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Mechanism of the MHC I chaperone TAPBPR and its role in promoting UGGT1-mediated quality control
(2022)
Information about the health status of most nucleated cells is provided through peptides presented on major histocompatibility complex I (pMHC I) on the cell surface. T cell receptors of CD8+ T cells constantly monitor these complexes and allow the immune system to detect and eliminate infected or cancerous cells. Antigenic peptides displayed on MHC I are typically derived from the cellular proteome and are translocated into the lumen of the endoplasmic reticulum (ER) by the ATP-binding cassette (ABC) transporter associated with antigen processing (TAP), which is part of the peptide-loading complex (PLC). In a process called peptide editing, the MHC I-dedicated chaperone tapasin (Tsn) selects peptides for their ability to form stable complexes with MHC I. While initial peptide loading is catalyzed in the confines of the PLC, the second quality control is mediated by TAPBPR, operating in the peptide-depleted cis-Golgi network. TAPBPR was shown to have a more fine-tuning effect on the presented peptide repertoire rather than initial peptide selection. The fundamental mechanism of peptide editing was illuminated by two crystal structures of TAPBPR in complex with peptide-receptive MHC I. Notably, one of these structures reported a structural element that inserted into the peptidebinding pocket. The so-called scoop loop was assumed to be involved in mediating peptide exchange but the underlying mechanism remained undefined. Additionally, latest results suggested that TAPBPR mediates the interaction of the glucosyltransferase UGGT1 with peptide-receptive MHC. To expand the current knowledge of quality control processes in the antigen presentation pathway, the contribution of the scoop loop in peptide editing and the role of TAPBPR in UGGT1-mediated quality control needs to be elucidated. In the first part of this study, TAPBPR proteins with various loop lengths were designed to scrutinize the contribution of the scoop loop in chaperoning peptidereceptive MHC I. In a light-driven approach, the ability of TAPBPR variants to form stable complexes with peptide-free MHC I was tested. These results demonstrated that in a peptide-depleted environment, the scoop loop is of critical importance for TAPBPR to chaperone intrinsically unstable, peptidereceptive MHC I clients. Moreover, fluorescence polarization-based assays allowed the pursuit of peptide exchange in different, native-like environments. Peptide displacement activities of TAPBPR variants illustrated that catalyzed peptide editing is primarily induced by structural elements outside the scoop loop. In a peptide-depleted environment, the scoop loop occupies the position of the peptide C-terminus and acts as an internal peptide surrogate. By combining complex formation and fluorescence polarization experiments, the scoop loop of TAPBPR was shown to be critically important in stabilizing empty MHC I and functions as an internal peptide selector. In the second part of this study, a novel in-vitro glucosylation assay was established to examine the role of TAPBPR in UGGT1-catalyzed re-glucosylation of TAPBPR-bound MHC I clients. Therefore, a peptide-free MHC I-TAPBPR complex with defined glycan species was designed which served as physiological substrate for UGGT1. By subjecting the recombinantly expressed HLA-A*68:02- TAPBPR complex and UGGT1 proteins to the new in-vitro system, UGGT1 was shown to catalyze the transfer of a glucose residue to the N-linked glycan of TAPBPR-bound Man9GlcNAc2-HLA-A*68:02. Moreover, a high-affinity, photocleavable peptide was applied to dissociate the MHC I-chaperone complex. However, in the absence of TAPBPR, no glucosyltransferase activity was observed. Generation of peptide-free MHC I through UV illumination also showed no activity, and only the addition of TAPBPR could restore UGGT1-mediated reglucosylation of the empty MHC I. Independent of the peptide status of HLAA*68:02, the combination of protein glycoengineering and LC-MS analysis implicated that UGGT1 exclusively acts on TAPBPR-chaperoned HLA-A*68:02. The newly established system provided insights into the function of TAPBPR during UGGT1-catalyzed re-glucosylation activity and quality control of MHC I. Taken together, the scoop loop allows TAPBPR to function as MHC I chaperone through stabilizing peptide-receptive MHC I. In a peptide-depleted environment, the loop structure serves as an internal peptide surrogate and can only be dislodged by a high-affinity peptide. Based on these findings, TAPBPR fulfills a dual function in the second level of quality control. On the one hand, TAPBPR functions as peptide editor, shaping the repertoire of presented peptides. On the other hand, TAPBPR mediates peptide-receptive MHC I clients to the folding sensor UGGT1. Here, TAPBPR is essential to promote UGGT1-catalyzed reglucosylation of the N-linked glycan, giving MHC I a second chance to be loaded with an optimal peptide cargo in the peptide loading complex.
Electrospinning is a versatile and promising drug delivery technology for the development of tailor-made drug delivery systems for various clinical applications. By applying high voltages to drug-loaded polymer solutions, solid polymeric nanofibers can be generated, which encapsulate active pharmaceutical ingredients (APIs) into their polymer matrix. During the electrospinning process, the fibers are deposited on a collector and form a nonwoven network of drug-loaded polymer fibers. These fibers are spatially distributed in aligned or random orientation, providing the opportunity to design highly tunable structural and mechanical properties, which can be adapted to the biological requirements of the intended application site. The mechanically flexible fiber networks can therapeutically be administered to a multitude of pharmaceutical application sites. Their highly porous fiber structure exhibits a large surface-to-volume ratio, which is ideal for controlled drug release kinetics from the polymer matrix upon contact with biological fluids, such as tear fluid, saliva, mucus, wound exudate or gastro-intestinal fluid. For application at the target site, fiber mats are cut into patches. As the patch size determines the quantity of applied API, the electrospinning process must ensure homogeneous distribution of the API throughout the entire fiber mat area.
In this thesis, electrospinning was established as a formulation technology for the rational fabrication of tailor-made multifunctional drug carrier systems for local and site-specific drug delivery to the epithelial interfaces skin, oral mucosa as well as cornea. For adequate characterization and analysis of the drug delivery systems, a broad panel of robust and predictive analytical tools, based of novel investigation techniques for physicochemical characterization of electrospun fibers, was developed.
The initial part of the thesis thematically focuses on the development of predictive analytical techniques, to determine fiber morphology and physicochemical properties, as well as fiber composition and drug release. By designing two model formulations with contrasting properties, and subsequent analysis and characterization with a set of newly developed techniques and state-of-the-art methods, a comprehensive toolset has been made available and evaluated, aiming at advancing and standardizing respective techniques in the scientific field of electrospun drug delivery systems.
Starting with the initiation of the electrospinning formulation process, which often relies on empirical data rather than analytical methods to predict successful processability, analysis of rheological properties of electrospinning solutions was used to rationally detect the minimum polymer concentration required for electrospinning.
For analysis of fiber morphology, scanning electron microscopy is a common technique. However, little attention is given to underlying readout parameters. By analyzing the fiber orientation and diameter of the respective fibers, predictive results regarding mechanical properties could be obtained, which were subsequently confirmed by measuring elongation force with tensile testing. Confocal Raman microscopy, a label-free method for chemically- selective imaging of the fiber samples, was introduced as a complementary visualization technique, enabling the detection of fiber composition and drug distribution.
A novel technique for investigation of water contact angles on the fiber surface of highly hydrophilic polymers was introduced, which provides predictive data regarding interaction with body fluids and the resulting drug release kinetics. Subsequent release testing in a newly developed setup for analyzing drug release from electrospun fibers in low-volume body compartments, confirmed the anticipated drug release kinetics from measurement of the surface hydrophilicity.
By combining complementary analytical methods, including spectral composition analysis, morphology visualization, characterization of physico-chemical properties and drug release kinetics, as well as the application of multivariate data analysis, a robust and predictive toolset has been established, which can support comparability of future electrospinning studies and the translation from the lab bench into clinics.
Based on the analytical toolset, the main part of the thesis focuses on the development and preparation of electrospun platform drug delivery systems for application on epithelial barriers. Electrospun fiber mats are thin, flat, and mechanically flexible, which allows close adherence to epithelial surfaces and reduction of diffusion paths, which enables efficient drug delivery to the skin, oral mucosa, as well as the cornea.
Electrospun fibers bear a high potential for application as wound dressings, while simultaneously controlling the local delivery of APIs to the wound area. Their close resemblance to the extracellular matrix of human skin provides a suitable microenvironment for cellular proliferation and migration for wound closure. In this work, insulin, a fragile proteohormone with growth factor characteristics, was successfully encapsulated into the core of coaxially electrospun fibers, thus maintaining bioactivity throughout and after the electrospinning process. The shell has been designed from biocompatible polymers, which, upon contact with aqueous wound exudate, partially dissolve and form pores through which bioactive insulin is released in a controlled manner. The shell layer provides a hydrophilic surface for interaction with body fluids and skin cells, and possesses substantial mechanical strength, flexibility, and high tensile elongation required for application on wounds. The biocompatibility of the wound dressing was investigated by interaction with primary human dermal fibroblasts and keratinocytes, which displayed healthy cell morphologies without indicating any elevated levels of cytotoxicity markers.
