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Gravitropism is a fundamental process in plants that allows shoots to grow upward and roots to grow downward. Protein phosphorylation has been postulated to participate in the intricate signaling cascade of gravitropism. In order to elucidate the underlying mechanisms governing the gravitropic signaling and unearth novel protein constituents, an exhaustive investigation employing microgravity-induced phosphoproteomics was undertaken. The significantly phosphorylated proteins unraveled in this study can be effectively divided into two groups through clustering analysis. Furthermore, the elucidation of Gene Ontology (GO) enrichment analysis disclosed the conspicuous overrepresentation of these clustered phosphoproteins in cytoskeletal organization and in hormone-mediated responses intimately intertwined with the intricate phenomenon of gravitropism. Motif enrichment analysis unveiled the overrepresentation of [-pS-P-] and [-R-x-x-pS-] motifs. Notably, the [-pS-P-] motif has been suggested as the substrate for the Casein kinase II (CK II) and Cyclin-dependent kinase (CDK). Kinase-inhibitor assays confirmed the pivotal role played by CK II and CDK in root gravitropism. Mutant gravitropism assays validated the functional significance of identified phosphoproteins, with some mutants exhibiting altered bending kinetics using a custom-developed platform. The study also compared phosphoproteomics data from different platforms, revealing variations in the detected phosphopeptides and highlighting the impact of treatment differences. Furthermore, the involvement of TOR signaling in microgravity-induced phosphorylation changes was uncovered, expanding the understanding of plant gravitropism responses.
To fulfill the large-scale verification of interesting candidates from the phosphoproteomics study, a novel root and hypocotyl gravitropism phenotyping platform was developed. This platform integrated cost-effective hardware, including Raspberry Pi, a high-quality camera, an Arduino board, a rotation stage (obtained from Prof. Dr. Maik Böhmer), and programmable green light (modified by Sven Plath). In addition, through collaboration with a software developer, machine-learning-based software was developed for data analysis. This platform tested the gravitropic response of candidate mutants identified in the phosphoproteomics study. Furthermore, the capabilities of this platform were expanded to investigate tropisms in other species and organs. To find novel proteins that might act as partners of a key protein that is involved in gravitropism signaling, ALTERED RESPONSE TO GRAVITY 1 (ARG1), immunoprecipitation coupled with Mass Spectrometry (IP-MS) was performed and identified ARG1-LIKE1 (ARL1) as a potential interacting protein with ARG1. This interaction was further confirmed through in vivo pull-down assays and bimolecular fluorescence complementation assays. In addition, the interaction between ARG1 and HSP70-1 was also validated.
Overall, this thesis sheds light on the molecular components and signaling events involved in plant gravitropism. It contributes to existing knowledge and opens up new ways to investigate this fascinating area of plant biology.
Electrospinning is an advanced method for the generation of polymer-based fibers. This fabrication technique has gained great interest in the biomedical field in recent years due to its straightforward application and significant versatility of the resulting fiber mats. The process is carried out by dissolving a (biologically or synthetically derived) polymer or a combination of several polymers in a suitable inorganic or organic solvent and transferring these solutions into a syringe with a needle tip as a spinneret. The power source is connected to the syringe tip, allowing for the application of a high voltage to the polymer solution, and a metallic collector, often a rotating drum cylinder on which the yielded polymer fibers are deposited. The usual fiber diameters range between nano- and micrometers. The yielded fiber mats have distinct characteristics, such as a large surface area, mechanical stability, and good encapsulation efficiency. Therefore, the fiber mats can be used as a topical dosage form for a multitude of diseases (e.g., conjunctivitis, keratitis), as they can be easily applied on or into the human body to release the drug for a prolonged period of time. In addition, the fibers exhibit a high degree of resemblance with the human extracellular matrix, which consists predominantly of collagen fibrils. Therefore, the obtained fiber mats can also be employed as innovative substrates for the cultivation of cells. As a result, electrospinning is suitable for a wide range of applications in the biomedical context, specifically for the targeted, topical delivery of bioactives and also as a cell culture substrate for the cultivation of cells in an enhanced in vivo relevant situation.
One objective of this work was the development and characterization of drug-loaded electrospun fibers for application to the inflamed and infected eye to complement the existing therapy of eye drops as well as systemic administration of anti-infectives. In particular, the focus of the project was the development of ocular implants to treat a herpes simplex infection affecting the human cornea. Additionally, electrospun fibers, which immediately dissolve in the tear fluid upon application and prolong the contact time of the bioactives at the eye, were developed as a topical dosage form to treat bacterial conjunctivitis. An additional objective of this work was the development of electrospun fiber mats as an innovative substrate for the cultivation of human induced pluripotent stem cells to mimic the human blood-brain barrier in vitro. The final objective of the present work was establishing an analytical concept for the comprehensive characterization of electrospun fibers to obtain a greater comparability and reproducibility of data and results from different laboratories.
Herpes simplex keratitis is a viral disease of the cornea that can potentially lead to blindness. This disease commonly occurs after corneal transplantation. As the cornea is the most transplanted tissue worldwide, the incidence of this disease varies from 4.9% to 12.6% (high- and low-income countries). The current therapy involves the application of eye drops as many as six times a day, and in severe cases, the systemic use of antiviral agents is necessary but can cause serious side effects (e.g., renal failure). To prevent the occurrence of herpes simplex keratitis after transplantation, a biodegradable electrospun nanofiber mat with a sustained release of acyclovir was established. The rational development of the fibers was facilitated by correlating the surface wettability with the release kinetics of the individual polymers, which allowed for the successful generation of fiber mats releasing the bioactive acyclovir over three weeks. The molecularly dispersed drug is present as an amorphous solid dispersion within the PLGA-based polymer matrix. Evaluating the cell viability in in vitro models proved that neither acyclovir nor the polymers or the generated fiber mats caused any cytotoxicity. The mechanical stability of the fiber mats was evaluated to ensure adequate handling of the fibers during implantation. The findings demonstrated that the fiber mats exhibit direction-independent mechanical properties, and their mechanical load-bearing capacity is greater than that of an excised human cornea. As a result, the fiber mats are suitable for surgical implantation into the anterior chamber of the eye. An in vitro model of human keratinocytes was infected with herpes simplex virus to demonstrate the antiviral efficacy of the electrospun fiber mats. Immunostaining for two specific viral proteins demonstrated the spread of infection in the model. Hereby, it was found that the placebo- and drug-loaded fibers significantly slowed the spread of infection, which was quantified by plaque assay determination. This experiment revealed that the electrospun fibers exert a synergistic antiviral effect by simultaneously releasing acyclovir, which is a virustatic agent that inhibits the replication of the virus in infected cells, and adsorbing released viral particles onto the surface of the polymer fibers. This reduces the overall burden of released viral particles, which is associated with the severity of the infection outbreak. Thus, with the aid of electrospinning, an ocular implant was successfully generated, which is biodegradable over time and significantly reduces the viral particle burden in vitro. Hence, the fibers represent a potential alternative for the prevention of herpes simplex keratitis after corneal transplantation...
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.
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.
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.
In this thesis, the early time dynamics in a heavy ion collision of Pb-Nuclei at LHC center-of-mass energies of 5 TeV is studied. Right after the collision the system is out-of-equilibrium and essentially gluon dominated, with their density saturating at a specific momentum scale Q_s. Based on a separation of scales for the soft and hard gluonic degrees of freedom, the initial state is given from an effective model, known as the Color Glass Condensate. Within this model, the soft gluons behave classical to leading order, making it possible to study their dynamics in gauge invariant fashion on a three dimensional lattice, solving Hamiltonian field equations of motion, keeping real time. Quark-Antiquark pairs are produced in the gluonic medium, known as the Glasma and manifest themselves as a source of quantum fluctuations.
They enter the dynamics of the gluons as a current, making the system semi-classical. In lattice simulations, the non-equilibrium system is tested for pressure isotropization, which is a necessary ingredient to reach a local thermal equilibrium (LTE), making a hydrodynamical description at a later stage possible. In addition, the occupation of energy modes is studied with its implications on thermalization and classicality.
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.
Understanding the brain's proactive nature and its ability to anticipate the future has been a longstanding pursuit in philosophy and scientific research. The predictive processing framework explains how the brain generates predictions based on environmental regularities and adapts to both predicted and unpredicted events. Prediction errors (PE) occur when sensory evidence deviates from predictions, triggering cognitive and neural processes that enhance learning and subsequent memory. However, the effects of PE on episodic memory have not been clearly explained. This dissertation aims to address three key questions to advance our understanding of PE and episodic memory. First, how does the degree of PE influence episodic memory, and how do expected and unexpected events interact in this process? Second, what insights can be gained from studying the electrophysiological activity associated with prediction violations, and what role does PE play in subsequent memory benefits? Lastly, how do memory processes change across the lifespan, and how does this impact the brain's ability to remember events? By answering these questions, this dissertation contributes to advancing our understanding of the cognitive and neural mechanisms underlying the relationship PE and episodic memory.
Das Experiment ALICE (A Large Ion Collider Experiment) am CERN (Conseil Européen pour la Recherche Nucléaire) LHC (Large Hadron Collider) fokussiert sich auf die Untersuchung stark wechselwirkender Materie unter extremen Bedingungen. Solche Bedingungen existierten wenige Mikrosekunden nach dem Urknall, als die Temperaturen so hoch waren, dass Partonen (Quarks und Gluonen) nicht zu farbneutralen Hadronen gebunden waren. In solch einem Quark-Gluon-Plasma können sich die Partonen frei bewegen, wobei sie allerdings mit anderen Partonen aus dem Medium stark wechselwirken. Am LHC werden Bleikerne auf ultra-relativistische Energien von bis zu 2.68 TeV beschleunigt und zur Kollision gebracht, wobei für weniger als 10 fm/c ein QGP entsteht, das schnell expandiert. Die Partonen hadronisieren, wenn das QGP sich auf Temperaturen von weniger als der Phasenübergangstemperatur von ≈155MeV abkühlt. Die finalen Teilchen- und Impulsverteilungen werden werden vom ALICE Detektor gemessen und geben Aufschluss auf elementare Prozesse im QGP.
Die TPC (Time Projection Chamber ) ist eines der wichtigsten Detektorsysteme von ALICE. Sie trägt maßgeblich zur Rekonstruktion von Teilchenspuren und zur Identifikation der Teilchensorten bei mittleren Rapiditäten bei. Die TPC ist eine große zylindrische Spurendriftkammer und besteht aus einem 88mˆ3 großen Gasvolumen, das von der zentralen Hochspannungselektrode in zwei Seiten geteilt wird. Durchquert ein Teilchen das Gasvolumen, ionisiert es entlang seiner Spur eine spezifische Menge von Gasatomen. Die Ionisationselektronen driften entlang des extrem homogenen elektrischen Feldes zu den Auslesekammern an den Endkappen auf beiden Seiten der TPC. Die Messung der Position und der Menge der Ionisationselektronen erlaubt die Rekonstruktion der Teilchenspur sowie, in Kombination mit der Impulsmessungen über die Krümmung der Teilchenspur im Magnetfeld, die Bestimmung der Teilchensorte über den spezifischen Energieverlust pro Wegstrecke im Gas. Das Gasvolumen der TPC war in LHC Run 1 (2010–2013) mit Ne-CO_2 (90-10) gefüllt. Die Gasmischung wurde zu Ar-CO_2 (88-12) für Run 2 (2015–2018) geändert. Als Auslesekammern wurden Vieldrahtproportionalkammern verwendet, die aus einer segmentierten Ausleseebene, einer Anodendrahtebene, einer Kathodendrahtebene und einem Gating-Grid (GG) bestehen. Das GG is eine zusätzliche Drahtebene, die durch zwei verschiedene Spannungseinstellungen transparent oder undurchlässig für Elektronen und positive Ionen geschaltet werden kann.
In den ersten Daten von Run 2 bei hohen Interaktionsraten wurden große Verzerrungen der gemessenen Spurpunkte beobachtet, die auf Grund von Verzerrungen des Driftfeldes auftreten und nicht von Daten aus Run 1 bekannt waren. Diese Verzerrungen treten nur sehr lokal an den Grenzen von manchen der inneren Auslesekammern (IROCs) auf. Zudem wurden auch große Verzerrungen in einer (C06) der äußeren Auslesekammern (OROCs) festgestellt, die sich bei einem bestimmten Radius über die ganze Breite der Kammer erstrecken. Die Ergebnisse dieser Arbeit befassen sich mit der Untersuchung jener Verzerrungen und ihrer Ursache, sowie mit der Entwicklung von Strategien um die Verzerrungen zu minimieren.
Messungen der Verzerrungen in den IROCs und Vergleiche mit Simulationen lassen darauf schließen, dass die Verzerrungen von positiver Raumladung hervorgerufen werden, die durch Gasverstärkung an sehr begrenzten Regionen der Auslesekammern entsteht und sich durch das Driftvolumen bewegt. Es werden charakteristische Abhängigkeiten von der Interaktionsrate sowie systematische Veränderungen bei Umkehrung der Orientierung des Magnetfeldes gemessen. Eine erneute Analyse von Run 1 Daten mit den Methoden aus Run 2 zeigt, dass die Verzerrungen bereits in Run 1 auftraten, jedoch durch die Ne-Gasmischung und niedrigere Interaktionsraten um eine Größenordnung kleiner waren. Neue Daten aus Run 2, für die die Gasmischung zeitweise wieder von Ar-CO_2 zu Ne-CO_2- N_2 geändert wurde, bestätigen die Ergebnisse der Run 1 Datenanalyse. Der Ursprung der Raumladung wird systematisch eingegrenzt. Es werden einzelne IROCs identifiziert, an deren Anodendrähten die Raumladung entsteht. Physikalische Modelle ermöglichen es, die Entstehung der Raumladung auf das Volumen zurückzuführen, das sich zwischen zwei IROCs befindet. Damit besteht die Vermutung, dass einzelne Spitzen von Anodendrähten am äußeren Rand dieser IROCs in das Gasvolumen hineinragen und somit hohe elektrische Felder erzeugen, an denen Gasverstärkung stattfindet. Die positiven Ionen können dann ungehindert in das Driftvolumen gelangen. Um diesen Effekt zu unterdrücken, wird das Potential der Cover-Elektroden angepasst, die sich auf den Befestigungsvorrichtungen der Drahtebenen an den Kammerrändern befinden. Dadurch kann die Menge von Ionisationselektronen, die in das Volumen zwischen zwei IROCs hineindriftet und vervielfacht wird, eingeschränkt werden. Über elektro-statische Simulationen und Messungen wird eine Einstellung für das Cover-Elektroden-Potential gefunden, mit der die Verzerrungen auf 30 % reduziert werden können. Die Verzerrungen in OROC C06 entstehen durch positive Ionen, die aus der Verstärkungsregion in das Driftvolumen gelangen, da an dieser bestimmten Stelle zwei aufeinanderfolgende GG-Drähte den Kontakt verloren haben. Die Verzerrungen werden um mehr als einen Faktor 3 reduziert, indem die Hochspannung der Anodendrähte um 50 V und somit der Gasverstärkungsfaktor um einen Faktor 2 verringert wird und indem das Potential der noch funktionierenden GG-Drähte erhöht wird.
Zusammenfassend konnten die lokalen Raumladungsverzerrungen für die letzte Pb−Pb Strahlzeit von Run 2 auf weniger als 1cm bei den höchsten Interaktionsraten verringert werden. Zudem wurde der Anteil des von Raumladungsverzerrungen betroffenen Volumens der TPC signifikant verringert, sodass die ursprüngliche Auflösung der Spurrekonstruktion wieder erreicht werden konnte.
Influenza is a contagious respiratory disease caused by influenza A and influenza B viruses. The World Health Organisation (WHO) reports that annual influenza epidemics result in approximately 1 billion infections, 3 to 5 million severe cases, and 300 to 650 thousand deaths. Understanding hidden mechanisms that lead to optimal vaccine efficacy and improvement antiviral treatment strategies remain continuous and central tasks. First, regarding the immune response to vaccines and natural infections, the antibody response echoes the dynamics of diverse immune elements such as B-cells, and plasma cells. Also, responses reflect the processes for B-cells to gain and adapt affinity for the virus. Antibodies (Abs) that respond to the virus surface proteins, particularly to the hemagglutinin (HA), have been identified to protect against infection. The Abs responses binding to HA can be broadly protective as this protein is considerably accessible on the virion. When following sequential infections with similar influenza strains, i.e. two infections with different strains of a subtype, an enhanced breadth and magnitude of Abs response is developed, mainly after the second infection. The effect of being effective to new strains is called Abs cross-reaction.
On the other hand, as for antiviral treatment, the WHO currently approves the use of neuraminidase inhibitors (NIs) such as zanamivir and oseltamivir. Diverse research areas such as system biology, learning-based methods, control theory, and systems pharmacology have guided the development of modern treatment schemes. To do so, mathematical models are used to describe a wide range of phenomena such as viral pathogenesis, immune responses, and the drug's dynamics in the body. Drug dynamics are usually expressed in two phases, pharmacokinetics (PK) and pharmacodynamics (PD) - the PK/PD approach. These schemes leverage pre-clinical and clinical data through modeling and simulation of infection and drug effects at diverse levels. Under such a framework, control-based scheduling systems seek to tailor optimal antiviral treatment for infectious diseases. Thus, influenza treatment can be theoretically studied as a control-based optimization duty (about systems stability, bounded inputs, and optimality). Finally, towards real-world implementation, learning-based methods such as neural networks (NNs) can guide solving issues on the control-based performance. Using NNs as identifiers provide a setting to deal with infrequent measures and uncertain parameters for the control systems.
This thesis theoretically explores central mechanisms in influenza infection via modeling and control approaches. In the first project, we explore how and to what extent antibody-antigen affinity flexibility could guide the Abs cross-reaction in two sequential infections using a hypothetical family of antigens. The set of antigens generally represent strains of influenza, such as those of a subtype. Each antigen is composed of a variable and a conserved area, generically representing the structures of the HA, head, and stalk, respectively. We test diverse scenarios of affinity thresholds in the conserved and variable areas of the antigens. The Abs response reaches a high magnitude when using equivalent affinity thresholds in the conserved and variable areas during the first infection. However, improved cross-reaction is developed when slightly increasing the affinity threshold of the variable area for the second infection. Key mutations via affinity maturation is a feature that, together with affinity flexibility between infections, guides Abs cross-reaction in the model outcome. These results could correlate with studies pointing out that broad responses might be dependent on reaching specific mutations for getting affinity to a newly presented antigen while broadly reaching related antigens. The general platform may serve as a proof-of-concept for exploring fundamental mechanisms that favor the Abs cross-reaction.
In a second project, theoretical schemes are developed to combine impulsive and inverse optimal control strategies to address antiviral treatment scheduling. We present results regarding stability, passivity, bounded inputs, and optimality using impulsive action. The study is founded on mathematical models of the influenza virus (target-cell limited model) adjusted to data from clinical trials. In these studies, participants were experimentally infected with influenza H1N1 and treated with NIs. Results show that control-based strategies could tailor dosage and reduce the amount of medication by up to 44%. Also, control-based treatment reaches the efficacy (98%) of the current treatment recommendations by the WHO. Monte Carlo simulations (MCS) disclose the robustness of the proposed control-based techniques. Using MCS, we also explore the applicability to the individualized treatment of infectious diseases through virtual clinical trials. Furthermore, bounded control strategies are applied directly in drug dose estimation accounting for overdose prevention. Finally, due to the limitations of the available technology intended for clinical practice, we emphasize the necessity of developing system identifiers and observers for real-world applications.
In the third project, the problem of data scarcity and infrequent measures in the real world is handled by means of learning-based methods. System identification is derived using a Recurrent High Order Neural Network (RHONN) trained with the Extended Kalman filter (EKF). Lessons learned from impulsive control frameworks are taken to develop a neural inverse optimal impulsive control --neurocontrol. The treatment efficacy is tested for early (one day post-infection) and late (2 to 3 days post-infection) treatment initiation. The neurocontrol reaches an efficacy of up to 95% while saving almost 40% of the total drug in the early treatment. Robustness is tested via virtual clinical trials using MCS.
Lastly, taking all together, the schemes developed in this thesis for modeling the Abs cross-reaction and control-based treatment tailoring can be extended and adapted to explore similar phenomena in different respiratory pathogens, such as SARS-CoV-2.
Molecular oxygen (O2) is essential for numerous metabolic processes. Not surprisingly, hypoxia and the resulting adaptations play a pivotal role in pathophysiology, e.g., in cancer or in inflammatory diseases. Of note, myeloid cells are known to accumulate in hypoxic regions such as tumor cores or rheumatoid arthritis joints and may contribute to disease progression. While most studies so far concentrated on transcriptional adaptation by the hypoxia-inducible factors (HIF) 1 and 2 under short term hypoxia, prolonged oxygen deprivation and alternative post-transcriptional regulation are rather poorly investigated.
Consequently, the aim of the study was to generate a comprehensive overview of mRNA de novo synthesis and degradation and its contribution to total mRNA changes in monocytic cells in the course of hypoxia.
To this end, I used thiol-linked alkylation for the metabolic sequencing of RNA (SLAM-Seq) to characterize RNA dynamics under hypoxia. Specifically, I labeled monocytic THP-1 cells under normoxia (N), acute hypoxia (AH; 8 h 1% O2), or chronic hypoxia (CH; 72 h 1% O2) with 4-thiouridine (4sU), which allows for transcriptome-wide identification of de novo synthesized mRNAs and estimation of their half-lives. Total mRNA expression analyses revealed that most changes occurred under CH. Considering that HIF accumulation and resulting transcriptional regulation was shown to decline again under CH, I further analyzed the impact of RNA stability on gene expression. I observed a global reduction in RNA half-lives under hypoxia, indicative for the attenuation of energy-consuming protein synthesis upon oxygen deprivation. Moreover, I observed a subgroup of hypoxic destabilized transcripts with resulting decreased mRNA expression under CH, which consisted of 59 nuclear-encoded mitochondrial mRNAs. This might prevent futile production of new mitochondria under conditions, where mitochondria are even actively degraded to prevent production of detrimental reactive oxygen species.
While stability-regulated transcripts were mainly destabilized under hypoxia, the vast majority of differentially de novo synthesized transcripts were upregulated.
Functional analyses revealed not only hypoxia, but also cholesterol homeostasis and inflammatory response as top enriched terms, corroborating findings on total mRNA level. Focusing on hypoxia-altered cholesterol metabolism, I observed an 9 accumulation of early and a decrease in late cholesterol precursors, which are separated by several oxygen-dependent enzymatic steps. Although total cholesterol levels were only slightly reduced, my data indicate locally lowered endoplasmic reticulum (ER) cholesterol levels under hypoxia, which cause feedback activation of the ER cholesterol-sensing transcription factor sterol regulatory element-binding protein 2 (SREBP2) and induction of cholesterol biosynthesis enzymes. Interestingly, a broad range of interferon-stimulated genes (ISGs), mainly known for their antiviral function, was also induced under hypoxia with similar kinetics as SREBP2 targets, suggesting an immunometabolic crosstalk. While the availability of certain cholesterol biosynthesis intermediates as well as a direct involvement of SREBP2 seemed rather unlikely to cause hypoxic ISG induction, changes in intracellular cholesterol distribution appeared crucial for the hypoxic induction of chemokine-ISGs. Mechanistically, I found that MyD88-dependent toll-like receptor 4 (TLR4) signaling contributes to enhanced hypoxic ISG induction, likely sensitized by changes in cholesterol dynamics. Importantly, hypoxia amplified induction of chemokine-ISGs in monocytes upon treatment with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein via TLR4 similarly as after addition of infectious virus, which might contribute to systemic inflammation in hypoxemic patients with severe coronavirus disease-2019 (COVID-19).
Taken together, I comprehensively analyzed RNA dynamics in hypoxic monocytes. Specifically, I identified RNA stability as a modulating mechanism to limit production of mitochondria under oxygen-restricted conditions. Moreover, I characterized the immunometabolic crosstalk between disturbed cholesterol homeostasis and spontaneous induction of interferon (IFN)-signaling in hypoxic monocytes, which might contribute to systemic inflammation in severe cases of COVID-19.
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.
The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death
(2023)
Every day, living organisms are challenged by internal and external factors that threaten to bring imbalance to their tightly regulated systems and disrupt homeostasis, leading to degeneration, and ultimately death. More than ever, we face the challenge of combating diseases such as COVID-19 caused by infection with the SARS-CoV-2 coronavirus. It is therefore crucial to identify host factors that control antiviral defense mechanisms. In addition, in the fight against cancer, it is becoming increasingly important to identify markers that could be used for targeted therapy to influence cellular processes and determine cell fate.
As a deubiquitylating enzyme, ubiquitin specific peptidase 22 (USP22) mediates the removal of the small molecule ubiquitin, which is post-translationally added to target proteins, thereby regulating several important processes such as protein degradation, activation or localization. Through its deubiquitylating function, USP22 controls several biological processes such as cell cycle regulation, proliferation and cancer immunoresistance by modulating key proteins involved in these pathways. Lately, USP22 was reported to positively regulate TNFα-mediated necroptosis, an inflammatory type of programmed cell death, in various human tumor cell lines by affecting RIPK3 phosphorylation. In addition, USP22 as a part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription complex is known to regulate gene expression by removing ubiquitin from histones H2A and H2B. However, little is known about the role of USP22 in global gene expression.
In this study, we performed a genome-wide screen in the human colon carcinoma cell line HT-29 and identified USP22 as a key negative regulator of basal interferon (IFN) expression. We further demonstrated that the absence of USP22 results in increased STING activity and ubiquitylation, both basally and in response to stimulation with the STING agonist 2'3'-cGAMP, thereby affecting IFNλ1 expression and basal expression of antiviral ISGs. In addition, we were able to establish USP22 as a critical host factor in controlling SARS-CoV-2 infection by regulating infection, replication, and the generation of infectious virus particles, which we attribute in part to its role in regulating STING signaling.
In the second part of the study, we connected the findings of USP22-dependent regulation of IFN signaling and TNFα-induced necroptosis and investigated the role of USP22 during necroptosis induced by the synergistic action of IFN and the Smac mimetic BV6 in caspase-deficient settings. We identified USP22 as a negative regulator of IFN-induced necroptosis, which does not depend on STING expression, but relies on a yet unknown mechanism.
In summary, we identify USP22 as an important regulator of IFN signaling with important implications for the defense against viral infections and regulation of the necroptotic pathway that could be exploited for devising targeted therapeutic strategies against viral infections and related diseases like COVID-19, and advancing precision medicine in cancer treatment.
Necroptosis is an immunogenic form of programmed cell death characterized by plasma membrane accumulation of activated mixed lineage kinase domain-like (MLKL) that eventually leads to membrane disruption and release of danger-associated molecular patterns (DAMPs). Necroptotic cell death is tightly controlled by checkpoints, including compartmentalization as well as post-translational modifications (PTMs), like phosphorylation and ubiquitination of receptor-interacting protein kinase (RIPK) 1, RIPK3 and MLKL. Removal of plasma membrane-located activated MLKL via endocytosis or exocytosis can counteract necroptosis, but up till now, the exact mechanisms by which necroptosis is regulated downstream of MLKL activation and oligomerization are not fully understood.
Ubiquitination is a key post-translational modification that regulates various cellular processes including cell survival and cell death signaling via ubiquitination of RIPK1, RIPK3 and MLKL. M1-linked (linear) poly-ubiquitination is mediated exclusively by the linear ubiquitin chain assembly complex (LUBAC) which critically regulates cell fate and immune signaling via death receptors such as TNF receptor 1 (TNFR1).
In this study, we demonstrate that M1 poly-Ubiquitin (poly-Ub) increases during necroptosis which can be blocked by inhibition of LUBAC activity with the small-molecule HOIL-1-interacting protein (HOIP) inhibitor HOIPIN-8 or by loss of LUBAC catalytic subunit HOIP. Intriguingly, HOIPIN-8, as well as the HOIP inhibitor gliotoxin, and HOIP knockdown effectively prevent TNFα/smac mimetic/zVAD.fmk-induced necroptotic cell death in cells of human origin, without affecting necroptotic RIPK1 and RIPK3 phosphorylation, necrosome formation and oligomerization of phosphorylated MLKL. We demonstrate that HOIPIN-8 treatment inhibits MLKL translocation to intracellular membranes and accumulation in plasma membrane hotspots as well as MLKL exocytosis. We further confirm that HOIPIN-8 treatment suppresses necroptotic cell death in primary human pancreatic organoids (hPOs). Using time-lapse imaging and live/dead staining, we demonstrate loss of organoid structure and hPO cell death induced by smac mimetics and caspase inhibitors, thus providing a novel platform to investigate necroptosis in near physiological settings. Inhibition of LUBAC activity with HOIPIN-8 prevents hPO collapse and extends cell viability. Of note, loss of the M1 Ub-targeting deubiquitinating enzymes (DUBs) OTU DUB with linear linkage specificity (OTULIN) and cylindromatosis (CYLD) in human cell lines does not affect necroptosis induction and HOIPIN-8-mediated rescue of necroptosis. Intriguingly, inhibition of LUBAC activity with HOIPIN-8 does not block necroptotic cell death in murine cell lines.
Using massive analyses of cDNA ends (MACE)-seq-based global transcriptome analysis we confirm that necroptosis induces a pro-inflammatory cytokine profile which is dependent on LUBAC function and necroptotic signaling. Loss of LUBAC activity prevents the MLKL-dependent production and release of pro-inflammatory cytokines and chemokines.
