Eine Aminosäure (AS) mit besonderer Relevanz für die Schweinefütterung ist Methionin (Met), welche in Form von DL-Met, L-Met oder DL-2-Hydroxy-4-(methylthio)buttersäure (DL-HMTBA) supplementiert werden kann. Es ist bekannt, dass diese Met-Supplemente den intestinalen Transport von Met sowie den Met-Metabolismus beeinflussen können. Die vorliegende Arbeit zielte darauf ab, die folgenden Hypothesen zu prüfen: Erstens, dass die Art des Met-Supplements nicht nur den Met-Transport, sondern auch den Transport weiterer AS beeinflusst und zweitens, dass sich verschiedene Met-Supplemente unterschiedlich auf das epitheliale Metabolom sowie die Expression von inflammatorischen Markern im porzinen Dünndarm auswirken. Im ersten Teil des Projektes wurde entweder eine Diät mit DL-Met, L-Met, DL-HMTBA oder keinem zusätzlichen Met-Supplement an junge Mastschweine verfüttert. Ihr Jejunum wurde verwendet, um den mukoserosalen Flux von [14C]-markiertem L-Glutamin, Glycin, LLeucin, L-Lysin, L-Met, L-Serin, L-Threonin, L-Tryptophan, L-Tyrosin und L-Valin mittels eines Ussing-Kammer-Modells zu untersuchen. Alle Fluxuntersuchungen wurde in An- und Abwesenheit von mukosalem Na+, sowie zwei mukosalen AS-Konzentrationen (50 μM/5 mM) durchgeführt. Weiterhin wurde eine Korrelationsanalyse der Na+-abhängigen AS-Fluxe angefertigt, sowie die cis-Inhibition des apikalen Transports durch mukosales L-Met untersucht. Für letzteres wurde ausschließlich eine DL-Met-Vorfütterung eingesetzt. Um funktionelle Unterschiede zwischen der vorliegenden und einer vorangegangenen Studie zu untersuchen, wurde die jejunale Genexpression neutraler apikaler AS-Transporter (SLC1A5, SLC6A19, SLC6A20, SLC6A14) sowie verschiedener Entzündungsmarker (NLR family pyrin domain containing 3 (NLRP3), Caspase1 (CASP1), Interleukin (IL)1β, IL8, IL18, Tumornekrosefaktor α, Transforming growth factor β (TGFβ)) und des Angiotensin-Konversionsenzyms II (ACE2) zwischen beiden Studien verglichen. Die Ergebnisse zeigten eine deutliche Na+-Abhängigkeit fast aller AS-Fluxe mit Ausnahme von Lysin und Tryptophan bei 5 mM AS-Konzentration. Diese Na+-abhängigen Fluxe korrelierten vielfach untereinander. Ausnahmen bildeten Glycin mit nur wenigen und Tryptophan mit keiner Korrelation zu weiteren AS. Die apikale Aufnahme aller AS außer Glycin und Lysin war einer cis-Inhibition durch mukosales Met unterworfen. Die vergleichenden Untersuchungen der Genexpression zeigte eine erhöhte Expression von SLC6A19 (B0AT1) und eine niedrigere Expression von SLC6A14 (ABT0,+) in der vorliegenden vs. der Vorgängerstudie. Zusätzlich konnte eine erhöhte Expression aller Entzündungsmarker außer NLRP3 und IL18 in der Vorgängerstudie nachgewiesen werden. Die Expression des Angiotensin-Konversionsenzyms II unterschied sich nicht signifikant zwischen den beiden Studien.

Im zweiten Teil des Projektes wurde das Metabolom des Duodenums, des proximalen und mittleren Jejunums sowie des Ileums von Schweinen mittels Ultrahochleistungsflüssigkeitschromatographie und Tandemmassenspektrometrie analysiert. Die Tiere erhielten entweder DL-Met, L-Met oder DL-HMTBA als Futtersupplemente. Weiterhin wurde dieses Gewebe verwendet, um den Einfluss der Supplemente auf die Expression verschiedener Entzündungsmarker (NLRP3, CASP1, IL1β, IL8, IL18, TGFβ) zu untersuchen. Die Supplemente hatten keine Auswirkung auf das globale intestinale Metabolom, allerdings zeigte eine Stoffwechselweg-Analyse einen signifikanten Effekt auf Stoffwechselwege des Lipidmetabolismus. So reduzierte L-Met die relative Konzentration von mehrfach ungesättigten Fettsäuren im Gewebe. Weiterhin waren sekundäre Gallensäuren im Jejunum der L-Met- und im Ileum in der DL-HMTBA-Gruppe angereichert. Im distalen Dünndarm zeigte sich eine signifikante Anreicherung von Monohydroxy-Fettsäuren sowie ein statistischer Trend in Richtung Anreicherung von Sphingosinen. Hingegen hatten die Supplemente eine weniger deutliche Auswirkung auf Metabolite, die mit dem antioxidativen System und dem inflammatorischen Status in Verbindung stehen. So führte insbesondere L- und DL-Met-Supplementierung zu einer Anreicherung einzelner oxidierter Met-Metabolite im Gewebe. Zusätzlich wurde ein statistischer Trend der Anreicherung von Metaboliten im Tocopherol- und im Histidin-Metabolismus beobachtet. Hierbei ging eine DL-Met- und DL-HMTBA-Supplementierung mit einem relativen jejunalen Anstieg von β- und γ-Tocopherolen sowie von α-Tocotrienolen im Gewebe einher. Zusätzlich waren Histidinmetabolite in der DL-Met-Gruppe angereichert. Die Expressionslevel der Entzündungsmarker unterschieden sich in dieser Studie nicht zwischen den Met-Supplementen. Zusammenfassend hatte die Met-Supplementierung in dieser Arbeit keine Auswirkung auf den intestinalen AS-Transport. Allgemein unterstützen die Daten jedoch das gegenwärtige Konzept des intestinalen neutralen AS-Transportes, welches B0AT1 eine prominente Rolle zuschreibt. Im intestinalen Metabolom beeinflussten die Met-Supplemente verschiedene Lipid-Stoffwechselwege, die Expression von Entzündungsmarkern unterschied sich in der vorliegenden Studie allerdings nicht zwischen den Met-Supplementen. Jedoch sprechen die Expressionsunterschiede der Entzündungsmarker zwischen den Studien dafür, dass die Effekte von Met-Supplementen auf den AS-Transport durch sekundäre Faktoren wie beispielsweise eine Entzündung beeinflusst werden können. Die vorliegende Arbeit zeigt praxisrelevante Aspekte der Met-Supplementierung auf und legt damit die Basis für zukünftige Studien zur detaillierten Charakterisierung des Einflusses externer Faktoren auf die Interaktion zwischen Met-Supplementen und dem AS-Transport sowie der Regulationsmechanismen von Met-Supplementierung im intestinalen Lipidmetabolismus.

