Arctic amplification is the phenomenon of accelerated warming of the Arctic polar regions in the context of climate change induced by anthropogenic greenhouse gas emissions. A number of recent cold episodes in midlatitudes in winter have raised the question of whether the Arctic amplification has led to the observed midlatitude cooling. The proposed mechanism of this Arctic-midlatitude linkage involves a stratospheric pathway. Accordingly, the horizontal temperature gradients are decreasing due to the enhanced polar warming at the surface. Consequently, the superjacent winds are weakened which results in slower and more meandering polar jet streams. The enhanced planetary waves propagate into the stratosphere, where they induce strong circulation anomalies, referred to as sudden stratospheric warmings (SSWs). These strong disturbances of the stratospheric polar vortex can potentially exert a downward influence on the troposphere, favouring local cold air outbreaks. Investigating this stratospheric pathway in a climate model requires an accurate representation of the middle atmosphere. Therefore, a comprehensive evaluation using the climate-chemistry model ECHAM/MESSy Atmospheric Chemistry (EMAC) was conducted in this thesis, with the additional aim of examining the impact of ozone chemistry on the stratospheric processes. The suggested steps of the stratospheric pathway were examined using a set of transient simulations and timeslice experiments. Although the Arctic amplification signal continued to rise, no cooling trend or cessation of warming was found in the transient simulations. In contrast, the frequency of SSWs has increased significantly under the influence of climate change. This increase could be attributed to a larger planetary wave input from the troposphere. The primary area of enhanced planetary wave propagation was identified as the Northern Pacific and the region spanning the North Atlantic and Europe. Moreover, the number of events corresponding to strong wave input into the stratosphere increased significantly in a warmer climate. The behaviour of the jet streams was evaluated using a jet detection scheme to determine the exact jet positions. The Eurasian region exhibited an increased occurrence of polar jets, showing a wavier path as well. In contrast, the western hemisphere was characterised by a reduced polar jet frequency. A straightforward connection between the wavier jets and temperature gradients in the lower troposphere was not established in this study. While the modifications in the lower layers imply reduced baroclinic instability and fewer atmospheric waves, an enhanced wave generation became evident in the upper troposphere. However, these anomalies coincide with changes in the static stability and in subtropical temperature changes. The release of additional waves into the stratosphere appears to be related to these upper-tropospheric anomalies. The findings of this thesis suggest that the step linking temperature gradients to upper-tropospheric wave propagation requires further clarification. This is critical for validating the stratospheric pathway. Moreover, an evaluation of the downward influence of SSWs was conducted. Despite more frequent SSWs in a warmer climate, no tendency towards more extreme cold events was found in the most affected regions. While significant cooling anomalies still existed after the SSW events, the temperature anomalies are considerably lower. Finally, the representation of ozone chemistry in the model was determined to have an insignificant impact on the results of the stratospheric pathway.

This work explores the potential of supercritical fluid chromatography for the analysis of peptide drugs. The regulatory aspects of peptides are more complex and challenging than for small drug molecules. The same applies to the analytical characterization of peptides. After technical innovations and the introduction of a new instrument generation, supercritical fluid chromatography nowadays is a suitable alternative to reversed-phase chromatography, which is considered the gold standard in peptide analysis. However, the scientific data on the analysis of peptides with supercritical fluid chromatography is limited. In the first part of this thesis, supercritical fluid chromatography is used to develop a method to separate the variety of cyclic and linear peptides characteristic for tyrothricin. A software-based design of experiments approach is utilized to optimize an analytical method that provides superior separation of cyclic peptides compared to the reversed-phase separation. Even isomeric peptide pairs are efficiently separated. Subsequently, the potential of a binary mixture of methanol and acetonitrile combined with additives as the modifier for the separations of other cyclic peptides is investigated. The proportion of the mixture is optimized to improve the resolution and control the elution order of the analytes. Paired with an aromatic stationary phase, this chromatographic parameter offers an effective method optimization opportunity. This knowledge is furthermore extended to larger and more hydrophilic peptides in the following experiment. A single method for the purity analysis of human insulin and six analogs is optimized. A crown ether is also investigated as a promising additive to control the elution order of the insulins in dependence to its concentration. In the last study, the applicability of an established chromatographic modeling software for the in-silico optimization of peptide separations in supercritical fluid chromatography is demonstrated. Optimizing a binary mixture of methanol and acetonitrile serves as an effective parameter of the model. Using such software tools offers enormous time savings and, at the same time, meets the current requirements for analytical method development according to the quality by design concept. These already published studies are finally put into the context of the life cycle concept of analytical methods that will soon be demanded by the ICH Q14 guideline "Analytical procedure development". Existing knowledge, in the form of scientific publications, is employed as the basis for a general risk assessment applicable to peptide separations with supercritical fluid chromatography. From this, a generic method development strategy is derived and presented. This way, the current state of science and technology is taken into account to define an efficient workflow. This is particularly relevant in early pharmaceutical development phases, from where only a small fraction of initial drug candidates reaches market maturity. At the same time, this approach enhances the drug knowledge in early development phases and ultimately serves patient safety. The present work makes an important contribution to demonstrating the potential of supercritical fluid chromatography for the analysis of peptide drugs. It serves as a concise example of how supercritical fluid chromatography can be interpreted as a new technique in the ICH Q14 guideline setting. The approach presented can be easily applied to any other analytical technique or analyte class.

In the skin sensitization assessment, the in vivo method, specifically the Local Lymph Node Assay (LLNA), is regarded as the gold standard. However, to adhere to the 3R principle (Reduction, Refinement, Replacement), which advocates for the Reduction, Refinement, and Replacement of animal testing, and to enhance the ethical standards of risk assessment, a greater number of in vitro and in silico testing methods have been developed. While these in vitro and in silico methods offer a higher throughput and a more ethical approach to risk assessment, they each come with their own set of limitations. For instance, most in vitro tests can only be used for a single readout. On the other hand, in silico methods lack physiological and biological feedback. The adverse outcome pathway (AOP) for skin sensitization, as defined by the Organization for Economic Cooperation and Development (OECD) guidelines, involves four key events, which are protein binding (Key Event 1 – KE1), keratinocyte activation (Key Event 2 – KE 2), dendritic cell activation (Key Event 3 – KE 3), and T-lymphocyte activation (Key Event 4 – KE 4) (OECD, 2014). While animal-based assays are reasonably accurate, their skin is physiologically different than human skin. Therefore, this thesis aimed to develop the ‘ImmuSkin-MT’, a 3D-human skin model with immune cells, designed to address the limitations inherent in in vitro skin sensitization assays. The thesis began with 2D co-culture of keratinocytes and immune cells, which have shown to fall short in detecting a skin sensitizer. In parallel to the development of the 2D co-culture assay, the advantages of the autologous in comparison to the allogenic 2D co-culture were investigated. The results indicated that within the donor pool used, allogenic 2D co-culture did not lead to an alloreactive response in the cells. Therefore, whether autologous or allogenic cell pairings were used in the co-culture, they had no impact on the development of the assay. Later, the project progressed and the 'ImmuSkin-MT', created from human hair follicle-derived keratinocytes (HFDK), fibroblasts (HFDF), and immune cells were developed. monocyte-derived Langerhans cells (MoLC) and CD4+ naïve T-lymphocytes were integrated into the reconstructed human skin models (RHS). ImmuSkin-MT were topically exposed to various substances, including non-sensitizers and contact sensitizers. Using the MoLC activation and the stimulation index (SI) of T-lymphocytes, results demonstrate the effectiveness of ImmuSkin-MT in replicating KE 3 and 4 of skin sensitization in response to the skin sensitizers. This model presents a promising alternative to animal testing for contact sensitizers, contributing to more ethical and precise skin sensitization assessment techniques. In conclusion, ImmuSkin-MT represents a significant advancement in skin sensitization assessment by capturing multiple key events simultaneously. The project's findings have the potential to optimize skin sensitization testing methods, providing more accurate, reliable, and ethical alternatives to traditional animal-based assays.

