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Regulatory required, classical toxicity studies for environmental hazard assessment are costly, time consuming, and often lack mechanistic insights about the toxic mode of action induced through a compound. In addition, classical toxicological non-human animal tests raise serious ethical concerns and are not well suited for high throughput screening approaches. Molecular biomarker-based screenings could be a suitable alternative for identifying particular hazardous effects (e.g. endocrine disruption, developmental neurotoxicity) in non-target organisms at the molecular level. This, however, requires a better mechanistic understanding of different toxic modes of action (MoA) to describe characteristic molecular key events and respective markers.
Ecotoxicgenomics, which uses modern day omic technologies and systems biology approaches to study toxicological responses at the molecular level, are a promising new way for elucidating
the processes through which chemicals cause adverse effects in environmental organisms. In this context, this PhD study was designated to investigate and describe MoA-characteristic
ecotoxicogenomic signatures in three ecotoxicologically important aquatic model organisms of different trophic levels (Danio rerio, Daphnia magna and Lemna minor).
Applying non-target transcriptomic and proteomic methodologies post chemical exposure, the aim was to identify robust functional profiles and reliable biomarker candidates with potential
predictive properties to allow for a differentiation among different MoA in these organisms. For the sublethal exposure studies in the zebrafish embryo model (96 hpf), the acute fish embryo toxicity test guideline (OECD 236) was used as conceptual framework. As different test compounds with known MoA, the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyrostatic 6-propyl-2-thiouracil (6-PTU), as well as six nerve- and muscle-targeting insecticides (abamectin, carbaryl, chlorpyrifos, fipronil, imidacloprid and methoxychlor) were evaluated. Furthermore, a novel sublethal immune challenge assay in early zebrafish embryos (48 hpf) was evaluated for its potential to assess immuno-suppressive effects at the gene expression level. Therefore, toxicogenomic profiles after an immune response inducing stimulus with and without prior clobetasol propionate (CP) treatment were compared. For the aquatic invertebrate D. magna, the study was performed with previously determined low effect concentrations (EC5 & EC20) of fipronil and imidacloprid according to the acute immobilization test in water flea (OECD 202). The aim was to compare toxicogenomic signatures of the GABA-gated chloride channel blocker (fipronil) and the nAChR agonist (imidacloprid). With similar low effect concentrations, a shortened 3 day version of the growth inhibition test with L. minor (OECD 221) was conducted to find molecular profiles differentiating between photosynthesis and HMG-CoA reductase inhibitory effects. Here, the biological interpretation of the molecular stress response profiles in L. minor due to the lack of functional annotation of the reference genome was particularly challenging. Therefore, an annotation workflow was developed based on protein sequence homology predicted from the genomic reference sequences.
With this PhD work, it was shown how transcriptomic, proteomic and computational systems biology approaches can be coupled with aquatic toxicological tests, to gain important mechanistic insights into adverse effects at the molecular level. In general, for the different investigated adverse effects for the different organisms, biomarker candidates were identified, which describe a potential functional link between impaired gene expressions and previously reported apical effects. For the assessed chemicals in the zebrafish embryo model, biomarker candidates for thyroid disruption as well as developmental toxicity targeting the heart and central nervous system were described. The biomarkers derived from nerve- and muscletargeting insecticides were associated with three major affected processes: (1) cardiac muscle cell development and functioning, (2) oxygen transport and hypoxic stress and (3) neuronal development and plasticity. To our knowledge, this is the first study linking neurotoxic insecticide exposure and affected expression of important regulatory genes for heart muscle (tcap, actc2) and forebrain (npas4a) development in a vertebrate model. The proposed immunosuppression assay found CP to affect innate immune induction by attenuating the response of genes involved in antigen processing, TLR signalling, NF-КB signalling, and complement activation ...
As one of the most widespread infectious diseases in the world, it is currently estimated that approximately 296 million people globally are chronically infected with Hepatitis B virus (HBV), the consequences of HBV infection cause more than 620,000 deaths each year. Although safe and effective HBV vaccines have reduced the incidence of new HBV infections in most countries, there are still around 1.5 million new infections each year. HBV remains a major health problem because there is no large-scale effective vaccination strategy in many countries with a high burden of disease, many people with chronic HBV infection are not receiving effective and timely treatment, and a complete cure for chronic infection is still far from being achieved.
Since its discovery, HBV has been identified as an enveloped DNA virus with a diameter of 42 nm. For efficient egress from host cells, HBV is thought to acquire the viral envelope by budding into multivesicular bodies (MVBs) and escape from infected cells via the exosome release pathway. It is clear that HBV hijacks the host vesicle system to complete self-assembly and propagation by interacting with factors that mediate exosome formation. Consequently, the overlap with exosome biogenesis, using MVBs as the release platform, raises the possibility for the release of exosomal HBV particles. Currently, virus containing exosomal vesicles have been described for several viruses. In light of this, this study explored whether intact HBV-virions wrapped in exosomes are released by HBV-producing cells.
First, this study established a robust method for efficient separation of exosomes from HBV virions by a combination of differential ultracentrifugation and iodixanol density gradient centrifugation. Fractionation of the density gradient revealed that two populations of infectious viral particles can be separated from the culture fluids of HBV-producing cells. The population present in the low-density peak co-migrates with the exosome markers. Whereas the population that appeared in the high-density fractions was the classical HBV virions, which are rcDNA-containing nucleocapsids encapsulated by the HBV envelope.
Subsequently, the characterization of this low-density population was performed, namely the highly purified exosome fraction was systematically investigated. Relying on the detergent sensitivity of the exosome membrane and the outer envelope of the HBV virus, disruption of the exosome structure by treatment with limited detergent revealed the presence of HBsAg in the exosomes. At the same time, mild and limited NP-40 treatment of highly purified exosomes and a further combination of density gradient centrifugation resulted in the stepwise release of intact HBV virions and naked capsids from the exosomes generated by HBV-producing cells. This implies the presence of intact HBV particles encapsulated by the host membrane.
The presence of exosome-encapsulated HBV particles was consequently also verified by suppressing the morphogenesis of MVBs or exosomes. Impairment of MVB- or exosome-generation with small molecule inhibitors has significantly inhibited the release of host membrane-encapsulated HBV particles as well. Likewise, silencing of exosome-related proteins caused a diminution of exosome output, which compromised the budding efficiency of wrapped HBV.
Moreover, electron microscopy images of ultra-thin sections combined with immunogold staining visualized the hidden virus in the exosomal structure. Additionally, the presence of LHBs on the surface of exosomes derived from HBV-expressing cells was also observed.
As expected, these exosomal membrane-wrapped HBV particles can spread productive infection in differentiated HepaRG cells. In HBV-susceptible cells, as LHBs on the membrane surface, this type of exosomal HBV appeared to be uptaken in an NTCP receptor-dependent manner.
Taken together these data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV. Exosomes hijacked by HBV act as a transporter impacting the dissemination of the virus.
Ischemic heart disease caused by occlusion of coronary vessels leads to the death of downstream tissues, resulting in a fibrotic scar that cannot be resolved. In contrast to the adult mammalian heart, the adult zebrafish heart can regenerate following injury, enabling the study of the underlying cellular and molecular mechanisms. One of the earliest responses that take place after cardiac injury in adult zebrafish is coronary revascularization. Previous transcriptomic data from our lab show that vegfc, a well-known regulator of lymphatic development, is upregulated early after injury and peaks at 96 hours post cryoinjury, coinciding with the peak of coronary endothelial cell proliferation. To test the hypothesis that vegfc is involved in coronary revascularization, I examined its expression pattern and found that it is expressed by coronary endothelial cells after cardiac damage. Using a loss-of-function approach to block Vegfc signaling, I found that it is required for coronary revascularization during cardiac regeneration. Notably, blocking Vegfc signaling resulted in a significant reduction in cardiomyocyte regeneration. Using transcriptomic analysis, I identified the extracellular matrix component gene emilin2a and the chemokine gene cxcl8a as effectors of Vegfc signaling. During cardiac regeneration, cxcl8a is expressed in epicardium-derived cells, while the gene encoding its receptor cxcr1 is expressed on coronary endothelial cells. I found that overexpressing emilin2a increases coronary revascularization, and induces cxcl8a expression. Using loss-of-function approaches, I observed that both cxcl8a and cxcr1 are required for coronary revascularization after cardiac injury.
Altogether, my findings indicate that Vegfc acts as an angiocrine factor that plays an important role in regulating cardiac regeneration in zebrafish. Mechanistically, Vegfc promotes the expression of emilin2a, which promotes coronary proliferation, at least in part by enhancing Cxcl8a-Cxcr1 signaling. This study helps in understanding the mechanisms underlying coronary revascularization during cardiac regeneration, with promising therapeutic applications for human heart regeneration.
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
Sensors for high rate charge particle tracking have to withstand the harsh radiation doses deposited by the particles to be sensed. This holds particularly for the novel CMOS Monolithic Active Pixel Sensors, which are considered a promising sensor technology for future vertex detectors due to their very light material budget and excellent spatial resolution. To resist the radiation doses expected close to the interaction regions of heavy-ion experiments, the sensors have to be hardened against radiation doses, which exceed the native tolerance of CMOS technology significantly. In this thesis, the results of non-ionizing radiation hardness studies at the IKF on sensor prototypes developed at the IPHC in Strasbourg are presented. Our results demonstrate that the CMOS sensors evaluated in the context of this thesis can withstand non-ionizing radiation of up to 5×10^14 neq/cm^2. This hardness qualifies them as promising candidates for use in future vertex detectors.
The stellar nucleosynthesis of elements heavier than iron can primarily be attributed to neutron capture reactions in the s and r process. While the s process is considered to be well understood with regards to the stellar sites, phases and conditions where it occurs, nucleosynthesis networks still need accurate neutron capture cross sections
with low uncertainties as input parameters. Their quantitative outputs for the isotopic abundances produced in the s process, coupled with the observable solar abundances, can be used to indirectly infer the expected r process abundances. The two stable gallium isotopes, 69Ga and 71Ga, have been shown in sensitivity studies to have considerable impact on the weak s process in massive stars. The available experimental data, mostly derived from neutron activation measurements for quasi-stellar neutron spectra at kBT = 25 keV, show disagreements up to a factor of three.
Determining the differential neutron capture cross section can provide input data for the whole range of astrophysically relevant energies. To that end, a neutron time of flight experimental campaign at the n_TOF facility at CERN was performed for three months, using isotopically enriched samples of both isotopes. The data taken at the EAR1 experimental area covered a wide neutron energy range from thermal to several hundred keV. The respective differential and spectrum averaged neutron capture cross sections for 69Ga and 71Ga were determined in this thesis. They show good agreement with the evaluated cross sections for 71Ga, but reproduce the deviations from the evaluated data that other, more recent activation measurements showed for 69Ga.
The main task of modern large experiments with heavy ions, such as CBM (FAIR), STAR (BNL) and ALICE (CERN) is a detailed study of the phase diagram of quantum chromodynamics (QCD) in the quark-gluon plasma (QGP), the equation of state of matter at extremely high baryonic densities, and the transition from the hadronic phase of matter to the quark-gluon phase.
In the thesis, the missing mass method is developed for the reconstruction of short-lived particles with neutral particles in their decay products, as well as its implementation in the form of fast algorithms and a set of software for prac- tical application in heavy ion physics experiments. Mathematical procedures implementing the method were developed and implemented within the KF Par- ticle Finder package for the future CBM (FAIR) experiment and subsequently adapted and applied for processing and analysis of real data in the STAR (BNL) experiment.
The KF Particle Finder package is designed to reconstruct most signal particles from the physics program of the CBM experiment, including strange particles, strange resonances, hypernuclei, light vector mesons, charm particles and char- monium. The package includes searches for over a hundred decays of short-lived particles. This makes the KF Particle Finder a universal platform for short-lived particle reconstruction and physics analysis both online and offline.
The missing mass method has been proposed to reconstruct decays of short-lived charged particles when one of the daughter particles is neutral and is not regis- tered in the detector system. The implementation of the missing mass method was integrated into the KF Particle Finder package to search for 18 decays with a neutral daughter particle.
Like all other algorithms of the KF Particle Finder package, the missing mass method is implemented with extensive use of vector (SIMD) instructions and is optimized for parallel operation on modern many-core high performance com- puter clusters, which can include both processors and coprocessors. A set of algorithms implementing the method was tested on computers with tens of cores and showed high speed and practically linear scalability with respect to the num- ber of cores involved.
It is extremely important, especially for the initial stage of the CBM experiment, which is planned for 2025, to demonstrate already now on real data the reliability of the developed approach, as well as the high efficiency of the current implemen- tation of both the entire KF Particle Finder package, and its integral part, the missing mass method. Such an opportunity was provided by the FAIR Phase-0 program, motivating the use in the STAR experiment of software packages orig- inally developed for the CBM experiment.
Application of the method to real data of the STAR experiment shows very good results with a high signal-to-background ratio and a large significance value. The results demonstrate the reliability and high efficiency of the missing mass method in the reconstruction of both charged mother particles and their neutral daughter particles. Being an integral part of the KF Particle Finder package, now the main approach for reconstruction and analysis of short-lived particles in the STAR experiment, the missing mass method will continue to be used for the physics analysis in online and offline modes.
The high quality of the results of the express data analysis has led to their status as preliminary physics results with the right to present them at international physics conferences and meetings on behalf of the STAR Collaboration.
In Europe, the sugar refinery is largely based on sugar beets. This route for obtaining household sugar results in a large amount of biomass waste, consisting mainly of the insoluble beet resi-dues, e.g., cell wall fragments. To a vast moiety this debris consists of the polymer pectin (up to 20% in the dry total solids). The structure of pectin is based on a backbone of D-galacturonic acid units (GalA), but also contains various other sugar monomers, predominantly L-arabinose, D-galactose, L-rhamnose and D-xylose. The amount of GalA adds up to a moiety of up to 70% with-in this sugar cocktail. So far, this debris is only fed to cattle or simply burnt. In nature, pectin is a common substrate for various organisms. The degradation of pectin-rich biomass is often per-formed by filamentous fungi like Hypocrea jecorina (also known as Trichoderma reesei) and As-pergillus niger, which evolved pectinases to degrade the pectin backbone and pathways to con-sume the monomer GalA as a sole carbon source. The fungal catabolism of pectin residues starts with the reduction of GalA to L-galactonate (GalOA) by a GalA-reductase. Even though filamen-tous fungi are native hosts of the GalA-catabolism and certain engineering approaches have al-ready been demonstrated, this class of organisms remains challenging with regard to bioreactor cultivation and tedious genetic accessibility. In contrast, the yeast S. cerevisiae is well known in fermentation processes and easily modified by a versatile set of genetic tools. So far, first ap-proaches have already been conducted to transfer the GalA utilization pathways into S. cerevisiae, but these approaches indicated limitations regarding GalA-uptake and redox cofac-tor replenishment due to the relatively high oxidative state of GalA compared to other sugars like glucose and galactose. Furthermore, the generally strongly increased demand for redox co-factors must be met by GalA reduction by finding new cofactor sources or redirecting reactions of the core metabolism.
This work aimed at the production of GalOA, which is the first intermediate of the fungal GalA catabolism. This compound shows an interesting range of potential applications, for instance as a food and cosmetic additive. To overcome the oxidized character of GalA, the presence of a more reduced co-substrate as a redox donor and as a carbon and energy source was required. To further enhance the reduction of GalA, modulation of the redox-cofactor supply and enzyme engineering were performed.
This work is about resumptive and non-resumptive relative clauses (RCs) in the three big Ibero-Romance languages: Spanish, Portuguese, and Catalan. In (1), the examined structures are exemplified for Spanish: (1a.) No conozco el hombre que viste _ ayer. “I don’t know the man that you saw yesterday.” (1b.) Es este el hombre que le enviaron el libro. “This is the man to whom they sent the book.” (1c.) Es este el hombre a quien le enviaron el libro. “This is the man to whom they sent the book.”
(1a.) displays a non-resumptive, or canonical, RC, which is characterized by the canonical use of a relativizing operator and a gap in the subordinate’s object position, a piece of evidence which has induced most of the generative literature to assume wh-movement of the relative operator in the sense of Chomsky (1977). The last two decades, however, have seen a big debate regarding the exact derivational analysis, starting with Kayne’s (1994) antisymmetry theory and the following focus on reconstruction and anti-reconstruction effects in RCs. This search for the correct starting site of the RC’s head noun has dismissed the original Head External Analysis (HEA) (Chomsky 1965, 1977) and led to the development of a Head Raising Analysis (RA) (Kayne 1994, Bianchi 1999, a.o.) and a Matching Analysis (MA) (Munn 1994, Sauerland 1998, a.o.). The discussion in this work argues that the data on reconstruction and anti-reconstruction effects are not sufficiently clear and reliable in order to adopt one of the head-internal analyses, i.e. a HEA or a MA. Instead, the work follows a variant of the HEA proposed for Portuguese by Rinke & Aßmann (2017), which adheres to standard assumptions about Romance syntax, and avoids the empirical problems that the other proposals have to face. Arguing that the HEA holds for all Ibero-Romance languages, this work also takes a stance in the debate around the categorical status of the relativizing element que and argues that it is always a D-element, and never of category C, i.e. there is no such thing as a relativizing complementizer (cf. also Kayne 2010, Kato & Nunes 2008, Poletto & Sanfelici 2018).
The work argues that wh-movement in a HEA fashion is the correct analysis also for resumptive relative clauses as in (1b., c.), which crucially lack a gap in argument position but show a resumptive pronominal element instead. Furthermore, it takes advantage of the fact that the choice of such genetically closely related languages like Spanish, Portuguese, and Catalan enables research to address the phenomenon under consideration from a microcomparative perspective, which is “the closest we can come … to a controlled experiment in comparative syntax” (Kayne 2005: 281-282). The descriptive literature suggests that, at least for Spanish and Catalan, there are two types of a resumptive RC structure available: a simple resumption as in (1b.), including mere que, and a complex resumption structure which displays a more complex relativizer like a quien in combination with a resumptive pronoun (1c.). However, a corpus study carried out for this work reveals that speakers of the three languages behave alike insofar as the only resumptive RC used in spontaneous speech is a simple-resumption structure, while complex resumption never occurs. Additionally, a multivariate analysis shows that in all three languages, grammatical case is the most important factor when it comes to the possibility of a resumptive structure in RCs: with a dative argument, simple resumption is obligatory, while for accusative and nominative arguments, resumption is optional. The discussion concludes that simple and complex resumption constitute different phenomena also on a structural level: the latter one is argued to be a subcase of clitic doubling, and therefore, receives an analysis along the lines of Pineda (2016), who argues against a dative alternation in Romance languages and locates the (non-)realisation of the dative clitic in a transitive clitic-doubling structure outside of syntax, it being a case of silent variation along the lines of Sigurðsson (2004) and Kayne (2005). From this perspective, it follows naturally that in Portuguese, complex resumption structures are ungrammatical. Simple resumption, on the other hand, which is a possible structure in all three languages, is argued to represent the phonological counterpart of “scattered deletion”, i.e. the preferred interpretation for an A’-chain according to Chomsky (2003): in the operator position SpecCP, every feature except for the operator feature is deleted, resulting in the phonological outcome que, while in the variable position, everything but the operator is interpreted, resulting in a pronominal element according to the argument’s phi-features.
This work takes a stance in the latest topics on generative analyses for relative clauses. Using not only theoretical considerations but conclusions drawn from empirical data on three languages, it offers a new perspective on pending questions and proposes to take a fresh look on supposedly outdated analyses.
Gait analysis as a clinical examination method has been increasingly used in recent years. In particular, the external knee adduction moment was often used as a surrogate measure for internal medial knee joint loading, e.g., in elderly individuals with medial knee osteoarthritis. Therefore, the knee adduction moment is also associated with the progression of knee osteoarthritis. Children and adolescents with valgus malalignment have been found to experience a reduced external knee adduction moment, but internal knee joint contact forces, particularly in the lateral compartment, were not previously studied.
First, medial and lateral knee joint contact forces were studied using muskulosceletal modeling in young individuals with and without valgus malalignment treated by guided growth. In addition, a systematic literature review was conducted to explore the relationship between external joint moments and internal joint contact forces. Finally, this relationship was investigated in children and adolescents with and without valgus malalignment. Furthermore, we examined whether statistical models could be determined to accurately predict internal knee joint contact forces by commonly used parameters from three-dimensional gait analysis, such as external knee joint moments.
It was found that guided growth normalized knee joint contact forces after treatment. In addition, the static radiographic mechanical axis angle correlated better after the treatment when the patients showed a typical limb alignment compared to the correlation before guided growth with the valgus malalignment due to compensating strategies during gait. Furthermore, the systematic review showed that the peak medial knee joint contact force was best predicted by the knee adduction moment and even better together with the knee flexion moment in the first half of stance. However, for the second half of stance of the medial knee joint contact force and the entire stance of the lateral knee joint contact force, only low correlations with knee adduction and/or flexion moment were found. Finally, statistical models could be determined with high accuracy for both medial and lateral knee joint contact force, for both peaks in the first and second half of stance, and for both study groups of children and adolescents with and without valgus malalignment by including knee adduction and flexion moment as predictors.
These results demonstrate the importance of examining not only the external knee adduction moment but also the knee flexion moment and, even better, the medial and lateral knee joint contact forces when evaluating knee joint loading. With these statistical models, clinicians can predict the medial and lateral knee joint contact forces without the need to perform musculoskeletal simulations and can therefore use standard three-dimensional gait analysis parameters such as knee adduction and flexion moment. This can improve guided growth treatment in children and adolescents with valgus malalignment with regard to implantation or explantation of the growth restricting plates or to rebound. Instrumented gait analysis could be particularly helpful in borderline cases, as kinematic compensation mechanisms during gait may play a role and the static radiograph alone does not provide information about dynamic joint loads.
Single-electron transport in focused electron beam induced deposition (FEBID)-based nanostructures
(2022)
Mit steigender Komplexität von integrierten Schaltungen im Nanometer-Maÿstab werden immer innovativere Techniken nötig, um diese zu fabrizieren. Dies erfordert einen starken Fokus auf die Kontrolle der Fabrikation akkurater Strukturen und der Materialreinheit, und dies im Zusammenhang mit einer skalierbaren Produktion. In diesem Kontext hat Elektronenstrahlinduzierte Abscheidung (engl. Focused Electron Beam Induced Deposition, FEBID) eine wachsende Aufmerksamkeit im Bereich der Nanostrukturierung gewonnen. Der FEBID-Prozess basiert auf der lokalen Abscheidung von Material auf einem Substrat. Das Deponat entsteht durch die Spaltung von Präkursor-Molekülen durch die Interaktion mit einem Elektronenstrahl entsteht. Als Beispiel sei hier der Präkursor Me3PtCpMe angeführt. Das auf dem Substrat abgelagerte Material besteht aus wenigen Nanometer großen Kristalliten aus Platin, welche in einer Matrix aus amorphem Kohlenstoff eingebettet sind. Die Pt-C FEBID Ablagerungen sind nano-granulare Metalle, deren elektrische Transporteigenschaften die Folge des Zusammenspiels von diffusivem Transport von Ladungen innerhalb der Pt-Kristalliten und temperaturabhängigen Tunneleffekten sind. Das größte Interesse an diesen Materialien liegt an der Möglichkeit, Strukturen für technische Anwendungen im Nanometerbereich herstellen zu können.
In dieser Arbeit wurden Anwendungen, die auf Einzelelektroneneffekten beruhen, ausgewählt, um die FEBID basierte Probenpräparation zu testen. Um Einzelelektronentransport zu ermöglichen, der auf dem Tunneln einzelner Elektronen basiert, müssen alle Parameter wie Grösse und Abstände der Strukturen genauestens definiert sein. Im Rahmen dieser Arbeit wurden Einzelelektronenbausteine entwickelt, die auf zwei unterscheidlichen Anwendungen des Pt-C FEBID-Prozesses basieren. Die beiden Anwendungen sind: 1) Arrays von Gold-Nanopartikeln (Au-NP), welche mittels Pt-Strukturen kontaktiert wurden, die mit FEBID präpariert und anschlieÿend aufgereinigt wurden; 2) Einzelelektronentransistoren (engl. Single-Electron Transistors, SET), deren Inseln aus elektronennachbestrahlten Pt-C FEBID Deponate bestehen. Die elektrischen Eigenschaften der präparierten Nanostrukturen wurden charakterisiert und mit der erzielten Auflösung und Materialqualität in Relation gesetzt. Es wurden Optimierungen an der Präparationsmethode durchgeführt, welche direkt die Leitfähigkeit des Pt-C FEBID-Materials erhöhen. Dies kann durch die Änderung der
Karbonmatrix oder die Erhöhung des metallischen Gehalts der Struktur geschehen. In dieser Arbeit wurde eine katalytische Aufreinigungsmethode von Pt-C FEBID Strukturen für zwei Anwendungen genutzt: zum Einen wurden die aufgereinigten Strukturen als Keimschichten für die nachfolgende ortsgenaue Atomlagenabscheidung (engl. Area-Selective Atomic Layer
Deposition, AS-ALD) von Pt-Dünnschichten genutzt. Zum Anderen wurde diese Technik dafür genutzt, Metallbrücken zwischen den bereits durch Auftropfen zufällig auf dem Substrat aufgebrachten NP-Gruppen und den zuvor aufgebrachten UV-Lithographie (UVL) präparierten Cr-Au Kontakten zu erzeugen. Eine NP-Gruppe ist ein periodisches, granulares Array von Partikeln, welche uniform in Größe und Form sind und einen unterschiedlichen Grad von Ordnung inne haben. Durch die Art des Aufbringens kann die Anordnung der Nanopartikel durch Lösen und Erzeugen der Verbindungen beeinflusst werden. Diese Systeme zeigen ein Verhalten wie Tunnelkontakte mit Coulombblockade und eine Verteilung der Schwellspannung. Die Ergebnisse der elektrischen Messungen bestätigen den Einzelelektronentransport durch die Nanopartikel in einem typischen Elektronentransportregime mit schwacher Kopplung. Trotz dieser Ergebnisse war die Anwendung dieser Technik für die SET Nanostrukturierung nicht erfolgreich. Die Ursache
konnte zurückgeführt werden auf das Vorhandensein von Pt-Partikeln in der Nähe der Kontakte zu den Au-NP-Arrays. Die Pt-Partikel sind durch den FEBID Fertigungsprozess in
der Nähe der vorgegebenen Struktur entstanden. Aus diesem Grund wurde das FEBID Co-Deponat in der folgenden SET-Nanofabrikation entfernt.
Ein SET basiert auf einer Nano-Insel, welche durch Tunnelkontakte mit Source- und Drain-Elektroden verbunden ist. Darüber hinaus besteht eine kapazitive Verbindung zu einer
oder mehreren Gate-Elektrode(n). Innerhalb der Insel gibt es eine feste Anzahl von Elektronen.
In dieser Arbeit wurden die Source-, Drain- und Gate-Kontakte durch Ätzen mittels eines fokussierten Gallium-Strahls erzeugt, was Abstände von 50nm ermöglichte, wohingegen die SET Insel mit Pt-C FEBID-Material erzeugt wurde. Die Leitfähigkeit der Insel aus Pt-C wurde mit anschließender Elektronenbestrahlung erhöht. Als letzter Präparationsmethode wurde ein neueartiges Argon-Ätzverfahren genutzt, um die durch FEBID erzeugten Co-Ablagerungen in der direkten Umgebung der Insel zu entfernen. Durch die Elektronennachbestrhalung kann die Kopplung der einzelnen metallischen Kristalliten angepasst werden. Die Auswirkungen unterschiedlicher starker Tunnelkontakte auf die elektronischen Eigenschaften der Insel und die daraus resultierende Performanz des SETs wurden in dieser Arbeit beobachtet ...
Folgend auf den ersten Realisierungen von Bose-Einstein Kondensaten erschienen weitere innovative Experimente, die sich in den optischen Gittern gefangenen Quantengasen widmeten. In diesen zahlreichen, wissenschaftlichen Untersuchungen konnten die Eigenschaften von Bose-Einstein Kondensaten besser verstanden werden. Das Prinzip von Vielteilchensystemen, gefangen in einem periodischen Potential, bot eine Plattform zur Untersuchung weiterer Quantenphasen.
Eine konzeptionell einfache Modifikation von solchen Systemen erhält man durch die Kopplung der Grundzustände der gefangenen Teilchen an hoch angeregten Zuständen mithilfe einer externen Lichtquelle. Im Falle dessen, dass diese Zustände nahe der Ionisationsgrenze des Atoms liegen, spricht man von Rydberg-Zuständen und Atome, welche zu diesen Zuständen angeregt werden, bezeichnet man als Rydberg-Atome. Eines der vielen charakteristischen Eigenschaften von Rydberg-Atomen ist die Fähigkeit über große Entfernungen jenseits der atomaren Längenskalen zu wechselwirken. Im Rahmen von Vielteilchensystemen wurden dementsprechend Kristallstrukturen aus gefangenen Rydberg-Atomen experimentell beobachtet.
Nun stellt sich die Frage, was mit einem gefangenen Bose-Einstein Kondensat passiert, dessen Teilchen an langreichweitig wechselwirkenden Zuständen gekoppelt sind. Gibt es ein Parameterregime, in dem sowohl Kristallstruktur als auch Suprafluidität in solchen Systemen koexistieren können? Dies ist die zentrale Frage dieser Arbeit, die sich mit der Theorie von gefangenen Quantengasen gekoppelt an Rydberg-Zuständen auseinandersetzt.
This thesis is concerned with the study of symmetry breaking phenomena for several different semilinear partial differential equations. Roughly speaking, this encompasses equations whose symmetries are not necessarily inherited by their solutions, which is particularly interesting for ground state solutions.
Die vorliegende Dissertation stellt eine Methode zur Löslichkeitsbestimmung vor, die für die Anwendung im Rahmen von BCS-Biowaiver Monografien entwickelt wurde. Der Methode und dem dafür konzipierten Studienprotokoll liegt das Prinzip der „Minimallöslichkeit“ zugrunde. Damit lässt sich einfach, kosteneffizient und wissenschaftlich verlässlich feststellen, ob ein Arzneistoff „hochlöslich“ gemäß den BCS-Biowaiver Richtlinien der Gesundheitsbehörden FDA, EMA und WHO ist und sich dementsprechend generische Produkte des Arzneistoffs grundsätzlich für das BCS-Biowaiver Zulassungsverfahren eignen.