The development of quantum technologies, such as quantum simulators, promises significant advancements in the study of complex systems, including many-body physics. One promising approach for realizing a quantum simulator is to use nitrogen-vacancy (NV) centers in diamond. A hybrid system, in which an NV center serves as a quantum probe for initialization, manipulation and readout, while nuclear spin arrays on the diamond surface function as qubits, presents a potential direction for quantum simulator development.

NV centers are widely used as quantum sensors due to their unique properties. However, their high sensitivity, while advantageous, also introduces challenges in the fabrication and stability of a diamond-based quantum simulator. Achieving a functional system requires careful consideration of structural complexity, material interactions, and environmental conditions.

This PhD thesis explores and addresses the challenges encountered during the fabrication process of an NV-based quantum simulator. From hyperfine interactions to limitations imposed by experimental equipment, this study provides a comprehensive overview of the complexities involved. Additionally, it presents potential strategies for mitigating these challenges, contributing to the ongoing development of scalable and reliable quantum simulation platforms.

A diamond-based quantum simulator offers promising advantages, including room-temperature operation, non-destructive measurements, and the potential for long-lived quantum memories. Advances in diamond growth, surface engineering, and quantum control techniques are steadily improving the stability and coherence of NV centers, bringing this technology closer to practical realization. While large-scale NV-based quantum simulators have yet to be implemented, this work represents an important step toward their feasibility, identifying key material and control challenges that must be addressed to unlock their full potential.

Background: Studies in mice infected with gastrointestinal (GI) nematodes, particularly Heligmosomoides polygyrus bakeri (H. bakeri), provide critical insights into the immune factors that govern resistance to infection. Resistance to H. bakeri is primarily mediated by high levels of GATA-3+ CD4+ Th2 cells in mesenteric lymph nodes (MLN). Two mouse strains are commonly studied: BALB/c mice, which efficiently clear the infection within weeks, and C57BL/6 mice, which are more susceptible, often carrying the infection for months, making them a useful model for chronic infection research. Age impacts resistance in BALB/c mice, with older mice showing reduced immunity due to slower effector cell migration to the infection site. This study explores whether resistance to H. bakeri infection is influenced by both genetic and age-related factors affecting effector cell recruitment to the infected gut. Additionally, the research examines the role of liver-draining lymph nodes (celiac lymph node; CLN, portal lymph node, PLN) in immune response modulation and the interplay between the liver and gut during infection, analyzing how these affect the liver metabolic microenvironment and shape immune response and infection outcome.

Aims: 1. To determine whether genotype and age influence resistance to H. bakeri infection 2. To investigate how these factors modulate the speed and efficiency of effector cell recruitment 3. To elucidate the role of liver-draining lymph nodes (LLNs) in the differentiation of classical Th2 and Th2/1 hybrid cells 4. To explore the potential impact of co-drainage between the MLN and LLNs on immune responses 5. To explore the interaction between liver and T cells during H. bakeri infection

Results: We show that in resistant BALB/c mice, rapid Th2 cell recruitment to the gut, driven by CCR9 expression and specific dendritic cells, effectively limits early H. bakeri larval development. In contrast, delayed Th2 responses in susceptible C57BL/6 mice allow larvae to mature into adult worms, while aging BALB/c mice also experience reduced Th2 homing, diminishing their resistance. Additionally, liver-draining lymph nodes contribute to immunity byexpanding Th2 cells (CLN) and supporting high-affinity IgG1 antibody production (PLN). Liver-resident T cells produce high levels of IL-5, IL-4, and IL-13 in a glucose-rich environment, which enhances their immune functionality.

Conclusion: This study demonstrates that BALB/c mice resist H. bakeri infection more effectively than C57BL/6 mice due to enhanced CCR9 expression and swift Th2 cell recruitment to the gut. In older BALB/c mice, reduced ALDH activity and delayed Th2 recruitment weaken resistance. Furthermore, despite limited Th2 activity, PLN compensates with strong TFH bias, forming more GCs that produce high-affinity antibodies. The liver also plays a key immune role by sustaining Th2/1 responses through IFN-γ, CXCR3, and metabolic adaptations. This highlights the liver’s potential as a target for optimizing immune response modulation in intestinal helminth infections.

The Periodic Event Scheduling Problem (PESP) serves as the foundational framework for optimizing periodic timetables in public transport. We here present advances in the theoretical understanding of PESP and its underlying geometries, as well as novel ways to model and optimize timetabling with integrated infrastructure aspects in practice. First, having formulated PESP as a mixed-integer program, we study the geometric structure of the problem. In the periodic timetable space we find a collection of polytropes, tropical polytopes that are also traditionally convex, wrapped onto a torus. Instead, in the space of cycle offsets, which are auxiliary integer variables, we find a special zonotope, of high combinatorial interest. We establish connections between these two geometries, and employ these newfound insights to develop a novel local improvement heuristic for PESP, Tropical Neighbourhood Search (\tns{}), which we extensively test and evaluate with excellent computational results. In the second half of the dissertation, we focus on the challenge of properly integrating infrastructure constraints with periodic timetabling. We construct novel MIP-based modelling strategies, relevant cutting planes, and other approaches using cycle orders and perfect matchings, to ensure that found solutions are safely operable on the available infrastructure, while also optimizing the assignment of vehicles to ground resources, making full use of previously untapped track capacity. The viability and practical relevance of our models, Infrastructure-Aware PESP and its extensions, is showcased over multiple case studies on real-world public transport networks.