Endogenous retroviruses (ERVs) are repetitive elements that constitute around 10 percent of mammalian genomes. ERVs need to be kept transcriptionally silenced during development and ERV upregulation is linked to loss of pluripotency and mouse embryonic lethality. Precise mechanistic understanding of this process remains incomplete. To investigate effects of ERV derepression, I utilized degradation tag (dTAG) system to acutely deplete endogenous levels of TRIM28, a heterochromatin protein that recruits silencing machinery to ERVs in mouse embryonic stem cells (mESCs). Nascent transcriptome sequencing indicated TRIM28 depletion induces acute transcriptomic changes: upregulation of ERVs and other targets of TRIM28-mediated repression and simultaneous downregulation of key pluripotency super enhancers (SEs) and associated genes. High-resolution microscopy data indicated ERV derepression reduces association of SEs with RNA polymerase II (RNAPII) and Mediator (MED1), the key components of transcriptional condensates. In turn, derepressed ERVs associated with RNAPII and MED1, indicating transcriptional condensate components are redistributed upon TRIM28 depletion. Presence of transcriptional machinery at derepressed ERV loci upregulated nearby genes, including Cthrc1. Ectopic overexpression of pluripotency factors enriched at SEs prevented the reduction in transcriptional condensate association with SEs. ERV knockdown rescued transcriptional condensate localization indicating RNAs produced at ERV play an important in transcriptional condensate redistribution. Evidence presented here shows derepressed ERVs have the capacity to ‘hijack’ transcriptional condensates from key pluripotency genes upon TRIM28 degradation. This may be the molecular mechanism contributing to embryonic lethality associated with ERV derepression in TRIM28 knockout mice.

We live in a structured world, where objects rarely exist in isolation but are often surrounded by similar environments. When objects consistently co-occur with certain objects and scene contexts, our neural systems can implicitly extract and learn such regularities in real-world environments. Predictive processing theories propose that our brains can use learned statistical regularities to predict the structure of incoming sensory input across space and time during visual processing. The predictions may allow us to efficiently recognize objects and understand scenes, thus forming coherent visual experiences in natural vision. In this dissertation, we conducted three studies to explore how our brains use real-world structures to create coherent visual experiences using neuroimaging techniques (EEG & fMRI) and multivariate pattern analyses (MVPA). Study 1 investigated how scene context affects object processing across time by recording EEG signals while participants viewed semantically consistent or inconsistent objects within scenes. The results reveal that semantically consistent scenes facilitate object representations, but this facilitation is task-dependent rather than automatic. In Study 2, we investigated how cortical feedback mediates the integration of visual information across space by manipulating the spatiotemporal coherence of naturalistic video stimuli shown in both visual hemifields. By analytically combining EEG and fMRI data, we demonstrated that spatial integration of naturalistic visual inputs is mediated by cortical feedback in alpha dynamics that fully traverse the visual hierarchy. In Study 3, we further investigated what level of spatiotemporal coherence is needed to trigger such integration-related alpha dynamics. The findings suggest that integration-related alpha dynamics have some flexibility so that they can accommodate information from videos belonging to the same basic-level category. Together, the dissertation provides multimodal evidence demonstrating that contextual information facilitates object perception and scene integration, highlighting the critical role of predictions related to real-world regularities in constructing coherent visual experiences.

Als eine bisher noch wenig für fremd- und zweitsprachliche Kontexte erarbeitete soziale Kategorie werden in der vorliegenden Arbeit „Zugehörigkeiten“ als lohnender Zugang für den Sprach-, Kultur- und Literaturunterricht vorgeschlagen. Am Beispiel brasilianischer Germanistikstudierender zeigt die Studie, wie die erworbene Zugehörigkeit zur deutschen Sprachgemeinschaft und die bestehende Zugehörigkeit zu Brasilien als fruchtbar und motivierend für fremdsprachliche Lehr-Lern-Prozesse genutzt werden können. Dafür wurde ein Vernetzungsmodell konzipiert, das einzelne Verdichtungspunkte deutsch-brasilianischer Literaturen betrachtet und sie nicht in Bezug auf Nationalphilologien, sondern im Kontext zueinander sieht. Konzeptionell wurde für das Modul die Perspektive einer doppelten Zugehörigkeitsorientierung entwickelt und deren unterrichtliche Ausarbeitung empirisch begleitet: So teilt sich das Konzept in eine subjektorientierte Zugehörigkeitsorientierung, die sich an den Zugehörigkeitskontexten der Teilnehmer*innen in Lehr- und Lernarrangements orientiert und in eine epistemische Zugehörigkeitsorientierung, die die Teilnehmer*innen anleitet, die Zugehörigkeiten von anderen verstärkt wahrzunehmen und die sich somit auf die soziale Verfasstheit unserer Gesellschaften richtet.

Resistive random-access memory (RRAM), one of the next-generation non-volatile memory technologies, offers scalability, enhanced performance, and lower power consumption compared to traditional memories, making it a promising solution for future memory architectures. In this work, we study different types of polycrystalline RMnO3 (R = Y, Er) thin films-based RRAM devices. This research starts with the synthesis and characterization of polycrystalline YMnO3 and ErMnO3 thin films with mixed hexagonal and orthorhombic phases. Films are prepared with radio frequency sputtering at room temperature and post-deposition annealing. The orthorhombic phase cannot always be discriminated by X-ray diffraction, as there is an overlap with the peaks of the hexagonal phase and its low volume fraction and the nano-sized grains lead to undetectable peaks. Employing a set of correlative spectroscopy and microscopy techniques, we develop a method to unambiguously identify the presence of the orthorhombic phase, locate it, and quantify it. Subsequently, we investigate different types of memristive devices using polycrystalline thin films with the presence of both hexagonal and orthorhombic phases. First, electrochemical metallization (ECM) memristive devices based on YMnO3 with Al active electrode are studied. These devices exhibit bipolar resistive switching with high ROFF/RON ratios (~ 104), low Set/Reset voltages (VSet ~ 1.7 V and VReset ~ -0.36 V), and good retention. The resistive switching mechanism is ascribed to the formation and rupture of an Al filament along oxygen-deficient boundaries between hexagonal and orthorhombic phases. These localized nanochannels for Al3+ migration effectively remove the randomness of the Al filament formation in the electrolyte. Second, we investigate bipolar resistive switching in Pt/ErMnO3/Ti/Au devices. They exhibit high ROFF/RON ratios (~ 105) and ultra-low resistances (~ 10 ohm) in the low resistance state (RON). The resistive switching is the result of the formation and rupture of an oxygen-vacancy-based conductive filament, which likely occurs either in the orthorhombic phase or at the boundary between the two polymorphs. An increased fraction of the orthorhombic phase strongly reduces the operating voltage in devices (down to VSet ~ -2.07 V) and the variability of VSet. The presence of hexagonal phase prevents large leakage currents in the devices, which otherwise would not show switching behavior. Finally, we present the first demonstration of electroforming-free threshold switching devices with ErMnO3. Pt/ErMnO3/Pt devices exhibit repeatable unipolar threshold switching with a memory window of 0.7 V, characterized by a S-shape current-controlled negative differential resistance (NDR). The devices show a high endurance up to 104 sweeps. We successfully model the threshold switching using Joule-heating-enhanced 3D Poole-Frenkel conduction mechanism. The conducting orthorhombic phase plays a key role in enabling the self-heating mechanism while the hexagonal phase prevents too large electrical and thermal conductivities. Adjusting the conductivity of o-ErMnO3 and engineering the two crystalline phase fractions are key knobs for tuning the threshold switching characteristics. The oscillatory behavior of the NDR devices is demonstrated. This new type of NDR devices based on two polymorphs with different electronic and thermal properties present advantages for tunability of the memory window compared to known NDR devices such as those based on an insulator-to-metal transition (e.g. VO2) or based on the formation of a filament (e.g. NbOx). In all studied devices, the coexistence of the hexagonal and orthorhombic phases of RMnO3 (R = Y, Er) provides unique functionalities. The possibility to engineer these two phases both in conductivity and content through synthesis, provide original routes to design new memristive devices and optimize their properties.