To investigate the effect of insulin on cell migration, in vitro scratch assays on human skin cells were performed. Increased cellular migration speed and wound closure could be observed, indicating improved wound healing. Bio relevance of in vitro wound healing potential results was advanced by development of 3D ex vivo human epidermal skin wound models from reduction surgery donor material. These complex wound models were treated with electrospun insulin fibers and analyzed by proteome analysis to reveal significant increases in wound healing-associated signaling pathways, which could be attributed to a material-driven remarkably positive impact on wound healing of the electrospun fibers...
Die digitale Revolution stellt viele traditionelle Industrien vor große Herausforderungen. Auf dem Finanzmarkt werden innovative Geschäftsmodelle geschaffen, die die Rahmenbedingungen, unter denen Finanzprodukte und -dienste angeboten werden, drastisch verändern. Infolgedessen entstehen rechtliche Unsicherheiten sowohl für die Marktakteure als auch für die Aufsicht. Diese Unsicherheiten weisen auf die Notwendigkeit hin, den Rechtsrahmen an die technologische und ökonomische Entwicklung anzupassen. Im Rahmen dieser Dissertation werden die Herausforderungen für das Aufsichts- und Wettbewerbsrecht untersucht, die die digitale Transformation des Finanzmarktes verursacht. Der Finanzmarkt wird vor allem durch die Entstehung von FinTechs, durch das Eintreten von BigTechs in den Finanzbereich und durch die Veränderung der Produkte und Dienstleistungen traditioneller Anbieter auf der Grundlage moderner Technologien transformiert. Die Arbeit gibt einen Überblick über die zentralen innovativen Geschäftsmodelle, der mit den zahlreichen praxisrelevanten Beispielen begleitet wird. Anschließend folgt eine Beurteilung des Umfanges und der Effizienz der vorhandenen und vorgeschlagenen aufsichtsrechtlichen Vorschriften. Diese Erkenntnisse dienen als Grundlage für die wettbewerbsrechtliche Analyse des Finanzmarktes mit dem Fokus auf die plattform- und algorithmenbasierten Geschäftsmodelle im zweiten Teil der Dissertation. Da werden wettbewerbsrechtliche Konstellationen betrachtet, die zwischen konkurrierenden innovativen Geschäftsmodellen entstehen. Die Verfasserin befasst sich mit der Problematik der Definition des relevanten Marktes bei den algorithmen- und plattformbasierten Geschäftsmodellen, der Beurteilung ihrer Marktmacht, den möglichen Auswirkungen der Netzwerkeffekte auf die mehrseitigen Plattformen. Ferner werden solche Aspekte wie Datenzugriff als wettbewerbsrechtlicher Faktor und die Rolle der Algorithmen für die Durchführung wettbewerbswidrigen Praktiken analysiert. Infolgedessen wird ein Überblick über mögliche kartellrechtliche Probleme gegeben, die im Rahmen der Digitalisierung im Finanzmarkt auftreten können, sowie die Vorstellungen darüber, wie sich Wettbewerbsfaktoren verändern sollten, wenn die Anwendung der traditionellen Konzepte des Wettbewerbsrechts nicht immer möglich oder sinnvoll ist. Es werden die Bereiche identifiziert, in denen das europäische und deutsche Wettbewerbsrecht derzeit nicht in der Lage ist, die von den innovativen Geschäftsmodellen stammenden Herausforderungen effektiv zu bewältigen. Im dritten Teil der Dissertation wird erörtert, wie sich das Aufsichts- und das Wettbewerbsrecht ergänzen und zusammen ein System der Ex-ante- und Ex-post-Regulierung bilden. Es wird dargestellt, wie die aufsichtsrechtlichen Anforderungen an die Finanzinstitute, FinTechs und BigTechs mit den ausgewählten Wettbewerbsergebnissen im Finanzsektor zusammenhängen. Darüber hinaus erklärt die Verfasserin, inwiefern einige aufsichtsrechtliche Vorschriften wettbewerbsnachteilig oder wettbewerbsfreundlich auswirken können und warum die Koordination zwischen den aufsichts- und wettbewerbsrechtlichen Instrumenten und Ansätzen essenziell ist. Schließlich werden die Vor- und Nachteile unterschiedlicher Optionen zur Regulierung innovativer Geschäftsmodelle auf dem Finanzmarkt auf einer abstrakteren Ebene erwogen.
People can describe spatial scenes with language and, vice versa, create images based on linguistic descriptions. However, current systems do not even come close to matching the complexity of humans when it comes to reconstructing a scene from a given text. Even the ever-advancing development of better and better Transformer-based models has not been able to achieve this so far. This task, the automatic generation of a 3D scene based on an input text, is called text-to-3D scene generation. The key challenge, and focus of this dissertation, now relate to the following topics:
(a) Analyses of how well current language models understand spatial information, how static embeddings compare, and whether they can be improved by anaphora resolution.
(b) Automated resource generation for context expansion and grounding that can help in the creation of realistic scenes.
(c) Creation of a VR-based text-to-3D scene system that can be used as an annotation and active-learning environment, but can also be easily extended in a modular way with additional features to solve more contexts in the future.
(d) Analyze existing practices and tools for digital and virtual teaching, learning, and collaboration, as well as the conditions and strategies in the context of VR.
In the first part of this work, we could show that static word embeddings do not benefit significantly from pronoun substitution. We explain this result by the loss of contextual information, the reduction in the relative occurrence of rare words, and the absence of pronouns to be substituted. But we were able to we have shown that both static and contextualizing language models appear to encode object knowledge, but require a sophisticated apparatus to retrieve it. The models themselves in combination with the measures differ greatly in terms of the amount of knowledge they allow to extract.
Classifier-based variants perform significantly better than the unsupervised methods from bias research, but this is also due to overfitting. The resources generated for this evaluation are later also an important component of point three.
In the second part, we present AffordanceUPT, a modularization of UPT trained on the HICO-DET dataset, which we have extended with Gibsonien/telic annotations. We then show that AffordanceUPT can effectively make the Gibsonian/telic distinction and that the model learns other correlations in the data to make such distinctions (e.g., the presence of hands in the image) that have important implications for grounding images to language.
The third part first presents a VR project to support spatial annotation respectively IsoSpace. The direct spatial visualization and the immediate interaction with the 3D objects should make the labeling more intuitive and thus easier. The project will later be incorporated as part of the Semantic Scene Builder (SeSB). The project itself in turn relies on the Text2SceneVR presented here for generating spatial hypertext, which in turn is based on the VAnnotatoR. Finally, we introduce Semantic Scene Builder (SeSB), a VR-based text-to-3D scene framework using Semantic Annotation Framework (SemAF) as a scheme for annotating semantic relations. It integrates a wide range of tools and resources by utilizing SemAF and UIMA as a unified data structure to generate 3D scenes from textual descriptions and also supports annotations. When evaluating SeSB against another state-of-the-art tool, it was found that our approach not only performed better, but also allowed us to model a wider variety of scenes. The final part reviews existing practices and tools for digital and virtual teaching, learning, and collaboration, as well as the conditions and strategies needed to make the most of technological opportunities in the future.
Protein biosynthesis is a fundamental process across all domains of life. Polypeptides are produced by translating the genetic information of the messenger RNA (mRNA) into amino acids. This elaborate procedure is divided into the four distinct phases: initiation, elongation, termination, and ribosome recycling. The phases are controlled and regulated by a multitude of translation factors. During initiation, the ribosome assembles on the mRNA. Initiation factors (IFs) bind to the small ribosomal subunit (SSU) and assist the recruitment of mRNA and initiator transfer RNA (tRNA), which delivers the first amino acid methionine. After positioning the SSU at the start codon of the mRNA, additional IFs support the joining of the large ribosomal subunit (LSU). Next, elongation factors (EFs) deliver amino-acylated tRNAs (aa-tRNAs) to the translating ribosome and assist kinetic proofreading and ribosome subunit translocation after the catalytic transfer of the polypeptide onto the aa-tRNA. When a stop codon is reached, translation is terminated by release factors (RFs) that hydrolyze the peptidyl-tRNA to release the nascent protein chain. Afterwards, the ribosome is recycled in Eukaryotes and Archaea by the conserved and essential factor ABCE1, which splits the ribosome into the LSU and SSU. ABCE1 remains bound to the SSU forming the post-splitting complex (post-SC). mRNA translation closes into a cycle by recruitment of IFs to the post-SC and the start of a new round of initiation. The post-SC presents the platform for translation initiation. However, the role of ABCE1 in initiation remains elusive. Therefore, the main goal of my thesis was to unravel the molecular mechanism of ABCE1 on the post-SC and during initiation complex (IC) assembly.