Dieses Verfahren für die Zulassung von Generika erlaubt die Beurteilung der Bioäquivalenz eines festen generischen Arzneimittels zur peroralen Anwendung auf Basis von in vitro-Freisetzungsuntersuchungen anstatt von in vivo-Studien wie z.B. pharmakokinetischen Studien am Menschen und erleichtert dadurch eine Marktzulassung sowohl durch Zeit- als auch Kosteneinsparung. Die Anwendung des Verfahrens ist von Vorteil, um die Verfügbarkeit von qualitativ hochwertigen, generischen (und damit kostengünstigen) Arzneimitteln zu erhöhen. Dies ist besonders wünschenswert für die Verfügbarkeit von gemäß der Weltgesundheitsorganisation essenziellen Arzneistoffen und unter denen gerade von solchen, die zur Bekämpfung von Krankheiten mit nur wenigen und/oder teuren therapeutischen Alternativen benötigt werden.
Entstanden ist die Löslichkeitsbestimmungsmethode im Rahmen von zwei Projekten, die beide zu diesem Ziel einer guten globalen Gesundheitsversorgung beitragen: die Erstellung der Biowaiver Monografien von Proguanilhydrochlorid (ein Malaria-Prophylaktikum) und Cefalexinmonohydrat (ein Antibiotikum aus der Gruppe der Cephalosporine) setzt die Publikationsreihe „Biowaiver Monograph Series“ der FIP Focus Group „Bioclassification/Biowaiver“ fort. Jede Monografie gibt eine umfassende wissenschaftliche Empfehlung zur Eignung eines Wirkstoffs der WHO „Model List of Essential Medicines“ und seiner generischen Produkte für das BCS-Biowaiver Verfahren hinsichtlich aller regulatorisch geforderten Aspekte ab. Proguanilhydrochlorid (BCS Klasse III – „hochlöslich“ und nicht „hoch permeabel“) und Cefalexinmonohydrat (BCS Klasse I – „hochlöslich“ und „hoch permeabel“) sind beide für dieses Zulassungsverfahren geeignet.
Im Zuge des anderen Projektes wurde die Löslichkeit und anschließend die BCS Klasse von Wirkstoffen bestimmt, die der 16. und 17. Version der WHO „Model List of Essential Medicines“ neu hinzugefügt wurden. Neun von 16 untersuchten Wirkstoffen, die in feste, perorale Arzneimittel formuliert werden können, sind im Hinblick auf ihre BCS Klasse für das eine Zulassung per BCS-Biowaiver geeignet. Eine umfangreichere Empfehlung könnte im Rahmen einer Biowaiver Monografie gegeben werden.
Die experimentelle Bestimmung der Löslichkeit über einen pH-Wert-Bereich von 1-6,8 war essenzieller Bestandteil beider Projekte, da Literaturdaten zur Löslichkeit der Wirkstoffe nicht oder nur unvollständig vorlagen. Die entwickelte Methode basiert auf einer im Kleinmaßstab angesetzten „Shake-Flask“-Methode zur Bestimmung der thermodynamischen Löslichkeit, wird jedoch in einem Zeitrahmen von 24 Stunden durchgeführt. Sie nutzt die höchste Dosis der Wirkstoffe als Substanzmenge, um zu bestimmen, ob dieser „hochlöslich“ gemäß den BCS-Biowaiver Richtlinien ist oder nicht. Die Methode bzw. das dazugehörige Studienprotokoll beinhalten Empfehlungen zu den einzelnen Schritten der Durchführung, der Auswahl der Medien und Herausforderungen wie Präzipitation (Fallbeispiel: Proguanilhydrochlorid) und Zersetzungsreaktionen (Fallbeispiel: Cefalexinmonohydrat). Löslichkeitsdaten, die mit dieser Methode erhoben werden, können für eine Zulassung per BCS-Biowaiver bei den Gesundheitsbehörden eingereicht werden, aber auch für ein Vorab-Screening genutzt werden, dass „hochlösliche“ Arzneistoffe aus einer Vielzahl von Substanzen herauszufiltern soll, um nähere Untersuchungen im Rahmen einer Biowaiver Monografie anzuschließen.
Machine learning (ML) techniques have evolved rapidly in recent years and have shown impressive capabilities in feature extraction, pattern recognition, and causal inference. There has been an increasing attention to applying ML to medical applications, such as medical diagnosis, drug discovery, personalized medicine, and numerous other medical problems. ML-based methods have the advantage of processing vast amounts of data.
With an ever increasing amount of medical data collection and large, inter-subject variability in the medical data, automated data processing pipelines are very much desirable since it is laborious, expensive, and error-prone to rely solely on human processing. ML methods have the potential to uncover interesting patterns, unravel correlations between complex features, learn patient-specific representations, and make accurate predictions. Motivated by these promising aspects, in this thesis, I present studies where I have implemented deep neural networks for the early diagnosis of epilepsy based on electroencephalography (EEG) data and brain tumor detection based on magnetic resonance spectroscopy (MRS) data.
In the project for early diagnosis of epilepsy, we are dealing with one of the most common neurological disorders, epilepsy, which is characterized by recurrent unprovoked seizures. It can be triggered by a variety of initial brain injuries and manifests itself after a time window which is called the latent period. During this period, a cascade of structural and functional brain alterations takes place leading to an increased seizure susceptibility.
The development and extension of brain tissue capable of generating spontaneous seizures is defined as epileptogenesis (EPG).
Detecting the presence of EPG provides a precious opportunity for targeted early medical interventions and, thus, can slow down or even halt the disease progression. In order to study brain signals in this latent window, animal epilepsy models are used to provide valuable data as it is extremely difficult to obtain this data from human patients. The aim of this study is to discover biomarkers of EPG using animal models and then to find the equivalent and counterparts in human patients' data. However, the EEG features for EPG are not well-understood and there is not a sufficiently large amount of annotated data for ML-based algorithms. To approach this problem, firstly, I utilized the timestamp information of the recorded EEG from an animal epilepsy model where epilepsy is induced by an electrical stimulation. The timestamp serves as a form of weak supervision, i.e., before and after the stimulation. Secondly, I implemented a deep residual neural network and trained it with a binary classification task to distinguish the EEG signals from these two phases. After obtaining a high discriminative ability on the binary classification task, I proposed to divide further the time span after the stimulation for a three-class classification, aiming to detect possible stages of the progression of the latent EPG phase. I have shown that the model can distinguish EEG signals at different stages of EPG with high accuracy and generalization ability. I have also demonstrated that some of the learned features from the network are clinically relevant.
In the task of detecting brain tumors based on MRS data, I first proposed to apply a deep neural network on the MRS data collected from over 400 patients for a binary classification task. To combat the challenge of noisy labeling, I developed a distillation step to filter out relatively ``cleanly'' labeled samples. A mixing-based data augmentation method was also implemented to expand the size of the training set. All the experiments were designed to be conducted with a leave-patient-out scheme to ensure the generalization ability of the model. Averaged across all leave-patient-out cross-validation sets, the proposed method performed on par with human neuroradiologists, while outperforming other baseline methods. I have demonstrated the distillation effect on the MNIST data set with manually-introduced label noise as well as providing visualization of the input influences on the final classification through a class activation map method.
Moreover, I have proposed to aggregate information at the subject level, which could provide more information and insights. This is inspired by the concept of multiple instance learning, where instance-level labels are not required and which is more tolerant to noisy labeling. I have proposed to generate data bags consisting of instances from each patient and also proposed two modules to ensure permutation invariance, i.e., an attention module and a pooling module. I have compared the performance of the network in different cases, i.e., with and without permutation-invariant modules, with and without data augmentation, single-instance-based and multiple-instance-based learning and have shown that neural networks equipped with the proposed attention or pooling modules can outperform human experts.
To this day, stroke is the leading cause of death and disability worldwide. Due to increasing age of the world population and poor lifestyle, the incidence is further rising. Besides mechanical thrombectomy as a surgical option, there is a lack of therapeutic options with recombinant tissue plasminogen activator (rt-PA) being the only approved drug for treatment for ischemic stroke. However, there are various problems that make the administration of rt-PA difficult. In particular, it can only be given for ischemic (not hemorrhagic) stroke, and there is a narrow time frame of 4.5 hours after onset of stroke, in which it can be successfully applied. While the success rates of combined thrombectomy with rt-PA are around 60%, less than 5% of patients receive this therapy.
ß-Hydroxybutyrate (BHB) is a ketone body that is formed in high amounts during fasting and lipolysis. Ketone bodes and the ketogenic diet have been shown to have neuroprotective properties in neurodegenerative diseases. In prior work of our group, the ketogenic diet was shown to have beneficial effects in mice after transient ischemia. In the present work, a single dose of BHB was tested for beneficial effects. For this purpose, microdialysis was used to demonstrate that BHB can cross the blood-brain barrier. For the next series of experiments, transient cerebral ischemia was induced in mice for 90 minutes by unilaterally occluding the middle cerebral artery (MCAO) with a silicone-covered filament. Behavioral tests one day after BHB administration showed that the moderate dose of 30 mg/kg, given immediately after reperfusion, improved the neurological score significantly whereas a lower (10 mg/kg) and a higher dose (100 mg/kg) had no effects The main part of the experiments focused on mitochondrial respiration as a potential mechanism of action for BHB. In isolated mitochondria from mouse brain, BHB (1-10 mM) was able to stimulate mitochondrial respiration stronger than pyruvate, but not as strong as succinate.. In the following experiments, MCAO was induced in vivo, and mitochondria were isolated and investigated ex vivo. Experiments were conducted 60 minutes, 24 hours, 72 hours, and 7 days after cerebral ischemia and reperfusion. Besides mitochondrial respiration (normalized to mitochondrial protein content or citrate synthase activity), several other parameters were monitored: the development of bodyweight throughout the experiment, citrate synthase activity, plasma metabolites and behavior to assess motor functions. Three behavioral tests were conducted: first, the Corner test, an experiment for measuring the extent of unilateral movement. Here, if a stroked mouse is put into a narrow corner (30°), it is most likely to turn unilaterally to the right, whereas an unimpaired mouse will turn to both sides randomly. From a total of 10 turns, a laterality index was calculated. Second, in the Chimney test, the mouse walks heads first into a tube. Once it reaches the end, the tube is tipped 90 degrees to stand on the table vertically. Motorically impaired animals have difficulties crawling backwards up to the top of the tube. The experiment was stopped if an animal did not reach the top of the tube within 60 seconds. Third, in the Rotarod test, the mouse is placed on a rotating beam on which it is supposed to walk for at least 60 seconds, and the time when the animal falls off the rotating tube is measured.
All animals that had undergone ischemia showed massive weight loss until 72 hours after reperfusion. Weight loss then stagnated and there was a trend of increasing weight 7 days after reperfusion. The behavioral analysis showed that 24 hours after reperfusion, BHB-treated animals performed significantly better in the Corner test, meaning their moving patterns were more heterogeneous than those of saline-treated animals and in the Chimney test. 72 hours after reperfusion, BHB-treated animals still performed significantly better in the Chimney test, but 7 days after reperfusion, the performances of BHB- and saline-treated animals were no longer different from each other in any of the behavioral tests. In separate experiments, the plasma metabolites glucose, lactate, and pyruvate were changed in the animals that had undergone ischemia but were not affected by BHB administration.
Mitochondrial respiration was tested at four time points after the administration of BHB after reperfusion – 60 minutes, 24 hours, 72 hours, and 7 days after transient cerebral ischemia. 60 minutes later, data showed an increase of oxygen consumption of the complexes I and II. OxPhos was also increased but the effect at this point, did not reach statistical significance. 24 hours after reperfusion, this effect was consolidated: complex I, complex II and OxPhos respiration were significantly improved in the BHB-treated group compared to saline...
This thesis examines the referential properties of prenominal possessive modifiers in Serbian. The focus of the investigation is on the configurations that have been claimed to violate Binding Principles B and C: lexical or pronominal possessives modifying a noun in subject position binding a pronoun or an R-expression in object position. Such constructions have been claimed to be ungrammatical in Serbian due to the alleged adjectival status of the possessive and its respective syntactic position as NP-adjoined (Despić 2013).
The present thesis takes up the ongoing debate about the categorial status of Serbian possessives as adjectives or determiners. Based on several arguments, such as word order, binding of anaphora, coordination, and the fact that they are typically represented by either nouns or pronouns, it is concluded that possessives rather behave like full noun phrases than adjectives. Therefore, I analyse possessives as DPs from a categorial point of view.
In a second step, the syntactic position of the possessives within the Serbian noun phrase has been investigated. Based on theoretical arguments (cf. Bašić 2004) and empirical evidence, I propose a structural position that would accommodate the binding facts and the referential possibilities in these configurations. In line with Kayne (1994), Bernstein and Tortora (2005) and Alexiadou et al. (2007), I assume that possessives occupy SpecAgrP in Serbian, where they move from their base position (SpecPossP). Thirdly, I question the (im)possibility of coreference with possessives in comparison to ‘typical’ binding constructions without possessives by providing empirical evidence from three experimental studies, showing that coreference between possessive modifiers and objects is indeed available in Serbian.
The results from Experiment 1 (a picture selection task) have shown that coreference between a lexical possessive and a (clitic or strong) pronoun is allowed in Serbian. Further, there is a tendency that the coreferential reading is preferred with clitics, while the disjoint reference is preferred with strong pronouns. The fact that coreference is possible, does not necessarily mean that it is always available as the only interpretation, but can be influenced by other (pragmatic) factors. The same is observed in Experiments 2 and 3 as coreference was chosen between pronominal possessives modifying a noun in subject position and R-expressions but rejected between pronouns and R-expressions in a forced-choice task, suggesting a structural difference – no c-command – in the former case. The results from the self-paced reading task corroborate this finding.
Importantly, the experimental results provide evidence that possessive configurations are not violating Binding Principles B and C. This implies that Serbian possessive constructions do not c-command out of the noun phrase, as predicted by the proposed syntactic analysis.
The findings from all experiments contribute to the bigger picture concerning the nature and behaviour of Serbian possessives and cast doubt on the cross-linguistic DG/AG parameter. Instead, the theoretical arguments and the empirical results from the experiments rather speak for a parallel structure of possessive noun phrases in Serbian and English and ultimately in favour of the Universal DP Hypothesis.
The present work deals with photoionization in the realm of the absorption of one single photon. The formal treatment of one-photon ionization usually employs a semi-classical approach, where the electron’s initial and final states are described as quantum-mechanical wave functions but the photon is treated as a classical electromagnetic wave. In the calculation of photoionization cross sections with this semi-classical method, there is an often used approximation which is called the electric dipole approximation. Mathematically, the application of the dipole approximation corresponds to truncating the series expansion of an exponential after the leading term. Physically, this means neglecting the linear photon momentum and the spatial dependence of the light field. The dipole approximation is valid if the wavelength of the light is much larger than the spatial extent of the target and if the photon momentum is small compared to the momenta of the reaction products, which is generally the case for photon energies short above the electron binding energy.
For the present work, we experimentally investigated nondipolar photoionization, i.e., one-photon ionization at high photon energies where the dipole approximation breaks down. In our experiments, we irradiated single atoms and molecules with such high-energetic photons and measured the three-dimensional momentum distributions of the reaction fragments to uncover the effects of the linear photon momentum and the spatially-dependent light field on photoionization. Our observations allow the first profound insight into photoionization that reveals all photon properties, i.e., photon energy, spin, linear momentum, and the speed of light. Hopefully, our efforts make a constructive contribution to the understanding and the further exploration of light-matter interaction.
Since Vietnamese is an isolating language, word order plays an important role in identifying the function of a particular word. Yet in some contexts word order may be flexible especially in the case of special information-structural settings. Discontinuous noun phrases constitute a specific case of non-canonical word order in Vietnamese.
I have conducted two read-speech experiments in order to find out whether there are prosodic or intonational effects in a comparison between continuous and discontinuous noun phrases in Vietnamese. In the first experiment, speakers from the Northern dialect were recorded and in the second experiment speakers from the Southern dialect. The results showed prosodic differences in the two word order conditions in both dialects. The duration of the classifier is significantly longer (p<0.001, ANOVA calculation) in the case of discontinuous noun phrases and the rising tone (sắc) is clearly articulated as rising. In the case of continuous noun phrases, the duration of the classifier is significantly shorter (p<0.001, ANOVA calculation) and a classifier with rising tone may lose its rising property. These prosodic effects are related to prosodic boundaries. In the case of discontinuous noun phrases, the classifier constitutes the prosodic boundary, whereas with continuous noun phrases, the (right) prosodic boundary occurs further to the right.
I assume that in Vietnamese there is generally a correspondence between syntactic and prosodic structure as in Selkirk (2011) and Féry (2017).
This means that for example the DP hai trái cam ‘two oranges’ (two CLF orange) is matched by a prosodic phrase, thus (hai trái cam)Φ. However, when the noun cam ‘orange’ is separated from the numeral-classifier complex, the noun and the classifier form a prosodic phrase on their own: (hai trái)Φ. It can thus be concluded that intonation effects in Vietnamese are not only present when expressing sentence modality and when changing the role of function words (Đỗ et al. 1998 and Hạ & Grice 2010), but they also play a role in word order change, as in discontinuous nominal phrases.
When it comes to syntactic aspects of discontinuous noun phrases, I discuss whether split constructions in Vietnamese involve movement as proposed by Trịnh (2011) or base-generation as put forward by Fanselow & Féry (2006). I argue for base-generation analysis since the second part of a discontinuous NP (remnant) may also occur outside of discontinuous noun phrases without its head noun and some discontinuous noun phrases do not have a continuous counterpart. My study confirms the connection between syntax and prosody.
The two parts of the discontinuous noun phrase form their own phrases syntactically as well as prosodically.
Acute myeloid leukemia (AML) is one of the most frequently occurring and fatal types of leukemia. Initiated by genetic alterations in hematopoietic stem and progenitor cells, rapidly proliferating cancer cells (leukemic blasts) infiltrate the bone marrow and damage healthy hematopoiesis. Subgroups of AML are defined by underlying molecular and cytogenetic abnormalities, which are decisive for treatment and prognosis. For AML patients that can be intensively treated, the first line treatment remains a combination of cytarabine and anthracycline, which was developed in the 1970s. While this treatment regimen clears the disease and reinstates normal hematopoiesis (complete remission, CR) in 60% to 80% of patients below the age of 60, CR rates in patients above the age of 60 are only 40% to 50%. Relapse and refractory disease are the major cause of death of AML patients, despite large efforts to improve risk-adjusted post-remission therapy with further chemotherapy cycles and, if possible, allogeneic bone marrow transplantation. Elderly patients are particularly difficult to treat because of age-related comorbidities and because their disease tends to relapse more often than the disease of younger patients. Thus, the cure rates of AML vary with age, with 5-year survival rates of about 50% in young patients, and less than 20% in patients above the age of 65 years. With the median age of AML patients being 68 years, the need for novel therapeutic options is immense. The recent approval of eight new agents (venetoclax, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, gemtuzumab ozogamicin and CPX-351 (liposomal cytarabine and daunorubicin)) has added considerably to the therapeutic armamentarium of AML and has increased cure rates in specific subgroups of AML. However, the high heterogeneity among patients, clonal evolution and commonly occurring drug resistance, which cause the high relapse rates, remain a substantial problem in the treatment of AML. Therefore, a better understanding of currently used therapeutics and further development of novel therapeutics is urgently needed.
In recent years, attention has increasingly focused on therapeutic strategies to interfere with the metabolic requirements of cancer cells. The last three decades have provided extensive insights into the diversity and flexibility of AML metabolism. AML cells use different sources of nutrients compared to normal hematopoietic progenitor cells and reprogram their metabolic pathways to fulfill their exquisite anabolic and energetic needs. As a result, they develop high metabolic plasticity that enables them to thrive in the bone marrow microenvironment, where oxygen and nutrient availability are subject to constant change.
Cancer cells, specifically AML cells, have a strong dependency for the amino acid glutamine. Glutamine serves in energy production, redox control, cell signaling as well as an important nitrogen source. The only enzyme capable of de novo glutamine synthesis is glutamine synthetase (GS). GS catalyzes glutamine production from glutamate and ammonium. In AML, the metabolic role and dependency of GS is poorly understood. Here, we investigated the effects of GS deletion on AML growth, and its functional relevance in AML metabolism. Genetic deletion of GS resulted in a significant decrease of cell growth in vitro, and impaired leukemia progression in vivo in a xenotransplantation mouse model. Interestingly, the dependency of AML cell growth on GS was shown to be independent of its functional role in glutamine synthesis. Glutamine starvation did not increase the dependency of the AML cells on GS, nor did increased glutamine availability rescue the GS-knockout-associated growth disadvantage. Instead, functional studies revealed the role of GS in the detoxification of ammonium. GS-deficient cells showed elevated ammonium secretion as well as a higher sensitivity towards the toxic metabolite. Exogenous provision of 15N-labeled ammonium was detoxified by GS-driven incorporation into glutamine. Studies on cells that had gained resistance to GS-knockout-mediated growth inhibition indicated enzymes involved in the urea cycle and the arginine biogenesis pathway to compensate for a loss of GS. Together, these findings unveiled GS as an important ammonium scavenger in AML.
Clinical studies on AML patients revealed increased ammonium concentrations in the blast-infiltrated bone marrow compared to peripheral blood. In line with this finding, proteome and transcriptome analysis of AML blasts showed a significant upregulation of GS in AML compared to healthy progenitors, further indicating its importance in ammonium detoxification.
Analyzing pathways that contribute to ammonium production revealed protein uptake followed by amino acid catabolism as a yet not identified mechanism supporting AML growth. Protein endocytosis and subsequent proteolytic degradation were shown to rescue AML cells from otherwise growth-inhibiting glucose or amino acid depletion. Furthermore, protein metabolization led to the reactivation of the mammalian target of rapamycin (mTOR) signaling pathway, which was deactivated upon leucine and glutamine depletion, revealing protein consumption as an important alternative source of amino acids in AML.
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The book deals with a comprehensive constellation of narrative and visual, often counterposed representations of the causes, course, and results of the assault on the Palace of Justice of Colombia by a guerrilla commando and the immediate counterattack launched by state security forces on November 6, 1985, as well as with the local memorial traditions in which the production, circulation and reproduction of these representations have taken place between 1985 and 2020. The research on which it is based was grounded in the method and perspective of classical anthropology, in as much as qualitative fieldwork and the search for the perspective of the actors involved have played a central role. Within that context, memory entrepreneurs belonging to diverse sectors, from the far-right to the human rights movement, were followed through multisited fieldwork in various locations of Colombia, as well as in various countries of America and Europe. The analyses of fieldwork data, documental sources, and visual representations that constitute the core of the argument are framed in the field of memory studies and mainly based on theoretical and methodological resources from Pierre Bourdieu’s Field Theory, Jeffrey Alexander’s theory of social trauma, and Ernst Gombrich’s characterization of iconological analysis.
The book is composed of four chapters preceded by an introduction and followed by the conclusions and documental appendices, and substantiates three main theses. The first is that the Palace of Justice events were a radio- and television-broadcasted dispersed tragedy that affected the lives of actors from different social sectors and regions of Colombia, who have launched since 1985 multiple memorial initiatives in different fields of culture, thereby contributing to the formation and intergenerational transmission of a widespread cultural trauma. The second is that the narrative and visual representations at the core of that trauma express a vast universe of local representational traditions that can be traced at least until the early 20th century, and therefore preexists the so-called Colombian “memory boom”, dated to the mid-1990s. As an example of the preexistence and longstanding impact of these traditions, the local usage of the figure of “holocaust” for representing the effects of politically motivated violence is analyzed regarding the Palace of Justice events, but also traced to other representations emerged in the decade of 1920. The third thesis is that analyzing the diverse, frequently counterposed accounts of political violence elaborated within these traditions provides an opportunity to explore a wide variety of understandings of the causes and characteristics of the longstanding Colombian social and armed conflict.
Keywords: Political violence, Cultural trauma, Collective Memory, Iconology, Holocaust, Colombia.
Characteristics of critical incident reporting systems in primary care: an international survey
(2022)
Aim: The aim of the study was to support the development of future critical incident reporting systems (CIRS) in primary care by collecting information on existing systems. Our focus was on processes used to report and analyse incidents, as well as strategies used to overcome difficulties.
Methods: Based on literature from throughout the world, we identified existing CIRS in primary care. We developed a questionnaire and sent it to operators of a purposeful sample of 17 CIRS in primary care. We used cross-case analysis to compare the answers and pinpoint important similarities and differences in the CIRS in our sample.
Results: Ten CIRS operators filled out the questionnaire, and 9 systems met the inclusion criteria. The sample of CIRS came from 8 different countries and was rather heterogeneous. The reporting systems invited a broad range of professions to report, with some also including reports by patients. In most cases, reporting was voluntary and conducted via an online reporting form. Reports were analysed locally, centrally, or both. The various CIRS used interesting ideas to deal with barriers. Some, for example, used confidential reporting modes as a compromise between anonymity and the need for follow-up investigations, whereas others used smartphone applications and call centres to speed up the reporting process.
Conclusion: We found multiple CIRS that have operated in primary care for many years, have received a high number of reports and were largely developed in accordance with recommendations found in literature. Although primary care in Germany differs from other countries, these CIRS could serve as an inspiration for CIRS in German primary care.
We present new results on nonlocal Dirichlet problems established by means of suitable spectral theoretic and variational methods, taking care of the nonlocal feature of the operators. We mainly address: First, we estimate the Morse index of radially symmetric sign changing bounded weak solutions to a semilinear Dirichlet problem involving the fractional Laplacian. In particular, we derive a conjecture due to Bañuelos and Kulczycki on the geometric structure of the second Dirichlet eigenfunctions. Secondly, we study a small order asymptotics with respect to the parameter s of the Dirichlet eigenvalues problem for the fractional Laplacian. Thirdly, we deal with the logarithmic Schrödinger operator. In particular, we provide an alternative to derive the singular integral representation corresponding to the associated Fourier symbol and introduce tools and functional analytic framework for variational studies. Finaly, we study nonlocal operators of order strictly below one. In particular, we investigate interior regularity properties of weak solutions to the associated Poisson problem depending on the regularity of the right-hand side.
The thesis is composed of four Chapters.
In the first Chapter, the boundary expression of the one-sided shape derivative of nonlocal Sobolev best constants is derived. As a simple consequence, we obtain the fractional version of the so-called Hadamard formula for the torsional rigidity and the first Dirichlet eigenvalue. An application to the optimal obstacle placement problem for the torsional rigidity and the first eigenvalue of the fractional Laplacian is given.
In the second Chapter, we introduce and prove a new maximum principle for doubly antisymmetric functions. The latter can be seen as the first step towards studying the optimal obstacle placement problem for the second fractional eigenvalue. Using the new maximum principle we derive new symmetry results for odd solutions to semilinear Dirichlet boundary value problems with Lipschitz nonlinearity.
In the third Chapter, we derive new integration by parts formula for the fractional Laplace operator with a general globally Lipschitz vector field and in particular, we obtain a new Pohozaev type identity generalizing the one obtained by X. Ros-Oton and J. Serra. As an application we obtain nonexistence results for semilinear Dirichlet boundary problems in bounded domains that are not necessarly starshaped.
In the last Chapter, we study symmetry properties of second eigenfunctions of annuli. Using results from the first Chapter and the maximum principle in Chpater 2, we extend the result on the optimal obstacle placement problem from the first eigenvalue to the second eigenvalue.
Standard cancer therapy research targets tumor cells while not considering the damage on the tumor microenvironment (TME) and its associated implications in impairing therapy response. Employing patients-derived organoids (PDOs) and matched stroma cells or a novel murine preclinical rectal cancer model of local radiotherapy, it was demonstrated that tumor cells-derived IL-1α polarizes cancer-associated fibroblasts towards an inflammatory (iCAFs) phenotype. While numerous studies in different tumor entities highlighted the molecular heterogeneity of CAFs, so far there are no clear findings on their functional heterogeneity and relevance in therapy resistance and response. The present study molecularly characterized iCAFs subpopulation among RCA patients as well as the preclinical mouse model and importantly unraveled the detailed molecular mechanism underlying their contribution to impair therapy response. Mechanistically, iCAFs were demonstrated to be characterized by an upregulation of nitric oxide synthase (iNOS) which triggered accumulation of reactive nitrogen species (RNS) and subsequently an oxidative DNA damage response (DDR). Such a baseline IL-1α-driven DNA damage further sensitized iCAFs to a p53-mediated therapy induced senescence (TIS) causing extensive extracellular matrix (ECM) changes and induction of senescence associated secretory phenotype (SASP) that favored tumor progression and hindered tumor cell death. Moreover, iCAFs reversibility and repolarization into more quiescent like phenotype was demonstrated upon IL-1 signaling inhibition by anakinra, a recombinant IL-1 receptor antagonist (IL1RA). Accordingly, treating mice with anakinra or specific deletion of Il1r1 in CAFs sensitized stroma-rich resistant tumors to chemoradiotherapy (CRT). Similarly, targeting CAFs senescence by senotherapy (venetoclax chemical) or employing Trp53 deficient mice reverted therapy resistance among non-responsive tumors in vivo by reducing ECM deposition and consequently favoring CD8+ T cells intratumoral infiltration posttherapy. Importantly, rectal cancer patients that do not completely respond to neoadjuvant therapy displayed an iCAFs senescence program post-CRT. Moreover, these patients presented a baseline increased CAFs content, a dominant iCAFs signature that correlated with poorer disease-free survival (DFS) and a significantly reduced circulating IL1RA serum levels. While reduced pretherapeutic IL1RN gene expression predicted poor prognosis among RCA patients, IL1RA serum levels were associated with rs4251961 (T/C) single nucleotide polymorphism (SNP) in the IL1RN gene. Finally, functional validation assays revealed that conditioned media of PDOs drove inflammatory polarization of fibroblasts and consequently rendered them sensitive to RNS-mediated DNA damage and TIS. Collectively, the study highlighted a crucial and novel role of a CAFs subset, iCAFs, in therapy resistance among RCA patients, shedding light on their functional relevance by identifying IL-1 signaling as an appealing target for their repolarization and successful targeting. Therefore, it makes sense to combine the newly demonstrated and thoroughly proven therapeutic approach of targeting IL-1 signaling in combination with conventional CRT and possibly immunotherapy. This might have a major impact on RCA therapy and be of immense relevance for other stroma-rich tumors.