Finally, we identify Flotillin-1 and -2 (FLOT1/2) as putative targets of necroptosis-induced M1 poly-Ub. Ubiquitin-binding in ABIN and NEMO (UBAN)-based pulldowns of M1 poly-ubiquitinated proteins revealed enrichment of FLOTs after necroptosis induction which is dependent on LUBAC activity and can be blocked with necroptosis inhibitors Nec-1s, GSK’872 and NSA, targeting RIPK1, RIPK3 and MLKL, respectively. Of note, loss of FLOT1/2 potentiates necroptosis suppression induced by LUBAC inhibition with HOIPIN-8.
Together, these findings identify LUBAC-mediated M1 poly-Ub as an important mediator of necroptosis and identify FLOTs as novel putative targets of LUBAC-mediated M1 poly-Ub during necroptosis. In addition, by modeling necroptosis in primary human organoids, we further expand the spectrum of experimental models to study necroptosis in human cellular settings.
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.
Um sich an ändernde Umwelteinflüsse und metabolische Bedürfnisse anpassen zu können, ist es für Zellen essenziell, dass Boten-RNA (engl. messenger RNA, mRNA) stetig und schnell nach der Translation abgebaut wird. In Prokaryoten ist dafür der Proteinkomplex Degradosom verantwortlich, in dem Endo- und Exoribonukleasen RNase E und PNPase das RNA-Transkript in kleinere Fragmente und schließlich einzelne Nukleotide spalten. Die DEAD-Box Helikase RhlB im Komplex dient zusätzlich dazu, mögliche Sekundärstrukturen in der RNA zu entfalten, welche sonst die weitere Degradation behindern würden. Es konnte gezeigt werden, dass RhlB’s sehr geringe katalytische Aktivität – gemessen durch ATP-Verbrauch und Rate an entwundener RNA – signifikant durch die allosterische Bindung an Komplexpartner RNase E erhöht wird. Gleichzeitig deuten andere Studien darauf hin, dass RhlB eine mögliche Selektivität für doppelsträngige RNA-Substrate mit 5‘-Einzelstrang-Überhängen aufweist.
Diese Arbeit liefert neue Erkenntnisse in Bezug auf die Kommunikation zwischen den Degradosom-Komponenten RhlB und RNase E aus E. coli, indem das potenzielle Wechselspiel zwischen RhlBs RNA-Selektivität und der allosterischen Aktivierung durch RNase E untersucht wurde. Der vielseitige Einsatz NMR-spektroskopischer Techniken sowie die Verwendung kurzer RNA-Substrate mit spezifischen Strang-Eigenschaften ermöglicht es, mit einen ungewöhnlichen, RNA-zentrierten Ansatz an diese unzureichend verstandene Protein-Interaktion heranzugehen.
Zunächst wurden hierzu eine Reihe kurzer doppelsträngiger RNA-Konstrukte hergestellt, die sich nicht nur in ihren Einzelstrang-Merkmalen unterscheiden, sondern auch die thermodynamischen Anforderungen eines DEAD-Box Helikase Substrats erfüllen, und gleichzeitig eine ausreichende NMR-spektroskopische Signal-Zuordnung erlauben. Die thermale Stabilität, das Faltungsverhalten sowie die 1H Imino-protonen- und 13C HSQC-Zuordnungen aller geeigneten Konstrukte wurden erfolgreich bestimmt.
Um den Einfluss spezifischer RNA-Substrate sowie die Bindung zweier verschiedener RNase E Fragmente auf RhlBs ATP-Umsatzrate zu untersuchen, wurde sich zunächst eines photometrischen Phosphat-Assays bedient. Damit konnte deutlich gezeigt werden, dass RhlB in Abwesenheit des Komplex-Partners nicht in der Lage ist, signifikante Mengen an ATP umzusetzen, unabhängig davon, welches RNA-Konstrukt eingesetzt wird. Die Bindung der RNase E Fragmente erhöhte signifikant die ATP-Hydrolyse-Rate der Helikase, wobei die größte Aktivierung für den RNA-Duplex mit 5‘-Einzelstrang sowie ein einzelsträngiges Substrat zu beobachten ist. Da diese Ergebnisse deutlich eine RNA-Abhängigkeit beim ATP-Umsatz der Helikase zeigen, wurde untersucht, ob diese Unterschiede ihren Ursprung bereits in der Bindung der spezifischen RNA-Substrate haben. Mittels einer Mischapparatur, die es erlaubt die enzymatische Reaktion direkt im Spektrometer zu initiieren sowie zeitaufgelöster 31P NMR-Experimente konnte die allosterische Aktivierung der ATP-Hydrolyse-Rate von RhlB auch unter NMR-spektroskopischen Messbedingungen nachgewiesen werden.
Da die Ergebnisse des ATPase Assays deutlich eine RNA-Abhängigkeit bei der ATP-Umsatz-Rate der Helikase zeigen, wurde zusätzlich untersucht, ob diese Unterschiede ihren Ursprung in den Affinitäten für die verschiedenen RNA-Substrate haben und ob diese durch die Bindung von RNase E and RhlB beeinflusst werden. Um im gleichen Zuge zu überprüfen, ob die Bindung der RNA an RhlB die RNA-Konformation oder Basenpaarung ändert, werden 1H NMR-Titrationsexperimente durchgeführt. Es konnte erstmals gezeigt werden, dass RhlB eine inhärente Präferenz für Duplexe mit 5‘-Überhang gegenüber Konstrukten mit 3‘-Überhang oder stumpfen Enden besitzt, was sich in einer erhöhten Affinität zeigt. Zusätzlich offenbaren die Messungen, dass RNase Es allosterische Bindung selektiv die Affinität gegenüber Konstrukten mit Einzelstrang-Überhang erhöht, während die Affinität zu RNA Duplexen ohne Überhang sogar verringert wird. Diese Ergebnisse liefern erstmals einen Nachweis, dass RNase E aktiv Einfluss auf RhlBs RNA-Bindung nimmt. Weder die Bindung der RNA and RhlB noch an den RhlB/RNase E Komplex scheint die Basenpaarung oder Konformation der RNA-Substrate zu beeinflussen, da lediglich eine homogene Peak-Verbreitung aller Imino-Protonen-Signale im 1H NMR-Spektrum beobachtet werden konnte.
The role of Apelin signaling and endocardial protrusions during cardiac development in zebrafish
(2023)
During cardiac development, cardiomyocytes (CMs) are delaminated from the compact muscle wall to increase the muscle mass of the heart. This process is also known as cardiac trabeculation. It has been shown that growth factors produced by endocardial cells (EdCs) are required for myocardial morphogenesis and growth. In particular, Neuregulin produced by EdCs promotes myocardial trabeculation. The deficiency of Neuregulin signaling leads to hypotrabeculation. Endocardial protrusions project from the endocardium to the myocardium are also essential for the trabeculae onset. Yet current studies only introduce the function of endocardial sprouts descriptively. This article first reports the mechanisms of endocardial sprouting during myocardial trabeculation. By living imaging, we first demonstrate that EdCs interact with CMs through membrane protrusions in zebrafish embryos. More interestingly, these protrusions stay in close contact with their target CMs in spite of the cardiac contraction. We utilize loss-of-function strategies to report the importance of myocardial apelin, which induces endocardial protrusion formation. Zebrafish lacking Apelin signaling exhibit defects in endocardial protrusion formation as well as excessive deposition of cardiac jelly and hypotrabeculation. Notably, we also present data that blocking protrusion formation in endocardial cells phenocopies the trabeculation defects in apelin mutants. Mechanistically, endocardial-derived Neuregulin requires Apelin signaling mediated endocardial protrusions, and Neuregulin dependent pERK expression is attenuated in the condition of reduced endocardial protrusion formation. Together, our data suggest that endocardial-myocardial communication through endocardial protrusions acts as an underlying principle allowing myocardial growth.
This research attempts to provide for an overview of the state of co-operation between the United Nations and regional organizations like the CoE, OSCE, EU and NATO during the last Yugoslav wars, considering the 1991-2008 period. In this case, the "reconstruction" of what the organisations did in each of the countries involved in the conflicts, the country-by-country approach used in writing the research and the consideration of both headquarters and field level should facilitate the understanding of the state of things at that time. The research further includes an analysis of the co-operative trends developed by the considered international organisations since the beginning of the 1990s and is concluded by a reflection on the normative relevance of the issue of "international cooperation". In this case, the intention of the author was to go beyond the general policy level approach used for the description of UN-regional organizations interaction and propose a re-consideration of the concept of "international co-operation" as a possible normative tool in guiding the so far nebulous division of tasks of international actors in conflict-related scenarios. In this case, the concise description of the general framework for co-operation under Chapter VIII of the UN Charter, already matter of wide debate by academics and practitioners, sets the frame for a more elaborate, and hopefully innovative, consideration of the notion of "international cooperation". This, of course, is to be contextualized to the lessons learned extrapolated from the case study.
Terahertz (THz) radiation lies between the micro and far-infrared range in the electromagnetic spectrum. Compared with microwave and millimeter waves, it has a larger signal bandwidth and extremely narrow antenna beam. Thus, it is easier to achieve high-resolution for imaging and detection applications. The unique properties, such as penetration for majority non-polar materials, non-ionizing characteristic and the spectral fingerprint of materials, makes THz imaging an appealing artifice in the military, biomedical, astronomical communications, and other areas. However, THz radiation’s current low power level and detection sensitivity block THz imaging system from including fewer optical elements than the visible or infrared range. This leads to imaging resolution, contrast, and imaging field of view degenerate and makes the aberration more serious. THz imaging based on the space Fourier spectrum detection is developed in this thesis to achieve high-quality imaging. The main concept of Fourier imaging is by recording the field distribution in the Fourier plane (focal plane) of the imaging system; the information of the target is obtained. The numerical processing method is needed to extract the amplitude and phase information of the imaged target. With additional process, three-dimensional (3D) information can be obtained based on the phase information. The novel recording and reconstructing ways of the Fourier imaging system enables it to have a higher resolution, better contrast, and broader field of view than conventional imaging systems such as microscopy and plane to plane telescopic imaging system.
The work presented in this thesis consists of two imaging systems, one is working at 300 GHz based on the fundamental heterodyne detection of the THz radiation, the other is operated at 600 GHz by utilizing the sub harmonic heterodyne detection technique. The realization and test of the heterodyne detection are based on the THz antenna-coupled field-effect transistor (TeraFET) detector developed by Dr. Alvydas Lisauskas. Both systems use two synchronized electronic multiplier chains to radiate the THz waves. One radiation works as the local oscillator (LO), the other works as illumination with a slight frequency shift, the radiations are mixed on the detector scanning in the Fourier plane to record the complex Fourier spectrum of the imaged target. The LO has the same frequency range as the illuminating radiation for fundamental heterodyne detection but half the frequency range for the sub-harmonic heterodyne detection. The 2-mm resolution, 60-dB contrast, and 5.5-cm diameter imaging area at 300 GHz and the of 500-μm resolution, 40-dB contrast, and 3.5-cm diameter imaging area at 600 GHz are achieved (the 300-GHz illuminating radiation has the approximate power of 600 μW , the 600-GHz illuminating radiation has the approximate power of 60 μW ).
The thesis consists of 6 parts. After the introduction, the second chapter expands on the topic of Fourier optics from a theoretical point of view and the simulations of the Fourier imaging system. First, the theory of the electromagnetic field propagation in free space and through an optical system are investigated to elicit the Fourier transform function of the imaging system. The simulation is used for theoretical considerations and the implementation of a Fourier optic script that allows for numerical investigations on reconstruction. The preliminary imaging field of view and resolution are also demonstrated. The third chapter describes the Fourier imaging system at 300 GHz based on the fundamental heterodyne detection, including the experimental setup, the 2D, and 3D imaging results. The following fourth chapter reports the integration of the TeraFET detector with two substrate lenses (one is a Si lens on the back-side Si substrate, the other is a wax/PTFE lens on the front side containing the bonding wires) for sub-harmonic heterodyne detection at 600 GHz. The characteristic of the wax/PTFE lens at THz range is presented. After that, the compared imaging results between the detector with and without the wax/PTFE lens are shown. The fifth chapter extends the demonstration on the lateral and depth resolution of the Fourier imaging system in detail and uses the experimental results at 600 GHz to validate the analytical predictions. The comparison of the resolution between the Fourier imaging system and the conventional microscopy system proves that the Fourier imaging system has better imaging quality under the same system configuration. The last chapter in this thesis concludes on the findings of the THz Fourier imaging and gives an outlook for the enhancement of the Fourier imaging system at THz range.
This dissertation investigates a special class of anaphoric form, yè, in Ewe known as the logophoric pronoun. This research makes a number of novel observations.
In the first chapter, I introduce the reader to the phenomenon under investigation as well as provide information on Ewe and its dialects and, methodology. In Chapter 2, I present the pronominal system of Ewe which is categorised into strong and weak forms following Cardinaletti & Starke (1994) and Agbedor (1996). The distribution of pronouns is outlined which sets the tone for an overview of logophoric marking. In this respect, I present variations in logophoric marking strategies cross linguistically and show that Ewe differs significantly from other pronouns in this category. In an effort to explain the deviant case of yè, I entertain the idea that yè is a pure logophoric pronoun in the sense of Clements (1975) and thus, its additional de re and strict interpretation does not imply non-logophoricity.
Chapter 3 demonstrates that yè is sensitive to contexts which portray the intention of an individual. Following Sells (1987), the antecedent of yè must have an intention to communicate. I broadly categorize logophoric contexts into reportative (direct-indirect speech) or non-reportative (speaker’s mental attitude, reporter’s observation or background knowledge of a situation). Based on this categorization, indirect speech report (Clements 1975), dis- course units such as a paragraph or an episode (Clements 1975), and sentential adjuncts such as purpose, causal and consequence clauses (Culy 1994a) are reviewed. The logophoric pro- noun occurs in the complement of attitude verbs (Clements 1975), also termed logocentric (à la (Stirling 1994)) or logophoric predicates (à la (Culy 1994a)) as well as with non-attitudinal verbs (e.g. va ‘come’ or wO ‘do’ as in sentential adjuncts). I argue contra Clements (1975) and Culy (1994a) that yè can occur with perception predicates. I further provide three new instances of non-reportative contexts which are compatible with yè namely, as-if clauses, benefactive na clauses and alesi ‘how’ clauses. I show, corroborating previous studies that contexts which are necessary for the licensing of yè include all of the aforementioned except causal clauses. Among these contexts, the complementizer be or regarding cases where there is no be, an element in C (due to the Doubly-Filled-Comp Filter (DFCF) c.f. Chomsky & Lasnik (1977)), is sufficient to license yè. Following Bimpeh & Sode (2021), yè is licensed by feature checking (in the spirit of von Stechow (2004)): be bears the interpretatble [log] feature which checks the uninterpretable [log] feature of yè. I include a redefinition of logophoricity as pertaining to Ewe.
Given the disparity found in the literature concerning the interpretation of yè: Ewedome (pronounce EVedome) has only de se readings (Bimpeh 2019); while ‘pure’ Ewe, Mina (variety of Ewe spoken in Togo) Pearson (2015), Danyi (O’Neill 2015) and Anlo (pronounced ANlO) (Satık 2019) has de re readings; chapter 4 aims at lending empirical support to the ungoing discussion by verifying the interpretation of yè. Two acceptability judgment tasks were conducted namely, truth value judgment task and binary forced choice task. The results corroborates Pearson (2012, 2015) and others’ discovery that yè has a de re interpretation in the Ewedome (contra Bimpeh (2019); Bimpeh et al. (2022)), Anlo and Tonu (pronounced TONu) dialects of Ewe.
In chapter 5, I discuss the relation between logophoricity (yè, yè a) and Control (PRO). I show that yè may be restricted to a set of verbs which obligatorily require the morpheme a ‘potential marker’ (Essegbey 2008), in subject position. This set of verbs are those that are known as control verbs c.f. (Landau 1999) in English. As a result of this restriction, research such as Satık (2019) claims that yè a is the overt instantiation of PRO in English. According to the Ewe facts, it appears as though on one hand, yè and PRO share similar properties in logophoric contexts and on the other hand, yè in combination with the potential marker, a also share properties with PRO in subject control environments. Against this background, I discuss the relation between yè, yè a and PRO and show that neither yè in isolation nor yè in combination with a, contrary to Satık (2019), is the overt instantiation of PRO. I clarify that the potential morpheme a is not cliticised or combined with the logophoric yè. The two forms are seperate morphemes. The potential marker a only shows up in control environments because a sub-class of verbs require it for grammaticality purposes. As such, the property of de se-ness does not come from yè by itself, yè a or a but rather from the sub-class of verbs which require the potential marker a...
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.
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.
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.
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.
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.
Precise tune determination and split beam emittance reconstruction at the CERN PS synchrotron
(2023)
In accelerator physics, the need to improve the performance and better control the operating point of an accelerator has become, year after year, an increasingly important need in order to achieve higher energies and brightness, as well as point-like particle beams. If this involves increasingly advanced technological developments (in terms, for example, of materials for more intense superconducting magnets), it can not take place in the absence of targeted studies of linear and non-linear beam dynamics. In the context of this Ph.D. thesis in physics, linear and non-linear dynamics of charged particles in circular accelerators is the topic that will be discussed and treated in detail. In particular, the presentation and discussion of the results will be divided in two main topics: the need to know the physical properties of a proton beam; and the development of innovative methods to determine and study the accelerator’s working point. With regard to the first topic, an innovative procedure will be presented to determine the transverse size of the PS beam in the beam extraction phase. Among the different ways the extraction occurs at the PS, the analysed one is based on the transverse splitting of the beam by means of non-linear fields. Thus, the knowledge of the transverse beam size is not trivial since resonant linear and non-linear beam structures (namely, core and islands) arise and, for each of them, the beam size has to be quantified. This parameter is crucial for two main reasons: the accelerator that will receive the beam exiting the upstream accelerator may have restrictions (physical or magnetic) that involve a partial or total loss of the incoming beam; and any experiments located downstream of the considered accelerator may need a beam with a transversal size as constant as possible; consequently, its monitoring and control are essential. The second topic concerns the accurate determination of the working point of an accelerator, defined as the number of transverse oscillations the particle beam travels per unit of accelerator circumference, both horizontally and vertically. This quantity is called horizontal and vertical tune, respectively. Their knowledge is also crucial to understand whether the beam will be stable or unstable. In fact, not all tune values are acceptable, as there are particular values that bring the beam into resonance. In this configuration, the amplitude of the transverse oscillations of the particles increases in an uncontrolled manner and leads to the loss of all or part of the beam. Note that, in particular operating conditions, the resonant conditions are sought and desired to model, in a suitable way, the transversal shape of the beam, such as the above mentioned PS extraction scheme. It is even clearer how much the determination of the machine working point is essential to determine the operating conditions of an accelerator. In this context, several methods (also taken from the field of applied mathematics) to calculate the tune will be demonstrated and tested numerically on different types of synthetic signals. At the end of this description, the use of experimental data will allow to obtain the benchmark of a new method for the direct calculation of some characteristic quantities of non-linear beam dynamics (namely, the amplitude detuning, i.e. the variation of tune as a function of intensity of the perturbation provided to the beam.
Bei den meisten erwachsenen Säugetieren führt ein Herzinfarkt zu Fibrose und Verlust von funktionellem Herzgewebe. Einige Wirbeltiere, wie der Zebrabärbling, besitzen jedoch die bemerkenswerte Fähigkeit, nach einer Schädigung ihres Herzgewebes verlorenes Gewebe zu regenerieren und so schädliche Folgen zu verhindern. Die lokale Immunantwort auf eine Verletzung wird zunehmend als eine wichtige Determinante für das regenerative Potential eines Gewebes gesehen. Das Komplementsystem ist Teil des humoralen Immunsystems. Historisch ist es als eine Sammlung von Protein bekannt, den Komplementkomponenten, die in der Leber synthetisiert werden und im Blutkreislauf zirkulieren. Bei Exposition gegenüber einem Auslöser, wie z. B. einem Pathogen, wird eine Komplementkomponentproteinspaltungskaskade initiiert, die dazu führen kann, dass Immunzellen rekrutiert werden, und, dass die Phagozytose erleichtert, ggf. die Zielzelle lysiert wird. Studien legen nahe, dass das Komplementsystem an zellulären Prozessen beteiligt sei, die für Entwicklungs- und Krankheitsprozesse entscheidend sind, wie etwa Proliferation und Dedifferenzierung. Es gibt Hinweise, dass das Komplementsystem eine Rolle bei Krebserkrankungen und bei regenerativen Prozessen spielen könnte. In verschiedenen Arten wurde eine lokale verletzungsinduzierte Expression von komplementkomponentkodierenden Genen in regenerierendem Gewebe beobachtet.
Einzelne Studien legen nahe, dass Funktionsverlust einzelner Komplementkomponenten regenerative Prozesse beeinträchtigt.
Offene Fragen bleiben jedoch: Ist die lokale Expression von mehreren komplementkomponentkodierenden Genen ein Merkmal von regenerierendem Gewebe, das sie von Geweben unterscheidet, welchem die Fähigkeit zur Regeneration fehlt? Und welche Rolle könnte das Komplementsystem und seine Komponenten während des regenerativen Prozesses spielen? Um diesen Fragen nachzugehen, wurde eine Expressionsanalyse von Zebrabärblingsgewebe nach Verletzung mittels RT-qPCR und in situ Hybridisierung durchgeführt: kardiale Kryoverletzung, Larvenrumpfamputation und Schwanzflossenamputation. Ich beobachtete, dass mehrere komplementkomponentkodierende Gene in diesen Geweben nach Verletzung induziert wurden. Die Interpretation veröffentlichter single cell RNAseq Datensätze legt nahe, dass diese komplementkomponentenkodierenden Gene von verschiedenen Zelltypen exprimiert werden, darunter Immunzellen, Epikardzellen und Fibroblasten. Um transkriptionelle Unterschiede zwischen regenerierendem und nicht regenerierendem Gewebe zu identifizieren, verwendete ich ein nicht regeneratives Zebrabärblingmodell, die il11ra- Mutante. Dieser Mutante fehlt die Fähigkeit, verschiedene Organe zu regenerieren, das ist der Fall beim Herzen, dem larvalen Rumpf, und der Schwanzflosse. Ich stellte fest, dass die Mehrheit der verletzungsinduzierten komplementkomponentkodierenden Gene il11ra nachgeschaltet war. Darüber hinaus zeigten Experimente unter Verwendung chemischer Inhibitoren, dass speziell die Expression der komplementkomponentkodierenden Gene c3a.1,
c4b und c7a im Larvenrumpfamputationsmodell durch den Il11-Stat3-Signalweg moduliert wird.
Zur Klärung der Frage, ob das Komplementsystem und/ oder seine Komponenten eine Rolle während der Regeneration spielen, wurden verschiede Funktionsverlustmodelle generiert und im larvalen Rumpfamputationsmodell auf mögliche Aberrationen getestet. Zum einen generierte ich Überexpressionslinien von endogenen Inhibitoren der Komplementproteinspaltungskaskade. Überexpression eines etablierten Komplementsysteminhibitors rca2.1/ tecrem führte zu einer im Vergleich zu Wildtyp- Geschwistern verringerten Regeneration des larvalen Rumpfs. Zum anderen generierte ich Funktionsverlustmutanten von individuellen Komplementkomponenten durch CRISPR/Cas9 vermittelter Mutagenese, und zwar für masp1, masp2, cfd, c1s, c4b, c5 und c9. Die larvale Rumpfregeneration war in diesen Mutanten unauffällig. Allerdings zeigten c4b Mutanten eine verringerte Kardiomyozytenproliferation und eine differenzielle Expression von einigen Markergenen, einschließlich einer erhöhten Expression von inflammatorischen Zytokinen.
Meine Studien führten zu neuen Einblicken in das Komplementsystem im Kontext der Regeneration. Ich fand heraus, dass mehrere komplementkomponentenkodierenden Gene in regenerierendem Zebrabärblinggewebe exprimiert werden, und zwar im Herzgewebe, im larvalen Rumpf und in der adulten Flosse. Darüber hinaus zeige ich, dass die verletzungsinduzierte Expression von komplementkodierenden Genen in regenerierendem Gewebe dem Regenerationsmasterregulator il11ra nachgeschaltet ist. Speziell c3a.1, c4b und c7a wurden durch il11/ stat3 reguliert...
Compaction and spheroid formation modulates stemness and differentiation of human pancreas organoids
(2023)
The incidence of diabetes type 1 (T1D) in children and young adults is increasing worldwide. T1D is well treated by insulin administration. However, there is currently no long-lasting cure for this ailment. The success rate of pancreatic islet transplantation to treat T1D is limited by the availability of patient-matched islets and the necessity of using life-long immunosuppressive medication. The difficulties caused by transplantation can be overcome by generating bio-engineered pancreatic islets from patient-derived progenitor cells. Aim of this thesis is to establish new strategies for the generation and analysis of pancreatic lineages derived from human progenitor cells. It reports on the optimization of a technique to form human pancreatic spheroids from hollow monolayered human pancreas organoids (hPOs) to investigate how cell-cell and cell-matrix interaction can be leveraged to induce endocrine differentiation of the pancreas progenitor cell organoids. We introduce cell aggregation protocols to generate endocrine pancreas cell lineages from ductal pancreatic cells. Next, we study the effect of co-culture with stromal and endothelial cells to promote cell differentiation toward a pancreatic fate enhancing β cells productivity.
This thesis has focused on identifying the differences in gene expression along with phenotypical transformation during differentiation of human pancreatic organoids (hPOs) towards human β cells to be used in the future of cellular therapeutics in treating T1D patients.
Structure-function relationships in substrate binding protein dependent secondary transporters
(2023)
This work provides new insights into the relevance of SBP dependent secondary transport systems, especially in the thus far under-researched subgroup of TAXI transporters. Importantly, we identified and characterized the TAXI transport system TAXIPm-PQM from Proteus mirabilis. We demonstrated that, in contrast to previously characterized SBP dependent secondary transport systems, TAXIPm-PQM is a proton coupled system and transports the C5-dicarboxylate α- ketoglutarate. Since initially the transport of α-ketoglutarate could only be demonstrated in vivo but not in vitro using established protocols (Mulligan et al. 2009), we investigated in detail the differences between the in vivo and in vitro assay. This resulted in a bioinformatic analysis of TRAP and TAXI signal peptides, which strongly implied that TAXIPm-P requires a transmembrane anchor to allow for transport. We then provided TAXIPm-P surface tethered to the membrane in in vitro transport assays and confirmed the prediction of our bioinformatic analysis that TAXIPm-PQM deploys a membrane-anchored instead of a soluble SBP. Furthermore, the TAXI transport system TAXIMh-PQM from Marinobacter hydrocarbonoclasticus transports fumarate only if both membrane domains Q and M are present. For further characterization, Michaelis-Menten kinetics and affinities were determined for both TAXI transport systems TAXIPm-PQM from Proteus mirabilis and TAXIMh-PQM from Marinobacter hydrocarbonoclasticus. In addition, nanobodies were selected for the membrane domain TAXIPm-QM from Proteus mirabilis to stabilize different conformations which can serve in subsequent structural elucidation studies. Furthermore, the TRAP SBP TRAPHi-SiaP from Haemophilus influenzae was shown to interact not only with its corresponding membrane domain TRAPHi-SiaQM but with at least one additional transporter. It was thereby excluded that TRAPHi- SiaP transfers N-acetylneuraminic acid to the only native E. coli TRAP transporter TRAPEc-YiaMNO and suggested to rather interact with a SBP dependent ABC transport system as this protein family represents the largest SBP dependent protein group in E. coli (Moussatova et al. 2008).
Baleen whales (Mysticeti) are a clade of highly adapted carnivorous marine mammals that can reach extremely large body sizes and feature characteristic keratinaceous baleen plates used for obligate filter feeding. From a conservation perspective, nearly all baleen whale species were hunted extensively over a roughly 100 years lasting time period that depleted many of the respective whale stocks with so far unknown consequences for e.g. their molecular viability. From an evolutionary perspective, the lack of fossil records together with conflicting molecular patterns resulted in a still unclear and debated phylogeny of modern baleen whales, particularly in rorquals (Balaenopteridae). In this dissertation, I will demonstrate the application of baleen whale genomes to tackle these open questions by using modern approaches of conservation and evolutionary genomics.
Conservation genomic aspects of baleen whales were addressed in two projects, both using whole genome data of either an Icelandic fin whale (Balaenoptera physalus) population or multiple blue whale (Balaenoptera musculus) populations to evaluate the impact of the industrial whaling era on their molecular viability. The results suggest a substantial drop in effective population size of both species but also a lack of manifestation in genotypes of the fin whale population when compared to the blue whale populations. Especially the rare and short runs of homozygosity (ROH), usually indicative for inbreeding, suggest frequent outcrossing in fin whales while all analyzed blue whale populations featured long and frequent ROH. In addition to these analyses, genome data of blue whale populations was further used to evaluate if northern hemisphere blue whales diverged into different subspecies. Population genetic and gene flow analyses showed clearly separated and well isolated populations in accordance with their assumed geographical distance. In contrast, the genome-wide divergence between all blue whale populations was low compared to other cetacean populations and to the next closely related sei whale species. Because this includes the morphologically different and well recognized pygmy blue whale subspecies, a proposal was made to equally categorize the two northern-hemisphere blue whale populations as subspecies.