Applying electron spin resonance (ESR) dating to sedimentary quartz poses a significant challenge due to the difficulty in bleaching the signals. The aim of this PhD thesis is to contribute to the improvement of a reliable ESR dating technique by quantifying and correcting ESR residual signals caused by insufficient bleaching prior to sediment deposition. Particularly within the realm of archaeology, there is a pressing need for a geochronological method capable of dating quartz and other materials well beyond the constraints of the already-established dating methods, e.g. luminescence dating. The initial study investigates the reliability of quartz ESR dating, focusing on the Ti centre. Loess from the Luochuan site on the Chinese Loess Plateau with a known reference age was used. By the use of the single-aliquot regenerative dose (SAR) protocol the equivalent dose (De) was determined for a sequence of five samples. For the two youngest samples the ESR ages were higher than the anticipated ages, indicating an incomplete signal resetting before sedimentation, whereas the oldest samples showed an approx. 20 % underestimation, indicating thermal signal loss with time. In the latter case, the apparent age could be successfully corrected for the signal loss. For the remaining samples the corrected ages were in satisfactory agreement with the expected ages. The second study focuses on the ESR residual signals caused by incomplete signal resetting before sedimentation. For this purpose, a series of early Holocene fluvial sediments with known optically stimulated luminescence (OSL) ages were analysed. Applying the SAR protocol to determine the residual doses for both the aluminium (Al) and titanium (Ti) impurity centres. It was revealed, that all investigated samples carried a considerable residual dose. To rule out methodological problems, the SAR protocol was tested for accuracy. The test revealed that the signal originating from the lithium-compensated Ti centre (Ti-Li) and a signal which originates from both the Ti-Li and the hydrogen-compensated Ti centre (Ti-H), referred to as Ti-mix, showed good results whereas the signal from the Al centre and the Ti-H signal showed that the heating and annealing steps throughout the process alter the samples ESR characteristics rendering the SAR protocol inappropriate to use in this two cases. Our findings suggest the necessity of conducting more direct comparisons between luminescence and ESR equivalent doses, with the subtraction of residual doses obtained from the difference being essential for obtaining reliable ESR ages if needed. This procedure was not used in previous work, as it was either assumed that the Ti centres were all completely bleached or, as long as the determined ages of the individual centres matched, complete bleaching was assumed. In order to apply the knowledge gained in the course of this work, the transition age from the Early Stone Age (ESA) to the Middle Stone Age (MSA) in archaeological sites near the Victoria Falls, Zambia, was dated since this technological change in stone tool making has been poorly understood by now. The combined advantages of both, single-grain OSL and single aliquot ESR dating on quartz, the good bleachability of the signals and the extended age range was used. For the same set of young samples, derived from sandy deposits bearing Stone tools, we applied both methods and found large differences in OSL and ESR equivalent doses. We estimated the mean ESR residual age by discerning the difference between OSL ages and the apparent ESR ages. Specifically emphasizing the SAR protocol, we successfully determined the mean ESR residual age for the Ti and Al centre, encompassing the non-bleachable signal component for the latter. We successfully determined the average residual age of ESR for both Ti and Al centre, incorporating the non-bleachable signal component for the latter. The size of these residual ages, ranging from 209 ± 13 ka to 268 ± 39 ka and 695 ± 23 ka to 742 ± 118 ka for the Ti centre and Al centre, which cannot be understated and must be taken into consideration. Consequently, the apparent ESR ages were adjusted by subtracting the residual age. By this we were able to get persistent residual subtracted ESR ages, which are within a 2- uncertainty when compared to the OSL ages. Eventually, we successfully pinpointed the end of the Early Stone Age at 590 ± 86 ka, establishing a maximum age for the transition to the Middle Stone Age in this specific region of south-central Africa. It should be noted that the residual ages depend on the respective geological setting, the climate and the deposition history of the sediments. Factors such as the transport route, the efficiency of bleaching by sunlight and possible rearrangements have a significant influence on whether a sediment grain was completely bleached before deposition or whether a residual signal or a residual age remains. In order to recognize and evaluate such influences, it is necessary to examine the deposition environment in detail, for example by analysing the grain size distribution, sediment structures or the composition of the sediment. Only through such sedimentological and geomorphological contextualization can ages obtained in this way be reliably interpreted. Nevertheless, we think that this approach gives a good example for the combined use of two related dating methods by carefully investigate the signal properties to unveil ESR residual signals. The age we published adds to the sparsely dated ESA/MSA transition in this geographic region.

„Sprachalchemisten“, „Barock-„ oder „Beatpoeten“, „DDR-Avantgarde“, „Kollaborateure unter der Maske der französischen Theorie“: Bereits diese schillernden Zitate aus Feuilleton und Forschung lassen erahnen, welche Mythen und Kontroversen sich bis heute um jene inoffizielle Lyrikszene ranken, die sich in der Spätphase der DDR im Ostberliner Stadtteil Prenzlauer Berg herausbildete. Nicht erst seit der Stasi-Affäre um Sascha Anderson 1991 ist rege darüber diskutiert worden, ob es sich bei der „Prenzlauer-Berg-Connection“ überhaupt um eine klar abgrenzbare Gemeinschaft oder nicht doch eher um eine konstruierte Zusammenschau chaotischer Einzelkämpfer handelte.

Die Studie von Lukas Nils Regeler verfolgt die These, dass sich die Gemeinschaftsbildung im Prenzlauer Berg der 1980er Jahre besonders dort gut nachvollziehen lässt, wo die Autor:innen als Leser:innen auftreten, indem sie etwa Theorieelemente, Schreibweisen und Motive aus literarischen und philosophischen Strömungen aufgreifen und produktiv machen, die im offiziellen Kulturbetrieb der DDR wenig Platz hatten. Anhand der emphatischen Bezugnahmen von Autor:innen wie Bert Papenfuß, Elke Erb, Johannes Jansen, Leonhard Lorek, Raja Lubinetzki oder Rainer Schedlinski auf die Literaturen der Frühen Neuzeit, die historischen Avantgarden, die US-amerikanische Beatgeneration und die Theorien des französischen Poststrukturalismus werden so Ein- und Ausschlussmechanismen der Gemeinschaftsbildung greifbar. Damit leistet die Untersuchung auch einen Beitrag zu einer Literaturgeschichte der DDR, die weniger in Generationen und Schulen argumentiert, sondern vielmehr transtemporale und internationale Verflechtungen in den Blick nimmt.