Acute myeloid leukemia (AML) is a neoplastic disease of an early myeloid precursor cell in hematopoiesis. It leads to the accumulation of monoclonal cells in the bone marrow and the peripheral blood, showing a differentiation block and deregulated self-renewal. Frequently, the leukemic cells exhibit genetic aberrations with reciprocal chromosomal translocations. These translocations induce the formation of a fusion protein, that can lead to new cellular functions and a transformation into a leukemic cell. Common chromosomal translocation in AML are t(8;21) or t(15;17), which cause the formation of the fusion proteins AML1/ETO and PML/RARα and determine the leukemic phenotype of the AML.
The translocation t(6;9) leads to the formation of the fusion protein DEK/CAN and is of special interest, because of its association with mostly young patients and a very aggressive course of the disease. The fusion product induces leukemia in a small subset of hematopoietic stem cells, but its mechanism of leukemogenesis is greatly unknown.
The intention of this work was to characterize the DEK/CAN-induced AML on a molecular genetic level to gain a deeper understanding of the disease pathogenesis. Therefore, gene expression analysis with polymerase chain reaction (PCR) and microarray analysis was performed.
To detect DEK/CAN in different cell lines by PCR and real-time quantitative PCR (qPCR), specific primers and probes were designed, and a standardized workflow was established. Emphasis was placed on the optimization of RNA isolation, DNase treatment, cDNA synthesis with following PCR and qPCR, which enabled the detection of the fusion product DEK/CAN in the cell lines 32B, Phoenix and FKH-1. To quantify the fusion product DEK/CAN, the method of qPCR with absolute and relative quantification was used. Absolute quantification enabled the calculation of an exact copy number of the fusion transcript DEK/CAN with a detection limit of 50 copies/µl at a sensitivity of 10-6, which is of importance in determining the minimal residual disease (MRD) of patients with DEK/CAN-positive AML. MRD detection by qPCR is a highly sensitive diagnostic method to identify leukemic cells, even in low cell counts. This enables a thorough evaluation of the treatment response and allows an early detection of changes in the MRD level as part of the remission control.
Additionally, a microarray gene expression analysis was performed to identify alterations in relevant target genes and associated signaling pathways in DEK/CAN-positive cells.
Because of DEK/CAN’s potential to induce leukemia in a subset of hematopoietic stem cells, Sca+/Lin- cells of the bone marrow of C57Bl/6 mice were used and transfected with the gene products DEK/CAN and PML/RARα. Microarray analysis led to the identification of 16 different genes of interest, which demonstrated significant alterations of gene expression in DEK/CAN-positive cells. They were validated and quantified with TaqMan assay assisted qPCR. The elevated expression of the transcription factors TRIM25, HIF1α and ATF2, in DEK/CAN-positive cells, indicated an altered transcription factor activity and interaction with DNA in the nucleus. The localization of DEK/CAN in the nucleus emphasizes this assumption. Also, the upregulated expression of the nuclear export receptor XPO1 suggested changes in nuclear transport processes and impaired export activity in DEK/CAN-positive cells.
Furthermore, the results demonstrated changes of gene expression in genes that are involved in the JAK/STAT signaling pathway. PTPRC, the Protein Tyrosine Phosphatase Receptor Type C, functions as a direct inhibitor of JAKs (Janus Kinases) and STATs (Signal Transducers and Activators of Transcription) and their associated signaling pathway.
It was shown that the gene expression of PTPRC was significantly reduced in DEK/CAN-positive cells. This allowed the assumption, that the reduced expression of PTPRC led to a loss of inhibition and thus a consecutive hyperactivation of the JAK/STAT signaling pathway. This hypothesis was supported by an independent activation of PIM1, a target gene of STAT5 and the activation of LMO2, a direct target gene of JAK2. In addition, the transmembrane receptor CSF1R, which is directly involved in STAT activation, also showed an upregulation in gene expression.
The results of this work show an activation of the JAK/STAT signaling pathway in DEK/CAN-positive cells, which may be a key mechanism in DEK/CAN-induced leukemogenesis.
Considering treatment options in the future, the addition of targeted therapy, such as pan-JAK inhibitors, to the standard therapy, could be a chance to improve the overall survival rate and the prognosis of t(6;9)-positive AML.
This work describes the development and characterization of two instruments and their data evaluation, which contributes to a better understanding of new particle formation and growth, as well as their interactions with clouds. Both instruments were characterized at the Cosmics Leaving Outdoor Droplets (CLOUD) experiment at the European Center for Nuclear Research (CERN).
The expansion of actors and instruments in sovereign debt markets through bond financing generated a coordination problem among bondholders during the debt restructuring process. There is a risk that an individual bondholder will be passive or act against the restructuring slowing down or even precluding the process of restructuring even though it is in the general interest of bondholders as a group, not to mention the population of the country experiencing the shortage of funds for public welfare. In particular, the disruptions to sovereign debt restructuring by frivolous litigation is considered as one of the main threats.
This dissertation is the first major study devoted to sovereign bonds structured through a trust arrangement and the promising features that such a legal structure possesses for an effective and efficient sovereign debt restructuring. It provides a comprehensive inquiry into the evolution of the mechanisms to coordinate creditors, with a focus on bondholders and institutional frameworks which facilitated this coordination. It examines intriguing primary sources from League of Nations archives and provides in-depth case studies on the functionality of the trustees in sovereign bond restructurings performed by Argentina in 2016 and Ecuador in 2008.
Assessing the utility of trust arrangements to address coordination problems, this thesis is driven by the puzzle: How to better balance (i) the need for smooth sovereign debt restructurings, which by definition entails some losses for creditors, with (ii) bondholders’ legitimate interests? What approach can be used in constructing a legal and institutional framework for trustees to promote the best interest of the bondholders in sovereign debt restructuring? As a solution, it seems that incentives for bond trustees to pursue debt sustainability will achieve both goals.
In this regard, recognition of the concept of debt sustainability, being in substance the IMF and WB debt sustainability assessment, as the best interest of bondholders in sovereign debt restructuring is beneficial from multiple aspects. It enables a bond trustee to excel in its role as a guardian of bondholders by following the best interest of bondholders in exercising its discretion. Moreover, it fosters an equilibrium between the interests of private creditors and a state taking into account its socio-political aspects.
Facial expression recognition is linked to clinical and neurofunctional differences in autism
(2022)
Background: Difficulties in social communication are a defining clinical feature of autism. However, the underlying neurobiological heterogeneity has impeded targeted therapies, and requires new approaches to identifying clinically relevant bio-behavioural subgroups. In the largest autism cohort to date, we comprehensively examined difficulties in facial expression recognition, a key process in social communication, as a bio-behavioural stratification biomarker, and validated them against clinical features and neurofunctional responses.
Methods: Between 255 and 488 participants aged 6-30 years with autism, typical development and/or mild intellectual disability completed the Karolinska Directed Emotional Faces task, the Reading the Mind in the Eyes Task and/or the Films Expression Task. We first examined mean-group differences on each test. Then we used a novel intersection approach that compares two centroid and connectivity-based clustering methods to derive subgroups based on the combined performance across the three tasks. Measures and subgroups were then related to clinical features and neurofunctional differences measured using fMRI during a fearful face-matching task.
Results: We found significant mean-group differences on each expression recognition test. However, cluster analyses showed that these were driven by a low-performing autistic subgroup (~30% of autistic individuals who performed below 2SDs of the neurotypical mean on at least one test), while a larger subgroup (~70%) performed within 1SD on at least 2 tests. The low-performing subgroup also had on average significantly more social-communication difficulties and lower activation in the amygdala and fusiform gyrus than the high-performing subgroup.
Limitations: Findings of autism expression recognition subgroups and their characteristics require independent replication. This is currently not possible, as there is no other existing data set that includes all relevant measures. However, we demonstrated high internal robustness (91.6%) of findings between two clustering methods with fundamentally different assumptions, which is a critical pre-condition for independent replication.
Conclusions: We identified a subgroup of autistic individuals with expression recognition difficulties and showed that this related to clinical and neurobiological characteristics. If replicated, expression recognition may serve as bio-behavioural stratification biomarker and aid in the development of targeted interventions for a subgroup of autistic individuals.
Protein biosynthesis is a fundamental process across all domains of life. Polypeptides are produced by translating the genetic information of the messenger RNA (mRNA) into amino acids. This elaborate procedure is divided into the four distinct phases: initiation, elongation, termination, and ribosome recycling. The phases are controlled and regulated by a multitude of translation factors. During initiation, the ribosome assembles on the mRNA. Initiation factors (IFs) bind to the small ribosomal subunit (SSU) and assist the recruitment of mRNA and initiator transfer RNA (tRNA), which delivers the first amino acid methionine. After positioning the SSU at the start codon of the mRNA, additional IFs support the joining of the large ribosomal subunit (LSU). Next, elongation factors (EFs) deliver amino-acylated tRNAs (aa-tRNAs) to the translating ribosome and assist kinetic proofreading and ribosome subunit translocation after the catalytic transfer of the polypeptide onto the aa-tRNA. When a stop codon is reached, translation is terminated by release factors (RFs) that hydrolyze the peptidyl-tRNA to release the nascent protein chain. Afterwards, the ribosome is recycled in Eukaryotes and Archaea by the conserved and essential factor ABCE1, which splits the ribosome into the LSU and SSU. ABCE1 remains bound to the SSU forming the post-splitting complex (post-SC). mRNA translation closes into a cycle by recruitment of IFs to the post-SC and the start of a new round of initiation. The post-SC presents the platform for translation initiation. However, the role of ABCE1 in initiation remains elusive. Therefore, the main goal of my thesis was to unravel the molecular mechanism of ABCE1 on the post-SC and during initiation complex (IC) assembly.
Using a reconstituted system, the high-resolution structure of the archaeal post-SC was solved by cryogenic electron microscopy (cryo-EM) following the native splitting route. It was the first complete model of an archaeal SSU at atomic resolution and revealed a previously undescribed ribosomal protein, which we termed eS21. The hinge 2 region of ABCE1 was identified to be the major interaction interface that anchors to the SSU. Functional characterization of single residue mutations in hinge 2 unraveled essential interactions with the ribosomal RNA backbone of the SSU. Sensing of SSU-binding was found to be allosterically transmitted to the nucleotide-binding sites (NBSs) for integration into the ATPase cycle of ABCE1.
Reconstitution of the archaeal translation apparatus allowed for dissection of IC assembly in the presence of ABCE1. Three different ICs were resolved by cryo-EM. The results were in accordance with recent structural findings of eukaryotic translation initiation and highlighted that the involvement of ABCE1 is conserved.
In a semi-native approach, recombinant ABCE1 was pulled-down from crenarchaeal cell lysates. Mass spectrometric analysis of co-immunoprecipitated ribosomal complexes identified the association of numerous translation factors to the post-SC in a cellular context. The establishment of the genetic toolbox of the acidothermophilic Sulfolobus acidocaldarius allowed the homologous expression of ABCE1. Pull-down of native ABCE1 revealed similar ribosomal complexes as the semi-native and reconstituted approaches. Together, my results gave first physiological relevance of ABCE1 involvement in mRNA translation initiation in Archaea. Native archaeal ABCE1-ICs were vitrified for structural analysis by cryo-EM. Thereby, future structural analysis will allow to analyze the interactions of ABCE1 on native ICs and identify its role in IC assembly.
To address the molecular process of IC assembly, the binding affinity of aIF1 to the SSU was determined by fluorescence polarization. Similar studies will allow for a detailed functional analysis on IF recruitment to the SSU in presence of ABCE1.
mRNA surveillance and ribosome-associated quality control (RQC) mechanisms evolved to ensure cell viability. The pathways overcome ribosome stalling and defective translation components. Stalled ribosomes are terminated by special RFs, which do not hydrolyze the peptidyl-tRNA, but allow dissociation of the ribosome by ABCE1. Faulty messages are degraded via mRNA decay pathways and the LSU is rescued by RQC factors. Recently, the bacterial RQC factor MutS2 was identified to specifically target collided di- and polysomes but its molecular mechanism remains unknown. In this thesis, initial functional analyses showed tri-phosphate specific nucleotide binding of MutS2. While the dissociation of collided disomes by MutS2 could not be observed, the results pave the way for future in vitro studies of bacterial RQC factors acting on specific ribosome populations.
In the future, mRNA translation research must focus on complex quality control processes to comprehensively understand this fundamental cellular process in a holistic context.
As part of two drilling campaigns of the International Continental Scientific Drilling Program (ICDP), several geophysical borehole measurements were carried out by the Leibniz Institute for Applied Geophysics (LIAG) in two lakes. The acquired data was used to answer stratigraphic and paleoclimatic research questions, including the establishment of robust age-depth models and the construction of continuous lithological profiles.
Lake Towuti is located on Sulawesi (Indonesia), within the "Indo-Pacific Warm Pool" (IPWP), a globally important region for atmospheric heat and moisture budgets. The lake exists for approximately one million years, but its exact age is uncertain. We present the first agedepth model for the approximately 100 m continuous sediment sequence from the central part of the lake. The basis for this model is the magnetic susceptibility measured in the borehole and a tephra layer with an age of about 797 ka at 72 m depth. Our age-depth model is inferred from cyclostratigraphic analysis of borehole data and covers a period from 903 ± 11 to 131 ± 67 ka. We suggest that orbital eccentricity and/or changes between global cold and warm periods are responsible for hydroclimatic changes in the IPWP, that these changes affect sedimentation processes in Lake Towuti, and that we can measure and observe this effect in the sediment properties today. Additionally, we created a continuous artificial lithological profile from a series of different borehole data using cluster analysis. This provides information from parts of the borehole where no sediment is available due to core loss.
Lake Ohrid is 1.36 million years old and is located on the Balkan Peninsula on the border between Albania and North Macedonia. The primary hole 'DEEP' in the central part of the lake has been the subject of several investigations, but information about sediments of the marginal locations 'Pestani' and 'Cerava' have not been published yet. In our study, we use natural gamma radiation (GR) measured in the borehole to generate an age-depth model for DEEP. This is performed using the correlation of GR to the global LR04 reference record of Lisiecki and Raymo (2005).
The age information is then transferred via prominent seismic marker horizons to the other two sites, Pestani and Cerava, where it provides the first age-control points for the construction of age-depth models from correlation of GR to LR04. The generated age-depth models are tested using cyclostratigraphic methods, but the limits of this approach are revealed. At DEEP, sedimentation rates (SR) from the cyclostratigraphic method and the correlative approach differ by 2.8 %, at Pestani this difference is 16.7 %, and at Cerava the quality of the data does not allow a reliable evaluation of SR using the cyclostratigraphic approach. We used cluster analysis to construct artificial lithological profiles at all three sites and integrated them into the respective age-depth models. This enables us to determine which sediment types were deposited at what time, and we recognize the change between warm and cold periods in the sediment properties at all three locations. The analyses in this study were all performed on borehole and seismic data and thus do not involve sediment core data. Especially at Pestani and Cerava, new insights into the sedimentological history of Lake Ohrid could be obtained.
In the last part we discuss the occurrence of the half-precession (HP) signal in the European region during the last one million years. The focus is on Lake Ohrid, but a range of other proxies, from the eastern Mediterranean, across the European continent, up to Greenland are analyzed in regards to HP. Applying filters, we focus on the frequency range with a period of 13-8.5 ka and only HP remains in the records. We use correlative methods to determine the clarity of the HP signal in proxies distributed across the European realm. Additionally, we determined the development of HP over time. The HP signal is clearest in the southeast and decreases toward the north. It is further more pronounced in interglacial periods and in the younger part (<621 ka) of most proxies. We suggest that there are mechanisms that transmit the HP signal from its origin near the equator to higher latitudes via different processes. In this context, for instance, the African monsoon, the Nile River and the Mediterranean outflow via the Strait of Gibraltar can be important factors.
People can describe spatial scenes with language and, vice versa, create images based on linguistic descriptions. However, current systems do not even come close to matching the complexity of humans when it comes to reconstructing a scene from a given text. Even the ever-advancing development of better and better Transformer-based models has not been able to achieve this so far. This task, the automatic generation of a 3D scene based on an input text, is called text-to-3D scene generation. The key challenge, and focus of this dissertation, now relate to the following topics:
(a) Analyses of how well current language models understand spatial information, how static embeddings compare, and whether they can be improved by anaphora resolution.
(b) Automated resource generation for context expansion and grounding that can help in the creation of realistic scenes.
(c) Creation of a VR-based text-to-3D scene system that can be used as an annotation and active-learning environment, but can also be easily extended in a modular way with additional features to solve more contexts in the future.
(d) Analyze existing practices and tools for digital and virtual teaching, learning, and collaboration, as well as the conditions and strategies in the context of VR.
In the first part of this work, we could show that static word embeddings do not benefit significantly from pronoun substitution. We explain this result by the loss of contextual information, the reduction in the relative occurrence of rare words, and the absence of pronouns to be substituted. But we were able to we have shown that both static and contextualizing language models appear to encode object knowledge, but require a sophisticated apparatus to retrieve it. The models themselves in combination with the measures differ greatly in terms of the amount of knowledge they allow to extract.
Classifier-based variants perform significantly better than the unsupervised methods from bias research, but this is also due to overfitting. The resources generated for this evaluation are later also an important component of point three.
In the second part, we present AffordanceUPT, a modularization of UPT trained on the HICO-DET dataset, which we have extended with Gibsonien/telic annotations. We then show that AffordanceUPT can effectively make the Gibsonian/telic distinction and that the model learns other correlations in the data to make such distinctions (e.g., the presence of hands in the image) that have important implications for grounding images to language.
The third part first presents a VR project to support spatial annotation respectively IsoSpace. The direct spatial visualization and the immediate interaction with the 3D objects should make the labeling more intuitive and thus easier. The project will later be incorporated as part of the Semantic Scene Builder (SeSB). The project itself in turn relies on the Text2SceneVR presented here for generating spatial hypertext, which in turn is based on the VAnnotatoR. Finally, we introduce Semantic Scene Builder (SeSB), a VR-based text-to-3D scene framework using Semantic Annotation Framework (SemAF) as a scheme for annotating semantic relations. It integrates a wide range of tools and resources by utilizing SemAF and UIMA as a unified data structure to generate 3D scenes from textual descriptions and also supports annotations. When evaluating SeSB against another state-of-the-art tool, it was found that our approach not only performed better, but also allowed us to model a wider variety of scenes. The final part reviews existing practices and tools for digital and virtual teaching, learning, and collaboration, as well as the conditions and strategies needed to make the most of technological opportunities in the future.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
This work ties in with the investigation of the intermediate valent states and valence fluctuations in certain europium based intermetallic systems. Valence fluctuations are a property of the electronic system of a compound that is possibly accompanied by structural effects, which, in some cases, are quite noticable. By assuming how the changes in the electronic system and in the crystal lattice are connected, valence _uctuations of europium are believed to be a possible probe for the theory of quantum critical elasticity, which is investigated on by the SFB TRR 288 (Frankfurt, Mainz, Karlsruhe, Bochum, Dresden).
Here, the proceedings in growing single crystals of di_erent compounds related to this _eld of research are reported. This includes the ThCr2Si2 (122) type compounds EuPd2Si2 as well as the doping series EuPd2(Si1-xGex)2, the Europium based ternary Phosphides EuFe2P2, EuCo2P2, EuNi2P2 and EuRu2P2, and attempts to grow compounds of a derived 1144 structure by ordered substitution of half the Europium, EuKRu4P4.
The largest part of this work focusses on the EuPd2Si2 system, which exhibits intermediate valent europium and a temperature dependent transition between two di_erent intermediate valent states of europium. Crystals of this system were grown using the Czochralski method with a levitating melt and an europium excess flux after a two step prereaction process. Also, explorations of a PdSi-rich flux and external flux methods are reported. Ten Czochralski grown experiments, in six generations iteratevely seeded by the previous generation, were prepared.
Thermodynamical and structural analyses of the crystals located the transition between the di_erent intermediate valent states of europium between 140K and 165 K, transitioning from a high temperature Eu2.3+ state to a low temperature Eu2.7+ state, and classified it as a second order transition. To this transition a lattice anomaly of the a-parameter collapsing about 2% is connected, while the c-parameter remains largely unaffected. Large differences between individual samples can be explained by combining thermodynamical and structural analyses with compositional analysis, revealing the valence transition temperature as strongly dependent on the sample composition and Pd-Si site interchanges.
Searching to change the character of the valence transition to first order, silicon was substituted by germanium to introduce negative pressure. Germanium substituted samples of EuPd2(Si1-xGex)2 were grown using the Czochralski method with the optimized parameters from the growth experiments for the undoped compound. Samples were prepared with a nominal substitution of x = 0.05, x = 0.10, x = 0.15, x = 0.20 (twice) and x = 0.30. For the EuPd2(Si1-xGex)2 system, a phase diagram for the europium valence states is derived from chemical and thermodynamical characterizations.
n ternary europium phosphides EuT2P2, the position of the compounds in the generalized phase diagram and the question of long range magnetic order or valence transition appear connected to an isostructural transition of the tetragonal crystal structure, drastically decreasing the length of the c-parameter while establishing covalent bonds between phosphorus atoms of different interlayers of the structure, the so called ‚collapse‘. While EuFe2P2, EuT2P2 and EuCo2P2 display both long range magnetic order and a non-collapsed crystal structure, EuNi2P2 shows both a valence transition between two intermediate valent states at a characteristic temperature of 36K - accompanied by a small lattice anomaly of the a-parameter shrinking about 0.2% - and a collapsed crystal structure. Samples of EuFe2P2, EuCo2P2 and EuNi2P2 were grown in tin flux and using solid-solid sintering approaches.
Single crystals of EuFe2P2, EuCo2P2 and EuRu2P2 were investigated at ESRF in Grenoble with single crystal X-ray di_ractometry on a pressure range up to 15GPa and at temperatures down to 15K to investigate the nature of the structural transitions in the compounds. While in EuCo2P2 the structural transition occurs as a transition of first order at all temperatures (e.g. at 2GPa for 15 K), in EuFe2P2 and EuRu2P2 the structural collapse evolves over a broad pressure range up to 8GPa and as a transition of second order troughout the temperature ranges, albeit seeming to sharpen at lower temperatures. From the crystallographic data, elastic constants of the compounds could be derived, revealing EuFe2P2 and EuRu2P2 as unexpectedly elastic materials.
In order to probe the structural collapse at more accessible pressures, crystals with a sturcture derived from the 122 structure, but with ordered 50% substitution of europium and hence altering the symmetry from I4/mmm to P4/mmm in a 1144 structure, were exploratively pursued. Different experiments to obtain EuAT4P4 (with A = K, Rb, Cs and T = Fe, Ru) from binary or ternary prereactants or directly from the elements remained largely unsuccessful.
Locomotion, the way animals independently move through space by active muscle contractions, is one of the most apparent animal behaviors. However, in many situations it is more beneficial for animals to actively prevent locomotion, for instance to briefly stop before reorienting with the aim of avoiding predators, or to save energy and recuperate from stress during sleep. The molecular and cellular mechanisms underlying such locomotion inhibition still remain elusive. So, the aim of this study was to utilize the practical genetic model organism Caenorhabditis elegans to efficiently tackle relevant questions on how animals are capable of suppressing locomotion.
Nerve cells, mostly called neurons, are known to control locomotion patterns by activating some and inhibiting other muscle groups in a spatiotemporal manner via local secretion of molecules known as neurotransmitters. This study particularly focuses on whether neuropeptides modulate such neurotransmission to prevent locomotion. Neuropeptides are small protein-like molecules that are secreted by specific neurons and that act in the brain by activating G protein-coupled receptors (GPCRs) expressed in other target neurons. They can act as hormones, neuromodulators or neurotransmitters. DNA sequences coding for neuropeptides and their cognate receptors are similar across diverse species and thus indicate evolutionary conservation of their molecular signaling pathways. This could potentially also imply that regulatory functions of specific neuropeptides are also similar across species and are thus meaningful to unravel more general mechanisms for instance underlying locomotion inhibition.
Specifically, we find that the modulatory interneuron RIS constitutes a dedicated stop neuron of which the activity is sufficient to initiate rapid locomotion arrest in C. elegans while maintaining its body posture. Similar to its known function in larval sleep, RIS requires RFamide neuropeptides encoded by the flp 11 gene for this activity, in addition to GABA. Furthermore, we find that spontaneous calcium activity transients in RIS are compartmentalized and correlated with locomotion stop. These findings illustrate that a single neuron can regulate both stopping and sleeping phenotypes.
Secondly, we show that C. elegans RPamide neuropeptides encoded by nlp-22 and nlp-2 regulate sleep and wakefulness, respectively. We unexpectedly find that these peptides activate gonadotropin-releasing hormone (GnRH)-like receptors dose dependently and we highlight their sequence resemblance to other bilaterian GnRH-like neuropeptides. In addition, we show that these receptors are expressed in distinct subsets of neurons that are associated with motor behavior. Finally, we show that nlp 22 encoded peptides signal through GNNR 6 receptors to regulate larval sleep and that nlp 2 encoded peptides require both GNRR 3 and GNRR 6 receptors to promote wakefulness.
In sum, we find that locomotion inhibition in C. elegans is regulated by multiple, but evolutionary conserved RFamide and GnRH-like RPamide neuropeptidergic signaling pathways.
We study the polarization of relativistic fluids using the relativistic density operator at global and local equilibrium. In global equilibrium, a new technique to compute exact expectation values is introduced, which is used to obtain the exact polarization vector for fields of any spin. The same result has been extended to the case of massless fields. Furthermore, it is demonstrated that at local equilibrium not only the thermal vorticity but also the thermal shear contribute to the polarization vector. It is shown that assuming an isothermal local equilibrium, the new term can solve the polarization sign puzzle in heavy ion collisions.
Heart development is a dynamic process modulated by various extracellular and intracellular cues. Cardiac progenitors in vertebrates such as the zebrafish, migrate over to the midline after differentiation from the epiblast (Bakkers, 2011; Rosenthal & Harvey, 2010; Stainier et al., 1996; Trinh & Stainier, 2004). These progenitors form a cardiac disc at the midline which elongates into the linear heart tube. The differentiation and migration of cardiac precursors is modulated by signaling interactions between cardiac precursor cells and their extracellular environment known as the Extracellular Matrix (ECM). Studies have shown that Cell-ECM interactions play a crucial role in sculpting the heart during early morphogenic events (Davis CL, 1924; Männer & Yelbuz, 2019; Rosenthal & Harvey, 2010). One key factor to these processes is the presence of a specialized ECM known as the Basement Membrane (BM). Extracellular basement membrane proteins such as Fibronectin have been shown to modulate these very early migration processes of the cardiomyocyte progenitors (Trinh & Stainier, 2004). As the heart develops further, the linear heart tube is composed of myocardial cells with an inner endothelial cell lining separated by a layer of thick jelly like substance called the cardiac jelly (Barry A, 1948; Davis CL, 1924; Little et al., 1989). The cardiac jelly also called the cardiac basement membrane, has been shown to regulate distinct developmental events during cardiogenesis. This early CJ contains components of the basal lamina such as laminins, fibronectin, hyaluronan as well as non-fibrillar collagens such as Collagen IV (Little et al., 1989). In this study, I aimed to identify ECM molecules of the Basement Membrane in the heart and identify their role in the modulation of cardiac development and regeneration using the zebrafish as my model organism.
I identified genes belonging to the Zebrafish Matrisome expressed during cardiac developmental and regeneration and performed CRISPR/Cas9 sgRNA mediated mutagenesis. I also developed overexpression tools for these genes.
Agrinp168 mutants exhibited no obvious gross morphology defects during cardiac development and were adult viable. Adult mutants exhibited reduced cardiomyocyte proliferation, but no significant difference in cardiomyocyte dedifferentiation post cardiac cryoinjury.
Decorin overexpression through mRNA injections led to increased myocardial wall thickness and DN dcn overexpression through mRNA injections led to loss of cardiac looping during early development.
Mutants for Small Leucine Rich Proteoglycan (SLRP) prelp generated using CRISPR/Cas9 mutagenesis exhibited cardiovascular defects. Close observation of prelp mutant hearts revealed a reduced heart rate and impaired fractional shortening of the ventricle. prelp mutants exhibited an enlarged atrium at 48 hpf and 72 hpf as well as a reduced ventricle size at 72 hpf. Chamber size in the mutant hearts were enlarged irrespective of contractility of the heart. Mutants showed an increased number of Atrial cardiomyocytes, but no change in cell size. On the molecular level, extracellular Laminin localization was disrupted in prelp mutants along with an increase in thickness and volume of the cardiac HA in the CJ suggesting a potential compensatory role, or retention of immaturity of the cardiac jelly in the prelp mutants. Transcriptomics analysis on the prelp mutant hearts revealed downregulation of ECM organization and ECM-Receptor interaction processes in the mutants. Gene Ontology analysis on prelp mutants hearts transcriptome revealed increased MAPK signaling. Interestingly, genes related to degradation of cardiac HA and maturation of cardiac jelly were downregulated, and genes related to epithelial identity of cardiomyocytes were upregulated. Analysis of the mutant hearts at single cell resolution revealed increased number of mutants exhibiting rounded up cardiomyocytes and loss of apical Podocalyxin. Truncated forms of prelp were generated to identify domain specific roles for Prelp, and reintroduction of N-terminal truncated Prelp into the mutants rescued the basal lamina localization and cardiac jelly volume phenotypes. Myocardium specific re-establishment of prelp expression revealed a marked rescue of the mutant cardiovascular phenotype suggesting that tissue specific expression of prelp is not required so long as Prelp is secreted into the CJ. With these data, I’ve elucidated the role of ECM SLRPs in modulation of cardiac chamber morphogenesis process and regeneration of the heart.
In order to understand the origin of the elements in the universe, one must understand the nuclear reactions by which atomic nuclei are transformed. There are many different astrophysical environments that fulfill the conditions of different nucleosynthesis processes. Even though great progress has been made in recent decades in understanding the origin of the elements in the universe, some questions remain unanswered. In order to understand the processes, it is necessary to measure cross sections of the involved reactions and constrain theoretical model predictions. A variety of methods have been developed to measure nuclear reaction cross sections relevant for nuclear astrophysics. In this thesis, two different experiments and their results, both using the well-established activation method, are presented.
A measurement of the proton capture cross section on the p-nuclide 96Ru was performed at the Institute of Structure and Nuclear Astrophysics ISNAP - Notre Dame, USA. The main goal of this experiment was to compare the results with those obtained by Mei et al. in a pioneering experiment using the method of inverse kinematics at the GSI Helmholtzzentrum für Schwerionenforschung GmbH - Darmstadt, Germany. Therefore, the activations were taken out at the same center of mass energies of 9 MeV, 10 MeV and 11 MeV. Another activation was taken out at an energy of 3.2 MeV to compare the result to a measurement of Bork et al. who also used the activation method. While the results at 3.2 MeV agree quite well with those of Bork et al., the results at higher energies show significantly smaller cross sections than those measured by Mei et al.. Experimental details, the data analysis and sources of uncertainties are discussed.