Using a reconstituted system, the high-resolution structure of the archaeal post-SC was solved by cryogenic electron microscopy (cryo-EM) following the native splitting route. It was the first complete model of an archaeal SSU at atomic resolution and revealed a previously undescribed ribosomal protein, which we termed eS21. The hinge 2 region of ABCE1 was identified to be the major interaction interface that anchors to the SSU. Functional characterization of single residue mutations in hinge 2 unraveled essential interactions with the ribosomal RNA backbone of the SSU. Sensing of SSU-binding was found to be allosterically transmitted to the nucleotide-binding sites (NBSs) for integration into the ATPase cycle of ABCE1.
Reconstitution of the archaeal translation apparatus allowed for dissection of IC assembly in the presence of ABCE1. Three different ICs were resolved by cryo-EM. The results were in accordance with recent structural findings of eukaryotic translation initiation and highlighted that the involvement of ABCE1 is conserved.
In a semi-native approach, recombinant ABCE1 was pulled-down from crenarchaeal cell lysates. Mass spectrometric analysis of co-immunoprecipitated ribosomal complexes identified the association of numerous translation factors to the post-SC in a cellular context. The establishment of the genetic toolbox of the acidothermophilic Sulfolobus acidocaldarius allowed the homologous expression of ABCE1. Pull-down of native ABCE1 revealed similar ribosomal complexes as the semi-native and reconstituted approaches. Together, my results gave first physiological relevance of ABCE1 involvement in mRNA translation initiation in Archaea. Native archaeal ABCE1-ICs were vitrified for structural analysis by cryo-EM. Thereby, future structural analysis will allow to analyze the interactions of ABCE1 on native ICs and identify its role in IC assembly.
To address the molecular process of IC assembly, the binding affinity of aIF1 to the SSU was determined by fluorescence polarization. Similar studies will allow for a detailed functional analysis on IF recruitment to the SSU in presence of ABCE1.
mRNA surveillance and ribosome-associated quality control (RQC) mechanisms evolved to ensure cell viability. The pathways overcome ribosome stalling and defective translation components. Stalled ribosomes are terminated by special RFs, which do not hydrolyze the peptidyl-tRNA, but allow dissociation of the ribosome by ABCE1. Faulty messages are degraded via mRNA decay pathways and the LSU is rescued by RQC factors. Recently, the bacterial RQC factor MutS2 was identified to specifically target collided di- and polysomes but its molecular mechanism remains unknown. In this thesis, initial functional analyses showed tri-phosphate specific nucleotide binding of MutS2. While the dissociation of collided disomes by MutS2 could not be observed, the results pave the way for future in vitro studies of bacterial RQC factors acting on specific ribosome populations.
In the future, mRNA translation research must focus on complex quality control processes to comprehensively understand this fundamental cellular process in a holistic context.
Einleitung: Knochenersatzmaterialien gewinnen in der regenerativen Medizin immer weiter an Bedeutung. Polylactid-co-Glycolid (PLGA) wird als etabliertes synthetisches Biomaterial bereits vielseitig in der medizinischen Versorgung eingesetzt, häufig in Form von Mikropartikeln oder Platzhaltern beladen mit Antibiotika oder immunaktiven Zellen zur topischen Antibiotikatherapie und Freisetzung von regenerativen Zellen in einer Defektzone. Ziel dieser Studie war die Charakterisierung einer neuen Darbietungsform von PLGA auf ihre physikalischen Eigenschaften, ihre immunologische Signatur und Zytokompatibilität. Dieses Material soll den Einsatz von PLGA in Form einer porösen Membran zur Abdeckung von großen Knochendefekten ermöglichen.
Material und Methoden: Das PLGA-Gemisch wurde in Form von scheibenförmigen Prüfkörpern und Membranen aufbereitet. Es wurde eine biomechanische Analyse des Materials auf Bruchfestigkeit, Biegesteifigkeit und seine Diffusionskapazität durchgeführt. Die Scaffolds wurden mit Knochenmarkstammzellen (BMC und MSC) besiedelt. Mittels MTT-Tests wurde die Lebensdauer und die Viabilität der Zellen auf der Prüfkörperoberfläche ermittelt. Die Zellen wurden durch PCR-Analyse auf osteogene Differenzierung untersucht. Zur Erfassung der immunologischen Signatur erfolgte ein Vollblutstimulationstest mit anschließender umfassender Auswertung des Sekretoms mittels Proteom Profiler-Analyse.
Ergebnisse: Das Material wies eine geringe mechanische Stabilität auf. Die PLGA-Membran zeigte sich für Proteine bis 11,4 kDa durchlässig. Es konnte eine stetige Degeneration des Materials in wässrigem Milieu festgestellt werden. In der Sekretom-Analyse des Vollblutstimulationstests zeigte sich eine für die PLGAMembran charakteristische immunologische Signatur mit Aktivierung von proinflammatorischen Faktoren. Das Zellüberleben auf der Prüfkörperoberfläche nahm während des Erfassungszeitraums konstant ab, während die Zellviabiliät auf dem Wellboden stieg. Es konnte keine signifikante osteogene Differenzierung der Stammzellen festgestellt werden.
Diskussion: Die schnelle Degeneration des Materials in wässriger Umgebung beeinflusste sowohl die Stabilität der Membran als auch die Zelladhäsion auf den Scaffolds negativ. Das Material wirkte dabei weder toxisch auf die Zellen, noch induzierte es eine osteogene Differenzierung. Die Analyse der Immunsignatur ließ eine frühe und starke Entzündungsreaktion nach Implantation des Materials vermuten.
Schlussfolgerung und Ausblick: Während das vorgelegte PLGA-Gemisch nicht die mechanischen Eigenschaften eines gewichttragenden Knochenersatzstoffes aufweist, so lässt es sich dennoch erfolgreich mit Stammzellen beladen, die nach Implantation gezielt in der Defektzone freigesetzt werden können. Die Anregung einer starken Immunantwort kann gerade in der frühen Heilungsphase hilfreich sein. Weitere in vivo-Untersuchungen am Tiermodell sind notwendig zur Optimierung möglicher therapeutischer Anwendungen.
Das akut-auf-chronische Leberversagen (ACLF) entsteht durch eine akute Dekompensation einer vorbestehenden Leberzirrhose mit begleitendem Multiorganversagen und ist durch eine sehr hohe Kurzzeitmortalität gekennzeichnet. Die Kriterien der European Association for the Study of Chronic Liver Failure (EASL-CLIF) definieren in diesem Zusammenhang ein Lungenversagen als Abfall des Horovitz-Quotienten unter 200mmHg oder als Abfall des Quotienten aus SpO2/FiO2 unter 214. Während Lungenfunktionsstörungen als unabhängiger Risikofaktor für Mortalität bei Patienten mit Leberzirrhose belegt sind, wurde die Bedeutung von mechanischer Beatmung und Lungenversagen bisher nicht isoliert untersucht. Ziel der Studie ist die Rolle von mechanischer Beatmung und Lungenversagen im ACLF zu analysieren und eine mögliche Unterinterpretation in den etablierten prädiktiven Modellen zu evaluieren.
Es wurden Daten aus 775 Hospitalisationen mit intensivmedizinischer Behandlung am Universitätsklinikum Frankfurt von insgesamt 498 Patienten mit Leberzirrhose im Zeitraum März 2015 bis Juli 2019 retrospektiv erfasst und ausgewertet. ACLF und Organversagen wurden gemäß EASL-CLIF Kriterien definiert. Es erfolgte die statistische Analyse verschiedener Kohorten, wobei ACLF-Patienten mittels Propensity Score hinsichtlich CLIF-C ACLF Score und Geschlecht gematcht und anhand der pulmonalen Beeinträchtigung in eine dreiarmige Testkohorte bestehend aus 49 Patienten mit Lungenversagen, 49 Patienten mit Schutzintubation und 49 Patienten ohne pulmonale Beeinträchtigung aufgeteilt wurden.
216 Patienten zeigten ein ACLF bei Aufnahme, 121 wurden mechanisch beatmet und 68 hatten ein Lungenversagen. In der gematchten Kohorte (n=147) konnten Lungenversagen (HR 3,0) und mechanische Beatmung (HR 1,7) als unabhängige Risikofaktoren für eine hohe 28-Tage-Mortalität identifiziert werden. Der CLIF-C ACLF Score konnte als bestes Modell für die Vorhersage der Kurzzeitmortalität bestätigt werden (AUROC 0,81), zeigte allerdingt deutliche Schwächen in der Subkohorte der Patienten mit Lungenversagen (AUROC 0,49) und mechanischer Beatmung (AUROC 0,68). Eine Kalibrierung des CLIF-C ACLF Scores für diese beiden Risikofaktoren liefert auch in der Gesamtkohorte aller zirrhotischer Patienten eine signifikant verbesserte prädiktive Performance (AUROC 0,87; p=0,001).
Mechanische Beatmung und Lungenversagen sind im Rahmen eines ACLF mit einer erhöhten Kurzzeitmortalität assoziiert und nur ungenügend in den etablierten Prädiktionsmodellen abgebildet. Die Kalibrierung des CLIF-C ACLF Scores für diese Risikofaktoren führt zu einer verbesserten Vorhersagegenauigkeit.
