain localization, the nature of the reaction (solid-solid, melt/fluid-rock) seems to play a subordinate role compared to its timing. Pre- to early syn-kinematic melt-triggered reactions result in strain localization along map-scale shear zones. Late stage syn-kinematic melt-rock or metamorphic reactions under high stress conditions are capable of localizing the deformation along discrete, sub-centimeter thick ultramylonites.
Blockchains in public administration : a RADIUS on blockchain framework for public administration
(2023)
The emergence of blockchain technology has generated a great deal of attention, as reflected in numerous scientific and journalistic articles. However, the implementation of blockchain for public administrations in Germany has encountered a setback owing to unsuccessful initiatives. Initial enthusiasm was followed by disillusionment. Nevertheless, technology continues to evolve. This paper examines whether the use of a blockchain can still optimize the processes of public administrations. Not only the failed projects are analysed, but also more current applications of the technology and their potential relevance for the administration, especially in the state of Hesse.
To answer if blockchains are promising to administrations, a Design Science Research (DSR) research approach is chosen. The DSR method is a research-based approach that aims to create new and innovative solutions to real-world problems through the development and evaluation of artefacts such as models, methods, or prototypes. For this work, the implementation of a framework to realize an Authentication, Authorization, and Accounting (AAA) system on the blockchain was identified as profitable. The framework aims to implement the aforementioned AAA tasks using a blockchain. The Remote Authentication Dial-In User Service (RADIUS) protocol has been identified as a potential protocol of the AAA system. The goal is to create a way to implement the system either entirely on a blockchain or as a hybrid system. Various blockchain technologies will be considered. Suitable for development, the framework AAA-me is named.
The development of AAA-me has shown that the desired framework for implementing RADIUS on the blockchain is possible in various degrees of implementation. Previous work mostly relied on full development. Additionally, it has been shown that AAA-me can be used to perform hybrid integration at different implementation levels. This makes AAA-me stand out from the few hybrid previous approaches. Furthermore, AAA-me was investigated in different laboratory environments. This was to determine the expected resilience against Single Point of Failure (SPOF). The results of the lab investigation indicated that a RADIUS system on top of a blockchain can provide benefits in terms of security and performance. In the lab environment, times were measured within which a series of authorization requests were processed. In addition, it was illustrated how a RADIUS system implemented using blockchain can protect itself against Man-in-the-Middle (MITM) attacks.
Finally, in collaboration with the Hessian Central Office for Data Processing (German: Hessische Zentrale für Datenverarbeitung) (HZD), another test lab demonstrated how a RADIUS system on the blockchain can integrate with the existing IT systems of the German state of Hesse. Based on these findings, this work reevaluated the applicability of blockchain technology for public administration processes.
The work has thus shown that the use of a blockchain can still be purposeful. However, it has also been shown that an implementation can bring many problems with it. The small number of blockchain developers and engineers also poses the risk of finding people to develop and maintain a system. In addition, one faces the problem of determining an architecture now that will be applied to many projects in the future. However, each project can, in turn, have an impact on the choice of architecture. Once one has solved this problem and a blockchain infrastructure is available, it can be established quickly and be more SPOF resistant, for example, for Public Key Infrastructure (PKI) systems.
AAA-me was only applied in lab and test environments. As a result, no real data ran over its own infrastructure. This allowed the necessary flexibility for development. However, system-related properties could appear in real situations that are not detectable here in this way. Furthermore, the initial stage of AAA-me’s development is still in its infancy. Many manual adjustments need to be made in order for this to integrate with an existing RADIUS system. Also, no system security effort in and of itself has been carried out in the lab environments. Thus, vulnerabilities can quickly open up on web servers due to misconfigurations and missing updates. For the above reasons, productive use should be discouraged unless major developments are carried out.
Discrepancies between knockdown and knockout animal model phenotypes have long stood as a perplexing phenomenon. Several mechanisms explaining such observations have been proposed, namely the toxicity or the off-target effects of the knockdown reagents, as well as, in certain cases, genetic robustness – an organism's ability to maintain its phenotype despite genetic perturbations. In addition to these explanations, transcriptional adaptation (TA), a phenomenon defined as an event whereby a mutation in one gene leads to transcriptional upregulation or downregulation of another, adapting, gene or genes expression, has been recently proposed as an alternative explanation for the conflicting knockdown and knockout phenotype paradox.
Since its discovery in 2015, TA's precise mechanism remains a subject of ongoing research. Majority of evidence suggests that mutant mRNA degradation plays a central in TA. Epigenetic remodeling is also thought to play a role, as evidenced by an increase in active histone marks at the transcription start sites of the adapting genes. Whether mRNA degradation is indeed the key player in TA remains debated. Furthermore, it is still unknown how exactly TA develops, what adapting genes it targets, and whether genomic mutations that render mutant mRNA sensitive to degradation are required for TA to occur.
