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In this thesis I explore the interplay of geometry and quantum information theory via the holographic principle, with a specific focus on geometric phases in quantum systems like two interacting qubits, and how they relate to entanglement measures and Hilbert space factorisation. I establish geometric phases as an indicator for Hilbert space factorsiation, both in an abstract sense using von Neumann operator algebras as well as applied to the eternal black hole within the AdS/CFT correspondence. For the latter case I show that geometric phases allow to diagnose non-factorisation from a boundary point of view. I also introduce geometric quantum discord as a second geometric measure for non-factorisation and reveals its potential implications for the study of black hole microstates.
Biochemical characterization of the TFIIH translocase XPB from \(Chaetomium\) \(thermophilum\)
(2024)
DNA repair and gene expression are two major cellular processes that are fundamental for the maintenance of biological life. Both processes require the enzymatic activity of the super family 2 helicase XBP, which is an integral subunit of the general transcription factor TFIIH. During transcription initiation, XPB catalyzes the initial melting of promoter DNA enabling RNA polymerase II to engage with the coding DNA strand and start gene transcription. In nucleotide excision repair, XPB acts in concert with the other TFIIH helicase XPD causing strand separation around a lesion site. Mutations within the genes encoding XPB or other TFIIH subunits are associated with different cancer types as well as with the autosomal recessive disorders Xeroderma Pigmentosum and trichothiodystrophy and rarely combined features of Xeroderma Pigmentosum and Cockayne syndrome.
In the last few years, great progress has been made towards unraveling the structure of TFIIH and its individual subunits including XPB. These structural insights tremendously improved our understandings with respect to the molecular interactions within this intriguing protein complex. However, the underlying regulation mechanisms that functionally control XPB during transcription and repair remained largely elusive. We thus executed the biochemical characterization of this protein to investigate the functional network that regulates XPB within the scaffold of TFIIH. Due to their enhanced stability compared to the human proteins, we utilized the proteins that originate from the thermophilic fungus Chaetomium thermophilum for this purpose as a model organism for eukaryotic TFIIH.
The present work provides novel insights into the enzymatic function and regulation of XPB. We could show that both, DNA and the TFIIH subunit p52 stimulate XPB’s ATPase activity and that the p52-mediated activity is further boosted by p8, another subunit within TFIIH. Surprisingly, DNA can activate XPB’s ATPase activity to a greater extent than its TFIIH interaction partners p52/p8, but when both, i.e. p52/p8 and DNA are present at the same time, p52 dominates the activation and the enzymatic speed is maintained at the level observed through the sole activation of p52/p8. We thus defined p52 as the master regulator of XPB that simultaneously activates and represses XPB’s enzymatic activity. Based on a correlative mutagenesis study of the main interface between p52 and XPB that was set into context with recent structural data, a model for the p52-mediated activation and speed limitation of XPB’s ATPase was proposed. The research on XPB’s ATPase was expanded with the investigation of the inhibition mechanism of XPB’s ATPase via the natural compound Triptolide. Furthermore, we investigated XPB’s DNA translocase function and could observe that XPB can only perform its translocase movement when it is fully incorporated into core TFIIH and this translocase movement is further enhanced by the nucleotide excision repair factor XPA. Fluorescence polarization measurements with nucleotide analogues revealed that XPB displays the highest affinity towards DNA in the ADP + Pi bound state and its binding is weakened when ADP is bound or the nucleotide is dissociated from the enzyme, suggesting a movement on the DNA during the distinct states of the ATPase cycle. Finally, the well-known and highly conserved RED motif was found to be the crucial element in XPB to enable this translocase movement. Combined, the data presented in this work provide novel insights into the intricate regulation network that controls XPB’s enzymatic activity within TFIIH and furthermore show that XPB’s enzymatic activity is tightly controlled by various factors.
This thesis is dedicated to construct a non-abelian holographic dynamical minimal composite Higgs model. We first build a non-abelian bottom-up AdS/YM model that can explain the QCD meson spectrum well. The model is made non-abelian by considering non-abelian DBI action in the top-down model. We then change the dual theory from the QCD to the minimal composite Higgs model U (4)/Sp(4). By adding a second explicit U (4) → Sp(4) breaking through the NJL interaction at the boundary, we managed to construct a composite Higgs phase and a technicolor phase in this model. The transition between the two phases is also realized, which is controlled by the NJL coupling. This thesis is based on the works [1, 2].
The present thesis is concerned with the automated computation of integrated and differential
cross sections of diboson production in proton–proton and electron–positron collisions at very
high energies, including a resummation of electroweak Sudakov logarithms to all orders in the
fine-structure constant using soft–collinear effective theory.
The search for new physics at future colliders such as the FCC–hh or the CLIC requires
precise predictions for scattering cross sections from the theoretical high-energy physics com-
munity. Electroweak Sudakov logarithms, which currently limit the accuracy of predictions in
the high-energy tails of differential distributions for LHC-like energies, are known to destroy the
convergence behaviour of the fixed-order perturbative series, once sufficiently high energies are
considered.
To resum these large corrections, soft–collinear effective theory has been applied to simple
processes, which permits analytic calculations. Within this work, we present an automated
computation within a Monte Carlo integration framework, thus facilitating the computation of
fully differential cross section to complicated processes. This requires the use of the Catani–
Seymour subtraction algorithm to treat the occurring infrared divergences. The machinery is
applied to all diboson processes with intermediate weak gauge bosons, including the photon-
induced W+ W− -production channel.
To this end we carefully study the validity of the necessary assumptions such as the double-
pole approximation and estimate the order of magnitude of neglected effects. Especially the
non-doubly-resonant contributions turn out to be sizeable in several interesting phase-space
regions.
For lepton collisions at 3 TeV we obtain the integrated cross sections of W-pair and Z-pair
production to be shifted by more than 20% with respect to the Born value, owing to the resum-
mation of the leading-logarithmic corrections These effects are partly cancelled by subleading
effects. For proton–proton collisions at √
s = 100 TeV we observe sizeable resummation effects
in the high-energy tails, while the integrated cross sections are dominated by interactions, for
which soft–collinear effective theory is not applicable.
Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent developmental disorders, affecting 5.9% children and adolescents and 2.5% adults worldwide. The core characteristics are age-inappropriate levels of hyperactivity, impulsivity and inattention, often accompanied by co-morbidities such as mood and conduct disorders as wells as learning deficits. In the majority of cases, ADHD is caused by an interplay of accumulated genetic and environmental risk factors. Twin studies report a very high heritability of 70–80%, however, common genetic variants in the population only explain a third of the heritability. The rest of the genetic predisposition is composed of rare copy number variations (CNVs) and gene x environment interactions including epigenetic alterations. Through genome wide association (GWAS) and linkage studies a number of likely candidate genes were identified. A handful of them play a role in dopamine or noradrenaline neurotransmitter systems, simultaneously those systems are the main targets of common drug treatment approaches. However, for the majority of candidates the biological function in relation to ADHD is unknown. It is crucial to identify those functions in order to gain a deeper understanding of the pathomechanism and genetic networks potentially responsible for the disorder. This work focuses on the three candidate genes GFOD1, SLC2A3 and LBX1 and their role in the healthy organism as well as in case of ADHD. The neuroanatomy was regarded through expression analysis and various behavioural assays of activity were performed to link alterations on the transcript level to phenotypes associated with the neurodevelopmental disorder. Zebrafish orthologues of the human risk genes were identified and extensive temporal and spacial expression characterisation performed via RNA in situ hybridisation. Through morpholino derived knock-down and mRNA overexpression zebrafish models with subsequent behavioural analysis, both hyper- and hypoactive phenotypes were discovered. Additional expression analysis through double in situ hybridisation revealed a co-localisation during zebrafish neurodevelopment of each gfod1 and slc2a3a together with gad1b, a marker for GABAergic neurons. Interestingly, both risk genes have previously been associated with glucose homeostasis and energy metabolism, which when disrupted could lead to alterations in signal transduction and neuron survival. Likewise, Lbx1 plays a pivotal role in GABAergic versus glutamatergic neuron specification during spinal cord and hindbrain development in mice and chicken. Preliminary results of this work suggest a similar role in zebrafish. Taken together, those findings on the one hand represent a sturdy basis to con- tinue studies of the function of the genes and on the other hand open up the opportunity to investigate novel aspects of ADHD research by exploring the role of the GABAergic neurotransmitter system or the connection between energy metabolism and psychiatric disorders.
Fusobacterium nucleatum is an emerging cancer-associated bacterium belonging to the Fusobacteriota phylum, which is evolutionary distant from all model bacteria. Recent analysis generated global fusobacterial RNA maps, which enabled the discovery of 24 small noncoding RNAs (sRNAs) in F. nucleatum. Notably, the σE-dependent sRNA FoxI and FoxJ act as a posttranscriptional regulator of several cell envelope proteins. The σE-dependent sRNAs in Escherichia coli and Salmonella require the RNA chaperone Hfq for their functions. Intriguingly, F. nucleatum seems to have no homologs of the three common RNA-binding proteins (RBPs) CsrA, Hfq and ProQ. However, it remains unclear if other families of RBPs act in concert with FoxI, FoxJ and other fusobacterial sRNAs.
This work has successfully established a 14-mer capture tagged-sRNA affinity purification procedure initially using 6S RNA as a proof-of-concept. Applying this method to 19 different F. nucleatum sRNAs led to a comprehensive mapping of sRNA-binding proteins in this bacterium. This screen identified a total of 75 proteins significantly enriched across all sRNAs and prominent in ribosomal proteins, uncharacterized proteins and enzymes associated with metabolism. This work further focused on the homologs of two KH domain proteins KhpA and KhpB, which were recently recognized as global RBPs in various Gram-positive bacteria such as Streptococcus pneumoniae, Clostridioides difficile, and Enterococcus faecalis.
Comparative analyses revealed conserved domain composition and gene synteny of KhpA and KhpB across F. nucleatum, S. pneumoniae, C. difficle and E. faecalis, indicating conserved roles of these proteins in bacteria. Further protein-protein interaction assays and global RNA targets profiling demonstrated that KhpA and KhpB form dimers and act together as broad RBPs, binding to sRNAs, mRNAs and tRNAs in F. nucleatum. Further functional characterizations unveiled that KhpA/B are required for the growth of F. nucleatum under nutrient limitation conditions and impact cell morphology. Additionally, the two RBPs also influence global gene expression in F. nucleatum affecting various bacterial physiological processes, including ethanolamine utilization.
In summary, this work established a sRNA-centric approach for screening sRNA-binding proteins in F. nucleatum. Further, the assay could be applied in other non-model organisms and is feasible to screen multiple sRNA baits in parallel for sRNA-interactors. By applying this procedure to nearly all known fusobacterial sRNAs, this work generated an extensive map of sRNA-interacting proteins in F. nucleatum. Molecular and genetic studies identified that KhpA/B act as major RBPs and gene regulators in F. nucleatum, representing important first steps in elucidating key players of post-transcriptional control at the root of the bacterial phylogenetic tree.
Opioid receptors (ORs) are among the most intensively studied members of the G protein-coupled receptor (GPCR) family due to their important role in pain management and their involvement in psychological and neurological disorders. However, currently available opioid drugs exhibit both serious drawbacks, such as addiction, and life-threatening side effects, such as respiratory depression. Contrary to the classic monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be exploited to develop innovative drugs. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological condition are missing. The focus of this thesis was the design, synthesis and characterization of new, highly subtype-selective OR fluorescent ligands to be used as tools for state-of-the-art microscopy methods, such as single molecule microscopy (SMM), in heterologous cells and potentially in native tissue, in order to investigate OR organization and mobility on the surface of intact, living cells, at low/physiological expression levels.
The μOR is the OR subtype which plays the most critical role in pain modulation, while mediating the effects of the most powerful analgesic drugs. Also, it is the OR subtype which is mostly responsible for the major adverse effects of the currently marketed opioid drugs. We aimed to develop a new μOR-selective fluorescent ligand with a potential irreversible binding mode. Although the approach was in principle successful, i.e. the labelled cells were visible and distinguishable; this initial attempt was not suitable for SMM due to the ligands’ poor selectivity and affinity as well as due to its high background noise. A second generation of the fluorescent ligand was designed; however the synthesis and characterization are part of another doctoral thesis.
Lately, δOR has received attention as a promising drug target, due to its distinct pharmacological profile which features low abuse liability and lack of physical dependence. In addition, δOR expression has been associated with cancer regulation in the periphery, thus further highlighting the interest of imaging tools for this receptor. In this thesis, the development and characterization of two new δOR-selective fluorescent probes with excellent optical properties, based on the well-studied ligand naltrindole (NTI) is presented. Their application in SMM studies is currently underway at the group of Prof. Dr. Davide Calebiro at the University of Birmingham.
The κOR is a subtype which has also emerged as a drug target due to its low abuse potential. Despite a growing interest in this receptor, κOR-selective fluorescent probes have been particularly scarce in literature. Herein, the design, synthesis and characterization of the first reported set of fluorescent κOR-selective probes with antagonistic properties, based on the established ligand 5’-guanidinonaltrindole (5’-GNTI) is presented. Two of these were employed for SMM experiments to investigate κOR homodimerization, localization and trafficking. Our findings do not support homodimerization of the κOR-bound probe complexes, while showing that the majority of them follow a normal Brownian diffusion on the cell surface.
The field of photopharmacology has attracted considerable attention due to applying the spatial and temporal precision of light to pharmacological systems. Photoswitchable biologically active compounds have proven useful in the field of G protein-coupled receptors (GPCRs), which are of tremendous therapeutic relevance. Generally, the pharmacology of GPCRs is complex, perhaps even more complex than originally thought. Suitable tools are required to dissect the different signalling pathways and mechanisms and to unravel how they are connected in a holistic image. This is reflected in the enormous scientific interest in CB2R, as the neuroprotective and immunomodulatory effects attributed to CB2R agonists have not yet translated into effective therapeutics. This work focused on the development of a novel photoswitchable scaffold based on the privileged structure of benzimidazole and its application in photoswitchable CB2R ligands as photopharmacological tools for studying the CB2R.