Untersuchungen zur Bedeutung selektiver Autophagie für Alterungsprozesse von Podospora anserina
(2022)
Das Ziel der vorliegenden Arbeit war, die Funktion und die Rolle von Autophagie-assoziierten Proteinen im Alternsmodell Podospora anserina zu untersuchen und einen Einblick in die nicht-selektive Autophagie, die Mitophagie und die Bildung und den Abbau von Autophagosomen im Zusammenhang zur Alterung von P. anserina zu analysieren. Dabei wurden folgende Erkenntnisse erhalten:
1. Die Untersuchungen zu ΔPaAtg8 bestätigen, dass die PaATG8-abhängige Autophagosomenbildung zur Aufrechterhaltung der Lebensspanne benötigt wird. In ΔPaAtg8 kommt es zu einem Verlust der nicht-selektiven Autophagie. Die Mitophagie hingegen ist auch ohne PaATG8 partiell möglich und es liegt ein PaATG8-unabhängiger Abbau von mitochondrialen Proteinen in P. anserina vor.
2. In P. anserina ist PaATG11 an der nicht-selektiven Autophagie beteiligt und auch die Mitophagie erfolgt in Abhängigkeit dieses Gerüstproteins. Während der PaAtg11-Deletionsstamm unter Normalbedingungen keinen zum Wildtyp veränderten Phänotyp zeigt, führt eine Kultivierung auf M2-Medium mit Glycerin als einziger Kohlenstoffquelle zu einer starken Verkürzung der Lebensspanne. Eine mikroskopische Untersuchung der Mitochondrien zeigte, dass im juvenilen Altersstadium von ΔPaAtg11 stark fragmentierte Mitochondrien vorliegen. Während der Alterung normalisiert sich die Mitochondrienmorphologie wieder. Der mitochondriale Funktionsverlust wird möglicherweise von den fragmentierten Mitochondrien ausgelöst, denn eine Kultivierung von älteren ΔPaAtg11-Stämmen auf M2-Medium mit Glycerin führt zu einer Normalisierung der Lebensspanne.
3. Die initialen Untersuchungen zur ΔPaAtg11/ΔPaAtg24-Doppelmutante zeigen, dass es bei der Kultivierung unter Normalbedingungen zu einem additiven Effekt der beiden Genverluste kommt. Bei der Anzucht auf M2-Medium mit Glycerin hingegen kann eine im Vergleich zum ΔPaAtg11-Stamm längere Lebensspanne festgestellt werden. Die Mikroskopie der Mitochondrien in ΔPaAtg11/ΔPaAtg24 zeigt, dass im juvenilen Alter zum Wildtyp vergleichbare filamentöse Mitochondrien vorhanden sind.
4. In P. anserina ist PaATG24 kein Mitophagierezeptorprotein, da im PaAtg24-Deletionsstamm eine Beeinträchtigung der nicht-selektiven Autophagie vorliegt. Auch die Mitophagie ist in diesem Stamm geschädigt. Die mikroskopische Betrachtung der Mitochondrien zeigt keinen Unterschied zum Wildtyp. Bei der Untersuchung zur Mitochondrienfunktion durch M2-Medium mit Glycerin ist wie unter Normalbedingungen eine verkürzte Lebensspanne feststellbar.
5. Der Abbau von GFP::PaATG8 ist in der PaAtg24-Deletionsmutante signifikant verringert und es kommt zu einer Akkumulation von Autophagosomen, somit liegt in diesem Stamm eine Beeinträchtigung des autophagosomalen Flusses vor. Bei der mikroskopischen Untersuchung von PaATG24 zeigt sich, dass dieses Protein in P. anserina im Bereich der Vakuolen lokalisiert ist. Die Analyse der Vakuole-Autophagosomen-Fusion zeigt jedoch, dass dieser Mechanismus unabhängig von PaATG24 ist. Die Vakuolenmorphologie und Vakuolengröße ist in ΔPaAtg24 beeinträchtigt und dadurch kommt es zu dem beobachteten Defekt der nicht-selektiven und selektiven Autophagie.
Protein kinases are key signalling molecules and transduce intracellular signals via the post-translational phosphorylation of substrate proteins, often other protein kinases. Dysregulation of this protein family has been linked to many diseases including neurodegenerative diseases, inflammation and cancer and amplifications of kinases play important roles as diagnostic biomarkers in a variety of cancers. Various strategies have been developed to treat dysregulated protein kinases. Most commonly, chemical small molecule inhibitors are used to modulate protein kinase activity in cancer cells. Many inhibitor and general research efforts have focused only on a small subset of protein kinases, resulting in a large portion of the kinome, the so-called “dark” kinome, remaining largely unexplored. As part of the strategy to develop inhibitors, it is crucial to understand the structure-activity-relationships (SAR) of small molecules to the activity towards the targets based on understanding small molecule-target affinities as determined by biophysical, biochemical, and cellular methods. However, not always do in vitro determined affinities, which are frequently used as basis for SAR considerations, correlate with the cellular affinity. For protein kinases in particular, it has been shown that the cellular concentration of the natural substrate adenosine-triphosphate (ATP) plays a critical role for the resulting small molecule affinity, as substrate and inhibitor frequently compete for the same binding site of the protein kinase. The cellular target engagement assay NanoBRET is a versatile assay that overcomes this problem and can be used to assess binding of a compound to the full-length protein kinase, in the presence of natural binding partners. Another important factor in inhibitor optimization is the selectivity of the molecule within the family of protein kinases. When comparing the selectivity profiles of small molecule kinase inhibitors in vitro and in cells, different profiles can be observed. Frequently, a compound, binds fewer protein kinases with high affinity in cells, indicating that cellular profiling of protein kinase inhibitors is necessary to understand the selectivity profile of an inhibitor.
The goal of this work was to understand cellular SARs of inhibitors for kinases and dark kinases in medicinal chemistry projects, and to understand the selectivity profiles of existing small molecules in cells, including already approved drugs and clinically used kinases inhibitors. The cellular potency and selectivity aspects guided optimization of the inhibitors towards selective small molecules ‘chemical probes’ or highly validated inhibitors with a narrow selectivity profile as part of ‘chemogenomic libraries’. One strategy to improve selectivity has been to use sterically restricted cyclic small molecules, called macrocycles, that allow fewer conformations of the molecule than their non-cyclic parent compound. In this thesis the dark kinase STK17A was investigated. Macrocyclization was used to develop a selective chemical probe molecule that is also selective in the cellular context. For another kinase, SIK2, a rational design approach was used to exclude off-targets bound by the lead structure, resulting in a chemical probe that selectively targets the SIK1/2/3 proteins. Assessing cellular potency of another series of inhibitors, a probe was developed for the PCTAIRE subfamily of the CDK kinases. This required co-expression of the binding partners of CDKs, the cyclins, in cells to obtain a functional assay. To identify new candidates for the neglected family of splicing kinases comprising the CLK, SRPK, DYRK and HIPK protein kinase subfamilies, a literature review was conducted, and the best small molecule candidates were compared for their target engagement in cells. This led to a series of small molecule inhibitors that may be used as a set or single agents to target the CLK proteins and SRPK proteins or in combination to target the remaining proteins. In search of new starting points for this subfamily of kinases, an initial screen with NanoBRET technology was performed using a library of over 2000 inhibitors, and new starting points were identified. Additionally, a set of clinical and approved small molecule kinase inhibitors was assessed for their selectivity in cells. Several highly selective molecules were identified that were much less selective in in vitro approaches. The set of data allowed for a comprehensive comparison of cellular potencies with published data using in vitro binding, in vitro activity and data obtained from cell lysates and identified several protein kinases that would need to be investigated in cells...
Mutational analysis of ribosomal DNA and maturation-scheme analysis of ribosomal RNA in A. thaliana
(2022)
Ribosome biogenesis is a fundamental cellular process beginning with long precursor rRNA transcription from multi-copies of repetitive 45S ribosomal DNAs. At the subunit level, the primary pre-rRNA transcript encapsuled in 90S protein-RNA complex undergoes decisive splitting in two chief ways for further maturation into large (LSU) and small (SSU) ribosomal subunit. The usage of specific rDNA copies from defined chromosomes and their selective role during growth and development have been a topic of interest owing to its contribution to specialized ribosome theory which proposes non-monolithic functions for ribosomes and thereby their mRNA translation potential. Dual-guide CRISPR/Cas9 mediated disruption of rDNA regions resulted in stable disruption of up to 2.5% and 5% of all rDNA copies in hetero- and homozygous (ploop KD) conditions, respectively. At the RNA level, the mutation excised a critical structural element, P-loop on the LSU 25S rRNA. Mutation caused a dosage dependent defect with homozygosity leading to severe developmental defects through vegetative and reproductive growth phases which is manifested in their proteome by means of disregulation through both increase and decrease of several gene ontological categories of proteins in mutants. Interestingly, the mutation on chromosome 4 triggered dosage compensation through rRNA expression from chromosome 2 further compounded by ectopic rRNA biogenesis defects. The mutated copies however are not incorporated in the translating ribosomes and as a direct or indirect consequence led to elevated basal autophagic levels in the mutants.