Throughout the experiments described in this Dissertation, I have designed an inducible TA system where TA can be triggered on demand and its effects on the cell’s transcriptome followed through time. I have demonstrated that degradation-prone transgenes, once induced and expressed, can be efficiently degraded, resulting in the protein loss-independent upregulation of adapting genes via TA. Adapting genes with higher degree of sequence similarity become upregulated faster than genes with lower degree of sequence similarity. Further functionality of this approach to study TA is limited by the leakiness of the inducible gene expression system; however, constitutively expressed degradation-prone transgenes were used to demonstrate TA in human cells.
In addition, I have developed an approach to target wild-type cytoplasmic mRNAs without altering the cell’s genome and reported a TA-like phenomenon, which manifested as adapting gene upregulation not relying on mutations in other genes. Cytoplasmic mRNA cleavage with CRISPR-Cas13d triggered a TA-like response in three different gene models: Actg1 knockdown, Ctnna1 knockdown, and Nckap1 knockdown. After comparing two different modes of triggering TA, CRISPR-Cas9 knockout versus CRISPR-Cas13d knockdown, I reported little overlap between the dysregulated genes and suggested that diverse mRNA degradation modes led to distinct TA responses. In addition, the transcriptional increase of Actg2 caused by CRISPR-Cas13d-mediated Actg1 mRNA cleavage did not require chromatin accessibility changes.
Experiments and genetic tools described in this dissertation investigated how TA develops from its earliest onset, how it affects the global transcriptome of the cell, as well as provided compelling evidence for an mRNA degradation-central TA mechanism. I have created tools to study both direct and indirect TA gene targets and unveiled important insights into the temporal dynamics of TA. Genes with higher sequence similarity were found to be upregulated more rapidly than those with lower similarity. Furthermore, it was revealed that the epigenetic properties of TA responses vary depending on the triggering mechanism. Cas13d-mediated degradation of wild-type mRNAs led to immediate transcriptional enhancement independent of epigenetic changes, which stood in contrast to previously measured alterations in chromatin accessibility in CRISPR-Cas9 mutants. This research has thus significantly advanced our knowledge of TA and provided valuable tools and findings that contribute to the broader understanding of gene expression regulation in response to mRNA degradation.
Type 1 diabetes (T1D) is precipitated by the autoimmune destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. Chemokines have been identified as major conductors of the islet infiltration by autoaggressive leukocytes, including antigen-presenting cells and islet autoantigen-specific T cells. We have previously generated a roadmap of the gene expression in the islet microenvironment during T1D in a mouse model and found that most of the chemokine axes are chronically upregulated during T1D. We focused our attention on CXCL10/CXCR3, CCL5/CCR5, CXCL16/CCR6, CX3CL1/CX3CR1, and XCL1/XCR1. First, we found that the absence of CCR6 and of CX3CR1 diminished T1D incidence in a mouse model for T1D. Further, the XCL1/XCR1 chemokine axis is of particular interest, since XCR1 is exclusively expressed on convention dendritic cells type 1 (cDC1) that excel by their high capacity for T cell activation. Here we demonstrate that cDC1 expressing XCR1 are present in and around the islets of patients with T1D and of islet-autoantibody positive individuals. Further, in an inducible mouse model for T1D, we show that XCL1 plays an important role in the attraction of highly potent dendritic cells expressing XCR1 to the islets. XCL1-deficient mice display a diminished infiltration of XCR1+ cDC1 and subsequently also a reduced magnitude and activity of islet autoantigen-specific T cells. XCR1-deficient mice display a reduced magnitude and activity of islet autoantigen-specific T cells. A 3D-visualization of the entire pancreas reveals that both XCL1-deficient mice and XCR1-deficient mice indeed maintain most of their functional islets after induction of the disease. Thus, the absence of XCL1 results in a profound decrease in T1D incidence. The XCR1-deficiency also reduces T1D incidence, even if in a less drastic way compared to XCL1-deficiency. An interference with the XCL1/XCR1 chemokine axis might constitute a novel target for the therapy for T1D.
The simultaneous inhibition of HDACs and BET proteins has shown promising anti-proliferative effects against different cancer types, including the difficult to treat pancreatic cancer. In this work, the strategy of concurrently targeting HDACs and BET proteins was pursued by developing different types of dual inhibitors.
By developing a novel scaffold that selectively inhibits HDAC1/2 together with BET proteins in cells, an effective tool for the investigation of pancreatic cancer, and other diseases which are sensitive to epigenetic processes, was created. The compound’s small size further gives the opportunity to further develop the inhibitor towards optimized pharmacokinetic properties, potentially resulting in a drug for cancer treatment.