The visible-light photoswitchable ligand 10d enables the investigation of CB2R activation with regard to βarr2 bias, exhibiting a unique pharmacological profile as a “cis-on” affinity switch at receptor level and as a “trans-on” efficacy-switch in βarr2-mediated receptor internalization. The novel photoswitchable scaffold developed in this work further serves as a guide for the development of novel photoswitchable GPCR ligands based on the privileged structure of benzimidazole. To obtain a different tool compound for studying CB2R activation and signalling mechanisms, a previously reported putatively dualsteric CB2R ligand was rendered photoswitchable, by linking the orthosteric agonist to a CB2R-selective PAM via photoswitchable azobenzene. Compound 27-para exhibits a desirable “cis-on” behaviour across all investigated assays with >10-fold higher potency compared to its trans-isomer and can be used as an efficacy-switch employing specific concentrations.
Cancer is one of the leading causes of death worldwide. Toxic contaminants in human food or medicinal products, such as substances like pyrrolizidine alkaloids (PAs), have been thought to contribute to cancer incidence. PAs are found in many plant species as secondary metabolites, and they may affect humans through contaminated food sources, herbal medicines, and dietary supplements. Hundreds of compounds belonging to PAs have been identified, differing in their chemical structures, either in their necine base moiety or esterification at their necic acid moiety. PAs undergo hepatic metabolism, and after this process, they can induce hepatotoxicity, genotoxicity, and carcinogenicity. However, the mechanism of inducing genotoxicity and carcinogenicity is still unclear and warrants further investigation.
Therefore, the present study aims to investigate the mechanism of genotoxicity induced by selected PAs with different chemical structures in in vitro systems. Primarily, human hepatoma HepG2 cells were utilized, and in co-culture, metabolically active HepG2 cells were combined with non-metabolically active human cervical HeLa H2B-GFP cells.
First, the genotoxicity of the PAs europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine, and lasiocarpine was investigated in the cytokinesis-block micronucleus (CBMN) assay. All seven selected PAs caused the formation of micronuclei in a dose-dependent manner, with the maximal increase of micronucleus formation ranging from 1.64 to 2.0 fold. The lowest concentrations at which significant induction of micronuclei was found were 3.2 µM for lasiocarpine and riddelliine, 32 µM for retrorsine and echimidine, and 100 µM for seneciphylline, europine, and lycopsamine. These results confirmed previously published potency rankings in the micronucleus assay.
The same PAs, with the exception of seneciphylline, were also investigated in a crosslink-modified comet assay, and reduced tail formation after hydrogen peroxide treatment was found in all diester-type PAs. Meanwhile, an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration. Thus, the crosslinking activity was related to the ester type.
Next, the role of metabolic enzymes and membrane transporters in PA-induced genotoxicity was assessed. Ketoconazole (CYP 450-3A4 inhibitor) prevented lasiocarpine-induced micronucleus formation completely, while furafylline (CYP 450-1A2 inhibitor) reduced lasiocarpine-induced micronucleus formation, but did not abolish it completely. This implies that the CYP 450 enzymes play an important role in PA-induced genotoxicity.
Carboxylesterase 2 enzyme (CES 2) is commonly known to be involved in the detoxification of xenobiotics. Loperamide (CES 2 inhibitor) yielded an increased formation of lasiocarpine-induced micronuclei, revealing a possible role of CES-mediated detoxification in the genotoxicity of lasiocarpine. Also, intracellular glutathione (GSH) plays an important role in the detoxification of xenobiotics or toxins in the cells. Cells which had been pretreated with L-buthionine sulfoximine (BSO) to reduce GSH content were significantly more sensitive for the induction of micronucleus formation by lasiocarpine revealing the importance of GSH in PA-induced genotoxicity.
Quinidine (Q) and nelfinavir (NFR) are OCT1 and OATP1B1 influx transporter inhibitors, respectively, which reduced micronucleus induction by lasiocarpine (only quinidine significantly), but not completely, pointing to a relevance of OCT1 for PA uptake in HepG2 cells. Verapamil (V) and benzbromarone (Bz) are MDR1 and MRP2 efflux transporter inhibitors, respectively, and they caused a slightly increased micronucleus induction by lasiocarpine (significant only for benzbromarone) thus, revealing the role of efflux transporters in PA-induced genotoxicity.
The mechanistic approach to PA-induced genotoxicity was further studied based on oxidative stress via the formation of reactive oxygen species (ROS) in HepG2 cells. Overproduction of ROS can cross-link cellular macromolecules such as DNA, leading to genomic damage. An equimolar concentration of 10 µM of lasiocarpine (open-diester PA), riddelliine (cyclic-diester PA), and europine (monoester) significantly induced ROS production, with the highest ROS generation observed after lasiocarpine treatment, followed by riddelliine and then europine. No significant increase in ROS production was found with lycopsamine (10 µM; monoester PA), even at a higher concentration (320 µM). The generation of ROS by these PAs was further analyzed for confirmation by using 5 mM of the thiol radical scavenger antioxidant N-acetyl cysteine (NAC) combined with lasiocarpine, riddelliine, or europine. This analysis yielded a significant decrease in ROS after combining NAC with lasiocarpine, riddelliine, and europine. In addition, lasiocarpine, riddelliine, and europine induced a loss of mitochondrial membrane potential, pointing to mitochondria as the source of ROS generation.
In vivo, hepatic sinusoidal epithelial cells (HSECs) are known to be damaged first by PAs after hepatic metabolization, but HSECs themselves do not express the required metabolic enzymes for activation of PAs. To mimic this situation, HepG2 cells were used to metabolically activate PA in a co-culture with HeLa H2B-GFP cells as non-metabolically active neighbours. Due to the green fluorescent GFP label the HeLa cells could be identified easily based in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronucleus formation in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of CYP 450 enzymes with ketoconazole abrogated micronucleus formation induced by the same PAs tested in the co-culture. The efflux transporter inhibitors verapamil and benzbromarone reduced the micronucleus formation in the co-culture. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured non-metabolically active green HeLa cells.
Finally, in HepG2 cells as well as the co-culture, combinations of PAs lasiocarpine and riddelliine favoured an additive effect rather than synergism. Thus, this study therefore provides support that the assumption of dose-addition can be applied in the characterization of the genotoxicity risk of PAs present in a mixture.
p97 uses the energy of ATP hydrolysis to unfold and thereby segregate proteins. It is involved in various cellular processes such as proteasomal degradation, DNA damage repair, autophagy, and endo-lysosomal trafficking. The specificity for these processes is controlled by more than 30 regulatory cofactors.