The primary 35S transcript is known to undergo two modes of initial cleavages at the pre-rRNA level that aid in their subsequent maturation. Root cell culture (RCC) studies shows that these cells contain a novel ITS2-first cleaved precursor even under control growth conditions, P-C2 adding a third maturation means for the 35S pre-rRNA. This maturation path is further known to be triggered under elevated growth temperature forming a novel adaptive response in Arabidopsis and two other crop plants, tomato, and rice. Taken together, the pulse-chase labeling analysis of control and stressed tissues uncovers the fine-tuned pre-rRNA schematics with crossovers between multiple maturation paths.
Restructuration des répertoires langagiers de migrant·e·s de la République du Congo en Lorraine
(2023)
Cette thèse étudie la complexité du plurilinguisme des migrant·e·s d’origine de la République du Congo en Lorraine à travers le prisme de la restructuration des répertoires langagiers. En affûtant la conceptualisation de la restructuration des répertoires langagiers par l’étude du plurilinguisme des migrant·e·s d’origine congolaise, cette recherche ouvre de nouvelles perspectives pour les recherches portant sur le plurilinguisme, notamment concernant les mobilités transgénérationnelles et la diversité des processus de restructuration façonnant les répertoires langagiers. En se focalisant sur les biographies langagières et migratoires de 15 individus migrants, sur leurs réseaux sociaux et sur leurs ressources langagières, cette étude révèle la diversité des processus et des facteurs au cœur des restructurations des répertoires langagiers à travers une étude ethnographique multi-située en Lorraine et au Congo. La compréhension de la diversité des dynamiques restructurant les connaissances langagières des enquêté·e·s passe par l’étude des situations de socialisation langagière au Congo dans leur historicité, des itinéraires de migration et des restructurations des réseaux sociaux ainsi que des répertoires langagiers dans l’installation en Lorraine. Les participations à la société lorraine et ses groupes sociaux imprègnent les identifications, les orientations sociales et les positionnements dans les réseaux sociaux et vice-versa.
Les répertoires langagiers apparaissent comme des enregistrements de la mobilité des individus et de celle des générations antérieures ainsi que de leur entourage. Les restructurations concernent entre autres les ressources associées au français, aux langues congolaises et à d’autres langues appropriées par la migration. Les ressources du français sont restructurées par les migrant·e·s en s’appropriant les ressources courantes dans différentes situations sociales en Lorraine, en marquant et/ou en dissimulant les ressources appropriées ailleurs et inappropriées dans ces situations. En même temps, un savoir de différenciation des ressources, dont font aussi partie les schémas de catégorisation et les stratégies communicatives, est développé et une (in)sécurité langagière se manifeste. Les ressources associées aux langues congolaises, leurs fonctions sociales et leurs représentations sont restructurées dans des processus d’attrition, d’actualisation, de transformation et d’élaboration langagière. Les ressources associées à d’autres langues européennes appropriées par la migration sont reléguées au second plan et se perdent lentement par manque d’usage. Enfin, les connaissances liées à la gestion du plurilinguisme, de la diversité culturelle et de l’altérité, appropriées dans les mêmes situations de diversité, aident au traitement interne des expériences des mobilités spatiales et sociales ainsi que des restructurations des répertoires langagiers.
In the human brain, the incoming light to the retina is transformed into meaningful representations that allow us to interact with the world. In a similar vein, the RGB pixel values are transformed by a deep neural network (DNN) into meaningful representations relevant to solving a computer vision task it was trained for. Therefore, in my research, I aim to reveal insights into the visual representations in the human visual cortex and DNNs solving vision tasks.
In the previous decade, DNNs have emerged as the state-of-the-art models for predicting neural responses in the human and monkey visual cortex. Research has shown that training on a task related to a brain region’s function leads to better predictivity than a randomly initialized network. Based on this observation, we proposed that we can use DNNs trained on different computer vision tasks to identify functional mapping of the human visual cortex.
To validate our proposed idea, we first investigate a brain region occipital place area (OPA) using DNNs trained on scene parsing task and scene classification task. From the previous investigations about OPA’s functions, we knew that it encodes navigational affordances that require spatial information about the scene. Therefore, we hypothesized that OPA’s representation should be closer to a scene parsing model than a scene classification model as the scene parsing task explicitly requires spatial information about the scene. Our results showed that scene parsing models had representation closer to OPA than scene classification models thus validating our approach.
We then selected multiple DNNs performing a wide range of computer vision tasks ranging from low-level tasks such as edge detection, 3D tasks such as surface normals, and semantic tasks such as semantic segmentation. We compared the representations of these DNNs with all the regions in the visual cortex, thus revealing the functional representations of different regions of the visual cortex. Our results highly converged with previous investigations of these brain regions validating the feasibility of the proposed approach in finding functional representations of the human brain. Our results also provided new insights into underinvestigated brain regions that can serve as starting hypotheses and promote further investigation into those brain regions.
We applied the same approach to find representational insights about the DNNs. A DNN usually consists of multiple layers with each layer performing a computation leading to the final layer that performs prediction for a given task. Training on different tasks could lead to very different representations. Therefore, we first investigate at which stage does the representation in DNNs trained on different tasks starts to differ. We further investigate if the DNNs trained on similar tasks lead to similar representations and on dissimilar tasks lead to more dissimilar representations. We selected the same set of DNNs used in the previous work that were trained on the Taskonomy dataset on a diverse range of 2D, 3D and semantic tasks. Then, given a DNN trained on a particular task, we compared the representation of multiple layers to corresponding layers in other DNNs. From this analysis, we aimed to reveal where in the network architecture task-specific representation is prominent. We found that task specificity increases as we go deeper into the DNN architecture and similar tasks start to cluster in groups. We found that the grouping we found using representational similarity was highly correlated with grouping based on transfer learning thus creating an interesting application of the approach to model selection in transfer learning.
During previous works, several new measures were introduced to compare DNN representations. So, we identified the commonalities in different measures and unified different measures into a single framework referred to as duality diagram similarity. This work opens up new possibilities for similarity measures to understand DNN representations. While demonstrating a much higher correlation with transfer learning than previous state-of-the-art measures we extend it to understanding layer-wise representations of models trained on the Imagenet and Places dataset using different tasks and demonstrate its applicability to layer selection for transfer learning.
In all the previous works, we used the task-specific DNN representations to understand the representations in the human visual cortex and other DNNs. We were able to interpret our findings in terms of computer vision tasks such as edge detection, semantic segmentation, depth estimation, etc. however we were not able to map the representations to human interpretable concepts. Therefore in our most recent work, we developed a new method that associates individual artificial neurons with human interpretable concepts.
Overall, the works in this thesis revealed new insights into the representation of the visual cortex and DNNs...
Bei Vorliegen eines metastasierten kastrationsresistenten Prostatakarzinoms ist die Radioligandentherapie mit 177Lu-PSMA-617 bzw. 177Lu-PSMA-I&T (hier abgekürzt 177Lu-PSMA) eine mögliche Therapieoption. Sie setzt eine ausreichende Expression von prostataspezifischem Membranantigen (PSMA) auf dem Tumorgewebe des Prostatakarzinoms voraus. Da PSMA auch auf physiologischem Gewebe exprimiert wird, sind bestimmte Organe einer erhöhten Dosis ausgesetzt. Die Niere ist aufgrund der PSMA-Expression und der renalen Eliminierung des Radionuklids eines dieser Risikoorgane.
Eine generelle Leitlinie zur Überwachung der Nierendosis unter Therapie mit 177Lu-PSMA existiert noch nicht, jedoch wird empfohlen, bei Durchführung mehrerer Therapiezyklen eine Dosimetrie der Risikoorgane durchzuführen. Meist erfolgt die Nierendosimetrie mittels SPECT/CT. Dieses Verfahren wird als Goldstandard angesehen, da es eine Korrektur der Schwächung, Streuung und des Hintergrundes zulässt.
Die Dosimetrie durch eine SPECT/CT-Bildgebung ist jedoch nicht immer möglich. Dementsprechend gibt es in Studien mehrere Ansätze der Nierendosimetrie ohne SPECT/CT.
In dieser Arbeit werden zwei unterschiedliche Verfahren zur Nierendosimetrie vorgestellt, die einzig mit konventionellen nuklearmedizinischen Modalitäten durchgeführt werden. Diese werden an einem großen Patientenkollektiv angewendet und anhand bereits in der Literatur bestehenden Studien und Ergebnissen evaluiert und mit diesen verglichen. Die Nierendosis in der Literatur bewegt sich dabei meist zwischen 0,4 und 0,8 Gy pro GBq applizierter Aktivität (Gy/GBq).