Diese Arbeit untersucht die im Dīwān luġāt at-Turk des Maḥmūd al-Kāšġarī aus dem 11. Jh. n. Chr. verstreuten Angaben zur Sprache der Qïfǰāq mit dem Ziel, die Beziehungen dieser frühesten bekannten kiptschakischen Wörter einschließlich ihrer Lautgestalt zu historischen Vorgänger- und Nachfolgersprachen und bestimmten Sprachregionen der gesamten Turcia herauszuarbeiten und sie so in einen sprachhistorischen Kontext zu stellen. Dazu wurden sämtliche Varietäteneinträge des 637 Manuskriptseiten umfassenden Dīwān luġāt at-Turk neu verzettelt und der so gebildete Auszug des Wortmaterials der Qïfǰāq in alphabetischer Reihenfolge in der Art eines Meta-Wörterbuches aufbereitet. Nach Klärung der Textüberlieferung, Wortherkunft, Wortbildung, Lautstruktur und Bedeutungsspannweite wurde eine Belegsammlung erstellt, die das betreffende Wort in der Gesamtheit seiner Erscheinungsformen in möglichst zahlreichen alttürkischen, mitteltürkischen und neutürkischen Sprachen und Varietäten registriert und die Überlieferungs- und wo möglich Entlehnungswege der spezifisch kiptschakischen Elemente nachzuverfolgen versucht. Im Auswertungsteil wurden die Ergebnisse dieser Nachverfolgung zusammengeführt und verglichen. Besonderes Augenmerk lag darüberhinaus auf der textkritischen Aufarbeitung der Stellen, auf der Rolle des Kopisten und besonders späterer Kommentatoren, auf der Frage nach der Verlässlichkeit der Angaben Maḥmūd al-Kāšġarīs auf dem Hintergrund der so erarbeiteten Daten und auf seinen Angaben zur Verbreitung bestimmter Merkmale im Kiptschakischen zugleich mit anderen Varietäten des 11. Jhs., insbesondere in den Ogusischen Sprachen. Die Nachverfolgung, Lokalisierung und Bewertung charakteristischer sich während der gesamten historischen Zeit ausbreitender Lautwandelerscheinungen erfolgte in einer ergänzenden Untersuchung. Das so gewonnene Bild wurde in einen durch muslimische Geschichtsquellen des 10. bis 12. Jhs. und moderne Untersuchungen an historischem Genmaterial aufgebauten Rahmen eingeordnet. Hier wurde auch versucht, Bezüge zu aus der Geschichtsschreibung bekannten mittelalterlichen Völkerbewegungen herzustellen. Als Arbeitsgrundlage dienten neben dem einzigen erhaltenen Autographen des Dīwān luġāt at-Turk, gelesen nach dem Faksimile von 1990, die Edition des Werks von Dankoff und Kelly (Maḥmūd al-Kāšγarī, Compendium of the Turkic dialects, drei Bände, 1982 bis 1985) und zahlreiche Wörterbücher der alttürkischen, mitteltürkischen und neutürkischen Sprachen, darunter auch historische Wörterbücher, außerdem eine Auswahl an mitteltürkischen literarischen Quellen, Editionen islamischer Geschichtsschreiber und moderne Sekundärliteratur, u.a. zu Geschichte, Klimageschichte und historischer DNA Eurasiens. Als Ergebnis des sprachwissenschaftlichen Teils der Arbeit mit ihrer Untersuchung der von Maḥmūd al-Kāšγarī als kiptschakisch bezeichneten Formen nebst aller anderen Varianten in ihrer zeitlichen und lokalen Verbreitung ergaben sich 68 Wortstammbäume. Diese Wortstammbäume wurden ihrerseits ggf. unter Berücksichtigung interner Kategorien des Dīwān luġāt at-Turk wie Verbreitung in mehreren Türksprachenvarietäten des 11. Jhs. hinsichtlich des Vor- und Weiterlebens der als Kiptschakisch gekennzeichneten Varianten zusammengeführt und ausgewertet und die Ergebnisse ggf. in Gruppen nach Art des Merkmals sortiert und einzeln besprochen. Für einzelne Wörter ergaben sich im Zuge der Aufarbeitung Beiträge zur Lehnwortforschung und Etymologie. Insgesamt konnte die Zweiteilung der kiptschakischen Siedlungsgebiete, wie sie auf der Karte des Dīwān luġāt at-Turk gezeigt ist, durch das Textmaterial bestätigt werden, es ergaben sich jeweils leicht unterschiedliche Bezüge zum West- und Ostalttürkischen. Für das 11. Jh. waren entsprechend charakteristische Sprachvarietätengruppierungen feststellbar. Hier zeichnete sich schwach eine Aufteilung in ein nordtürkisches und ein südtürkisches Sprachareal ab, das allerdings nicht mit dem im Text des Dīwān luġāt at-Turk vorgestellten Nord- und Südriegel der Türkstämme deckungsgleich ist – diese Anordnung im Dīwān luġāt at-Turk stellt eine Momentaufnahme der Wanderbewegungen diverser Türkvölker nach Westen dar. In den Kapiteln zum Erhalt des kiptschakischen Wortmaterials des Dīwān luġāt at-Turk in den mittel- und neutürkischen Sprachen ergaben sich vereinzelt Einblicke in die Entstehungsgeschichte der zeitgenössischen Familie der Kiptschaksprachen. Hierbei waren Überlegungen zu Lautsystemverschiebungen für die Auswertung unerlässlich. Für das 11. Jh. wurde dahingehend eine grobe Dreiteilung der türkischen Sprachgemeinschaft rekonstruiert und diese für die Folgejahrhunderte in ihrer weiteren Entwicklung nachverfolgt. Auch die Vertretung der türkischen Klusile im Altuigurischen von Xinjiang und die Häufigkeit von Frikativen in der Aralo-kaspischen Region spielen in diesem Rahmen eine Rolle. Zwei Kapitel zur Bewertung der Fähigkeiten und Leistungen des Autors Maḥmūd al-Kāšġarī, eines ohne und eines mit Einbeziehung der Ergebnisse der Wortstammbäume, liefern ein mehrschichtiges Profil des Autors und seiner Leistungen. Ein Ergebnis der textkritischen Arbeit ist die versuchte Identifikation des bisher namentlich unbekannten aktivsten nachträglichen Kommentators und Glossenschreibers des Dīwān luġāt at-Turk, "the later hand", mit Aḥmad Ḫaṭīb Dārayyā und damit die Datierung wichtiger Glossen. Schließlich wurde eine Auswertung der muslimischen Quellen zur Geschichte der Kiptschaken mit einer Sichtung historischen genetischen Materials kombiniert und über die aus der sprachwissenschaftlichen Untersuchung gewonnenen Daten geblendet. Im Ergebnis ergaben sich Vermutungen zum Ablauf des mehrphasigen Wanderungsgeschehens der Kiptschaken, Kimäk und Yemäk unter Beteiligung von Bevölkerungsgruppen östlicher (darunter C-Y15550 und C-F12970 unter C-M86) und westlicher (darunter R1b-M73) Herkunft.