A second novel approach that was pursued, was the development of a small-molecule degrader, targeting HDACs and BET proteins. Through synthesizing a variety of different molecules, a compound that was capable of lowering BRD4 levels and, at the same time, increasing histone acetylation was developed. While additional mechanistic investigations are needed to verify the degradation, the potent antiproliferative effects in pancreatic cancer cells encourage further studies following this alternative new strategy.
Aim: The cytochrome P450 reductase (POR) along with the cytochrome P450 enzymes (CYP) are responsible for the metabolism of a multitude of metabolites important for the maintenance of tissue function. Defects in this system have been associated with cardiovascular diseases. These enzymes are known to produce vasoactive lipids that modulate vascular tone. The aim of this study was to identify the consequence of a loss in endothelial POR for vascular function.
Methods and Results: To identify the endothelial contribution of the POR/CYP450 system to vascular function, we generated an endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR-/-). Under basal condition ecPOR-/- already exhibited endothelial dysfunction in aorta and mesenteric vessels (acetylcholine-dependent relaxation, LogEC50 -7.6M for CTR vs. -7.2M for ecPOR-/- in aorta) and lower nitric oxide levels in the plasma (CTR: 236.8 ±77.4; ecPOR-/- 182.8 ±34.1 nmol/L). This dysfunction was coupled to attenuated eNOS function detected by the heavy arginine assay and decreased eNOS phosphorylation on S1177. Furthermore, insulin-induced phosphorylation of the eNOS activator, AKT, was also attenuated in the aorta from ecPOR-/- mice as compared to control mice. CYP450-dependent EET production was lower in plasma, lung and aorta of ecPOR-/- mice and this was accompanied with increased levels of vasoconstriction prostanoids (lipidomics of aorta, plasma and lung freshly isolated from CTR and ecPOR-/- mice). MACE-RNAseq from these aortas also showed a significant increase in genes annotated to eicosanoid production. In an in vivo angiotensin II model, acute deletion of POR increased the blood pressure as measured by telemetry and tail cuff (137.4 ± 15.9 mmHg in WT; 152.1 ± 7.154 mmHg in ecPOR-/-). In a rescue experiment using the NSAID naproxen, the increase in blood pressure induced by deletion of endothelial POR was abolished.
Conclusion: Collectively, in endothelial cells POR regulates eNOS activity and orchestrates the metabolic fate of arachidonic acid towards the vessel dilating EETs and away from deleterious prostanoids. In the absence of POR this endothelial regulation is compromised leading to vascular dysfunction.
This thesis develops a naturalist theory of phenomenal consciousness. In a first step, it is argued on phenomenological grounds that consciousness is a representational state and that explaining consciousness requires a study of the brain’s representational capacities. In a second step, Bayesian cognitive science and predictive processing are introduced as the most promising attempts to understand mental representation to date. Finally, in a third step, the thesis argues that the so-called “hard problem of consciousness” can be resolved if one adopts a form of metaphysical anti-realism that can be motivated in terms of core principles of Bayesian cognitive science.
In this thesis, the flow coefficients vn of the orders n = 1 − 6 are studied for protons and light nuclei in Au+Au collisions at Ebeam = 1.23 AGeV, equivalent to a center-of-mass energy in the nucleon-nucleon system of √sNN = 2.4 GeV. The detailed multi-differential measurement is performed with the HADES experiment at SIS18/GSI. HADES, with its large acceptance, covering almost full azimuth angle, combined with its high mass-resolution and good particle-identification capability, is well equipped to study the azimuthal flow pattern not only for protons, deuterons, and tritons but also for charged pions, kaons, the φ-mesons, electrons/positrons, as well as light nuclei like helions and alphas. The high statistics of more than seven billion Au-Au collisions recorded in April/May 2012 with HADES enables for the first time the measurement of higher order flow coefficients up to the 6th harmonic. Since the Fourier coefficient of 7th and 8th order are beyond the statistical significance only an upper bound is given. The Au+Au collision system is the largest reaction system with the highest particle multiplicities, which was measured so far with HADES. A dedicated correction method for the flow measurement had to be developed to cope with the reconstruction in-efficiencies due to occupancies of the detector system. The systematical bias of the flow measurement is studied and several sources of uncertainties identified, which mainly arise from the quality selection criteria applied to the analyzed tracks, the correction procedure for reconstruction inefficiencies, the procedures for particle identification (PID) and the effects of an azimuthally non-uniform detector acceptance. The systematic point-to-point uncertainties are determined separately for each particle type (proton, deuteron and triton), the order of the flow harmonics vn, and the centrality class. Further, the validity of the results is inspected in the range of their evaluated systematic uncertainties with several consistency checks. In order to enable meaningful comparisons between experimental observations and predictions of theoretical models, the classification of events should be well defined and in sufficiently narrow intervals of impact parameter. Part of this work included the implementation of the procedure to determine the centrality and orientation of the reaction.
