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This thesis focusses on the synthesis of functional chiral molecules using carbo- or hetero[7]helicenes as a chiral element, combined with multiple helicenes, phthalocyanines, and 1,4-azaborine units. The objective is to achieve properties that surpass those of the parent compounds.
In the first project, an enantiopure, propeller-shaped multi-helicene polycyclic aromatic hydrocarbon containing three (P)-[7]helicene units and three (M)-[5]helicene units was stereospecifically synthesized and can be obtained in gram quantities. Leveraging the configurational stability of [7]helicene and the configurational instability of [5]helicene, we exclusively obtained the most thermodynamically stable enantiomer out of 10 possible enantiomeric pairs. The effects of the multi-helicene structure on optical rotation, UVVis absorption, fluorescence, and electronic circular dichroism (CD) spectroscopy were investigated.1
Building on the success of the first project, the second project used the configurationally stable [7]helicene again. Zinc-[7]helicenocyanine (Zn-7HPc) was stereospecifically synthesized by directly conjugating [7]helicenes with a phthalocyanine (Pc) core. Zn-7HPc demonstrates a CD signal in the near-infrared region, indicating efficient chirality transfer from the helicenes to the Pc core. Zn-7HPc forms stable, discrete homochiral dimers over a wide range of concentrations in tetrahydrofuran and dimethyl sulfoxide, as well as in the solid state. These homochiral dimers are formed even within the racemic mixture due to the interlocking of two homochiral monomers. The large comproportionation constant and the observed intervalence charge transfer band that appeared in spectroelectrochemistry experiments indicate strong communication between the two Pc monomers in the dimer.2
In the third project, aza[7]helicenes were incorporated with a 1,4-azaborine unit, which exhibits a multiple-resonance effect, to achieve narrow-band emission, high fluorescence quantum yield (FL), and a small Stokes shift. These properties are essential for ultrahigh-definition organic light-emitting diodes that emit circularly polarized light (CP-OLEDs). The synthesized series of molecules demonstrate small Stokes shifts (0.06–0.07 eV), exceptionally narrow fluorescence and circularly polarized luminescence bands with small full width at half maximum (FWHM, 17–28 nm, 0.07–0.13 eV), and high FL (72–85%).3
In conclusion, the synthesis of functional chiral molecules based on carbo- or hetero[7]helicenes was successfully achieved. The efficient synthetic strategies and improved properties of these molecules provide valuable insights for further investigations into helicenes with advanced structures and enhanced properties.
Axon growth, a fundamental process of neuron development, is regulated by both intrinsic and external guidance signals. Impairment of axon growth and maintenance is implicated in the pathogenesis of neurodegenerative disorders such as Amyotrophic Lateral Sclerosis and Alzheimer’s disease (AD). Axon growth is driven by several post-transcriptional RNA processing mechanisms, including alternative splicing, polyadenylation, subcellular localization, and translation. These mechanisms are controlled by RNA-binding proteins (RBPs) through interacting with their target RNAs in a sequence-dependent manner. In this study, we investigate the cytosolic functions of two neuronal RBPs, Ptbp2 and hnRNP R, which are essential for axon growth in motoneurons.
Polypyrimidine tract binding protein 2 (Ptbp2) contributes to neuronal differentiation and axonogenesis by modulating different splicing programs to adjust the level of proteins involved in these processes. While the nuclear functions of Ptbp2 in alternative splicing have been studied in more detail, the cytosolic roles of Ptbp2 associated with axon growth have remained elusive. In the first part of the study, we show that Ptbp2 is present in cytosolic fractions of motoneurons including axons and axon terminals. Depletion of Ptbp2 impairs axon growth and growth cone maturation in cultured embryonic mouse motoneurons. Moreover, Ptbp2 knockdown affects the level of piccolo protein in the growth cone of cultured motoneurons. We detect Ptbp2 as a top interactor of the 3' UTR of the Hnrnpr transcript encoding the RBP hnRNP R. This interaction results in axonal localization of and thereby local translation of Hnrnpr mRNA in motoneurons. Consequently, axonal synthesis of hnRNP R was diminished upon depletion of Ptbp2 in motoneurons. We present evidence that Ptbp2 through cooperation with translation factor eIF5A2 controls hnRNP R synthesis. Additionally, we observe that re-expression of hnRNP R in Ptbp2-deficient motoneurons rescued axon growth defect while Ptbp2 overexpression failed to normalize the axon elongation defect observed in hnRNP R-deficient motoneurons. Our findings pinpoint axonal synthesized hnRNP R as a mediator of Ptbp2 functions in axon growth.