Tight junctions (TJs) seal the paracellular space between epithelial and endothelial cells, and are essential for tissue compartmentalization and barrier formation. The backbone of the TJ is formed by a group of transmembrane proteins, called claudins. Claudins polymerize into strands and meshes through cis- and trans-interactions, and are expressed in different combinations, based on the needs of the respective tissue. The large protein family can be divided into pore- and barrier-formers claudins, with the former being selectively permeable to ions or water. Structural insight in claudin polymerization is limited; models have been created, but only of a few homopolymers. Recent studies have found only half of the claudin family to form homopolymers, whereas co-expression can promote polymerization of certain claudins. This thesis thus focuses on regulation of claudin polymerization. In the first part of the thesis, intrinsic regulation was examined by a comparison of claudin-3 and claudin-4. These proteins have high sequence homology, yet only claudin-3 polymerizes by itself in fibroblasts. With sequence alignment and molecular dynamics simulations, we predicted a motif in the second extracellular segment key to stable trans-interaction and polymerization. We mutated the two residues in claudin-4 into their claudin-3 equivalents and found that they promote polymerization in fibroblasts. Moreover, the mutated claudin-4 could (partially) rescue TJ localization, as well as the formation of a TJ barrier with ruffled morphology in TJ-deficient epithelial cells. Extrinsic regulation of claudin polymerization, specifically how co-expression of claudins can influence polymerization, was investigated next. Our focus was on claudin-16 and claudin-19, that form a cation-permeable copolymer in the thick ascending limb (TAL) of the kidney. Mutations in either protein cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, emphasizing their essential role in kidney Ca2+ transport. We also investigated claudin-14, that can block this cation-pore upon its hypercalcemia-induced expression, and is associated with kidney stone formation. It is known that non-polymerizing claudin-16 integrates into the claudin-19 meshwork, but how this is affected by claudin-14 is still poorly understood. Super-resolution microscopy in fibroblasts revealed that claudin-14 can replace claudin-16 in a strand-specific manner. We further validated this in epithelial cells with endogenous claudin-16 and in the TAL of hypercalcemic mice. Claudin-14 expression by transduction in epithelial cells, and in response to hypercalcemia in the mice, again led to claudin-16 replacement. Overall, we have identified a motif that intrinsically regulates claudin polymerization and have shown that claudin-14 replaces claudin-16 to regulate Ca2+ transport in the kidney. These insights form a basis to understand and manipulate claudin polymerization, for instance to improve drug delivery or to prevent pathogen entry. More directly, the knowledge of the claudin-14 regulatory mechanism can be used to develop treatments for kidney stones and other pathologies involving hypercalciuria.

Chemotherapy and radiation are standard-of-care cancer treatments, but their effectiveness is often hampered by therapy resistance within the tumor. Numerous studies have demonstrated that tumor cells can evade cell death triggered by genotoxic therapies by activating IKK/NF-κB pathway, thereby preventing apoptosis. The direct targeting of IKKs using pharmacological interventions is not a viable option due to the significant adverse effects caused by the essential role of IKK/NF-κB signaling in various physiological processes. To circumvent this, previous work by our group identified structurally unrelated small molecule inhibitors, MW01 and MW05, that selectively inhibit IKK/NF-κB solely in response to DNA double strand breaks induced by chemotherapy and radiation. Importantly, these compounds do not interfere with IKK/NF-κB activation triggered by other physiological stimuli. Initial work began by confirming the genotoxic stress-specific inhibition by the compounds before moving onto target identification studies. Considering the similar cellular effects of both compounds within the DNA damage-induced NF-κB pathway, comparative target identification studies including kinase assay panels, structural derivatization, and molecular signaling characterization were performed, seeking targets shared between both lead compounds. Common regulators shared by other NF-κB stimuli were first excluded as potential targets of the compounds before investigation of the several identified shared targets revealed a previously unknown regulators of genotoxic stress-induced NF-κB activity, Cdc-like kinases (CLK) 2 and 4, as the functional target of MW01 and MW05. Silencing of the CLK2 and 4 revealed that they are essential for DNA damage-induced NF-κB activity and promote the phosphorylation of IKK at Ser-85, a genotoxic stress specific ATM-dependent phosphosite, critically localizing the CLKs within the cascade between ATM and IKK. CLK2 and 4 were also confirmed as the target of active structural derivatives of MW01 and MW05 and were spared by inactive derivatives, confirming CLK2 and 4’s role in genotoxic stress-induced NF-κB. In addition, CLK inhibitor MU-1210 also inhibited NF-κB following DNA damage, suggesting that CLK inhibitors could be used to potentiate the tumor killing effect of standard cancer treatments. MW01 and MW05 were tested in co-treatment with DNA damaging agents, in the context of on-going DNA damage, and in patient derived glioblastoma cells to assess their potential clinical applications. Critically, neither MW01 nor MW05 exhibit general toxicity; instead, they notably enhance apoptosis specifically in tumor cells following genotoxic stress. In BRCA1-deficient cells and in co-treatment with PARP inhibitor Olaparib, both characterized by on-going DNA damage, MW01 and MW05 potentiated DNA damage and p53 levels, suggesting that the compounds unbalance the NF-κB/p53 axis in favor of apoptosis. This approach introduces a novel therapeutic strategy to curb NF-κB activity induced by DNA damage in cancer cells without impacting its essential functions in healthy cells.

Cell-type specification is guided by transcription factors (TFs) that control specificity and activity of transcription by binding to regulatory DNA elements, such as enhancers. TFs are modular proteins, consisting of structured DNA-binding domains, which allow binding to TF-specific DNA motifs, and intrinsically disordered regions (IDRs), which often harbor “minimal activation domains” that control the activity of the TF. Both, DNA-binding specificity and transcriptional activity of TFs have been extensively studied using ChIP-sequencing and activation domain screens. However, the mechanisms by which TFs establish specific gene expression programs remain poorly understood, as TF-binding or the presence of a minimal activation domain within a TF IDR do not necessarily correlate with target gene expression. Recent studies suggest that non-linear sequence features of TF IDRs facilitate the formation of transcriptional condensates, contributing to both the activity and specificity of TFs, thus indicating a relationship between these two features.

In the following, I provide evidence for an evolutionary trade-off between the activity and specificity in human transcription factors encoded as submaximal dispersion of aromatic residues in their IDRs. I identified multiple human TFs that display significant dispersion of aromatic residues in their IDRs, resembling imperfect prion-like sequences. Mutation of dispersed aromatic residues reduced transcriptional activity, while increasing aromatic dispersion in multiple human TFs enhanced transcriptional activity. Furthermore, sequence optimization by increasing aromatic dispersion enhanced in vitro reprogramming efficiency, promoted liquid-like features of condensates formed in vitro, and led to more promiscuous DNA-binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. I propose that engineering of amino acid features that alter condensation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.