In the conclusion the experimental results are discussed, including various scaling properties of the flow harmonics. It is found that the ratio v4/v2 for protons and light nuclei (deuterons and tritons) at midrapidity for all centrality classes approaches values close to 0.5 at high transverse momenta, which was suggested to be indicative for an ideal hydrodynamic behaviour. A remarkable scaling is observed in the pt dependence of v2 (v4) at mid-rapidity of the three hydrogen isotopes, when dividing by their nuclear mass number A (A^2) and pt by A. This is consistent with naive expectations from nucleon coalescence, butraises the question whether this mass ordering can also be explained by a hydrodynamical-inspired approach, like the blast-wave model. The relation of v2 and v4 to the shape of the initial eccentricity of the collision system is studied. It is found that v2 is independent of centrality for all three particle species after dividing it by the averaged second order participant eccentricity v2/⟨ε2⟩. A similar scaling is shown for v4 after division by ⟨ε2⟩^2.
This thesis contains three theoretical works about certain aspects of the interplay of electronic correlations and topology in the Hubbard model.
In the first part of this thesis, the applicability of elementary band representations (EBRs) to diagnose interacting topological phases, that are protected by spatial symmetries and time-reversal-symmetry, in terms of their single-particle Matsubara Green’s functions is investigated. EBRs for the Matsubara Green’s function in the zero-temperature limit can be defined via the topological Hamiltonian. It is found that the Green’s function EBR classification can only change by (i) a gap closing in the spectral function at zero frequency, (ii) the Green’s function becoming singular i.e. having a zero eigenvalue at zero frequency or (iii) the Green’s function breaking a protecting symmetry. As an example, the use of the EBRs for Matsubara Green’s functions is demonstrated on the Su-Schriefer-Heeger model with exact diagonalization.
In the second part the Two-Particle Self-Consistent approach (TPSC) is extended to include spin-orbit coupling (SOC). Time-reversal symmetry, that is preserved in the presence of SOC, is used to derive new TPSC self-consistency equations including SOC. SOC breaks spin rotation symmetry which leads to a coupling of spin and charge channel. The local and constant TPSC vertex then consists of three spin vertices and one charge vertex. As a test case to study the interplay of Hubbard interaction and SOC, the Kane-Mele-Hubbard model is studied. The antiferromagnetic spin fluctuations are the leading instability which confirms that the Kane-Mele-Hubbard model is an XY antiferromagnet at zero temperature. Mixed spin-charge fluctuations are found to be small. Moreover, it is found that the transversal spin vertices are more strongly renormalized than the longitudinal spin vertex, SOC leads to a decrease of antiferromagnetic spin fluctuations and the self-energy shows dispersion and sharp features in momentum space close to the phase transition.
In the third part TPSC with SOC is used to calculate the spin Hall conductivity in the Kane-Mele-Hubbard model at finite temperature. The spin Hall conductivity is calculated once using the conductivity bubble and once including vertex corrections. Vertex corrections for the spin Hall conductivity within TPSC corresponds to the analogues of the Maki-Thompson contributions which physically correspond to the excitation and reabsorption of a spin, a charge or a mixed spin-charge excitation by an electron. At all temperatures, the vertex corrections show a large contribution in the vicinity of the phase transition to the XY antiferromagnet where antiferromagnetic spin fluctuations are large. It is found that vertex corrections are crucial to recover the quantized value of −2e^2/h in the zero-temperature limit. Further, at non-zero temperature, increasing the Hubbard interaction leads to a decrease of the spin Hall conductivity. The results indicate that scattering of electrons off antiferromagnetic spin fluctuations renormalize the band gap. Decreasing the gap can be interpreted as an effective increase of temperature leading to a decrease of the spin Hall conductivity.
Trait-dependent effects of biotic and abiotic filters on plant regeneration in Southern Ecuador
(2024)
Tropical forests have always fascinated scientists due to their unique biodiversity. However, our understanding of ecological processes shaping the complexity of tropical rainforests is still relatively poor. Plant regeneration is one of the processes that remain understudied in the tropics although this is a key process defining the structure, diversity and assembly of tropical plant communities. In my dissertation, I combine experimental, observational and trait-based approaches to identify processes shaping the assembly of seedling communities and compare associations between environmental conditions and plant traits across plant life stages. By working along a steep environmental gradient in the tropical mountains of Southern Ecuador, I was able to investigate how processes of plant regeneration vary in response to biotic and abiotic factors in tropical montane forests.