Evolutionary aspects were addressed in a third project, by constructing the genome of the pygmy right whale (Caperea marginata) and testing its potential in phylogenetics and cancer research. Phylogenomic analyses using fragments of a whole-genome alignment featuring nearly all extant baleen whales, allowed the revision of the complex evolutionary relationships of rorquals by quantifying and characterizing the amounts of conflicts in early diverging branches. These relationships were further used to identify phylogenetically independent pairs of baleen whales with a maximum of diverging body size differences to compare rates of positive selection between their genomes. The results suggest nearly evenly distributed frequencies of alternative topologies which supports the representation of the early divergence of rorquals as a hard polytomy with high amounts of introgression and incomplete lineage sorting. Within the set of available genomic data, three independent pairs of baleen whales with diverging body sizes were found and comparisons of positive selection rates resulted in many potentially body size and cancer related genes. The lack of conserved selection patterns, however, suggest a more convergent evolution of size and cancer resistance like previously discussed in paleontology.
In conclusion, the application of whole genome data using methods of conservation genetics allowed for a comprehensive estimation about the molecular viability of blue and fin whales as well as an assessment of the taxonomic status of northern-hemisphere blue whale populations. The rather different results between blue and fin whales underlines the importance of genomic monitoring of baleen whales because different species show rather different molecular consequences of their potentially varying depletions. Furthermore, as showcased for the northern-hemisphere blue whale, many important isolated populations of baleen whales may still be unknown to conservation management and genome-wide comparisons will most likely contribute to overcome this under-classification problem. The application of whole genome data in evolutionary research allowed the characterization of the complex patterns of molecular conflicts within baleen whales and especially rorquals that will contribute to the still rather unclear understanding of their evolution. The here found molecular support for the idea of convergent evolution of gigantism in whales will further guide the search for molecular patterns responsible for Peto’s paradox.
Electrospinning is a versatile and promising drug delivery technology for the development of tailor-made drug delivery systems for various clinical applications. By applying high voltages to drug-loaded polymer solutions, solid polymeric nanofibers can be generated, which encapsulate active pharmaceutical ingredients (APIs) into their polymer matrix. During the electrospinning process, the fibers are deposited on a collector and form a nonwoven network of drug-loaded polymer fibers. These fibers are spatially distributed in aligned or random orientation, providing the opportunity to design highly tunable structural and mechanical properties, which can be adapted to the biological requirements of the intended application site. The mechanically flexible fiber networks can therapeutically be administered to a multitude of pharmaceutical application sites. Their highly porous fiber structure exhibits a large surface-to-volume ratio, which is ideal for controlled drug release kinetics from the polymer matrix upon contact with biological fluids, such as tear fluid, saliva, mucus, wound exudate or gastro-intestinal fluid. For application at the target site, fiber mats are cut into patches. As the patch size determines the quantity of applied API, the electrospinning process must ensure homogeneous distribution of the API throughout the entire fiber mat area.
In this thesis, electrospinning was established as a formulation technology for the rational fabrication of tailor-made multifunctional drug carrier systems for local and site-specific drug delivery to the epithelial interfaces skin, oral mucosa as well as cornea. For adequate characterization and analysis of the drug delivery systems, a broad panel of robust and predictive analytical tools, based of novel investigation techniques for physicochemical characterization of electrospun fibers, was developed.
The initial part of the thesis thematically focuses on the development of predictive analytical techniques, to determine fiber morphology and physicochemical properties, as well as fiber composition and drug release. By designing two model formulations with contrasting properties, and subsequent analysis and characterization with a set of newly developed techniques and state-of-the-art methods, a comprehensive toolset has been made available and evaluated, aiming at advancing and standardizing respective techniques in the scientific field of electrospun drug delivery systems.
Starting with the initiation of the electrospinning formulation process, which often relies on empirical data rather than analytical methods to predict successful processability, analysis of rheological properties of electrospinning solutions was used to rationally detect the minimum polymer concentration required for electrospinning.
For analysis of fiber morphology, scanning electron microscopy is a common technique. However, little attention is given to underlying readout parameters. By analyzing the fiber orientation and diameter of the respective fibers, predictive results regarding mechanical properties could be obtained, which were subsequently confirmed by measuring elongation force with tensile testing. Confocal Raman microscopy, a label-free method for chemically- selective imaging of the fiber samples, was introduced as a complementary visualization technique, enabling the detection of fiber composition and drug distribution.
A novel technique for investigation of water contact angles on the fiber surface of highly hydrophilic polymers was introduced, which provides predictive data regarding interaction with body fluids and the resulting drug release kinetics. Subsequent release testing in a newly developed setup for analyzing drug release from electrospun fibers in low-volume body compartments, confirmed the anticipated drug release kinetics from measurement of the surface hydrophilicity.
By combining complementary analytical methods, including spectral composition analysis, morphology visualization, characterization of physico-chemical properties and drug release kinetics, as well as the application of multivariate data analysis, a robust and predictive toolset has been established, which can support comparability of future electrospinning studies and the translation from the lab bench into clinics.
Based on the analytical toolset, the main part of the thesis focuses on the development and preparation of electrospun platform drug delivery systems for application on epithelial barriers. Electrospun fiber mats are thin, flat, and mechanically flexible, which allows close adherence to epithelial surfaces and reduction of diffusion paths, which enables efficient drug delivery to the skin, oral mucosa, as well as the cornea.
Electrospun fibers bear a high potential for application as wound dressings, while simultaneously controlling the local delivery of APIs to the wound area. Their close resemblance to the extracellular matrix of human skin provides a suitable microenvironment for cellular proliferation and migration for wound closure. In this work, insulin, a fragile proteohormone with growth factor characteristics, was successfully encapsulated into the core of coaxially electrospun fibers, thus maintaining bioactivity throughout and after the electrospinning process. The shell has been designed from biocompatible polymers, which, upon contact with aqueous wound exudate, partially dissolve and form pores through which bioactive insulin is released in a controlled manner. The shell layer provides a hydrophilic surface for interaction with body fluids and skin cells, and possesses substantial mechanical strength, flexibility, and high tensile elongation required for application on wounds. The biocompatibility of the wound dressing was investigated by interaction with primary human dermal fibroblasts and keratinocytes, which displayed healthy cell morphologies without indicating any elevated levels of cytotoxicity markers.
To investigate the effect of insulin on cell migration, in vitro scratch assays on human skin cells were performed. Increased cellular migration speed and wound closure could be observed, indicating improved wound healing. Bio relevance of in vitro wound healing potential results was advanced by development of 3D ex vivo human epidermal skin wound models from reduction surgery donor material. These complex wound models were treated with electrospun insulin fibers and analyzed by proteome analysis to reveal significant increases in wound healing-associated signaling pathways, which could be attributed to a material-driven remarkably positive impact on wound healing of the electrospun fibers...
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.
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.
Synaptic transmission is a fundamental process that involves the transfer of information from a presynaptic neuron to a target cell through the release of neurotransmitters. The SV cycle is a complex series of events that enables the recycling of SVs, allowing for the sustained release of neurotransmitters. This process is mediated by a variety of proteins and enzymes, and its regulation is critical for maintaining proper synaptic function. Despite extensive research efforts, many aspects of the SV cycle and the underlying synaptic proteins remain poorly understood, highlighting the need for continued investigation into this important process. During this work, multiple aspects of synaptic transmission were studied by performing
behavioural, pharmacological, optogenetic, electrophysiological and ultrastructural assays on Caenorhabditis elegans. First, the role of two proteins (ERP-1 and RIMB-1) were analysed in the synaptic vesicle cycle. Second, a new optogenetic tool, the pOpsicle assay was described, which enables the direct visualization of synaptic vesicle (SV) release.
Activity-dependent bulk endocytosis (ADBE) enables the endocytosis of SV membrane and proteins in a fast manner during intense stimulation, resulting in bulk endosomes (also so-called large vesicles, LVs). Recycling proteins can be characterized by its site of action, whether they act at the plasma membrane (participating at the LV formation), or at the LV membrane (participating at the SV formation). ERP-1 (the C. elegans ortholog of Endophilin B) was recently identified as a possible SV recycling factor, its contribution to synaptic transmission has not been analysed before. During this project the function and possible cooperation of three proteins, ERP-1, UNC-57 (the C. elegans ortholog of Endophilin A) and CHC-1 (the C. elegans ortholog clathrin heavy chain) were studied, with a special emphasis of the site of action. It has been confirmed that these proteins participate together in synaptic vesicle recycling. Endophilins (ERP-1 and UNC-57) act both at the PM and the LV level, but while UNC-57 has been identified as the main player, ERP-1 rather has a minor role and acts as a back-up protein. CHC-1 functions the LV level in the first place, but it can compensate for the loss of UNC-57 and acts as a back-up protein at the PM.
RIM-binding protein is an evolutionarily conserved active zone protein, which interacts directly with RIM and N, P/Q, as well as L-type Ca2+ channels. RIM-BP and RIM have redundant functions in different model organisms including C. elegans, however, while the loss of UNC-10 (the C. elegans ortholog of RIM) led to drastic behavioural defects, the loss of RIMB-1 (the C. elegans ortholog of RIM-BP) led only to mild phenotypes. During this work the synaptic function of RIMB-1 and its interaction with UNC-10 and UNC-2 (C. elegans ortholog of the CaV2 1 subunit) were extensively investigated. It has been shown that RIMB-1 contributes to the precise localization of VGCCs in cooperation with UNC-10. Furthermore, it has been demonstrated, that RIMB-1 plays different roles in cholinergic and GABAergic neurons, thus it contributes to maintain a proper excitation/inhibition balance.
There are numerous available assays, which enable the indirect analysis of synaptic transmission, however, a tool, that enables the direct visualization of SV release, is highly desired. pOpsicle is a method which combines the optogenetic stimulation of cholinergic neurons with real-time visualization of SV release. A pH-sensitive fluorescence protein, pHuji, was inserted into the second intravesicular loop of the synaptic vesicle membrane protein, synaptogyrin (SNG-1). The fluorescence of pHuji is quenched inside the vesicles, but once they are released, the pH increases and pHuji can be detected. pOpsicle enables not only the direct visualization of SV exo-, and endocytosis events, but also the identification of putative SV recycling proteins.
Semi-arid African ecosystems influence trends and variability in global terrestrial carbon dynamics. However, there are uncertainties in potential effects of future climates for semi-arid ecosystems, especially for niche ecosystems. At the same time, African ecosystems provide the livelihoods and ecosystem services for around 1.4 billion people. Future population growth and associated changes in land use pose a challenge for the protection of African biodiversity. Therefore, this work focussed on future impacts of climate change on African ecosystems and carbon dynamics and also for African protected areas (PAs), where they may cooccur with other global change factors. Another focus was on uncertainties associated with future projections and with modelling the Nama Karoo, as an example of a semi-arid niche ecosystem. Dynamic vegetation models (DVMs) were the main research tool.
In Chapter 2, we analysed climate change impacts on African ecosystems and carbon pools until the end of the 21st century and associated uncertainties based on an ensemble of vegetation simulations with the DVM adaptive dynamic vegetation model (aDGVM). We investigated the impact of increased atmospheric CO2 concentrations and two climate change scenarios (medium (RCP4.5) and high emissions (RCP8.5); RCP - representative concentration pathway) on vegetation changes. Differences in the simulated vegetation were primarily driven by assumptions about the influence of CO2 on plants. Elevated CO2 concentrations led to increased total aboveground vegetation biomass and shrub encroachment into grasslands and savannas for both climate scenarios. In simulations without the direct influence of CO2 on plants, there was hardly any shrub encroachment and vegetation biomass decreased or varied between a slight decrease in some cases and a slight increase in others. Based on these results, biome changes due to climate change are likely in Africa in the future. Due to the large uncertainties in future projections, strategies to adapt to climate change must be flexible.
The simulated vegetation in Chapter 2 represented potential, natural vegetation and is particularly suitable to investigate PAs. However, PAs do not exist isolated from their environment and social developments. In Chapter 3, the vegetation projections with CO2 effect from Chapter 2 were combined with projections for population density and land use. Except for many PAs in North Africa, most PAs were adversely affected by at least one of the three drivers by the end of the 21st century in both investigated scenarios ("middle-of-the-road" and "fossil-fuelled development"). Cooccurrence of the drivers varied by region and scenario for PAs. Both scenarios implied increasing challenges for the conservation of African biodiversity in PAs. The impact of climate change on vegetation is likely to be exacerbated by socio-economic change for most African PAs. Strong mitigation of future climate change together with equitable societal development may facilitate successful ecosystem conservation.
The simulations in Chapters 2 and 3 showed large-scale patterns of vegetation change, but their low resolution makes them unsuitable for local analyses. In Chapter 4, the challenges of simulating smaller scale, semi-arid ecosystems and their carbon cycle were analysed for the Nama Karoo with the aDGVM2 and its shrub module. The aDGVM2 is based on the aDGVM, but represents plants more flexibly. In all tested aDGVM2 configurations, the carbon fluxes improved compared to initial simulations but still overestimated them. The measured morphology of the dwarf shrubs and soil water dynamics were not reproduced in aDGVM2. Semi-arid soil water dynamics and coping strategies of semi-arid dwarf shrubs under drought stress are not adequately implemented in the aDGVM2. Further field research on semi-arid water and carbon dynamics of vegetation is necessary to parameterise the aDGVM2 for dwarf shrubs. If these challenges are overcome, DVMs can be a powerful tool for much-needed research on the impacts of climate change on the Nama Karoo.
The analyses have shown that climate change under medium to high emission scenarios is likely to lead to large-scale changes in ecosystems and the carbon balance in Africa. Because lower emissions scenarios come with less uncertainty, climate change adaptation strategies likely need to be less complex or extensive if climate change is minimised. For African PAs, the challenges of climate change may be exacerbated by socio-economic factors to a regionally varying extent. This research suggests that successful ecosystem conservation depends on climate change mitigation measures and ensuring equitable, sustainable development. The shown uncertainties, e.g., in the implementation of the CO2 effect on plants or vegetation dynamics in more niche ecosystems, help to focus future research efforts and increase our understanding of the range of plausible futures we may need to adapt to.
The impact of the Covid-19 pandemic called for rapid responses in face of unprecedented challenges. In this context, earning more about the causative agent SARS-CoV-2 becomes imperative. Therefore, clinical virus isolates were studied with focus on infectivity, replication kinetic, and caspase activity.
Firstly, clinical specimens collected from patients were tested for infectivity in cell culture. Combined with polymerase chain reaction results, a formula predicting infectivity in cell culture based on abundance of viral RNA was developed. Additionally, analysis of different specimen types, sources, and material, elucidate the question of infectivity. Here, infectivity was demonstrated in specimens derived from different parts of the respiratory tract, including specimens collected from deceased persons. A protocol for virus isolation on human airway epithelium in air-liquid interface culture was established.
Secondly, replication kinetics of 20 clinical isolates were compared, including a subset of seven sequenced isolates. All isolates replicated in the colon epithelial cell culture model. Within the subset, differences between isolates carrying the D614G amino acid exchange and with original spike protein were observed.
Lastly, elevated caspase activity was demonstrated in two cell culture models including human airway epithelium in air-liquid interface culture.
Subsequently, caspase inhibition by small-molecule compound Emricasan and its effects on the cytopathic effect observed in cell culture were studied. Here, increased cell survival in a colon epithelial cell line was shown with unimpaired virus replication. Elevated caspase activity was identified as early marker of infection and validated by testing across 20 clinical virus isolates.
This study offers information on infectivity that can help shape the understanding of transmission risk. As such, parts of the data collected here were used for validation of rapid antigen tests. The insights gained by studying caspase activity contributed in part to the development of a drug screening method by Bojkova et al.,41 thus aiding routine laboratory workflow. It was demonstrated that Emricasan exhibits no antiviral effect, while the finding of increased cell survival in cell culture could give rise to further research on prevention of tissue damage.
This dissertation is about case competition in headless relatives. Case competition is a situation in which two cases are assigned but only one of them surfaces. One of the constructions in which case competition takes place is in headless relatives, i.e. relative clauses that lack a head. This dissertation has two goals: (i) to give an overview of the data, and (ii) to provide an account for the observed data.
The grammaticality of a headless relative is determined by two aspects. The first aspect concerns which case wins the case competition. In all languages with case competition that I am aware of, this is determined by the case scale in NOM < ACC < DAT. A case more to the right on the scale wins over a case more to the left on the scale. This scale is not specific to case competition in headless relatives, but it can also be observed in syncretism patterns and morphological case containment. I show that that the case scale can be derived from assuming the cumulative case decomposition (cf. Caha 2009). A case wins over another case when it contains all features that the other case contains.
The second aspect of case competition in headless relatives concerns whether the winner of the case competition is allowed to surface when it wins the case competition. The winning case can be either the internal case required by the predicate in the relative clause, or the external case required by the predicate in the main clause. It differs from language to language whether they allow the internal and the external case to surface.
All language types I discuss allow for a headless relative when the internal and the external case match. The unrestricted type of language allows both the internal case and the external case to surface when either of them wins the case competition. Examples of this language type are Old High German, Gothic and Ancient Greek. The internal-only type of language allows only the internal case to surface when it wins the case competition, and it does not allow the external case to do so. An example of this language type is Modern German. The external-only type of language allows only the external case to surface when it wins the case competition, and it does not allow the internal case to do so. To my knowledge, there is no language that behaves like this. The matching type of language allows neither the internal nor the external case to surface when either of them wins the case competition. An example of this language type is Polish.
To account for the data, I set up a proposal that generates the attested patterns and excludes the non-attested ones. I let the variation between languages follow from properties of languages that can be independently observed. By investigating the morphology of the languages, I suggest differences between the lexical entries in the different languages. These different lexical entries ultimately lead languages to be of different types. In my proposal, I assume that headless relatives are derived from light-headed relatives. Light-headed relatives contain a light head and a relative pronoun. In a headless relative either the light head or the relative pronoun is deleted. The necessary requirement for deletion is that the deleted element (either the light head or relative pronoun) is structurally or formally contained in the other element.
I motivate the analysis for the internal-only type of language for Modern German, for the matching type of language for Polish and for the unrestricted type of language for Old High German. I first identify the morphemes that the light heads and relative pronouns in the languages consist of, and then I show to which features each of the morphemes correspond. The crucial difference between the internal-only type of language Modern German and the matching type of language Polish is how the phi and case features are spelled out. In Modern German they are spelled out by a phi and case feature portmanteau, and, in Polish, the same features are spelled out by a phi feature morpheme and a case feature morpheme. Old High German differs from the other two languages in that it has light heads and relative pronouns that are syncretic. I show how these differences in the morphology of the languages ultimately leads to different grammaticality patterns in headless relatives.
Comparing my account to others shows that all proposals account for the case facts using some kind of case hierarchy. The proposals differ in how they model the variation, both in the technical details of the proposal, but more importantly, also in empirical scope and predictions they make.
Experiments on Vibrational Energy Transfer (VET) in proteins contribute to our understanding of fundamental biological processes such as allostery, dissipation of excess energy, and possibly enzymatic catalysis. While these processes have been studied for a long time, many questions remain unanswered. The aim of this work was to expand the application of existing spectroscopic techniques to investigate VET, seeking tailored solutions for the diversity of proteins and amino acid environments. Additionally, new target proteins were to be established to broaden the spectrum of VET experiments towards the role of VET and low-frequency protein modes (LFMs).
To test their suitability as VET sensors, the non-canonical amino acids (ncAAs) Azidoalanine (N3Ala), azido-L-Homoalanine (Aha), p-azido-Phenylalanine (N3Phe), p-cyano-Phenylalanine (CNPhe), and 4-cyano-Tryptophan (CNTrp) were coupled to the VET donor β-(1-azulenyl)-L-Alanine (AzAla) in dipeptides. Their spectral properties were compared using FTIR and VET spectra in H2O, dimethyl sulfoxide, and tetrahydrofuran.
The solvent strongly influences the measured VET signals, which can be explained by the direct interaction of the solvent with the dipeptides. Additionally, the peak time within the subgroups of azide and nitrile sensors increased with the size of the side chain, indicating the dependence between peak time and the distance between VET donor and sensor. When incorporated into a protein, solvent interactions are less dominant. Therefore, Aha, N3Phe, and CNPhe were additionally incorporated at two different positions in the PDZ protein domain and investigated. Due to Fermi resonances, signals from azide sensors are challenging to predict, unlike those of the nitrile sensors.
Overall, the experiments showed that nitrile groups can serve well as VET sensors, as their lower extinction coefficient is compensated for by a narrower bandwidth. This expands the number of potential target proteins, and sensor incorporation can be less disruptive at various protein locations.
Since the VET donor AzAla can inject the energy of a photon into a protein as vibrational energy at a specific location, it can also be used for the targeted excitation of LFMs. If these modes are involved in an enzymatic reaction, a direct influence on activity is expected. This hypothesis has long existed but has not been definitively verified. Some studies have found evidence for the involvement of LFMs in formate dehydrogenase (FDH) catalysis. Therefore, FDH was chosen for the investigation of LFMs in enzymes. This specific system additionally allows the use of a natural VET sensor: it forms a stable complex with NAD+ and N3-, an excellent IR marker. Thus, it provided the opportunity to test low-molecular-weight non-covalent ligands as VET sensors.
After ensuring sufficient AzAla supply through the internal establishment of an enzymatic synthesis, AzAla could be incorporated at various positions in FDH. Despite spectral overlap between free and bound N3-, the latter could be identified by its narrower FWHM. For some variants, no binding could be observed. Circular dichroism spectra showed that these variants structurally deviate slightly from other variants and the wild type (WT). VET could be observed over 22 Å from two regions of the protein to the N3- bound in the active center, at protein concentrations of below 2 mM. Unbound N3- did not generate signals, allowing it to be added in excess ensuring the saturation of the protein in VET experiments.
The activity of FDH WT and four AzAla mutants was investigated under substrate saturation without and with AzAla excitation. In these experiments, a slight reduction in activity under illumination was observed, even for the WT, who is not expected to interact with the excitation light. So far, a difference in sample temperature cannot be excluded as the cause for this decline.
The presented experiments with FDH illustrate the potential of low-molecular-weight ligands as VET sensors, with N3- being particularly attractive due to its simple structure (preventing Fermi resonances) and its high extinction coefficient. Its use can add many metalloproteins as potential targets for VET experiments and allows investigation without a VET sensor ncAA. Additionally, initial experiments were conducted to measure light-dependent FDH activity. By specifically exciting protein LFMs, this project could contribute in the future to answering longstanding questions about the extraordinary catalytic efficiency of enzymes.
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.
This thesis comprises the usage of two commonly known hinge-binding moieties in drug discovery. First, the quinazoline scaffold of gefitinib (5) was utilized in a macrocyclization strategy to introduce selectivity. In general, the quinazoline hinge-binding moiety is a commonly used scaffold which can be found in 14% of approved kinase inhibitors. The most familiar applications are EGFR inhibitors such as gefitinib (5), erlotinib (6), afatinib, or dacomitinib for the treatment of NSCLC. But other kinases like CDK2, CDK4, or p38 are reported targets as well.
The N-phenylquinazolin-4-amine moiety of gefitinib (5) was conserved however, the residues at the aromatic ring in the linker were modified, the residue targeting the solvent-exposed region was varied, and the linker at the C6 position of the quinazoline was adjusted to enable the macrocyclization. An overview of the structural modifications is shown in Figure 35A.
Kinome-wide screening of gefitinib (5) revealed several off-targets besides EGFR (Figure 35B), making it an excellent starting point for a macrocyclization strategy. Introducing a linker to the N phenylquinazoline-4-amine scaffold and retaining the residues on the aromatic ring as well as the methoxy group targeting the solvent-exposed region improved the selectivity profile and the efficacy towards EGFR WT and its mutants. Truncation of the linker moiety led to the mutant selective macrocycle 26f with an excellent kinome-wide selectivity profile (Figure 35B). An inhibitor that is effective on EGFR mutations while ineffective on the EGFR WT could represent an enhancement of patient treatment, as it potentially causes less side effects. Further studies could determine the effect of the most promising macrocycles in lung cancer cell lines. Additionally, the pharmacokinetic properties could be optimized, e.g. by introducing solubilizing groups, targeting the solvent-exposed region.
The second scaffold comprises the 3-aminopyrazole-based hinge-binding moiety. It is a privileged scaffold in medicinal chemistry for the development of kinase inhibitors. Previous publications report the anti-proliferative and anti-cancer potential of pyrazole-based molecules. They play a crucial role in the treatment of various diseases and cancer types like inflammation disorders, lymphoma, or breast cancer. This scaffold can be found e.g. in the aurora kinase inhibitor tozasertib or in the promiscuous kinase inhibitor 23, published by Statsuk et. al. Rescreening compound 23 in a comprehensive kinase panel against 468 human protein kinases confirmed the unselective behavior with a selectivity score of S35 = 0.56 (Figure 36B), making it a great starting point for further optimizations. The N-(1H-pyrazol-3-yl)pyrimidin-4-amine scaffold was conserved however, the residues targeting the solvent-exposed region were varied and different linkers were attached.
The introduction of different residues at the pyrazole dramatically influenced the selectivity profile of the desired kinases. Ester moieties caused to a favorable combination of selectivity and potency towards the kinase of interest CDK16. The removal of additional residues at the pyrimidine, targeting the solvent-exposed region, increased the efficiency towards CDK16. Further optimization led to the highly potent and selective CDK16 inhibitor 98d (IC50 = 33 nM). NanoBRETTM screening against the complete CDK family revealed a preferred inhibition of the PCTAIRE and PFTAIRE subfamily with cellular IC50 values of 20 nM – 120 nM and 50 nM – 180 nM, respectively. A FUCCI cell cycle assay and viability assessment of 98d confirmed previously published results, reporting a G2/M cell cycle arrest followed by apoptosis and accumulation of p27 through knockout of CDK16 in SCC cells. Consequently, further studies could evaluate the anti-tumor activity of 98d in SCC and NSCLC or elucidate the effect of 98d in AMPK-related macroautophagy. 98d represents a novel tool compound to investigate the understudied kinases of the PCTAIRE family and enable to enlighten the biological role of those kinases.
Macrocyclization of the N-(1H-pyrazol-3-yl)pyrimidin-4-amine core resulted in the selective BMPR2 inhibitor 110a. It showed a good binding affinity towards BMPR2 with a KD value of 205 nM as well as a good potency with an IC50 value of 506 nM. A comprehensive selectivity screen against 468 kinases revealed an excellent selectivity profile with S35 = 0.01. As no BMPR2 inhibitors have been published so far, 110a represents a novel compound that may provide further insights into the canonical BMP pathway, noncanonical signaling, or its impact on BMPR2-associated diseases like PAH.
The introduction of additional residues targeting the solvent-exposed region shifted the selectivity towards the MST kinases. The exchange from the pyrimidine to a quinazoline moiety resulted in the highly potent and selective macrocyclic MST3 inhibitor 113c. NanoBRETTM measurements demonstrated the preferred inhibition of MST3 with IC50 values of 210 nM and 30 nM for intact and lysed cells, respectively. A weaker activity could be seen for MST4 with 1.8 µM and 510 nM, while MST1 and MST2 were not affected. To date, no selective MST3 inhibitors have been published, making 113c a valuable tool compound for further functional studies. As MST3 is influencing the cell cycle progression, 113c could be tested in a further cell cycle assay to elucidate the inhibitory effect of 113c on MST3 and consequently on the cell cycle. Furthermore, the anti-tumor activity of 113c in breast cancer could be determined, as Madsen et. al. reported a high MST3 and MST4 activity triggered by FAM40B mutations.
Protein kinases are key signalling molecules and transduce intracellular signals via the post-translational phosphorylation of substrate proteins, often other protein kinases. Dysregulation of this protein family has been linked to many diseases including neurodegenerative diseases, inflammation and cancer and amplifications of kinases play important roles as diagnostic biomarkers in a variety of cancers. Various strategies have been developed to treat dysregulated protein kinases. Most commonly, chemical small molecule inhibitors are used to modulate protein kinase activity in cancer cells. Many inhibitor and general research efforts have focused only on a small subset of protein kinases, resulting in a large portion of the kinome, the so-called “dark” kinome, remaining largely unexplored. As part of the strategy to develop inhibitors, it is crucial to understand the structure-activity-relationships (SAR) of small molecules to the activity towards the targets based on understanding small molecule-target affinities as determined by biophysical, biochemical, and cellular methods. However, not always do in vitro determined affinities, which are frequently used as basis for SAR considerations, correlate with the cellular affinity. For protein kinases in particular, it has been shown that the cellular concentration of the natural substrate adenosine-triphosphate (ATP) plays a critical role for the resulting small molecule affinity, as substrate and inhibitor frequently compete for the same binding site of the protein kinase. The cellular target engagement assay NanoBRET is a versatile assay that overcomes this problem and can be used to assess binding of a compound to the full-length protein kinase, in the presence of natural binding partners. Another important factor in inhibitor optimization is the selectivity of the molecule within the family of protein kinases. When comparing the selectivity profiles of small molecule kinase inhibitors in vitro and in cells, different profiles can be observed. Frequently, a compound, binds fewer protein kinases with high affinity in cells, indicating that cellular profiling of protein kinase inhibitors is necessary to understand the selectivity profile of an inhibitor.