The second part of this thesis describes a neutron capture cross section experiment. At the Institut für Kernphysik - Goethe Universtität Frankfurt an experimental setup allows to produce quasi maxwell-distributed neutron fields to measure maxwell-averaged cross sections (MACS) relevant for s-process nucleosynthesis. The setup was upgraded by a fast electric linear guide to transport samples from the activation to the detection site. The cyclic activation of the sample allows to increase the signal-to-noise ratio and to measure neutron captures that lead to nuclei with
half-lives on the order of seconds. In a first campaign, MACS of the reactions 51V(n,γ), 107,109Ag(n,γ) and 103Rh(n,γ) were measured. The new components of the setup aswell as the data analysis framework are described and the results of the measurements are discussed.
Tinnitus is a symptom experienced by most people at least once in their lifetime. In most documented cases, a new onset of chronic tinnitus can be chronologically correlated with hearing loss. However, tinnitus can also occur in people with (apparently) normal hearing and remains without a traceable preceding cause. Despite the frequency of occurrence of tinnitus, the pathophysiological mechanisms are still not fully understood. A currently proposed hypothesis focuses on a "hidden" hearing loss called synaptopathy as a pathomechanism of tinnitus in normal hearing subjects. In the present study, the objective was to test whether finestructure audiometry or measurement of otoacoustic emissions can reveal possibly overlooked hearing impairment in presumed normalhearing individuals with chronic tinnitus. Thus, a hearing loss not audiologically detectable by the usual methods would supplement or replace the presumed synaptopathic pathomechanism. Another objective was to attempt to replicate the existing findings of another research group on synaptopathy as cause for tinnitus in normal hearing people. Schaette and McAlpine (2011) were able to demonstrate a significant difference in wave I amplitudes between groups of normal hearing subjects with and without chronic tinnitus by deriving clickevoked auditory brainstem potentials, thus supporting the hypothesis of synaptopathy18.
For the present study, a cohort of normal-hearing subjects consisting of a group of tinnitus subjects (N = 15) and a control group (N = 14) was tested. Manual puretone audiometry with 11 test frequencies was conducted to determine hearing performance. Inclusion criteria were defined as air conducted hearing thresholds of 10 dB HL or lower. A deviation at a test frequency of 15 dB HL or less was tolerated. Data of tinnitus characteristics, such as pitch and intensity, were collected by presentation and matching of comparative tones, quality and subjective disturbance by questionnaire. Furthermore, data was obtained from both test groups by Békésy gliding frequency audiometry (794 test frequencies), as well as DPOAE measurement (36 test frequencies) and auditory brainstem response (ABR) audiometry (derivation of early auditory evoked potentials). The results showed a correlation of the determined tinnitus comparison pitch with the frequency location of the largest deviation (impairment) from the normal hearing curve in the Békésy gliding frequency audiometry (p = 0.032). All further analyses of the finestructure hearing curve (steepness of hearing loss, slope, number of hearing loss dips) showed no statistically significant relationship between the morphology of the fine-structure hearing curve and tinnitus characteristics. Finestructure measurement revealed areas of hearing loss that were not mapped in manual puretone audiometry. These "undetected" hearing losses would have led to the exclusion of 12 of 29 subjects (41.4 %) if the finestructure hearing curve had been used as an inclusion criterion. A direct comparison of the mean finestructure hearing curves of both test groups showed a statistically significant better mean hearing performance of the tinnitus group (p < 0.05) in 3 different test frequency ranges (1.5 kHz, 3 kHz, 7 kHz) with a maximum of 4 dB HL. Analy-sis of the mean amplitudes of wave I of the ABRs showed, contrary to expectation, a weak trend toward higher amplitudes in the tinnitus group (p = 0.06). According to Schaette and McAlpine (2011), synaptopathy pathogenesis should have resulted in an opposite trend, i.e., a decrease in wave I amplitude in the tinnitus group. As a secondary finding, a weak trend between wave I amplitude and subjectively perceived disturbance of tinnitus was demonstrated (p = 0.06). Statistical analysis of the parameters determined from the DPOAE measurements did not reveal any significant differences between the tinnitus group and control group. Direct comparison of the DPOAE and finestructure hearing curves, revealed a significant difference in the differences of the frequencyspecific measurements around 2.4 kHz (p = 0.007).
The results of the study suggest that in previous studies with supposedly normal hearing tinnitus subjects there were unrecognized hearing losses that either went unrecognized by the screening by manual puretone audiometry, or subjects with previously aboveaverage hearing experienced a subtle spontaneous decrease in their hearing as tinnitus pathogenesis. This assumption is also supported by the fact that there is a significant correlation between the frequency range of the greatest hearing loss in the finestructure hearing curves and the tinnitus frequency.
The suspected pathomechanism of synaptopathy in "normal hearing" subjects with tinnitus could not be confirmed. The correlation between wave I amplitudes and subjectively perceived disturbance by tinnitus, indicated by the data of this study, should be investigated in more detail in future studies. Further research with more accurate measurement methods and larger subject groups is needed to clarify the hypothesis "Genesis of chronic subjective tinnitus without hearing loss".
Chapter I of this work addressed the piggyBac (PB) transposon system, a non-viral genome engineering tool that is capable of efficiently performing stable integration of DNA sequences into a target cells genome and has already been used in clinical trials. However, the PB transposase has the problematic property of preferentially integrating transposons near transcriptional start sites (TSSs). This increases the likelihood of causing genotoxic effects, limiting its potential use as a tool in clinical applications. It has been shown in the past that the PB transposase shows physical interactions with BET proteins (e.g. BRD4) through Co-IP experiments. Representatives of these proteins are part of the transcriptional activation complex and are abundant at TSSs. Accordingly, it was previously proposed that this interaction is the underlying cause for the biased integration preference. For the first chapter of this thesis, the goal was to disrupt this interaction potentially modifying said integration preference. A secondary structure hypothesized to be mainly responsible for said interaction was extensively mutated resulting in several PB variants that were analyzed for their interaction capacity through a series of Co-IP experiments with BRD4. In total, seven substitutions were identified (E380F, V390K, T392Y, M394R, K407C, K407Q, and K407V) which exhibited reduced interaction capacity with BRD4. Each of the aforementioned mutants were used to generate integration libraries and, through NGS, it was determined if the integration preferences of the respective mutants had changed. In the immediate range 200 base pairs up- and downstream from known TSSs all mutants used exhibited a reduced integration bias. At a wider observation window 3 kbp up- and downstream from TSSs, further mutants with the substitutions M394R, T392Y and V390K showed a reduction in integration frequency of 17.3%, 1.5% and 5.4%, respectively, compared to the wildtype. Of particular note was the M394R mutant, which showed a reduction in all window sizes analyzed with a maximum of 65% less integration preference in the immediate vicinity of TSSs, theoretically generating a safety advantage over the wildtype transposase.
Chapter II was dedicated to the overall safety improvement for transposon-based gene modification and addresses the time point after the transgene has already been integrated and serious side effects may not be preventable. With this in mind, the aim was to develop a novel suicide-switch that can be stably introduced into cells via transposition, and reliably leads to cell death of the modified cells once activated. A system based on CRISPR/Cas9 was developed, where single guide RNAs were used to guide the Cas9 nuclease to Alu elements. These are short, repetitive sequences, which are distributed over the human genome in more than one million copies. Inducing double strand breaks within these elements would lead to genomic fragmentation and cell death. To be inducible, a transcriptional as well as post- translational control mechanism was added. Transcription of the Cas9 nuclease was regulated using a tet-on system, making expression dependent on doxycycline (DOX) supplementation. Furthermore, a version of the Cas9 nuclease called arC9 was used that allows double strand break generation only in the presence of 4-Hydroxytamoxifen (4-HT). Together with an expression cassette for the Alu-specific guide RNA and an expression cassette for the reverse tetracycline controlled transactivator all components were arranged between transposase-specific recognition sequences on a plasmid to allow transposon-system based gene transfer. The system was tested in HeLa cells. First, conditional expression of the arC9 nuclease was confirmed by addition of 1 μg/ml DOX. Second, the suicide-switch was further induced by adding 200 nM 4-HT and protein extracts were assayed for the KAP1 phosphorylation. Only upon induction with DOX and 4-HT phosphorylated KAP1 was detected, indicating DNA damage. Further, extensive growth and survival experiments were conducted to determine the effect of suicide-switch induction on cell proliferation and survival. Between 24 and 48 hours after induction, a halt in cell division was detected, after which extensive cell death was observed. Within 5 days post induction, >99% of all cells were eliminated. In the absence of both inducers, no significant differences in survival were observed compared to control cells line lacking Alu-specific guide RNAs. Microscopic examinations of the <1% surviving cell fraction revealed a senescence-associated phenotype and showed no signs of resumption of the cell division process. Accordingly, the second chapter of this thesis also achieved its goal in developing a functional suicide-switch that can be inserted into human cells via transposition, is highly dependent on the necessary induction signals, and exhibits excellent elimination capabilities in the context tested.
Many countries have restricted public life during the SARS-CoV2 pandemic. As related measures limited the access to sports facilities, this dissertation aimed (1) to examine changes in physical activity (PA) and well-being in affected countries, and (2) to determine the effectiveness of a digital home exercise program in this context.
Part 1 (PA/well-being) of the dissertation was a digital survey administered in 14 countries. Participants reported a 41 - 42% reduction of PA (NPAQ-SF) during restrictions (n=13,503 valid responses). Compliance with international PA guidelines decreased by nearly 19%. Mental well-being declined substantially (n=14,975 responses; 68.1 to 51.9 points on the WHO5 index) and the proportion of individuals at risk of depression tripled (14.2% to 45.2%). Physical well-being (SF-36 Pain) decreased slightly (85.8% to 81.3%). About two thirds (68.1%) of the respondents reported being interested in digital home exercise.
For Part 2 (digital home exercise) of the dissertation, an international multicenter randomized, controlled trial was performed allocating healthy adults (n=763; 33±12 years) to an intervention (IG) or control (CG) group. In contrast to the CG, the IG was offered live-streamed home exercise for four weeks. Subsequently, both groups had access to pre-recorded workouts for another four weeks. Outcomes were measured weekly using validated questionnaires. Mixed-models data analyses revealed an up to 1.65-fold (95% CI: 1.4-1.94; week 1) increase of PA relative to the CG. Moreover, small improvements in exercise motivation (SKK scale), psychological well-being (WHO-5 index), sleep quality (MOS Sleep Scale), and anxiety symptoms (GAD-7 Scale) were observed for IG.
The results of this dissertation suggest that public life restrictions associated with the pandemic had significant adverse effects on movement behavior and well-being. Digital home exercise can help to maintain and/or increase health- beneficial PA and well-being and may hence represent a supportive element of viral containment efforts.
Mechanism of the MHC I chaperone TAPBPR and its role in promoting UGGT1-mediated quality control
(2022)
Information about the health status of most nucleated cells is provided through peptides presented on major histocompatibility complex I (pMHC I) on the cell surface. T cell receptors of CD8+ T cells constantly monitor these complexes and allow the immune system to detect and eliminate infected or cancerous cells. Antigenic peptides displayed on MHC I are typically derived from the cellular proteome and are translocated into the lumen of the endoplasmic reticulum (ER) by the ATP-binding cassette (ABC) transporter associated with antigen processing (TAP), which is part of the peptide-loading complex (PLC). In a process called peptide editing, the MHC I-dedicated chaperone tapasin (Tsn) selects peptides for their ability to form stable complexes with MHC I. While initial peptide loading is catalyzed in the confines of the PLC, the second quality control is mediated by TAPBPR, operating in the peptide-depleted cis-Golgi network. TAPBPR was shown to have a more fine-tuning effect on the presented peptide repertoire rather than initial peptide selection. The fundamental mechanism of peptide editing was illuminated by two crystal structures of TAPBPR in complex with peptide-receptive MHC I. Notably, one of these structures reported a structural element that inserted into the peptidebinding pocket. The so-called scoop loop was assumed to be involved in mediating peptide exchange but the underlying mechanism remained undefined. Additionally, latest results suggested that TAPBPR mediates the interaction of the glucosyltransferase UGGT1 with peptide-receptive MHC. To expand the current knowledge of quality control processes in the antigen presentation pathway, the contribution of the scoop loop in peptide editing and the role of TAPBPR in UGGT1-mediated quality control needs to be elucidated. In the first part of this study, TAPBPR proteins with various loop lengths were designed to scrutinize the contribution of the scoop loop in chaperoning peptidereceptive MHC I. In a light-driven approach, the ability of TAPBPR variants to form stable complexes with peptide-free MHC I was tested. These results demonstrated that in a peptide-depleted environment, the scoop loop is of critical importance for TAPBPR to chaperone intrinsically unstable, peptidereceptive MHC I clients. Moreover, fluorescence polarization-based assays allowed the pursuit of peptide exchange in different, native-like environments. Peptide displacement activities of TAPBPR variants illustrated that catalyzed peptide editing is primarily induced by structural elements outside the scoop loop. In a peptide-depleted environment, the scoop loop occupies the position of the peptide C-terminus and acts as an internal peptide surrogate. By combining complex formation and fluorescence polarization experiments, the scoop loop of TAPBPR was shown to be critically important in stabilizing empty MHC I and functions as an internal peptide selector. In the second part of this study, a novel in-vitro glucosylation assay was established to examine the role of TAPBPR in UGGT1-catalyzed re-glucosylation of TAPBPR-bound MHC I clients. Therefore, a peptide-free MHC I-TAPBPR complex with defined glycan species was designed which served as physiological substrate for UGGT1. By subjecting the recombinantly expressed HLA-A*68:02- TAPBPR complex and UGGT1 proteins to the new in-vitro system, UGGT1 was shown to catalyze the transfer of a glucose residue to the N-linked glycan of TAPBPR-bound Man9GlcNAc2-HLA-A*68:02. Moreover, a high-affinity, photocleavable peptide was applied to dissociate the MHC I-chaperone complex. However, in the absence of TAPBPR, no glucosyltransferase activity was observed. Generation of peptide-free MHC I through UV illumination also showed no activity, and only the addition of TAPBPR could restore UGGT1-mediated reglucosylation of the empty MHC I. Independent of the peptide status of HLAA*68:02, the combination of protein glycoengineering and LC-MS analysis implicated that UGGT1 exclusively acts on TAPBPR-chaperoned HLA-A*68:02. The newly established system provided insights into the function of TAPBPR during UGGT1-catalyzed re-glucosylation activity and quality control of MHC I. Taken together, the scoop loop allows TAPBPR to function as MHC I chaperone through stabilizing peptide-receptive MHC I. In a peptide-depleted environment, the loop structure serves as an internal peptide surrogate and can only be dislodged by a high-affinity peptide. Based on these findings, TAPBPR fulfills a dual function in the second level of quality control. On the one hand, TAPBPR functions as peptide editor, shaping the repertoire of presented peptides. On the other hand, TAPBPR mediates peptide-receptive MHC I clients to the folding sensor UGGT1. Here, TAPBPR is essential to promote UGGT1-catalyzed reglucosylation of the N-linked glycan, giving MHC I a second chance to be loaded with an optimal peptide cargo in the peptide loading complex.
KMT2A-rearrangements are causative for 70-80% all infant acute lymphoblastic leukemias (Pieters et al., 2019, 2007). Among these, the translocation t(4;11)(q21;23) generating the oncogenic fusion genes KMT2A::AFF1 and AFF1::KMT2A is the most frequent one, accounting for almost every second case of KMT2A-r infant ALL (Meyer et al., 2018). Despite passing a multimodal chemotherapy, 64% of patients achieve an event including relapse or death within four years from diagnosis, and overall survival three years from relapse remains poor with only 17% (Driessen et al., 2016; Pieters et al., 2019, 2007). Vari-ous studies have shown that relapse and therapy resistance were not mediated by chemotherapy-induced mutagenesis as there was no accumulation of secondary mutations in the dominant leukemic clone between diagnosis and relapse (Agraz-Doblas et al., 2019; Andersson et al., 2015; Bardini et al., 2011; Dobbins et al., 2013; Driessen et al., 2013; Mullighan et al., 2007).
Intriguingly, exclusively infant t(4;11) ALL patients were reported to subdivide in two groups depending on the level of HOXA gene cluster expression (Trentin et al., 2009). The HOXAlo group displayed a high expression of IRX1 and the HOXAhi group a low expression of IRX1 (Symeonidou and Ottersbach, 2021; Trentin et al., 2009). Importantly, the HOXAlo/IRX1hi group was characterized to possess a strongly ele-vated relapse incidence compared to the HOXAhi/IRX1lo group (Kang et al., 2012; Stam et al., 2010). IRX1 was identified to upregulate the Early growth response genes EGR1, EGR2 and EGR3 (Kühn et al., 2016).
The doctoral project “EGR-mediated relapse mechanisms in infant t(4;11) acute lymphoblastic leuke-mia” aimed to investigate a potential correlation between the HOXAlo-IRX1-EGR axis and relapse development in infant t(4;11) ALL. The primary objective was to clarify through which molecular mechanism(s) relapse development despite continuous chemotherapy could be achieved. In this context, the role of the EGR genes has been investigated. In addition, this project aimed to disclose molecular targets which could offer novel therapeutic interventions to interfere with therapy resistance and relapse formation.
This thesis has two main parts.
The first part is based on our publication [1], where we use perturbation theory to calculate decay rates of magnons in the Kitaev-Heisenberg-Γ (KHΓ) model. This model describes the magnetic properties of the material α-RuCl 3 , which is a candidate for a Kitaev spin liquid. Our motivation is to validate a previous calculation from Ref. [2]. In this thesis, we map out the classical phase diagram of the KHΓ model. We use the Holstein-Primakoff
transformation and the 1/S expansion to describe the low temperature dynamics of the Kitaev-Heisenberg-Γ model in the experimentally relevant zigzag phase by spin waves. By parametrizing the spin waves in terms of hermitian fields, we find a special parameter region within the KHΓ model where the analytical expressions simplify. This enables us to construct the Bogoliubov transformation analytically. For a representative point in the special parameter region, we use these results to numerically calculate the magnon damping, which is to leading order caused by the decay of single magnons into two. We also calculate the dynamical structure factor of the magnons.
The second part of this thesis is based on our publication [3], where we use the functional renormalization group to analyze a discontinuous quantum phase transition towards a non-Fermi liquid phase in the Sachdev-Ye-Kitaev (SYK) model. In this thesis, we perform a disorder average over the random interactions in the SYK model. We argue that in the thermodynamic limit, the average renormalization group (RG) flow of the SYK model is identical to the RG flow of an effective disorder averaged model. Using the functional RG, we find a fixed point describing the discontinuous phase transition to the non-Fermi liquid phase at zero temperature. Surprisingly, we find a finite anomalous dimension of the fermions, which indicates critical fluctuations and is unusual for a discontinuous transition. We also determine the RG flow at zero temperature, and relate it to the phase diagram known from the literature.
Oceanic islands only comprise a small amount of the Earth’s land area but harbour a disproportionate amount of global biodiversity. This vast diversity is not only reflected in the taxonomic uniqueness of island biota but also in the remarkable evolution of functional traits. Functional traits, i.e. measurable characteristics that strongly influence the fitness of species, determine how a species responds to its environment and can help to gain more insights into the biogeographical, ecological and evolutionary processes that have shaped island biodiversity. However, research in island biogeography has primarily focused on species richness, and knowledge of functional trait patterns on oceanic islands is scarce. Hence, in this dissertation, I have explored how trait-based approaches can increase our understanding of how biodiversity on oceanic islands assembles and how it is driven by the environment. The Canary Islands (Spain) are a particularly suitable model system to investigate patterns and drivers of biodiversity. The archipelago is characterised by a high variation in environmental heterogeneity and inhabits a unique and well-described native flora. Therefore, I have investigated five principal research questions using the flora (Spermatophytes) of the Canary Islands as a study object. First, I have analysed how climate and biogeography shape the assembly of the Canary Islands flora using a novel trait-based approach. Second, the question of whether rare climates link to functional trait distinctiveness in the native Canary Islands flora was addressed. Third, I have examined how intraspecific trait variation is represented in the native flora of oceanic islands focusing on the succulent scrub of La Palma (Canary Islands). Fourth, this dissertation investigated whether scientific floras can be reliable sources for trait data of plants native to oceanic islands. Finally, I have explored how climate change may impact the native Canary Islands flora by analysing possible climate change-induced shifts in plant species distribution and plant traits.
The results of my dissertation expand the understanding of the importance of biogeography and the environment in determining the functional composition of island floras. I have assessed that traits of endemic plant species did not expand the functional trait space of the Canary Islands but were packed with the ones of non-endemic species. This result hints at a trait convergence in endemic species, possibly driven by non-adaptive speciation processes. Moreover, I have evidenced that humidity is a critical driver of functional diversity in native plant assemblages and particularly leads to a high trait convergence in arid environments via environmental filtering. In contrast, alien species have expanded the Canary Islands flora’s functional trait space. I further have shown that in contrast to native species assemblages, alien species assemblages are characterised by an increasing functional diversity with increasing aridity. This contrasting pattern of functional diversity could pose a potential risk to the native flora of the Canary Islands as a low functional diversity is expected to reduce the resilience of species assemblages to the establishment of more functionally diverse alien plant species. However, in this dissertation, I also have revealed that endemic plant species on the Canary Islands show a high intraspecific variation in arid environments, possibly as an adaptation to environmental stress. Intraspecific variation could help endemic plant species have a competitive advantage over alien species and be more resilient to environmental changes. Furthermore, in this dissertation, I have shown that scientific floras and taxonomic monographs could be used to gain information on quantitative functional traits of plants native to oceanic islands. This finding is particularly relevant for advances in trait-based research, as coverage of trait data for oceanic island floras is extremely poor in global trait databases. Hence, for some of the studies included in this dissertation, trait data were retrieved from scientific floras and taxonomic monographs and used to answer novel scientific research questions. Thus, I have used trait data from the literature to analyse the effect of climate change on the range size of plants native to the Canary Islands. Identifying plant species of particular conservation concern is critical on oceanic islands as many island species have limited distributions and small population sizes, and their niche tracking is impeded by insularity. I have revealed that single-island endemic plants gain less and lose more climatically suitable areas than archipelago endemic and non-endemic native plants due to a climate change-induced decrease in precipitation until 2100...
The relevant field of interest in High Energy Physics experiments is shifting to searching and studying extremely rare particles and phenomena. The search for rare probes requires an increase in the number of available statistics by increasing the particle interaction rate. The structure of the events also becomes more complicated, the multiplicity of particles in each event increases, and a pileup appears. Due to technical limitations, such data flow becomes impossible to store fully on available storage devices. The solution to the problem is the correct triggering of events and real-time data processing.
In this work, the issue of accelerating and improving the algorithms for reconstruction of the charged particles' trajectories based on the Cellular Automaton in the STAR experiment is considered to implement them for track reconstruction in real-time within the High-Level Trigger. This is an important step in the preparation of the CBM experiment as part of the FAIR Phase-0 program. The study of online data processing methods in real conditions at similar interaction energies allows us to study this process and determine the possible weaknesses of the approach.
Two versions of the Cellular Automaton based track reconstruction are discussed, which are used, depending on the detecting systems' features. HFT~CA Track Finder, similar to the tracking algorithm of the CBM experiment, has been accelerated by several hundred times, using both algorithm optimization and data-level parallelism. TPC~CA Track Finder has been upgraded to improve the reconstruction quality while maintaining high calculation speed. The algorithm was tuned to work with the new iTPC geometry and provided an additional module for very low momentum track reconstruction.
The improved track reconstruction algorithm for the TPC detector in the STAR experiment was included in the HLT reconstruction chain and successfully tested in the express production for the online real data analysis. This made it possible to obtain important physical results during the experiment runtime without the full offline data processing. The tracker is also being prepared for integration into a standard offline data processing chain, after which it will become the basic track search algorithm in the STAR experiment.
In this thesis we discuss the group Out(Gal_K) of outer automorphism of the absolute Galois group Gal_K of a p-adic number field K. Using results about the mapping class group of a surface S, as well as a result by Jannsen--Wingberg on the structure of the absolute Galois group Gal_K, we construct a large subgroup of Out(Gal_K) arising as images of certain Dehn twists on S.
Background: Increasing numbers of patients surviving malignant bone tumors around the knee joint have led to an increasing importance to investigate long-term results. This study assessed the long-term results of rotationplasty after resection of malignant bone tumors regarding functional outcome and quality of life to allow better comparison with other treatment options in bone cancer treatment.
Procedure: 60 participants who underwent rotationplasty due to bone cancer took part in this multicentric questionnaire- based study. The long-term functional outcome was measured by the Musculoskeletal tumor society score (MSTS) and the Tegner activity level scale. The health-related quality of life (HRQL) was assessed by using the Short Form Health Survey (SF-36).
Results: Patients treated with rotationplasty (median follow- up of 22 years, range 10–47 years) regained a high level of activity (median MSTS score of 24). Even a return to high level sports was possible (mean Tegner activity level scale of 4). Duration of follow-up did not influence the functional outcome. HRQL scores were comparable to the general German popula tion. Concerns of psychological problems due to the unusual appearance of the rotated foot have not been confirmed.
Conclusion: Rotationplasty can be a good alternative to en- doprosthetic replacement or amputation, either as primary surgery or as a salvage procedure. Especially for growing children and very active patients rotationplasty should be considered.
Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with a multifarious clinical presentation. Even though many genetic risk factors have been identified and studied in mouse models, the neurophysiological mechanisms underlying the autistic phenotype are still unclear. Based on the high rates of comorbidity with epilepsy, it was hypothesized that the balance between excitation and inhibition in neural circuits may be disrupted in autistic individuals.
In this dissertation, synaptic and network activity was measured in three different genetically modified mouse models that exhibit the characteristic behavioral abnormalities of the disorder: the Neurobeachin (Nbea) haploinsufficient mouse, the Neuroligin-3 (Nlgn3) knockout (KO) mouse, and the Neuroligin-4 (Nlgn4) KO mouse. Each of the affected proteins is involved in the formation and/or function of synapses in the central nervous system. Therefore, it was posited that the reduction or deletion of these proteins might alter the balance of excitatory to inhibitory synaptic transmission in individual neurons and in neural circuits. Extracellular recordings in the hippocampal dentate gyrus of anesthetized mice revealed that the excitation-inhibition (E-I) balance was reduced in Nbea haploinsufficient and Nlgn4 KO mice, but unchanged in Nlgn3 KO mice despite a reduction in excitatory synaptic transmission to dentate granule cells. Unexpectedly, the intrinsic excitability of dentate granule cells was altered in all three mouse models. These results imply that a homeostatic increase in the intrinsic excitability is able to compensate for the decreased excitatory transmission in Nlgn3 KO mice, whereas the decreased intrinsic excitability in the Nbea haploinsufficient and Nlgn4 KO mice leads to a reduction in the E-I balance. Taken together, these findings suggest that the influence of genetic factors on the E-I balance might be a potential common mechanism underlying the development of ASD.
Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
A promising strategy to reduce the dependency from fossil fuels is to use the yeast Saccharomyces cerevisiae to bioconvert renewable non-food feedstocks or waste streams, like lignocellulosic biomass, into bioethanol and other valuable molecule blocks. Lignocellulosic feedstocks contain glucose and significant fractions of the pentoses xylose and arabinose in varying proportions depending on the biomass type. S. cerevisiae is an efficient glucose consumer, but it cannot metabolize xylose and arabinose naturally. Therefore, extensive research using recombinant DNA techniques has been conducted to introduce and improve the biochemical pathways necessary to utilize these non-physiological substrates. However, any functional pathway capable of metabolizing D xylose and L arabinose in S. cerevisiae requires the transport of these sugars across the plasma membrane. The endogenous sugar transport system of S. cerevisiae can conduct a limited uptake of D-xylose and L-arabinose; this uptake enables only basal growth when the enzymatic pathways are provided. For this reason, the uptake of D xylose and L-arabinose has been recognized as a limiting step for the efficient utilization of these non-physiological substrates.
Gal2, a member of the major facilitator superfamily, is one of the most studied hexose transporters in S. cerevisiae. Although its expression is repressed in the presence of glucose, it also transports this sugar with high affinity when constitutively expressed. Recent efforts to engineer yeast strains for the utilization of plant biomass have unraveled the ability of Gal2 to transport non-physiological substrates like xylose and arabinose, among others. Improving Gal2 kinetic and substrate specificity, particularly for pentoses, has become a crucial target in strain engineering. The main goal of this study is to improve the utilization of xylose and arabinose by increasing the cell permeability of these non physiological substrates through the engineering of the galactose permease Gal2.
GAL2 gene expression depends on galactose, which acts as an inducer; nevertheless, even in the presence of galactose, glucose act as a strict repressor; consequently, GAL2 gene is usually placed under the control of a constitutive promoter. However, the presence of glucose additionally triggers the Gal2 degradation, which is mediated by the covalent attachment of the small 76 amino acid protein ubiquitin (Ub) to the targeted transporter; in a multi-step process called ubiquitination.
Ubiquitination of hexose permeases involves the activation of the Ub molecule by the E1 Ub-activating enzyme using ATP; then, the activated Ub is transferred to a specific Ub-conjugating enzyme E2, which donates the Ub indirectly through a specific HECT E3 enzyme (Rsp5) to a lysine residue of the substrate, with the aid of an adaptor protein which recognizes the target (Rsp5-adaptor). Ubiquitinated permeases are sent by membrane invagination to early endosomes, where they encounter ESCRTs (endosomal sorting complex required for transport). The targeted permeases are sorted in intralumenal vesicles (ILV) inside of the endosome, which after several cycles, turns into a multivesicular body (MVB) that subsequently fuses with the vacuole to expose the protein content of the ILVs to lumenal hydrolases for degradation.
Gal2 contains 30 lysine residues that may accept the ubiquitin molecule, which targets its degradation. It is known that mono-ubiquitination by Rsp5 on multiple lysine residues is necessary to internalize Gal2 (Horak & Wolf, 2001). However, the authors did not identify the specific lysine residues involved in the ubiquitination processes. This study screened several Gal2 variants where lysine residues were mutated or removed from the protein sequence to discover which lysine residues are likely involved in ubiquitination and consequent turnover of the transporter. The results of the screening showed that mutation of the N terminal lysine residues 27, 37, and 44 to arginine (Gal23KR) produced a functional transporter that, when fused with GFP (Gal23KR_GFP), showed an exclusive localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b).