T-cell-based immunotherapies aim to treat various malignancies by targeting intracellular protein-derived peptides processed by the proteasome and presented on MHC class I molecules. A critical step in the development of these therapies is the accurate selection of target MHC ligands. This selection is often guided by computational prediction algorithms, particularly in the context of vaccine design, rather than experimental validation of antigen presentation. However, these algorithms frequently fail to accurately reflect the complexity of natural antigen processing and presentation, potentially leading to false-positive results and misdirected therapeutic strategies. To systematically evaluate these predictions, this thesis employs a COS-7 monoallelic cell system in liason with immunopeptidomics to screen 150 combinations of individual neoantigens/tumor-associated antigens and HLA alleles. Surprisingly, only 21.6% of predicted neoepitopes were experimentally validated, revealing a 78% false-positive rate of computational prediction algorithms. Similarly, these computational tools are frequently used to establish links between public neoepitope presentation and downstream biological effects including immune evasion through HLA loss of heterozygosity (HLA-LOH). However, this link was also disrupted in 77% of HLA-LOH cases, where the predicted neoepitopes showed no evidence of presentation on the corresponding HLA alleles, thereby challenging the assumption that HLA-LOH is mainly driven by public neoantigen presentation. Importantly, we identified 24 novel public neoepitopes across 11 HLA alleles, derived from recurrent driver mutations (e.g., TP53, EGFR) and resistance-related alterations (e.g., CTNNB1, ESR1), alongside 13 novel tumor-associated epitopes (e.g., derived from MAGE-A4, PRAME, CT83, NY-ESO-1) across 4 common HLA alleles. Two epitopes, KVDELAHFL (MAGE-A4) and KVLEHVVRV (MAGE-A4), were each presented on two common HLA alleles, offering a promising strategy to broaden patient eligibility for TCR-based therapies. Furthermore, when testing sequences used for personalized mRNA neoepitope vaccines, we observed that clinical responses were independent of the presentation status of the neoepitopes, highlighting the risk of measuring false-positive responses during T-cell monitoring. Even a presumably neoepitope-specific TCR clone from a neoantigen vaccination study could not be linked to a successfully presented neoepitope. In contrast, we observed cross-reactivity of the TCR’s targets with a CMV-specific epitope as a potential explanation for the T-cell response measured during T-cell monitoring. Overall, these data challenge current assumptions about neoantigen responses, immune evasion, and the reliability of computational predictions.

Heute weitgehend in Vergessenheit geraten, war Johann Wilhelm Überfeld um 1700 ein Mann, der polarisierte: Der gelernte Kaufmann inszenierte sich als religiöser Virtuose mit unmittelbarem Zugang zu göttlicher Weisheit. Vom niederländischen Leiden aus schickte er unzählige Unterweisungsschreiben an Bewunderer und Interessierte quer durch das protestantische Mitteleuropa. Theologische Widersacher erkannten in ihm das Oberhaupt einer gefährlichen außerkirchlichen Gruppierung – der Gemeinschaft der Engelsbrüder. Lennart Gard befasst sich in seiner Studie erstmals eingehend mit Überfeld und den Menschen, die sich mit ihm austauschten und auseinandersetzten. Auf Basis umfassender Quellenbestände geht er exemplarisch der Frage nach, wie religiöses Miteinander jenseits kirchlicher Strukturen in der Frühen Neuzeit funktionierte und gedeutet wurde. Die Untersuchung beschäftigt sich dazu mit Vorstellungen, Sozialbeziehungen, Lebenswelten und Erinnerungskulturen religiöser Menschen des 17. und 18. Jahrhunderts. Zugleich wird verdeutlicht, wie in jener Zeit polemische Gemeinschaftsbilder mit Auswirkungen bis in die Gegenwart entstanden. Als Kultur- und Sozialgeschichte religiöser Kommunikation vermittelt die Studie neue Perspektiven auf die Vielfalt des frühneuzeitlichen Protestantismus und deren historische und historiographische Wahrnehmung.

Aging is a complex biological process associated with systemic and cellular dysfunctions. Both fasting (i.e., time-restricted feeding) and dietary protein restriction (PR) are among the most promising interventions to promote healthy aging. Since the two paradigms are hardly compatible, it has remained unclear whether they may exert synergistic effects. However, recent studies have shown that the endogenous polyamine spermidine increases during fasting in both the fruit fly, Drosophila, and humans. In my dissertation, I therefore treated Drosophila with a combination of dietary spermidine supplementation (SPD) and PR to assess whether their effects are additive or synergistic. My observations confirmed that both interventions act through orthogonal mechanisms, which encourages future clinical studies in this direction. The Drosophila study included behavioral analyses (lifespan, locomotion, and fecundity) combined with polyamine quantification and proteome profiling. Both SPD and PR alone promoted healthspan and lifespan in flies, while their combination provided additional benefits — including extended lifespan, improved locomotion, and prolonged fecundity in aging flies. SPD (but not PR) increased putrescine and spermidine levels in PR-fed flies and reprogrammed the proteome toward specific metabolic pathways — including mitochondrial metabolism, autophagy, and hypusination. Hypusination is a posttranslational modification of the eukaryotic initiation factor 5A (eIF5A), in which spermidine donates an aminobutyl group to a specific lysine residue, forming hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine). ____________________________________________________________________________________________________ In the second part of this work (ImmuneAge trial in human participants), we investigated the role of SPD in immune rejuvenation and conducted a comprehensive analysis of polyamine biokinetics in blood fractions (plasma, serum, and cellular components) in collaboration Charité – Universitätsmedizin Berlin, German Institute of Human Nutrition Potsdam-Rehbrücke (DIfE), Leibniz Institute for Analytical Sciences – ISAS – e.V., and The Longevity Labs GmbH (TLL). The ImmuneAge Trial used a multi-parameter molecular profiling approach, including ELISA for inflammatory markers, LC-MS for polyamine quantification, proteomics, western blot analysis for autophagy and hypusination markers, and flow cytometry to evaluate immune responses. We showed that a 20-day SPD significantly enhanced autophagy and hypusination in peripheral blood mononuclear cells (PBMCs), particularly in younger participants (20–40 years), with similar trends observed in older individuals (60–90 years). These effects correlated with SPD’s strong ability to counteract the age-related decline of spermidine levels in PBMCs. Moreover, SPD reduced inflammaging and improved aspects of both innate and adaptive immune responses to the SARS-CoV-2 spike peptide, with stronger effects in younger participants. Plasma proteomics revealed that SPD strengthened the immune system, lowered thrombosis risk, and reduced inflammation in older participants, while in younger participants, it improved lipid metabolism and triggered changes considered cardioprotective.