The goal of this work was to understand cellular SARs of inhibitors for kinases and dark kinases in medicinal chemistry projects, and to understand the selectivity profiles of existing small molecules in cells, including already approved drugs and clinically used kinases inhibitors. The cellular potency and selectivity aspects guided optimization of the inhibitors towards selective small molecules ‘chemical probes’ or highly validated inhibitors with a narrow selectivity profile as part of ‘chemogenomic libraries’. One strategy to improve selectivity has been to use sterically restricted cyclic small molecules, called macrocycles, that allow fewer conformations of the molecule than their non-cyclic parent compound. In this thesis the dark kinase STK17A was investigated. Macrocyclization was used to develop a selective chemical probe molecule that is also selective in the cellular context. For another kinase, SIK2, a rational design approach was used to exclude off-targets bound by the lead structure, resulting in a chemical probe that selectively targets the SIK1/2/3 proteins. Assessing cellular potency of another series of inhibitors, a probe was developed for the PCTAIRE subfamily of the CDK kinases. This required co-expression of the binding partners of CDKs, the cyclins, in cells to obtain a functional assay. To identify new candidates for the neglected family of splicing kinases comprising the CLK, SRPK, DYRK and HIPK protein kinase subfamilies, a literature review was conducted, and the best small molecule candidates were compared for their target engagement in cells. This led to a series of small molecule inhibitors that may be used as a set or single agents to target the CLK proteins and SRPK proteins or in combination to target the remaining proteins. In search of new starting points for this subfamily of kinases, an initial screen with NanoBRET technology was performed using a library of over 2000 inhibitors, and new starting points were identified. Additionally, a set of clinical and approved small molecule kinase inhibitors was assessed for their selectivity in cells. Several highly selective molecules were identified that were much less selective in in vitro approaches. The set of data allowed for a comprehensive comparison of cellular potencies with published data using in vitro binding, in vitro activity and data obtained from cell lysates and identified several protein kinases that would need to be investigated in cells...
How the brain evolved remains a mystery. The goal of this thesis is to understand the fundamental processes that are behind the evolutionary history of the brain. Amniotes appeared 320 million years ago with the transition from water to land. This early group bifurcated into sauropsids (reptiles and birds) and synapsids (mammals). Amniote brains evolved separately and display obvious structural and functional differences. Although those differences reflect brain diversification, all amniote brains share a common ancestor and their brains show multiple derived similarities: equivalent structures, networks, circuits and cell types have been preserved during millions of years. Finding these differences and similarities will help us understand brain historical evolution and function. Studying brain evolution can be approached from various levels, including brain structure, circuits, cell types, and genes. We propose a focus on cell types for a more comprehensive understanding of brain evolution. Neurons are the basic building blocks and the most diverse cell types in the brain. Their evolution reflects changes in the developmental processes that produce them, which in turn may shape the neural circuits they belong to. However, there is currently a lack of a unified criteria for studying the homology of connectivity and development between neurons. A neuron’s transcriptome is a molecular representation of its identity, connectivity, and developmental/evolutionary history. Hence the comparison of neuronal transcriptomes within and across species is a new and transformative development in the study of brain evolution. As an alternative, comparing neuronal transcriptomes across different species can provide insights into the evolution of the brain. We propose that comparing transcriptomes can be a way to fill this gap and unify these criteria. In previous studies, published in Science (Tosches et al., 2018) and Nature (Norimoto et al., 2020), we leveraged scRNAseq in reptiles to re-evaluate the origins and evolution of the mammalian cerebral cortex and claustrum. Motivated by the success of this approach, in this thesis we have now expanded single-cell profiling to the entire brain of a lizard species, the Australian dragon Pogona vitticeps, with a special focus in thalamus and prethalamus of. This approach allowed us to study the evolution of neuron types in amniotes. Therefore, we aimed to build a multilevel atlas of the lizard brain based on histology and transcriptomic and compare it to an equal mouse dataset (Zeisel et al., 2018).
Our atlas reveals a general structure that is consistent with that for other amniote brains, allowing us to make a direct comparison between lizard and mouse, despite their evolutionary divergence 320 million years ago. Through our analysis of the transcriptomes present in various neuron types, we have uncovered a core of conserved classes and discovered a fascinating dichotomy of new and conserved neuron types throughout the brain. This research challenges the traditional notion that certain brain regions are more conserved than others.
Our research also has uncovered the evolutionary history of the lizard thalamus and prethalamus by comparing them to homologous brain regions of the mouse. This pioneering research sheds new light on our understanding of the evolutionary history of the lizard brain. We propose a new classification of the lizard thalamic nuclei based on
transcriptomics. Our research revealed that the thalamic neuron types in lizards can be grouped into two large, conserved categories from the medial to lateral thalamus. These categories are encoded by a common set of effector genes, linking theories based on connectivity and molecular studies of these areas. In our data we have seen that there is a conservation of the medial-lateral transcriptomic axis in mouse and lizard, this conservation was most likely already present in the common ancestor. Although there is a shared medial-lateral axis, a deeper study of the thalamic cell types has allowed us to see the existence of a partial diversification of the thalamic population, specifically in the sensory-related lateral thalamus; in opposition, the medial thalamic nuclei neuron-types have been preserved.
On the other hand, the comparison with the mammalian prethalamus allowed us to confirm that the lizard ventromedial thalamic neuron types are homologous to mouse reticular thalamic neuron types (Díaz et al., 1994), even if they do not express the classical Reticular thalamic nucleus (RTn) marker PV/pvalb. We also discovered that there has been a simplification in the mammalian prethalamic neuron types in favor of an increase in the number of Interneurons (IN) types within their thalamus. We suggest that the loss of GABAergic neuronal types in the mammalian prethalamus is linked to the need for a more efficient control of the thalamo-pallial communication in mammals, while in lizards, where thalamo-pallial communication is probably simpler, the diversity prethalamus presents a higher diversity.
G-protein-coupled receptors (GPCRs) from the largest family of receptors in the human body. They contain seven transmembrane helices. There are roughly 800-900 GPCR genes expressed in humans encoded by 4-5% of the human genome. These receptors are the most important signal transducers and play a crucial role in cell physiology and pathology, by using various extracellular stimuli to start complex intracellular signaling. GPCRs interact with a wide variety of stimuli from small molecules (photons, ions, amines) to large molecules (peptides, small proteins), and trigger downstream cascade effects by interacting with G-proteins, GPCR kinases, and ß-arrestin. Because of their crucial roles in many cellular functions, GPCRs are the most important drug targets for the pharmaceutical industry. Approximately 30% of the clinically approved drugs available in the market are against GPCRs. In this work achieved successful expression and purification of GPCRs from class-C and class-A families. Combined with biochemical experiments, DNP-ssNMR, and molecular simulation helped to decipher the mechanism of crosstalk between the allosteric modulator, and the orthosteric binding sites of the peptide receptor. The main findings and major highlights of this dissertation are outlined in the following paragraphs.
The calcium-sensing receptor (CaSR) belongs to the GPCR class-C family and contains a large extracellular domain. This receptor regulates Ca2+ homeostasis in blood and its absorption in the kidney and bone. To understand the molecular and structural mechanisms of these receptors their cDNAs were cloned into the pPICZ and pOET1 vectors to express them in Pichia pastoris and in Sf9 insect cells respectively. The CaSR was successfully expressed heterologously in Pichia pastoris and in the insect cell with high yield. The purified receptor purified in LMNG shows no aggregation in a monomeric state. Further optimization was performed to use it for cryo-EM sample preparation and structure determination. In 2nd part of the thesis, different mini G (mini Gs, mini Gi, mini Gqs, and mini Gsi) DNA constructs were made and expressed in E. coli. It's challenging to obtain active GPCR structures due to the instability of G-protein or G-protein-bound receptors. In this work, all mini-G proteins and chimera mini-G-protein-maltose binding protein (MBP) were cloned and expressed in E. coli and purified with a His-trap column with high purity.
In the last part of the thesis, to decipher the mechanism of allosteric modulation of orthosteric binding sites in the bradykinin receptor was produced and characterized in insect cells. Angiotensin I converting enzyme inhibitors (ACEIs), are very important drugs and are widely used for the treatment of hypertension, congestive heart failure, and diabetic neuropathy. These drugs target primarily the catalytic zinc center of the ACE. It has been shown that enalaprilat, a well-known ACEI, binds to a proposed zinc-binding site on hB1R and even directly activates the receptor. To obtain information on the influence of ACEIs on the receptor-peptide complex, and to have a better understanding of the molecular mechanism and structural plasticity of the bradykinin receptor and PAM, we used the three commercially available ACEIs captopril, enalaprilat, and lisinopril for our studies. An important result of this thesis is that though enalaprilat, captopril, and lisinopril all have similar functional properties in humans, each one regulates the orthosteric binding site of hB1R in a unique way. These findings provide atomic insights into the allosteric modulation of the bradykinin receptor. This study along with the effects of ACEI on the binding sites of receptors also deciphers the effects of the Zn2+ as well as the crosstalk between zinc binding sites and ACEI compounds. The binding of allosteric modulators induces distinct endogenous binding, which might aid in creating new possibilities in the pharmaceutical field.
Epithelial cells enable essential physiological functions, including absorption, morphogenesis, secretion, and transport. To execute these functions, epithelial cells often form three-dimensional shapes that include curved sheets of cells surrounding a pressurized fluid-filled lumen. These three-dimensional tissues (called domes) are essential for organ function, but when they are not working properly, developmental defects, inflammation, and cancer can ensue. Recently, it has been shown that the cells that form domes show active superelasticity on micropatterned plates.
We show here that the immortalized renal proximal tubule epithelial cell line, LLC-PK1, stereotypically forms tubules in 10 days. Tubule formation takes place in 4 stages. When cells are plated on a culture dish, they form a monolayer on the 1st day; on the 3rd day, three-dimensional structures are formed, called domes; and after the 4.5th day, these domes start fusing to begin the transition stage and transit to the tubule stage. At the end of the 10th day, differentiated, elongated, and matured tubes form (Figure 3.1). Therefore, tubule formation is a self-organized, stereotypic morphogenetic program under long-term, unperturbed tissue culture conditions.
We propose that tubulogenesis is a two-step process in proximal tubules by doming and wrapping. The process begins with dome formation, and as the cell layers come together in the transition stage at the edge of the dome, this leads to the formation of the lumen of the eventual tubule. We also found that F-actin provides the mechanical strength during the formation of these three-dimensional structures during tubule formation. To better understand this 4-step process on a molecular level, we performed proteomics of tubule formation to identify the different proteins that play a significant role in proximal tubule development. Importantly, we identified proximal tubule markers like synaptopondin, angiotensin 1-10, collectrin, polycystin 1, and polycystin 2. These proteins play an important role in renal tube formation and differentiation.
Cell division is carried out by highly conserved cyclin-CDK complexes, which phosphorylate various cellular components. Cyclin-CDKs act differently depending on the cell cycle phase and work cooperatively to create DNA replication and cytokinesis. Therefore, we identified that cyclin-B1, marker of proliferation Ki-67, the RAD51 recombinase, and proliferating cell nuclear antigen (PNCA) are upregulated in the monolayer stage, and the expression decreases as tubule formation takes place. The proximal tubule reabsorbs 60-65% of the glomerulus filtrate. Therefore, it requires a lot of energy generated by using the fatty acid oxidation (FAO) pathway. In our model, we found FAO expression is higher than that of the other metabolic pathways.
We found expression of an intricate protein network in mitochondria, which we interpret as a sign of mitochondrial homeostasis being vital for the FAO pathway to work. Furthermore, we also identified different types of transporters at each stage of proximal tubule formation, and we could recognize different cytoskeletal components playing a significant role in each stage of proximal tubule formation, for instance, at the monolayer stage, vimentin expression is high, and its expression is reduced as tubules form. Hence, this 2D system, at this step of characterization, seems suitable to use to study differential transport protein expression and how this might relate to physiological functions and syndromes.
Next, we inhibited different transporters using specific inhibitors and analyzed the effect on dome and tubule formation. We identified that Na+/K+ ATPase and vacuolar H+ ATPase play a significant role in the process of epithelial dynamics. Digoxin (a Na+/K+ ATPase inhibitor) treatment inhibits dome and tubule formation. Bafilomycin (a v-ATPase inhibitor) treatment demonstrated a delay in dome and tube formation. Therefore, this study shows that this 2D proximal tubule novel system can be used for screening of pharmacological leads in the context of specific aspects of kidney physiology.
Despite the recent success in growing kidney organoids, they are not well suited to investigate various pathophysiological conditions in vitro for several reasons: They grow in 3D and form a tissue that later needs to be dissected/cleared and stained to investigate pathophysiological changes. Moreover, organoids require complex and expensive protocols for generation and are challenging to use in screening approaches. Therefore, we set out to demonstrate feasibility for our 2D system using normal renal epithelial cells, which are the origin of various pathological conditions, to study pathophysiological conditions.
RNAs are key players in life as they connect the genetic code (DNA) with all cellular processes dominated by proteins. The dynamics study of RNA modifications has become an important part of epitranscriptomics field, as they are reversible and dynamically regulated far more than originally thought. Several evidences portrait a catalog of RNA modifications and their links to neurological disorders, cancers, and other diseases. Therefore, a deeper investigation of RNA modifications dynamics including their specific profile, biosynthesis, maturation and degradation is required for pioneering disease diagnostics and potential therapeutics development.
Mammalian tissues reveal diverse physiology and functions, despite sharing identical genomes and overlapping transcription profiles. So far, most research on this diversity were referred to variable transcriptomic processing among tissues and differential post-translational modifications that tune the activity of ubiquitous proteins to each tissue’s needs. However, study of epitranscriptome dynamics relevance to tissues’ functions is not yet revealed. There are a few reports on mouse RNA modification profiles, which are focused on only one type of RNA and limited types of modifications. The first part of my dissertation aims to generate a comprehensive tissue-specific as well as RNA species-specific investigation of all existing RNA modifications, as well as investigating potential codon as an effector of translation diversity among tissues. Using isotope dilution mass spectrometry, I created a library including absolute quantification of 24 tRNA modifications, and up to 22 rRNA modifications. I find an almost identical pattern of modifications in 28S- and 18S-rRNA subunits, but different levels of most modifications in 5.8S-rRNA or tRNA among highly metabolic active organs to e.g. heart or spleen. The findings suggest a high degree of similarity between quantities of modifications between presented data to all previous literature, confirming that it is a suitable model to study the tissue-based RNA modification patterns.
The most noticeable difference exhibited was tRNA modifications, which suggests a discerning tRNA engagement in translation between different organs. This can be a good start for investigation of codon bias in enriched genes of specific tRNA modifications among different tissues that may cause differential translation pattern, causing organs diversity. Moreover, 5.8S rRNA data showed an organ-specific pattern, which proposes functional diversity of this rRNA subunit among different organs. Future studies must investigate the possible implications of organ-specific 5.8S rRNA modifications functions, to elucidate the core of the observed variations.
Abundance of RNA modifications is carefully regulated in cells. Part of this regulation is achieved by activity of enzymes removing RNA modifications, named RNA erasers. Literature has provided proof of demethylation activity of AlkBH family on different types of RNA. For instance, AlkBH5 is known to remove m6A in mRNA, and both AlkBH3 and AlkBH1 are reported to demethylate m1A and m3C in tRNA. So far, RNA erasers are mainly studied in vitro and direct in vivo studies are missing.
Mass spectrometry is a promising approach in the identification and quantification of many RNA modifications. However, mass spectrometric analysis by nature, offers only a static view of nucleic acid modifications, and fails to account for their cellular dynamics. Nucleic Acid Isotope Labeling coupled Mass Spectrometry (NAIL-MS) was developed as a powerful technique which differentiates among remaining, co-transcriptional and post-transcriptional incorporation of a target RNA modification. This temporal resolution captures the dynamic nature of RNA modifications, and offers absolute and relative quantification of all existing nucleosides in any given RNA sequence, including different isotopologues and isotopomers.
The objective of this study was to uncover the first “direct” iv vivo data on AlkBH1, 3 and 5 activities in demethylating each of their specific substrates. I investigated the RNA modification changes through pulse-chase experiments in collaboration with my colleagues Dr. Kayla Borland and Dr. Felix Hagelskamp. A remarkable observation was that AlkBH3 protein -but not AlkBH1- was overexpressed under methylating reagent treatment in vivo. These findings suggest that AlkBH3 -but not AlkBH1- is a methylation damage induced enzyme, that potentially triggers ASCC-AlkBH3 alkylation repair complex after aberrant methylation damage by MMS treatment. However, using NAIL-MS method, we could not detect any significant effect on demethylation activity of the enzymes in tRNA, rRNA or mRNA towards the possible substrates m6A, m1A, m3C, m5C and m7G in vivo. These distinct outcomes can be partially explained by probable existence of other unidentified demethylases that compensate for AlkBHs demethylation activity; or more probably, demethylation may still arise by remaining active AlkBHs to restore the original levels of the observed RNA modifications, since a stronger KD or a complete knockout of AlkBHs genes was not possible. Further research on fully knocked out AlkBHs genes can provide stronger evidence on unidentified demethylation activities in HEK cells.
Neutron stars are unique laboratories for the investigation of the high density properties of bulk matter. In this work, the astrophysical constraints for a phase transition from hadronic matter to deconfined quark matter are examined thoroughly. A scheme for relating known astrophysical observables such as mass, radius and tidal deformability to the parameter space of such a transition is devised and applied to the set of data currently available.
In order to span a wide parameter space, a highly parameterizable relativistic mean field equation in compliance with chiral effective field theory results is used, where the stiffness of the equation of state can be varied via the effective mass at saturation density. The phase transitions are modelled using a Maxwell construction and assumed to be of first order, with a constant speed of sound quark matter model. The resulting equations of state are analyzed and divided into four categories, which can be used to constrain the parameter space that allows phase transition. It is highlighted, that a subset of this parameter space would even be detectable without the need of higher precision measurements. A phase transition at high densities is shown to be particularly promising in this regard. Finally, the groundwork is laid to apply the equation of state used in this work for supernova or merger simulations, by extending it to non-zero temperatures.
RNA research is very important since RNA molecules are involved in various gene regulatory mechanisms as well as pathways of cell physiology and disease development.1 RNAs have evolved from being considered as carriers of genetic information from DNA to proteins, with the three major types of RNA involved in protein synthesis, including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).2 In addition to the RNAs involved in protein synthesis numerous regulatory non-coding RNAs (ncRNAs) have been discovered in the transcriptome. The regulatory ncRNAs are classified into small ncRNAs (sncRNAs) with transcripts less than 200 nucleotides (nt) and long non-coding RNAs (lncRNAs) with more than 200 nt.3
LncRNAs represent the most diverse and versatile class of ncRNAs that can regulate cellular functions of chromatin modification, transcription, and post-transcription through multiple mechanisms.4 They are involved in the formation of RNA:protein, RNA:RNA and RNA:DNA complexes as part of their gene regulatory mechanism.4,5 The RNA:DNA interactions can be divided into RNA:DNA heteroduplex formation, also called R-loops, and RNA:DNA:DNA triplex formation. In triplex formation, RNA binds to the major groove of double-stranded DNA through Hoogsteen or reverse Hoogsteen hydrogen bonding, resulting in parallel or anti-parallel triplexes, respectively. In vitro studies have confirmed the formation of RNA:DNA:DNA triplexes.6 However, the extent to which these interactions occur in cells and their effects on cellular function are still not understood, which is why these structures are so exciting to study (Chapter I RNA:DNA:DNA Triplexes).
This cumulative thesis investigates several functional and regulatory important RNAs. The first project involves the improved biochemical and biophysical characterization of RNA:DNA:DNA triplex formation between lncRNAs of interest and their target genes. Triplex formation was confirmed by a series of experiments including electromobility shift assays (EMSA), thermal melting assays, circular dichroism (CD), and liquid state nuclear magnetic resonance (NMR) spectroscopy. The following is a summary of the main findings of these publications.
In research article 5.1, the oxygen-sensitive HIF1α-AS1 was identified as a functionally important triplex-forming lncRNA in human endothelial cells using a combination of bioinformatics techniques, RNA/DNA pulldown, and biophysical experiments. Through RNA:DNA:DNA triplex formation, endogenous HIF1α-AS1 decreases the expression of several genes, including EPH receptor A2 (EPHA2) and adrenomedullin (ADM), by acting as an adaptor for the repressive human silencing hub (HUSH) complex, which has been studied by our collaborators in the groups of Leisegang and Brandes.
2) Triplex formation between HIF1α-AS1 and the target genes EPHA2 and ADM was investigated in biochemical and biophysical studies. The EMSA results indicated that HIF1α-AS1 forms a low mobility RNA:DNA:DNA triplex complex with the EPHA2 DNA target sequence. The CD spectrum of the triplex showed distinct features compared to the EPHA2 DNA duplex and the RNA:DNA heteroduplex. Melting curve analysis revealed a biphasic melting transition for triplexes, with a first melting point corresponding to the dissociation of the RNA strand with melting of the Hoogsteen hydrogen bonds. The second, higher melting temperature corresponds to the melting of stronger Watson-Crick base pairing. Stabilized triplexes were formed using an intramolecular EPHA2 DNA duplex hairpin construct in which both DNA strands were attached to a 5 nucleotide (nt) thymidine linker. This approach allowed improved triplex formation with lower RNA equivalents and higher melting temperatures. By NMR spectroscopy, the triplex characteristic signals were observed in the 1H NMR spectrum, the imino signals in a spectral region between 9 and 12 ppm resulting from the Hoogsteen base pairing. To elucidate the structural and sequence specific Hoogsteen base pairs 2D 1H,1H-NOESY measurements of the EPHA2 DNA duplex and the HIF1α-AS1:EPHA2 triplex were performed. The 1H,1H-NOESY spectrum of the HIF1α-AS1:EPHA2 triplex with a 10-fold excess of RNA was semi-quantitatively analyzed for changes in the DNA duplex spectrum. We discovered, strong and moderate attenuation of cross peak intensities in the imino region of the NOESY spectrum. This attenuation was proposed to result from weakening of Watson-Crick base pairing by Hoogsteen hydrogen bonding induced by RNA binding. The Hoogsteen interactions can be mapped based on the analysis of the cross peak attenuation in the NOESY spectra, which we used to generate a structural model of the RNA:DNA:DNA triplex. These biophysical results support the physiological function of HIF1α as a triplex-forming lncRNA that recruits the HUSH-epigenetic silencing complex to specific target genes such as EPHA2 and ADM, thereby silencing their gene expression through RNA:DNA:DNA triplex formation.
In the recent past, we are making huge progress in the field of Artificial Intelligence. Since the rise of neural networks, astonishing new frontiers are continuously being discovered. The development is so fast that overall no major technical limits are in sight. Hence, digitization has expanded from the base of academia and industry to such an extent that it is prevalent in the politics, mass media and even popular arts. The DFG-funded project Specialized Information Service for Biodiversity Research and the BMBF-funded project Linked Open Tafsir can be placed exactly in that overall development. Both projects aim to build an intelligent, up-to-date, modern research infrastructure on biodiversity and theological studies for scholars researching in these respective fields of historical science. Starting from digitized German and Arabic historical literature containing so far unavailable valuable knowledge on biodiversity and theological studies, at its core, our dissertation targets to incorporate state-of-the-art Machine Learning methods for analyzing natural language texts of low-resource languages and enabling foundational Natural Language Processing tasks on them, such as Sentence Boundary Detection, Named Entity Recognition, and Topic Modeling. This ultimately leads to paving the way for new scientific discoveries in the historical disciplines of natural science and humanities. By enriching the landscape of historical low-resource languages with valuable annotation data, our work becomes part of the greater movement of digitizing the society, thus allowing people to focus on things which really matter in science and industry.
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.
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.
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.
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.
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.
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.
Binary neutron star mergers represent unique observational phenomena because all four fundamental interactions play an important role at various stages of their evolution by leaving imprints in astronomical observables. This makes their accurate numerical modeling a challenging multiphysics problem that promises to increase our understanding of the high-energy astrophysics at play, thereby providing constraints for the underlying fundamental theories such as the gravitational interaction or the strong interaction of dense matter. For example, the first and so far only multi-messenger observation of the binary neutron star merger GW170817 resulted in numerous bounds on the parameters of isolated non-rotating neutron stars, e.g., their maximum mass or their distribution in radii, which can be directly used to constrain the equation of state of cold nuclear matter. While many of these results stem from the observation of the inspiral gravitational-wave signal, the postmerger phase of binary neutron star mergers encodes even more details about the extreme physics of hot and dense neutron star matter. In this Thesis we focus on the exploration of dissipative and shearing effects in binary neutron star mergers in order to identify novel approaches to constrain hot and dense neutron star matter.
The first effect is the well-motivated dissipation of energy due to the bulk viscosity which arises from violations of weak chemical equilibrium. We start by exploring the impact of bulk viscosity on black-hole accretion. This simplified problem gives us the opportunity to develop a test case for future codes taking into account the effects of dissipation in a fully general-relativistic setup and build intuition in the physics of relativistic dissipation. Next, we move on to isolated neutron stars and binary neutron star mergers by developing a robust implementation of bulk-viscous dissipation for numerical relativity simulations. We test our implementation by calculating the damping of eigenmodes of isolated neutron stars and the violent migration scenario. Finally, we present the first results on the impact of bulk viscosity on binary neutron star mergers. We identify a number of ways how bulk viscosity impacts the postmerger phase, out of which the suppression of gravitational-wave emission and dynamical mass ejection are the most notable ones.
In the last part of this Thesis we investigate how the shearing dynamics at the beginning of the merger affects the amplification of different initial magnetic-field topologies. We explore the hypothesis that magnetic fields which are located only in a small region near the stellar surface prior to merger lead to a weaker magnetic-field amplification. We show first evidence which confirms this hypothesis and discuss possible implications for constraining the physics of superconduction in cold neutron stars.
The EMT-transcription factor ZEB1 has been intensively studied in solid cancers, where it is expressed at the invasive front and in cancer-associated fibroblasts (CAFs). In tumour cells, ZEB1 has been involved in multiple steps of cancer progression including stemness, metastasis and therapy resistance, yet its role in the tumour-microenvironment is largely unknown. Here, the role of Zeb1 in CAFs was investigated using mouse models reflecting different tumour stages in immunocompetent fibroblast specific Zeb1 KO mice. Fibroblast-specific depletion of Zeb1 accelerated tumour growth in the inflammation driven AOM/DSS tumour initiation model, reduced tumour growth and invasion in the sporadic AOM/P53 model and reduced liver metastasis in a progressed orthotopic transplantation model. Immunohistochemical and single cell RNA-sequencing analysis showed that Zeb1 ablation resulted in attenuated expression of the myofibroblast marker aSMA and reduced ECM deposition, indicating a shift among fibroblast subpopulations. Modulation of CAFs was furthermore associated with increased inflammatory signaling in fibroblasts resulting in immune infiltration into primary tumours and exaggerated inflammatory signaling in T cells, B cells and macrophages. These changes in the tumour microenvironment were associated with increased efficacy of immune checkpoint inhibition therapy. In summary, Zeb1 expression in CAFs was identified as a potential target to block immunosuppression and metastatic dissemination in colon cancer.
In this dissertation, different aspects of turbulent transport and thermally driven flows over complex terrain are investigated. Two publications concentrate on the vertical heat and moisture exchange in the convective boundary layer over mountainous terrain. To study this, Large-Eddy Simulation (LES) is used. Both turbulent and advective transport mechanisms are evaluated over the simple orography of a quasi-two-dimensional, periodic valley with prescribed surface fluxes. Here, terrain elevation varies along only one of the horizontal coordinate axes. Even a relatively shallow orography, possibly unresolved in existing numerical weather prediction models, modifies the domain-averaged moisture and temperature profiles. For the analysis, the flow is decomposed into a local turbulent part, a local mean circulation, and a large-scale part. An analysis of the turbulent kinetic energy and turbulent heat and moisture flux budgets shows that the thermal circulation significantly contributes to the vertical transport. It is found that thermal upslope winds are important for the moisture transport from the valley to the mountain tops. In total, moisture export out of the valley is mostly accomplished by the mean circulation. On the temperature distribution, which is horizontally relatively homogeneous, the thermal circulation has a weaker impact. If an upper-level wind is present, it interacts with the thermal circulation. This weakens the vertical transport of moisture and thus reduces its export out of the valley. The heat transport is less affected by the upper-level wind because of its weaker dependence on the thermal circulation. These findings were corroborated in a more realistic experiment simulating the full diurnal cycle using radiation forcing and an interactive land surface model.
Based on these results, coherent turbulent structures in the convective boundary layer over non-flat terrain are studied in further detail. A conditional sampling method based on the concentration of a decaying passive tracer is implemented in order to identify the boundary-layer plumes objectively. Conditional sampling allows to quantify the contribution of plume structures to the vertical transport of heat and moisture. In case of the idealized valley, vertical transport by coherent structures is the dominant contribution to the turbulent components of both heat and moisture flux. It is comparable in magnitude to the advective transport by the mean slope-wind circulation, although it is more important for heat than for moisture transport. A set of less idealized simulations considers the flow over three-dimensional terrain. In this case, conditional sampling is carried out by using a simple domain-decomposition approach. We demonstrate that thermal updrafts are generally more frequent on hill tops than over the surroundings, but they are less persistent on the windward sides when large-scale winds are present in the free atmosphere.