Interactions of p97 with cofactors and target proteins are known to be highly dynamic and transient. To identify new interaction partners and to uncover novel cellular functions of p97, the interactome of endogenous p97 was determined by using in cellulo crosslinking followed by immunoprecipitation and mass spectrometry. Myoferlin (MYOF) was identified as a novel interactor of p97 and the interaction was validated in reciprocal immunoprecipitation experiments for different cell lines.
The ferlin family member MYOF is a tail-anchored membrane protein containing multiple C2 domains. MYOF is involved in various membrane repair and trafficking processes such as the endocytic recycling of cell surface receptors. The MYOF interactome was determined by mass spectrometry. Among others, the p97 cofactor PLAA, CD71 and Rab14 were identified as common interactors of p97 and MYOF. Immunoprecipitation experiments with PLAA KO cells revealed that the interaction between MYOF and p97 depends on PLAA. Immunofluorescence microscopy showed a co-localization of MYOF with Rab14 and Rab11, which are both involved in endocytic recycling pathways. Furthermore, immunofluoroscence experiments revealed that MYOF and the p97 cofactor PLAA are localized to Rab14- and Rab5-positive endosomal compartments.
Using p97 inhibitors and p97 trapping mutants, the presence of p97 at MYOF-positive and Rab14-positive structures could be demonstrated. Consistent with this finding, the endocytic recycling of transferrin was delayed upon inhibition of p97. Taken together, this work identified MYOF as a novel interactor of p97 and suggests a role for p97 in the recycling of endocytic cargo.
The Macroeconomic Dimensions of Credit: A Comprehensive Analysis of Finance, Inequality and Growth
(2024)
Schumpeter's monetary growth theory is particularly influential for the modern understanding of the macroeconomic role of banks and credit. Based on this theory, this dissertation examines the macroeconomic role of the financial system, especially credit, in (1) generating economic growth, (2) directing economic resources and (3) distributing wealth.
Chapter 3 first shows empirically that 1) there is a positive correlation between the growth of credit and economic growth, even for developed countries, 2) no empirical correlation between household saving and economic growth can be established, and 3) there are both positive, negative and insignificant effects of credit on economic growth at country-specific level. Thus, there is broad empirical support for Schumpeter's monetary hypotheses.
A particularly interesting application of Schumpeter's growth theory can be seen in China. The results of the empirical study suggest that there is generally a positive correlation between credit and economic growth in China, that is, however, not linear in terms of regions, time and size of the financial system. Furthermore, the results in Chapter 4 suggest that credit-financed industrial policy in China may have contributed to more investment and GDP growth, although there are non-linearities between individual industries and types of companies.
Finally, Chapter 5 raises the question of the role of the financial system in the distribution of wealth. While credit to households and companies, together with indicators of working and saving behavior and the age structure of the population, are the most important determinants of wealth inequality, there are also various non-linearities in the relationship between credit and wealth inequality, including in relation to the level of development of financial systems and home ownership ratios.
Binge Eating Disorder (BED) is a common, early-onset mental health condition characterised by uncontrollable episodes of overeating followed by negative emotions such as guilt and shame. An improved understanding of the neurocognitive mechanisms underlying BED is central to the development of more targeted and effective treatments. This thesis comprises a systematic review and three empirical studies contributing to this endeavour.
BED can be thought of as a disorder of cognitive-behavioural control. Indeed, self-report evidence points towards enhanced impulsivity and compulsivity in BED. However, retrospective self-reports do not capture the mechanisms underlying impulsive and compulsive lapses of control in the moment. The systematic review therefore focussed on the experimental literature on impulsivity and compulsivity in BED. The evidence was very mixed, although there was some indication of altered goal-directed control and behavioural flexibility in BED. We highlight poor reliability of experimental paradigms and the failure to properly account for weight status as potential reasons for inconsistencies between studies. Moreover, we propose that impulsivity and/or compulsivity may be selectively enhanced in negative mood states in BED and may therefore not be consistently detected in lab-based studies.
In the empirical studies, we explored the role of behavioural flexibility in BED using experimental and neuroimaging methods in concert with computational modelling. In the first empirical study, we assessed the reliability of a common measure of behavioural flexibility, the Probabilistic Reversal Learning Task (PRLT). We demonstrate that the behavioural and computational metrics of the PRLT have sufficient reliability to justify past and future applications if calculated using hierarchical modelling. This substantially improves reliability by reducing error variance. The results support the use of the PRLT in the second and third empirical studies on development and BED.
Because a majority of patients develop BED as adolescents or young adults, we speculated that it may emerge as a consequence of disrupted or deficient
maturation of behavioural flexibility. Little is known about typical development in this domain. We therefore investigated normative development of reversal learning from adolescence to adulthood in the second empirical study. Typically- developing adolescents exhibited less adaptive and more erratic and explorative behaviour than adults. This behaviour was accounted for by reduced sensitivity to positive feedback in a reinforcement learning model, and partially mediated by reduced activation reflecting uncertainty in the medial prefrontal cortex, a region known to mature substantially during adolescence.
In the third empirical study, we investigated reversal learning in BED, paying special attention to potential biases associated with learning from wins vs learning from losses. We speculated that negative urgency could make it more difficult for BED patients to learn and make decisions under pressure to avoid losses. To dissociate between effects of excess weight and BED, we collected data from obese individuals with and without BED as well as normal-weight controls. As hypothesised, there were subtle neurocognitive differences between obese participants with and without BED with regard to learning to obtain rewards and to avoid losses. Obese individuals showed relatively impaired learning to obtain rewards, while BED patients showed relatively impaired learning to avoid losses. This was reflected in differential learning signals in the brain and associated with BED symptom severity.
In sum, this thesis shows that the evidence on impulsivity and compulsivity in BED is inconsistent and offers potential explanations for this inconsistency. It highlights the need for reliability in interindividual difference research and indicates ways to improve it. Further, it charts the typical development of reversal learning from adolescence to adulthood and underscores the relevance of exploration in the context of learning and decision-making in adolescence. Finally, it demonstrates qualitative differences between BED and obesity, hinting at a pivotal role of aversive states in loss of control in BED.