Die hier vorgestellte Methode 1 arbeitet mit Messwerten aus der Gammasonde, Ganzkörperszintigraphie und abdominalem SPECT. Messwerte werden an der Gammasonde 0 und 48 Stunden nach Applikation von 177Lu-PSMA erhoben, die Szintigraphie wird 48 Stunden und die SPECT 24, 48 und 72 Stunden post iniectionem angefertigt.
Methode 2 der Nierendosimetrie dagegen benötigt nur die 24, 48 und 72 Stunden nach Applikation durchgeführte SPECT sowie die durch eine CT-Bildgebung messbaren Abstände der Nieren zur Körperoberfläche. Durch zuvor durchgeführte Messungen mit einem Torso-Phantom ist die Bestimmung einer patientenindividuellen Effektivität möglich, die die Bestimmung der Nierendosis in Abhängigkeit zur Lage der Nieren möglich macht.
Die gemittelte Nierendosis von 196 Patienten, die eine Therapie mit 177Lu-PSMA erhielten, betrugt 0,29 ± 0,14 Gy/GBq bei Methode 1 und 0,54 ± 0,2g Gy/GBq bei Methode 2. Im Schnitt wurden 3,54 ± 2,63 Therapiezyklen je Patient durchgeführt.
Da bisher nur Studien mit einer kleinen Patientenkohorte vorliegen, besteht die Notwendigkeit zur Durchführung von Arbeiten mit mehr Patienten. Die vorliegende Arbeit schließt 196 Patienten mit 926 durchgeführten Therapiezyklen ein, wodurch Ergebnisse erzielt wurden, die valide Aussagen treffen lassen.
Nach Vergleich der Ergebnisse mit bereits vorliegenden Studien sollte Methode 1 der Nierendosimetrie angesichts der fehlenden Möglichkeit zur Schwächungskorrektur verworfen werden, da dies die Gefahr der Unterschätzung der Nierendosis mit sich bringt. Stattdessen sollte Methode 2 präferiert werden, deren Ergebnisse sich mit den anderen zum Vergleich herangezogenen Studien decken.
Eine Durchführung mit Methode 2 ist dementsprechend sicher und ist in Betracht zu ziehen, sollte die Nierendosimetrie mit einem SPECT/CT nicht möglich sein.
Oceanic islands only comprise a small amount of the Earth’s land area but harbour a disproportionate amount of global biodiversity. This vast diversity is not only reflected in the taxonomic uniqueness of island biota but also in the remarkable evolution of functional traits. Functional traits, i.e. measurable characteristics that strongly influence the fitness of species, determine how a species responds to its environment and can help to gain more insights into the biogeographical, ecological and evolutionary processes that have shaped island biodiversity. However, research in island biogeography has primarily focused on species richness, and knowledge of functional trait patterns on oceanic islands is scarce. Hence, in this dissertation, I have explored how trait-based approaches can increase our understanding of how biodiversity on oceanic islands assembles and how it is driven by the environment. The Canary Islands (Spain) are a particularly suitable model system to investigate patterns and drivers of biodiversity. The archipelago is characterised by a high variation in environmental heterogeneity and inhabits a unique and well-described native flora. Therefore, I have investigated five principal research questions using the flora (Spermatophytes) of the Canary Islands as a study object. First, I have analysed how climate and biogeography shape the assembly of the Canary Islands flora using a novel trait-based approach. Second, the question of whether rare climates link to functional trait distinctiveness in the native Canary Islands flora was addressed. Third, I have examined how intraspecific trait variation is represented in the native flora of oceanic islands focusing on the succulent scrub of La Palma (Canary Islands). Fourth, this dissertation investigated whether scientific floras can be reliable sources for trait data of plants native to oceanic islands. Finally, I have explored how climate change may impact the native Canary Islands flora by analysing possible climate change-induced shifts in plant species distribution and plant traits.
The results of my dissertation expand the understanding of the importance of biogeography and the environment in determining the functional composition of island floras. I have assessed that traits of endemic plant species did not expand the functional trait space of the Canary Islands but were packed with the ones of non-endemic species. This result hints at a trait convergence in endemic species, possibly driven by non-adaptive speciation processes. Moreover, I have evidenced that humidity is a critical driver of functional diversity in native plant assemblages and particularly leads to a high trait convergence in arid environments via environmental filtering. In contrast, alien species have expanded the Canary Islands flora’s functional trait space. I further have shown that in contrast to native species assemblages, alien species assemblages are characterised by an increasing functional diversity with increasing aridity. This contrasting pattern of functional diversity could pose a potential risk to the native flora of the Canary Islands as a low functional diversity is expected to reduce the resilience of species assemblages to the establishment of more functionally diverse alien plant species. However, in this dissertation, I also have revealed that endemic plant species on the Canary Islands show a high intraspecific variation in arid environments, possibly as an adaptation to environmental stress. Intraspecific variation could help endemic plant species have a competitive advantage over alien species and be more resilient to environmental changes. Furthermore, in this dissertation, I have shown that scientific floras and taxonomic monographs could be used to gain information on quantitative functional traits of plants native to oceanic islands. This finding is particularly relevant for advances in trait-based research, as coverage of trait data for oceanic island floras is extremely poor in global trait databases. Hence, for some of the studies included in this dissertation, trait data were retrieved from scientific floras and taxonomic monographs and used to answer novel scientific research questions. Thus, I have used trait data from the literature to analyse the effect of climate change on the range size of plants native to the Canary Islands. Identifying plant species of particular conservation concern is critical on oceanic islands as many island species have limited distributions and small population sizes, and their niche tracking is impeded by insularity. I have revealed that single-island endemic plants gain less and lose more climatically suitable areas than archipelago endemic and non-endemic native plants due to a climate change-induced decrease in precipitation until 2100...
A promising strategy to reduce the dependency from fossil fuels is to use the yeast Saccharomyces cerevisiae to bioconvert renewable non-food feedstocks or waste streams, like lignocellulosic biomass, into bioethanol and other valuable molecule blocks. Lignocellulosic feedstocks contain glucose and significant fractions of the pentoses xylose and arabinose in varying proportions depending on the biomass type. S. cerevisiae is an efficient glucose consumer, but it cannot metabolize xylose and arabinose naturally. Therefore, extensive research using recombinant DNA techniques has been conducted to introduce and improve the biochemical pathways necessary to utilize these non-physiological substrates. However, any functional pathway capable of metabolizing D xylose and L arabinose in S. cerevisiae requires the transport of these sugars across the plasma membrane. The endogenous sugar transport system of S. cerevisiae can conduct a limited uptake of D-xylose and L-arabinose; this uptake enables only basal growth when the enzymatic pathways are provided. For this reason, the uptake of D xylose and L-arabinose has been recognized as a limiting step for the efficient utilization of these non-physiological substrates.
Gal2, a member of the major facilitator superfamily, is one of the most studied hexose transporters in S. cerevisiae. Although its expression is repressed in the presence of glucose, it also transports this sugar with high affinity when constitutively expressed. Recent efforts to engineer yeast strains for the utilization of plant biomass have unraveled the ability of Gal2 to transport non-physiological substrates like xylose and arabinose, among others. Improving Gal2 kinetic and substrate specificity, particularly for pentoses, has become a crucial target in strain engineering. The main goal of this study is to improve the utilization of xylose and arabinose by increasing the cell permeability of these non physiological substrates through the engineering of the galactose permease Gal2.
GAL2 gene expression depends on galactose, which acts as an inducer; nevertheless, even in the presence of galactose, glucose act as a strict repressor; consequently, GAL2 gene is usually placed under the control of a constitutive promoter. However, the presence of glucose additionally triggers the Gal2 degradation, which is mediated by the covalent attachment of the small 76 amino acid protein ubiquitin (Ub) to the targeted transporter; in a multi-step process called ubiquitination.
Ubiquitination of hexose permeases involves the activation of the Ub molecule by the E1 Ub-activating enzyme using ATP; then, the activated Ub is transferred to a specific Ub-conjugating enzyme E2, which donates the Ub indirectly through a specific HECT E3 enzyme (Rsp5) to a lysine residue of the substrate, with the aid of an adaptor protein which recognizes the target (Rsp5-adaptor). Ubiquitinated permeases are sent by membrane invagination to early endosomes, where they encounter ESCRTs (endosomal sorting complex required for transport). The targeted permeases are sorted in intralumenal vesicles (ILV) inside of the endosome, which after several cycles, turns into a multivesicular body (MVB) that subsequently fuses with the vacuole to expose the protein content of the ILVs to lumenal hydrolases for degradation.
Gal2 contains 30 lysine residues that may accept the ubiquitin molecule, which targets its degradation. It is known that mono-ubiquitination by Rsp5 on multiple lysine residues is necessary to internalize Gal2 (Horak & Wolf, 2001). However, the authors did not identify the specific lysine residues involved in the ubiquitination processes. This study screened several Gal2 variants where lysine residues were mutated or removed from the protein sequence to discover which lysine residues are likely involved in ubiquitination and consequent turnover of the transporter. The results of the screening showed that mutation of the N terminal lysine residues 27, 37, and 44 to arginine (Gal23KR) produced a functional transporter that, when fused with GFP (Gal23KR_GFP), showed an exclusive localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b).