This study furthermore evaluated upstream signals caused by phosphorylation which triggers ubiquitination and consequent turnover of the targeted protein; using similar screening approaches to assess the stabilization of Gal2 by lysine residue modifications, it was possible to identify that N terminal serine residues 32, 35, 39, 48, 53, and 55 are likely involved in the internalization of Gal2, since a Gal2 construct where all these serines were mutated to alanine residues and tagged with GFP (Gal26SA_GFP) exhibited practically complete localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b)...
Mutational analysis of ribosomal DNA and maturation-scheme analysis of ribosomal RNA in A. thaliana
(2022)
Ribosome biogenesis is a fundamental cellular process beginning with long precursor rRNA transcription from multi-copies of repetitive 45S ribosomal DNAs. At the subunit level, the primary pre-rRNA transcript encapsuled in 90S protein-RNA complex undergoes decisive splitting in two chief ways for further maturation into large (LSU) and small (SSU) ribosomal subunit. The usage of specific rDNA copies from defined chromosomes and their selective role during growth and development have been a topic of interest owing to its contribution to specialized ribosome theory which proposes non-monolithic functions for ribosomes and thereby their mRNA translation potential. Dual-guide CRISPR/Cas9 mediated disruption of rDNA regions resulted in stable disruption of up to 2.5% and 5% of all rDNA copies in hetero- and homozygous (ploop KD) conditions, respectively. At the RNA level, the mutation excised a critical structural element, P-loop on the LSU 25S rRNA. Mutation caused a dosage dependent defect with homozygosity leading to severe developmental defects through vegetative and reproductive growth phases which is manifested in their proteome by means of disregulation through both increase and decrease of several gene ontological categories of proteins in mutants. Interestingly, the mutation on chromosome 4 triggered dosage compensation through rRNA expression from chromosome 2 further compounded by ectopic rRNA biogenesis defects. The mutated copies however are not incorporated in the translating ribosomes and as a direct or indirect consequence led to elevated basal autophagic levels in the mutants.
The primary 35S transcript is known to undergo two modes of initial cleavages at the pre-rRNA level that aid in their subsequent maturation. Root cell culture (RCC) studies shows that these cells contain a novel ITS2-first cleaved precursor even under control growth conditions, P-C2 adding a third maturation means for the 35S pre-rRNA. This maturation path is further known to be triggered under elevated growth temperature forming a novel adaptive response in Arabidopsis and two other crop plants, tomato, and rice. Taken together, the pulse-chase labeling analysis of control and stressed tissues uncovers the fine-tuned pre-rRNA schematics with crossovers between multiple maturation paths.
Lipopolysaccharide (LPS) is a major glycolipid component in the outer leaflet of the outer membrane of Gram-negative bacteria and known as endotoxin exhibited by the lipid A moiety, which serves as a membrane anchor. The effective permeability barrier properties of the outer membrane contributed by the presence of LPS in the extracellular layer of the outer membrane confer Gram-negative bacteria a high resistance against hydrophobic compounds such as antibiotics, bile salts and detergents to survive in harsh environments. The biogenesis of LPS is well studied in Escherichia coli (herewith E. coli) and the LPS transport (Lpt) is carried out by a transenvelope complex composed of seven essential proteins (LptABCDEFG), which are located in the three compartments of the cell such as the outer membrane, the inner membrane and the periplasm. The Lpt system also exists in Anabaena sp. PCC 7120 (herewith Anabaena sp.), however, homologues of LptC and LptE are still missing. BLAST search failed to identify a homologue of LptC, in contrast, the secondary structure analysis using the Pfam database based on the existing ecLptC secondary structure identified one open reading frame All0231 as the putative Anabaena sp. homologue of LptC, which is designated anaLptC. Despite the low sequence similarity, the secondary structure alignment between anaLptC and ecLptC using the HHpred server showed that both proteins share high secondary structural similarities. The genotypic analysis of the insertion mutant anaLptC did not identify a fully segregated genome and its phenotypic analysis revealed that it was sensitive against chemicals, suggesting that the analptC gene is essential for the growth of Anabaena sp. and involved in the outer membrane biogenesis. This is further supported by the observation of the small cell phenotype in the anaLptC mutant via transmission electron microscopy. Moreover, physical interactions between the anaLptC periplasmic domain with anaLptA as well as with anaLptF were established, indicating that the anaLptC periplasmic domain is correctly folded and alone functional and that the transmembrane helix is not required for the interaction with anaLptA and anaLptF. Furthermore, the reduction of the O-antigen containing LPS was observed in the insertion mutant anaLptC and the dissociation constant Kd of the anaLptC periplasmic domain for ecLPS was determined.The three-dimensional structure of the periplasmic domain of anaLptC was solved by X-ray crystallography with a resolution of 2.8 Å. The structural superposition between the ecLptC crystal structure (PDB number 3my2) and the crystal structure of anaLptC periplasmic domain obtained by this study showed the similarity in the folding of the two proteins with a Cα r.m.s.d value of about 1 Å and confirmed that the length of anaLptC is more than two times longer than that of ecLptC. The structural comparison also revealed that both structures share the typical β-jellyroll fold and conserved amino acids, which were shown in ecLptC to bind to LPS in vivo and found in anaLptC. Overall, these data strongly suggest that anaLptC is involved in the transport of LPS and support the model whereby the bridge spanning the inner membrane and the outer membrane would be assembled via interactions of the structurally conserved β-jellyroll domains shared by five (LptACDFG) out of seven Lpt proteins.
The majority of B-cell precursor acute leukemias in infants are associated with the chromosomal translocation t(4;11)(q21;q23), resulting in the fusion of the mixed-lineage leukemia (MLL) and ALL1-fused gene of chromosome 4 (AF4) genes. While the fusion protein MLL-AF4 is expressed in all t(4;11) patients and essential for leukemia progression, the distinct role of the reciprocal fusion protein AF4-MLL, that is expressed in only 50-80% of t(4;11) leukemia patients (Meyer et al., 2018), remains unclear. In addition, t(4;11) leukemia could so far exclusively be generated in vivo in the presence of AF4-MLL and independent of the co-expression of MLL-AF4 (Bursen et al., 2010).
In a multifactorial approach inhibiting histone deacetylases (HDACs) and expressing the dominant negative mutation of Taspase1 (dnTASP1), both MLL fusion proteins were targeted simultaneously to evaluate a possible cooperative effect between MLL-AF4 and AF4-MLL during the progression of leukemia. Of note, neither HDACi nor dnTASP1 expression negatively affect endogenous MLL, but rather endorse its function hampered by the MLL fusion proteins (Ahmad et al., 2014; Bursen et al., 2004; Zhao et al., 2019). The mere expression of dnTASP1 failed to induce apoptosis, whereas dnTASP1 could elevate apoptosis levels significantly in HDACi-treated t(4;11) cells underlining the therapeutic potential of co-inhibiting both MLL fusion proteins.
Next, the impact of inhibiting either MLL-AF4 or AF4-MLL in vivo was resolved using whole transcriptome analysis. In PDX cells obtained by the Jeremias Laboratory (Völse, 2020) that co-expressed both t(4;11) fusion proteins, the knock-down of MLL-AF4 revealed the down-regulation of pivotal hemato-malignant factors. The expression of dnTASP1 led to massive deregulation of cell-cycle genes in vivo. Considering that the inhibition of particularly MLL-AF4 but not AF4-MLL impaired leukemic cell growth in vivo (Völse, 2020), the results of this work suggest a cooperative effect between both fusion proteins, while the loss of AF4-MLL during leukemia progression appears not essential.
Thereafter, a possible short-term role of AF4-MLL during the establishment of t(4;11) leukemia was analyzed. For this purpose, an in vitro t(4;11) model was constructed to investigate the transforming potential of transiently expressed AF4-MLL in cells constitutively expressing MLL-AF4, putatively reflecting the situation in vivo. Due to the lack of a leukemic background of the applied cell line, the aim was to investigate the long-term potential of AF4-MLL to significantly alter the epigenome rather than mimicking the development of leukemia. Strikingly, short-term-expressed AF4-MLL in cooperation with MLL-AF4 exerted durable epigenetic effects on gene transcription and chromatin accessibility. The here obtained in vitro data suggest a clonal evolutionary process initiated by AF4-MLL in a cooperative manner with MLL-AF4. Importantly, no long-term changes in chromatin accessibility could be observed by the transient expression of either MLL-AF4 or AF4-MLL alone.
All in all, considering endogenous MLL, MLL-AF4 and AF4-MLL in a targeted treatment is a promising approach for a more tailored therapy against t(4;11) leukemia, and AF4-MLL is suggested to act in a cooperative manner with MLL-AF4 especially during the development of a t(4;11) leukemia.
High-resolution, compactness, scalability, efficiency – these are the critical requirements which imaging radar systems have to fulfil in applications such as environmental monitoring, cloud mapping, body sensing or autonomous driving. This thesis presents a modular millimetre-wave frequency modulated continuous-wave (FMCW) radar front-end solution intended for such applications. High-resolution is achieved by enlarging the operating frequency band of the radar system. This can be realized at millimetre-wave frequencies due to the large spectrum availability. Furthermore, the size of components decreasing with increasing frequency makes millimetre-wave systems a good candidate for compactness. However, the full integration of radar front-ends is a challenge at millimetre-wave frequencies due to poor signal integrity and spectral purity, which are essential for imaging applications. The proposed radar uses an alternative technique and tackles this limitation by featuring highly-integrable architectures, specifically the Hartley architecture for signal conversion and enhanced push-pull amplifier for harmonic suppression. The resolution of imaging radars can be further improved by increasing the number of transmitters and receivers. This has spurred the investigation of spectrum, time and energy-efficient multiplexing techniques for multi-input multi-output (MIMO) radar systems. The FMCW radar architecture proposed in this thesis is based on code-division technique using intra-pulse, also called intra-chirp modulation. This advanced scalable and non-complex solution, made possible by the latest achievements on direct digital synthesis for signal generation, guarantees signal integrity and compact size implementation. The proposed architecture is investigated by a thorough system analysis. A transmitter module and a receiver module for a 35 GHz imaging radar prototype are designed, fabricated and fully characterized to validate the feasibility of our novel approach for high-resolution highly-integrated MIMO front-ends.
The health status of every nucleated cell in the human body is monitored through peptides presented by major histocompatibility complex class I (MHC I) to T-cell receptors of CD8+ T-cells. Thereby, the adaptive immune system ensures the recognition and elimination of infected or cancerous cells. MHC I molecules comprise the polymorphic heavy chain (hc) and the light chain β2-microglobulin (β2m). More than 13,000 allomorphs of the MHC I hc have been identified. All MHC I hcs associate with β2m but differ in their binding preferences for peptides, ensuring the presentation of a large peptide pool. After maturation of MHC I hc/β2m heterodimers in the endoplasmic reticulum (ER), most of the peptide-deficient MHC I molecules are recruited to the peptide-loading complex (PLC). There, they go through peptide loading and editing before they are released as stable peptide-MHC I (pMHC I) complexes and traffic to the cell surface for antigen presentation.
During the stringent quality control of MHC I peptide loading and editing within the PLC, the chaperone tapasin in conjunction with the oxidoreductase ERp57 stabilizes peptide-receptive MHC I molecules and alters the peptide cargo for high immunogenicity by catalyzing peptide-exchange. The tapasin-homologue TAP-binding protein related (TAPBPR) is involved in downstream quality control, editing the peptide repertoire of MHC I molecules that slipped through peptide proofreading by tapasin. Both chaperones were shown to adopt similar binding-modes for MHC I, suggesting related mechanisms of peptide editing. Nevertheless, the MHC I specific chaperones operate in different subcellular locations with differing assistance. While TAPBPR mediates peptide-exchange solely in the peptide-poor environment of the cis-Golgi and ER-Golgi intermediate compartment (ERGIC), tapasin functions mainly within the PLC together with ERp57 and the lectin-like chaperone calreticulin. Calreticulin with its lectin-, arm- and C-terminal domain contacts the MHC I heterodimer, ERp57 and the C-terminal domain of tapasin, respectively. Notably, the interaction site between calreticulin and tapasin has not yet been elucidated experimentally at molecular detail. The depletion of tapasin leads to a compromised immune response and a change in the pool of peptide cargo. The numerous MHC I allomorphs vary in their plasticity and their dependence on tapasin for the loading of optimal peptides. Moreover, the conformational plasticity of MHC I correlates with their dependence on tapasin. However, the molecular basis on how tapasin edits the various MHC I allomorphs and the structural features that are essential for peptide exchange catalysis at atomic resolution remained elusive.
In the first part of this thesis, the trimeric complex of tapasin–ERp57/calreticulin was analyzed. To this end, laser induced liquid bead ionization mass spectrometry (LILBID-MS) was performed as part of a collaboration and revealed the trimeric assembly for tapasin–ERp57 and calreticulin. Furthermore, additional to a wildtype construct of calreticulin, a second construct, lacking the acidic helix of calreticulin that was found to come to close contact with tapasin, was utilized for isothermal titration calorimetry (ITC). A micromolar affinity of wildtype calreticulin to tapasin–ERp57 was determined. Previous biochemical and NMR studies utilizing the P-domain of calreticulin and solely ERp57 provided a micromolar affinity for the complex of calreticulin and ERp57. In this study, no interaction of calreticulin lacking the acidic helix with tapasin–ERp57 could be measured by ITC. However, these results undergo with findings that calreticulin lacking the acidic helix impairs the function of the PLC. Most likely, the negatively charged acidic helix is located in a groove of tapasin, carrying a more positive charge. Taken together, the functional data demonstrates the importance of the acidic helix of calreticulin for assembly of the trimeric subunit of calreticulin/tapasin–ERp57.
In the main part of this study an MHC I–tapasin–ERp57 complex was structurally analyzed. Therefore, a photo-triggered approach was chosen to assemble the transient complex of MHC I–tapasin–ERp57. Various allomorphs were screened for complex formation with the tapasin–ERp57 heterodimer after photocleavage by size exclusion chromatography (SEC), resulting in mouse MHC I H2-Db as the suited allomorph. Microseed matrix screening was performed. Crystals diffracting X-rays to a resolution of 2.7 Å were obtained showing one tetrameric tapasin–ERp57–MHC I complex per asymmetric unit.
The MHC I-chaperone structure shows molecular rearrangements upon MHC I engagement and unveils structural features of tapasin, involved in peptide-exchange catalysis...
In the last twenty years, there has been splendid progress in energy conversion technologies to have sustainable energy sources. For example, solar cells contribute significantly to energy production as the sun is an enormous source for renewable energy. Currently, the most common commercialized photovoltaic devices are silicon-based. The scientists' main targets are high efficiency, low cost, environmentally friendly, and easy to synthesize new semiconductor materials to replace silicon. Furthermore, understanding the photophysical properties of these materials is very important for designing high efficient photoconversion systems.
This thesis investigates the photophysics of lead-based wide-bandgap perovskites with different dimensionality (2D, 3D) and how they can be optimized for optoelectronic applications. In chapter 1, we present the background and progress in perovskite research. The basic concepts of semiconductor and spectroscopic methods of the applied techniques in this work are discussed in chapter 2.
In the first project (chapter 3.1), we used our time-resolved techniques to study the ultrafast dynamics of energy transfer from the inorganic to the organic layer in a series of three lead-based mixed-halide 2D perovskites containing benzyl ammonium (BA), 1-naphthyl methyl ammonium (NMA), and 1-pyrene methyl ammonium (PMA) thin films.
In the second project (chapter 3.2), we used time-resolved spectroscopic techniques to study the effect of adding 5% of Cs on the dynamics of a mixed-cation wide bandgap bromide-based 3D perovskite.
In another side project (chapter 4), we present the photophysics properties of newly synthesized new Schiff bases containing indole moieties using piperidine as an organic base catalyst and Au@TiO2 as a heterogeneous catalyst. Finally, the results of this work are summarized in Chapter 5 with an outlook and a discussion of open questions for further research.
Ceramide synthase (CerS) is the enzyme responsible for the de novo synthesis of ceramide. In this process, the different CerS isoforms are substrate-specific and produce ceramides of different chain lengths. Ceramides form the backbone for other sphingolipids and are enriched in membrane microdomains called lipid rafts. Lipid rafts are important signaling platforms for many transmembrane proteins, but can also act as bioactive lipids. Depending on the chain length, the effects on signaling pathways can vary. The aim of this work was to further investigate the chain length-specific effects by CerS4 on the progression of inflammatory colon cancer. To understand the tissue-specific effects of CerS4 deficiency on the progression of acute colitis and colitis-associated cancer (CAC), CerS4 knockout models were used. Disease progression of wild-type CerS4 (WT) was compared with that of mice with global CerS4 knockout (CerS4 KO) and mice in which CerS4 deficiency was restricted to T cells (CerS4 LCK/Cre) or intestinal cells (CerS4 Vil/Cre). Acute colitis was induced with sodium dextran sulfate (DSS), whereas azoxymethane (AOM)/DSS combinations were used to induce CAC in mice. The results showed a different disease progression depending on the specific knockout. While CerS4 KO mice were sensitive to DSS. AOM/DSS treatment was lethal for these mice, indicating an important role of CerS4 in other tissues. CerS4 Vil/Cre mice were protected from tumor formation. In contrast, CerS4 LCK/Cre mice experienced increased tumor formation and pan-inflammation. The mechanism behind this is due to the absence of cytotoxic T cells and the increase of regulatory T cells in the CerS4 LCK/Cre mice, demonstrating that CerS4 is critical for T cell function and development. To understand the role of CerS in humans, organoids were prepared from patients and the CerS profile in the different organoids was elucidated. This work provides, for the first time, insights into the CerS profile in human organoids and demonstrates a link between differentiation markers and stem cell markers with CerS. In addition, the role of CerS4 was investigated in vitro using three different colon cell lines-Caco-2 cells, HCT116 cells, and HCT15 cells. Hypoxia induced downregulation of CerS4 in all cell lines. Using the luciferase promoter assay, hypoxia-induced downregulation could already be detected at the promoter. Downregulation of CerS4 and CerS5 in Caco-2 cells and HCT116 cells resulted in different metabolic changes and mitochondrial dynamics after hypoxia. In conclusion, the results show that the role of CerS4 depends on the tissue cell type and stage of colorectal carcinoma, which complicates the consideration of CerS4 as a target in patients.
In the human brain, the incoming light to the retina is transformed into meaningful representations that allow us to interact with the world. In a similar vein, the RGB pixel values are transformed by a deep neural network (DNN) into meaningful representations relevant to solving a computer vision task it was trained for. Therefore, in my research, I aim to reveal insights into the visual representations in the human visual cortex and DNNs solving vision tasks.
In the previous decade, DNNs have emerged as the state-of-the-art models for predicting neural responses in the human and monkey visual cortex. Research has shown that training on a task related to a brain region’s function leads to better predictivity than a randomly initialized network. Based on this observation, we proposed that we can use DNNs trained on different computer vision tasks to identify functional mapping of the human visual cortex.
To validate our proposed idea, we first investigate a brain region occipital place area (OPA) using DNNs trained on scene parsing task and scene classification task. From the previous investigations about OPA’s functions, we knew that it encodes navigational affordances that require spatial information about the scene. Therefore, we hypothesized that OPA’s representation should be closer to a scene parsing model than a scene classification model as the scene parsing task explicitly requires spatial information about the scene. Our results showed that scene parsing models had representation closer to OPA than scene classification models thus validating our approach.
We then selected multiple DNNs performing a wide range of computer vision tasks ranging from low-level tasks such as edge detection, 3D tasks such as surface normals, and semantic tasks such as semantic segmentation. We compared the representations of these DNNs with all the regions in the visual cortex, thus revealing the functional representations of different regions of the visual cortex. Our results highly converged with previous investigations of these brain regions validating the feasibility of the proposed approach in finding functional representations of the human brain. Our results also provided new insights into underinvestigated brain regions that can serve as starting hypotheses and promote further investigation into those brain regions.
We applied the same approach to find representational insights about the DNNs. A DNN usually consists of multiple layers with each layer performing a computation leading to the final layer that performs prediction for a given task. Training on different tasks could lead to very different representations. Therefore, we first investigate at which stage does the representation in DNNs trained on different tasks starts to differ. We further investigate if the DNNs trained on similar tasks lead to similar representations and on dissimilar tasks lead to more dissimilar representations. We selected the same set of DNNs used in the previous work that were trained on the Taskonomy dataset on a diverse range of 2D, 3D and semantic tasks. Then, given a DNN trained on a particular task, we compared the representation of multiple layers to corresponding layers in other DNNs. From this analysis, we aimed to reveal where in the network architecture task-specific representation is prominent. We found that task specificity increases as we go deeper into the DNN architecture and similar tasks start to cluster in groups. We found that the grouping we found using representational similarity was highly correlated with grouping based on transfer learning thus creating an interesting application of the approach to model selection in transfer learning.
During previous works, several new measures were introduced to compare DNN representations. So, we identified the commonalities in different measures and unified different measures into a single framework referred to as duality diagram similarity. This work opens up new possibilities for similarity measures to understand DNN representations. While demonstrating a much higher correlation with transfer learning than previous state-of-the-art measures we extend it to understanding layer-wise representations of models trained on the Imagenet and Places dataset using different tasks and demonstrate its applicability to layer selection for transfer learning.
In all the previous works, we used the task-specific DNN representations to understand the representations in the human visual cortex and other DNNs. We were able to interpret our findings in terms of computer vision tasks such as edge detection, semantic segmentation, depth estimation, etc. however we were not able to map the representations to human interpretable concepts. Therefore in our most recent work, we developed a new method that associates individual artificial neurons with human interpretable concepts.
Overall, the works in this thesis revealed new insights into the representation of the visual cortex and DNNs...
In this thesis, we cover two intimately related objects in combinatorics, namely random constraint satisfaction problems and random matrices. First we solve a classic constraint satisfaction problem, 2-SAT using the graph structure and a message passing algorithm called Belief Propagation. We also explore another message passing algorithm called Warning Propagation and prove a useful result that can be employed to analyze various type of random graphs. In particular, we use this Warning Propagation to study a Bernoulli sparse parity matrix and reveal a unique phase transition regarding replica symmetry. Lastly, we use variational methods and a version of local limit theorem to prove a sufficient condition for a general random matrix to be of full rank.
Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway
(2022)
The human body is permanently exposed to its environment and thus to viruses and other pathogens, which require a flexible response and defense. Alongside to the innate immune system, the adaptive immune system provides highly specialized protection against these threats. The major histocompatibility complex class I (MHC I) antigen presentation system is a cornerstone of the adaptive immune system and a major constituent of cellular immunity. Pathogens such as viruses that invade a cell will leave traces in the form of proteins and peptides which are degraded and loaded onto MHC I molecules. MHC I peptide loading is performed by peptide loading complex (PLC) in the membrane of the endoplasmic reticulum as part of a multifaceted and comprehensive quality control machinery. Monitored by multiple layers of quality assurance, the MHC I molecules consequently display the immune status of the cell on its surface. In this context, the captured fragment of the virus serves as a call for help issued by the cell, alerting the adaptive immune system to the infection to mount an appropriate immune response.
The three-dimensional structure as well as the mechanistic details of parts of this complex machinery were characterized in the context of this dissertation. Among other tools, light-modulable nanotools were developed in this thesis, which permit external regulation of cellular processes in temporal and spatial resolution. Furthermore, methods and model systems for the biochemical characterization of cellular signaling cascades, proteins, as well as entire cell organelles were developed, which are likely to influence the field of cellular immunity and protein biochemistry in the future.
This cumulative work comprises a total of six publications whose scientific key advances will be briefly outlined in this abstract. In the introduction, the scientific background as well as the current state of research and methodological background knowledge are conveyed. The results section condenses the main aspects of the publications and links them to each other. Further details can be retrieved from the attached original publications.
In “Semisynthetic viral inhibitor for light control of the MHC I peptide loading complex, Winter, Domnick et al., Angew Chem Int Ed 2022” a photocleavable viral inhibitor of the peptide loading complex was produced by semi-synthesis. This nanotool was shown to be suitable for both purifying the PLC from human Raji cells as well as reactivating it in a light-controlled manner. Thus, this tool establishes the isolation of a fully intact and functional peptide loading complex for biochemical characterization. In addition, a novel flow cytometric analysis pipeline for microsomes was developed, allowing cellular vesicles to be characterized with single organelle resolution, similar to cells.
In “Molecular basis of MHC I quality control in the peptide loading complex, Domnick, Winter et al., Nat Commun 2022” the peptide loading complex was reconstituted into large nanodiscs, and a cryo-EM structural model of the editing module at 3.7 Å resolution was generated. By combining the structural model with in vitro glycan editing assays, an allosteric coupling between peptide-MHC I assembly and glycan processing was revealed, extending the known model of MHC I loading and dissociation from the PLC. These mechanisms provide a prototypical example for endoplasmic reticulum quality control.
In a related context, in “Structure of an MHC I–tapasin–ERp57 editing complex defines chaperone promiscuity, Müller, Winter et al., Nat Commun 2022” a recombinantly assembled editing module comprised of MHC I-tapasin-ERp57 was crystallized for X-ray structural biology. The resulting crystal structure at a resolution of 2.7 Å permitted the precise identification of characteristic features of the editing module and particularly of the peptide proofreading mechanism of tapasin. This study provided pivotal insights into the tapasin-mediated peptide editing of different MHC I allomorphs as well as similarities to TAPBPR-based MHC I peptide proofreading.
In “TAPBPR is necessary and sufficient for UGGT1-mediated quality control of MHC I, Sagert, Winter et al. (in preparation)” novel insights concerning the peptide proofreader TAPBPR and its close interplay with the folding sensor and glucosyltransferase UGGT1 were obtained. It was shown that TAPBPR is an integral part of the second level of endoplasmic quality control and is indispensable for effective MHC I coordination by UGGT1.
In “Light-guided intrabodies for on-demand in situ target recognition in human cells, Joest, Winter et al., Chem Sci 2021” intracellular nanobodies were equipped with a photocaged target recognition domain by genetic code expansion via amber suppression. These intrabodies, acting as high-affinity binding partners endowed with a fluorophore, could be used in a light-triggered approach to instantaneously visualize their target molecule...
Recent advances in artificial neural networks enabled the quick development of new learning algorithms, which, among other things, pave the way to novel robotic applications. Traditionally, robots are programmed by human experts so as to accomplish pre-defined tasks. Such robots must operate in a controlled environment to guarantee repeatability, are designed to solve one unique task and require costly hours of development. In developmental robotics, researchers try to artificially imitate the way living beings acquire their behavior by learning. Learning algorithms are key to conceive versatile and robust robots that can adapt to their environment and solve multiple tasks efficiently. In particular, Reinforcement Learning (RL) studies the acquisition of skills through teaching via rewards. In this thesis, we will introduce RL and present recent advances in RL applied to robotics. We will review Intrinsically Motivated (IM) learning, a special form of RL, and we will apply in particular the Active Efficient Coding (AEC) principle to the learning of active vision. We also propose an overview of Hierarchical Reinforcement Learning (HRL), an other special form of RL, and apply its principle to a robotic manipulation task.
This work investigated the influence of the CRISPR/Cas9 mediated knockout of 5-lipoxygenase (5-LO) on different adherent tumour cell lines derived from solid tumours. For this, the 5-LO expressing tumour cell lines HCT-116, HT-29, and U-2 OS were transiently transfected using a plasmid carrying the CRISPR/Cas9 complex sequence to the ALOX5 gene. Subsequently, cells were selected using Puromycin and analysed via Western blotting and DNA Sanger sequencing. Cells that were transfected with a control plasmid missing the guide RNA sequence, were used as a control for all experiments.
Differential gene expression analysis, performed after next-generation RNA sequencing, revealed that the expression of various genes was altered after the knockout of 5-LO. In HCT-116 cells, 28 genes were expressed differentially in all 5-LO knockout single-cell clones, while in HT-29 cells the expression of 18 genes and in U-2 OS cells of 234 genes was influenced by the knockout of 5-LO. These findings were validated by real-time qPCR. A lot of the genes that were influenced by the 5-LO knockout are known to be connected to epithelial-mesenchymal-transition (EMT), a process necessary for tumour metastasis. The results from RNA sequencing were the starting point for further investigations. In the following, different aspects of the tumour cell lines were examined. In HT-29, as
well as in U-2 OS cells, it was shown that knockout of the 5-LO resulted in impaired cell proliferation. Also, the formation of three-dimensional tumour spheroids was altered. In HT-29 cells, the knockout of 5-LO increased the number of cells in spheroids. In contrast, in U-2 OS cells, the number of cells per spheroid was decreased, even though the diameter of the spheroids was increased, due to more loosely packed spheroids. The difference between 5-LO positive and negative U-2 OS cells became even more obvious after embedding the spheroids in an artificial extracellular matrix. In that scenario, cells lacking the 5-LO formed smaller spheroids that did not have the same ability to grow into the extracellular matrix as 5-LO positive cells did. Also, directed cell migration was strongly influenced by the knockout of 5-LO. In both, HCT-116 and U-2 OS cells, directed cell migration towards a serum gradient was increased in 5-LO knockout single-cell clones. Pharmacological inhibition of the enzyme was used to investigate, whether canonical or non-canonical functions were responsible for the previously mentioned effects.
Therefore, vector control cells were treated with the 5-LO inhibitors Zileuton and CJ-13610 in different concentrations. Interestingly, only some of the effects mediated by the complete knockout of 5-LO could be reproduced by inhibiting the enzyme, leading to the suggestion, that canonical, as well as non-canonical functions of 5-LO, play a role in these tumour cells.
To conclude, it was shown in this study, that 5-LO affects various cellular functions when expressed in adherent tumour cell lines. These cell line-dependent effects result in altered gene expression, enhanced proliferation, and spheroid formation, as well as impaired cell motility, and can be mediated by enzymatic activity as well as other non-canonical functions.
Oxidative stress is thought to be a driver for several diseases. However, many data to support this concept were obtained by the addition of extracellular H2O2 to cells. This does not reflect the dynamics of intracellular redox modifications. Cells actively control their redox-state, and increased formation of ROS is a response to cellular stress situations such as chronic inflammation.
In this study, it was shown that different types of ROS lead to different metabolic and transcriptomic responses of HUVECs. While 300 μM extracellular H2O2 led to substantial metabolic and transcriptomic changes, the effects of DAO-derived H2O2 and menadione were low to moderate, indicating that the source and the concentration of ROS are important in eliciting changes in metabolism and gene expression.