Allogeneic hematopoietic cell transplantation (Allo-HCT) is the main and only treatment for many malignant and non-malignant haematological disorders. Even though the treatment has improved through the years and patient life expectancy has increased, graft versus host disease (GvHD) is still considered the main obstacle and one of the main reasons for increased mortality. Furthermore, improved patient’s survival and life expectancy brought into question the late post-HCT complications. The leading cause of late death after allo-HCT is the relapse of primary disease as well as chronic GvHD (cGvHD). However, a clear relationship was also described with pulmonary complications, endocrine dysfunction and infertility, and cataracts in post-HCT patients. In the last years big concern regarding a cumulative cardiovascular incidence in long-term survivors has been raised. Severe cardiovascular disease (CVD) is caused by atherosclerosis which is considered a chronic inflammatory disease of blood vessels. As such, it takes a long time from endothelial damage, as the onset event, and followed plaque formation to a manifestation of severe consequences, such as stroke, coronary heart disease, or peripheral arterial disease. Endothelial damage is well documented in patients post-HCT. In the context of allo-HCT, the endothelial damage is induced by the conditioning regimen with or without total body irradiation (TBI). Furthermore, endothelial cells (ECs) have been documented as a target of GvHD and increased concentration of circulating endothelial cells (CEC) coinciding with an increase in the number of circulating alloreactive T cells. According to 2021 ESC Guidelines on CVD prevention, the main atherosclerotic CVD (ASCVD) risk factors are blood apolipoprotein B (ApoB)-containing lipoproteins (of which low-density lipoprotein (LDL) is the most abundant), high blood pressure, cigarette smoking and diabetes mellitus (DM). GvHD is considered a high-risk factor for the onset of dyslipidaemia, hypertension, and DM. Overall, the risk of premature cardiovascular death is 2.7 fold increased in comparison to the general population, while the cumulative incidence of cardiovascular complications was shown to be up to 47% at ten years after reduced intensity conditioning (RIC), post-HCT. However, up to date, there are no available studies elucidating the interconnection between GvHD and atherosclerosis. The goal of this study was, therefore, to investigate the involvement of GvHD in the progression of atherosclerosis as well as to elucidate whether cytotoxic, CD8+ T cells that were shown to play a significant role in endothelial damage during the course of skin GvHD on one hand, and inducers of formation of unstable plaque on the other, are involved in this interconnection. For that purpose we established a novel minor histocompatibility anti gens (miHAg) allo-HCT Western diet (WD)-induced atherosclerosis mouse model. We were able to show that GvHD has a significant impact on atherosclerosis development in B6.Ldlr−/− recipient mice even in the absence of overt clinical disease activity. It seems that the impact is at least partly induced by CD8+ T cells, that showed significantly increased infiltration of aortic lesions in mice facing subclinical GvHD. As studies have shown in regular atherosclerotic mouse models as well as in humans, these CD8+ T cells exhibited not only increased expression of genes involved in activation, survival and differentiation to cytotoxic phenotype, but also some genes pointing out their exhaustion, that were absent in CD4+ T cell cluster. When anti-CD8β antibody was applied once per week along with WD feeding for eight weeks, the plaque formation was significantly reduced in aorta and aortic root pointing out the importance of these cells in an alloreactivity induced lesion formation. Furthermore, anti-CD8β treatment led to significantly decreased necrotic core formation followed by overall increase in plaque stability. Strikingly, bone marrow plus T cells (BMT) recipients fed WD showed significantly increased serum cholesterol levels in comparison to bone marrow (BM) (a group lacking alloreactive T cells that induce GvHD). This effect was reversed when anti-CD8β treatment was applied, suggesting, at least partly, an impact of alloreactive CD8+ T cells on cholesterol levels. Expression of genes responsible for lipid metabolism pointed out the tendency of the liver to regulate the increased cholesterol levels, however, the mechanism behind this phenotype still remains to be revealed. On the other hand, the impact of obesity, induced by chronic high-fat diet (HFD) feeding, has been shown to be an independent risk factor for gastrointestinal GvHD. Similarly, in major histocompatibility complex (MHC) disparate allo-HCT mouse model, we have noticed that even short-term WD intake leads to a significant decrease in survival of mice post-HCT. When the concentration of transplanted alloreactive T cells was reduced, the survival was improved, pointing out the involvement of these cells in the pathogenesis. Additionally, bioluminescence imaging (BLI) during initiation and effector phase of acute GvHD (aGvHD) revealed increased infiltration of alloreactive T cells in mice fed WD. Studies in an obesity model, we could confirm the involvement of specifically CD4+ T cells in WD induced impact, as the relative number of these cells was significantly increased in small intestine on day six post-HCT in mice fed WD. This increased intestinal infiltration was preceded by increase in the number of alloreactive T cells expressing intestine homing receptor (α4β7 integrin) in peripheral lymph nodes (LNs). Even though the number of T cells was not changed in the spleen of WD fed mice, the subset of CD4+ and CD8+ T cells that were highly secreting TNFα was increased as well as the expression of genes regulating pro-inflammatory cytokines such as IL-6 and interferon (IFN)γ pointing out significant WD-induced inflammation. Moreover, slight tendency towards increased intestinal permeability and load of translocated luminal bacteria, that we observed, could induce severe endotoxemia and dysregulated systemic immune response that could lead to detrimental induction of cell death. Justifying our speculations, we noted increased levels of transaminases and an increase in lactate dehydrogenase (LDH) levels (pointing out significant tissue damages). However, the exact mechanism behind this detrimental WD impact still remains to be elucidated.
The evolutionary success of higher plants is largely attributed to their tremendous developmental
plasticity, which allows them to cope with adverse conditions. However, because these adaptations
require investments of resources, they must be tightly regulated to avoid unfavourable trade-offs.
Most of the resources required are macronutrients based on carbon and nitrogen. Limitations in the
availability of these nutrients have major effects on gene expression, metabolism, and overall plant
morphology. These changes are largely mediated by the highly conserved master kinase SNF1-RELATED
PROTEIN KINASE1 (SnRK1), which represses growth and induces catabolic processes. Downstream of
SnRK1, a hub of heterodimerising group C and S1 BASIC LEUCINE ZIPPER (bZIP) transcription factors has
been identified. These bZIPs act as regulators of nutrient homeostasis and are highly expressed in
strong sink tissues, such as flowers or the meristems that initiate lateral growth of both shoots and
roots. However, their potential involvement in controlling developmental responses through their
impact on resource allocation and usage has been largely neglected so far. Therefore, the objective of
this work was to elucidate the impact of particularly S1 bZIPs on gene expression, metabolism, and
plant development.
Due to the high homology and suspected partial redundancy of S1 bZIPs, higher order loss-of-function
mutants were generated using CRISPR-Cas9. The triple mutant bzip2/11/44 showed a variety of robust
morphological changes but maintained an overall growth comparable to wildtype plants. In detail
however, seedlings exhibited a strong reduction in primary root length. In addition, floral transition
was delayed, and siliques and seeds were smaller, indicating a reduced supply of resources to the shoot
and root apices. However, lateral root density and axillary shoot branching were increased, suggesting
an increased ratio of lateral to apical growth in the mutant. The full group S1 knockout
bzip1/2/11/44/53 showed similar phenotypes, albeit far more pronounced and accompanied by
growth retardation. Metabolomic approaches revealed that these architectural changes were
accompanied by reduced sugar levels in distal sink tissues such as flowers and roots. Sugar levels were
also diminished in leaf apoplasts, indicating that long distance transport of sugars by apoplastic phloem
loading was impaired in the mutants. In contrast, an increased sugar supply to the proximal axillary
buds and elevated starch levels in the leaves were measured. In addition, free amino acid levels were
increased in bzip2/11/44 and bzip1/2/11/44/53, especially for the important transport forms
asparagine and glutamine. The increased C and N availability in the proximal tissues could be the cause
of the increased axillary branching in the mutants.