Verletzbarkeit als Fähigkeit und als dezidiert politische und feministische Haltung: Diesen Ansatz verfolgt eine Reihe radikal autobiografischer Schreibweisen, die in den letzten Jahren aus den sozialen Medien in den Literaturbetrieb eingezogen sind. Anhand von Beispielen aus dem deutsch-, englisch- und chinesischsprachigen Internet beschreibt Lea Schneider Ästhetiken radikaler Verletzbarkeit, ihre medialen Affordanzen sowie die Wertungskämpfe, die sie beim Eintritt in den etablierten Literaturbetrieb auslösen – bis hin zu einer Neudefinition des Literaturbegriffs selbst.

Polnische Bürgermeister waren eine wichtige Beamtengruppe im Verwaltungsapparat des Generalgouvernements. Gemeinsam mit den deutschen Kreis- und Stadthauptmännern gestalteten sie Kommunalpolitik und waren an der Verfolgung und Ermordung polnischer und europäischer Juden sowie an der Ausbeutung des Generalgouvernements maßgeblich beteiligt. Auf der Grundlage umfangreicher Archivrecherchen und neuer Kontextualisierungen stellt dieses Buch ausgewählte polnische Bürgermeister vor und zeigt, wie diese sich während des Zweiten Weltkriegs verhielten. In die Analyse werden kleine Städte wie Otwock, mittelgroße wie Tschenstochau und Metropolen wie Warschau einbezogen. Mit dieser Studie legt Grzegorz Rossoliński-Liebe ein Standardwerk vor, das eine unverzichtbare Grundlage für die Erforschung von Kommunalverwaltungen im Holocaust darstellt.

The Internet is a complex system of autonomous but cooperating networks that constitute a critical Infrastructure with a vast socio-economic significance. Any disruption of the Internet and its services has detrimental effects to its users, be it in the private sector or the industry. This is why Internet research aims for observing, mitigating, and ultimately preventing attacks.

In this thesis, we provide methodologies to evaluate and extend the coverage of attack observations, we assess the efficacy of current and emerging attack mitigation solutions, and we identify new opportunities for attack prevention. We do so by utilizing two major vantage point positions, the Internet core and the Internet edge. Our contributions have an operational impact on today's Internet but also its future deployment.

Climate change presents significant challenges for agriculture. The emission of greenhouse gases calls for a reassessment that influences the manufacture and processing of animal feed. Sustainable solutions, such as cultivating domestic grains as protein sources and adopting techniques like grazing livestock on grasslands and agroforestry, will gain importance. However, certain plants that are increasing in significance possess SPM. SPM serve various significant functions and have diverse effects on their surroundings. In addition to benefiting the plant and its environment, the primary motivation for producing these compounds is to provide a growth advantage and protection against herbivore predators, ensuring plant survival and reproduction. Nevertheless, ingestion of plants with SPM still occurs resulting in a possible risk to both animals and consumers if there is a transfer of SPM via feed into food of animal origin, especially into milk.

For some SPM transfer into the milk is already investigated while for others there is still lack of data as summarized in Chapter I.

Chapter I reviews the current literature on two secondary plant metabolites for which there is still a lack of data on their occurrence and possible transfer to milk, although there are indications that transfer may be possible. The QA are naturally occurring alkaloids in Fabaceae. Their best-known representative with a wide range of applications in animal nutrition, due to their beneficial protein content, are lupins, which are subdivided into sweet and bitter lupins depending on their QA content. QAs have multiple toxicological effects that result in a so-called anticholinergic syndrome causing among other coordination disorders, respiratory paralysis, tachyarrhythmia or cardiac arrest. Due to its chemical structure, a transfer into the milk of cows was suspected, but there are no data so far. Sapindaceae, a different botanical family, possesses recognized toxic properties. Within this family, certain SPM, namely HGA, MCPrG, and HGB, have led to significant toxic effects in humans, horses, and wild ruminants. Important representatives of the Sapindaceae are sycamore maple trees, which can be found in meadows and fields with contents on HGA, MCPrG and HGB in their seeds and seedlings. Initial studies suggest that these SPM may be transferred into the milk of mares or cows after ingestion of seeds or seedlings. Nonetheless, thorough research on the effects of these substances and their excretion in milk has not been conducted in dairy cows.

Chapter II therefore explains the aims and hypothesis of the current thesis. The main part of this thesis consists of two published manuscripts summarized in Chapter III and IV. The primary objective was on increasing knowledge on the transfer QA into the milk of dairy cows as well as on the intake of maple toxins HGA, MCPrG and HGB and their subsequent Transfer into the milk of dairy cows. These efforts aimed to provide a more comprehensive assessment of the risks posed to both animals and consumers. Furthermore, a toxicokinetic model was derived to predict the feed to food transfer for QAs (published in Engel et al. 2022, Chapter III).

The first study was conducted as a feeding trial in four Holstein-Friesian dairy cows. During the trial rapeseed meal was switched for either one or two kg of narrow-leafed lupins (L. angustifolius variety Boregine) for seven days as experimental periods with respective depuration periods. During these periods milk was sampled twice daily and analyzed on their respective QA content with an in-house validated novel LC/MS-MS method. Furthermore, milk ingredients were monitored regularly. Based on the data three-compartment toxicokinetic model was derived to predict feed to food transfer. The results reveal that an intake of 1’774 mg QA per cow per day had no effects on animal health. Thereby, the pattern of the used lupin was like those already reported for narrow-leafed lupins even though total QA content was in the upper range of reported QA contents. Already the administration of 1 kg of lupins resulted in a transfer of QA into milk with different transfer rates for all QAs. Administration of twice the number of lupins (2 kg) showed a significant dosedependent transfer of QA into milk. Calculation of individual transfer rates revealed transfer rates differing from 1.05% for isolupanine to 3.74% for multiflorine (Chapter III). With maximum QA contents in milk a preliminary risk assessment was made for high consumers (P95) indicating a potential risk for consumers in this scenario (Chapter III). Nevertheless, data on toxicokinetic and occurrence of QA in feed and food is lacking.

Chapter IV aimed to investigate if there is an intake of sycamore seedlings by dairy cows while grazing and if so, if there is a transfer of their SPM into milk without the occurrence of clinical signs as known for other herbivores like horses after SPM ingestion. For that, five cows were subjected to an observational study over 4 days. Cows had access to a pasture with numerous seedlings growing between grass over a defined period. Additionally, they received a partial mixed ration in the barn ad libitum and concentrate feed suitable for their respective milk yields. Milk of individual cows was sampled twice daily as well as bulk tank milk of the whole herd (n=87) and analyzed on their content of HGA, MCPrG, HGB and their respective metabolites with a novel validated LC/MS-MS method. Experimental plots were placed on the pasture and seedlings were counted and photographed daily before cows were allowed to graze. Additionally, cows were observed by two independent observers and intake was captured if possible. Already on the first day, intake of sycamore maple seedlings was observed in dairy cows as a by-product of grazing. Noteworthily, only respective conjugated metabolites of HGA and MCPrG were measured in milk samples already on day 1 after grazing. Urine samples revealed MCPA-G contents above contents measured in diseased Peré David’s deer without the appearance of clinical symptoms. Statistical analysis revealed an increasing trend in MCPA-G contents in milk. There is still lack of data on toxicological effects of conjugated metabolites, but cows may be in general less susceptible to maple toxin intoxication.