The tools for flow decomposition and budget analysis are also applied in another idealized case with a quasi-two-dimensional valley featuring the stable boundary layer. Here, the formation of a low stratus cloud is investigated. The main driver for the cloud formation is radiative cooling due to outgoing longwave radiation. Despite a purely horizontal flow, the advection terms in the prognostic equations for heat and moisture produce vertical mixing across the upper cloud edge leading to a loss of cloud water content. However, this behavior is not due to any kind of thermally-driven circulation. Instead, this spurious mixing is caused by the diffusive error of the advection scheme in regions where the sloping surfaces of the terrain-following vertical coordinate intersect the cloud top. It is shown that the intensity of the (spurious) numerical diffusion strongly depends on the horizontal resolution, the order of advection, and the choice of the scalar advection scheme. A LES with 4 m horizontal resolution serves as a reference. For horizontal resolutions of a few hundred meters, carried out with a model setup as it is used in Numerical Weather Prediction, a strong reduction of the simulated liquid-water path is observed. In order to keep the (spurious) numerical diffusion at coarser resolutions small, at least a fifth-order advection scheme should be used. In the present case, a WENO scalar advection scheme turns out to increase the numerical diffusion along a sharp cloud edge compared to an upwind scheme. Furthermore, the choice of the vertical coordinate has a strong impact on the simulated liquid-water path over orography. With a modified definition of the terrain-following sigma coordinate, it is possible to produce cloud water where the classical sigma coordinate does not allow any cloud formation.
The capacity of pathogenic bacteria to adhere to host cells and to avoid subsequent clearance by the host´s immune response is the initial and most decisive step leading to infections. Human pathogenic bacteria circulating in the bloodstream need to find ways to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, particularly fibronectin (Fn). Trimeric autotransporter adhesins (TAA) have been described as important pathogenicity factors of Gram-negative bacteria. The TAA from human pathogenic Bartonella henselae, Bartonella adhesin A (BadA), is one of the longest and best characterised adhesin and represents a prototypic TAA due to its domain architecture. B. henselae, the causative agent of cat scratch disease, endocarditis, and bacillary angiomatosis, adheres to ECs and ECM proteins via BadA interaction.
In this research, it was determined that the interaction between BadA and Fn is essential for B. henselae host cell adhesion. BadA interactions were identified within the heparin-binding domains of Fn, and the exact binding sites were revealed by mass spectrometry analysis of chemically crosslinked whole-cell bacteria and Fn. It turned out that specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs. These data were confirmed by using BadA-deficient bacteria and CRISPR-Cas FN1 knockout ECs. It was also identified that BadA binds to Fn from both cellular and plasma origin, suggesting that B. henselae binding to Fn might possibly take part in other infection processes apart from bacterial adherence, e.g. evasion from the host cell immune system.
Interactions between TAAs and Fn represent a key step for adherence of B. henselae to ECs. Still, Fn-mediated binding is of more significant importance for pathogenic bacteria than broadly recognised. Fn removal from the ECM environment of ECs, also reduced adherence of Staphylococcus aureus, Borrelia burgdorferi, and Acinetobacter baumannii to host cells Interactions between adhesins and Fn might therefore represent a crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection or as means for persistence.
This research demonstrated that combining large-scale analysis approaches to describe protein-protein interactions with supportive functional readouts (binding assays) allows for the discrimination of crucial interactions involved in bacterial adhesion to the host. The herein-described experimental approaches and tools might guide future research for other pathogenic bacteria and represent an initial point for the future generation of anti-virulence strategies to inhibit bacterial binding to host cells.
In our rapidly changing world, land use has been recognized as having one of the strongest impacts on species and genetic diversity. The present state of temperate forests in Europe is a product of decisions made by former and current management and policy actions, rather than natural factors. Alterations of crown projection areas, structural complexity of the forest stand caused by thinning and cuttings, and changes in tree species composition caused by regeneration or plantings not only affect forest interior buffering against warming, but also the understorey light environment and nutrient availability. Ultimately, current silvicultural management practices have deep impact on the forest ecosystems, microenvironmental changes and forest floor understorey herbs. In response to environmental changes, plants rely on genetically heritable phenotypic variation, an important level of variation in the population, as it is prerequisite for adaptation. However, until now most studies on plant adaptation to land use focus on grassland management. Yet, studies on the adaptation of forest understorey herbs to forest management have been absent so far. This is important because understanding adaptation of understorey herbs is crucial for biodiversity conservation, forest restoration, and climate change mitigation. Studying current adaptation of understorey herbs to forest management yields insights into the evolutionary consequences of management practices, which could be employed to improve sustainable use of forest habitat.
In sum, my conducted experiments complement each other well and managed to fill in research gaps on the topic of genetically heritable phenotypic variation in understorey herbs and how it is affected by forest management and related microenvironmental variables. I showed that forest management has direct evolutionary consequences on the genetic basis of understorey herbs, but also indirectly through the microenvironment. Furthermore, I revealed that local adaptation and phenotypic plasticity of understorey herbs to forest structural attributes act along continuous gradients. And lastly, I highlighted the important role of intra-individual variation by revealing plastic responses to drought and shading, urging researchers to not ignore this important level of trait variation. Ultimately, understorey herbs in temperate forests employ phenotypic plasticity as a flexible strategy to adapt to varying environmental conditions. By adjusting their leaf characteristics, reproductive investment, and phenology, they can optimize their fitness and survival in response to changes in light availability, resource availability, and seasonal cues. The anthropogenic impact on temperate forests and understorey herbs will continue and likely increase in the future. This should urge foresters to adapt their silvicultural management decisions towards the long-term preservation of genetic diversity and, through this, the evolvability and adaptability of forest understorey herbs and associated organisms. Based on the results shown in my dissertation, variation in forest management regimes and types could be beneficial for promoting genetic diversity within several species of forest understorey herbs. Lastly, in the face of future climatic changes, the mechanisms by which plants can cope with increasing stressful environmental conditions might very well rely heavily on intra-individual variation, providing the necessary rapid plastic adjustment to changing microclimatic conditions within populations and thus increase climate change resilience.
A synchrotron is a particular type of cyclic particle accelerator and the first accelerator concept to enable the construction of large-scale facilities [10], such as the largest particle accelerator in the world, the 27-kilometre-circumference Large Hadron Collider (LHC) by CERN near Geneva, Switzerland, the European Synchrotron Radiation Facility (ESRF) in Grenoble, France for the synchrotron radiation, the superconducting, heavy ion synchrotron SIS100 under construction for the FAIR facility at GSI, Darmstadt, Germany and so on. Unlike a cyclotron, which can accelerate particles starting at low kinetic energy, a synchrotron needs a pre-acceleration facility to accelerate particles to an appropriate initial value before synchrotron injection. A pre-acceleration can be realized by a chain of other accelerator structures like a linac, a microtron in case of electrons, for example, Proton and ion injectors Linac 4 and Linac 3 for the LHC, UNLAC as the injector for the SIS18 in GSI and in future the SIS18 as injector for the SIS100. The linac is a commonly used injector for the ion synchrotron and consists of some key components. The three main parts of a linac are: An ion source creating the particles, a buncher system or an RFQ followed by the main drift tube accelerator DTL. In order to meet the energy and the beam current requirement of a synchrotron injector linac, its cost is a remarkable percentage of the total facility costs.
However, the normal conducting linac operation at cryogenic temperatures can be a promising solution in improving the efficiency and reducing the costs of a linac. Synchrotron injectors operate at very low duty factor with beam pulse lengths in 1 micros to 100 micros range, as most of the time is needed to perform the synchrotron cycle. Superconducting linacs are not convenient, as they cannot efficiently operate at low duty factor and high beam currents.
The cryogenic operation of ion linacs is discussed and investigated at IAP in Frankfurt since around 2012 [1, 37]. The motivation was to develop very compact synchrotron injectors at reduced overall linac costs per MV of acceleration voltage. As the needed beam currents for new facilities are increasing as well, the new technology will also allow an efficient realization of higher injector linac energies, which is needed in that case. Operating normal conducting structures at cryogenic temperature exploits the significantly higher conductivity of copper at temperatures of liquid nitrogen and below. On the other hand, the anomalous skin effect reduces the gain in shunt impedance quite a bit[25, 31, 9]. Some intense studies and experiments were performed recently, which are encouraging with respect to increased field levels at linac operation temperatures between 30 K and 70 K [17, 24, 4, 23, 5, 8]. While these studies are motivated by applications in electron acceleration at GHz-frequencies, the aim of this paper is to find applications in the 100 to 700 MHz range, typical for proton and ion acceleration. At these frequencies, a higher impact in saving RF power is expected due to the larger skin depth, which is proportional to the frequency to the power of negative half with respect to the normal skin effect. On the other hand, it is assumed that the improvement in maximum surface field levels will be similar to what was demonstrated already for electron accelerator cavities. This should allow to find a good compromise between reduced RF power needs for achieving a given accelerator voltage and a reduced total linac length to save building costs.
A very important point is the temperature stability of the cavity surface during the RF pulse. This is of increasing importance the lower the operating temperature is chosen: the temperature dependence of the electric conductivity in copper gets rather strong below 80 K, as long as the RRR - value of the copper is adequate. It is very clear, that this technology is suited for low duty cycle operated cavities only - with RF pulse lengths below one millisecond. At longer pulses the cavity surface will be heated within the pulse to temperatures, where the conductivity advantage is reduced substantially. These conditions fit very well to synchrotron injectors or to pulsed beam power applications.
H – Mode structures of the IH – and of the CH – type are well-known to have rather small cavity diameters at a given operating frequency. Moreover, they can achieve effective acceleration voltage gains above 10 MV/m even at low beam energies, and already at room temperature operation[29]. With the new techniques of 3d – printing of stainless steel and copper components one can reduce cavity sizes even further – making the realization of complex cooling channels much easier.
Another topic are copper components in superconducting cavities – like power couplers. It is of great importance to know exactly the thermal losses at these surfaces, which can’t be cooled efficiently in an easy way.
Brain development is a complex and highly organized process that relies on the coordinated interaction between neurons and vessels. These cell systems form a neurovascular link that involves the exchange of oxygen, ions, and other physiological components necessary for proper neuronal and vascular function. This physiologically coupled process is executed through analogous structural and molecular signaling mechanisms shared by both cell types. At the neurovascular interface, the cellular crosstalk via these shared signaling mechanisms allows for the synchronized expansion and integration of neurons and vessels into complex cellular networks. This study investigated the role of VEGFR2, a receptor for vascular endothelial growth factor (VEGF), during postnatal neuronal development in the mouse hippocampus. Prior studies have revealed physiological roles of VEGF, a pro-angiogenic morphogen, in nervous system development. However, it was unclear if VEGF signaling had a direct effect on neuronal physiology and function through neuronal-expressing receptors. In this investigative work, we identified a previously unknown function of VEGFR2, whereby VEGF-induced signaling coordinates the development and circuitry integration of CA3 pyramidal neurons in the early postnatal mouse hippocampus. Mechanistically, we found that VEGFR2 signaling requires receptor endocytosis, a process mediated by ephrinB2. We also found that VEGF-induced cooperative signaling between VEGFR2 and ephrinB2 is functionally required for the dendritic arborization and spine maturation of developing CA3 neurons during the first few postnatal weeks. Moreover, in a collaborative effort with the research group of Carmen Ruiz de Almodovar, formerly at the University of Heidelberg, we simultaneously studied VEGF-induced VEGFR2 signaling in CA3 axonal development. Together, we aimed to gain a comprehensive understanding of the complex interplay between VEGF and VEGFR2 signaling during the early postnatal development of CA3 neurons. Ruiz de Almodovar’s research group found that, unlike the branch and spine development of CA3 dendrites, VEGF-VEGFR2 signaling promotes axonal development through mechanisms that are independent of ephrinB2 function. Our findings on CA3 dendritic development are reported in the published manuscript, Harde et al. (2019), and the complementary work on CA3 axonal development from Ruiz de Almodovar's group is presented in the co-published manuscript, Luck et al. (2019). Although the totality of Ruiz de Almodovar's group's work on CA3 axons is not fully discussed here, it is referenced where noted to provide biological context for our findings on CA3 dendritic development.
VEGFR2 signaling within neurovascular niches is known to play a role in the neurogenesis of neural progenitor cells during embryonic development and within the adult brain. However, the precise localization of neuronal VEGFR2 expression and functional role within the nervous system during postnatal brain development was unknown. To investigate this, we used immunohistochemistry to identify the spatial expression of VEGFR2 within the mouse hippocampus during the first few weeks after birth. Our results showed that VEGFR2 was predominantly expressed within the hippocampal vasculature, consistent with prior studies. However, we also observed localized VEGFR2 expression in pyramidal cell neurons of the hippocampal CA3 region by postnatal day 10 (P10). This spatially restricted postnatal expression of VEGFR2 in CA3 neurons suggested a potential role in the development of these neurons during this developmental stage.
The first two weeks after birth in the mouse hippocampus is a critical period for the development of neuronal circuits, as neurons undergo extensive dendritic arborization and spine formation. To explore the role of VEGFR2 in the postnatal nervous system, we used a Nes-cre VEGFR2lox/- mouse line to target the deletion of VEGFR2 expression within the nervous system while preserving normal receptor expression in all other cell types. We also generated corresponding control mice that were negative for Nes-cre. By breeding these mice with Thy1-GFP reporter mice, we could analyze the functional consequences of VEGFR2 by assessing the morphologies of CA3 dendritic trees and spine density and maturation at P10 and P15, respectively. Our analysis showed that CA3 neurons in Nes-cre VEGFR2lox/- mice had less complex dendritic arbors compared to control mice. There were significant reductions in total length and branch points, particularly in areas located 100-250 μm from the cell soma within the stratum radiatum layer. Additionally, Nes-cre VEGFR2lox/- mice exhibited a significant decrease in spine density accompanied by an increased proportion of immature spines. These findings suggest that VEGFR2 plays a crucial role in the proper development of CA3 dendrites and spines during the early postnatal weeks.
Cyber Physical Systems (CPS) are growing more and more complex due to the availability of cheap hardware, sensors, actuators and communication links. A network of cooperating CPSs (CPN) additionally increases the complexity. This poses challenges as well as it offers chances: the increasing complexity makes it harder to design, operate, optimize and maintain such CPNs. However, on the other side an appropriate use of the increasing resources in computational nodes, sensors, actuators can significantly improve the system performance, reliability and flexibility. Therefore, self-X features like self-organization, self-adaptation and self-healing are key principles for such systems.
Additionally, CPNs are often deployed in dynamic, unpredictable environments and safety-critical domains, such as transportation, energy, and healthcare. In such domains, usually applications of different criticality level exist. In an automotive environment for example, the brake has a higher criticality level regarding safety as the infotainment. As a result of mixed-criticality, applications requiring hard real-time guarantees compete with those requiring soft real-time guarantees and best-effort application for the given resources within the overall system. This leads to the need to accommodate multiple levels of criticality while ensuring safety and reliability, which increases the already high complexity even more.
This thesis deals with the question on how to conveniently, effectively and efficiently handle the management and complexity of mixed-critical CPNs (MC-CPNs). Since this cannot be done by the system developer without the assistance of the system itself any longer, it is essential to develop new approaches and techniques to ensure that such systems can operate under a range of conditions while meeting stringent requirements.
Based on five research hypothesis, this thesis introduces a comprehensive adaptive mixed-criticality supporting middleware for Cyber-Physical Networks (Chameleon), which efficiently and autonomously takes care of the management and complexity of CPNs with regard to the mixed-criticality aspect.
Chameleon contributes to the state-of-art by introducing and combining the following concepts:
- A comprehensive self-adaption mechanism on all levels of the system model is provided.
- This mechanism allows a flexible combination of parametric and structural adaptation actions (relocation, scheduling, tuning, ...) to modify the behavior of the system.
- Real-time constraints of mixed-critical applications (hard real-time, soft real-time, best-effort) are considered in all possible adaptation conditions and actions by the use of the importance parameter.
- CPNs are supported by the introduction of different scopes (local, system, global) for the adaptation conditions and actions. This also enables the combination of different scopes for conditions and actions.
- The realization of the adaptation with a MAPE-K loop instantiated by a distributed LCS allows for real-time capable reasoning of adaptation actions which also works on resource-spare systems.
- The developed rule language Rango offers an intuitive way to specify an initial rule set for LCS in the context of CPS/CPNs and supports the system administrators in the process of rule set generation.
For thousands of years, S. cerevisiae has been employed by humans in brewing and baking. Nowadays, this budding yeast is more than that: it is a well investigated model organism and an established workhorse in biotechnology. S. cerevisiae serves as a production host for various applications such as i) bioethanol production ii) the biosynthesis of hormones including insulin or iii) cannabinoid biosynthesis. Hereby, the robustness of S. cerevisiae and its high tolerances regarding pH and salt concentrations qualifies it for a wide range of industrial applications. Moreover, products of S. cerevisiae are generally recognised as safe (GRAS), enabling diverse biotechnological applications. Various mechanisms for genetic engineering of S. cerevisiae are applicable and the engineering process itself is straightforward since methods are established and widely known. Due to the wide range of industrial applications of S. cerevisiae, this organism is an ideal candidate for applied research and implementation of the recombinant biosynthesis of tocochromanols in this study.
Tocochromanols encompass tocotrienols and tocopherols, which are lipid-soluble compounds that are commonly associated with vitamin E activity. Hereby, α-tocopherol is the most prevalent form, as it is an essential nutrient in the diet of humans and animals. Naturally, tocochromanols are almost exclusively synthesised by photoautotrophic organisms such as plants or cyanobacteria. They consist of an aromatic head group and a polyprenyl side chain which is saturated in tocopherols and 3-fold unsaturated in tocotrienols. The methylation status of the chromanol ring distinguishes α-, β-, γ- and δ-tocochromanol. All forms of tocochromanols represent a group of powerful antioxidants, scavenging reactive oxygen species (ROS) and preventing the propagation of lipid oxidation in lipophilic environments. Recently, attention has been drawn to tocotrienols, due to their benefits in neuroprotection as well as cholesterol-lowering and anti-cancer properties. Consequently, tocochromanols are valuable additives in the food, feed, cosmetic and pharmaceutical industries.
The metabolic engineering strategy of S. cerevisiae to enable tocochromanol biosynthesis was started in a preceding master thesis with the provision of the aromatic moiety, homogentisic acid (HGA), from the aromatic amino acid biosynthesis. Hereby, the upregulation and redirection of the native pathway was essential. Therefore, a strain with an engineered aromatic amino acid pathway for improved 4 hydroxyphenylpyruvate (HPP) production (MRY33) was utilised from Reifenrath and Boles (2018). Furthermore, a heterologous hydroxyphenylpyruvate dioxygenase (HPPD) was required to convert HPP into HGA. Thus, several heterologous HPPDs were expressed and characterised regarding their HGA production within the previous study. The best variant originated from Yarrowia lipolytica, YlHPPD, and was integrated into the genome of MRY33. The resulting strain JBY2, produced 435 mg/L HGA in a shake flask fermentation.
This work was started with the genetically highly modified strain JBY2, whose genome already contained a large number of genes artificially expressed behind strong promoters. For further strain development, it was advantageous to maintain a high degree of sequence variability in order to prevent genomic instabilities due to sequence homologies. Thus, 17 artificial promoters (AP1-AP17) were characterised regarding their strength of expression by the yellow fluorescent protein (YFP). These sequences were also part of a patent that was filed during this work (WO2023094429A1).
The key point of this study was the development of a metabolic engineering strategy for the strain JBY2. First, the sufficient supply of the second precursor, the polyprenyl side chain, was investigated. Natively, S. cerevisiae produces the precursor, geranylgeranyl diphosphate (GGPP), from the isopentenyl diphosphate pathway. However, without further engineering, GGPP was barely detectable in JBY2 (< 0.1 mg/L). Thus, engineering of the isopentenyl diphosphate biosynthesis was necessary. The limiting enzyme of the mevalonate pathway was the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which is encoded by HMG1. Therefore, a truncation for feedback-resistance and its overexpression by a promoter exchange was performed. Furthermore, the promoter of the gene for the squalene synthase (pERG9) was exchanged by the ergosterol sensitive promoter pERG1 to limit the metabolic flux of the mevalonate pathway into the ergosterol pathway. The native GGPP synthase (BTS1) was another limitation that was observed throughout this study. To overcome this bottleneck, plasmid-based and integrative overexpression of the native BTS1 and a codon optimised BTS1 were investigated. Other strategies to improve GGPP production were the deletion of the gene for the diacylglycerol pyrophosphate phosphatase (DPP1) to prevent excessive dephosphorylation of GGPP to geranylgeraniol (GGOH), and the overexpression of the farnesyl pyrophosphate synthetase, encoded by ERG20. However, the best improvements of the GGPP biosynthesis, inferred through GGOH measurements, were achieved from the screening of several heterologous GGPP synthases in S. cerevisiae. The best performing strain was JBY61 (JBY2, hmg1Δ::pTDH3-HMG1tr[1573–3165], pERG9Δ::pERG1, ChrIV-49293-49345Δ::pTDH3-XdcrtE-tSSA1_LEU2), bearing the heterologous GGPP synthase crtE of Xanthophyllomyces dendrorhous and produced 64.23 mg/L GGOH. Consequently, this engineering strategy improved the GGOH production by a factor of 642 compared to the parent strain JBY2.
In the last twenty years, a variety of unexpected resonances had been observed within the charmonium mass region. Although the existence of unconventional states has been predicted by the quantum chromodynamics (QCD), a quantum field theory describing the strong force, a clear evidence was missing. The Y(4260) is such an unexpected and supernummerary state, first observed at BaBar in 2005, and aroused great interest, because it couples much stronger to hidden charm decays (charm-anticharm states like J/Psi or h_c) instead of open charm decays (D meson pairs). This is unusual for states with masses above the D anti-D threshold. Furthermore, it decays into a charged exotic state Y(4260)->Z_c(3900)^+- pi^-+. The charge of the Z_c(3900)^+- is an indication that it comprises of two more quarks than the charm-anticharm pair, and could therefore be assumed to be a four-quark state. Due to these still not understood properties of these QCD-allowed states, they are referred to as exotic XYZ states to emphasize their particularity.
In 2017, the collaboration of the Beijing Spectrometer III (BESIII) investigated the production reaction of the Y(4260) resonance based on a high-luminosity data set. This significantly improved precision of the measurement of the cross-section sigma(e+e- -> J/Psi pi^+ pi^-) permitted a resolution into two resonances, the Y(4230) and the Y(4360). The Z_c(3900)^+- had been discovered by the BESIII collaboration in 2013, thus this experiment at the Beijing Electron-Positron Collider II (BEPCII) is a top-performing facililty to study exotic charmonium-like states.
In this work, an inclusive reconstruction of the strange hyperon Lambda in the charmonium mass region is performed to study possible decays of Y states in order to provide further insight into their nature. Finding more states or new decay channels may provide crucial hints to understand the strong interaction beyond nonperturbative approaches.
Three resonances are observed in the energy dependent cross-section: the first with a mass of (4222.01 +- 5.68) MeV and a width of (154.26 +- 28.16) MeV, the second with a mass of (4358.88 +- 4.97) MeV and a width of (49.58 +- 13.54) MeV and the third with a mass of (4416.41 +- 2.37) MeV and a width of (23.88 +- 7.18) MeV. These resonances, with a statistical significance Z > 5sigma, can be interpreted as the states Y(4230), Y(4360) and psi(4415).
Additionally, a proton momentum-dependent analysis strategy has been used in terms of the inclusiveness of the reconstruction and to address the momentum discrepancies between generic MC and measured data.
Artificial intelligence in heavy-ion collisions : bridging the gap between theory and experiments
(2023)
Artificial Intelligence (AI) methods are employed to study heavy-ion collisions at intermediate collision energies, where high baryon density and moderate temperature QCD matter is produced. The experimental measurements of various conventional observables such as collective flow, particle number fluctuations, etc. are usually compared with expensive model calculations to infer the physics governing the evolution of the matter produced in the collisions. Various experimental effects and processing algorithms can greatly affect the sensitivity of these observables. AI methods are used to bridge this gap between theory and experiments of heavy-ion collisions. The problems with conventional methods of analyzing experimental data are illustrated in a comparative study of the Glauber MC model and the UrQMD transport model. It is found that the centrality determination and the estimated fluctuations of the number of participant nucleons suffer from strong model dependencies for Au-Au collisions at 1.23 AGeV. This can bias the results of the experimental analysis if the number of participant nucleons used is not consistent throughout the analysis and in the final model-to-data comparison. The measurable consequences of this model dependence of the number of participant nucleons are also discussed. In this context, PointNet-based AI models are developed to accurately reconstruct the impact parameter or the number of participant nucleons in a collision event from the hits and/or reconstructed track of particles in 10 AGeV Au-Au collisions at the CBM experiment. In the last part of the thesis, different AI methods to study the equation of state (EoS) at high baryon densities are discussed. First, a Bayesian inference is performed to constrain the density dependence of the EoS from the available experimental measurements of elliptical flow and mean transverse kinetic energy of mid rapidity protons in intermediate energy collisions. The UrQMD model was augmented to include arbitrary potentials (or equivalently the EoSs) in the QMD part to provide a consistent treatment of the EoS throughout the evolution of the system. The experimental data constrain the posterior constructed for the EoS for densities up to four times saturation density. However, beyond three times saturation density, the shape of the posterior depends on the choice of observables used. There is a tension in the measurements at a collision energy of about 4 GeV. This could indicate large uncertainties in the measurements, or alternatively the inability of the underlying model to describe the observables with a given input EoS. Tighter constraints and fully conclusive statements on the EoS require accurate, high statistics data in the whole beam energy range of 2-10 GeV, which will hopefully be provided by the beam energy scan programme of STAR-FXT at RHIC, the upcoming CBM experiment at FAIR, and future experiments at HIAF and NICA. Finally, it is shown that the PointNet-based models can also be used to identify the equation of state in the CBM experiment. Despite the uncertainties due to limited detector acceptance and biases in the reconstruction algorithms, the PointNet-based models are able to learn the features that can accurately identify the underlying physics of the collision. The PointNet-based models are an ideal AI tool to study heavy-ion collisions, not only to identify the geometric event features, such as the impact parameter or the number of participant nucleons, but also to extract abstract physical features, such as the EoS, directly from the detector outputs.
Seed dispersal is a key ecosystem function for plant regeneration, as it involves the movement of seeds away from the parental plants to particular habitats where they can germinate and transition to seedlings and ultimately adult plants. Seed dispersal is shaped by a diversity of abiotic and biotic factors, particularly by associations between plants and climate and between plants and other species. Due to the ongoing loss of biodiversity and changing global conditions, such interactions are prone to change and pose a severe threat to plant regeneration. One way to address this challenge is to study associations between plant traits and abiotic and biotic factors to understand the potential impacts of global change on plant regeneration. Plant communities have long been analyzed through the lens of vegetative traits, mainly ignoring how other traits interact and respond to the environment. For instance, while associations between vegetative traits (e.g., specific leaf area, leaf nitrogen content) and climate are well studied, there are few case studies of reproductive traits in relation to trait-environment associations in the context of global change.
Thus, the overarching aim of this dissertation is to explore how trait-environment associations, with a special focus on reproductive traits, can improve our understanding of the effect that global change may have on seed dispersal, and ultimately on plant regeneration. To this end, my research focuses on studying associations between plant traits and abiotic and biotic factors along an elevational gradient in both forests and deforested areas of tropical mountains. This dissertation addresses three principal research objectives.
First, I investigate the extent to which reproductive (seed and fruit traits) and vegetative traits (leaf traits) are related to abiotic and biotic factors for communities of fleshy-fruited plants in the Ecuadorian Andes. I used multivariate analyses to test associations between four (a)biotic factors and seven reproductive traits and five vegetative traits measured on 18 and 33 fleshy fruited plant species respectively. My analyses demonstrate that climate and soil conditions are strongly associated with the distribution of both reproductive and vegetative traits in tropical tree communities. The production of “costly” vs. “cheap” seeds, fruits and leaves, i.e., the production of few rewarding fruits and acquisitive leaves versus the production of many less-rewarding fruits and conservative leaves, is primarily limited by temperature, whereas the size of plant organs is more related to variation in precipitation and soil conditions. My findings suggest that associations between reproductive and vegetative traits and the abiotic environment follow similar principles in tropical tree communities.
Second, I assess how climate and microhabitat conditions affect the prevalence of endozoochorous plant species in the seed rain of tropical montane forests in southern Ecuador. I analyzed seed rain data for an entire year from 162 traps located across an elevational gradient spanning of 2000 m. I documented the microhabitat conditions (leaf area index and soil moisture next to each seed trap) at small spatial scale as well as the climatic conditions (mean annual temperature and rainfall in each plot) at large spatial scale. After a one-year of sampling, I counted 331,838 seeds of 323 species/morphospecies. My analyses demonstrate that the prevalence of endozoochorous plant species in the seed rain increases with temperature across elevations and with leaf area index within elevations. These results show that the prevalence of endozoochory is shaped by the interplay of both abiotic and biotic factors at large and small spatial scales.
Third, I examine the potential of seed rain to restore deforested tropical areas along an elevational gradient in southern Ecuador. For this chapter, I collected seed rain using 324 seed traps installed in 18 1-ha plots in forests (nine forest plots) and in pastures (nine deforested plots) along an elevational gradient of 2000 m. After a sampling period of three months, I collected a total of 123,039 seeds of 255 species/morphospecies from both forests and pastures along the elevational gradient. I did not find a consistent decrease in the amount and richness of seed rain between forests and pastures, but I detected a systematic change in the type of dispersed seeds, as heavier seeds and a higher proportion of endozoochorous species were found in forests compared to pastures at all elevations. This finding suggests that deforestation acts as a strong filter selecting seed traits that are vital for plant regeneration.
Understanding the role that trait-environment associations play in how plant communities regenerate today could serve as a basis for predicting changes in regeneration processes of plant communities under changing global conditions in the near future. Here, I show how informative the measurement of reproductive traits and trait environment associations are in facilitating the conservation of forest habitats and the restoration of deforested areas in the context of global change.