Specifically, it was identified that acute increases in ROS transiently inactivate the enzyme ω-amidase/NIT2 of the glutaminase II pathway, which supplies cells with anaplerotic α-ketoglutarate. The pathway has not been studied systematically because, as noted above, the major intermediate, KGM, is not commercially available. In the present study, an internal standard for targeted detection of KGM in cells and blood plasma/serum was used. Deletion of NIT2 by CRISPR/Cas9 significantly reduced α-ketoglutarate levels in HUVECs and elevated KGM levels. It appears that in cell culture conditions, hydrolysis of KGM to α-ketoglutarate is very efficient. Knockout of the glutamine transaminases significantly reduced methionine, suggesting that the glutaminase II pathway is an important source of amino acid replenishment.
Similar to genetic silencing of GLS1 [91,92], HUVECs lacking NIT2 showed reduced proliferation and angiogenic sprouting. Furthermore, our results indicate that, at least in HUVECs, the enzyme also locates in the mitochondria where it interacts with key enzymes of glutamine/glutamate/α-ketoglutarate metabolism.
The data of the present work indicate that the glutaminase II pathway is an underappreciated, redox-sensitive pathway for glutamine utilization in HUVECs. Genetic deletion of NIT2 has considerable physiological effects highlighting the importance of glutamine for ECs.
Navigating a complex environment is assumed to require stable cortical representations of environmental stimuli. Previous experimental studies, however, show substantial ongoing remodeling at the level of synaptic connections, even under behaviorally and environmentally stable conditions. It remains unclear, how these changes affect sensory representations on the level of neuronal populations during basal conditions and how learning influences these dynamics.
Our approach is a joint effort between the analysis of experimental data and theory. We analyze chronic neuronal population activity data – acquired by out collaborators in Mainz – to describe population activity dynamics during basal dynamics and during learning (fear conditioning). The data analysis is complemented by the analysis of a circuit model investigating the link between a neural network’s activity and changes in its underlying structure.
Using chronic two-photon imaging data recorded in awake mouse auditory cortex, we reproduce previous findings that responses of neuronal populations to short complex sounds typically cluster into a near discrete set of possible responses. This means that different stimuli evoke basically the same response and are thus grouped together into one of a small set of possible response modes. The near discrete set of response modes can be utilized as a sensitive and robust means to detect and track changes in population activity over time. Doing so we find that sound representations are subject to a significant ongoing remodeling across the time span of days under basal conditions. Auditory cued fear conditioning introduces a bias into these ongoing dynamics, resulting in a differential generalization both on the level of neuronal populations and on the behavioral level. This means that sounds that are perceived similar to the conditioned stimulus (CS+) show an increased co-mapping to the same response mode the CS+ is mapped to. This differential generalization is also observed in animal behavior, where sounds similar to the CS+ result in the same freezing behavior as the CS+, whereas dissimilar sounds do not. These observations could provide a potential mechanism of stimulus generalization, which is one of the most common phenomena associated with post-traumatic stress disorder, on the level of neuronal populations.
To investigate how the aforementioned changes in neuronal population activity are linked to changes in the underlying synaptic connectivity, we devised a circuit model of excitatory and inhibitory neurons. We studied this firing rate model to investigate the effect of gradual changes in the network’s connectivity on its activity. Apart from an input dominated uni-stable regime (one response per stimulus independent of the network) and a network dominated uni-stable regime (one response per network independent of the stimulus), we also find a multi-stable regime for strong recurrent connectivity and a high ratio of inhibition to excitation. In this regime the model reproduces properties of neural population activity in mouse auditory cortex, including sparse activity, a broad distribution of firing rates, and clustering of stimuli into a near discrete set of response modes. This clustering in the multi-stable regime means that, not only can identical stimuli evoke different responses, depending on the network’s initial condition, but different stimuli can also evoke the same response.
Applying gradual drift to the network connectivity we find periods of stable responses, interrupted by abrupt transitions altering the stimulus response mapping. We study the mechanism underlying these transitions by analyzing changes in the fixed points of this network model, employing a method to numerically find all the fixed points of the system. We find that such abrupt transitions typically cannot be explained by the mere displacement of existing fixed points, but involve qualitative changes in the fixed point structure in the vicinity of the response trajectory. We conclude that gradual synaptic drift can lead to abrupt transitions in stimulus responses and that qualitative changes in the network’s fixed point topology underlie such transitions.
In summary we find that cortical networks display ongoing representational drift under basal conditions that is biased towards a differential generalization during fear conditioning. A circuit model is able to reproduce key characteristics of auditory cortex, including a clustering of stimulus responses into a near discrete set of response modes. Implementing synaptic drift into this model leads to periods of stable responses interrupted by abrupt transitions towards new responses.
Die vorgelegte Dissertation behandelt den Einfluss homöostatischer Adaption auf die Informationsverarbeitung und Lenrprozesse in neuronalen Systemen. Der Begriff Homöostase bezeichnet die Fähigkeit eines dynamischen Systems, bestimmte interne Variablen durch Regelmechanismen in einem dynamischen Gleichgewicht zu halten. Ein klassisches Beispiel neuronaler Homöostase ist die dynamische Skalierung synaptischer Gewichte, wodurch die Aktivität bzw. Feuerrate einzelner Neuronen im zeitlichen Mittel konstant bleibt. Bei den von uns betrachteten Modellen handelt es sich um eine duale Form der neuronalen Homöostase. Das bedeutet, dass für jedes Neuron zwei interne Parameter an eine intrinsische Variable wie die bereits erwähnte mittlere Aktivität oder das Membranpotential gekoppelt werden. Eine Besonderheit dieser dualen Adaption ist die Tatsache, dass dadurch nicht nur das zeitliche Mittel einer dynamischen Variable, sondern auch die zeitliche Varianz, also die stärke der Fluktuation um den Mittelwert, kontrolliert werden kann. In dieser Arbeit werden zwei neuronale Systeme betrachtet, in der dieser Aspekt zum Tragen kommt.
Das erste behandelte System ist ein sogennantes Echo State Netzwerk, welches unter die Kategorie der rekurrenten Netzwerke fällt. Rekurrente neuronale Netzwerke haben im Allgemeinen die Eigenschaft, dass eine Population von Neuronen synaptische Verbindungen besitzt, die auf die Population selbst projizieren, also rückkoppeln. Rekurrente Netzwerke können somit als autonome (falls keinerlei zusätzliche externe synaptische Verbindungen existieren) oder nicht-autonome dynamische Systeme betrachtet werden, die durch die genannte Rückkopplung komplexe dynamische Eigenschaften besitzen. Abhängig von der Struktur der rekurrenten synaptischen Verbindungen kann beispielsweise Information aus externem Input über einen längeren Zeitraum gespeichert werden. Ebenso können dynamische Fixpunkte oder auch periodische bzw. chaotische Aktivitätsmuster entstehen. Diese dynamische Vielseitigkeit findet sich auch in den im Gehirn omnipräsenten rekurrenten Netzwerken und dient hier z.B. der Verarbeitung sensorischer Information oder der Ausführung von motorischen Bewegungsmustern. Das von uns betrachtete Echo State Netzwerk zeichnet sich dadurch aus, dass rekurrente synaptische Verbindungen zufällig generiert werden und keiner synaptischen Plastizität unterliegen. Verändert werden im Zuge eines Lernprozesses nur Verbindungen, die von diesem sogenannten dynamischen Reservoir auf Output-Neuronen projizieren. Trotz der Tatsache, dass dies den Lernvorgang stark vereinfacht, ist die Fähigkeit des Reservoirs zur Verarbeitung zeitabhängiger Inputs stark von der statistischen Verteilung abhängig, die für die Generierung der rekurrenten Verbindungen verwendet wird. Insbesondere die Varianz bzw. die Skalierung der Gewichte ist hierbei von großer Bedeutung. Ein Maß für diese Skalierung ist der Spektralradius der rekurrenten Gewichtsmatrix.
In vorangegangenen theoretischen Arbeiten wurde gezeigt, dass für das betrachtete System ein Spektralradius nahe unterhalb des kritischen Wertes von 1 zu einer guten Performance führt. Oberhalb dieses Wertes kommt es im autonomen Fall zu chaotischem dynamischen Verhalten, welches sich negativ auf die Informationsverarbeitung auswirkt. Der von uns eingeführte und als Flow Control bezeichnete duale Adaptionsmechanismus zielt nun darauf ab, über eine Skalierung der synaptischen Gewichte den Spektralradius auf den gewünschten Zielwert zu regulieren. Essentiell ist hierbei, dass die verwendete Adaptionsdynamik im Sinne der biologischen Plausibilität nur auf lokale Größen zurückgreift. Dies geschieht im Falle von Flow Control über eine Regulation der im Membranpotential der Zelle auftretenden Fluktuationen. Bei der Evaluierung der Effektivität von Flow Control zeigte sich, dass der Spektralradius sehr präzise kontrolliert werden kann, falls die Aktivitäten der Neuronen in der rekurrenten Population nur schwach korreliert sind. Korrelationen können beispielsweise durch einen zwischen den Neuronen stark synchronisierten externen Input induziert werden, der sich dementsprechend negativ auf die Präzision des Adaptionsmechanismus auswirkt.
Beim Testen des Netzwerks in einem Lernszenario wirkte sich dieser Effekt aber nicht negativ auf die Performance aus: Die optimale Performance wurde unabhängig von der stärke des korrelierten Inputs für einen Spektralradius erreicht, der leicht unter dem kritischen Wert von 1 lag. Dies führt uns zu der Schlussfolgerung, dass Flow Control unabhängig von der Stärke der externen Stimulation in der Lage ist, rekurrente Netze in einen für die Informationsverarbeitung optimalen Arbeitsbereich einzuregeln.
Bei dem zweiten betrachteten Modell handelt es sich um ein Neuronenmodell mit zwei Kompartimenten, welche der spezifischen Anatomie von Pyramidenneuronen im Kortex nachempfunden ist. Während ein basales Kompartiment synaptischen Input zusammenfasst, der in Dendriten nahe des Zellkerns auftritt, repräsentiert das zweite apikale Kompartiment die im Kortex anzutreffende komplexe dendritische Baumstruktur. In früheren Experimenten konnte gezeigt werden, dass eine zeitlich korrelierte Stimulation sowohl im basalen als auch apikalen Kompartiment eine deutlich höhere neuronale Aktivität hervorrufen kann als durch Stimulation nur einer der beiden Kompartimente möglich ist. In unserem Modell können wir zeigen, dass dieser Effekt der Koinzidenz-Detektion es erlaubt, den Input im apikalen Kompartiment als Lernsignal für synaptische Plastizität im basalen Kompartiment zu nutzen. Duale Homöostase kommt auch hier zum Tragen, da diese in beiden Kompartimenten sicherstellt, dass sich der synaptische Input hinsichtlich des zeitlichen Mittels und der Varianz in einem für den Lernprozess benötigten Bereich befindet. Anhand eines Lernszenarios, das aus einer linearen binären Klassifikation besteht, können wir zeigen, dass sich das beschriebene Framework für biologisch plausibles überwachtes Lernen eignet.
Die beiden betrachteten Modelle zeigen beispielhaft die Relevanz dualer Homöostase im Hinblick auf zwei Aspekte. Das ist zum einen die Regulation rekurrenter neuronaler Netze in einen dynamischen Zustand, der für Informationsverarbeitung optimal ist. Der Effekt der Adaption zeigt sich hier also im Verhalten des Netzwerks als Ganzes. Zum anderen kann duale Homöostase, wie im zweiten Modell gezeigt, auch für Plastizitäts- und Lernprozesse auf der Ebene einzelner Neuronen von Bedeutung sein. Während neuronale Homöostase im klassischen Sinn darauf beschränkt ist, Teile des Systems möglichst präzise auf einen gewünschten Mittelwert zu regulieren, konnten wir Anhand der diskutierten Modelle also darlegen, dass eine Kontrolle des Ausmaßes von Fluktuationen ebenfalls Einfluss auf die Funktionalität neuronaler Systeme haben kann.
Extreme convective precipitation events are among the most severe hazards in central Europe and are expected to intensify under global warming. However, the degree of intensification and the underlying processes are still uncertain. In this thesis, recent advances in continuous, radar-based precipitation monitoring and convection-permitting climate modeling are used to investigate Lagrangian properties of convective rain cells such as precipitation intensity, cell area, and precipitation sum and their relationship to large-scale, environmental conditions.
Firstly, convective precipitation objects are tracked in a gauge-adjusted radar-data set and the properties of these cells are related to large-scale environmental variables to investigate the observed super-Clausius-Clapeyron (CC) scaling of convective extreme precipitation. The Lagrangian precipitation sum of convective cells increases with dew point temperature at rates well above the CC-rate with increasing rates for higher dew point temperatures. These varying, high rates are caused by a covarying increase of CAPE with dew point temperature as well as the effect of high vertical wind shear causing an increase in cell area and thus precipitation sum. At the same time, cells move faster at high vertical wind shear so that Eulerian scaling rates are lower than Lagrangian but still above the CC-rate. The results show that wind shear and static instability need to be taken into account when transferring precipitation scaling under current climate conditions to future conditions. Secondly, the representation of convective cell properties in the convection-permitting climate model COSMO-CLM is evaluated. The model can simulate the observed frequency distributions of cell properties such as lifetime, area, mean and maximum intensity, and precipitation sum. The increase of area and intensity with lifetime is also well captured despite an underestimation of the intensity of the most severe cells. Furthermore, the model can represent the temperature scaling of intensity, area, and precipitation sum but fails to simulate the observed increase of lifetime. Thus, the model is suitable to study climatologies of convective storms in Germany. Thirdly, two COSMO-CLM projections at the end of the century under emission scenario RCP8.5 were investigated. While the number of convective cells and their lifetime remain approximately constant compared to present conditions, intensity and area increase strongly. The relative increase of intensity and area is largest for the highest percentiles meaning that extreme events intensify the most. The characteristic afternoon maximum of convective precipitation is damped, and shifted to later times of day which leads to an increase of nighttime precipitation in the future. Scaling rates of cell properties with dew point temperature are nearly identical in present and future in the simulation driven by the EC-Earth model which means that the upper limit of cell properties like intensity, area, and precipitation sum could be predicted from near-surface dew point temperature. However, this result could not be reproduced by the simulation driven by MIROC5 and needs further investigation.
We discuss aspects of the phase structure of a three-dimensional effective lattice theory of Polyakov loops derived from QCD by strong coupling and hopping parameter expansions. The theory is valid for the thermodynamics of heavy quarks where it shows all qualitative features of nuclear physics emerging from QCD. In particular, the SU(3) pure gauge effective theory also exhibits a first-order thermal deconfinement transition due to spontaneous breaking of its global Z₃ center symmetry. The presence of heavy dynamical quarks breaks this symmetry explicitly and consequently, the transition weakens with decreasing quark mass until it disappears at a critical endpoint. At non-zero baryon density, the effective theory can be evaluated either analytically by the so-called high-temperature expansion which does not suffer from the sign problem, or numerically by standard Monte-Carlo methods due to its mild sign problem. The first part of this work devotes to a systematic derivation of the effective theory up to the 6th order in the hopping parameter κ. This method combined with the SU(3) link update algorithm provides a way to simulate the O(κ⁶) effective theory. The second part involves a study of the deconfinement transition of the pure gauge effective theory, with and without static quarks, at all chemical potentials with help of the high-temperature expansion. Our estimate of the deconfinement transition and its critical endpoint as a function of quark mass and all chemical potentials agrees well with recent Monte-Carlo simulations. In the third part, we investigate the N ſ ∈ {1,2} effective theory with zero chemical potential up to O(κ⁴). We determine the location of the critical hopping parameter at which the first-order deconfinement phase transition terminates and changes to a crossover. Our results for the critical endpoint of the O(κ²) effective theory are in excellent agreement with the determinations from simulations of four-dimensional QCD with a hopping expanded determinant by the WHOT-QCD collaboration. For the O(κ⁴) effective theory, our estimate suggests that the critical quark mass increases as the order of κ-contributions increases. We also compare with full lattice QCD with N ſ = 2 degenerate standard Wilson fermions and thus obtain a measure for the validity of both the strong coupling and the hopping expansion in this regime.
Although everyone is familiar with using algorithms on a daily basis, formulating, understanding and analysing them rigorously has been (and will remain) a challenging task for decades. Therefore, one way of making steps towards their understanding is the formulation of models that are portraying reality, but also remain easy to analyse. In this thesis we take a step towards this way by analyzing one particular problem, the so-called group testing problem. R. Dorfman introduced the problem in 1943. We assume a large population and in this population we find a infected group of individuals. Instead of testing everybody individually, we can test group (for instance by mixing blood samples). In this thesis we look for the minimum number of tests needed such that we can say something meaningful about the infection status. Furthermore we assume various versions of this problem to analyze at what point and why this problem is hard, easy or impossible to solve.
In this thesis, the emission of protons as well as the production of Λ hyperons, Κ0S mesons and 3ΛH hypernuclei are analyzed multi-differentially as a function of transverse momentum, rapidity and centrality. Therefore, the 3.03 billion 30 % most central Ag(1.58A GeV)+Ag events recorded by HADES are used. Furthermore, the lifetimes of Λ hyperons, Κ0S mesons and 3ΛH hypernuclei are measured. The obtained 3ΛH lifetime of (253 ± 24 ± 42) ps is compatible with the lifetime of free Λ hyperons, as predicted by theoretic calculations due to its low binding energy. Finally, also the double strange Ξ– hyperons are reconstructed. Unfortunately, the fully optimized signals lie below the confidence threshold of 5σ, which is why both an production rate and an upper production limit are estimated using averaged acceptance and efficiency corrections. Never before, 3ΛH or Ξ– were successfully reconstructed and analyzed in heavy-ion collisions at such low energies. The obtained results are compared to previous measurements and put in context with world data form different energies and collision systems.
Non-ribosomal peptide synthetases (NRPSs) are modular biosynthetic megaenzymes producing many important natural products and refer to a specific set of peptides in bacteria’s and fungi’s secondary metabolism. With the actual purpose of providing advantages within their respective ecological niche, the bioactivity of the structurally highly diverse products ranges from, e.g., antibiotic (e.g., vancomycin) to immunosuppressive (e.g., cyclosporin A) to cytostatic (e.g., echinomycin or thiocoralin) activity.
An NRPS module consists of at least three core domains that are essential for the incorporation of specific substrates with the 'multiple carrier thiotemplate mechanism' into a growing peptide chain: an adenylation (A) domain selects and activates a cognate amino acid; a thiolation (T) domain shuffles the activated amino acid and the growing peptide chain, which are attached at its post-translationally 4ʹ-phosphopantetheine (4'-PPant) group, between the active sites; a condensation (C) domain links the upstream and downstream substrates. NRPS synthesis is finished with the transfer of the assembled peptide to the C-terminal chain-terminating domain. Accordingly, the intermediate is either released by hydrolysis as a linear peptide chain or by an intramolecular nucleophilic attack as a cyclic peptide.
The NRPS’s modular character seems to imply straightforward engineering to take advantage of their features but appears to be more challenging. Since the pioneering NRPS engineering approaches focused on the reprogramming and replacement of A domains, several working groups developed advanced methods to perform a complete replacement of subdomains or single or multiple catalytic domains.
The first part of this work focusses parts of the publication with the title 'De novo design and engineering of non-ribosomal peptide synthetases', which follows up assembly line engineering with the development of a new guideline. Thereby, the pseudodimeric V-shaped structure of the C domain is exploited to separate the N-terminal (CDSub) and C-terminal (CASub) subdomains alongside a four-AA-long linker. This results in the creation of self-contained, catalytically active CASub-A-T-CDSub (XUC) building blocks. As an advantage over the previous XU concept, the characteristics (substrate- and stereoselectivity) assigned to the C domain subunits are likewise exchanged, and thus, no longer represent a barrier. Furthermore, with the XUC concept, no important interdomain interfaces are disrupted during the catalytic cycle of NRPS, allow to expect much higher production titers. Moreover, the XUC concept shows a more flexible application within its genus origin of building blocks to create peptide libraries. Additionally, with this concept only 80 different XUC building blocks are needed to cover the entire proteinogenic amino acid spectrum.
The second part of this work addresses the influence of the C domain on activity and specificity of A domains. In a comprehensive analysis, a clear influence of different C domains on the in vitro activation rate and the in vivo substrate spectrum could be observed. Further in situ and in silico characterizations indicate that these influences are neither the result of the respective A domains promiscuity nor the C domain’s proofreading, but due to an 'extended gatekeeping' function of the C domain. This novel term of an 'extended gatekeeping' function describes the very nature of interfaces that C domains can form with an A domain of interest. Therefore, the C-A interface is assumed to have a more significant contribution to a selectivity filter function.
The third part of this work combines the NRPS engineering with phylogenetic/evolutionary perspectives. At first, the C-A interface could be precisely defined and further identified to encode equivalent information corresponding to the complete C-A didomain. Moreover, the comparison of NRPSs topology reveals hints for a co-evolutionary relatedness of the C-A didomain and could be shown to reassemble even after separation. In this regard, based on a designed CAopt.py algorithm, the reassembling-compatibility of hybrid interfaces could be determined by scoring of the co-expressed NRPS hybrids. This algorithm also enables the randomization of the interface sequences, thus, leading to the identification of more functional interface variant, which cause significantly higher peptide production and could even be applied to other native and hybrid interfaces.
Mitochondria are important for cellular health and their dysfunction is linked to a variety of diseases, especially neurodegeneration. Thus, the renewal and degradation of dysfunctional mitochondria is crucial for the well-being of organisms. The selective digestion of damaged mitochondria via the lysosome (mitophagy), is the main pathway to do so.
In my dissertational work, I investigated the connection between protein misfolding, protein import into mitochondria and the degradation of mitochondria via mitophagy. Here, I present a new model for the initiation of mitophagy without collapse of the membrane potential. This model provides the link between protein import into mitochondria, stress signal transduction to the cytosol and the mitochondrial stress sensor PINK1. To comprehensively examine how mitophagy can be triggered, I performed a genome-wide CRISPR knockout screen utilizing the mitophagy reporter mitochondrial mKEIMA. Thereby, I observed numerous novel gene deletions that induce mitophagy. Prominently, I identified an accumulation of gene deletions of the protein import and of protein quality control factors. I validated several of those and examined HSPA9 (mitochondrial HSP70) and LONP1 (a mitochondrial matrix AAA protease) in more detail, regarding their effect on mitophagy and protein import. For this, I used an established fluorescence-based, mitochondrial-targeted EGFP, as well as a newly-developed pulsed-SILAC mass spectrometry approach (mePRODmt). Depletions of both genes resulted in reduced protein import and PINK1-dependent mitophagy. Strikingly, I did not observe any loss of mitochondrial membrane potential, which was hitherto believed to be essential for activation of PINK1-mediated mitophagy. Literature shows that certain mitochondrial stressors can also induce mitophagy without mitochondrial membrane depolarization, which I confirmed with my assays. Next, I characterized the impact of LONP1 and HSPA9 depletion, which are involved in proteostasis maintenance, and the mtHSP90 inhibitor GTPP on mitochondrial protein folding in more detail. GTPP treatment and LONP1 depletion both resulted in the accumulation of an insoluble protein fraction, as judged by proteomic analysis. This insoluble protein fraction enriched several components of the presequence translocase-associated motor PAM, including TIMM44. TIMM44 acts as a link between the translocon, the import pore of the inner mitochondrial membrane (TIM) complex and the PAM complex. Thus, I hypothesized that TIMM44 dissociates from the TIM complex upon protein folding stress, when it becomes part of the insoluble protein fraction. To validate this model, I measured the TIMM44 interactome upon proteostasis disturbance using proximity labeling. Indeed, interaction of TIMM44 with the import pore was almost completely abolished, explaining the loss of matrix-targeted import upon protein folding stress. From these findings, I reasoned that an import reduction mediated by the PAM complex would likely also inhibit the degradation of PINK1. Consistent with this hypothesis, I observed that mitophagy induced by HSPA9 or LONP1 deletion was prevented when PINK1 was genetically deleted. In comparison, non-processed PINK1 was stabilized on mitochondria in wild type cells when mitochondrial protein import was impaired. On this basis, I drew the conclusion that the loss of mitochondrial import was the stress signal, which leads to the stabilization of PINK1, as it could not be processed anymore via the inner mitochondrial membrane protease PARL. PINK1 auto-activates itself upon accumulation and signals to the cytosol that this mitochondrion is damaged. Mitophagy is subsequently initiated by the ubiquitin kinase activity of PINK1. As a result, the autophagy apparatus gets activated, damaged mitochondria are engulfed by a double membrane and removed via lysosomal digestion. This proposed model is, to the best of my knowledge, the first to provide an explanation for protein folding stress-induced and protein import inhibition-triggered mitophagy without mitochondrial depolarization. The model thus extends the PINK1/PARKIN-dependent mitophagy pathway to milder stresses and clears some of the open questions in the field. Furthermore, this work is also important, because protein misfolding stress and dysfunctional mitochondria are two hallmarks of neurodegeneration. In particular, mitochondrial protein import inhibition during Parkinson’s and Huntington disease might be driver of mitochondrial dysfunction. Hence, I hope and anticipate that the newly developed protein import method, mePRODmt, and the proposed model will be beneficial to further characterize underlying processes and to establish which factors prevent or drive these disorders on molecular level.
The intensive use of the North Sea area through offshore activities, sand mining, and the spreading of dredged material is leading to increasing pollution of the ecosystem by chemicals such as hydrophobic organic contaminants (HOCs). Due to their toxicological properties and their ability to accumulate in the environment, HOCs are of particular concern. The contaminants partition between aqueous (pore water, overlying water) and solid phases (sediment, suspended particulate matter, and biota) within these systems. The accumulated contaminants in the sediment are of major concern for benthic organisms, who are in close contact with sediment and interstitial water. It is thus particularly important to better understand how contaminants interact with biota, as these animals may contribute to trophic transfer through the food web. Furthermore, sediments are a crucial factor for the water quality of aquatic systems. They not only represent a sink for contaminants but also determine environmental fate, bioavailability, and toxicity. The Marine Strategy Framework Directive (MSFD) was introduced to protect our marine environment across Europe and includes the assessment of pollutant concentrations in the total sediment, which, however, rarely reflects the actual exposure situation. The consideration of the pollutant concentrations in the pore water is not implemented, although this is needed for the evaluation of bioavailability and risk assessment. For this reason, special attention is given to further development, implementation, and validation of pollutant monitoring methods that can determine the bioavailable fraction in sediment pore water. For risk assessment purposes, it is furthermore important to use biological indicators in addition to classical analytics to determine the effect of pollutants on organisms. The main objective of this thesis was to gain insight into the pollution load and the potential risk of hydrophobic organic chemicals (HOCs) in the sediment of the North Sea and to evaluate these results with regard to possible risks for benthic organisms and the ecosystem. The following five aims are covered within these studies to gain a holistic assessment of sediment contamination:
1. Assessment of the pore water concentrations of PAHs and PCBs
2. Determination of the bioturbation potential by macrofauna analysis
3. Application of the SPME method on biological tissue
4. Assessment of recreated environmental mixtures in passive dosing bioassays
5. Development of SPME method for DDT in sediments
The thesis is comprised of three main studies supported by three additional studies ...
The increasing demand of the high value ω-3 fatty acids due to its beneficial role for human health, explains the huge need for alternative production ways of ω-3 fatty acids. The oleaginous alga Phaeodactylum tricornutum is a prominent candidate and has been investigated as biofactory for ω-3 fatty acids, e.g. the synthesis of eicosapentaenoic acid (EPA). In general, the growth and the lipid content of diatoms can be enhanced by genetic engineering or are influenced by environmental factors, e.g. nutrients, light or temperature.
In this study, the potential of P. tricornutum as biofactory was improved by heterologously expressing the hexose uptake protein 1 (HUP1) from the Chlorophyte Chlorella kessleri.
An in situ localization study revealed that only the full length HUP1 protein fused to eGFP was correctly targeted to the plasma membrane, whereas the N-terminal sequence of the protein is only sufficient to enter the ER. Protein and gene expression data displayed that the gene-promoter combination was relevant for the expression level of HUP1, while only cells expressing the protein under the light-inducible fcpA promoter showed a significant expression. In these mutants an efficient glucose uptake was detectable under mixotrophic growth condition, low light intensities and low glucose concentrations leading to an increased cell dry weight.
In a second approach, the growth and lipid content of wildtype cells were analyzed in a small 1l photobioreactor. Here, a commercial F/2 medium and a common culture medium, ASP and modified versions were compared. There was neither a significant impact on the growth and lipid content in P. tricornutum cells due to the supplemention of trace elements nor due to elevated salt concentrations in the media. In a modified version of ASP medium, with adapted nitrate and phosphate concentration a constantly high biomass productivity was achieved, yielding the highest value of 82 mg l-1 d-1 during the first three days. This was achieved even though light intensity was reduced by 40%. The differences in biomass productivity as well as the lipid content and the lipid composition underlined the importance of the choice of culture medium and the harvest time for enhanced growth and EPA yields in P. tricornutum.
The climate system is one of the classical examples of a complex dynamical system consisting of interacting sub-systems through mass, momentum, and energy exchange across various spatial and temporal scales. This thesis aims to detect and quantify sub-component interactions from an information exchange (IE) perspective. For this purpose, IE estimators derived from information theory are explored and applied to the available climate data obtained from observations, reanalysis, global and regional climate models. Specifically, this thesis investigates the usefulness of information theory methods for process-oriented climate model evaluation.
Firstly, methods derived from the concepts of information theory such as transfer entropy and information flow along with their linear and non-linear estimation techniques are initially tested and applied to idealized two-dimensional dynamical systems. The results revealed an expected direction and magnitude of IE providing insights into underlying dynamics. However, as expected the linear estimators are robust for linear systems but fail for non-linear systems. Though the non-linear estimators (kernel and kraskov) showed expected results for all the idealized systems, their free tuning parameters are to be tested for consistent results. Moreover, these methods are sensitive to the available time series length.
A real world example case study involving the dynamics between the Indian and Pacific oceans revealed a physically consistent bi-directional IE. However, unexpected IE was detected in the example of North Atlantic and European air temperatures indicating hidden drivers. Though IE provides insights into system dynamics, the availability of time series length and the system at hand must be carefully taken into account before inferring any possible interpretations of the results.