Driving fundamentally challenges the established equilibrium paradigms of interacting quantum many-body systems. Quantum spin systems exposed to periodic, quasiperiodic and direct-current driving can enter non-equilibrium phases of matter with no equilibrium analog. A striking example is a Floquet time crystal, a phase of matter stabilized by periodic driving, that breaks time-translation symmetry and is thus at odds with thermal equilibrium. The time crystal owes its robustness against perturbations to the interactions between the particles. It is thus a representative case that highlights the important role of interactions in the presence of driving.

This thesis employs the quantum Ising model as a platform to explore driven many-body dynamics. First, we study the dynamics of correlation functions of the periodically driven quantum Ising model. In the open chain, periodic driving stabilizes Floquet Majorana zero modes (MZMs) and Majorana π modes (MPMs) at its boundaries, implying characteristic level pairings throughout the many-body spectrum. We show that the level pairing statistics differ markedly between MZMs and MPMs in the presence of random symmetry-breaking fields, with implications for the boundary spin correlations. In the coexisting regime of MZMs and MPMs, we construct a composite boundary mode as their operator product. We analyze the resilience of the composite mode against integrability-breaking perturbations, surpassing the stability of the individual boundary modes. Next, we apply a quasiperiodic Fibonacci drive to the quantum Ising chain. The boundaries can host Majorana golden-ratio modes (MGMs) unique to the quasiperiodic setting. We map out the dynamic phase diagram which contains self-similar structures under time evolution. Returning to periodic drives, we revisit the relation of the Floquet time crystal to the spectral π pairings of the many-body Floquet operator. Our work shows that the level pairing statistics provides analytical expressions for the temporal spin correlations in Floquet time crystals.

Finally, we turn to magnetic impurities in superconductors. We treat the spins of a Yu-Shiba- Rusinov dimer fully quantum in a zero-bandwidth approximation, accounting for the complex interplay of screening, superconducting correlations, magnetic anisotropies and substrate-mediated interactions. The dimer shows rich phases and excitation spectra which underscore the role of interactions between spins under direct-current driving in a solid-state platform.

This dissertation explores how macroeconomic policy and conditions interact with expectations, institutional constraints, and country, as well as household heterogeneity. In four chapters, I study how monetary policy reaches the public via the media, how fiscal rules shape governments' ability to respond to shocks, and how we can track changes in income inequality in real time. Chapter 1 examines how media coverage of the European Central Bank (ECB) shapes consumer inflation expectations. The media act as a key intermediary between central banks and the public; I investigate which monetary policy topics in media reporting matter most to consumers. I identify seven core topics - interest rates, inflation, economic growth, purchase programs, uncertainty, fiscal policy, and financial markets - and measure their prominence in leading economic newspapers in the euro area’s four largest economies using Latent Semantic Indexing with factor rotation. To isolate the impact of topic-specific coverage, I construct media topic shifts around ECB press conferences in an event-study framework and estimate their effect using local projections. The results suggest that media coverage significantly influences inflation expectations: discussions on inflation and economic growth raise expectations, while talk about the financial topic dampens them. Consumers respond more strongly to the media's interpretation of ECB messages than to the messages themselves. Furthermore, the media generally reinforce ECB messaging, with the exception of the fiscal topic, where consumer expectations move in opposing directions depending on the source. These findings offer new insights into how monetary policy (communication) is filtered through the media and received by the public. Chapter 2, co-authored with Vegard H. Larsen and Nicolò Maffei-Faccioli, studies how ECB-related inflation news affect consumer inflation expectations heterogeneously across the four largest euro area countries. Using Latent Dirichlet Allocation, we measure the intensity of inflation coverage in the national media and estimate its effects in a Structural Vector Autoregressive model. We find that German and Italian consumers respond significantly to ECB inflation news, while no clear effect emerges for Spain and France. These results point to substantial cross-country heterogeneity in how central bank communication is received, highlighting the importance of national media in shaping policy transmission in a diverse monetary union. Chapter 3, joint with Christoph Große-Steffen and Malte Rieth, examines how fiscal rules affect macroeconomic stabilization in response to exogenous shocks. Using the unpredictability of natural disasters in an instrumental-variable approach, we construct a shock measure that is exogenous and comparable across countries. We combine the resulting shock series with quarterly macroeconomic data for 89 countries over nearly fifty years in a dynamic panel model. The results suggest that countries with fiscal rules absorb shocks better, with stronger recovery of GDP and private demand. These effects are linked to more expansionary fiscal policy and depend on available fiscal space, suggesting that rules can enhance countercyclical capacity. We further explore the role of rule design in a sovereign default model, focusing on the interplay between rule tightness and flexibility. The model shows that tight fiscal rules with escape clauses can support countercyclical responses and generate welfare gains, even under market discipline. Overall, our findings offer new evidence that well-designed fiscal rules can enhance resilience to economic shocks rather than constrain the policy response. Chapter 4, co-authored with Nina Maria Brehl and Geraldine Dany-Knedlik, examines whether macroeconomic developments shape the labor income distribution. Accurate and timely data on income dynamics are essential for informed policy responses, yet such information is typically published only annually and with substantial delays. We propose a method to nowcast the income distribution using dynamic factor models that combine high-frequency macroeconomic indicators with low-frequency household survey data from the German Socio-Economic Panel. A pseudo-real time evaluation shows that incorporating macroeconomic signals and inter-decile dynamics substantially improves forecast accuracy over a univariate benchmark - particularly for the middle and lower parts of the distribution. We apply the best-performing model to project income growth through 2024 and estimate inequality under the assumption of a generalized Pareto distribution. The results highlight a heterogeneous response of different income groups to macroeconomic shifts and show that inequality in Germany has likely risen in the aftermath of the European energy crisis. Our approach offers a practical framework for real-time monitoring of distributional developments.