My dissertation comprises three complementary chapters, each addressing an individual research question. First, I studied how trait composition in plant communities varies in relation to the broad- and local-scale environmental conditions and across the plant life cycle. I measured key traits reflecting different ecological strategies of plants that correspond to three stages of the plant life cycle (i.e., adult trees, seed rain and recruiting seedlings). I worked on 81 subplots along an elevational gradient covering a large climatic gradient at three different elevations (1000, 2000 and 3000 m a.s.l.). In addition, I measured soil and light conditions at the local spatial scale within each subplot. My findings show that the trait composition of leaves, seeds and seedlings changed similarly across the elevational gradient, but that the different life stages responded differently to the local gradients in soil nutrients and light availability. Consequently, my findings highlight that trait-environment associations in plant communities differ between large and small spatial scales and across plant life stages.
Second, I investigated how seed size affects seedling recruitment in natural forests and in pastures in relation to abiotic and biotic factors. I set up a seed sowing experiment in both habitat types and sowed over 8,000 seeds belonging to seven tree species differing in seed size. I found that large-seeded species had higher proportions of recruitment in the forests compared to small-seeded species. However, small-seeded species tended to recruit better in pastures compared to large-seeded species. I showed that high surface temperature was the main driver of differences in seedling recruitment between habitats, because it limited seedling recruitment of large-seeded species. The results from this experiment show that pasture restoration requires seed addition of large-seeded species and active protection of recruiting seedlings in order to mitigate harmful conditions associated with high temperatures in deforested areas.
Third, I examined the associations between seedling beta-diversity and different abiotic and biotic factors between and within elevations. I applied beta-diversity partitioning to obtain two components of beta-diversity: species turnover and species richness differences. I associated these components of beta-diversity with biotic pressures by herbivores and fungal pathogens and environmental heterogeneity in light and soil conditions. I found that species turnover in seedling communities was positively associated with the dissimilarity in biotic pressures within elevations and with environmental heterogeneity between elevations. Further, I found that species richness differences increased primarily with increasing environmental heterogeneity within elevations. My findings show that the associations between beta-diversity of seedling communities and abiotic and biotic factors are scale-dependent, most likely due to differences in species sorting in response to biotic pressures and species coexistence in response to environmental heterogeneity.
My dissertation reveals that studying processes of community assembly at different plant life stages and spatial scales can yield new insights into patterns and processes of plant regeneration in tropical forests. I investigated how community assembly processes are governed by abiotic and biotic filtering across and within elevations. I also experimentally explored how the process of seedling recruitment depends on seed size-dependent interactions, and verified how these effects are associated with abiotic and biotic filtering. Identifying such processes is crucial to inform predictive models of environmental change on plant regeneration and successful forest restoration. Further exploration of plant functional traits and their associations with local-scale environmental conditions could effectively support local conservation efforts needed to enhance forest cover in the future and halt the accelerating loss of biodiversity.
In this work I investigate two different systems - spin systems and charge-density-waves. The same theoretical method is used to investigate both types of system. My investigations are motivated by experimental investigations and the goal is to describe the experimental results theoretically. For this purpose I formulate kinetic equations starting from the microscopical dynamics of the systems.
First of all, a method is formulated to derive the kinetic equations diagrammatically. Within this method an expansion in equal-time connected correlation functions is carried out. The generating functional of connected correlations is employed to derive the method.
The first system to be investigated is a thin stripe of the magnetic insulator yttrium-iron-garnet (YIG). Magnons are pumped parametrically with an external microwave field. The motivation of my theoretical investigations is to explain the experimental observations. In a small parameter range close to the confluence field strength where confluence processes of two parametrically pumped magnons with the same wave vector becomes kinematically possible the efficiency of the pumping is reduced or enhanced depending on the pumping field strength. Because it is expected that that confluence and splitting processes of magnons are essential for the experimental observations I go beyond the kinetic theories that are conventionally applied in the context of parametric excitations in YIG and investigate the influence of cubic vertices on the parametric instability of magnons in YIG.
Furthermore, the influence of phonons is investigated. Usually in the literature these are taken into account as heat bath. Here, I want to explain experiments where an accumulation of magnetoelastic bosons - magnon-phonon-quasi-particles - has been observed. I employ the method of kinetic equations to investigate this phenomenon theoretically. The kinetic theory is able to reproduce the experimental observations and it is shown that the accumulation of magnetoelastic bosons is purely incoherent.