To identify bZIP target genes that might cause the observed shifts in metabolic status, RNAseq
experiments were performed. Strikingly, clade III SUGARS WILL EVENTUALLY BE EXPORTED (SWEET)
8
genes were abundant among the differentially expressed genes. As SWEETs are crucial for sugar export
to the apoplast and long-distance transport through the phloem, their reduced expression is likely to
be the cause of the observed changes in sugar allocation. Similarly, the reduced expression of
GLUTAMINE AMIDOTRANSFERASE 1_2.1 (GAT1_2.1), which exhibits glutaminase activity, could be an
explanation for the abundance of glutamine in the mutants. Additional experiments (ATAC-seq, DAPseq, PTA, q-RT-PCR) supported the direct induction of SWEETs and GAT1_2.1 by S1 bZIPs. To confirm
the involvement of these target genes in the observed S1 bZIP mutant phenotypes, loss-of-function
mutants were obtained, which showed moderately increased axillary branching. At the same time, the
induced overexpression of bZIP11 in axillary meristems had the opposite effect.
Collectively, a model is proposed for the function of S1 bZIPs in regulating sink tissue development. For
efficient long-distance sugar transport, bZIPs may be required to induce the expression of clade III
SWEETs. Thus, reduced SWEET expression in the S1 bZIP mutants would lead to a decrease in apoplastic
sugar loading and a reduced supply to distal sinks such as shoot or root apices. The reduction in longdistance transport could lead to sugar accumulation in the leaves, which would then increasingly be
transported via symplastic routes towards proximal sinks such as axillary branches and lateral roots or
sequestered as starch. The reduced GAT1_2.1 levels lead to an abundance of glutamine, a major
nitrogen transport form. The combined effect on C and N allocation results in increased nutrient
availability in proximal tissues, promoting the formation of lateral plant organs. Alongside emerging
evidence highlighting the power of bZIPs to steer nutrient allocation in other species, a novel but
evolutionary conserved role for S1 bZIPs as regulators of developmental plasticity is proposed, while
the generation of valuable data sets and novel genetic resources will help to gain a deeper
understanding of the molecular mechanisms involved
Forests are essential sources of tangible and intangible benefits, but global climate change associated with recurrent extreme drought episodes severely affects forest productivity due to extensive tree die-back. On that, it appeals to an urgency for large-scale reforestation efforts to mitigate the impact of climate change worldwide; however, there is a lack of understanding of drought-effect on sapling growth and survival mechanisms. It is also challenging to anticipate how long trees can survive and when they succumb to drought. Hence, to ensure success of reforestation programs and sustainable forest productivity, it is essential to identify drought-resistant saplings. For that, profound knowledge of hydraulic characteristics is needed. To achieve this, the study was split into two phases which seek to address (1) how the hydraulic and anatomical traits influence the sapling’s growth rate under drought stress. (2) how plant water potential regulation and physiological traits are linked to species’ water use strategies and their drought tolerance.
The dissertation is assembled of two study campaigns carried out on saplings at the Chair of Botany II, University of Würzburg, Germany. The first study involved three ecologically important temperate broadleaved tree species — saplings of 18-month (Acer pseudoplatanus, Betula pendula, and Sorbus aucuparia) — grown from seeds in contrasting conditions (inside a greenhouse and outside), with the latter being subjected to severe natural heat waves. In the second study, two additional temperate species (Fagus sylvatica and Tilia cordata) were added. The drying-out event was conducted using a randomised blocked design by monitoring plant water status in a climate-controlled chamber and a greenhouse.
In campaign I, I present the result based on analysed data of 82 plants of temperate deciduous species and address the juvenile growth rate trade-off with xylem safety-efficiency. Our results indicate biomass production varies considerably due to the contrasted growing environment. High hydraulic efficiency is necessary for increased biomass production, while safety-efficiency traits are decoupled and species-specific. Furthermore, productivity was linked considerably to xylem safety without revealing a well-defined pattern among species. Moreover, plasticity in traits differed between stressed and non-stressed plants. For example, safety-related characteristics were more static than efficiency-related traits, which had higher intra-specific variation. Moreover, we recorded anatomical and leaf traits adjustments in response to a stress condition, but consistency among species is lacking.
In campaign II, I combined different ways to estimate the degree of isohydry based on water potential regulation and connected the iso-anisohydric spectrum (i.e., hydroscape area, HSA) to hydraulic traits to elucidate actual plant performance during drought. We analysed plant water potential regulation (Ψpd and Ψmd) and stomatal conductance of 28-29 month saplings of five species. I used a linear mixed modelling approach that allowed to control individual variations to describe the water potential regulation and tested different conceptual definitions of isohydricity. The combined methods allowed us to estimate species' relative degree of isohydry. Further, we examined the traits coordination, including hydraulic safety margin, HSM; embolism resistance, P88; turgor loss, Ψtlp; stomata closure, Ps90; capacitance, C; cuticular conductance, gmin, to determine time to hydraulic failure (Thf). Thf is the cumulative effect of time to stomata closure (Tsc) and time after stomatal closure to catastrophic hydraulic failure (Tcrit).
Our results show the species' HSA matches their stomatal stringency, which confirms the relationship between stomatal response and leaf water potential decline. Species that close stomata at lower water potential notably had a larger HSA. Isohydric behaviour was mostly associated with leaf hydraulic traits and poorly to xylem safety traits. Species' degree of isohydry was also unrelated to the species' time to death during drying-out experiments. This supports the notion that isohydry behaviours are linked to water use rather than drought survival strategies. Further, consistent with our assumptions, more isohydric species had larger internal water storage and lost their leaf turgor at less negative water potentials. Counter to our expectations, neither embolism resistance nor the associated hydraulic safety margins were related to metrics of isohydry. Instead, our results indicate traits associated with plant drought response to cluster along two largely independent axes of variation (i.e., stomatal stringency and xylem safety). Furthermore, on the temporal progression of plant drought responses, stomatal closure is critical in coordinating various traits to determine species' hydraulic strategies. Desiccation avoidance strategy was linked to Tsc and coordinated traits response of Ps90, Ψtlp, and HSA, whereas desiccation tolerance was related to Tcrit and traits such as lower P88 value, high HSM, and lower gmin. Notably, the shoot capacitance (C) is crucial in Thf and exhibits dichotomous behaviour linked to both Tsc and Tcrit.
In conclusion, knowledge of growth rate trade-offs with xylem safety-efficiency combined with traits linked to species’ hydraulic strategies along the isohydry could substantially enhance our ability to identify drought-resistant saplings to ensure the success of reforestation programs and predicting sensitivity to drought for achieving sustainable forest ecosystems.