Humans are remarkably fast at processing scenes and making decisions based on the information they contain. Within a few hundred milliseconds of viewing a scene, our brain can extract the most important information through a hierarchical cascade starting with perceptual attributes (color, edges, etc.) and ending with abstract properties (category, relationship between objects, etc.), eventually supporting decision-making. Despite the central role of scene processing, many aspects of how it unfolds in the brain remain poorly understood. In particular, the intermediate stages linking perceptual and abstract scene understanding, i.e., mid-level feature processing, are largely unresolved. Moreover, the link between neural activity and behavior, i.e., when, where and what kind of scene information arising in the brain influences decision-making, remains unclear. This thesis addresses these gaps through three studies implementing empirical and computational methods. In Study 1, we used a novel stimulus set to reveal that various mid-level features of scenes are processed in humans between ∼100 ms and ∼250 ms after stimulus onset, bridging low- and high-level feature representations, and with a temporal hierarchy that is mirrored by convolutional neural networks (CNNs). In Study 2, we showed that neural representations of scenes are suitably formatted for behavioral readout of scene naturalness between ∼100 ms and ∼200 ms, i.e., in the intermediate processing stages, and that intermediate CNN layers best correlated with the neural representations in this time-window, suggesting that mid-level features underlie behaviorally-relevant representations. In Study 3, we showed that neural representations of scenes are suitably formatted for behavioral readout of scene naturalness in the early visual cortex and in the object-selective high-level cortex, and that intermediate CNN layers best explain this brain-behavior relationship, indicating that behaviorally-relevant representations in these areas are driven by mid-level features. Taken together, the studies included in this thesis revealed the timing, spatial localization, and behavioral relevance of mid-level feature representations in scene processing, contributing to a better understanding of how the human brain extracts information from the surrounding world.

The assessment of skin sensitization is a crucial aspect of toxicology and regulatory safety testing, particularly for industries involving pharmaceuticals, chemicals, and cosmetics. Traditional animal-based assays, such as the Local Lymph Node Assay have been widely used for this purpose. However, these models face ethical concerns, regulatory restrictions, and limited predictive accuracy due to interspecies differences. To address these challenges, in vitro and in silico assays have been developed, but current methods only assess individual key events in the Adverse Outcome Pathway for skin sensitization, lacking a comprehensive evaluation of the sensitization process. This thesis aimed to develop a fully immunocompetent human skin model derived entirely from induced pluripotent stem cells (iPSC) to provide a more physiologically relevant and scalable alternative for skin sensitization assessment. The skin model was built using iPSC-derived fibroblasts, keratinocytes, and dendritic cells (iPSC-FB, iPSC-KC and iPSC-DC), integrated into a three-dimensional skin structure. This innovation allows for the simultaneous evaluation of multiple key events in the sensitization process, including keratinocyte activation, dendritic cell maturation, and cytokine secretion, making it a more robust and mechanistically relevant tool for the detection of skin sensitizing substances. To achieve this, hair follicle-derived keratinocytes were reprogrammed into iPSC using non-integrative Sendai virus vectors, ensuring genomic stability and ethical sourcing. These iPSC were then efficiently differentiated into functional skin-resident cells, including fibroblasts, keratinocytes, and dendritic cells. The differentiated cells exhibited characteristics comparable to their primary cell counterparts, with iPSC-FB demonstrating robust collagen secretion and extracellular matrix formation, and iPSC-KC expressing key epidermal differentiation markers. Additionally, iPSC-DC displayed antigen-presenting capabilities, as confirmed by the expression of CD86, HLA-DR, and CD209, and were able to induce allogeneic T-cell proliferation, confirming their immune functionality. The developed iPSC-derived immunocompetent skin models were functionally evaluated using a Lucifer Yellow permeability assay to confirm epidermal barrier integrity, as well as assays evaluating the cell viability. Furthermore, a skin sensitization assay was conducted, where the model was exposed to sensitizers of varying potencies, including dinitrochlorobenzene, p-phenylenediamine, isoeugenol, resorcinol and the non-sensitizer glycerol. The results demonstrated increased dendritic cell maturation and cytokine secretion (IL-8, IL-1β, MIP-1β, IL-18, TSLP, and TGF-β1) in response to sensitizers, confirming the model’s ability to distinguish between sensitizing and non-sensitizing compounds. Notably, the immunocompetent skin model outperformed conventional skin models by integrating both key event 2 (keratinocyte activation) and key event 3 (dendritic cell activation), aligning with the Adverse Outcome Pathway framework and improving predictive accuracy. This thesis represents a significant advancement in skin model development by creating a fully human, reproducible, and scalable system that eliminates the need for primary cell sourcing and animal testing. The iPSC-derived immunocompetent skin model holds broad applications in toxicology, the assessment of skin sensitizers, and disease modeling, offering a powerful platform for studying inflammatory skin conditions such as atopic dermatitis and psoriasis. Furthermore, the ability to generate patient-specific iPSC lines enables the development of personalized in vitro models for precision medicine and drug screening in the future. By providing an ethically responsible and human-relevant alternative to current testing models, this study contributes to the ongoing efforts to replace, reduce, and refine (3R principles) the use of animal testing in toxicology. The fully integrated iPSC-derived skin model presents a scalable and standardized approach for evaluating chemical sensitization, advancing both scientific research and regulatory safety assessment. Future research should focus on further refining the model for clinical applications, integrating additional immune components, and expanding its use in genetic disease modeling and personalized therapeutic testing.

Chemical synapses are the points of contact through which information flows between neurons. The fusion of neurotransmitter-filled vesicles with the plasma membrane is at the center of this cellular process. This fusion is orchestrated by three soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, among which Syntaxin-1 (STX1) is paramount. Anchored to the plasma membrane through a transmembrane domain (TMD), STX1 interacts with the other two SNARE proteins (Synaptobrevin-2 and SNAP-25) through its SNARE domain to form the SNARE complex. Regulatory proteins including Synaptotagmin-1, complexin and Munc18-1, also interact with STX1 through the SNARE domain. Additionally, STX1 has many other regulatory domains such as the N-peptide, the Habc-domain, the linker region, and the juxtamembrane domain (JMD). Understanding the individual roles of these domains can help us unravel the complex process that is vesicle fusion. In this compilation of studies, we make use of a mouse hippocampal neuronal model lacking STX1 (STX1-null). In these STX1-null neurons we can express genetically modified constructs of STX1 which target different domains and perform structure-function analysis by studying the electrophysiological responses of these neurons. Our goal is to elucidate the specific roles of distinct STX1 domains. First, we characterized the role of the N-peptide of STX1, which has been reported to be indispensable for the function of STX1 in vesicular release, in the natural and constitutively open conformations of STX1, which has been reported to be indispensable for the function of STX1 in vesicular release. For this we created increasing deletions of the N-peptide up until the Habc-domain of STX1 and expressed these mutant constructs in our STX1-null neuron model. Our results concluded that the N-peptide is non-essential for neurotransmitter release, however it has a regulatory role in the Ca2+-triggered release. Second, we performed an in-depth analysis of the role of the juxtamembrane domain (JMD) and the transmembrane domain (TMD) of STX1, and the role of palmitoylation of these two domains in vesicular release. In order to do this first we created STX1 constructs which carry elongations at either side of the JMD, to understand the precise function of the continuity between SNARE domain and JMD and JMD and TMD. Our results showed that the structural continuity between SNARE domain and JMD and JMD and TMD is essential for spontaneous and Ca2+-evoked vesicular release. Additionally, mutating residues that are targets of palmitoylation of these domains showed that the palmitoylation of the TMD (which is regulated by the JMD) is an important regulatory mechanism in spontaneous release. Finally, we studied the function of the SNARE domain of STX1. For this we performed a chimeric approach using STX1 and an isoform, STX2. This analysis consisted on interchanging the Nterminal half, the C-terminal half or the entire SNARE domain between the two isoforms. Our results showed that the C-terminal half of the SNARE complex of STX1 has a regulatory function on the stability of the primed vesicles and in the spontaneous release of vesicles. Furthermore, we created point mutations in the C-terminal half of the SNARE domain of residues that differed between STX2 and STX1 and found that residues D231 and R232 seemed to be especially important for these two functions. This comprehensive study contributes to our understanding of the nuanced mechanisms governing synaptic vesicle fusion.