This study furthermore evaluated upstream signals caused by phosphorylation which triggers ubiquitination and consequent turnover of the targeted protein; using similar screening approaches to assess the stabilization of Gal2 by lysine residue modifications, it was possible to identify that N terminal serine residues 32, 35, 39, 48, 53, and 55 are likely involved in the internalization of Gal2, since a Gal2 construct where all these serines were mutated to alanine residues and tagged with GFP (Gal26SA_GFP) exhibited practically complete localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b)...
Terahertz (THz) radiation lies between the micro and far-infrared range in the electromagnetic spectrum. Compared with microwave and millimeter waves, it has a larger signal bandwidth and extremely narrow antenna beam. Thus, it is easier to achieve high-resolution for imaging and detection applications. The unique properties, such as penetration for majority non-polar materials, non-ionizing characteristic and the spectral fingerprint of materials, makes THz imaging an appealing artifice in the military, biomedical, astronomical communications, and other areas. However, THz radiation’s current low power level and detection sensitivity block THz imaging system from including fewer optical elements than the visible or infrared range. This leads to imaging resolution, contrast, and imaging field of view degenerate and makes the aberration more serious. THz imaging based on the space Fourier spectrum detection is developed in this thesis to achieve high-quality imaging. The main concept of Fourier imaging is by recording the field distribution in the Fourier plane (focal plane) of the imaging system; the information of the target is obtained. The numerical processing method is needed to extract the amplitude and phase information of the imaged target. With additional process, three-dimensional (3D) information can be obtained based on the phase information. The novel recording and reconstructing ways of the Fourier imaging system enables it to have a higher resolution, better contrast, and broader field of view than conventional imaging systems such as microscopy and plane to plane telescopic imaging system.
The work presented in this thesis consists of two imaging systems, one is working at 300 GHz based on the fundamental heterodyne detection of the THz radiation, the other is operated at 600 GHz by utilizing the sub harmonic heterodyne detection technique. The realization and test of the heterodyne detection are based on the THz antenna-coupled field-effect transistor (TeraFET) detector developed by Dr. Alvydas Lisauskas. Both systems use two synchronized electronic multiplier chains to radiate the THz waves. One radiation works as the local oscillator (LO), the other works as illumination with a slight frequency shift, the radiations are mixed on the detector scanning in the Fourier plane to record the complex Fourier spectrum of the imaged target. The LO has the same frequency range as the illuminating radiation for fundamental heterodyne detection but half the frequency range for the sub-harmonic heterodyne detection. The 2-mm resolution, 60-dB contrast, and 5.5-cm diameter imaging area at 300 GHz and the of 500-μm resolution, 40-dB contrast, and 3.5-cm diameter imaging area at 600 GHz are achieved (the 300-GHz illuminating radiation has the approximate power of 600 μW , the 600-GHz illuminating radiation has the approximate power of 60 μW ).
The thesis consists of 6 parts. After the introduction, the second chapter expands on the topic of Fourier optics from a theoretical point of view and the simulations of the Fourier imaging system. First, the theory of the electromagnetic field propagation in free space and through an optical system are investigated to elicit the Fourier transform function of the imaging system. The simulation is used for theoretical considerations and the implementation of a Fourier optic script that allows for numerical investigations on reconstruction. The preliminary imaging field of view and resolution are also demonstrated. The third chapter describes the Fourier imaging system at 300 GHz based on the fundamental heterodyne detection, including the experimental setup, the 2D, and 3D imaging results. The following fourth chapter reports the integration of the TeraFET detector with two substrate lenses (one is a Si lens on the back-side Si substrate, the other is a wax/PTFE lens on the front side containing the bonding wires) for sub-harmonic heterodyne detection at 600 GHz. The characteristic of the wax/PTFE lens at THz range is presented. After that, the compared imaging results between the detector with and without the wax/PTFE lens are shown. The fifth chapter extends the demonstration on the lateral and depth resolution of the Fourier imaging system in detail and uses the experimental results at 600 GHz to validate the analytical predictions. The comparison of the resolution between the Fourier imaging system and the conventional microscopy system proves that the Fourier imaging system has better imaging quality under the same system configuration. The last chapter in this thesis concludes on the findings of the THz Fourier imaging and gives an outlook for the enhancement of the Fourier imaging system at THz range.
Einführung: In den letzten Jahren wurden mobile Endgeräte in zunehmenden Maße zur Messung biometrischer Daten wie z.B. Herzfrequenz, Blutdruck, Sauerstoffsättigung und EKG Messungen wie z.B. Rhythmusstörungen verwendet. In den meisten Fällen kann bezüglich eines EKG hierbei nur eine einzige Ableitung gemessen werden. Mit Ausnahme von EKG-Brustgürteln sind Messungen nur in Ruhe möglich. Letztere sind häufig nicht als Medizinprodukte zugelassen. Veränderungen in einem vektoriell, aus einer reduzierten Elektrodenzahl, errechneten EKG unter Belastung, welche mit einem mobilen Endgerät gemessen wurden, sind bisher nicht evaluiert worden. Einige Pathologien werden nur unter Stressbedingungen demaskiert, so dass mit den aktuellen Systemen eine diagnostische Lücke im häuslichen Umfeld existiert. Es wurde ein mit einem mobilen Endgerät (Smartphone/Tablet) vektoriell abgeleitetes 4 Elektroden EKG System mit dem aktuellen Goldstandard 12-Kanal EKG sowohl in Ruhe als auch unter Belastung verglichen.
Methoden und Ergebnisse: 428 Patienten wurden während einer fahrradergometrischen Untersuchung simultan mit beiden EKG Systemen untersucht. Das vektorielle EKG wurde als errechnetes 12-Kanal EKG dargestellt und bezüglich EKG-Qualität und diagnostischer Genauigkeit mit dem Standard EKG verglichen. Alle EKGs wurden unabhängig durch 2 verblindete und in der EKG Interpretation versierten Ärzte untersucht. Die EKG Qualität in beiden Verfahren war gut. In Ruhe gab es eine ausgezeichnete Übereinstimmung zwischen beiden EKG Technologien in Hinsicht auf Rhythmus (98%), AV-Überleitung (97%), Wellendauer (90%) und elektrischer Achse (88-97%). Während der Belastung gab es eine Übereinstimmung in 90% der Fälle. Der positiv prädiktive Wert lag bei 48.5% und der negativ prädiktive Wert bei 94%. ST-Deviationen >0.2 mV zeigten eine 97% Übereinstimmung während des Stresstests. Rhythmusstörungen und intraventrikuläre Leitungsstörungen (Links- und Rechtsschenkelblock) konnten in jeweils >90% der Fälle übereinstimmend identifiziert werden.
Schlussfolgerung: Ein mobiles Endgerät kann benutzt werden, um vektoriell errechnete 12-Kanal EKGs unter Alltagsbedingungen zuverlässig darzustellen. Die diagnostische Aussagekraft dieser EKGs in Ruhe und unter Belastung ist gut.
Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway
(2022)
The human body is permanently exposed to its environment and thus to viruses and other pathogens, which require a flexible response and defense. Alongside to the innate immune system, the adaptive immune system provides highly specialized protection against these threats. The major histocompatibility complex class I (MHC I) antigen presentation system is a cornerstone of the adaptive immune system and a major constituent of cellular immunity. Pathogens such as viruses that invade a cell will leave traces in the form of proteins and peptides which are degraded and loaded onto MHC I molecules. MHC I peptide loading is performed by peptide loading complex (PLC) in the membrane of the endoplasmic reticulum as part of a multifaceted and comprehensive quality control machinery. Monitored by multiple layers of quality assurance, the MHC I molecules consequently display the immune status of the cell on its surface. In this context, the captured fragment of the virus serves as a call for help issued by the cell, alerting the adaptive immune system to the infection to mount an appropriate immune response.
The three-dimensional structure as well as the mechanistic details of parts of this complex machinery were characterized in the context of this dissertation. Among other tools, light-modulable nanotools were developed in this thesis, which permit external regulation of cellular processes in temporal and spatial resolution. Furthermore, methods and model systems for the biochemical characterization of cellular signaling cascades, proteins, as well as entire cell organelles were developed, which are likely to influence the field of cellular immunity and protein biochemistry in the future.
This cumulative work comprises a total of six publications whose scientific key advances will be briefly outlined in this abstract. In the introduction, the scientific background as well as the current state of research and methodological background knowledge are conveyed. The results section condenses the main aspects of the publications and links them to each other. Further details can be retrieved from the attached original publications.
In “Semisynthetic viral inhibitor for light control of the MHC I peptide loading complex, Winter, Domnick et al., Angew Chem Int Ed 2022” a photocleavable viral inhibitor of the peptide loading complex was produced by semi-synthesis. This nanotool was shown to be suitable for both purifying the PLC from human Raji cells as well as reactivating it in a light-controlled manner. Thus, this tool establishes the isolation of a fully intact and functional peptide loading complex for biochemical characterization. In addition, a novel flow cytometric analysis pipeline for microsomes was developed, allowing cellular vesicles to be characterized with single organelle resolution, similar to cells.