In view of a growing world population and the finite nature of fossil resources, the development of eco-friendly production processes is essential for the transition towards a sustainable industry. Methanol, which can be produced both petrochemically and from renewable resources, offers itself as bridging technology and attractive alternative raw material for biotechnological processes. This work describes developments for the progress of the well-studied methylotrophic α proteobacterium Methylorubrum extorquens AM1 towards an efficient methylotrophic cell factory. Although many homologous and heterologous production routes have already been described and realized for M. extorquens in a laboratory scale, no industrial process has yet been realized. Three major reasons can be identified for this: (1) A limited choice of tools for genetic modifications, (2) a lack of understanding of carbon fluxes and side reactions occurring in modified strains, such as product reimports, and (3) the lack of tailored production strains for profitable target products and optimized bioprocessing protocols. The aim of the present work was to achieve developments for the mentioned areas. As a model application, the high-level production of chiral dicarboxylic acids from the substrate methanol was chosen. Enantiomerically pure chiral compounds are of great interest, e.g., as building blocks for chiral drugs. The ethylmalonyl CoA metabolic pathway (EMCP) which is part of the primary metabolism of M. extorquens, harbors unique chiral CoA-ester intermediates. Their acid derivatives can be released by cleavage of the CoA-moiety using heterologous enzymes. The dicarboxylic acids 2 methylsuccinic acid and mesaconic acid were produced in a previous study by introducing the heterologous thioesterase YciA into M. extorquens. In the said study, a combined product titer of 0.65 g/L was obtained in shake flask experiments. These results serve as the basis for the developments in the present work.
First, the previously described reuptake of products was thoroughly investigated and dctA2, a gene encoding for an acid transporter, was identified as target for reducing the product reuptake. In addition, reuptake of mesaconic acid was prevented by converting it to (S)-citramalic acid, a product not metabolizable by M. extorquens, by the introduction of a heterologous mesaconase. Together with 2-methylsuccinic acid, for which a high enantiomeric excess of (S)-2-methylsuccinic acid was determined, a second chiral molecule was thus added to the product spectrum. For the release of dicarboxylic acid products, YciA, a broad-range thioesterase that accepts a variety of CoA-esters with different chain lengths as substrates, was chosen. The enzyme should theoretically be able to hydrolyze all CoA-esters of interest present in the EMCP. However, in culture supernatants of M. extorquens strains that were overexpressing the corresponding yciA gene, only mesaconic acid and 2 methylsuccinic acid could be detected. To expand the substrate spectrum of YciA thioesterase with respect to other EMCP intermediates, semi-rational enzyme engineering was attempted. Screening of the corresponding strains carrying the respective YciA variants did not result in strains capable of producing new dicarboxylic acid products. However, the experiments revealed an amino acid position that strongly affected the production of mesaconic acid and 2-methylsuccinic acid in vivo. By substituting the according amino acid in YciA, the maximum titers of mesaconic acid and 2-methylsuccinic acid could be increased substantially. Application of an improved thioesterase variant in a second E. coli-based process confirmed the enhanced activity of the enzyme. The desired extension of the product spectrum by another chiral molecule (2-hydroxy-3-methylsuccinic acid, presumably the (2S,3R)-form) was finally achieved by using an alternative thioesterase. Tailored fermentation strategies were developed for the high-level production of the above-mentioned products.
As second part of the work, two novel genetic tools for M. extorquens were developed and characterized. The pBBR1-derived plasmid pMis1_1B was shown to be stably maintained in M. extorquens cells. In addition, its suitability for co-transformations with other plasmids was demonstrated. The second tool, the cumate-inducible promoter Ps6, is tailored for expression of pathways with toxic products, as the transcription of genes controlled by Ps6 is strongly repressed in the absence of an inducer.
Overall, the present work demonstrates the enormous potential of using M. extorquens as a methylotrophic cell factory. In the applications shown, the biotechnological production of high-priced chiral molecules is combined with the use of an attractive alternative substrate. In addition, new achievements and approaches are presented to facilitate the development of future M. extorquens production strains.
In the past decade, the optogenetic toolbox for the manipulation of ion currents and cNMP levels in Caenorhabditis elegans (C. elegans) expanded. However, the implemented tools for cAMP generation were soluble enzymes (euPAC, bPAC, IlaC22 k27 and PaaC) and thus they do not precisely mimic physiological cAMP signalling occurring in microdomains in close proximity to the plasma membrane. Here, cAMP is predominantly generated by membrane-bound adenylyl cyclases, that are located in microdomains together with G protein-coupled receptors (GPCRs), protein kinase A (PKA) and their targets, enabling spatially and temporal regulation of cAMP signalling. For this reason, one aim of this study was to develop and implement membrane bound photoactivatable adenylyl cyclases for the manipulation of cAMP mediated signalling in close proximity to the plasma membrane. For this purpose, the guanylyl cyclase domains of the Blastocladiella and Catenaria Cyclase Opsins (CyclOps) were mutated to adenylyl cyclases either by introducing the mutations E497K and C566D (abbreviated as (A-2x)) or by the mutations E497K, H564D, and C566T (abbreviated as (A-3x)).
To determine the nucleotide specificity switch from GTP to ATP and the extent of light-dependent cAMP generation, the engineered enzymes were expressed in body wall muscle cells of C. elegans and in vitro cNMP measurements using C. elegans extracts were performed. Here, the highest levels of light induced cAMP generation during sustained stimulation (0.5 mW/mm2; 470 nm, 15 min) were detected for the variants BeCyclOp(A-2x), YFP-BeCyclOp(A-2x), and YFP-CaCyclOp(A-2x) (39, 57, 40 nM, respectively), though they did not reach the extent produced by the soluble bPAC (142 nM). In contrast, low magnitudes of generated cAMP were measured for the versions BeCyclOp(A-3x) and CaCyclOp(A-2x) (8 and 7 nM, respectively). Importantly, no obvious residual cGMP and basal activity was ascertained for any of the engineered enzymes.
To assess their potential to trigger and modulate cAMP mediated cholinergic neurotransmission, and to evaluate the influence of cytosolic and membrane proximal optogenetic cAMP generation, the enzymes were expressed in cholinergic motor neurons and compared to the implemented soluble bPAC via locomotion behaviour analysis on solid and in liquid media. Photoactivation of BeCyclOp(A-2x), YFP-BeCyclOp(A-2x), and YFP-CaCyclOp(A-2x) caused similarly enhanced or even more potent behavioural changes (swimming and crawling) as bPAC, whereas a more rapidly decaying response was observed for the bPAC evoked effects. Moreover, an increased diversity of the behavioural output was detected for cytosolic cAMP production by bPAC, i.e. increased bending angles and a decreased body length.
Confocal fluorescence microscopy was performed to examine the expression levels of YFP-tagged enzymes in cholinergic neurons, whereas both YFP-CyclOp(A-2x)s were expressed at similar levels, but 1.4-fold lower relative to the soluble bPAC-YFP. To compare the amount of light-dependent cAMP generation bPAC and BeCyclOp(A-2x) at light conditions that match the conditions of the behavioural experiments (30 s), cAMP measurements using C. elegans extracts were performed, whereas BeCyclOp(A-2x) depicted a 4-fold lower amount of optogenetic cAMP production than the soluble bPAC.
In sum, local (membrane proximal) cAMP generation by the membrane-bound photoactivatable adenylyl cyclases may more specifically activate cAMP dependent neurotransmission of cholinergic motor neurons than cytosolic cAMP generation, i.e. an increased mobilization and priming/docking of synaptic vesicles and an increased filling of the synaptic vesicles with the neurotransmitter acetylcholine and thus an increase in locomotion behaviour.
The optogenetic toolbox for the manipulation of cGMP mediated signalling in C. elegans consisted of the natural membrane-bound BeCyclOp and the artificial soluble bPGC. The latter generates cGMP with low efficiency and slow kinetics (~0.2 cGMP s-1), whereas BeCyclOp enables the production of much larger amounts of cGMP (L/D = 5000) at a high turnover rate (~17 cGMP s-1). Thus, one aim of this thesis was to implement a tool with features in between those of BeCyclOp and bPGC. Several orthologous CyclOps were assessed by Gao et al., 2015 for light-regulated cGMP production by in vitro assays based on the measurement of the cNMP content from CyclOp containing oocyte membranes. Here, CaCyclOp showed the highest ratio of light versus dark activity (L/D = 230) after BeCyclOp, and thus was selected for characterization in C. elegans...
The role of lncRNAs in the CVS and the endothelium is highly diverse and has been subject to a substantial amount of research over the last decade. The identification of lncRNAs as clinically relevant biomarkers and as co-regulatory molecules let to the appreciation of the functional relevance of lncRNAs.
In the present study, LINC00607 was identified as an endothelial-enriched, human-specific lncRNA. With its distinct functions, LINC00607 maintains and supports the endothelial homeostasis especially in response to VEGF-A signalling.
In the first part of this study, LINC00607 was functionally characterized in human endothelial cells. LINC00607 is highly and specifically expressed in endothelial cells and is differentially regulated in CVDs. Depletion of LINC00607 resulted in decreased angiogenic sprouting, reduced integration of ECs in a newly formed vascular network in vivo, enhanced endothelial migration and differential expression of many important genes for endothelial cell homeostasis. Functionally, LINC00607 maintains ERG-driven endothelial gene expression programs through BRG1. BRG1 secures stably accessible enhancer regions as well as TSS of ERG target genes, thus enabling transcription of endothelial gene programs.
The second part of this study proposes an additional mode of action for LINC00607. The strongly impaired response to VEGF-A after LINC00607 KO can only be partially explained by its’ expression control of ERG target genes. It rather appears that LINC00607 is involved in the control of alternative splicing of VEGF receptor FLT1. The differential splicing of FLT1 produces the anti-angiogenic soluble isoform of FLT1. Even though further validation is needed to uncover the underlying mechanism, there is the potential of a more general role of LINC00607 in splicing control through BRG1. As AS of FLT1 is a clinical marker in preeclampsia, LINC00607 might qualify to be an additional marker for the onset and manifestation of the pregnancy disorder.
Taken together, LINC00607 is a target in future for molecular therapy in CVD to restore a healthy endothelial phenotype and has the potential to serve as a biomarker in preeclampsia.
This study examines the political contestation among Malay Professional Satirists (MPS) through their selected political satire works between 2011-2018. Political satire challenges those in power and is often regarded as fake news and libel. Therefore, political satirists tend to be frequently subject to legal action and are accused of disrupting national harmony. However, there is another group within the social and cultural community, which I call Social and Cultural Professionals (SCP), who also use satire but are supported by the government. This group frequently received financial benefits from the state and are at lower risk of suffering legal consequences. These contrasting conditions raise several important questions: who are the Malaysian Professional Satirists? Who are the targets of MPS in their satirical work? Why do MPS satirise them? And why do the MPS still produce political satire despite the potential legal consequences? Therefore, this study attempts to identify the characters, themes, and issues the MPS highlight; it also considers the reasons and motivations that political satirists have for creating such allegedly controversial works. Malaysia’s Reformasi movement and the booming use of the internet in 1998 mobilised multiple alternative social movements, mainly through art-related activities. Art workers, NGOs, as well as musical and cultural groups, protested creatively against the UMNO-BN ruling regime. Creative protests that employed satire and humour somehow succeeded in attracting a significant proportion of the public to follow political and current issues, especially youths in universities who had been depoliticised with the inception of the University and University College Act (AUKU 1979). This study establishes a point of view that political satire is a fun, loose, free form of resistance, contrasting with formal procedural democracy. The previous literature proposes that the study of Malaysia’s political system should focus on formal political procedures, especially election and representation. However, the study of political satire vis-à-vis democratisation is often neglected and thus such studies are scarce, which might have resulted from how satire is strictly discussed in terms of language and media. There has been a growing interest in how satirist and satirical works are regarded; hence, this study attempts to fill a gap in research on political satire in Malaysia. In contrast, democratisation is often discussed in terms of history, politics, anthropology, sociology, and economics. This qualitative study presents a comprehensive account of interviews with four (4) art workers identified as MPS, as based on appropriate criteria. Each informant had either partaken in alternative social movements or faced legal action from authorities or, indeed, both. In this study, the Theory of Contestation and Two-Social Reality serves as a primary framework to lead to an understanding of the contestation of power in Malaysia through political satire. This study further intends to broaden the knowledge of political satire and humour in the study of democratisation, adding to the existing literature, particularly outside formal political procedures.
"Autonomy is the condition under which what one does reflects who one is" (Weinrib, 2019, p.8). This quote encapsulates the core idea of autonomy, namely the correspondence of one’s inner values with one’s actions. This is a beautiful idea. After all, who wants their actions to be determined or controlled from the outside?
The classical definition of autonomy is precisely about this independence from external circumstances, which Murray (1938) primarily coined. Among other things, Murray characterizes autonomy as resistance to influence and defiance of authority. Similarly, Piaget (1983) describes individuals as autonomous, independent of external influences, in their thinking and actions, and foremost, adult authority. Subsequent work criticized this equation of autonomy with separation or independence (Bekker, 1993; Chirkov et al., 2003; Hmel & Pincus, 2002). In lieu thereof, autonomy is defined as an ability (Chirkov, 2011; Rössler, 2017) and as an essential human need (Ryan & Deci, 2006). Focus is now
on self-governing while relying on rationally determined values to pursue a happy life (Chirkov, 2011). According to Social Determination Theory (SDT), autonomy is about a sense of initiative and responsibility for one’s own actions. The experience of interest and appreciation can strengthen autonomy, whereas experiences of external control, e.g., through rewards or punishments, limit autonomy (Ryan & Deci, 2020). In the psychological discourse of autonomy, SDT is strongly represented (Chirkov et al., 2003; Koestner & Losier, 1996; Weinstein et al., 2012). Notably, SDT distinguishes between autonomy and independence as follows. While a person can autonomously ask for help or rely on others, a person can also be involuntarily alone and independent. Interestingly, these definitions are again closer to its etymological meaning as self-governing, originating from Greek αυτòνoμζ (autonomous).
The two strands of autonomy as independence and autonomy as self-determination are also reflected in the vital differentiation into reactive and reflective autonomy by Koestner and Losier (1996). Resisting external influence, particularly interpersonal in fluence, is what reactive autonomy entails. This interpretation is closely related to the classical concept of autonomy as separation and independence from others (Murray, 1938). On the other hand, reflective autonomy concerns intrapersonal processes, such as self-governing or self-regulation, as defined in Self-Determination Theory (Ryan et al., 2021). In this dissertation, we investigated the concept in three different approaches while focusing on its assessment and operationalization: To begin, in Article 1, we compared the layperson’s and the scientific perspective to each other to gain insight into the characteristics of autonomy. Then, in Articles 2 and 3, we experimentally tested behavioral autonomy as resistance to external influences. Simultaneously, we investigated the link between various autonomy trait measures and autonomous behavior. As a result, in Article 2, we looked at how people reacted to the effects of message framing and sender authority on social distancing behavior during the early COVID-19 pandemic. Finally, in Article 3 we investigated the resistance to a descriptive norm in answering factual questions, in the context of autonomous personality. In our first article, we used a semi-qualitative bottom-up approach to gain insights into the laypersons’ perspective on autonomy and compare it to the scientific notion. We followed a design proposed by Kraft-Todd and Rand (2019) on the term heroism. We derived five components from philosophical and psychological literature: dignity, independence from others, morality, self-awareness, and unconventionality. In three preregistered online studies, we compared these scientific components to the laypersons’ understanding of autonomy. In Study 1, participants (N = 222) listed at least three and up to ten examples of autonomous (self-determined) behaviors. Here, the participants named 807 meaningful examples, which we systematically categorized into 34 representative items for Study 2. Next, new participants (N = 114) rated these regarding their autonomy. Finally, we transferred the five highest-rated autonomy and the five lowest-rated autonomy items to Study 3 (N = 175). We asked participants to rate how strongly the items represented dignity, independence from others, morality, self-awareness, and unconventionality. We found all components to distinguish between high and low autonomy items but not for unconventionality. Thus, we conclude that laypersons’ view corresponds with the scientific characteristics of dignity, independence from others, self-awareness, and morality. A qualitative analysis of the examples also showed that both reactive and reflective definitions of autonomy are prevalent.
This work focuses on the investigation of K+, K- and ϕ-meson production in Ag(1.58 A GeV)+Ag collisions. The energetically cheapest channel for direct K+ production in binary NN-collisions NN→NΛK+ lies at exactly this energy. For the remaining K- and ϕ-mesons, an excess energy of 0.31 GeV and 0.34 GeV in the centre of mass system has to be provided by the system. This makes these particles an excellent probe for effects inside the medium.
K+ and K- mesons can be reconstructed directly as they possess a cτ of approximately 3.7 m. Using the approximately 3 billion recorded Ag(1.58 A GeV)+Ag 0-30% most central collision events, all reconstructed K+ and K- within the detector acceptance are investigated for their kinematic properties and their particle production rates compared to a selection of existing models.
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.
This cumulative dissertation examines learning in chemistry laboratories, focusing on the challenges and benefits of problem-based learning (PBL) for novices in the lab. It addresses the lack of consistent understanding about what should be learned in labs and why it's important. The research aims to understand what students learn, how they learn, and how lab learning can be improved.
A central concept in PBL labs is Information Literacy, defined as a sociocultural practice enabling learners to identify and use information sources within a specific context as legitimized by the practice community.
The first publication, Wellhöfer and Lühken (2022a), investigates the relationship between PBL and learner motivation. It identifies factors that can foster students' intrinsic motivation in a PBL lab. Autonomy is found to be a key factor, increasing student motivation and presenting a model of the autonomous scientific process. This model involves four steps: information acquisition, designing and applying experimental procedures, experimental feedback, and autonomous process optimization. The results suggest that intrinsic motivation in PBL labs can be enhanced by enabling students to independently execute these steps.
The second publication, Wellhöfer and Lühken (2022b), examines the information process students undergo during their first PBL lab. Using a sociocultural framework, it explores Information Literacy to understand students' handling of information and their perceptions of the information process. The findings reveal that in PBL labs, developing a practical, applicable experimental procedure is crucial for problem-solving and significantly shapes the information-acquisition process. This process is iterative, influenced by new information, leading to more precise information needs. Students assess information quality based on its usefulness for their problem, implementability (considering cognitive understanding, available equipment, and psychomotor skills), and safety.
Furthermore, the role of privileged knowledge forms in evaluating the quality of text sources is explored. Students viewed non-scientific sources as "poor" and scientific sources as "good," yet used both for information gathering. There were discrepancies between their assessment of source quality and actual use, indicating that perception of source quality doesn't always affect their practical decisions.
The third publication, Wellhöfer, Machleid, and Lühken (2023), investigates students' information practices in the lab, focusing on discourse between novice learners and experienced assistants. It shows that theoretical knowledge isn't sufficient for independent practical action, and students need actionable social information from experienced community members. The results highlight that information literacy in the lab for newcomers to a community of practice has distinctive features, and physical experience and tacit knowledge are crucial for learning the methods and group-specific knowledge of the practice community. The article demonstrates how learning information literacy in a practice community requires a social and physical experience and provides insights on how educators can support this process.
Mechanistic characterization of photoisomerization reactions in organic molecules and photoreceptors
(2023)
In dieser Arbeit wurden verschiedene Einflüsse auf die Dynamik von Photoisomerisierungen in Phytochromen und indigoiden Photoschaltern untersucht. Beide Forschungsgebiete teilen wesentliche Aspekte wie die Kontrolle durch sterische Wechselwirkungen und den starken Einfluss der Polarität oder der ionischen Umgebung.
Auf dem Gebiet der Phytochrome wurde die relative Positionierung der knotenlosen Phytochrome innerhalb der Superfamilie der Phytochrome in Bezug auf ihre Photodynamik und den Effekt von Grundzustandsheterogenität herausgearbeitet. Es wurde anhand von ultraschnellen, zeitaufgelösten Anrege-Abtast-Experimenten der einzelnen GAF-Domäne All2699g1 im Vergleich mit dem vollständigen knotenlosen Phytochrom All2699g1g2 und dem strukturell ähnlichen knotenlosen Phytochrom SynCph2 gezeigt, dass knotenlose Phytochrome in ihrer Vorwärtsdynamik eine komplexe mehrphasige Kinetik mit einem langlebigen angeregten Zustand (~100 ps) aufweisen. Die beobachtete mehrphasige Kinetik konnte einer initialen Chromophordynamik sowie einer nicht exponentiellen Reorganisation der chromophor-umgebenden Proteinmatrix zugeordnet werden. Dies steht im starken Kontrast zur im Gebiet der Phytochrome etablierten Beschreibung derartiger mehrphasiger Kinetiken mittels heterogener Grundzustände. Stattdessen wurde ein konserviertes kinetisches Muster identifiziert, welches die mehrphasige Dynamik beschreibt und in allen in dieser Arbeit untersuchten Phytochrome beobachtet wurde. Zudem konnte dieses Muster in einem Phytochrom der Gruppe I und einem Phytochrom der Gruppe III, die einen ähnlichen Pr Dunkelzustand aufweisen, gezeigt werden, was eine breite Anwendbarkeit des damit verbundenen Mechanismus vermuten lässt. Weiterhin konnte die zentrale Rolle eines konservierten Tyrosins in der Photoisomerisierung anhand von Mutationsstudien in All2699g1 herausgearbeitet werden. Diese konservierte Aminosäure muss im Rahmen der Reorganisation der Proteinmatrix vom Chromophor weggezogen werden, damit die sterische Blockade abgebaut werden kann, die die Isomerisierung des Chromophors zunächst verhindert. Da diese Bewegung von diversen Faktoren in der den Chromophor umgebenden Proteinmatrix abhängt, weist sie eine nicht exponentielle Kinetik auf, die je nach Phytochrom, der spezifischen Flexibilität und dem vorhandenen Raum in der Bindetasche unterschiedliche Lebenszeiten aufweist.
Die Rückreaktion knotenloser Phytochrome konnte ebenfalls im Rahmen dieser Arbeit charakterisiert werden, welche im Pikosekundenbereich abläuft, und damit signifikant schneller ist als die Vorwärtsreaktion. Im Gegensatz zur Vorwärtsreaktion nimmt Grundzustandsheterogenität in der Rückreaktion eine weitaus bedeutendere Rolle ein. Hier weisen die in All2699g1 vorhandenen heterogenen Grundzustandspopulationen jeweils eine eigene Kinetik ihres angeregten Zustands auf, während die homogenen Grundzustände von All2699g1g2 und SynCph2 jeweils nur einen Zerfall des angeregten Zustands zeigen. Der Ursprung dieser Heterogenität konnte im Wasserstoffbrückennetzwerk des Chromophors lokalisiert und mit dem konservierten Tyrosin und einem konservierten Serin in der PHY-Domäne verknüpft werden. Die Anwesenheit der PHY-Domäne sorgt demnach für eine Verringerung der Grundzustandsheterogenität und des vorhandenen Raums in der Bindetasche, wodurch die Effizienz der Photoreaktion optimiert wird.
Zuletzt konnte die Millisekundendynamik knotenloser Phytochrome und der Einfluss der PHY-Domäne auf diese aufgeklärt werden. Die PHY-Domäne sorgt hierbei durch den verringerten Raum in der Bindetasche dafür, dass die zunächst stattfindende thermische Relaxation des Chromophors signifikant verlangsamt wird, während spätere Änderungen im Photozyklus nur wenig beeinflusst werden.
Auf dem Gebiet der indigoiden Photoschalter konnte, anhand eines sterisch überladenen Hemithioindigo Photoschalters, der Photoisomerisierungsmechanismus des Hula-Twists beobachtet und eine starke Lösungsmittelabhängigkeit der entsprechenden Kinetik aufgezeigt werden. Aus den durchgeführten zeitaufgelösten Anrege-Abtast-Experimenten in verschiedenen Lösungsmitteln konnte ein Modell für die Photodynamik des verwendeten Hemithioindigo Photoschalters entwickelt werden. In unpolaren Lösungsmitteln muss eine hohe Barriere zur produktiven konischen Durchschneidung überwunden werden, was zu Lebenszeiten des angeregten Zustands im Nanosekundenbereich führt. Der Weg zur produktiven konischen Durchschneidung folgt dabei dem Hula-Twist Mechanismus. Dieser Pfad ist in polaren Lösungsmitteln unerreichbar, weshalb eine schnelle Relaxation über eine unproduktive konische Durchschneidung stattfindet.
Im zweiten Projekt auf dem Gebiet der indigoiden Photoschalter wurde anhand der neuartigen Klasse der Iminothioindoxyl Photoschalter ein Schwingungsenergiedonor für Schwingungsenergietransferstudien entwickelt. Das daraus entwickelte Modellsystem, bestehend aus einer künstlichen Aminosäure auf Basis des Iminothioindoxyl Photoschalters und einem daran gekoppelten Schwingungsenergiesensor, wurde charakterisiert und die primäre Photoreaktion untersucht. Es konnte gezeigt werden, dass der angeregte Zustand des Modellsystems kurzlebig ist und unter Abgabe von großen Mengen an Schwingungsenergie zerfällt, unabhängig von der Anregungswellenlänge und dem verwendeten Lösungsmittel. Somit zeigt das entwickelte System vorteilhafte Eigenschaften für Schwingungsenergietransferstudien.
Insgesamt konnten somit die Mechanismen der Photoisomerisierungsreaktionen in knotenlosen Phytochromen und indigoiden Photoschaltern aufgeklärt und daraus die Relevanz der Umgebung für derartige Reaktionen herausgearbeitet werden.
Our mind has the function of representing the physical and social world we are in, so that we can efficiently interact with it. This results in a constant and dynamic interaction between mind and world that produces a balance when representations are at the same time accurate with respect to what the world is communicating to our organism, but also compatible with how our mind works.
A paradigmatic case of this interaction is offered by perception, which is the mental function that represents contingent aspects of the world built from what is captured by our senses. Indeed, the dominant philosophical view in cognitive science is that our perceptual states are representations of the world and not direct access to that world. These representational perceptual states therefor include the aspects of the world they represent and that initiate the perception by stimulating our sensory organs.
Perceptual representations are built using information from the sensory system, i.e., bottom-up information, but are also integrated with information previously acquired, i.e., top-down information, so that perception interacts with memory through language and other mental functions. Such organization is believed to reflect a general mechanism of our mind/brain, which is to acquire and use information to make efficient predictions about the future, continuously updating older information with present information.
This predictive processing works because the world is not random, but shows a regular structure from which reliable expectations can be built. One way that our minds make these predictions is by adapting to the structure of the world in an implicit, automatic and unconscious way, a process that has been called Implicit Statistical Learning (ISL). ISL is a learning process that does not require awareness and happens in an incidental and spontaneous way, with mere exposure to statistical regularities of the world. It is what happens when we learn a language during early childhood, and that allows us to be implicitly sensitive to the phonological structure of speech, or to associate speech patterns with objects and events to learn word meaning.
A specific case of ISL is the learning of spatial configuration in the visual world, which we apply to abstract arrays of items, but most importantly, also to more ecological settings such as the visual scenes we are immersed in during our everyday life. The knowledge we acquire about the structure of visual scenes has been called “Scene Grammar”, because it informs about presence and position of objects in a similar way to what linguistic grammar tells us about the presence and position of words. So, we implicitly acquire the semantics of scenes, learning which objects are consistent with a certain scene, as well as the syntax of scenes, learning where objects are positioned in a consistent way within a certain scene.
More recent developments have proposed that scene grammar knowledge might be organized based on a hierarchical system: objects are arranged in the scene, which offers the more general context, but within a scene we can identify different spatial and functional clusters of objects, called “phrases”, that offer a second level of context; within every phrase, then, objects have different status, with usually one object (“anchor object”) offering strong prediction of where and which are the other objects within the phrase (“local objects”). However, these further aspects of the organization of objects In scenes remain poorly understood.
Another problem relates to the way we measure the structure of scenes to compare the organization of the visual world with the organization in the mind. Typically, to decide if an object appears or not in a certain scene, and whether or not it appears in a certain position within a scene, researchers based their decision on intuition and common-sense, maybe validating those decisions with independent raters. But it has been shown that often these decisions can be limited and more complex information about objects’ arrangement in scenes can be lost.
A potential solution to this problem might be using large set of real-world images, that have annotations and segmentations of objects, to measures statistics about how objects are arranged in the environment. This idea exploits the nowadays larger availability of this kind of datasets due to increasing developments of computer vision algorithms, and also parallels with the established usage of large text corpora in language research.
The goals of the current investigation were to extract object statistics from this image datasets and test if they reliably predict behavioural responses during object processing, as well as to use these statistics to investigate more complex aspects of scene grammar, such as its hierarchical organization, to see if this organization is reflected in the organization of objects in our mind.
IL-38 is the latest discovered cytokine of the IL-1 family and has been added to the IL-36 subfamily. Since its discovery in 2001, increasing evidence suggests predominantly anti-inflammatory properties of IL-38, which are most likely exerted through three potential receptors, the IL-1 Receptor 1 (IL-1R1), IL-36 Receptor (IL-36R) and the IL-1 Receptor Accessory Protein Like 1 (IL-1RAPL1). However, to this date detailed knowledge of IL-38 functioning remains to be examined. Importantly, how IL-38 is processed, secreted from cells and the exact mechanisms of target receptor binding and intracellular signaling are not fully understood. Further, IL-38 has been associated with regulatory functions in autoimmune diseases like systemic lupus erythematosus (SLE) and psoriasis. At the same time however, connections between B cells as indispensable part of immunity and IL-38 remain rare.