Finally, charge-density waves (CDW) in quasi-one-dimensional materials will be investigated. Charge-density waves emerge from a Peierls-instability and are a prime example for spontaneous symmetry breaking in solids. Again, the motivation for my theoretical investigations are an experiment where the spectrum of amplitude and phase phonon modes has been measured. Starting from the Fröhlich-Hamiltonian I derive kinetic equations and from these kinetic equations the equations of motion for the CDW order parameter can be derived. The frequencies and damping rates of amplitude and phase phonon modes will be derived from the linearized equations of motion. I compare my theory with existing methods. Furthermore, I also investigate the influence of Coulomb interaction.
This thesis investigates exotic phases within effective models for strongly interacting matter.
The focus lies on the chiral inhomogeneous phase (IP) that is characterized by a spontaneous breaking of translational symmetry and the moat regime, which is a precursor phenomenon exhibiting a non-trivial mesonic dispersion relation.
These phenomena are expected to occur at non-zero baryon densities, which is a parameter region that is mostly non-accessible to first-principle investigations of Quantum chromodynamics (QCD).
As an alternative approach, we consider the Gross-Neveu (GN) and Nambu-Jona-Lasinio (NJL) model within the mean-field approximation, which can be regarded as effective models for QCD.
We focus on two aspects of the moat regime and the IP in these models.
First, we investigate the influence of the employed regularization scheme in the (3+1)-dimensional NJL model, which is nonrenormalizable, i.e., the regulator cannot be removed.
We find that the moat regime is a robust feature under change of regularization scheme, while the IP is sensitive to the specific choice of scheme.
This suggests that the moat regime is a universal feature of the phase diagram of the NJL model, while the IP might only be an artifact of the employed regulator.
Second, we study the influence of the number of spatial dimensions on the emergence of the IP.
To this end, we investigate the GN model in noninteger spatial dimensions d.
We find that the IP and the moat regime are present for d < 2, while they are absent for d > 2.
This demonstrates the central role of the dimensionality of spacetime and illustrates the connection of previously obtained results in this model in integer number of spatial dimensions.
Moreover, this suggests that the occurrence of these phenomena in three spatial dimensions is solely caused by the finite regulator.
In summary, this thesis contributes to advancing our understanding of the phase structure of QCD, particularly regarding the existence and characteristics of inhomogeneous phases and the moat regime.
Even though the investigations are performed within effective models, they provide valuable insight into the aspects that are crucial for the formation of an inhomogeneous chiral condensate in fermionic theories.
By combining two unique facilities at the Gesellschaft fuer Schwerionenforschung (GSI), the Fragment Separator (FRS) and the Experimental Storage Ring (ESR), the first direct measurement of a proton capture reaction of stored radioactive isotopes was accomplished. The combination of well-defined ion energy, an ultra-thin internal gas target, and the ability to adjust the beam energy in the storage ring enables precise, energy-differentiated measurements of the (p,gamma) cross sections. The new setup provides a sensitive method for measuring (p,gamma) reactions relevant for nucleosynthesis processes in supernovae, which are among the most violent explosions in the universe and are not yet well understood. The cross sections of the 118Te(p,gamma) and 124Xe(p,gamma) reactions were measured
at energies of astrophysical interest. The heavy ions were stored with energies of 6 MeV/nucleon and 7 MeV/nucleon and interacted with a hydrogen gas-jet target.
The produced proton-capture products were detected with a double-sided silicon strip detector. The radiative recombination process of the fully stripped ions and electrons from the hydrogen target was used as a luminosity monitor.
Additionally, post-processing nucleosynthesis simulations within the NuGrid [1] research platform have been performed. The impact of the new experimental results on the p-process nucleosynthesis around 124Xe and 118Te in a core-collapse supernova was investigated. The successful measurement of the proton capture cross sections of radioactive isotopes rises the motivation to proceed with experiments in lower energy regions.
[1] M. Pignatari and F. Herwig, “The nugrid research platform: A comprehensive simulation approach for nuclear astrophysics,” Nuclear Physics News, vol. 22, no. 4, pp. 18–23, 2012.
This dissertation is concerned with the task of map-based self-localization, using images of the ground recorded with a downward-facing camera. In this context, map-based (self-)localization is the task of determining the position and orientation of a query image that is to be localized. The map used for this purpose consists of a set of reference images with known positions and orientations in a common coordinate system. For localization, the considered methods determine correspondences between features of the query image and those of the reference images.
In comparison with localization approaches that use images of the surrounding environment, we expect that using images of the ground has the advantage that, unlike the surrounding, the visual appearance of the ground is often long-term stable. Also, by using active lighting of the ground, localization becomes independent of external lighting conditions.
This dissertation includes content of several published contributions, which present research on the development and testing of methods for feature-based localization of ground images. Our first contribution examines methods for the extraction of image features that have not been designed to be used on ground images. This survey shows that, with appropriate parametrization, several of these methods are well suited for the task.