Quantifying the IE from El-Ni\~{n}o southern oscillation (ENSO) and Indian Ocean Dipole (IOD) to the Indian Summer Monsoon Rainfall (ISMR) with the observational and reanalysis data sets revealed that both ENSO and IOD are synergistic predictors for the inter-annual variability of the ISMR over central India i.e., the monsoon core region. Though the investigated three Global Climate Models (GCM) could not reveal the underlying IE dynamics of ENSO, IOD, and ISMR, a Regional Climate Model (RCM) simulation downscaling one of the GCMs with realistic large scale signals across the lateral boundaries showed good agreement with the observations.
Evaluating a coupled regional climate modeling system driven by two different global data sets with IE estimators revealed significant differences between the process chains linking the north-west Mediterranean sea surface temperatures, evaporation, wind speed, and the Vb-cyclone induced precipitation over Danube, Odra, and Elbe catchments in the historical period (1951-2005). Detailed investigation revealed that the north-west Mediterranean Sea in the coupled regional simulation driven by ERA-20C reanalysis corresponded to the Vb-cyclone precipitation over the three catchments while no such correspondence is noted in the EC-EARTH driven simulation. This discrepancy is attributed to the inheritance of the simulation biases from GCM into the RCM. In the future period (1965-2099), no significant changes in the processes are noted from the simulation.
Overall, this thesis used IE estimators in investigating the underlying dynamics of climate system and climate models. The estimators proved useful in providing insights into climate system dynamics assisting in a process based climate model evaluation.
Individualization can be defined as the adaptation of instructional parameters to relevant characteristics of a specific learner. This definition raises several questions, however: Which characteristics are actually relevant? Which parameters of instruction need to be adjusted, and in which way, to positively interact with those characteristics? In a classroom context, additional questions arise: how can information about the relevant learner characteristics be delivered to the teacher? How can individualized instruction be delivered to each learner in a context that has originally been designed for whole-class instruction? By focusing on the measurement and modelling of learner characteristics and instructional adaptations, this dissertation aims to provide an insight into each of these issues.
This dissertation is divided into two parts. The first part is concerned with the theoretical (Paper 1) and statistical (Paper 2) modeling of learner characteristics in the context of individualized instruction. The second part is concerned with the measurement (Paper 3) and implementation (Paper 4) of individualized instruction in the classroom context.
Paper 1 summarizes existing research on individualization from different research traditions. From this summary I derive the need for a dynamic conceptualization of learner characteristics (acknowledging that learners change during and in interaction with the learning process) and synthesize a dynamic framework that details the opportunities for individualization on three different timescales. Paper 2 reports results from an exploratory study that investigated the potential benefits of utilizing person-centered analysis for the assessment of multivariate learner prerequisites and their interaction with instruction. We found that latent profiles over several reading related abilities could explain differential effectiveness of self-reported teaching foci in German third grade reading lessons. These findings indicate not just a need for stronger individualization of teaching but also an advantage of multivariate conceptualizations of learner characteristics. Additionally, they show the utility of person-centered approaches for the investigation of such multivariate learner characteristics and their interaction with instruction.
In the second part, I investigate possible approaches to the implementation and measurement of individualization in a classroom context. Paper 3 investigates whether teacher-, student- and observer perspectives converge when rating the amount of individualization present in regular classroom instruction. We found considerable agreement between the perspectives, indicating a common understanding of the construct at the classroom level as well as providing some evidence for the validity of the used measurement instruments. Paper 4 replicates findings concerning the effectiveness of formative assessment procedures for fostering reading education, supplemented by a moderator analysis showing that only children with low performance at the beginning of the school-year profited from its implementation. This indicates that the information provided by formative assessment procedures helps teachers to identify struggling readers but does not seem to be utilized for adapting instruction to specific deficits of average or high performing children.
In sum, this dissertation contributes to research on individualized instruction by demonstrating necessary conditions for its effectiveness. It posits the need for a dynamic conceptualization of learner characteristics, demonstrates the advantage of multivariate learner profiles, and points out ways towards the successful implementation of individualized instruction in the classroom.
The European Community has set a milestone in the European water policy in 2000: all water directives and policies were united into one comprehensive document – the European Water Framework Directive (EU WFD). The EU WFD requires the monitoring of 45 priority substances, primarily in the water phase, which is not related to a substantial amount of chemicals available on the market worldwide (about 50,000). About 60% of these are human and environmentally toxic. Hence, the currently monitored 45 priority substances are not even close to being sufficient to provide a comprehensive picture of the actual chemical pollution in the aquatic environment.
Furthermore, the EU WFD in its original shape paid less attention to sediments as an important source and sink for chemical contamination. Under stable hydrological conditions, polluted old sediments are covered by less polluted younger sediments preventing erosion of deeper sediment layers and, therefore, the release of particle-bound contaminants. However, urbanization, deforestation, flooding, dredging, riverbed renaturation, and stormwater overflow basin releases can lead to an unpredictable release of particle-bound pollutants. Therefore, in 2008, sediments were added to the EU WFD as a monitoring matrix for substances that tend to accumulate there. As a result, after 18 years of the EU WFD, less than half of all European waterbodies reached a good ecological (40%) and chemical (38%) status.
One of the primary pollution sources in aquatic ecosystems are wastewater treatment plants (WWTPs). Advanced wastewater treatment by ozonation is promising to remove most micropollutants. However, the knowledge about the possible improvement of the receiving waterbody is rare. The latter aspects were the main reasons for the start of the DemO3AC project in 2014. The study area was located in the federal state of North Rhine-Westphalia (Germany). The study area included the Wurm River and its tributary, the Haarbach River. Both waterbodies act as receiving waterbodies for WWTPs. One of them is the Aachen-Soers WWTP (receiving waterbody: Wurm River), upgraded by full stream ozonation as an advanced effluent treatment. Therefore, the extensive investigation program within the DemO3AC project included an investigation of the ecological and chemical status of both receiving waterbodies and the investigation of a possible improvement of the Wurm River after implementing advanced effluent treatment.
The current study was a part of the DemO3AC project and covered the sediment toxicity and a possible impact of the ozonation on aquatic organisms in the receiving waterbody. Time-resolved sampling campaigns allowed investigations under different hydrological conditions, mainly determined by the weather. The first sampling campaign took place in June 2017 during a prolonged dry period with low water flow in the receiving waterbodies. The second sampling campaign was performed exactly one year later (June 2018) after a long rainy period and corresponding high-water levels. Full-stream ozonation at the Aachen-Soers WWTP had been in operation for half a year. Furthermore, a wide range of organic micropollutants was investigated in the effluent of the studied WWTPs to assess a possible hazard emerging from contaminants released into the receiving waterbody.
The study design was developed based on the holistic approach to assessing the ecotoxicological pollution of surface waterbodies. It included the detection of chemical compounds combined with effect-based methods to identify possible drivers of toxicity. The sediment's ecotoxicological assessment included studies on endocrine-disrupting activity, genotoxic and embryotoxic potentials. These endpoints were evaluated using in vitro and in vivo bioassays. In addition, sediments’ chemical profiling was performed using modern analytical chemistry techniques.
The genotoxic potential was investigated using the Ames fluctuation assay with Salmonella typhimurium bacterial strains TA98, TA100, YG1041, and YG1042, sensitive to different classes of compounds, and the Micronucleus assay as a eukaryotic assay with mammalian cells. A unique feature of the present study was the implementation of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay. Moreover, a comprehensive genotoxicity ranking of chemical compounds identified in sediments was used and combined with statistical analysis to identify the drivers of genotoxicity. The results of this study were published in Shuliakevich et al. (2022a) (see also Annex 1), describing the mutagenic potential of all sampling sites, which was primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. In addition, the rainwater overflow basin was identified as a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay to assess the different classes of mutagenic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.
The sediments' endocrine-disruptive activity was investigated using the cell-based reporter gene CALUX® assay. A simultaneous launch of the full-scale effluent ozonation at the Aachen-Soers WWTP was used for investigation of the entrance of the ozonated effluent into the Wurm River and the endocrine-disrupting activity in the water phase. A particular focus of the present study was the unique investigation of PAHs as possible drivers of the endocrine-disrupting activity in sediments of the Wurm River. The results of this study were laid down in the publication by Shuliakevich et al. (2022b) (see also Annex 2), describing variations in endocrine-disrupting activity in the Wurm River under different weather conditions. Briefly, under stable hydrological conditions in June 2017, the estrogenic and the antiandrogenic activities in sediments of the Wurm River were within the range of 0.03-0.1 ng E2 equivalents (eq.)/g dry weight sediment equivalents (dw SEQ) and 3.0-13.9 µg Flu eq./g dw SEQ, respectively. After extensive rain events in June 2018, the sediments' estrogenic and antiandrogenic activities were detected within the range of 0.06-0.2 ng E2 eq./g dw SEQ and 1.7-39.2 µg Flu eq./g de SEQ, respectively. Increased endocrine-disruptive activity (up to 0.2 ng E2 eq./g dw SEQ in ERα- and 39.2 µg Flu eq./g dw SEQ in anti-AR-CALUX® assays) in sediments downstream of the rainwater overflow basin suggested it as a possible source of pollution. A unique result of the second study was finding a positive correlation between measured particle-bound antiandrogenic activity and detected polyaromatic hydrocarbons (PAHs) ...
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
The heart is the first functional organ that develops in the embryo. To become a functional organ, it undergoes several morphogenetic processes. These morphogenetic events involve different cell types, that interact with each other and respond to the surrounding extracellular matrix, as well as intrinsic and extrinsic mechanical forces, assuming different behaviors. Additionally, transcription factor networks, conserved among vertebrates, control the development.
To have a better understanding of cell behavior during development, it is necessary to find a model system that allows the investigation in vivo and at single-cell resolution. Thanks to the common evolutionary origin of the different cardiac structures, together with the conserved molecular pathways, the two-chambered zebrafish heart offers many advantages to study cell behavior during cardiac morphogenesis. Here, using the zebrafish heart as a model system, I uncovered the cell behavior behind two of the main cardiac morphogenetic events: cardiac wall maturation and cardiac valve formation.
In the first part of this study, I investigated how the cardiac wall is maintained at the molecular level. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required for myocardial wall integrity. Global loss of snai1b leads to the extrusion of CMs away from the cardiac lumen, a process we show is dependent on cardiac contractility. Examining CM junctions in snai1b mutants, we observed that N-cadherin localization was compromised, thereby likely weakening cell-cell adhesion. In addition, extruding CMs exhibit increased actomyosin contractility basally, as revealed by the specific enrichment of canonical markers of actomyosin tension - phosphorylated myosin light chain (active myosin) and the α-catenin epitope α-18. By comparing the transcriptome of wild-type and snai1b mutant hearts at the early stages of CM extrusion, we found the dysregulation of intermediate filament genes in mutants including the upregulation of desmin b. We tested the role of desmin b in myocardial wall integrity and found that CM-specific desmin b overexpression led to CM extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 is a critical regulator of intermediate filament gene expression in CMs and that it maintains the integrity of the myocardial epithelium during embryogenesis, at least in part by repressing desmin b expression.
In the second part of this study, I focused on the behavior of valve cells during cardiac development. Using the zebrafish atrioventricular valve, I focus on the valve interstitial cells which confer biomechanical strength to the cardiac valve leaflets. We find that initially AV endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the extracellular matrix (ECM) between the two EC monolayers, undergo an endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a pro-migratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b, a well-known regulator of epithelial-to-mesenchymal transition. This study shows for the first time that Nfatc1 regulates zebrafish VICs formation regulating valve EMT in part by regulating twist1b expression. Moreover, it proposes the zebrafish valve as an excellent model to study the cellular and molecular process that regulate VIC development and dysfunction.
In conclusion, my work: 1) identified an unsuspected role of Snai1 in maintaining the integrity of the myocardial epithelium, opening new avenues in its role in regulating cellular contractility; 2) uncovered the function of Nfatc1 in the establishment of the VIC, establishing a new model to study valve development and function.
The most versatile tool for visualizing endogenous RNA is molecular beacons (MBs). MBs are modified oligonucleotides that consist of a stem-loop structure equipped with a fluorophore and a quencher at the opposite ends. They only give a fluorescent signal when hybridized to the target RNA. Here we present our recent efforts to enhance the spatiotemporal resolution of RNA visualization by refining MBs.
We first asked if we could refine MBs to visualize defined subcellular populations of RNA in living neurons. To achieve this, we utilize visible light-activatable Q-dye MBs to allow only a subcellular fraction to be activated. Here, the fluorophore at the 5’-end was linked to a second quencher via a photolabile coumarin protecting group. Therefore, the MB only gives a fluorescent signal, when activated with visible light and hybridized to the target. This architecture allowed local activation of a hybridized subpopulation in a defined area of the cell. Knowing the exact origin of the activated RNA, we were able to increase the available monitoring time for neuronal mRNA from several minutes (literature known MBs) to more than 14 hours.
We next asked if it would be possible to gain spatiotemporal control over where the MB hybridization events occur. Therefore, we developed photo-tethered MBs where two phosphates in the loop backbone are covalently linked to each other via two photocages. This prevents the MB from hybridization to the target RNA. Only when light is applied, the photo-tethers are cleaved, and the inherent hybridization function of the MB is activated. This architecture allowed us to control the hybridization of photo-tethered MBs in primary cultured neurons.
Macroautophagy, herein referred to as autophagy, is an evolutionarily conserved homeostatic process that normally occurs inside eukaryotic cells which involves degradation of cytoplasmic substances via lysosomes. It can be induced by various conditions such as starvation and drug exposure, as well as be inhibited by numerous compounds. Under normal conditions, the doublemembrane autophagosomes engulf the cytosolic substrates and deliver them to lysosomes for digestion. These substrates include unnecessary or dysfunctional cell components, such as faulty macromolecules, organelles and even invading pathogens. Autophagosomes are formed through the co-operative work of various autophagy-related (ATG) proteins organized into complexes. Upon closure of the autophagosomes, they fuse with the acidic lysosomes, resulting in formation of autolysosomes and the delivery of lysosomal hydrolases to degrade the engulfed contents. The fusion of the autophagosome with lysosome is carried out by specific SNARE proteins, small GTPases and their effectors including tethers, adaptors and motor proteins. Autophagy is impaired in many human diseases including cancer, neurodegenerative diseases, aging and inflammation. Therefore, manipulation of autophagy pathway holds a great promise for new therapeutic applications ...
The aim of this thesis is to provide a complete and consistent derivation of second-order dissipative relativistic spin hydrodynamics from quantum field theory. We will proceed in two main steps. The first one is the formulation of spin kinetic theory from quantum field theory using the Wigner-function formalism and performing an expansion in powers of the Planck constant. The essential ingredient here is the nonlocal collision term. We will find that the nonlocality of the collision term arises at first order in the Planck constant and is responsible for the spin alignment with vorticity, as it allows for conversion between spin and orbital angular momentum.
In the second step, this kinetic theory is used as the starting point to derive hydrodynamics including spin degrees of freedom. The so-called canonical form of the conserved currents follows from Noether’s theorem.
Applying an HW pseudo-gauge transformation, we obtain a spin tensor and energy-momentum tensor with obvious physical interpretation. Promoting all components of the HW tensors to be dynamical, we derive
second-order dissipative spin hydrodynamics. The additional equations of motion for the dissipative currents are obtained from kinetic theory generalizing the method of moments to include spin degrees of freedom.
Attributive participle constructions in German behave like adjectives in terms of inflection and position, but keep their verbal arguments. They can be extended by adjuncts or arguments and these extended attributive present participles mainly occur in written language (Weber, 1994). As the same content can also be expressed in a relative clause (RC), I compare both constructions in order to find out under which conditions a participle construction could lead to processing difficulties and how this relates to RC processing.
Based on previous assumptions for production (e.g. Weber, 1971; Fabricius-Hansen, 2016), three potential factors on the comprehension of prenominal modifiers and RCs are investigated: modifier length, the internal structure and multiple levels of embedding. The hypotheses for an effect on modifier length are mainly based on two processing accounts that make opposite predictions under specific circumstances: memory-based accounts such as the dependency locality theory (DLT) (e.g. Gibson, 2000) and expectation-based accounts such as surprisal (e.g. Levy, 2008). An increase in modifier length results in more intervening material between determiner and noun for the participle construction, contrary to RCs where these elements are adjacent. This separation of the DP could increase memory load. Therefore, longer participles would slow down processing of the noun, while there should be no difference for RCs. Two acceptability judgment experiments showed a tendency for longer participle phrases to receive lower ratings. The modifier length was further investigated in online processing. Contrary to the predicted locality effect, self-paced reading data reveals an anti-locality effect for participle phrases, with lower RTs on the noun when additional material was present inside the modifier. This experiment was followed up by an eye-tracking experiment which replicated the anti-locality effect, but at the participle instead at the noun.
The second factor that was investigated is the argument structure of the participle (or RC verb). My hypothesis is that more “prototypical” adjectives in terms of syntactic structure and semantics are more acceptable and easier to process. Attributive participles are considered hybrids between verbs and adjectives (e.g. Fuhrhop & Teuber, 2000; L¨ubbe & Rapp, 2011) due to their modifier internal verbal function, but adjectival position and agreement with the noun. This double role could lead to difficulties, in particular with a more complex verbal structure. Therefore, the prediction was that the presence of an accusative object inside a participle phrase would lead to lower acceptability ratings and higher reading times in online processing. In the first two acceptability experiments, this prediction was borne out. In addition, an SPR experiment was conducted which manipulated the presence of either an accusative object or adjunct for participles (of verbs that could be used intransitively and transitively) and the corresponding RCs. The experiment showed an effect of the presence of an accusative object on the participle, with higher reading times if an object was present, compared to an adjunct. No such difference was found for the RC verb, which indicates that only participles are processed more slowly when there is an accusative object. An alternative explanation for this finding is the inherent imperfective aspect of the present participle: a direct object could change the event structure in such a way that the aspect no longer matches.
The third factor I investigate is an effect of double embedding on the acceptability of participle phrases and RCs. While double embedded participles are rated lower than double embedded RCs, there is a smaller decrease from single to double embedding for participles than for RCs, contrary to the predictions calculated by the metric of the DLT.
Overall, the results provide evidence for experience-based processing, but they cannot be explained by either memory- or experience-based accounts alone. The effect concerning the presence of an accusative object suggest that properties of the participle distinguish the construction from RCs and affect its processing. The thesis suggests that the latter effect needs to be investigated further in future research. Furthermore, the findings have implications for the role of attributive present participles in German and for hypotheses about similar constructions in other languages.
Leukemia is a cancer of the blood and bone marrow characterized by an uncontrolled proliferation and accumulation of abnormal white blood cells. Leukemia can be classified based on the course of the disease (acute or chronic) and the blood cell type involved (myeloid or lymphocytic), leading to four main subtypes: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Leukemia represents 2.5% of all new cancer cases per year, and survival rates in some leukemias remain low at 40%.
The bone marrow microenvironment (BMM) is a system within the bone marrow comprising cellular and acellular components, all of which play a major role in hematopoiesis, providing the physical space where hematopoietic stem cells (HSCs) reside. The BMM interacts with HSCs, offering a “niche” for those cells and in case of leukemia, the BMM has a supportive role in disease maintenance and progression by supporting Leukemia stem cells (LSCs). One of the components of the BMM are calcium ions. Calcium is the most abundant mineral in the body, a key component of bones and is released by parathyroid hormone (PTH) induced bone remodeling. Calcium ions play a role in the localization, engraftment and adhesion of normal HSC to extracellular matrix (ECM) proteins in the BMM via the calcium sensing receptor (CaSR), thereby maintaining normal hematopoiesis. In addition of a major regulator of calcium homeostasis, CaSR contribute to the development of different cancers, functioning as either tumor suppressor or oncogene, depending on the involved tissue. However, the role of CaSR and its associated pathways in the local BMM for the development of leukemia is poorly understood. We hypothesized that calcium ions released from bone, subject to a fine balance between osteoblasts and osteoclasts, and/or CaSR, contribute to development, progression and response to therapy.
We have shown that the local calcium concentration forms a gradient in the bone marrow niche and in mice with CML is similarly low as in control mice, but significantly higher in mice suffering from BCR ABL1 driven B ALL or MLL AF9 driven AML. Similarly, the calcium concentration in the human BMM was found to be higher in AML than in other leukemias. Regarding the function of calcium in leukemia cells, we found that AML and CML cells respond differently to calcium exposure, with AML cells exhibiting regulation of cellular processes such as adhesion to the ECM protein fibronectin and migration toward CXCL 12, whereas CML cells remained mostly unaltered. Using genetic deletion or overexpression of CaSR in murine models of leukemia, we observed that CaSR acts as tumor suppressor in BCR-ABL1 driven CML and B ALL and as oncogene in AML.
Focusing on AML, our data shows that deficiency of CaSR on LICs leads, on one hand to increased apoptosis, and on the other hand to reduced cell cycle, reactive oxygen species (ROS) production and DNA damage in vivo, which may explain the observed prolongation of survival of mice. Complementary, in vitro experiments demonstrated that cells overexpressing CaSR have a distinct, cancer promoting phenotype compared to wildtype cells. Overexpression of CaSR led to an increase in proliferation, cell cycle, ROS production, DNA damage and reduced apoptosis. We have identified CaSR mediated pathways in AML and shown that CaSR enhances leukemia progression by activating MAPK/ERK and Wnt β catenin signaling. In addition, the CaSR interacting protein filamin A (FLNA) was shown to contribute to aggressive disease in vitro and in vivo. Furthermore, the mechanism underlying the role of CaSR in AML pathogenesis and possible regulation of LSCs was studied. Our findings demonstrated that CaSR ablation reduces myeloid progenitor function and proved that CaSR is required for maintenance of LSC pool by regulating its frequency and function. Further supporting the role of CaSR in LSC maintenance, genes associated with AML stemness and self renewal capacity were upregulated when CaSR was overexpressed and downregulated when CaSR was depleted. Given the role of CaSR in AML, the CaSR antagonist NPS 2143 was tested in vivo. The combination treatment of NPS 2143 with the standard of care, ara C, significantly reduced the tumor burden and prolonged the survival of mice with AML in syngeneic and xenotransplantation experiments. Based on the finding that CaSR functions as a tumor suppressor in CML, treatment of mice with the CaSR agonist cinacalcet in combination with imatinib prolonged survival of mice with CML compared to treatment with the mice given vehicle.
Our results suggest that calcium ions stemming from the calcium-rich BMM via CaSR strongly and differentially influence leukemia progression. As an adjunct to existing treatment therapies, targeting of CaSR with specific pharmacologic antagonists may prolong survival of patients with AML.
While B-cell acute lymphoblastic leukaemia (B-ALL) can be described as the leukaemia of childhood, chronic myeloid leukaemia (CML) mostly develops in elderly individuals. Understanding and utilising mechanisms involved in the development and persistence of these leukaemias as possible targets for treatment strategies has received particular interest. Processes that happen in the vicinity of the cancerous cells themselves could influence cancer growth and behaviour and hence can serve as novel targets, leading to the development of two-pronged therapies that act both on leukaemic cells directly as well as their niche. The niche in the case of leukaemia is the bone marrow microenvironment (BMM) where these cells are not only generated but also instructed and protected. As the BMM is situated inside bones that undergo drastic changes and growth processes during the ageing process, the BMM itself is also being altered throughout life. These alterations and the very process of expansion itself may therefore also provide distinct regulatory influences on the cells (healthy or malignant) that are generated inside this niche, leading to the question: Does the age of the bone marrow microenvironment differentially influence the development of (“childhood”) B-ALL versus (“adult”) CML by the release of cytokines?
In previous studies by the host-laboratory the age distribution of B-ALL versus CML in a murine transduction/ transplantation model could be recapitulated; young mice which received the same number of leukaemia-initiating cells as their old counterparts died significantly earlier of B-ALL while showing a significantly delayed clinical course, when they were suffering from CML. The tumour load and other leukaemia-associated parameters also showed a clear disposition towards preferential induction of CML in elderly and B-ALL in younger mice.
In this project we could support the hypothesis that the age of the BMM differentially influences the proliferation of leukaemic cells and thereby the development and persistence of different types of leukaemias by utilising different in vitro culture experiments. Specifically, we could show that young (compared to old) bone marrow
11 stroma cells (BMSC) support the growth of (BCR-ABL1+) B-ALL cells both in a direct, cell on cell co-culture setting, as well as in young BMSC-derived conditioned medium. This supports the hypothesis that varying factors are differentially released from a young versus an old BMM and influence the growth of the leukaemia cells. The opposite might be true for CML cells (BCR-ABL1+ 32D cells); BMSC obtained from old animals showed a tendency to support their growth more profoundly than cells acquired from young animals.
Possible proteins responsible for the distinct regulation of myeloid versus lymphatic leukaemic cells by young versus old BMM have also been studied. We investigated C-X-C motif chemokine 13 (CXCL13) and growth differentiation factor 11 (GDF11) in their effect on leukaemia cells, as both proteins having previously been described to have tumour-modelling properties and age-dependent levels (see below).
We identified an increased secretion of CXCL13, a B-cell chemotactic factor, into conditioned medium from young versus old BMSC. In accordance with this we found migration of B-ALL cells towards BMSC from young compared to old mice to be improved, while adhesion of both B-ALL and CML cells to young versus old BMSC did not show any differences. By blocking CXCL13 the proliferation-supporting effect of young BMSC on B-ALL cells could be diminished. Similar effects could be demonstrated by blocking GDF11.
In the case of CML cells we could observe the opposite effect; blocking CXCL13 and GDF11 increased their proliferation in a co-culture with BMSC. This supported our hypothesis that both cytokines differentially regulate B-ALL and CML behaviour. After the completion of this thesis, another member of the host-laboratory convincingly demonstrated the role of BMM age in the regulation of B-ALL via CXCL13 signalling (see discussion).
Carbon is an element that controls planetary habitability, and is fundamental for life on Earth. Its behaviour has important consequences for the global climate system, the origin and evolution of life on Earth. While the biosphere and atmosphere’s carbon cycle only accounts for less than 1% of the global carbon budget, hidden reservoirs of deep carbon in the Earth’s interior comprise the predominant storage of carbon on the planet. At the Earth’s surface, 60-70 % of carbon is hosted by carbonate minerals, which are then transported to the Earth’s interior, mainly in the form of sediments, by subduction of the oceanic lithosphere. Subducting plates are subjected to decarbonation, dehydration, and melting with CO2 release via supra-subduction volcanism. Nevertheless, part of the subducted carbonates’ may survive and be further transported to the deep mantle. Direct evidence of the existence of carbonates in the Earth’s interior, possibly reaching down to the lower mantle, comes from the finding of syngenetic inclusions of carbonates in diamonds and mantle xenoliths. The presence of carbonates in the deep Earth has a critical effect on the physical properties of the mantle. Melting and chemical speciation of the mantle are strongly affected by the form of C and carbonate stability. Therefore, the study of the stability and physical properties of carbonates at high pressures and temperatures is fundamental, because understanding the processes involved in the deep carbon cycle helps to improve our picture of the whole mantle.
The systematic characterization of the elastic properties of carbonates as a function of their structure and chemical composition is of great importance because it may allow to identify their presence and distribution by seismology. Inverting seismic observations to successfully constrain the chemical composition and mineralogy of the Earth’s interior requires knowledge of the physical properties of all possible Earth’s materials at pressures and temperatures applicable to the Earth’s interior. Up to now, a multitude of studies has focused on the construction of phase diagrams and structural transitions by means of X-ray diffraction and vibrational spectroscopy experiments.
Few studies are available on the complete elastic tensor of carbonates, however most of the datasets are not accompanied by an accurate characterization of the samples, which are often solid solutions and the exact chemical composition, density or the details about the experimental methods used are not presented. The aim of this thesis is to study the effect of chemical composition on the elastic properties of carbonates, providing a reliable dataset on the elasticity of the main carbonates. In particular, the elastic properties of crystalline aragonite, CaCO3, and Fe-dolomite, (Ca, Mg, Fe)(CO3)2, with different compositions were studied by Brillouin spectroscopy at ambient conditions. Brillouin spectroscopy was also used to investigate the elastic behaviour of amorphous calcium carbonate samples with different water contents (up to 18 wt%) at high pressures, up to 20 GPa.
Furthermore, the importance of cationic substitution on the structure and high pressure behaviour of carbonates was investigated by studying a synthetic CaCO3-SrCO3 solid solution at ambient conditions and at high pressures, up to 10 GPa, by single crystal X-ray diffraction. Finally, the study of the effect of composition on the elastic properties of families of isostructural solids was also extended to a different class of materials, the metal guanidinium formates. The elasticity of a family of perovskite metal organic frameworks, metal guanidinium formates C(NH2)3MII(HCOO)3, with MII =Mn, Zn, Cu, Co, Cd and Ca was investigated by combining Brillouin spectroscopy, resonant ultrasound spectroscopy, density functional theory and thermal diffuse scattering analysis.
Pulsed dipolar (PD) EPR spectroscopy is an established and reliable tool for the investigation of biomolecules. In terms of long distance and orientation measurements, it is one of the leading methods and further fields of application are constantly being explored. The distances that can be detected with PD EPR also correspond to the range in which almost all important biomolecule interactions occur. In the transition from in vitro spectroscopy to in-cell spectroscopy, the power of PD EPR spectroscopy is particularly evident. It is non-invasive, more sensitive than NMR, and does not exhibit background signals from diamagnetic molecules. In particular, the absence of background signals is of great importance given the high density of molecules within cellular environment. However, like any other spectroscopic method, PD EPR has certain limitations. Owing to the intrinsically fast electron spin echo dephasing at higher temperature, these experiments are commonly carried out in frozen solutions at about 50 K. This temperature is far away from the physiological conditions and the freezing additives used, e.g. glycols, can further influence the structure. To enable measurements with and within living organisms, it is therefore necessary to ascend from the cold depths of the frozen state. At the same time, one has to adapt the spin tags for the desired application. Established nitroxides commonly used for EPR studies are typically susceptible to reduction. Thus, for studies under physiological conditions, e.g. in the cell, one has to fight against the reductive environment in the cell and somehow protect the spin labels. Initial published in-cell experiments within the research group and investigations of homogeneously distributed labeled double-stranded (ds) ‐DNA samples in solid matrices showed promising results and enabled pulsed measurement in the temperature range of 50‐ 295 K. It could also be demonstrated that spherical shielded nitroxides have a significantly longer life span in cellular environments than non-protected ones and first nuclear acids were measured in cell. Based on these results, we have gone further to overcome the standing limitations and developed the use of PD EPR spectroscopy. This work addresses these challenges with the overall goal of advancing the applications of PD EPR spectroscopy for studying biomolecules under physiological conditions.
We have focused on four different approaches. The results of these studies were published in various publications. They are presented and discussed together with further studies and put into the context of research conducted before and after the authors' publications.