In this study we examined the influence of IL-38 in peripheral human blood B cells differentiating into antibody secreting cells using a three-step in vitro differentiation process. We first show that all potential IL-38 binding receptors are present on peripheral blood B cells on a gene expression level and remain detectable throughout B cell differentiation. Next, while B cells treated with exogenous IL-38 depict no differences in early B cell activation markers, the process of B cell differentiation revealed significant alterations in B cell phenotype created by IL-38 treatment. Predominantly on day 7 of the differentiation process, IL-38 treated B cells showed significantly reduced CD38 expression which depicts an important step in development towards plasma cells. We hypothesize that IL-38 acts antagonistically on the IL-1R1 pathway reducing Nuclear factor kappa B (NFκB) expression and consequently decreasing CD38 expression. Further IL-38 reduced early antibody production while increasing IgM secretion at the end stages of differentiation. Next, we repeated the differentiation assays under the influence of additional IL-21 stimulation to further enhance plasma cell development. In these experiments, the impact of IL-38 on B cell differentiation and immunoglobulin production were reduced, indicating a comparatively moderate relevance of IL-38 for B cell differentiation. We then examined how proliferation and cell death were impacted by exogenous IL-38 during B cell differentiation. IL-38 treatment alone significantly reduced B cell survival which was further augmented by IL-21 stimulation. We conclude that IL-38 and IL-21 act synergistically in promoting B cell apoptosis, also depicting an anti-inflammatory property of IL-38. Finally, using a siRNA we successfully performed an IL-38 knockdown experiment of human blood B cells reducing IL-38 expression to 44% measured on day 4 of B cell differentiation. In these experiments we observed reversed tendencies of CD38 expression compared to exogenous IL-38 treatment. Here, IL-38 knockdown cells showed increased CD38 expression indicating endogenous regulatory properties of IL-38 in B cell differentiation.
Our project, for the first time proves direct effects of IL-38 on human B cells. The results support previous research of IL-38 to act anti-inflammatory as it seems to modulate B cell differentiation, survival, and immunoglobulin production in a down-regulatory manner. These findings pave way for more detailed research on the connection between B cell homoeostasis and IL-38 function.
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.
Biological membranes serve as physical barriers in cells and organelles, enabling the maintenance of chemical or ionic gradients that are essential for triggering various integral, peripheral, or lipid-anchored membrane proteins, necessary for their life-essential functions. The study of membrane proteins has unique challenges due to their hydrophobic nature, limited expression levels, and inherent flexibility. Single-particle analysis (SPA) enables the determination of high-resolution three-dimensional structures using minimal amounts of specimen without the need for crystallization. Additionally, cryogenic electron tomography (cryo-ET) and subtomogram averaging (StA) offer the ability to study membrane protein complexes, cellular architecture, and molecular interactions while preserving close-to-life conditions. With ongoing improvements in cryo-EM technologies, obtaining high-resolution structures of membrane proteins in vitro can allow people to understand their mechanisms and functions, and to facilitate the design and optimization of new therapeutic agents. Furthermore, there has been significant growth in the structural characterization of membrane proteins in situ, as studying biomolecules within their physiological context is an ultimate goal in structural biology for a comprehensive understanding of molecular networks in cells.
Due to the amphipathic nature of membrane proteins, their production, purification, and isolation pose significant challenges compared to soluble proteins. To maintain the membrane protein fold in an aqueous buffer after disrupting lipid membranes, the use of detergents, amphipols, lipid nanodiscs, saposin-lipoprotein (salipro), styrene-maleic acid co-polymer lipid particles (SMALPS) is common and often essential. A limitation of the membrane-mimetic systems is the absence of an actual lipid bilayer environment. To address this issue, membrane proteins can be reconstituted into liposomes, and this closed membrane environment closely mimics the physiological conditions of the proteins. The use of liposomes for structure determination is expected to significantly expand in the in vitro study of membrane proteins and membrane-associated proteins, particularly for capturing transient complexes in specific functional states.
Resolving the structures of membrane proteins in their native cellular context is considered the ideal approach for understanding their functions and associated molecular networks. While single-particle cryo-EM can achieve higher resolution than subtomogram averaging, it often requires at least partial purification of the target molecules from their native environment inside cells and tissues. By combining averaging tools on subvolumes obtained through cryo-ET, structures can currently be determined at resolutions of 10-30 Å. With ongoing advancements and refinements in cryo-ET methodologies, routine high-resolution structure determination in situ is poised to become a valuable tool for both structural and cell biologists in the long run, and the field holds great promise for further expanding our understanding of cellular structures and processes at the molecular level.
The main aim of this thesis is to further our knowledge of the structure and function of a small prokaryotic voltage-gated sodium ion channel, NaChBac in liposomes, and a large knob complex found on the surface of Plasmodium falciparum-infected human erythrocyte by cryo-ET and StA.
Chapter 2 presents the first StA map of the 120-kDa NaChBac embedded in liposomes under a resting membrane potential at a modest resolution of 16 Å. The approach presented in this study, which can be widely applied to cryo-EM analysis of membrane proteins, with a specific focus on membrane proteins with small soluble domains, lays the foundation for cryo-ET and StA of integral or peripheral membrane proteins whose functions are affected by transmembrane electrochemical gradients and/or membrane curvatures. Chapter 3 shows the first cryo-EM structure of the supramolecular knob complex in P. falciparum-infected human erythrocyte. While a previous study provided an overall architectural view of knobs using negative stain tomography, the in situ structure bridges this gap, guiding future investigations into the molecular composition and the role of these native knobs in Plasmodium infection and immunity.
This thesis opens up several promising lines for future studies of membrane proteins in vitro and in situ, where other membrane proteins can be studied in physiologically relevant environments. Already with the present generation of cryo-EM hardware and software, this thesis represents pioneering research in the field of membrane protein structural biology.
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.
With the rise of digitalization and ubiquity of media use, both opportunities and challenges emerge for academic learning. One prevalent challenge is media multitasking, which can become distracting and hinder learning success. This thesis investigates two facets of this issue: the enhancement of data tracking, and the exploration of digital interventions that support self-control.
The first paper focuses on digital tracking of media use, as a comprehensive understanding of digital distractions requires careful data collection to avoid misinterpretations. The paper presents a tracking system where media use is linked to learning activities. An annotation dashboard enabled the enrichment of the log data with self-reports. The efficacy of this system was evaluated in a 14-day online course taken by 177 students, with results confirming the initial assumptions about media tracking.
The second paper tackles the recognition of whether a text was thoroughly read, an issue brought on by the tendency of students to skip lengthy and demanding texts. A method utilizing scroll data and time series classification algorithms is presented and tested, showing promising results for early recognition and intervention.
The third paper presents the results of a systematic literature review on the effectiveness of digital self-control tools in academic learning. The paper identifies gaps in existing research and outlines a roadmap for further research on self-control tools.
The fourth paper shares findings from a survey of 273 students, exploring the practical use and perceived helpfulness of DSCTs. The study highlights the challenge of balancing between too restrictive and too lenient DSCTs, particularly for platforms offering both learning content and entertainment. The results also show a special role of media use that is highly habitual.
The fifth paper of this work investigates facets of app-based habit building. In a study over 27 days, 106 school-aged children used the specially developed PROMPT-app. The children carried out one of three digital activities each day, each of which was supposed to promote a deeper or more superficial processing of plans. Significant differences regarding the processing of plans emerged between the three activities, and the results suggest that a child-friendly planning application needs to be personalized to be effective.
Overall, this work offers a comprehensive insight into the complexity and potentials of dealing with distracting media usage and shows ways for future research and interventions in this fascinating and ever more important field.
Global analysis of halogenated trace gases in the UTLS: from long-lived to short-lived substances
(2023)
In this dissertation, the distribution of chlorinated and brominated substances in the upper troposphere and lower stratosphere is investigated. These substances contribute significantly to the catalytic decomposition of ozone and are involved in the recurrent formation of the polar ozone hole in the Antarctic winter and spring. The Montreal Protocol, a multilateral environmental treaty to protect the ozone layer, has successfully reduced emissions of long-lived chlorine- and bromine-containing substances. Short-lived chlorinated and brominated substances, some of which are natural and anthropogenic in origin, are not regulated by the Montreal Protocol and it can be assumed that their relative contribution to the stratospheric halogen budget will increase, while the contribution of long-lived compounds will steadily decrease. The distribution of long- and short-lived halogenated substances are part of current research. For the upper troposphere and lower stratosphere, the very short-lived substances are particularly important. The lower stratosphere needs special investigation in this respect, since its composition is influenced by different transport processes. The influences on ozone trends in the lower stratosphere are subject to great uncertainties. Especially in the Southern Hemisphere, the number of observations is very limited.
In this work, the GhOST (Gas chromatograph for Observational Studies using Tracers) instrument was used during the SouthTRAC measurement campaign on the German HALO (High Altitude and LOng range) research aircraft, providing observations of halogenated hydrocarbons in Antarctic late winter to early spring 2019, a generally poorly sampled region. The polar vortex was, compared to previous years, significantly weaker and shifted towards the eastern South Pacific and South America. From the airborne measurements of chlorinated source gases, inorganic chlorine (the sum of active chlorine and reservoir gases; Cly) could be inferred with the result that Cly within the vortex increased up to 1687 ± 19 ppt at 385 K potential temperature, accounting for about 50 % of the total chlorine within the vortex and only 15 % of the total chlorine in the southern mid-latitudes. A comparison with the Northern Hemisphere could be made using the PGS measurement campaign in the Arctic winter 2015/2016. Under comparable conditions (season and distance from the tropopause), only 40 % of the total chlorine was in the inorganic form within the Arctic polar vortex and about 20 % was found in the mid-latitudes of the Northern Hemisphere. In addition, about 540 ppt more Cly was present in the Antarctic vortex than in the Arctic vortex, exceeding the annual variations previously reported for Antarctica.
The mean age of air plays an essential role in the derivation of Cly via the organic source gases, as was done in this work. A new method for determining the mean age of air from observational data has been introduced that accounts for extra-tropical input to the stratosphere in addition to tropical input. This new method was compared with the previously used method, which considered only the tropical input. The new method shows more realistic values especially near the tropopause. On average, the air of the lower stratosphere in the Northern Hemisphere was older than in the Southern Hemisphere by about 0.5 ± 0.3 years. About 65 K above the tropopause, the pattern changed with older air in the mid-latitudes of the Northern Hemisphere, but older air in high latitudes of the Southern Hemisphere, which implies differences in the strength and isolation of the respective polar vortex as well as the wave forcing in the shallow branch of the Brewer-Dobson circulation of the respective hemisphere. This is in good agreement with the distribution of Cly. The difference in the lower stratosphere was not clearly evident with the old method and it can be assumed that investigations of the differences in Cly of Northern and Southern Hemisphere will benefit from the new method.
Finally, the global and seasonal distribution of the two most important representatives of the short-lived brominated substances, CH2Br2 and CHBr3, was investigated. For this purpose, two additional HALO measurement campaigns have been used, the 2012 TACTS measurement campaign and the 2017 WISE measurement campaign, as well as the HIAPER Pole-to-Pole Observations (HIPPO) and Atmospheric Tomography (ATom) measurement campaigns. Observations of CH2Br2 show a pronounced seasonality in the free and upper troposphere of both hemispheres with slightly larger values in the Northern Hemisphere. CHBr3, on the other hand, shows a generally higher variability and lower seasonality with larger mixing ratios at mid and high latitudes in the northern hemispheric winter and autumn. A comparison of the lower stratosphere is limited to autumn and spring of both hemispheres due to the limited data basis of the observations. The distributions in each spring are similar (less than 0.1 ppt differences for e.g., CH2Br2). In hemispheric autumn, larger differences are evident with substantially smaller mixing ratios in the southern hemispheric lower stratosphere. This suggests that the transport processes of the two hemispheres may be different and implies that the input of tropospheric air (flushing) to the Northern Hemisphere lowest stratosphere is more efficient than in the Southern Hemisphere. Vertical profiles of CH2Br2 and CHBr3 in the mid-latitudes of both hemispheres and resulting vertical gradients support this conjecture. However, the Southern Hemisphere data set is insufficient to quantify this difference and further measurements are needed.
Cytochrome P450 enzymes are a large superfamily of membrane-bound heme-containing monooxygenases. They are essential for the oxidative metabolism of endogenous substrates such as steroids and fatty acids, and biotransformation of xenobiotic substrates such as pollutants and drugs. Although the highest expression of CYPs is found in the liver, their cardiovascular expression is not negligible with CYP450 subfamilies being responsible for the production of vasoactive lipids. Of importance, the enzymatic activity of all microsomal CYP450 isoenzymes is dependent on the cytochrome P450 reductase (POR), an electron donor.
In the first part of this work, the role of cytochrome P450 monooxygenases on the biotransformation of organic nitrates was investigated. Recombinant SupersomesTM were selected and incubated with NTG and PETN, where nitrite release was measured as a nitric oxide (NO) footprint. The capacity of the recombinant POR/CYP450 system to release nitrite from NO prodrugs was shown to be CYP-specific and dose-dependent. To study the involvement of CYP450 enzymes in the vascular biotransformation of organic nitrates in vivo, a smooth muscle-cell specific, inducible knockout model of POR (smcPOR-/-) was generated. Organ chamber experiments revealed that the vascular POR/CYP450 system had no impact on the dilator response of NTG and PETN. In line with previous publications, inhibition of ALDH2, known as the main enzyme responsible for the activation of NTG and PETN, and/or abolishment of the endogenous NO production did not reveal a contribution of the POR/CYP450 system to the dilator response of NTG and PETN. To better understand these results, we looked at the expression of the hepatic and vascular expression of the POR/CYP450 system where the hepatic was increased by 10- to 40-fold as shown by Western blot analysis. We concluded that due to insufficient vascular expression of CYP450 enzymes their contribution to the bioactivation of NTG and PETN is only minor.
The second part of this work focused on the cardiac relevance of endothelial isoenzymes. For that purpose, an endothelial cell-specific, tamoxifen-inducible knockout model of POR was generated and characterized in the present study. RNA-sequencing of the heart of healthy mice revealed that the CYP450 expression is cell-specific with cardiac endothelial cells (ECs) exhibiting an enrichment in the expression of the Cyp4 family (ω-oxidation of fatty acids) and of the Cyp2 family (production of EETs). Under non-stredded conditions (i.e. 30 days after inducing the knockout by tamoxifen feeding), endothelial deletion of POR was associated with cardiac remodelling as observed by an increase in the ratio of heart weight to body weight and an increase in the cardiomyocyte area. RNA-sequencing of cardiac ECs suggested that loss of POR might alter ribosomal biogenesis and protein synthesis, which could potentially affect the cardiac contractility in ecPOR-/- mice. Metabolomics from cardiac tissue of CTL and ecPOR-/- mice were not indicative for an important metabolic function of the endothelial POR/CYP450 system in the heart. The combination of transverse aortic constriction (TAC) with endothelial deletion of POR accelerates the development of heart failure in mice as detected by a reduction in cardiac output and stroke volume. These effects were mediated most likely by a reduction in vascular EETs production, which increases vascular stiffness, resulting in cardiac remodeling.
Proteins are biological macromolecules playing essential roles in all living organisms.
Proteins often bind with each other forming complexes to fulfill their function. Such protein complexes assemble along an ordered pathway. An assembled protein complex can often be divided into structural and functional modules. Knowing the order of assembly and the modules of a protein complex is important to understand biological processes and treat diseases related to misassembly.
Typical structures of the Protein Data Bank (PDB) contain two to three subunits and a few thousand atoms. Recent developments have led to large protein complexes being resolved. The increasing number and size of the protein complexes demand for computational assistance for the visualization and analysis. One such large protein complex is respiratory complex I accounting for 45 subunits in Homo sapiens.
Complex I is a well understood protein complex that served as case study to validate our methods.
Our aim was to analyze time-resolved Molecular Dynamics (MD) simulation data, identify modules of a protein complex and generate hypotheses for the assembly pathway of a protein complex. For that purpose, we abstracted the topology of protein complexes to Complex Graphs of the Protein Topology Graph Library (PTGL). The subunits are represented as vertices, and spatial contacts as edges. The edges are weighted with the number of contacts based on a distance threshold. This allowed us to apply graph-theoretic methods to visualize and analyze protein complexes.
We extended the implementations of two methods to achieve a computation of Complex Graphs in feasible runtimes. The first method skipped checks for contacts using the information which residues are sequential neighbors. We extended the method to protein complexes and structures containing ligands. The second method introduced spheres encompassing all atoms of a subunit and skipped the check for contacts if the corresponding spheres do not overlap. Both methods combined allowed skipping up to 93 % of the checks for contacts for sample complexes of 40 subunits compared to up to 10 % of the previous implementation. We showed that the runtime of the combined method scaled linearly with the number of atoms compared to a non-linear scaling of the previous implementation We implemented a third method fixing the assignment of an orientation to secondary structure elements. We placed a three-dimensional vector in each secondary structure element and computed the angle between secondary structure elements to assign an orientation. This method sped up the runtime especially for large structures, such as the capsid of human immunodeficiency virus, for which the runtime decreased from 43 to less than 9 hours.
The feasible runtimes allowed us to investigate two data sets of MD trajectories of respiratory complex I of Thermus thermophilus that we received. The data sets differ only by whether ubiquinone is bound to the complex. We implemented a pipeline, PTGLdynamics, to compute the contacts and Complex Graphs for all time steps of the trajectories. We investigated different methods to track changes of contacts during the simulation and created a heat map put onto the three-dimensional structure visualizing the changes. We also created line plots to visualize the changes of contacts over the course of the simulation. Both visualizations helped spotting outstandingly flexible or rigid regions of the structure or time points of the simulation in which major dynamics occur.
We introduced normalizations of the edge weights of Complex Graphs for identi-fying modules and predicting the assembly pathway. The idea is to normalize the number of contacts for the number of residues of a subunit. We defined five different normalizations.
To identify structural and functional modules, we applied the Leiden graph clustering algorithm to the Complex Graphs of respiratory complex I and the respiratory supercomplex. We examined the results for the different normalizations of the weights of the Complex Graphs. The absolute edge weight produced the best result identifying three of four modules that have been defined in the literature for respiratory complex I.
We applied agglomerative hierarchical clustering to the edges of a Complex Graph to create hypotheses of the assembly pathway. The rationale was that subunits with an extensive interface in the final structure assemble early. We tested our method against two existing methods on a data set of 21 proteins with reported assembly pathways. Our prediction outperformed the other methods and ran in feasible runtimes of a few minutes at most.
We also tested our method on respiratory complex I, the respiratory supercomplex and the respiratory megacomplex. We compared the results for the different normalizations with an assembly pathway of respiratory complex I described in the literature. We transformed the assembly pathways to dendrograms and compared the predictions to the reference using the Robinson-Foulds distance and clustering information distance. We analyzed the landscape of the clustering information distance by generating random dendrograms and showed that our result is far better than expected at random. We showed in a detailed analysis that the assembly prediction using one normalization was able to capture key features of the assembly pathway that has been proposed in the literature.
In conclusion, we presented different applications of graph theory to automatically analyze the topology of protein complexes. Our programs run in feasible runtimes even for large complexes. We showed that graph-theoretic modeling of the protein structure can be used to analyze MD simulation data, identify modules of protein complexes and predict assembly pathways.
Die Kernspinresonanz(NMR)-Spektroskopie ist ein leistungsstarkes analytisches Werkzeug. Allerdings ist ihre Empfindlichkeit aufgrund geringer Wechselwirkungs-energie zwischen den Kernspins und dem externen Magnetfeld begrenzt. Die dynamische Kernpolarisation (DNP) erhöht DNP die Empfindlichkeit der NMR, indem sie die Polarisation von ungepaarten Elektronenspins auf die benachbarten Kernspins überträgt. In den letzten Jahrzehnten hat die DNP bei hohen Magnetfeldern erneut an Aufmerksamkeit gewonnen, bedingt durch die Verfügbarkeit leistungsstarker Gyrotron-Mikrowellen(mw)-Quellen. Jedoch wurde die Anwendung von DNP für Flüssigkeiten im Vergleich zu Festkörperproben bei niedrigen Temperaturen (≈100 K) weit weniger erforscht. Zwei Gründe können dafür hauptsächlich benennt werden. Bei hohen Magnetfeldern (entsprechend hohen mw-Frequenzen) wird die mw-Strahlung sehr stark von Flüssigkeiten absorbiert, was zu einer starken Erwärmung führt. Darüber hinaus sind die Translations- und Rotationsdynamik der Radikale und Target-Molekülen nicht schnell genug, um Spectraldichten bei den hohen mw-Frequenzen zu erzeugen, die für eine Overhauser-Effekt (OE) DNP Verstärkung benötigt werden. In dieser Arbeit wird gezeigt, Flüssigzustands-DNP bei hohen Magnetfeldern, insbesondere bei 9,4 T, mit hocheffizienten DNP-Probenköpfen möglich ist.
Der von skalaren Hyperfein-Wechselwirkung (hfWW) angetriebene OE ist für Flüssigzustands-DNP-Forschungen von besonderem Interesse, da der von der Theorie vorhergesagte Mechanismus auch bei hohen Magnetfeldern noch effizient ist. In der vorliegenden Arbeit wurde eine Methode zur Vorabprüfung potenzieller DNP-Kandidaten durch Messungen ihrer paramagnetischen NMR-Verschiebungen vorgeschlagen und untersucht. Wir beobachtete signifikante 13C-skalare OE DNP-Verstärkungen bis zu 50 bei den ausgewählten kleinen Biomolekülen, einschließlich Imidazol, Indol, verschiedene Aminosäuren und Kohlenhydraten. Das Lösungssystem wurde auch von organischen Lösungsmitteln auf Wasser erweitert.
Im Kontext von dipolarer OE DNP haben wir den Beitrag der Rotation des Radikals neben der Translationsbewegung zwischen Radikal und Target-Molekül zur OE DNP-Effizienz systematisch untersucht, indem wir verschiedene Nitroxidderivate mit unterschiedlichen Ringgeometrien und Substituenten verwendet haben. Mithilfe eines Models, das eine 'out-sphere' Translationsbewegung und eine 'inner-sphere' Rotationsbewegung des Radikal-Lösungsmittel-Komplexes enthält, konnte unsere Beobachtungen quantitativ simuliert werden. Außerdem wurde ein anderes Model untersucht, das eine Translationsbewegung mit der Rotation von Radikalen, bei denen das ungepaarte Elektron nicht im Zentrum sitzt, kombiniert.
Eine weitere neue Entdeckung in der DNP bei hohen Magnetfeldern waren der beobachtete SE (Solid-Effekt) an Lipidmolekülen mit BDPA-Radikal oberhalb der Lipidphasen-übergangstemperatur. Die neue Anwendung von SE DNP bietet einen alternativen Mechanismus zur OE DNP in Flüssigkeiten bei hohen Magnetfeldern und könnte möglicherweise auf Makromoleküle mit relativ langsamer Rotationsbewegung angewendet werden.
Wir haben zusätzliche Untersuchungen an den Lipiddoppelschichten mit Nitroxid-radikale durchgeführt, basierend auf dem beobachteten 1H DNP-Verstärkungen in einer viskosen Lipidumgebung bei 9,4 T . Durch Messung des Feldprofils wurden DNP-Verstärkungen durch OE und SE in Abhängigkeit ihrer relativen Verschiebungen von der Elektronen-Larmor-Frequenz bestimmt. Die individuelle OE DNP-Effizienzen für Protonen des Wassers, der Lipid-Cholin-Kopfgruppen oder der Lipid-Acylketten wurde bestimmt. Dadurch wird ein quantitativer Vergleich mit MD-Simulationen ermöglicht. Obwohl die von der MD-Simulationen vorhergesagten DNP Kopplungsfaktoren noch deutliche Abweichungen von den experimentellen Beobachtungen aufweisen, wird die schnelle Dynamik nahe der Elektronen-Larmor-Frequenz, die für einen erfolgreichen OE DNP Transfer erforderlich ist, von den MD-Simulationen gut erfasst.
In der Arbeit wurden auch zwei unterschiedliche Dreifachresonanz-DNP-Experimente durchgeführt. Zum einen wurde 13C OE DNP unter 1H-Entkopplung in wässriger Natriumpyruvatlösung, und zum anderen 13C-NMR von Glycin, verstärkt durch SE DNP an 1H zusammen mit einem 1H-13C INEPT-Polarisationstransfer, im Rahmen dieser Doktorarbeit durchgeführt.
In this thesis, we use lattice QCD to study a part of the QCD phase diagram, specifically the QCD phase transition at mu=0, where the QCD matter changes from hadron gas to quark-gluon plasma (QGP) with increasing temperature.
This phase transition takes place as a crossover, but when theoretically changing the masses of the quarks, the order of the phase transition changes as well.
We focus on the region of heavy quark masses with Nf=2 flavours, where we investigate the critical quark mass at the second order phase transition in the form of a Z2 point between the first-order and the crossover region.
The first-order region is positioned at infinitely heavy quarks. As the quark masses decrease, the associated Z3 centre symmetry breaks explicitly, causing the first-order phase transition to weaken until it turns into the Z2 point and finally into a crossover.
We study this Z2 point using simulations at Nf=2 and lattices of the sizes Nt = {6, 8, 10, 12}, partially building on previous work, in which the simulations for Nt = {6, 8, 10} were started.
The simulations for Nt=12 are not finished yet though, but we were able to draw some preliminary conclusions. These simulations are run on GPUs and CPUs, using the codes Cl2QCD and open-QCD-FASTSUM, respectively. Afterwards, the data goes through a first analysis step in the form of the Python program PLASMA, preparing it for the two techniques we use to analyse the nature of the phase transition.
As a first, reliable analysis method, we perform a finite size scaling analysis of the data to find the location of the Z2 point. Since we are using lattice QCD, performing a continuum extrapolation is necessary to reach the continuum result.
In regard to this, the finite size scaling analysis method is hampered by the excessive amount of simulated data that is needed regarding statistics and the total number of simulations, which is why this thesis is only an intermediate step towards the continuum limit.
This also leads to the second analysis technique we explore in this thesis.
We start to design a Landau theory which describes the phase boundary for heavy masses at Nf=2 based on the simulated data.
We develop a Landau functional for every Nt we have simulation data for.
Albeit the results are not at the same precision as the ones from the finite size scaling analysis, we are able to reproduce the position of the Z2 point for every Nt.
Even though we are not able to take a continuum extrapolation right now, after more development takes place in future works, this approach might, in the long run, lead to a continuum result that won't need as many simulations as the finite size scaling analysis.
Im Rahmen dieser Arbeit wurde ein verbessertes Buncher-System für Hochfrequenzbeschleuniger mit niedrigem und mittlerem Ionenstrom entwickelt. Die entwickelte Methodik hat ermöglicht, ein effektives, vereinfachtes Buncher-System für die Injektion in HF-Beschleuniger wie RFQs, Zyklotrons, DTLs usw. zu entwerfen, welches kleine Ausgangsemittanzen und beträchtliche Strahltransmissionen erzielt. Um einen mono-energetischen und kontinuierlichen Strahl aus einer Ionenquelle für den Einschuss in eine Hochfrequenz-Beschleunigerstruktur anzupassen, wird eine Energiemodulation benötigt, die im weiteren Verlauf (Driftstrecke) zur Längsfokussierung des Strahls führt. Durch eine Sägezahnwellenform wird die ideale Energiemodulation aufgrund der linearen Abhängigkeit zwischen der Energie der Teilchen und ihren relativen Phasen erreicht. Dies ist jedoch technologisch nicht möglich, da Teilchenbeschleuniger Spannungsniveaus im Bereich kV bis 100 kV benötigen. Dagegen ist für eine solche Zielsetzung eine räumliche Trennung der sinusförmigen Anregung mit der Grundfrequenz und höheren Harmonischen möglich.
Daher wurde in dieser Arbeit ein verbesserter harmonischer Buncher, der sogenannte „Double Drift Harmonic Buncher - DDHB“ entwickelt, welcher zahlreiche Vorteile hat. Eine geringe longitudinale Emittanz sowie finanzielle Aspekte sprechen für diesen Lösungsansatz. Die Hauptelemente eines DDHB Systems sind zwei Kavitäten, die durch eine Driftlänge L1 getrennt sind, wobei der erste Resonator mit der Grundfrequenz bei -90° synchroner Phase und angelegter Spannung V1 und der zweite Resonator bei der zweiten harmonischen Frequenz mit +90 synchroner Phase und angelegter Spannung V2 betrieben werden. Schließlich ist eine zweite Drift L2 am Ende des Arrays für eine longitudinale Strahlfokussierung am Hauptbeschleunigereingang erforderlich. Somit erfüllt ein solcher Aufbau das angestrebte Ziel einer hohen Einfangseffizienz und einer kleinen longitudinalen Emittanz durch Anpassen der vier Designparameter V1, L1, V2 und L2.