Based on this insight, we develop and examine methods for various subtasks of map-based localization in the following contributions. We examine global localization, where all reference images have to be considered, as well as local localization, where an approximation of the query image position is already known, which allows for disregarding reference images with a large distance to this position.
In our second contribution, we present the first systematic comparison of state-of-the-art methods for ground texture based localization. Furthermore, we present a method, which is characterized by its usage of our novel feature matching technique. This technique is called identity matching, as it matches only those features with identical descriptors, in contrast to the state-of-the-art that also matches features with similar descriptors. We show that our method is well suited for global and local localization, as it has favorable scaling with the number of reference images considered during the localization process. In another contribution, we develop a variant of our localization method that is significantly faster to compute, as it applies a sampling approach to determine the image positions at which local features are extracted, instead of using classical feature detectors.
Two further contributions are concerned with global localization. The first one introduces a prediction model for the global localization performance, based on an evaluation of the local localization performance. This allows us to quickly evaluate any considered parameter settings of global localization methods. The second contribution introduces a learning-based method that computes compact descriptors of ground images. This descriptor can be used to retrieve the overlapping reference images of a query image from a large set of reference images with little computational effort.
The most recent contribution included in this dissertation presents a new ground image database, which was recorded with a dedicated platform using a downward-facing camera. In addition to the data, we also explain our guidelines for the construction of the platform. In comparison with existing databases, our database contains more images and presents a larger variety of ground textures. Furthermore, this database enables us to perform the first systematic evaluation of how localization performance is affected by the time interval between the point in time at which the reference images are recorded and the point in time at which the query image is recorded. We find out that for outdoor areas all ground texture based localization methods have reliability issues, if the time interval between the recording of the query and reference images is large, and also if there are different weather conditions. These findings point to remaining challenges in ground texture base localization that should be addressed in future work.
Matroids are combinatorial objects that generalize linear independence. A matroid can be represented geometrically by its Bergman fan and we compare the symmetries of these two objects. Sometimes, the Bergman fan has additional automorphisms, which are related to Cremona transformations in projective space. Their existence depends on a combinatorial property of the matroid, as has been shown by Shaw and Werner, and we study the consequences for the structure of such matroids. This allows us to gain a better understanding of the so-called Cremona group of a matroid and we apply our results to root system matroids.
A central concern in genetics is to identify mechanisms of transcriptional regulation. The aim is to unravel the mapping between the DNA sequence and gene expression. However, it turned out that this is extremely complex. Gene regulation is highly cell type-specific and even moderate changes in gene ex- pression can have functional consequences.
Important contributors to gene regulation are transcription factors (TFs), that are able to directly interact with the DNA. Often, a first step in understanding the effect of a TF on the gene’s regulation is to identify the genomic regions a TF binds to. Therefore, one needs to be aware of the TF’s binding preferences, which are commonly summarized in TF binding motifs. Although for many TFs the binding motif is experimentally validated, there is still a large number of TFs where no binding motif is known. There exist many tools that link TF binding motifs to TFs. We developed the method Massif that improves the performance of such tools by incorporating a domain score that uses the DNA binding domain of the studied TF as additional information.
TF binding sites are often enriched in regulatory elements (REMs) such as promoters or enhancers, where the latter can be located megabases away from its target gene. However, to understand the regulation of a gene it is crucial to know where the REMs of a gene are located. We introduced the EpiRegio webserver that holds REMs associated to target genes predicted across many cell types and tissues using STITCHIT, a previously established method. Our publicly available webserver enables to query for REMs associated to genes (gene query) and REMs overlapping genomic regions (region query). We illus- trated the usefulness of EpiRegio by pointing to a TF that occurs enriched in the REMs of differential expressed genes in circPLOD2 depleted pericytes. Further, we highlighted genes, which are affected by CRISPR-Cas induced mutations in non-coding genomic regions using EpiRegio’s region query. Non-coding genetic variants within REMs may alter gene expression by modifying TF binding sites, which can lead to various kinds of traits or diseases. To understand the underlying molecular mechanisms, one aims to evaluate the effect of such genetic variations on TF binding sites. We developed an accurate and fast statistical approach, that can assess whether a single nucleotide polymorphism (SNP) is regulatory. Further, we combined this approach with epigenetic data and additional analyses in our Sneep workflow. For instance, it enables to identify TFs whose binding preferences are affected by the analyzed SNPs, which is illustrated on eQTL datasets for different cell types. Additionally, we used our Sneep workflow to highlight cardiovascular disease genes using regulatory SNPs and REM-gene interactions.
Overall, the described results allow a better understanding of REM-gene interactions and their interplay with TFs on gene regulation.