Soil restoration strategies are integrated management practices to recover soil health and enhance plant growth without compromising future demands. Despite land management practices being generally well-studied , effects of joint application of soil restoration practices with more than three factors are rarely addressed. A main reason might be the complex interactions between individual practices, such as two-way interactions. Such interactions can be affected by the fluctuation of external environmental patterns and other additional restoration factors, making the joint effect hard to predict. This doctoral work investigates the joint effects of multiple restoration factors on soil properties and plant growth. First, a laboratory study explores the joint effects of restoration practices (up to 8) for different factor numbers. This experiment acts as a proof- of- concept study to investigate the multiple restoration practices effects on soil properties. Next, two climate chamber experiments investigate the joint effects of restoration amendments on soil properties and plant growth. These two experiments focus on the effect of factor diversity for restoration amendments. One experiment investigates the carbon diversity effects by applying five diverse organic amendments, while for the other experiment nine restoration amendments were allocated into three distinct functional groups according to their physicochemical properties and effect mechanisms- namely, organic , inorganic and bio-fertilizer amendments. Both experiments unravel the importance of jointly applying diverse restoration practices under well-watered conditions, whereas applying a single factor may result in better outcomes to address a specific challenge, such as drought stress. Last but not least, we conducted an experiment to investigate restoration effects on soil properties and the plant community. Previous laboratory works showed that, in contrast to multiple global change factors, a higher factor number often cannot result in better performance in comparison to single factors. Therefore, we assessed the full spectrum of all two factor combinations with replicates to investigate the pairwise interactions and diagnosed the interaction type. We found that an increasing factor number can enhance soil properties, such as soil water holding capacity and soil pH, whereas the plant community composition is influenced mainly by the dissimilarity between factors. Moreover, we showed that pairwise synergistic interactions may enhance plant growth. Together, this work reports findings of joint effects from multiple restoration practices with various analytical approaches across different scales. Our results offer valuable insights into the complex interactions under multiple restoration contexts and provide solid suggestions on integrated restoration strategies for farmers, researchers and decision makers.

As plastic production and consumption continue to increase, so does its impact on the environment. The extent to which plastic is present in our ecosystems and the manner in which it affects them has not yet been sufficiently investigated. Therefore, the following thesis focuses on the investigation of possi- ble micro- and nanoplastics entry paths into the environment, the optimization of its analysis methods in environmental samples, and the approach to generate a reference material for future toxicity studies of nano-sized plastics in organisms.

Antimicrobial resistance (AMR) is one of the most pressing global public health challenges, posing a significant and immediate threat to both human and animal health. The use of antimicrobials in human medicine, veterinary medicine, and agriculture is a major driver of resistance. Regulation (EU) 2019/6 aims to curb the development and spread of AMR by introducing stricter controls on the use of antibiotics in animals. While the regulation harmonizes the rules for veterinary antimicrobial products across the EU, there is currently no coordinated European system for monitoring AMR in bacterial pathogens from diseased animals. As part of the HKP-Mon project, this study aimed to establish baseline data on the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) in dogs and cats in Germany from 2019 to 2021, and to assess the potential of retrospective laboratory data for continuous AMR monitoring. The analysis was based on a large dataset of routine diagnostic results provided by Laboklin, an accredited veterinary diagnostic laboratory. Samples originated from 3,491 veterinary practices and clinics, representing approximately one-third of small animal practices in Germany. Out of 175,171 total samples, 25.6% (5,526) were identified as S. aureus and 3.2% (44,880) as S. pseudintermedius. Data were stratified by year, animal species, and sample type. Phenotypic methicillin resistance was detected in 17.8% of S. aureus and 7.5% of S. pseudintermedius isolates. MRSA prevalence was lower in cats (15.6%) than in dogs (20.4%), while MRSP prevalence was higher in cats (16.1%) compared to dogs (7.1%). In contrast to veterinary findings, the average MRSA prevalence in human medicine during the same period was lower at 5.4%. For both MRSA and MRSP, the highest prevalences were observed in wound samples, with S. aureus exceeding 30% in dogs and 20% in cats, and S. pseudintermedius exceeding 15% in dogs and 20% in cats. Notably, feline urogenital tract samples also showed high MRSP prevalence, exceeding 20%. MRSA isolates exhibited the highest resistance to clindamycin (59.8%) and enrofloxacin (36.4%), while resistance to sulfamethoxazole-trimethoprim and gentamicin was moderate (13–14%), and resistance to chloramphenicol, doxycycline, and rifampicin remained below 6%. MRSP isolates showed even higher resistance rates, particularly to clindamycin (85.2%), enrofloxacin (50.5%), and sulfamethoxazole-trimethoprim (66.3%). The results confirm that MRSA and MRSP remain among the most relevant resistant pathogens in companion animals and highlight their concerning resistance profiles across multiple sample types. Comparisons with other studies revealed substantial variability in methodology, pathogens, and sample sizes, underscoring the need for harmonized monitoring approaches. Our findings demonstrate the value of routine diagnostic data as a scalable and sustainable resource for passive AMR monitoring and highlight its potential for integration into active surveillance systems and broader One Health monitoring frameworks.