Das Verständnis der Fokussierung, ausgehend von einem Gleichstromstrahl, einschließlich der Raumladungskräfte, ist einer der wesentlichen Bestandteile der Strahlphysik. Viele kommerzielle Codes bieten Simulationsmöglichkeiten in diesem Anwendungsbereich. Ihre Ansätze bleiben jedoch dem Anwender meist verborgen, oder es fehlen wichtige Details zur genauen Abbildung des vorliegenden Konzepts. Daher bestand eine Hauptaufgabe dieser Arbeit darin, einen speziellen Multi-Particle-Tracking-Beam-Dynamics-Code (BCDC) zu entwickeln, bei dem der Raumladungseffekt während des Bunch-Vorgangs, ausgehend von einem DC-Strahl berechnet wird. Der BCDC - Code enthält elementare Routinen wie Drift und Beschleunigungsspalt oder magnetische Linse für die transversale Strahlfokussierung und Raumladungsberechnungen unter Berücksichtigung der Auswirkungen der nächsten Nachbar-Bunche (NNB). Der Raumladungsalgorithmus in BCDC basiert auf einer direkten Coulomb- Gitter-Gitter-Wechselwirkung und Berechnungen des elektrischen Feldes durch Lokalisierung der Ladungsdichte auf einem kartesischen Gitter. Um Genauigkeit zu erreichen, werden die Feldberechnungen in Längsrichtung symmetrisch um das zentrale Bucket (βλ-Größe) erweitert, so dass das Simulationsfeld dreimal so groß ist. Die zentrale Teilchenverteilung wird dann nach jedem Schritt in die benachbarten Buckets kopiert. Anschließend werden die resultierenden Felder im Hauptgitterfeld neu berechnet, indem die elektrischen Felder im Hauptgitterfeld mit denen aus den benachbarten Regionen überlagert werden. Ohne diese Methode würde z. B. ein kontinuierlicher Strahl, welcher jedoch in der Simulation nur innerhalb einer Zelle der Länge βλ definiert ist, zu einer resultierenden Raumladungsfeldkomponente Ez an beiden Rändern der Zelle führen. Ein solches unphysikalisches Ergebnis konnte durch die Anwendung der NNB-Technik bereits weitgehend eliminiert werden. Zusätzlich zum NNB-Feature verfügt das BCDC über eine weitere Besonderheit nämlich die sogenannte Raumladungskompensation (SCC). Aufgrund der Ionisierung des Restgases kommt es entlang des Niederenergiestrahltransports zu einer teilweisen Raumladungskompensation, und zwar am und hinter dem Bunchersystem mit unterschiedlichen Prozentsätzen. Eines der Hauptziele des DDHB-Konzepts besteht darin, es für Hochstromstrahlanwendungen zu entwickeln. Dabei ermöglicht die teilweise Raumladungskompensation, dass das Design in der Praxis höhere Stromniveaus erreicht. Dadurch ist das BCDC-Programm ein leistungsstarkes Werkzeug für Simulationen in künftigen, stromstarken Projekten. Proof-of-Principle-Designs wurden in dieser Arbeit entwickelt.
This work addresses the investigation of the biosynthesis mechanisms of type II polyketide synthase (PKS) and fatty acid synthase (FAS) derived specialized metabolites (SMs) from Photorhabdus laumondii.
The elucidation of the biosynthetic pathway of the bacterial 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) was one of the major topics of this thesis. IPS exhibits several bioactive characteristics as it inhibits the phenoloxidase of insects, acts antibacterial, but also influences the soluble epoxide hydrolase which is involved in inflammatory reactions. It was recently approved as a treatment against psoriasis by the FDA and is the first Photorhabdus derived drug.
The stilbene generation in Photorhabdus requires the formation of the two acyl-carrier-protein (ACP) bound 5-phenyl-2,4-pentadienoyl- and isovaleryl-β-ketoacyl-moieties. The ketosynthase (KS)/cyclase StlD catalyzes a ring formation via a Michael-addition between the two intermediates which is then further processed by an aromatase. The formation of 5-phenyl-2,4-pentadienoyl-ACP was shown via in vitro assays with purified proteins by proving the influence of the KS FabH, ketoreductase FabG and dehydratase FabA or FabZ of the fatty acid metabolism. While E. coli was able to complement most of these enzymes in attempts to produce IPS in the heterologous host, the Photorhabdus derived FabH was not replaceable despite 73 % sequence identity with the E. coli based isoenzyme, acting as a gatekeeper enzyme for cinnamic acid (CA) moieties. Furthermore, the ability to incorporate meta-substituted halogenated CA-derivatives was shown in order to produce 3-chloro- and 3-bromo-IPS. While studying the stilbene biosynthesis, the ability of Photorhabdus and Xenorhabdus to produce hydrazines was also discovered.
The second investigated biosynthesis was the formation of benzylideneacetone (BZA). BZA is produced by Photorhabdus and Xenorhabdus strains acting as a suppressor for the immune cascade of insects and has also antibiotic activities towards Gram-negative bacteria. Due to its structural similarity towards CA and the intermediates during the stilbene formation, a shared mechanism for Photorhabdus and Xenorhabdus budapestensis was proposed due to their ability to produce CA. The production of BZA was also dependent on the stilbene related CoA-ligase, the ACP and FabH. It was verified in vitro and in vivo in E. coli yielding a 150-fold increase of the BZA production compared to the Photorhabdus and Xenorhabdus wildtype (WT) strains.
The second part of this work deals with the optimization of P. laumondii strains regarding the production titers of IPS. Therefore, several deletions of other SM related genes as well as promoter exchanges in front of stilbene related genes were carried out. These approaches were combined with the upregulation of the phenylalanine by heterologous plasmid expression, since it is the precursor of CA. Another approach applied in parallel was the optimization of the cultivation conditions with different media and supplementation with XAD-resins. It was proved that media rich on fatty acids or peptides led to higher optical densities of the cultures and thus to higher titers of stilbenes. Since IPS is inhibiting the phenoloxidase, an enzyme important for the insect immunity, it was hypothesized that cultivation in media containing insects might enhance the output of this SM. Starting from 23 mg/l of IPS in the P. laumondii WT strain, it was possible to increase the production levels to more than 860 mg/l by utilizing the mentioned approaches.
The last topic of this thesis focuses on the production of epoxidated IPS (EPS) and its derivatives. Under laboratory conditions, only a low titer of EPS was observed for the wildtype strain. However, the optimized IPS strains and IPS-production conditions could also be applied for EPS which led to higher productions and also to the detection of many new derivatives. Most of the EPS derivatives were amino acid or peptide derived acting as nucleophiles to open the epoxide ring and yielding β-amino-alcohols. However, purification and chemical synthesis attempts to obtain EPS failed due to its poor stability. Epoxides were utilized in in vitro assays with amino acids, peptides and proteins to get insights whether epoxidations might act as posttranslational modification in Photorhabdus. The reactions were performed with styrene oxide and stilbene oxide replacing EPS based on their structural similarity. The modifications were executed successfully although proteomics approaches with in vivo data are required to confirm these findings. During the purification attempts of EPS, further derivatives were detected. The structures of dimerized stilbenes, a cis-isomer of IPS and another derivative that might incorporate an amino-group in the resveratrol ring were proposed on the basis of the HPLC-MS data.
Anthropogenic interventions have altered all ecosystems around the world. One of those ecosystems are forests, the main resource for timber. They have been strongly transformed in their structure with large consequences on forest biodiversity. Especially the decrease in dead-wood volume due to the timber extraction and alternation of natural forest structures with even-aged stands of less diverse tree species composition has put especially saproxylic, i.e., dead-wood dependent species, under threat, which comprise about 20% of all forest species. Beetles, fungi and bacteria are three functional important groups for decomposition processes but we still lack much information about their sampling and the drivers of their diversity, thus it is difficult to comprehensively protect their diversity. Saproxylic fungi are a highly diverse species group and the main drivers of dead-wood decomposition; hence they play a major role in the global carbon cycle. Due to their cryptic lifestyle, many species are still unknown, but the recent advances in environmental DNA barcoding methods (metabarcoding) shed light on the formerly underestimated diversity. Yet, this method's accuracy and suitability in detecting specific species have not been assessed so far, limiting its current usefulness for species conservation. On the other hand, these methods are a convenient tool to study highly diverse areas with high numbers of unknown species, enabling the study of global diversity and its drivers, which are unknown for saproxylic fungi, but important to assess to predict the future impacts of global change. Since nature conservation concepts are usually not applied on a global scale, the drivers of diversity must also be assessed on smaller scales. Besides understanding the drivers of diversity, to identify focus scales to create comprehensive, evidence-based conservation concepts must utilize multi-taxonomic studies since saproxylic species are differently sensitive towards environmental variables and closely interact with each other. Filling these knowledge gaps is utterly needed to protect the high saproxylic diversity and ensure the functional continuity of decomposition processes, especially regarding the global change.
To address the usefulness of metabarcoding for fungal species conservation, I compared the traditional method of fruit body sampling with metabarcoding and their efficiency in detecting threatened fungal species in the first chapter of this thesis. Both methods have advantages and disadvantages. Their ability to detect threatened saproxylic fungal species and their dependencies on detecting specific fungal groups have not been compared, albeit they are important to inform species conservation like Red Lists properly. I found metabarcoding to generally detect more threatened fungal species than fruit body sampling with a higher frequency than fruit body sampling. Moreover, fruit body sampling detected a unique set of species, while fruit body sampling missed large parts of fungal diversity due to species-specific fruiting characteristics. Metabarcoding with high sampling intensity is thus a viable method to assess threatened saproxylic fungal diversity and inform nature conservation like Red Lists about distribution and abundances. Nevertheless, a complementary approach with fruit body sampling is indispensable for assessing all threatened fungal species.
In order to analyse the global diversity of saproxylic fungi and its drivers, I examined whether fungal species richness increases from the poles towards the equator and thus follows the latitudinal diversity gradient already found in many other species groups. I further investigated whether such an increase is caused by increasing ecological specialisation, i.e., niche partitioning, or local tree diversity, i.e., niche space. Gamma diversity per biome increased from the boreal, over the temperate to the tropics and thus confirmed the latitudinal diversity for saproxylic fungi. Contrastingly, alpha diversity at the log level did not significantly increase towards the tropics, suggesting a grain size dependency of the observed pattern and an equal niche space within dead-wood across latitudes. Ecological specialisation on the plot level was globally on a high level but did not increase significantly towards the equator. Additionally, I found local tree species richness to drive plot-based fungal diversity. Further analysis of gamma diversity against the total number of sampled tree species strengthened the assumption that tree species diversity and not increased ecological specialisation was the main driver of the latitudinal diversity gradient, as there was no significant difference between the gamma diversity of the temperate and tropical biome. Nonetheless, as the gamma diversity of the boreal biome was still significantly smaller, my results do not allow a complete neglection of the ecological specialisation hypothesis. The overall results indicate a strong dependency of saproxylic fungi diversity with host tree species diversity and that the global loss of tree species threatens saproxylic fungi with an unpredictable impact on carbon and nutrient cycling.
To support saproxylic conservation, I conducted two analyses. First, I compared the beta diversity of the three main decomposer groups (beetles, fungal fruit bodies, mycelial fungi (metabarcoding), and bacteria (metabarcoding)) across different scales to assess the impact of different environmental variables on their overall diversity. I used an experimental design to disentangle two different spatial scales, influenced by differences in macroclimate, forest microclimate and spatial distance, and two host scales, driven by differences between tree lineages and tree species. I set these beta diversities in relation to the gamma diversity of the three main decomposer groups to identify whether a unified conservation concept could be applied to one scale to optimally protect the diversity of all three species groups. Second, I identified whether diversity and community composition of fungi and bacteria differed among climate and land use gradients. Further I explored whether specialisation and niche packing could explain the expected pattern. To do so I used an experimental design disentangling climate and land use across a large gradient in Germany. The results differed among the species groups, denying a unified conservation concept focusing on one scale. Saproxylic beetle and fruit body beta diversity was equally high on each scale, as they are more sensitive towards environmental factors like macro- and microclimate. On the other hand, mycelial fungi and bacteria beta diversity was highest on the host scale, especially the host tree scale, indicating a high host specificity of the two groups. The second study also identified tree species as the main driver of diversity and community composition of these two study groups. Specialisation of fungi was not influenced by land use or climate. Bacterial specialisation and diversity were under a strong influence of mean precipitation. Comprehensive conservation of multi-taxonomic diversity across regions thus requires the integration of several scales. Within different macroclimatic regions, forests of varying microclimates, i.e., forest management, must be implemented. In these forests, dead-wood of different tree lineages, i.e., angio- and gymnosperms and tree species, must be provided.
Taken together, I could demonstrate that metabarcoding is an efficient method to sample threatened fungal species and identify differing drivers of fungal diversity present as fruit bodies or mycelium. Its usefulness will further increase due to the ongoing improvement of sequencing databases and thus better inform conservation concepts. Using metabarcoding, I could demonstrate that high host specialisation of saproxylic fungi is not a European but a global phenomenon and identify tree species loss under global change as one major concern for saproxylic diversity. My dissertation further highlighted the importance of multi-taxonomic studies for evidence-based nature conservation, as different species groups require varying concepts. These results were especially important for saproxylic bacteria as the drivers of their diversity are still largely unknown. Howbeit, large research gaps still exist regarding the impacts of global change on species and processes. Moreover, the spatial coverage of studies is needed to confirm or neglect the generality of current research especially concerning the highly diverse tropical areas. An increased focus on the drivers of diversity in these areas is crucial to ensure a globally comprehensive saproxylic conservation and the various ecosystem functions they control.
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all "cancer genes" were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: proximity of genes able to produce prematurely terminated transcripts, which leads to the production of transspliced fusion RNAs, and finally, the induction of DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced.
Lysosomes are major degradative organelles that contain enzymes capable of breaking down proteins, nucleic acids, carbohydrates, and lipids. In the last decade, new discoveries have traced also important roles for lysosomes as signalling hubs, affecting metabolism, autophagy and pathogenic infections. Therefore, maintenance of a healthy lysosome population is of utmost importance to the cell to respond to both stress conditions and also homeostatic signalling. For example, for minor perturbations to the lysosomal membrane, the cell activates repair processes which seal membrane nicks. For more extensive damage, autophagy is activated to remove damaged organelles from the cell. on the other hand, during pathogen invasion host cells have also evolved mechanisms to hijack the endolysosomal pathway to facilitate their own growth and replication in host cells.
The first part of the thesis work focuses on a lysosomal regeneration program which is activated under conditions where the entire lysosomal pool of the cell is damaged. Upon extensive membrane damage induced by the lysosomotropic drug LLOMe, the cell activates a regeneration pathway which helps in the formation of new functional lysosomes by recycling damaged membranes. I have identified the molecules important for this novel pathway of lysosomal regeneration and showed how the protein TBC1D15 orchestrates this process to regenerate functional organelles from completely damaged membrane masses in the first 2 hours following lysosomal membrane damage. This process resembles the process of auto- lysosomal reformation (ALR)- involving the formation of lysosomal tubules which are extended along microtubules and cleaved in a dynamin2 dependent manner to form proto-lysosomes which develop into fully functional mature lysosomes. These lysosomal tubules are closely associated with ATG8 positive autophagosomal membranes and require ATG8 proteins to bind to the lysophagy receptor LIMP2 on damaged membranes. This process is physiologically important under conditions of crystal nephropathy where calcium oxalate crystals induce damage to lysosomal membranes in nephrons in kidney disease.
The second part of the thesis shows how the endolysosomal system of the cell is hijacked by the bacteriaLegionella pneumophila. During Legionella infection the formation of conventional ATG8 positive autophagosomes are blocked due to the protease activity of the bacterial effector protein RavZ which cleaves lipidated ATG8 proteins from autophagosomal membranes. The SidE effectors of Legionella modify STX17 and SNAP29 by the process of non-canonical ubiquitination called phosphoribose-linked serine ubiquitination (PR-Ub). These proteins are essential for the formation of the autophagosomal SNARE complex which is used for fusion of the autophagosome with the lysosome. Upon Legionella infection, PR-UB of STX17 aids in formation of autophagosome-like replication vacuoles. ThesevacuolesdonotfusewiththelysosomebecauseSNAP29isalsoPR-Ubmodified. PR-UbofSTX17 and SNAP29 sterically blocks the formation of the autophagosomal-SNARE complex thereby preventing fusion of the autophagosome with the lysosome. As a result, Legionella can replicate in autophagosome- like vacuoles which do not undergo lysosomal degradation. In absence of PR-Ub modified STX17, bacterial replication is compromised when measured by bacterial replication assays in lung epithelial (A549) cells.
Taken together, this thesis highlights two important aspects of the autophagy-lysosomal system- how it responds to extensive membrane damage and its importance in Legionella pneumophila infection. Extensive damage to lysosomal membranes triggers a rapid regeneration process to partially restore lysosomal function before the effects of TFEB dependent lysosomal biogenesis becomes apparent. On the other hand, Legionella pneumophila infection segregates the lysosomes from the rest of the endo-lysosomal system by blocking autophagosome-lysosome fusion. Though lysosomes remain active, they are incapable of degrading pathogens since pathogen containing vacuoles do not fuse with the lysosome.
Climatology of morphology and cloud-radiative properties of marine low-level mixed-phase clouds
(2023)
Marine stratocumuli cover about 40 - 60% of the ocean surface. They self-organize into different morphological regimes. The two organized cellular regimes are called open and closed mesoscale-cellular convective (MCC) clouds. In mid-to-high latitudes, open and closed cells are the two most frequent types of MCC clouds. In particular, many MCC clouds consist of a mixture of vapor, liquid droplets, and ice particles, referred to as mixed-phase clouds (MPCs). Even for the same cloud fraction, the albedo of open cells is, on average, lower than that of closed MCC clouds. Cloud phase and morphology individually influence the cloud radiative effect. Thus, this thesis investigates the relationships between the cloud phase, MCC organization, cell size, and differences regarding the cloud-radiative effect.
This thesis focuses on space-borne retrievals to achieve extensive temporal and spatial coverage. The liDAR-raDAR (DARDAR) version 2 product collocates two active and one passive satellite: CloudSat, Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO), and Moderate Resolution Imaging Spectroradiometer (MODIS). The cloud phase of DARDAR is vertically integrated to establish a single cloud phase at each data point. The MCC classification data set based on the liquid water path (LWP) of MODIS scenes is collocated with the DARDAR product to determine the MCC organization. Cell-size statistics of both MCC clouds are obtained using a marker-based image segmentation method on MODIS reflectance scenes. In addition, based on MODIS reflectance scenes, a convolutional neural network (CNN) is developed to classify open and closed MCC scenes to avoid missing mature MPCs with a low LWP.
The first part of this thesis explores the relationships between cloud phase, morphology, and cloud albedo in the Southern Ocean (SO). At a given cloud-top temperature (CTT), seasonal changes in the mixed-phase fraction, defined as the number of MPCs divided by the sum of MPC and supercooled liquid cloud (SLC) pixels, are stronger than the morphological changes. Therefore, external factors seem to influence these changes instead of morphology. The dependence of cloud phase on cloud-top height (CTH) is more substantial than on CTT in clouds with CTHs below 2.5 km. The previously observed acceleration of closed-to-open transition in MPCs, known as preconditioning, is not the primary driver of climatological cloud morphology statistics in the SO. The morphological differences in cloud albedo are more pronounced in SLCs than in MPCs. This change in albedo alters the cloud radiative effect in the SO by 21Wm−2 to 39Wm−2 depending onseason and cloud phase.
Open and closed MCC clouds exhibit larger equivalent cell diameters in the MPCs than in SLCs in austral summer, whereas, in austral winter, the SLCs are larger. The cell’s aspect ratio accounts for varying CTHs. Closed cells have smaller aspect ratios than open cells, so their cell diameter is smaller, independent of CTH. While the seasonal differences in closed cells are due to changes in CTH, the seasonal aspect ratio differences in open cells are mainly caused by MPCs. With increasing aspect ratios, the cloud albedo decreases in both open and closed MCC clouds, with the most substantial decrease in open MPCs clouds. This leads to cloud-radiative changes of 60 - 75Wm−2 in the SO, depending on cloud phase and aspect ratio.
The established CNN exhibits a good accuracy of 80.6%, with even higher accuracies in the Open (85.5%) and Closed (87.3%) categories. The global MCC climatology based on the CNN generally agrees well with previous MCC distributions. The most notable difference occurs in the Northern Hemisphere (NH) in boreal winter, with a higher occurrence frequency of closed and open MCC clouds. This might indicate missing MPCs in previous studies based on the LWP and some restricted to warm cloud scenes. Thus, the developed CNN seems to better represent the different morphologies in MPCs than in previous classifications.
In conclusion, this thesis shows that understanding the dependencies of cloud phase, cloud morphology, and cell size is important to enhance predictions of the cloud-radiative effect and thus, it is important to evaluate how cloud phase, cloud morphology, and cellsize change in a warming climate.
N6-methyladenosine (m6A) is the most abundant and well understood modification in eukaryotic mRNA and was first identified in polyadenylated parts of the mRNA.The distinct distribution of m6A in the transcriptome with special enrichment in long internal exons, 39UTRs and around stop codons was uncovered by early biochemical work and later on antibody based sequencing techniques. The so called m6A writer, reader and eraser machinery is responsible for the dynamic and with that regulatory nature of the m6A modification. As m6A writer, the human N6-methyltransferase complex (MTC) cotranscriptionally methylates the central adenine within a RRACH (preferably GGACU) sequence context to form m6A in the nascent RNA chain.9–15 The catalytic core of the complex is formed by the two proteins METTL3 and METTL14, with the active site located in the methyltransferase domain (MTD) of METTL3.16–18 The DPPW motif near the methyl donor S-adenosylmethionine (SAM) binding site in this MTD was postulated to bind the target adenine during catalysis. Moreover, a positively charged groove in the METTL3-METTL14 interface, the C-terminal RGG domain in METTL14 and the zinc finger motifs in METTL3 were identified as important domains for RNA binding. However, to date there are no full-length or substrate-RNA-bound structures of the catalytic METTL3-METTL14 complex.
In addition, a set of accessory proteins assembles to the METTL3-METTL14 heterodimer to form the full MTC, mediated by WTAP that firmly binds to the N-terminal leader helix in METTL3.20 WTAP was shown to locate the whole complex to the nuclear speckles and can modulate m6A deposition to specific sites in the RNA. Moreover, WTAP acts as binding platform for other accessory proteins including VIRMA, RBM15, ZC3H13 and HAKAI that are mostly identified to mediate position specific methylation. For example, RBM15 was shown to mediates region-selective methylation in a WTAP dependent manner, directing specificity towards U-rich sequences.
The observed specificity of the methyltransferase complex to methylate only site specific DRACH sequenced is still poorly understood. Some possible modulators like the role of the accessory proteins are under investigation, however, the structural context of the RNA methylation sites or a structural preference of the complex have been mainly neglected so far. Moreover, the structural dynamics of this methylation process still remain elusive. This thesis contributes to the afore-mentioned aspects by analysis of the methylation process regarding RNA structure sensitivity with enzymatic activity assays and its dynamic nature by implementing a smFRET approach.
We hypothesized the target RNA secondary structure to be an additional important modulator of methylation efficiency, based on the RNA binding elements of the complex (positively charged binding groove, zinc finger domain, RGG domain) and the supposed target adenine binding in the active site. Here, we postulated the possibility for a flipped-out adenine to be of special relevance, which is closely related to the local stability of the target adenine containing structure. Moreover, efficient binding of the protein complex to the RNA should require the ability to anchor the RNA on both sides of the target sequence.
This work focused on the biosynthesis and characterization of esterified lipid mediators. Lipid mediators were generally thought to exert their effects as free molecules, and their esterification was regarded as a storage mechanism. However, more recent studies indicate that esterified lipid mediators are a distinct class of mediators. When this thesis started back in 2017, the idea of esterified lipids as a new class of mediators was relatively new so that respective compounds were either quite expensive or not commercially available at all. Therefore, a biosynthetic approach had to be established first to enable the study of the new lipid mediator class. Within the cell, esterified lipids are produced by activation and subsequent incorporation of polyunsaturated fatty acids. These steps are enzymatically catalyzed by members of the acyl-CoA synthetase family and the lysophosphatidylcholine acyltransferase family, respectively. Therefore, the enzymes acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 2 (LPCAT2) were selected for a biosynthetic approach due to their broad substrate acceptance.
In a first attempt, recombinant protein expression in E. coli was studied. While the expression and purification of C-terminally His6x-tagged ACSL4 resulted in a pure and active protein, the expression of LPCAT2 turned out quite troublesome. Although several expression and purification parameters were varied, including purification tags, buffer compositions, and chromatography strategies, successful purification of LPCAT2 was not achieved.
Instead, a second approach was studied. This time, stably transfected cells overexpressing ACSL4 and/or LPCAT2 were generated from the human embryonal kidney (HEK) 293T cell line. Stably transfected cell lines were characterized on protein level and regarding their oxylipin profile. After confirming the overexpression and functionality of the enzymes, lipoxygenases (LOs) were co-expressed in a doxycycline-inducible manner to prevent premature cell death due to increased oxidative stress. As a result, LO product formation was enhanced and enabled the investigation of specific oxylipins. Since increased lipid peroxidation is also a key component of the ferroptosis cell death mechanisms, cell lines were investigated towards their cell viability. Indeed, expression of ACSL4 and/or LPCAT2 promoted cell death when treated with the ferroptosis inducers erastin or RSL3, even in the absence of LO expression. Furthermore, analysis by laser scanning confocal microscopy revealed that the localization of 15-LO1 was altered in the presence of LPCAT2, similar to treatment with RSL3 in vector control cells.
In conclusion, a stable overexpression system of ACSL4 and/or LPCAT2 was successfully established in HEK293T cells, which enabled the synthesis and characterization of esterified oxylipins. Interestingly, characterization of the cell lines revealed a correlation with the cell death mechanism ferroptosis. Although the expression of ACSL4 has already been reported as a biomarker for ferroptosis, this is the first time that a potential connection of LPCAT2 with ferroptosis was demonstrated. As a result, this may provide new therapeutic options for ferroptosis-related pathologies such as neurodegeneration, autoimmune diseases, or tumorigenesis.
Polyunsaturated fatty acids (PUFAs) play essential roles in mediating inflammation and its resolution. PUFA metabolites generated by the cytochrome P450 (CYP) - soluble epoxide hydrolase (sEH) axis are known to regulate macrophage activation/polarization but little is known about their role in the resolution of inflammation. Monocytes were isolated from murine bone marrow or human peripheral blood and differentiated to naïve macrophages (M0). Thereafter cells were polarized using LPS and IFNγ (M1), IL-4 (M2a), or TGFβ1 (M2c). Gene expression was analyzed by RNA sequencing, RT-qPCR and Western blotting. Phagocytosis of zymosan and oxo-LDL were also assessed in vitro. Zymosan-induced peritonitis combined with immune cell profiling was used to evaluate the resolution of inflammation in vivo. The expression of sEH was comparable in M0, M1 and M2a macrophages but markedly elevated in M2c polarized cells. The increase in sEH expression elicited by TGFβ relied on the TGFβ receptor ALK5 and the phosphorylation of SMAD2, which was able to bind to the sEH promoter. In macrophages lacking sEH, M2c polarization was incomplete and characterized by lower levels of pro-resolving phagocytosis associated receptors (Tlr2 and Mrc1), as well as higher levels of the pro-inflammatory markers; Nlrp3, IL-1β and TNFα. Fitting with the failure to upregulate phagocytosis associated receptors, the uptake of zymosan and ox-LDL was less efficient in M2c macrophages from sEH-/- mice. The latter animals also demonstrated a retarded resolution of inflammation (zymosan-induced peritonitis) in vivo with fewer resident macrophages and recruited macrophages. PUFA profile analysis indicated decreased sEH substrates e.g., 11, 12-EET, as well as increased sEH products e.g., 11, 12-DHET, indicating an increased sEH activity in M2c macrophages. Taken together, our data indicates that sEH expression is required for the effective M2c polarization of macrophages and thus the resolution of inflammation.
A Large Ion Collider Experiment (ALICE) is a high-energy physics experiment, designed to study heavy ion collisions at the European Organization for Nuclear Research (CERN)Large Hadron Collider (LHC). ALICE is built to study the fundamental properties of matter as it existed shortly after the big bang. This requires reading out millions of sensors with high frequency, enabling high statistics for physics analysis, resulting in a considerable computing demand concerning network throughput and processing power. With the ALICE Run 3 upgrade [14], requirements for a High Throughput Computing
(HTC) online processing cluster increased significantly, due to more than an order of magnitude more data than in Run 2, resulting in a processing input rate of up to 900 GB/s. Online (real-time) event reconstruction allows for the compression of the data stream to 130 GB/s, which is stored on disk for physics analysis.
This thesis presents the implementation of the ALICE Event Processing Node (EPN) compute farm, to cope with the Run 3 online computing challenges. Building a Data Centre tailored to ALICE requirements for the Run 3 and Run 4 EPN farm. Providing the operational conditions for a dynamic compute environment of a High Performance Computing (HPC) cluster, with significant load changes in a short time span, when starting or stopping a data-taking run. EPN servers provide the required computing resources for online reconstruction and data compression. The farm includes network connectivity towards First Level Processors (FLPs), requiring reliable throughput of 900 GB/s between FLPs and EPNs and connectivity from the internal InfiniBand network to the CERN Exabyte Object Storage (EOS) Ethernet network, with more than 100 GB/s.
The results of operating the EPN computing infrastructure during the first year of Run 3 LHC collisions are described in the context of the ALICE experiment. The EPN farm was delivering the expected performance for ALICE data-taking. Data Centre environmental conditions remained stable during the last more than two years, in particular during starting and stopping runs, which include significant changes in IT load. Several unforeseen external circumstances lead to increasing demands for the Online Offline System (O2). Higher data rates than anticipated required network performance to exceed the initial design specifications, for the throughput between FLPs and EPNs. In particular, the high throughput from an internal EPN InfiniBand network towards the storage Ethernet network was one of the challenges to overcome.