In this thesis, we present a detailed consideration of both qualitative and quantitative properties of static spherically symmetric solutions of the Einstein equations with self-interacting scalar fields. Our focus is on solutions with naked singularities. We study the qualitative properties of the solutions of the Einstein equations with real static self-interacting $N$ scalar fields, making some assumptions on self-interaction. We provide a rigorous proof that the corresponding solutions will be regular up to $r=0$. Furthermore, we find the rigorous form of asymptotic solutions near the singularity and at spatial infinity. We construct some examples of spherical-like naked singularities at $r=r_s\neq0$ in curvature coordinates.
We analyze the stability of the previously considered solutions against odd-parity gravitational perturbations and also examine the fundamental quasi-normal modes spectra. For the general class of the self-interaction potential, we demonstrate well-posedness of the initial problem and stability for positively defined potentials. As an example, we numerically study the case of the scalar field with power-law self-interaction potential and find the fundamental quasi-normal modes frequencies. We demonstrate that they differ from the standard Schwarzschild black hole case.
We study in detail the motion of particles in the vicinity of previously considered solutions. Mainly, we are interested in considering properties of the distribution of stable circular orbits around the corresponding configurations and images of the accretion disk for a distant observer. For all cases, we find possible types of stable circular orbit distributions and domains of parameters where they are realized.
We also demonstrate that the presence of self-interaction can lead to a new type of circular orbit distributions, which is absent in the linear massless scalar field case. We build Keplerian disk images in the plane of a distant observer and demonstrate the possibility to mimic the shadows of black holes.
This cumulative dissertation contains four self-contained chapters on stochastic games and learning in intertemporal choice.
Chapter 1 presents an experiment on value learning in a setting where actions have both immediate and delayed consequences. Subjects make a series of choices between abstract options, with values that have to be learned by sampling. Each option is associated with two payoff components: One is revealed immediately after the choice, the other with one round delay. Objectively, both payoff components are equally important, but most subjects systematically underreact to the delayed consequences. The resulting behavior appears impatient or myopic. However, there is no inherent reason to discount: All rewards are paid simultaneously, after the experiment. Elicited beliefs on the value of options are in accordance with choice behavior. These results demonstrate that revealed impatience may arise from frictions in learning, and that discounting does not necessarily reflect deep time preferences. In a treatment variation, subjects first learn passively from the evidence generated by others, before then making a series of own choices. Here, the underweighting of delayed consequences is attenuated, in particular for the earliest own decisions. Active decision making thus seems to play an important role in the emergence of the observed bias.
Chapter 2 introduces and proves existence of Markov quantal response equilibrium (QRE), an application of QRE to finite discounted stochastic games. We then study a specific case, logit Markov QRE, which arises when players react to total discounted payoffs using the logit choice rule with precision parameter λ. We show that the set of logit Markov QRE always contains a smooth path that leads from the unique QRE at λ = 0 to a stationary equilibrium of the game as λ goes to infinity. Following this path allows to solve arbitrary finite discounted stochastic games numerically; an implementation of this algorithm is publicly available as part of the package sgamesolver. We further show that all logit Markov QRE are ε-equilibria, with a bound for ε that is independent of the payoff function of the game and decreases hyperbolically in λ. Finally, we establish a link to reinforcement learning, by characterizing logit Markov QRE as the stationary points of a game dynamic that arises when all players follow the well-established reinforcement learning algorithm expected SARSA.
Chapter 3 introduces the logarithmic stochastic tracing procedure, a homotopy method to compute stationary equilibria for finite and discounted stochastic games. We build on the linear stochastic tracing procedure (Herings and Peeters 2004), but introduce logarithmic penalty terms as a regularization device, which brings two major improvements. First, the scope of the method is extended: it now has a convergence guarantee for all games of this class, rather than just generic ones. Second, by ensuring a smooth and interior solution path, computational performance is increased significantly. A ready-to-use implementation is publicly available. As demonstrated here, its speed compares quite favorable to other available algorithms, and it allows to solve games of considerable size in reasonable times. Because the method involves the gradual transformation of a prior into equilibrium strategies, it is possible to search the prior space and uncover potentially multiple equilibria and their respective basins of attraction. This also connects the method to established theory of equilibrium selection.
Chapter 4 introduces sgamesolver, a python package that uses the homotopy method to compute stationary equilibria of finite discounted stochastic games. A short user guide is complemented with discussion of the homotopy method, the two implemented homotopy functions logit Markov QRE and logarithmic tracing, and the predictor-corrector procedure and its implementation in sgamesolver. Basic and advanced use cases are demonstrated using several example games. Finally, we discuss the topic of symmetries in stochastic games.