In the second part of this study, we identify hnRNP R binds to the 3' UTR of microtubule-associated tau (Mapt) transcript encoding tau protein and regulates the axonal translocation and translation of Mapt mRNA. Tau protein has a central role in neuronal microtubule assembly and stability. However, in AD, the accumulation of abnormally hyperphosphorylated tau protein leads to axon outgrowth defects. Loss of hnRNP R reduces axonal tau protein but not the total level of tau. We observe that the brains of 5xFAD mice, as a mouse model of AD, deficient for hnRNP R contain lower phospho-tau and amyloid-β plaques. Likewise, Neurons treated with blocking antisense oligonucleotides (ASO) to prevent binding of hnRNP R to Mapt mRNA show reduced axonal Mapt mRNA and consequently newly synthesized tau protein levels. We show that blocking Mapt mRNA transport to axons impairs axon elongation. Our data thus suggest that reducing tau levels selectively in axons, a major subcellular site of tangle formation, might represent a novel therapeutic approach for the treatment of AD.
In this work, two techniques, based on the established method of pump--probe spectroscopy were used to investigate the properties of molecular systems in the liquid phase within the visible spectral wavelength range.
The first technique is standard transient absorption (TA) spectroscopy which was applied to a diazo-precursor to identify the formation of a biradical in an inert solvent after UV excitation. With the combination of EPR spectroscopy and quantum chemical calculations, the formation of a biradical in an unpolar and non-protic solvent was proven. Besides, in the presence of air or a polar and protic solvent, the biradical reacts ultrafast to various side products.
The second technique is time-resolved circular dichroism (TRCD) spectroscopy, which was performed in two different ways. The first approach based on a pulse-enantiomer (PE) setup, where an initially circularly polarized pulse was split into two pulses, of which one was mirrored under normal incidence, to flip its polarization. The result was two pulses with mirrored polarization states that propagate collinearly to the sample as left and right circularly polarized probe pulses. The alignment procedure as well as the drawbacks of this setup are described in detail.
However, a new TRCD setup was built that used a polarization grating to get left and right circularly polarized pulses. With the experiences of working with the PE setup, the new TRCD setup could be optimized so that TRCD spectra of a chiral squaraine polymer could be measured. With the help of quantum chemical calculations, the signals were assigned to exciton dynamics that describe spatial and energetic rearrangements of the excitation energy. The alignment and the measurement procedures to perform TRCD spectroscopy with the new setup are described in detail for future experiments.
Climate change and associated extreme weather events are a threat not only for agricultural
yields but the plant kingdom in general. Therefore, there is a great necessity to better
understand the plants' intrinsic mechanisms to combat heat stress. The plant heat stress
response already has been investigated in many studies, including the role of HSFA1
transcription factors as the central regulators. Other aspects such as the initial perception of
heat and the role of heat-induced changes in plant metabolism are rather unknown.
In this thesis, the natural variation of 250 different accessions of Arabidopsis thaliana was
investigated regarding the temperature-dependent accumulation of raffinose and
triacylglycerols. A connection between these phenotypes and respective genotypes was
established using genome-wide association studies. As a result, the candidate gene
TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1), was identified. Enzymatic TPS1 is responsible
for the synthesis of trehalose 6-phosphate (T6P), which serves as an indicator and regulator
of sucrose homeostasis.
Subsequent analyses using tps1 tilling mutants demonstrated a link between T6P metabolism
and an increased accumulation of various soluble carbohydrates and starch, including
raffinose both under control conditions and during heat exposure. Furthermore, the mutant
lines displayed enhanced thermotolerance and survival rates following long-term heat stress.
Transcriptome analyses, however, did not show any difference in the regulation of canonical
heat stress-associated genes. Instead, genes related to photosynthesis were overrepresented
among the differentially upregulated genes in tps1 tilling lines during heat exposure. In this
work, a direct connection of T6P signaling, sucrose homeostasis, and thermotolerance is
shown for the first time.
In a second project, two Arabidopsis thaliana accessions (Oberursel-0, accession ID: 7276;
Nieps-0, accession ID: 7268) showing distinct capacities to acquire short-term
thermotolerance were compared to identify the putative causative regulators or mechanisms
that lead to the different levels of thermotolerance.
An examination of the transcriptomes of 7268 and 7276 showed that several hundreds of
genes were already differentially regulated within 10 minutes of exposure to 32 °C or 34 °C.
Among these, several genes associated with sulfur metabolism were more highly induced in
the more thermotolerant accession 7268. However, experimental as well as genetic
manipulation of sulfur availability and metabolism did not result in altered thermotolerance.
In addition to sulfur-related genes, most of the canonical heat stress-associated genes were
more highly expressed in 7268 than in 7276. While we could not identify a causative regulator
or mechanism of differential thermotolerances, the data strongly suggests that 7268 either
has a higher overall sensitivity, i.e., the heat stress response is initiated at lower temperatures,
or stronger overall heat stress response when exposed to a certain elevated temperature.