In conclusion, the present thesis highlights that a transfer of the investigated secondary plant metabolites into milk is possible. Current developments in relation to climate change call for a fundamental rethink of the agricultural sector. The significance of local forage and agroforestry methods is increasingly acknowledged. Further investigations are imperative to appraise the potential risk to consumers and to provide suggestions for farm management, feeding, and grazing practices. In the case of lupins, preliminary risk assessment revealed a possible Risk for certain consumer groups. However, the risk to the consumer can be further investigated and reduced by testing the lupins available on the market for their QA content, adapting the recommendations for use and carrying out additional toxicological studies. For SPM found in Sapindaceae including A. pseudoplatanus is remains uncertain whether the conjugated metabolites of HGA and MCPrG found in the milk of individual cows as well as bulk tank milk represent a potential risk to consumers. Nevertheless, both uptake by cows with apparently reduced susceptibility and the possibility of transfer were demonstrated which emphasizes the necessity to produce additional data. To promote sustainable agriculture, it is necessary to enhance the use of indigenous legumes like lupins and pasture farming. This progression necessitates a comprehensive analysis ofthe linked hazards. By establishing these potential threats, we can advance our knowledge of farming and agriculture and create positive environmental impacts that counteract climate change.

In den letzten Jahrzehnten ist durch gezielte Zucht die Wurfgröße bei Sauen gestiegen und mit dieser Entwicklung hat die Anzahl der Ferkel pro Wurf mit niedrigem Geburtsgewicht (LBW) zugenommen. Höhere Mortalitäts- und geringere Wachstumsraten werden bei LBW-Ferkeln beobachtet. Die hohen Mortalitätsraten werfen Fragen zur ethischen Rechtfertigung der Schweinehaltung auf und stehen im Kontrast zu einer ressourceneffizienten Tierhaltung. Die geringeren Wachstumsraten sind mit ökonomischen Verlusten verbunden und werden mit einer dysfunktionalen Dünndarmentwicklung von LBW-Ferkeln assoziiert. Bei abgesetzten Ferkeln wurden positive Effekte einer Glutamin (Gln)-Supplementierung auf Wachstum und Parameter der intestinalen Entwicklung beschrieben. Da die konditionell essentielle Aminosäure Gln zu einem großen Umfang in den Epithelzellen des Dünndarms verstoffwechselt wird, besteht die Möglichkeit, dass durch Gln die Entwicklung des Dünndarms bei LBW-Ferkeln normalisiert werden könnte.

Ziel dieser Studie war es sowohl die akuten als auch potentielle persistente Effekte einer neonatalen Gln-Supplementierung bei männlichen unkastrierten Ferkeln auf Wachstum und verschiedener Parameter der jejunalen Entwicklung zu untersuchen.

Hierfür wurden männlich unkastrierte Saugferkel in einem Fütterungsversuch untersucht. Die Ferkel von Jungsauen wurden direkt nach der Geburt auf Grund ihres Geburtsgewichtes in Paaren selektiert, wobei die Selektionsgrenzen für LBW bei 0,8-1,2 kg und für normalgewichtige Ferkel (NBW) bei 1,4-1,8 kg lagen. Selektierte Ferkel wurden entweder mit 1 g/kg Körpergewicht (BW) Gln oder der isonitrogenen Menge Alanin (Ala) (1,22 g/kg BW) zwischen dem ersten und dem zwölften Lebenstag oral supplementiert. Durch den Versuchsaufbau ergaben sich vier Versuchsgruppen (LBW-Gln; NBW-Gln; LBW-Ala; NBW-Ala (n =36/Versuchsgruppe)). Die Versuchstiere hatten stets die Möglichkeit, bei der Muttersau zu saugen und erhielten zusätzliches Beifutter ab dem 14. Lebenstag. Das Wachstum der Tiere wurde durch regelmäßiges Wiegen und zusätzliche zootechnische Messungen überwacht. Jeweils ein Drittel einer Versuchsgruppe wurde am fünften, zwölften sowie sechsundzwanzigsten (n=12) Lebenstag euthanasiert und das distale Jejunum inklusive Chymus wurde für weitere Analysen beprobt. Den Tieren aller Versuchsgruppen wurde eine Stunde vor der Euthanasie Bromdesoxyuridin (BrdU) zur Bestimmung der Zellproliferationsrate und L-2H5-Phenylalanin zur Bestimmung der fraktionellen Proteinsyntheserate (FPSR) intraperitoneal verabreicht. Zusätzlich wurde die Milchaufnahme mittels der Deuteriumoxid-Methode am elften bzw. am fünfundzwanzigsten. Lebenstag in entsprechenden Gruppen bestimmt.

Die ausgewerteten Parameter der jejunalen Entwicklung wurden sowohl durch die Gln-Supplementierung als auch durch das niedrige Geburtsgewicht nur geringfügig beeinflusst. Interessanterweise unterschied sich die Konzentration der supplementierten Aminosäuren im beprobten Jejunum-Gewebe und im Chymus zwischen den Supplementierungsgruppen nicht. Entsprechend wurden keine Unterschiede im Gewebe des Jejunums im Hinblick auf Morphologie, Immunzellpopulation und FPSR festgestellt. Weiterhin wurde kein Einfluss von Gln auf mRNA-Abundanz von Genen, welche den Aminosäurentransport, Aminosäurenstoffwechsel und Glutathion-Stoffwechsel regulieren, festgestellt. Für entsprechende Parameter wurden nur geringe Unterschiede zwischen den Geburtsgewichtsgruppen beobachtet. Das Alter der Ferkel hatte den stärksten Einfluss auf die Parameter der jejunalen Entwicklung. Folglich wurden Unterschiede in Bezug auf Morphologie, Aminosäurenmuster, Zellpopulation, Nukleinsäure-Konzentration und mRNA-Abundanz von Genen des Glutathionmetabolismus zwischen den zwölf und fünf Tage alten Ferkeln festgestellt.

Das BW von LBW-Gln-Ferkeln war sowohl während der akuten Supplementierung als auch darüber hinaus höher als bei LBW-Ala-Ferkeln. Jedoch war das BW am 26. Lebenstag zwischen LBW-Gln-Ferkeln und LBW-Ala-Ferkeln nicht unterschiedlich. Das höhere BW der LBW-Gln-Ferkel kann mit einer vergrößerten jejunalen Absorptionsfläche assoziiert werden. Das Wachstum von NBW und folglich das BW waren höher im Vergleich zu LBW. Das schnellere Wachstum beruht dabei nicht auf Unterschieden der intestinalen Entwicklungsparameter, da negative Effekte von einem niedrigen Geburtsgewicht auf die Darmentwicklung aus den gemessenen jejunalen Entwicklungsparametern nicht abzuleiten sind. Es wurden in Bezug auf jejunale Entwicklungsparameter, wie Zellproliferationsrate, Zellpopulation und Morphologie, Unterschiede zwischen den zwölf und 26 Tage alten Ferkeln festgestellt.

Abschließend lässt sich feststellen, dass eine orale Gln-Supplementierung das Wachstum von LBW-Ferkeln nur kurzfristig verbesserte, jedoch nur in sehr geringem Umfang Parameter der jejunalen Entwicklung beeinflusste. Außerdem wurde festgestellt, dass LBW nicht mit einer Entwicklungsstörung des Jejunums assoziiert ist und folglich nicht für das verlangsamte Wachstum verantwortlich ist. Schließlich wurde aufgezeigt, dass besonders ontogenetische Faktoren die Parameter der jejunalen Entwicklung beeinflussen.