In this thesis we examine the vector boson scattering (VBS) process \(\mathrm p \mathrm p \to \mathrm e^+ \nu_\mathrm e\mu^-\bar\nu_\mu\mathrm j\mathrm j +X\) (short: \(\mathrm W^+\mathrm W^-\) scattering) at NLO accuracy in two experimental setups by performing a Monte Carlo analysis of a \(13\,\mathrm{TeV}\) LHC run. \(\mathrm W^+\mathrm W^-\) scattering shows similarities and differences compared to the scattering of other vector bosons. We present a detailed description of the types of appearing subprocesses and background processes. We give insight into our code which solves the problems we are faced within \(\mathrm W^+\mathrm W^-\) scattering. This is especially the presence of the Higgs-boson resonance in the fiducial phase-space region. Particular attention is dedicated to the permutation of resonances. The integrated signal cross section at LO \(\mathcal O(\alpha^6)\) amounts to \(2.6988(3)\,\mathrm{fb}\) and \(1.5322(2)\,\mathrm{fb}\), respectively, in the two experimental setups. The LO QCD-induced background of \(\mathcal O(\alpha_\mathrm s^2\alpha^4)\) amounts to \(6.9115(9)\,\mathrm{fb}\) and \(1.6923(3)\,\mathrm{fb}\). The EW corrections to the signal are \(-11.4\%\) and \(-6.7\%\), the QCD corrections amount to \(-5.2\%\) and \(-23.0\%\). The EW corrections to the background are \(-8.3\%\) and \(-5.3\%\), the QCD corrections amount to \(-30.3\%\) and \(-77.6\%\). Our results for the QCD corrections and the QCD-induced background include a large uncertainty from varying the renormalisation and factorisation scale, and we discuss improvements for future calculations. We show the differential cross sections with unique features of \(\mathrm W^+\mathrm W^-\) scattering compared to other VBS processes and investigate in particular the subprocess of Higgs-boson production by using a modified version of our setups.
The Role of Sphingosine 1-phosphate and S1PR1-3 in the Pathophysiology of Meningococcal Meningitis
(2024)
Neisseria meningitidis (N. meningitidis) is an obligate human pathogen which causes live-threatening sepsis and meningitis. The fatality rate after meningococcal infection is high and surviving patients often suffer from severe sequelae. To cause meningitis, N. meningitidis must overcome the endothelium of the blood-brain barrier. The bacterium achieves this through the interaction with endothelial surface receptors leading to alternations of the cellular metabolism and signaling, which lastly results in cellular uptake and barrier traversal of N. meningitidis. Sphingosine 1-phosphate (S1P) is a lipid mediator that belongs to the class of sphingolipids and regulates the integrity of the blood-brain barrier through the interaction with its cognate receptors S1P receptors 1-3 (S1PR1-3).
In this study, high performance liquid chromatography coupled with mass spectrometry (LC-MS/MS) was used to generate a time-resolved picture of the sphingolipid metabolism in a brain endothelial cell line (hCMEC/D3) upon meningococcal infection. Among various changes, S1P was elevated in the cellular compartment as well as in the supernatant of infected hCMEC/D3s. Analysis of mRNA expression in infected hCMEC/D3s with quantitative real-time polymerase chain reaction (RT-qPCR) revealed that the increase in S1P could be attributed to the enhanced expression of the S1P-generating enzyme sphingosine kinase 1 (SphK1). Antibody-based detection of SphK1 protein or phosphorylation at SphK1 residue Serine 225 in hCMEC/D3 plasma membrane fractions via Western Blot revealed that N. meningitidis also induced SphK1 phospho-activation and recruitment to the plasma membrane. Importantly, recruitment of SphK1 to the plasma membrane increases the probability of substrate encounter, thus elevating SphK activity. Enhanced SphK activity was also reflected on a functional level, as detected by a commercially available ATP depletion assay used for measuring the enzymatic activity of SphK. Infection of hCMEC/D3 cells with pilus-deficient mutants resulted in a lower SphK activation compared to the N. meningitidis wild type strain. hCMEC/D3 treatment with pilus-enriched protein fractions showed SphK activation similar to the infection with living bacteria and could be ascribed to pilus interaction with the membrane-proximal domain of cellular surface receptor CD147. Inhibition of SphK1 or SphK2 through pre-treatment with specific inhibitors or RNA interference reduced uptake of N. meningitidis into hCMEC/D3 cells, as measured with Gentamicin protection assays. Released S1P induced the phospho-activation of epidermal growth factor receptor (EGFR) via S1PR2 activation, whose expression was also increasing during infection. Furthermore, S1PR2 blockage had a preventive effect on bacterial invasion into hCMEC/D3 cells. On the contrary, activation of S1PR1+3 also reduced bacterial uptake, indicating an opposing regulatory role of S1PR1+3 and S1PR2 during N. meningitidis uptake. Moreover, SphK2 inhibition prevented inflammatory cytokine expression as well as release of interleukin-8 after N. meningitidis infection. Taken together, this study demonstrates the central role of S1P and its cognate receptors S1PR1-3 in the pathophysiology of meningococcal meningitis.
In this thesis, the Josephson effect in mercury telluride based superconducting quantum point contacts (SQPCs) is studied. Implementing such confined structures into topological superconductors has been proposed as a means to detect and braid Majorana fermions. For the successful realization of such experiments though, coherent transport across the constriction is essential. By demonstrating the Josephson effect in a confined topological system, the presented experiments lay the foundation for future quantum devices that can be used for quantum computation. In addition, the experiments also provide valuable insights into the behavior of the Josephson effect in the low-channel limit (N<20). Due to the confinement of the weak link, we can also study the Josephson effect in a topological insulator, where the edge modes interact.
In conclusion, this thesis discusses the fabrication of, and low-temperature measurements on mercury telluride quantum point contacts embedded within Josephson junctions. We find that the merging of the currently used fabrication methods for mercury telluride quantum point contacts and Josephson junctions does not yield a good enough device quality to resolve subbands of the quantum point contact as quantization effects in the transport properties. As we attribute this to the long dry etching time that is necessary for a top-contact, the fabrication process was adapted to reduce the defect density at the superconductor-semiconductor interface. Employing a technique that involves side contacting the mercury telluride quantum well and reducing the size of the mercury telluride mesa to sub-micrometer dimensions yields a quantized supercurrent across the junction. The observed supercurrent per mode is in good agreement with theoretical predictions for ballistic, one-dimensional modes that are longer than the Josephson penetration depth. Moreover, we find that oscillatory features superimpose the plateaus of the supercurrent and the conductance. The strength of these oscillatory features are sample-dependent and complicate the identification of plateaus. We suggest that the oscillatory features originate mainly from local defects and the short gate electrode. Additionally, resonances are promoted within the weak link if the transparency of the superconductor-HgTe interface differs from one.
Furthermore, the research explores the regimes of the quantum spin Hall effect and the 0.5 anomaly. Notably, a small yet finite supercurrent is detected in the QSH regime. In samples fabricated from thick mercury telluride quantum wells, the supercurrent appears to vanish when the quantum point contact is tuned into the regime of the 0.5 anomaly. For samples fabricated from thin mercury telluride quantum wells, the conductance as well as the supercurrent vanish for strong depopulation. In these samples though, the supercurrent remains detectable even for conductance values significantly below 2 e²/h.