The seasonal snow cover in the European Alps plays a crucial role in the region's climate, ecology, and economy. It affects the local climate through its high albedo, protects permafrost, provides habitats, and acts as a water reservoir that feeds European rivers. However, these functions are threatened by climate change. Analyzing snow cover dynamics is essential to predict future developments and assess related ecological and economic impacts.
This study explores the potential of long Earth Observation (EO) time series for modeling and predicting the snow line elevation (SLE) in the Alps. Based on approximately 15,000 Landsat satellite images, SLE time series were generated for the years 1985 to 2022. Various univariate forecasting models were evaluated, with the best results achieved by Random Forests, Telescope, and Seasonal ARIMA. A newly developed approach combines the best models into a robust ensemble, achieving an average Nash-Sutcliffe efficiency (NSE) of 0.8 in catchments with strong seasonal signals.
Forecasts for 2030 indicate significant upward shifts in the SLE, particularly in the Western and Southern Alps. Given the variability in results, a multivariate modeling approach using climate variables is recommended to improve prediction accuracy. This study lays the groundwork for future models that could potentially project SLE dynamics through the end of the 21st century under various climate scenarios, which is highly relevant for climate policy in the Alpine region.
The TRAF-binding receptor CD40 belongs to the TNFR superfamily and is broadly expressed on healthy cells, mainly on antigen-presenting cells, but also on other immune cells and non-immune cells. CD40 is bound by its ligand CD40L, which is essential for a wide range of immunological responses by inducing or inhibiting different pathways that are essential for a variety of cellular processes, including immune activation and maturation. (1,2) Dysregulated CD40 signalling has been implicated in inflammatory diseases, such as hyper-IgM syndrome, psoriasis, and cancer. (3–6) Due to its broad expression across various tumour types, it can serve as a tumour-associated antigen and has therefore been proposed as a target for antibodies for cancer treatment. (2,7,8)
Agonistic anti-CD40 antibodies have been demonstrated to induce anti-tumoural immune responses as well as therapeutic immunity. (2) Furthermore, prolonged stimulation of CD40 in tumour cells in vitro has been shown to decrease proliferation, increase expression of cytotoxic TNFSFLs and induce apoptosis. (9,10) Their effect on anti-tumoral responses has been well studied and anti-tumoral responses by DC maturation and suppression of malignant growth of B-cells have been confirmed and were found to induce cell death in tumours in vitro. (11–14)
Many agonistic anti-CD40 antibodies specifically have been reported to require secondary crosslinking by binding to either activating or inhibitory FcγRs to be agonistic in vitro, while in vivo studies have indicated inhibitory FcƴR2B expression as critical factor. (15–17) However, FcƴR independent agonism has also been reported for anti-CD40 antibodies. (18,19) While agonistic anti-CD40 IgG1, IgG3 and IgG4 antibodies have been shown to display FcƴR dependent agonism, agonistic anti-CD40 IgG2 antibodies have shown to display FcƴR independent agonism. Conversion of anti-CD40 IgG1 antibodies into IgG2 has also been shown to convert the antibody’s agonism into FcƴR independent agonism. (20)
To overcome FcƴR dependency, bispecific antibody fusion proteins containing a scFv as anchoring domain allowing for crosslink independent of FcƴR binding have been designed before. This approach has been found to display strong agonism for other antibody fusion proteins when bound to both targets, with response levels resembling that of FcƴR bound antibodies. (21,22)
The relevance of antibody isotype and idiotype for FcƴR-dependent agonism as well as the relevance of valency and antibody oligomerization for FcƴR-independent agonism were investigated in this study on a panel of different anti-CD40 antibodies. Several clinically investigated anti-CD40 antibodies (ADC-1013(23), APX005M(24), ChiLob7.4(25) and CP-870,893(26)) and one preclinical antibody (G28.5(27,28)) were considered. Selected antibodies were then cloned onto an IgG1, IgG1(N297A), IgG2 and IgG4 backbone. The IgG1(N297A) isotype is an IgG1 antibody with a point mutation (N297A) that is known to strongly reduce binding to FcƴR1, while reducing the binding affinity to FcƴR2B to undetectable levels. (29,30) In this work it is demonstrated that the investigated anti-CD40 antibody variants across different isotypes activate both the classical and alternative NFκB pathway by stimulating U2OS cells in an FcƴR dependent manner. Stimulation in the presence of both human FcƴRs as well as murine FcƴRs resulted in CD40 stimulation. A difference in binding competition was observed for the various anti-CD40 IgG1 antibodies, but no indication of a CRD-dependent mechanism responsible for their agonistic activity was found. Moreover, this FcƴR dependency could be overcome by creation of tetravalent antibody fusion proteins.