Angiogenesis is the dynamic process of building new blood vessels from pre-existing ones. During sprouting angiogenesis, tip cells are mainly responsible for migration, stalk cells for proliferation and phalanx cells for maturation of the new vessel. Major research effort was directed to anti- and pro-angiogenic therapy. Therefore, in vitro models are frequently used. However, these assays face problems regarding reproducibility based on single stage assays and the inhomogeneous character of endothelial cells (ECs). When establishing and performing the all-in-one assay, which covers all stages of in vitro angiogenesis, differences regarding angiogenic potency was detected resulting in a classification of ECs into angiogenic and non-angiogenic. Proteome expression profiles of both classes exhibited one protein only found in non-angiogenic ECs, i.e. adenosylmethionine synthetase isoform type 2 (MAT2A), and seven proteins exclusively in angiogenic ECs. MAT2A represents a highly conserved enzyme being mainly involved in regulatory functions and suggested to have anti-angiogenic effects. Among the seven proteins found in angiogenic ECs, vimentin (VIM) and triosephosphate isomerase (TPI) are hypothesized to have pro-angiogenic impact on ECs. By VIM being a type III intermediate filament protein, it is highly involved in cell shape and motility. TPI is a glycolic enzyme generating energy and mainly influencing cell proliferation.

Both studies aimed to determine whether the expression of VIM, TPI and MAT2A is related to angiogenesis in vitro in human dermal microvascular endothelial cells (HDMECs). Therefore, two batches of HDMECs were long-term cultivated using pro-angiogenic media. Quantification of in vitro angiogenesis was carried out using phase-contrast microscopy twice a week. Knockdown groups got infected with lentiviral particles initiating a knockdown of VIM or TPI respectively. Additionally, a non-coding sequence was used for the infection of control groups. At days 1, 5, 25 and 50, cells of all groups were harvested and used for mRNA and Protein expression analysis. The mRNA expression of vascular endothelial growth factor 1 (VEGFR-1), vascular endothelial growth factor 2 (VEGFR-2), and mRNA and protein expression of VIM, TPI and MAT2A were determined by RT-qPCR and Western Blot.

In native cells, VIM was shown to be expressed mainly in beginning stages of sprouting and migrating, which are suggested to be enabled by VIM’s influence on the cytoskeleton. By knocking down VIM, cell death and a deceleration of in vitro angiogenesis was observed leading to the conclusion of VIM being an essential protein for HDMECs survival and having pro-angiogenic effects on ECs. For TPI, native cells showed an overall increase in Expression over the cultivation period. By TPI providing energy for cells, it is suggested to be essential for most angiogenic stages, i.e. cell migration, proliferation, and tube formation. By knocking down TPI, a deceleration of in vitro Angiogenesis was observed, leading to the assumption of TPI increasing the angiogenic potency of ECs. Furthermore, native TPI expression was shown to be consistent. The single decrease of TPI expression was connected to cell death, showing the essential character of TPI for HDMECs survival. Native MAT2A expression was highest at the beginning of cultivation, followed by a significant decrease. Moreover, a final increase was detected. With MAT2A owning regulatory functions via methylations, it is suggested to have anti-angiogenic effects, mainly expressed in quiescent cells and involved in maturation of HDMECs. In both knockdown studies, MAT2A displayed an overall tendency of being increased when lowering the angiogenic potency, supporting the assumption of MAT2A impacting anti-angiogenic events. By characterizing both batches of HDMECs via VEGFR-1 and VEGFR-2 expression, it was shown that HD1 comprised a higher amount of stalk cells. The difference in distribution of cell differentiation led to a divergence in angiogenic, compensatory and expressional behaviour of the batches. HD1 inherited a higher proliferative power which was visible in them having a higher cell density and higher values of sums of assigned stages. With HD1 owning more stalk cells, VIM expression was lower and TPI was significantly higher in contrast to HD2. Furthermore, HD2 displayed an acceleration of in vitro angiogenesis, which was connected to higher MAT2A Ct-values. Additionally, knockdown cells of HD1 were able to recover from infection by increasing their cell density and progressing to further angiogenic stages, while knockdown cells of HD2 were either stagnating their differentiation or displaying cell death. Further, control group of HD2 displayed a deceleration of in vitro angiogenesis while control group of HD1 was unaffected. Overall, it has been shown that the characterization of cell differentiations is of immense importance for EC application and the interpretation of in vitro angiogenesis assays.

Experiments should get repeated including cells from different distributors involving nonangiogenic cells, cell characterization should be extended by an additional detection method and specific assays should get employed for validating protein effects. For comprehension to living systems, subsequent experiments should involve more than one cell type, followed by in vivo studies. MAT2A knockdown should be initiated and further proteins influencing the angiogenic potency should be examined. Furthermore, upcoming investigations should Focus on developing and optimizing the characterization of EC populations to increase reproducibility and reliability of studies in the field of in vitro angiogenesis.

Die zunehmende Unwirksamkeit gängiger Medikamente gegenüber immer mehr auftretende multiresistente Bakterien erhöht den Bedarf an neuen Behandlungsmöglichkeiten. Das gramnegative Bakterium Pseudomonas aeruginosa ist ein Beispiel dieser Bakterien, welches für verschiedene nosokomiale Infektionen, insbesondere für Pneumonien, und den rasanten Erwerb von multiplen Resistenzen bekannt ist. Eine vielversprechende mögliche Alternative oder Ergänzung zu herkömmlichen Therapien sind Bakteriophagen, auch bekannt als Phagen. Diese Viren infizieren ausschließlich Bakterienzellen, vermehren sich in ihnen und lysieren sie. Obwohl allgemein anerkannt ist, dass Phagen unter Laborbedingungen in der Lage sind, (antibiotikaresistente) Bakterien zu lysieren, gibt es noch wenig Wissen darüber, welche Immunreaktionen sie im Zielgewebe, wie z. B. der Lunge, auslösen. Die Kenntnis darüber ist jedoch von entscheidender Bedeutung, um die Sicherheit der Phagentherapie beurteilen und sie erfolgreich beim Menschen einsetzen zu können. Das Ziel dieses Promotionsvorhabens war es, in ausgewählten Aspekten die Sicherheit und Verträglichkeit eines Bakteriophagencocktails in der Behandlung gegen eine Lungeninfektion mit P. aeruginosa präklinisch zu evaluieren. Dafür wurden die Bakteriophagen gegen P. aeruginosa ex vivo in einem Lungengewebekultur-Modell und in vivo in einem murinen Pneumonie-Modell untersucht.

Die Applikation der Einzelphagen JG005 und JG024 und eines Phagencocktails, bestehend aus JG005, JG024 und BHZ17, führten im Ex-vivo-Lungeninfektionsmodell zur Hemmung des bakteriellen Wachstums. Dieses Ergebnis konnte nur mit der MOI von 1000 oder höher erzielt werden. Der Einzelphage BHZ17 war bei keiner Dosierung in der Lage, die bakterielle Last in den Lungenstücken zu reduzieren. Die Applikation des Phagencocktails führte bei den mit DSM 107574, einem klinischen Isolat von Pseudomonas aeruginosa, infizierten Lungenstücken zu einer Erhöhung und bei den mit DSM 107570, einem weiteren klinischen Isolat von Pseudomonas aeruginosa, infizierten Lungenstücken zu einer Erniedrigung der IL-1β Ausschüttung.