This study aims to understand the position and path of the North Korean Kim Jong Un regime on the issue of reunification of the two Koreas and to explore desirable alternatives to the unification of the Korean Peninsula. Applying the analytical tools, East Germany has taken a Two-state policy based on the following factors: Structural Influence of Socialist Suzerainty, External system supported by statehood recognition, Cooperative Relationship with other divided Party, while North Korea has pursued a One-state policy, influenced by the following factors such as relatively superior economic power, strong nationalism, relatively superior military power and strong leader’s perception of unification issues. This study also attempts to derive policy implications by analyzing the situation in which the ‘One-state theory’ and the ‘Two-state theory’ have been raised as unification discourse in South Korean society. Considering that it is not easy to establish a single unified nation-state, achieved when one side is absorbed, and to form a federal system for two countries with different political and ideological systems, the possibility of forming a ‘Confederative Structure’ could be examined as an alternative measure for unification.

Following the 26 December 2004 Sumatra-Andaman (Mw 9.3) earthquake, back-projection became a widely used technique for remote imaging of large earthquake ruptures. We introduce a new teleseismic back-projection method that uses multiple seismic arrays and combines both P and pP seismic phases. For earthquakes deeper than 40 km, we incorporate pP backprojections, particularly when the pP amplitude is at least 40% of the P wave amplitude. The contribution of each array to the rupture image is controlled by its azimuthal distribution. This approach allows us to algorithmically determine key rupture parameters, including rupture length, directivity, speed, and aspect ratio.

Multi-array and multi-phase back-projection enhances resolution, facilitating the tracking and analysis of short-period earthquake rupture complexities. Early developments and applications include imaging the 23 January 2018 Gulf of Alaska (Mw 7.9) intraplate rupture, the 24 January 2020 Doğanyol-Sivrice (Mw 6.7) earthquake (Türkiye), and the 30 October 2020 Néon-Karlovásion (Mw 7.0) earthquake (Greece). The finalized method was also used to characterize the 12 August 2021 South Sandwich tsunamigenic earthquake (Mw > 8.2; South Atlantic) and the 6 February 2023 Türkiye seismic sequence (Mw 7.7 and 7.6).

We applied the newly developed back-projection method to characterize all large earthquakes with magnitudes Mw ≥ 7.5 and depths less than 200 km that occurred between 01/2010 and 12/2022 (56 events). For subduction megathrust earthquakes, we observed complex short-period ruptures (0.5-2.0 Hz) outlining megathrust asperities. Our results confirmed the prevalence of short-period radiation from the central and down-dip parts of the megathrust. Notably, we found that up-dip emissions from the main asperity are more common than previously reported. We also evaluated the prevalence of supershear ruptures and established new magnitude-rupture length scaling relationships for thrust, normal, and strike-slip earthquakes, consistent with previously published relationships based on aftershocks and total slip estimates.

We observed asperity encircling short-period ruptures of the 14 November 2007 Tocopilla (Mw 7.7) earthquake in Northern Chile using teleseismic and local strong-motion backprojections. The complex rupture was attributed to several factors: 1) the high-stress gradient caused by a kink in the slab interface, which had previously been proposed as the main mechanism arresting the trenchward rupture propagation; 2) the down-dip limit of the rupture, coinciding with the depth of the Continental Moho; and 3) the high-stress gradient surrounding the asperities.

Finally, we explore back-projection applications for locating volcano-induced landslides and assessing tsunami-warning strategies in Indonesia, focusing on the 22 December 2018 flank collapse of Anak Krakatau. Using long-period back-projection (40-70 s) of surface wave envelopes from the Indonesian seismic network, we demonstrated the flank collapse localization with two minutes of data after its initiation. Spectral analysis of the first 100 s of seismic data can distinguish the flank collapse from typical tectonic earthquakes, using stations at epicentral distances of 1.0°-2.5°. The results showed that massive landslides can be distinguished from earthquakes using a simple frequency ratio. We conclude by discussing some practical aspects for tsunami early warning systems, particularly for detecting and locating volcanic collapses and landslide-triggered tsunamis using real-time seismic data.

The conversion of atmospheric gases in organisms represents a crucial interface between the inanimate and animate nature, facilitated by biological catalysts called enzymes. These enzymes bear the potential to reduce air pollution emitted by a highly industrialized society. For this geoengineering purpose, the molybdenum-dependent formate dehydrogenase from Rhodobacter capsulatus might represent a promising candidate, due to its ability to reversibly convert formate into carbon dioxide by withdrawing two electrons. Withdrawn electrons are transferred via an electron transfer chain consisting of iron-sulfur (FeS) clusters within the enzyme to another protein domain, where an electron acceptor becomes reduced. However, the reaction mechanism at the molybdenum cofactor (MoCo) acting as the catalytic center and the electron transfer mechanism are not precisely described. A well-established method for investigating such metalloproteins is the electron paramagnetic resonance (EPR) spectroscopy, which is utilized in this work to examine the binding site of formate as a substrate and azide as a competitive inhibitor, potentially adopting similar orientations close to the MoCo. The application of pulse EPR spectroscopy revealed that carbon dioxide is already released in an azide-inhibited paramagnetic Mo(V) state, while the formate proton resides in close proximity to the MoCo. By employing a combined approach of density functional theory (DFT) and pulse EPR spectroscopy, the precise binding site of the formate proton was identified and the orientation of the azide inhibitor elucidated in proximity to the formate proton. Furthermore, the redox potentials of the electron transferring moieties were investigated using EPR-mediated redox titration elucidating the electron transfer path through the enzyme. This investigation along with the results from the analyses of the azide and formate proton positions lead to the proposal that MoCo acts as an electron transfer transducer, converting the two-electron transfer into a sequential one-electron transfer over the FeS clusters. This effect is reversed in the FdsGB domain by a further proposed electron transfer transducer. Additionally, it was demonstrated in this work that the investigation of such metalloproteins with multiple paramagnetic centers is only feasible after extensive prior analysis and assignment of the signals to their corresponding centers. To simplify such analyses for similar research subjects, a computational method based on deep learning was developed, separating EPR signals with respect to their associated lifetimes. This algorithm was extensively assessed using a defined test set to estimate its applicability.