Numerical calculation reproduce the transport behavior of the superconducting quantum point contacts.
Additionally, the topological nature of the weak link is thoroughly investigated using the supercurrent diffraction pattern and the absorption of radio frequency photons. The diffraction pattern reveals a gate independent, monotonous decay of $I_\text{sw}(B)$, which is associated with the quantum interference of Andreev bound states funneled through the quantum point contact. Interestingly, the current distribution in the weak link appears unaffected as the quantum point contact is depleted. In the RF measurements, indications of a 4π periodic supercurrent are observed as a suppression of odd Shapiro steps. The ratio of the 4π periodic current to the 2π periodic current appears to decrease for smaller supercurrents, as odd Shapiro steps are exclusively suppressed for large supercurrents. Additionally, considering the observation that the supercurrent is small when the bulk modes in the quantum point contact are fully depleted, we suggest that the re-emerging of odd Shapiro steps is a consequence of the group velocity of the edge modes being significantly suppressed when the bulk modes are absent. Consequently, the topological nature of the superconducting quantum point contact is only noticeable in the transport properties when bulk modes are transmitted through the superconducting quantum point contact.
The shown experiments are the first demonstration of mercury telluride superconducting quantum point contacts that exhibit signatures of quantization effects in the conductance as well as the supercurrent. Moreover, the experiments suggest that the regime of interacting topological edge channels is also accessible in mercury telluride superconducting quantum point contacts. This is potentially relevant for the realization of Majorana fermions and their application in the field of quantum computation.
In this work we expanded upon a study from our group where a ligand-based TNF-α mutein was developed to engage specifically TNFR2 and not TNFR1 activating Tregs and expanding them, which in an allo-HCT context conferred protection from GvHD. Fusing TNF trimers to the heavy chain of an Fc-dead and mouse irrelevant antibody, a new generation of this agonist was developed called NewSTAR2. It is believed that other members of the TNFSF can also target Tregs, therefore additional agonists against DR3 and GITR were developed under the same principles as for NewSTAR2. Phenotyping analysis of the expression of these three receptors were done to confirm their specificity for Tregs before in vitro and in vivo testings with mice or murine splenic cells. A potent expansion of Tregs was seen with NewSTAR2 and the other agonists as well as upregulation of activation markers on Tregs. Thorough analyses with NewSTAR2-treated mice showed how Tregs in several immune and non-immune organs were expanded and upregulated immunomodulatory receptors. A miniature suppressive assay and other cocultures with responder cells confirmed their enhanced suppression over unstimulated Tregs through contact dependent and independent mechanisms. Despite other myeloid cells also being increased after treatment, no undesired effects were observed under steady-state and prophylactic administration of a single dose of NewSTAR2 improved survival frequencies and lessened development of clinical symptoms. Prophylactic treatment with the other TNFRSF agonists showed similar protection yet Fc(DANA)-muTL1A was superior in in terms of less death events and lower clinical score. It was found that not all the three TNFSF members have redundant functions as development of skin lesions was observed with GITRL-based agonist Fc(DANA)-muGITRL, although its expansion of Tregs in steady-state was remarkable with no apparent adverse effects. Neither agonist had an impact on donor cell engraftment or allorective T cell response, however NewSTAR2-treatmend proved to reduce inflammation in small intestine and liver. This work is proof of concept of the effectivity of selectively engaging TNFSF to activate Tregs and expand them systemically allowing them to control strong and complex immune interactions like those governing GvHD.
Site-directed bioorthogonal conjugation techniques have substantially advanced research in numerous areas. Their exceptional value reflects in the extent of applications, that have been realized with spacial-controlled bioorthogonal reactions. Specific labeling of surfaces, proteins, and other biomolecule allows for new generations of drug delivery, tracking, and analyzing systems. With the continuous advance and refinement of available methods, this field of research will become even more relevant in the time to come. Yet, as individual as the desired purpose is, as different can be the most suitable modification strategy. In this thesis, two different bioconjugation approaches, namely CuAAC and factor XIIIa mediated ligation, are used in distinct application fields, featuring eGFP as a model protein showcasing the advantages as well as the challenges of each technique.
The introduction of a unique accessible functionality is the most critical feature of a site-specific reaction, and the first considerable hurdle to clear. While most surfaces, peptides, or small molecules might require less expenditure to modulate, equipping large biomolecules like proteins with additional traits requires careful consideration to preserve the molecule’s stability and function. Therefore, the first section of this project comprises the engineering of eGFP via rational design. Initially, wild-type eGFP was subcloned, expressed, and characterized to serve as a reference value for the designed variants. Subsequently, eGFP was mutated and expressed to display a recognition site for factor XIIIa. Additionally, a second mutant harbored a TAG-codon to enable amber codon suppression and consequently the incorporation of the alkyne bearing unnatural amino acid Plk to support a CuAAC reaction. Fluorescence spectroscopy was used to confirm that the fluorescent properties of all expressed muteins were identically equal to wild-type eGFP, which is a reliable marker for the intact barrel structure of the protein. Trypsin digestion and HPLC were deployed to confirm each protein variant's correct sequence and mass.
The second part of this work focuses on the conjugation of cargo molecules deploying the chosen approaches. Solid-phase peptide synthesis was used to create a peptide that served as a lysine donor substrate in the crosslinking mechanism of FXIIIa. Additionally, the peptide was provided with a cysteine moiety to allow for highly flexible and simple loading of desired cargo molecules via conventional thiol-Michael addition, thus establishing an adaptive labeling platform. The effective ligation was critically reviewed and confirmed by monitoring the exact mass changes by HPLC. Protocols for attaching payloads such as biotin and PEG to the linker peptide were elaborated. While the biotin construct was successfully conjugated to the model protein, the eGFP-PEG linkage was not achieved judging by SDS-PAGE analysis. Furthermore, featuring isolated peptide sequences, the properties of the FXIIIa-mediated reaction were characterized in detail. Relative substrate turnover, saturation concentrations, by-product formation, and incubation time were comprehensively analyzed through HPLC to identify optimal reaction conditions. CuAAC was successfully used to label the Plk-eGFP mutein with Azide-biotin, demonstrated by western blot imaging.
Within the last part of this study, the application of the conjugation systems was extended to different surfaces. As regular surfaces do not allow for immediate decoration, supplementary functionalization techniques like gold-thiol interaction and silanization on metal oxides were deployed. That way gold-segmented nanowires and Janus particles were loaded with enoxaparin and DNA, respectively. Nickel and cobalt nanowires were modified with silanes that served as linker molecules for subsequent small molecule attachment or PEGylation. Finally, the eGFP muteins were bound to a particle surface in a site-specific manner. Beads displaying amino groups were utilized to demonstrate the effective use of FXIIIa in surface modification. Moreover, the bead’s functional moieties were converted to azides to enable CuAAC “Click Chemistry” and direct comparison. Each modification was analyzed and confirmed through fluorescence microscopy.