Durch die Applikation des Phagencocktails konnten keine negativen Einflüsse auf das klinische Bild der Versuchstiere beobachtet werden. Die Phagentherapie führte zu einem verbesserten Körpertemperaturverlauf der infizierten Mäuse und zu weniger Bakterienlast 12 Stunden p.i. in der Lunge und BALF der infizierten Mäuse. Auf die Lungengefäßpermeabilität hatte die Behandlung mit Phagen keinen Einfluss. Mit Hilfe der Phagen konnte die systemische Erhöhung der Leukozytenanzahl durch die Infektion verhindert werden. Auf lokaler Ebene betrachtet, erhöhte die Behandlung mit Phagen den Prozentsatz polymorphkerniger Leukozyten in der bronchioalveolären Lavageflüssigkeit bei scheininfizierten Tieren zu allen Untersuchungszeitpunkten. Die Befunde der lokalen Immunreaktion wurden weiterhin durch die Ergebnisse der histopathologischen Auswertung bestätigt. Dort zeigten die scheininfizierten phagenbehandelten und placebobehandelten Tiere minimale bis geringgradige neutrophile alveoläre sowie perivaskuläre und peribronchiale Infiltrate. Die histopathologische Untersuchung zeigte außerdem keine signifikanten Unterschiede im Hinblick auf Grad der Pneumonie und Ödembildung zwischen der P. aeruginosa-infizierten und phagenbehandelten Gruppe im Vergleich zu der P. aeruginosa-infizierten und Placebo behandelten Gruppe. Durch die Applikation der Phagen kam es teilweise zur Verringerung und teilweise zur Erhöhung von Zytokinen auf lokaler und auf systemischer Ebene. TNF-α und GM-CSF waren in scheininfizierten mit Phagen behandelten Tieren 12 Stunden p.i. auf lokaler Ebene erhöht. Die Applikation der Phagen führte zu einer Reduktion der IL-1β Ausschüttung 12 Stunden p.i. auf lokaler und systemischer Ebene in den mit P. aeruginosa infizierten Tieren.

Die Ergebnisse dieser Studie zeigen die Fähigkeit der Phagen, Bakterienlast zu reduzieren, und damit ihr Potenzial als mögliches Therapeutikum. Trotz des Auftretens von leichten Immunreaktionen in den verschiedenen Modellen konnten keine klinisch sichtbaren unerwünschten Wirkungen im Mausmodell beobachtet werden. Die vorliegende experimentelle Studie unterstützt die Entwicklung eines Cocktails von Bakteriophagen für die klinische Therapie von P. aeruginosa Infektionen der Lunge.

The three studies in this dissertation investigate the interdependent influence of various environmental features in Urban Green Space (UGS) on human thermal comfort at the microscale. This research pertains to microscale urban climatology. Urban climatology is an interdisciplinary field focused on advising urban construction to achieve the preferred urban climate. Microscale urban climatological studies have more application potential than those at meso- and local scales. Yet, current limitations in microscale urban climatological research hinder realizing this potential. Firstly, the costs of microscale studies are generally high. Secondly, it is difficult to apply the research results elsewhere. As a results, the high-cost microscale studies must be continuously repeated in various locations. The challenge of generalizing research results is a common limitation in urban climatology. Approaches have been proposed to alleviate this limitation in urban climatological studies at meso- and local scales. However, there is a lack of such attempts at the microscale. This research aims to propose an approach contributing to the generalization of research results in microscale urban climatological studies. This approach upgrades from the previous single-parameter approach to a multi-parameter approach. The multi-parameter approach views the UGS holistically by exploring the interdependent influence of various environmental features on human thermal comfort. The interdependent influence can enhance the comprehensiveness of results, improve the results generalization, and facilitate the knowledge transfer into specific guidelines for climate-adaptative UGS design and planning. To explore the interdependent influence, the multi-parameter approach utilizes the factorial experimental design. The microclimate model ENVI-met is used to simulate scenarios in the factorial experiments. The simulations are conducted on idealized and simplified scenarios to minimize the research costs. To control the research cost, the Latin Hypercube Sampling (LHS) design is adopted to sample a subset of scenarios from the factorial experiment for field measurement and simulation. For result applicability, this research evaluates UGS cooling effects using both climatic parameters and biometeorological index -- Physiological Equivalent Temperature (PET). PET values in this research are calculated using the RayMan model. This thesis comprises three independent studies on the interdependent influence of various environmental features of UGS on human thermal comfort. The following common conclusions can be drawn from these three studies. Firstly, different environmental features influence the cooling effects of UGS in an interdependent way. Specifically, the correlation between an environmental feature and the PCI effect depends on various environmental features. Secondly, the multi-parameter approach is significantly superior to the single-indicator approach in comprehensively and accurately capturing the interdependent influence. Additionally, these three studies provide specific and generalizable suggestions for the climate-adaptative UGS design and planning, demonstrating the capability of the multi-parameter approach to enhance the applicability and generalizability of findings in urban climatological studies. The characteristics of the multi-parameter approach include the following three aspects. At the practical level, it can provide specific guidelines for practical UGS design and planning; at the research level, it explores the interdependent influence of multiple environmental features on human thermal comfort; and at the philosophical level, it adopts the holistic view. This research is not only an attempt to enhance methodology and promote the application of research results, but also a manifestation of the philosophical paradigm shift in urban climatology.

During the last decades virus infections posed a serious threat to humanity and have been studied intensively. However, the inadequate knowledge was evident during the global outbreak of the SARS-CoV-2 pandemic in recent years and demonstrated the need for further research and drug development. 3C and 3CL proteases of Riboviria are validated drug targets for the treatment of virus infections with a unique substrate specificity for glutamine in the S1 site. Numerous reported and approved protease inhibitors are peptidomimetics, however, they display limitations in pharmacokinetic and pharmacodynamic properties. Here, I present the design and evaluation of small molecule inhibitors against two proteases of Riboviria species using fragment-based methods. Furthermore, two different glutamine mimetics are validated for binding into the S1 pocket, namely the benzamide and the pyridine providing similar interactions as the native glutamine residue. The enterovirus D68 3C protease was used as a template for the evolution of Michael acceptors through a protein-templated Knoevenagel reaction and presented the first reported protein-templated carbon-carbon coupling. Further analysis of the resynthesized Michael acceptors regarding their binding modes, stability, and kinetics with enzyme assays, isothermal titration calorimetry, biolayer interferometry, HPLC-QTOF-MS, and protein MS suggested covalent reversible inhibitors with prolonged residence time at the protein to be the most active antivirals. Further investigations of chemical and kinetic stabilities of prominently reported and newly synthesized acylating fragment inhibitors of the main protease of SARS-CoV-2 confirmed the importance of the stability of the protease-inhibitor complexes. Despite improvements in the chemical stability, synthesized pyridinyl carbonates could not inhibit the proliferation of SARS-CoV-2 in infected cells probably due to the high vulnerability against hydrolysis of the acylated enzymes. Therefore, a library of pyridine fragments with different electrophiles was synthesized and tested for their inhibitory activity, producing a more stable covalent fragment inhibitor with aldehyde function and high ligand efficiency as a promising starting point for the evolution into a drug-like molecule. Finally, protein-templated Ugi reactions were investigated with fluorescence-based enzyme assays, HPLC-QTOF-MS, and native MS using the SARS-CoV-2 main protease as template, however, without the finding of a protein-catalyzed effect on the multicomponent reaction. With the obtained knowledge, an in-situ screening for the development of noncovalent inhibitors against 3C and 3CL proteases was generated and validated with reported Ugi product inhibitors of the main protease of SARS-CoV-2. With the high significance of multicomponent reactions (MCRs) in drug synthesis and the limited knowledge about protein-templated MCRs, this work constitutes an important contribution to the understanding of these reactions. The summarized results in this thesis present new and important insights into the development of fragment-based inhibitors and can be used to obtain potent protease inhibitors.