In “Molecular basis of MHC I quality control in the peptide loading complex, Domnick, Winter et al., Nat Commun 2022” the peptide loading complex was reconstituted into large nanodiscs, and a cryo-EM structural model of the editing module at 3.7 Å resolution was generated. By combining the structural model with in vitro glycan editing assays, an allosteric coupling between peptide-MHC I assembly and glycan processing was revealed, extending the known model of MHC I loading and dissociation from the PLC. These mechanisms provide a prototypical example for endoplasmic reticulum quality control.
In a related context, in “Structure of an MHC I–tapasin–ERp57 editing complex defines chaperone promiscuity, Müller, Winter et al., Nat Commun 2022” a recombinantly assembled editing module comprised of MHC I-tapasin-ERp57 was crystallized for X-ray structural biology. The resulting crystal structure at a resolution of 2.7 Å permitted the precise identification of characteristic features of the editing module and particularly of the peptide proofreading mechanism of tapasin. This study provided pivotal insights into the tapasin-mediated peptide editing of different MHC I allomorphs as well as similarities to TAPBPR-based MHC I peptide proofreading.
In “TAPBPR is necessary and sufficient for UGGT1-mediated quality control of MHC I, Sagert, Winter et al. (in preparation)” novel insights concerning the peptide proofreader TAPBPR and its close interplay with the folding sensor and glucosyltransferase UGGT1 were obtained. It was shown that TAPBPR is an integral part of the second level of endoplasmic quality control and is indispensable for effective MHC I coordination by UGGT1.
In “Light-guided intrabodies for on-demand in situ target recognition in human cells, Joest, Winter et al., Chem Sci 2021” intracellular nanobodies were equipped with a photocaged target recognition domain by genetic code expansion via amber suppression. These intrabodies, acting as high-affinity binding partners endowed with a fluorophore, could be used in a light-triggered approach to instantaneously visualize their target molecule...
This work investigated the influence of the CRISPR/Cas9 mediated knockout of 5-lipoxygenase (5-LO) on different adherent tumour cell lines derived from solid tumours. For this, the 5-LO expressing tumour cell lines HCT-116, HT-29, and U-2 OS were transiently transfected using a plasmid carrying the CRISPR/Cas9 complex sequence to the ALOX5 gene. Subsequently, cells were selected using Puromycin and analysed via Western blotting and DNA Sanger sequencing. Cells that were transfected with a control plasmid missing the guide RNA sequence, were used as a control for all experiments.
Differential gene expression analysis, performed after next-generation RNA sequencing, revealed that the expression of various genes was altered after the knockout of 5-LO. In HCT-116 cells, 28 genes were expressed differentially in all 5-LO knockout single-cell clones, while in HT-29 cells the expression of 18 genes and in U-2 OS cells of 234 genes was influenced by the knockout of 5-LO. These findings were validated by real-time qPCR. A lot of the genes that were influenced by the 5-LO knockout are known to be connected to epithelial-mesenchymal-transition (EMT), a process necessary for tumour metastasis. The results from RNA sequencing were the starting point for further investigations. In the following, different aspects of the tumour cell lines were examined. In HT-29, as
well as in U-2 OS cells, it was shown that knockout of the 5-LO resulted in impaired cell proliferation. Also, the formation of three-dimensional tumour spheroids was altered. In HT-29 cells, the knockout of 5-LO increased the number of cells in spheroids. In contrast, in U-2 OS cells, the number of cells per spheroid was decreased, even though the diameter of the spheroids was increased, due to more loosely packed spheroids. The difference between 5-LO positive and negative U-2 OS cells became even more obvious after embedding the spheroids in an artificial extracellular matrix. In that scenario, cells lacking the 5-LO formed smaller spheroids that did not have the same ability to grow into the extracellular matrix as 5-LO positive cells did. Also, directed cell migration was strongly influenced by the knockout of 5-LO. In both, HCT-116 and U-2 OS cells, directed cell migration towards a serum gradient was increased in 5-LO knockout single-cell clones. Pharmacological inhibition of the enzyme was used to investigate, whether canonical or non-canonical functions were responsible for the previously mentioned effects.
Therefore, vector control cells were treated with the 5-LO inhibitors Zileuton and CJ-13610 in different concentrations. Interestingly, only some of the effects mediated by the complete knockout of 5-LO could be reproduced by inhibiting the enzyme, leading to the suggestion, that canonical, as well as non-canonical functions of 5-LO, play a role in these tumour cells.
To conclude, it was shown in this study, that 5-LO affects various cellular functions when expressed in adherent tumour cell lines. These cell line-dependent effects result in altered gene expression, enhanced proliferation, and spheroid formation, as well as impaired cell motility, and can be mediated by enzymatic activity as well as other non-canonical functions.
Der Zusammenhang zwischen kognitiver Emotionsregulation, positivem Aufmerksamkeitsbias und Resilienz
(2022)
Das Verständnis von Faktoren, die die Widerstandsfähigkeit gegen Stress fördern, ist entscheidend für die Entwicklung von Stresspräventionsprogrammen und für die Verbesserung der Behandlung stressbedingter Störungen. Zum einen war es Ziel der vorliegenden Forschungsarbeit, den Einfluss der kognitiven Emotionsregulation (ER) auf die psychische Gesundheit zu untersuchen und bereits vorhandene Ergebnisse zum Einfluss der Emotionsregulation auf die Resilienz zu replizieren. Es wird zunehmend anerkannt, dass die meisten psychiatrischen Erkrankungen mit Emotionsdysregulation einhergehen und dass klinische Interventionen davon profitieren, wenn sie auf einem empirischen Verständnis der Emotionsprozesse beruhen.
Der Hauptfokus lag zudem darauf, zu untersuchen, ob es einen Zusammenhang zwischen Resilienz und einem Aufmerksamkeitsbias auf positive Informationen, einen Positivitätsbias, gibt.
In der vorliegenden Studie wurde eine Stichprobe psychisch gesunder Teilnehmer (n=229) im Alter von 18 bis 55 Jahren herangezogen, denen Bilder aus dem „International Affective Picture System“ präsentiert wurden. Dabei bekamen sie die Anweisung, negative Emotionen zu Fotos durch kognitives Umbewerten oder Distanzieren herunterzuregulieren. Die Reaktionszeiten sowie die Erregungsbewertungen wurden für die Bedingungen Neubewertung, Distanzieren, negatives passives Betrachten sowie neutrales passives Betrachten erhoben. Zudem wurde die Aufmerksamkeitsverzerrung gegenüber positiven und negativen Reizen mithilfe einer Visual Dot-Probe Aufgabe untersucht. Gemessen wurden die Reaktionszeiten der Studienteilnehmer bei Reaktionen auf einen Stimulus, der auf die Präsentation emotionaler Gesichter folgt, im Vergleich zu Reaktionen auf einen Stimulus, der an die Stelle von neutralen Gesichtern rückt. Hieraus wurden Aufmerksamkeitsverzerrungen abgeleitet.
Die anschließende Datenanalyse und statistische Auswertung konnten zeigen, dass die Neubewertung im Vergleich zum passiven Betrachten der negativen Bilder eine längere Reaktionszeit aufweist, was darauf hinweist, dass die kognitive Emotionsregulation möglicherweise anstrengendere kognitive Kontrollprozesse aktiviert. Hinsichtlich des Zusammenhangs mit der Resilienz konnte eine signifikante positive Korrelation der Reaktionszeitdifferenzen der Emotionsregulationsstrategien Distanzieren und Neubewerten mit dem Resilienz-Score beobachtet werden.
Bei den Erregungsbewertungen der Emotionsregulation zeigte sich weder ein signifikanter Effekt der Bedingung noch ein Zusammenhang mit dem Resilienz-Score.
Die Ergebnisse der Visual Dot-Probe zeigten, dass es keine Unterschiede in den Reaktionszeiten nach der Präsentation des neutralen sowie des emotionalen Stimulus gab. Auch zeigten sich keine Zusammenhänge mit der Resilienz.
Der erwartete Zusammenhang zwischen Resilienz und einem Aufmerksamkeitsbias auf positive Informationen konnte in den Ergebnissen nicht gefunden werden. In den vorliegenden Daten zeigte sich hingegen eine signifikante negative Korrelation der Fähigkeit der kognitiven Neubewertung mit der Höhe des negativen Aufmerksamkeits Bias-Scores.
Somit liefert die vorliegende Arbeit einen Hinweis darauf, dass Personen mit einem hohen Maß an Emotionsregulationskapazitäten eine höhere Aufmerksamkeitslenkung weg von emotional negativen Stimuli aufweisen.
Bezüglich der Anwendung auf klinische Stichproben besteht noch Raum zu
analysieren, ob es sich um ein stabiles Phänomen handelt, das auf diese übertragen werden kann.