CRISPR-Cas systems are a versatile tool in genetic engineering because they can be easily reprogrammed to cut a specific chromosomal region or RNA transcript. The choice of nuclease, gRNA design, and target region all influence targeting efficiency, so the appropriate CRISPR components should be chosen depending on the desired application. This thesis examines factors that influence targeting in both DNA- and RNA-targeting CRISPR systems. Chapter 1 discusses the importance of target RNA abundance in shaping the immunity of type VI CRISPR systems. In bacteria, the Cas13 nuclease is known to degrade RNA specifically and non-specifically, leading to cell growth arrest, also known as dormancy. In this chapter, the factors that determine dormancy are investigated by targeting genome- and plasmid-encoded transcripts in E. coli. The observations are extended to a gRNA library targeting the entire coding genome and gRNA design rules are extrapolated. Finally, the role of Cas13 in defense is investigated by testing how the system behaves during viral infection or plasmid transformation. Chapter 2 also looks at the factors that characterize targeting efficiency, but focuses on the Cas12a DNA-targeting system in K. pneumoniae. The ultimate goal is to develop CRISPR antimicrobials as alternatives to antibiotics to eliminate multidrug-resistant and hypervirulent bacteria. Several nucleases are tested for antimicrobial activity, the Cas12a nuclease is selected and the same gRNAs are used against different strains to understand the robustness of the method. Rules for gRNA design are also investigated by looking at secondary structure and testing a gRNA library across several genomic regions in two different strains. This information is used to develop a machine-learning algorithm to predict gRNA activity. In addition, the CRISPR-Cas systems are also packaged in a T7-like phage with engineered tail fibers and delivered to K. pneumoniae. While Chapter 2 uncovers various factors that improve targeting efficiency, Chapter 3 aims to reduce targeting by the Cas9 and Cas12a nucleases to favor homology-directed repair for genome editing in E. coli. Targeting is slowed down so that some copies of the chromosomes remain intact, allowing the bacterium to survive and integrate the desired edit. To reduce targeting, different gRNA formats or nuclease variations are used, gRNA expression is modulated, or gRNAs with attenuated targeting are designed. Attenuated gRNAs are tested to introduce point mutations as well as whole gene deletions and substitutions, and the method is extended to Klebsiella oxytoca and Klebsiella pneumoniae, where it is applied to block transcription of an antibiotic resistance gene in the genome, restoring sensitivity to ampicillin. Overall, this work discusses how changing the CRISPR components alters the outcome of targeting and highlights strategies to achieve efficient or attenuated targeting depending on the desired application.
Hematopoietic stem cell transplantation (HSCT) is a promising therapy for various malignancies and immune deficiency diseases, but it is often associated with graft versus host disease (GvHD), a life-threatening complication arising from immunological incompatibility between donor T cells and host tissues. Current standard therapies for GvHD involve the use of calcineurin inhibitors (CNIs) such as cyclosporine A (CsA) and tacrolimus (FK506), which effectively suppress T cell activation and proliferation. However, these drugs also impair the graft versus leukemia (GvL) effect, which is the advantageous ability of donor T cells to eliminate malignant cells.
Our previous studies demonstrated that the selective deletion of one or two members of the nuclear factor of activated T cells (NFAT) transcription factor family in donor T cells effectively prevented harmful GvHD without compromising GvL activity. This finding highlighted the potential of NFAT as a therapeutic target for GvHD.
In this study, we developed and evaluated novel treatment strategies that specifically target NFAT during allogeneic HSCT. We focused on the development of small molecules that mimic the PxIxIT motif of NFAT, thereby competitively inhibiting its binding to CN (CN) without affecting CN phosphatase activity. We identified two promising candidates, C17 and MRD37, and evaluated their efficacy in inhibiting NFAT and suppressing pro-inflammatory cytokine production. Among these molecules, MRD37 demonstrated the highest potency in selectively inhibiting NFAT at a sub-IC50 concentration without compromising the functional capacity of regulatory T cells (Tregs) in vitro. Furthermore, we demonstrated that MRD37 could effectively protect mice from major mismatch GvHD in vivo. This protection was initially predicted to be due to the enhanced presence of Tregs and Tr1-type cells but when pretreated T cells devoid of Tregs were transplanted it unraveled an additional increase of Th2-like cytokine release. Finally, our in vitro studies on human T cells confirmed that MRD37 could specifically inhibit NFAT while preserving the Treg population, suggesting its potential as a novel therapeutic strategy for GvHD.
Our findings provide compelling evidence for the development of MRD37 as promising alternative to CNIs in mitigating GvHD.
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.