In approach 1, we fought against the two main obstacles for using pulsed dipolar spectroscopy at ambient conditions – minimizing phase memory time T2 and averaging of the anisotropic dipolar coupling by rotational diffusion. We focused on an immobilization approach, while using rigid spin labels at same time. Besidesto the distance information, the incorporated rigid spin labels will give additional angular constrains and information about the molecular dynamics.
In approach 2, we focused on the on-site and on-demand formation of nitroxide spin labels using light-sensitive alkyl protection groups. This a very mild and efficient procedure that will hardly interfere with sensitive functional groups present in oligonucleotides or peptides. By establishing this method and using coumarin protecting groups plus two-photon excitation, this property may offer the potential to generate spin labels with very high levels of spatial and temporal resolution.
For approach 3, we used paramagnetic Gd3+ -ions as intrinsically stable labels, which are not reducible within a cellular environment. Easy to mix and bound to encodable lanthanide binding tags within the molecule Interleucin 1β, we were able to measure distances between two tags with PELDOR spectroscopy. We tested the extent to which this system is suitable for in-cell measurements.
Finally, we focus on methods for easier labeling by using non-covalentlabeling techniques. One of these is the novel nitroxide G´ for site-directed spin labeling of nucleic acids, especially for RNA. This spin label is sterically hindered, easy to build and binding occurs in seconds by simply mixing the spin label with the target. For large RNAs, another easy-to-mix and noncovalent spin-labeling strategy will be experimentally accompanied and presented.
The approaches and results described here are intended to demonstrate that the study of the biological functions of biomolecules under physiological conditions by pulsed EPR spectroscopy is feasible and operational. In combination, they will enable the life sciences to make further and faster progress in the search for the molecular master plan.
Die Vorläuferform der eukaryotischen mRNA (prä-mRNA) durchläuft, eine Reihe von Prozessierungs-Schritte, die schließlich zu der Synthese einer „reifen“ und Exportkompetenten mRNA führt. prä-mRNA Spleißen ist ein essentieller Teilschritt dieser Reifung bei der intragene Sequenzen, sogenannte Introns, von der prä-mRNA entfernt werden, während Exons legiert werden. Das prä-mRNA Spleißen wird durch das Spleißosom katalysiert. Dieser Mega-Dalton Komplex, besteht aus fünf Sub-Komplexen, die sich wiederum aus katalytisch aktiven „kleinen nukleären Ribonukleinsäuren“ (snRNAs) und einer Vielzahl von proteinogenen Faktoren zusammensetzen. Diese Subkomplexe, bezeichnet als snRNPs (small nuclear Ribonucleoprotein Particles), binden die prä-mRNA an charakteristischen Sequenzen und richten die prä-mRNA durch eine Reihe von Konformations-Änderungen so aus, dass benachbarte Exons in Kontakt treten und über eine biochemische Ligations-Reaktion verbunden werden können.
Die Exon- bzw Intronerkennung der snRNPs wird durch zahlreiche Spleißfaktoren reguliert. Eine Proteinfamilie, die essentiell für die Regulierung des Spleißens ist, sind Serin/Arginin-reiche Proteine (SR-Proteine). Diese binden vorzugsweise an das 3‘ oder 5’ Ende von Exons, rekrutieren snRNPs und stimulieren dadurch die Exon-Inklusion. Durch diese Stimulierung können Spleiß-Events reguliert und gezielt spezifische Exons ausgeschlossen oder eingeschlossen werden. Dieser Prozess, der als alternatives Spleißen (AS) bezeichnet wird, tritt in 95% des menschlichen Transkriptoms auf und erweitert die Diversität eines Organismus, da verschiedene Transkripte von demselben Gen erzeugt werden können und folglich die Translation unterschiedlicher Proteine mit distinkten Funktionen ermöglicht wird.
Darüber hinaus verfügt die Zelle durch das AS über eine weitere posttranskriptionale Genregulationsebene, die insbesondere unter zellulären Stressbedingungen zur Expression von alternativen Protein-Isoformen von der Zelle genutzt wird. Eine in medizinischer Hinsicht besonders relevante Stressbedingung ist die sogenannte Hypoxie, die eine Sauerstoff-Unterversorgung von Zellen oder Gewebebereichen beschreibt. Hypoxie bzw. hypoxische Bereiche finden sich in Krebszellen und treten in 90% aller soliden Tumoren auf. Als Teil der Hypoxie Stress-Antwort, verfügt die Zelle über einen Adaptations-Mechanismus, der durch Hypoxieinduzierbare Faktoren (HIF) vermittelt wird. Diese Faktoren induzieren die Transkription zahlreicher Gene und stimulieren die Expression von Stressfaktoren, die an der zellulären Adaption der Hypoxie beteiligt sind. Einer dieser Faktoren ist der vaskuläre endotheliale Wachstumsfaktor A (VEGFA), welcher unter hypoxischen Bedingungen sekretiert wird und dadurch die Proliferation von Endothelzellen, die Neubildung von Blutgefäßen und damit die Vaskularisation des hypoxischen Bereichs stimuliert.
Die zelluläre Anpassung ist jedoch nicht nur auf die transkriptionelle Regulation des HIF-vermittelten Hypoxie Signalwegs beschränkt, sondern wird auf multiplen Genexpressions-Ebenen reguliert. Obwohl bekannt ist, dass tausende Transkripte unter hypoxischen Bedingungen alternativ gespleißt werden, sind die Faktoren, die die zelluläre Stress-Antwort durch AS regulieren, sowie deren molekularer Mechanismus jedoch weitestgehend unbekannt.
Diese Arbeit umfasst die Identifizierung und Charakterisierung von AS Events, sowie den Einfluss und die Regulation von Spleißfaktoren auf AS unter hypoxischen Bedingungen. Hierzu führten wir globale Genexpressions- und AS-Analysen in HeLaKarzinomzelllinien unter Normoxie (21% O2) und Hypoxie (0.2% O2) durch und zeigen, dass 7962 Gene nach 24h Hypoxie unterschiedlich exprimiert werden. Über AS-Analysen konnten 4434 Transkripte identifiziert werden, die bei Hypoxie über AS reguliert sind. Dabei trat „Exon-Skipping“ als das am häufigsten auftretende AS-Events auf. Über PCR basierte Validierungs-Experimente konnten 5 regulierte Transkripte nachgewiesen werden. Dabei weisen Exon 3 und 4 in BORA, Exon 6 in MDM4 und Exon 4-5 in CSSP1 Exon-Skipping Events auf, während Exon-Inklusionen in CEP192 Exon 28 und in der 3’UTR von EIF4A2 validiert werden konnten.
Darüber hinaus wurde im Rahmen der AS-Analyse die Regulation des sogenannten „backsplicings“ bei Hypoxie untersucht. Im Gegensatz zum linearen Spleißens, wird beim backsplicing das 5’Ende und das 3’Ende von Exons verbunden, was die Bildung von sogenannten zirkulären RNAs (circRNAs) zufolge hat. Obwohl nur wenige Funktionen dieser RNA-Klasse bekannt sind, wurde die Regulation von circRNAs während der Zell-Differenzierung sowie in diversen Krebszellen beschrieben. Dabei können circRNAs als microRNA- oder Protein-Schwämme fungieren oder dienen als Protein-Interaktion Plattform und regulieren dabei die Genexpression.
Aortic valve (AV) and root replacement with composite graft and re-implantation of coronary arteries described first by Bentall and de Bono in 1968, is considered as a standard operation for treatment of different pathologies of the AV and aortic root. In centres where aortic valve and root repair techniques and Ross operation are well established, generally severely diseased patients remain indicated for this procedure. The aim of this study was to evaluate the early and long-term outcomes after Bentall-De Bono (BD) procedures in high-risk population with complex pathologies and multiple comorbidities.
Between 2005 and 2018, a total of 273 consecutive patients (median age 66 years; 23 % female) underwent AV and root replacement with composite-graft in so called button technique. We divided our population in the following groups: 1. acute type A aortic dissection group (ATAAD) (n = 48), 2. endocarditis group (n = 99) and 3. all other pathologies group (n = 126). The surgery has been per- formed emergent/urgent in 131 patients (49 %) and in 109 cases (40%) as a reoperation. Concomitant surgery was required in 97 patients (58%) and 167 pa- tients (61%) received a biological composite-graft.
Follow-up was completed in 96% (10 patients lost to follow-up) with a mean of 8.6 years (range 0.1-15.7 years), counting a total of 1450 patient-years. Thirty- day mortality was 17% (46 patients). The overall estimated survival in 5 and 10 years was 64% ± 3%) and 46% ±4 %). Group comparison showed a significant difference in favour of patient from the dissection group (p = 0.008). Implantation of a biological valve graft was associated with lower survival probability (p < 0.001). There was no significant difference in the freedom of reoperation rate between the groups. The same applies for freedom of postoperative endocarditis, thromboembolic events, and aortic prosthesis dysfunction. According to the uni- variate and multivariate logistic regression analysis primarily postoperative neu- rological dysfunction (OR 5.45), hypertension (OR 4.8) peripheral artery disease (OR 4.4), re-exploration for bleeding (OR 3.37) and postoperative renal replace- ment therapy (OR 3.09) were identified as leading predictors of mortality.
In conclusion, the BD operation can be performed with acceptable short- and long-term results in high-risk patients with complex aortic pathologies in a centre with well-established AV repair and Ross operation program.
The dissertation explores to what extent the post-financial crisis EU resolution regime, based on equity/debt write-down and conversion powers and bail-in tools will be effective in maintaining the stability of bank groups. To arrive at its unique angle, it first asks why bank groups are considered complex, thereby explaining the reasons for their proliferation and instability, and how this may inform the view regarding a desired regulatory framework. The main observation the dissertation makes is that, notwithstanding of other factors already pointed out in the literature, bank groups adopt complex structures with multiple entities, as it allows them, inter alia, to use double-leverage financing structures and internal capital markets.
Double-leverage financing structures allow bank groups to optimise the combination of their debt/equity funding from external parent entity investors with a combination of debt/equity funding downstreamed internally to subsidiaries and other entities in the bank group. An important component within this structure is also that the allocation of the bank group’s resources takes place through the internal capital market (ICM). The allocation of resources via the ICM allows bank groups to manage their liquidity constraint either to undertake activities that are more profitable, or to stabilise the financial position of the group as a whole.
While both double leverage and ICMs can optimise the funding and allocation of resources of the bank group, respectively, they can also generate perils to the stability of the bank group. In particular, this is because double-leverage can result in excessive risk taking and regulatory arbitrage. Moreover, the allocation of the intra-group resources in the ICM may not maintain the financial health of all subsidiaries in the bank group, which can prove to be incompatible with the financial stability goals of the regulators in the countries where those subsidiaries conduct their business.
Within this context, the dissertation argues that the current EU resolution regime does not clearly address issues of double leverage when setting out capital and other liability requirements, i.e. the ‘Total Loss Absorbing Capacity’ (TLAC) and ‘Minimum Requirement for Eligible Liabilities’ (MREL) requirements. Moreover, the dissertation emphasis that it is equally relevant to clarify the way in which the bank group resources are available ahead of, and in financial distress. It is argued that to this end, bank groups need to be allowed to make use of the ICM as it is often uncertain what may be the cause of the financial distress and how the resources of the bank group could be used to stabilise it. To this end, the dissertation highlights that there is lack of clarity in both the ex-ante provisions on intra-group support framework and in the ex-post provisions governing the allocation of any surplus TLAC/MREL resources.
Besides the ‘intra-group’ issues within the bank group, the third point the dissertation makes relation to the bank group’s presence in multiple jurisdictions. This transnational element adds to the complexity of the intra-group issues resulting from sub-optimal cooperation between home and host authorities. In this regard, the dissertation underlines that the current framework could adopt a more balanced way in which the regulatory fora will take into account the interest of the authorities of all parts of the bank group.
Zika-virus (ZIKV), a flavivirus mainly transmitted by Aedes mosquitoes, is a single-stranded, positive-sense RNA virus. The viral genome is surrounded by a nucleocapsid and a lipid bilayer, in which membrane and envelope proteins are embedded. ZIKV disease is mainly characterized by mild symptoms, such as fever, rash as well as pain in head and joints. However, after epidemics it caused in the Americas in 2015/16, ZIKV infections were also associated with severe neurological complications like the Guillain-Barré syndrome (GBS) and microcephaly in fetuses and newborns. So far there are no specific antiviral treatments or vaccines available against ZIKV. This strengthens the need for a detailed understanding of the viral life cycle and virus-host interactions.
The antiviral host factor tetherin (THN) is an interferon-stimulated protein and therefore part of the cellular innate immune response. It comprises an N-terminal cytoplasmic domain, followed by a transmembrane helix, an extracellular coiled-coil domain and a C-terminal glycosylphosphatidylinositol (GPI) anchor. Containing two sites for membrane insertion linked by a flexible structure, THN is able to integrate into the membrane of budding viruses, thereby attaching them to each other and to the cell membrane and preventing their further release and spread.
In this study, the crosstalk of ZIKV and THN was analyzed. Previous gene expression analyses by microarray and quantitative polymerase chain reaction (qPCR) had revealed a strong upregulation of the BST2 gene encoding for THN in ZIKV-infected cells. However, this enhanced expression did not correlate with an enhanced THN protein level. On the contrary, the amount of THN in THN-overexpressing cells was after infection even heavily reduced. Furthermore, immunofluorescence analyses revealed a loss of THN membrane localization in these cells. By performing a cycloheximide assay, this loss could be traced back to a reduced protein half-life of THN in infected versus uninfected cells. Treatment with inhibitors of different protein degradation pathways as well as colocalization analyses with markers of several subcellular compartments indicated an involvement of the endo-lysosomal route. A knock-down of the ESCRT-0 protein HRS however prevented the sorting of THN for lysosomal degradation and led to a stabilization of THN protein levels. After HRS depletion, the release and spread of viral particles was reduced in THN-overexpressing compared to wildtype cells.
Taken together, the data obtained in this study revealed the potential of THN to restrict ZIKV release and spread. The enhanced degradation of THN in ZIKV-infected cells via the endo-lysosomal pathway could therefore be explained as an effective viral escape strategy. This could be circumvented by knockdown of the ESCRT-0 protein HRS, which highlighted HRS as a potential target for the development of antiviral treatments.
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in early childhood. Despite recent advances in the treatment regimes of rhabdomyosarcoma, the 5-year survival is still alarmingly low for the more aggressive metastasizing alveolar rhabdomyosarcoma subtype. Novel treatment strategies are needed in order to increase the overall survival rate. Hallmarks of cancer include evade cell death induction and evade immune system surveillance. This is mediated in part by up-regulation of inhibitor of apoptosis (IAP) proteins. With the development of Smac mimetic compounds mimicking the endogenous IAP antagonist Smac, this tumor evasion mechanism became exploitable.
In this PhD thesis, a combinatory approach for a putative treatment option of RMS will be presented. Here, the Smac mimetic compound BV6 will be used as a pre-treatment of RMS cells. This leads to a sensitizing effect within the tumor cells, increasing the killing efficacy of natural killer (NK) cells.
Subtoxic concentrations of BV6 were chosen to sensitize RMS cells. To remodel the solid tumor characteristics of RMS, a multicellular RMS tumor spheroid culture model was used.
In both tumor spheroids and conventional monolayer cell culture BV6 induced the degradation of IAP proteins (cIAP1, cIAP2, in spheroids XIAP). Further, BV6 led to the activation of both, the canonical and non-canonical NF-κB signaling pathways.
This was demonstrated by an increased IκBα and p65 phosphorylation, and nuclear translocation of p-p65, indicative for an active canonical NF-κB signaling. On the other side, cIAP degradation led to the stabilization and accumulation of NIK and downstream partial degradation of p100 to p52 and its nuclear translocation, indicating non-canonical NF-κB signaling pathway activity. A bulk RNA sequencing approach of BV6 treated RH30 cells validated the NF-κB signaling involvement and identified 182 differentially expressed genes. Among the interesting target genes are NFKBIA (IκBα),BIRC3 (cIAP2), NFKB2 (p100), CCL5 and SSTR2. SSTR2 was thoroughly validated as being up-regulated on a transcriptional and on protein level. Here, SSTR2A, one of the two alternative splicing variants, is up-regulated and opens a hypothetical targeted treatment strategy, as SSTR2 expression is not associated with RMS, but rather described with neuroendocrine tumor entities. In addition, CCL5 was thoroughly validated as a BV6 induced target. Again, the up-regulated mRNA transcription was validated by an increased translation and by increased secretion of CCL5. As CCL5 being associated as pro-migratory and activating of NK cells, CRISPR/Cas9 mediated CCL5 knock-out studies were performed to evaluate the influence of CCL5 within a BV6 pre-treatment and NK cell co-cultivation setting. It was shown that CCL5 knock-out does not rescue BV6 pre-treated RMS spheroids from NK cell attack and killing.
The previous mentioned transcriptional activity by BV6 stimulation was NIK mediated as knock-down of NIK reduced the mRNA transcription of several interesting genes.
However, NIK mediated down-stream signaling had no influence on the BV6 induced sensitizing effect towards NK cell mediated attack. A NIK knock-down had no rescue effect upon BV6 pre-treatment and NK cell co-treatment.
As cIAP proteins are present in receptor bound complexes, e.g. complex I at the TNF receptor 1 (TNFR1), a putative involvement of death receptors in general was evaluated.
Indeed, BV6 treatment of RMS cells could increase the surface presentation of DR5, a death receptor ligating TRAIL. Functionally, co-treatment of BV6 with TRAIL led to an additive cell death inducting effect. However, within the NK cell co-cultivation setting, addition of a neutralizing TRAIL anitbody could not rescue BV6 pre-treated RMS spheroids from NK cell killing. A similar effect was observed when neutralizing TNFα by adding Enbrel during the NK cell co-cultivation. BV6 sensitization of RMS spheroids seems to be independent of death receptors.
In addition to activating NF-κB, BV6 as a Smac mimetic is supposed to be able to release caspases bound by IAP proteins. Indeed, BV6 pre-treatment of RMS spheroids and co-cultivation with NK cells could cleave and thereby activate the executioner caspase-3. Further, treatment with a pan-caspase inhibitor, zVAD.fmk, could reduce the BV6 mediated sensitizing effect towards NK cell attack in RD spheroids.
Taken together, BV6 does induce a thoroughly validated NF-κB signaling pathway, leading to a NIK mediated transcriptional signature change. However, the NF-κB activation might not be responsible for the observed sensitization. Further, BV6 in combination with NK cells led to a seemingly death receptor independent, caspase dependent cell death induction of RMS spheroids. Although the mechanism remains partially con-cealed, a therapeutic benefit by combining a cell death sensitizing compound, i.e. BV6, with cytotoxic lymphocytes is evident.
Membrane proteins are a diverse group of proteins that serve a multitude of purposes with one of the most important ones being transport. All kinds of substrates are shuffled over biological membranes with the help of dedicated proteins enabling the transport along and against a concentration gradient. Within the group of actively transporting proteins a diverse set of proteins that rely on an electrochemical gradient to facilitate transport of a substrate against its concentration gradient can be found. Those so-called secondary active
transporters are a group on integral membrane proteins ubiquitous to all cells. They allow the transport of all kinds of substrates like nutrients, ions, other metabolites and drugs over the hydrophobic barrier created by the cellular and organellar membrane. The gradients that provide the main driving force for most of the transporters are either sodium ions or protons, although transporters utilizing other ions or organic compounds are found as well. In case of exchangers two very similar substrates are transported in opposing direction over the membrane, one against its electrochemical gradient driven by the other.
Along with a structural diversity of the transporters concerning overall shape, oligomerization and number of transmembrane elements comes a mechanistic variety though still following the principle of alternating access. In humans the malfunction of secondary active transporters can lead to a physiological disorders such as epilepsy, depression or obesity.
The focus of this thesis was the structural and functional characterization of the secondary active transporter SeCitS from Salmonella enterica, a symporter of the 2-hydroxycarboxylate family. The transport of citrate as a bivalent ion is facilitated by the flux of sodium ions that have an inward-facing gradient over the inner membrane of Salmonella enterica. Transport experiments showed that the transport ratio is two sodium ions per citrate molecule, netting in an electroneutral transport. Compared to other members of the family the specificity of the transporter towards its main substrate is very high.
Structural information on the protein was initially obtained through 2D electron crystallography, which allowed the identification of the oval shaped dimer and a first hint towards a significant conformational change that the protein undergoes during its transport cycle. Using 3D crystallography, the X-ray structure of the transporter was solved. The protein crystalizes as a stable, but conformationally asymmetric dimer. As bound citrate can be readily identified in both protomers they can be assigned into an outward- and an inward-facing conformation, with the main citrate binding site in the outward-facing conformation.
One interesting feature of the crystal structure was the large surface available for multimerization, providing a platform for tight dimerization of the two protomers. On the other hand, SeCitS did not show a true cooperativity of transport. With those two aspects taken into account the question arose if any potential crosstalk between the monomers within the dimer takes place and influences transport (negative cooperativity) or the conformational distribution within the dimer (stabilization of the protein within the membrane).
The functional approach in answering this question was the use of mutated variants of the protein for cross-linking within one monomer. Two residues were chosen respectively to lock one of either conformation to be able to test for transport activity in the remaining protomer. The suitability of the residues was derived from the crystal structure (D112 – R205 to lock the inward-facing conformation and L337 – S412 for the outward-facing conformation). After initial promising results the final variants were not stable enough to be analyzed in transport assays.
To analyze the distribution of relative conformations within the dimer the protein was reconstituted into native-like lipid environment such as nanodiscs or saposin nanoparticles to be analyzed by cryo-electron microscopy. The first images were recorded and did yield promising 2D classes where the general features of the transporter were identified. Yet, an improved preparation is required to obtain a high resolution structure.
The key functional aspects of a transporter are its ability to bind and transport its substrates. In a set of experiments those features were investigated by a radioligand transport assay and by isothermal titration calorimetry (ITC). The transport properties of the protein were assessed in a filter assay using a radioactively labeled citrate as a read-out. The protein was reconstituted into proteoliposomes and subjected to different substrate conditions. Different ions were tested in its ability to drive or inhibit transport, but only sodium ions were able to drive transport and also not hindered by the presence of other ions...
Diseases such as cardiac arrhythmias, CPVT and other issues of the human heart still remain largely unexplored. To contribute to this field of research, it is necessary to create tools to control the spatial and temporal release and reuptake of Ca2+ from the sarcoplasmic/endoplasmic reticulum (SR/ER). Ca2+ release and uptake by the ryanodine receptor (RyR) and Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), respectively, are essential for the function of excitable cells. In this process, the rapid Ca2+ release from the SR/ER and the associated contraction in muscle cells is modulated by RyR. However, diseases due to calcium leakage, such as cardiac arrhythmias, seizures and contractile dysfunction, are also caused by RyR. The resting Ca2+ concentration in the cytosol, which is important for the cell, is kept in balance by Ca2+ release and reuptake into the SR/ER. This reuptake is controlled quite considerably by SERCA. SERCA is important for development and muscle function in both nematodes such as C. elegans and mammals, though there is also a great need for tools that can help study precise function.
To advance towards the goal of developing tools for optogenetic stimulation of intracellular Ca2+ release from the SR/ER, the model organism C. elegans was chosen. Its advantages are the fully sequenced genome and the neural network connectome. In addition, the ease of maintenance, self-fertilisation, transparency and rapid generation cycles, as well as the fact that it is a eutelic animal, are advantages for the application of the optogenetic approach.
So far, tools for light-induced Ca2+ release (LICR) have already been developed, involving the creation of ChR2 versions with higher Ca2+ conductivity based on the "CatCh" variant and further improving their conductivity through several established mutations. In addition, the pharynx of C. elegans was modified to produce an optogenetically stimulated muscle pump that resembles mammalian cardiac muscle cells. In this work, both optoUNC-68 (optically excitable RyR) and SERCA/LOV2 were generated in different variants by CRISPR/Cas9 and plasmid-based genome editing to achieve light-driven manipulation of calcium homeostasis in C. elegans. Here, LICR was triggered by LOV2 domains in an opto-mechanical manipulation of RyR as well as SERCA. This approach was made possible by recently published high-resolution cryoEM structural images. In addition, alternative approaches using Ca2+ conductance-optimised channelrhodopsin variants were tested in C. elegans body wall muscle cells.
By inserting ChR-XXM into C. elegans and subsequent fluorescence microscopy of the co-introduced GFP, an expression in body wall muscle cells could be detected. Furthermore, in contraction assays, ChR-XXM was demonstrated to induce contractions of the animals of up to 16% compared to the original body length in both medium (0.8mW/mm²) and high (1.4mW/mm²) stimulation at 470nm. ChR-XXM was thus identified as an excellent candidate for the development of an optogenetic tool, as it exhibits significantly increased Ca2+ conductivity compared to other ChR2 variants.
The use of CRISPR/Cas9 to insert AsLOV2 domains (L404-L546) into different insertion sites of RyR allowed the generation of a transgenic strain of C. elegans that could be stimulated to elongate during 0.3mW/mm² photostimulation. This demonstrated that RyR can be manipulated by photostimulation, spatiotemporally through conformational changes in the LOV2 domain and the resulting disruption of the pore region.
The CRISPR/Cas9 method was also used to insert LOV2 domains into SERCA. Here it could be demonstrated that a conformational change of the LOV2 domains induced by photostimulation leads to a stop or impairment of Ca2+ ion translocation by SERCA from the cytosol into the SR/ER. In contrast to LOV2 in RyR, this resulted in a contraction of C. elegans body length.
The data presented here indicate that the intracellular Ca2+ cycle involving the SR/ER and cytosol can be successfully manipulated by the introduction of optogenetic tools. It turned out that the manipulation/impairment of individual components of this system, such as RyR or SERCA, is usually insufficient to achieve a clear response. Therefore, simultaneous manipulation of the two main actors RyR and SERCA is arguably the best way to take another step towards creating optogenetic tools for light-stimulated manipulation of Ca2+ release and reuptake from the SR/ER.
The development of the designs of the superconducting CH cavities of the HELIAC project from CH0 [27] to CH1 and CH2 [1] has undergone permanent improvements and adaptations based on the learned experiences of each previous cavity. For example, the design of CH1 and CH2 focused on mechanical stabilization and optimization of performance by minimizing peak electric and magnetic fields. As a result, the changes made there were already able to increase stability and performance compared to CH0 by simplifying the design in different ways. The process of designing both cavities was time reasonable, since they are identical in construction and thus only one design had to be developed. However, for both the development and manufacturing of an entire accelerator of individual CH cavities, this type of design would become too time consuming and costly. In order to reduce this time-consuming design process and accelerate the fabrication of superconducting CH cavities, and also reduce costs, a modular cavity design for mass production of superconducting CH cavities was developed as presented in this thesis. In the following section, the conclusions gained in this work and the results already presented will be summarized once again.
So in the first chapters of this thesis the theoretical foundations were laid, which are necessary for the description of superconducting cavities and for their development process, like a theoretical description of superconductivity itself (see chapter 2), the physical basics of RF-acceleration and of the CH cavity (see chapter 3), but also the effects that limit the superconducting cavities in terms of acceleration (see chapter 4) or the properties and laws from structural mechanics needed in later measurements and simulation (see chapter 5). Based on the theoretical foundations given in these sections, all measurements, evaluations and simulations made in the following sections were made.
Zika virus (ZIKV) is a member of the Flaviviridae family that received public attention and scientific interest after the outbreak in French Polynesia (2013-2014) and the epidemic in the Americas (2015-2016). Even though only 20% of infected people exhibit clinical manifestations and they are predominantly flu-like symptoms, these events unveiled neurological complications associated with ZIKV infection, such as the Guillain-Barré syndrome in adults and microcephaly in newborns. Lacking a preventive vaccine and a specific antiviral therapy against ZIKV allied to the fact that this pathogen is a re-emerging virus, uncovering and comprehending novel virus-host interactions is crucial to the identification of new antiviral targets and the development of innovative antiviral approaches. Previous research work uncovered that the Chinese hamster ovary (CHO) cells do not support ZIKV infection.459 As this cell line does not express endogenous epidermal growth factor receptor (EGFR), this study aimed to investigate whether EGFR and EGFR-dependent signaling are relevant for the ZIKV life cycle in vitro.
In the first part of the study, viral infection was investigated in CHO cells and compared to A549 cells, a highly ZIKV permissive cell line. After performing binding and entry assays, ZIKV entry, but not the attachment, was significantly decreased in CHO cells in comparison to A549 cells. Additionally, in A549-EGFR KO cells, ZIKV entry was diminished relatively to the off-target control. These results show the clear impact that the absence of EGFR has on viral entry, implicating EGFR during this process. Even though EGFR overexpression in CHO cells could not render these cells permissive to ZIKV infection, as demonstrated by the lack of viral infection after electroporation with in vitro transcribed capped ZIKV-Renilla luciferase RNA, it was possible to rescue ZIKV entry. These findings suggest that there are additional elements, which are not expressed in CHO cells, required for viral replication.
Furthermore, the impact of ZIKV infection on EGFR mRNA and protein levels as well as on the EGFR subcellular localization and distribution was evaluated. The relative number of EGFR specific transcripts continuously increased with ZIKV infection, whereas the EGFR protein level diminished at later times of infection. Moreover, changes in the subcellular localization of EGFR and its colocalization with the early endosomal marker EEA1 in ZIKV-infected cells revealed that ZIKV triggers EGFR internalization. The relevance of EGFR in the ZIKV entry process was further corroborated by the observation of EGFR internalization at 30 min post-infection (mpi) and to less extent at 60 mpi, which concurs with the expected time of ZIKV entry into the host cells.
In the remaining part of the study, the influence of ZIKV infection in EGFR-dependent signaling as well as the contribution of EGFR and EGFR signaling for viral infection were studied. Activation of EGFR and the MAPK/ERK signaling cascade was detected as early as 5 mpi and ceased within 30 mpi in ZIKV-infected cells. Taking into account that EGFR internalization was observed at 30 mpi in infected cells, the activation of EGFR and ERK and subsequent dephosphorylation within this period go along with this previous observation. Vice-versa, inhibition of the activation of EGFR and the MAPK/ERK pathway declines ZIKV infection. On the one hand, inhibition of EGFR activation by Erlotinib affected ZIKV entry, as a consequence of impaired EGFR internalization. On the other hand, Raf and MEK inhibitors reduced ZIKV infection without disturbing viral replication or viral entry. These data suggest that the activation of the MAPK/ERK signaling cascade is necessary for a step of the viral life cycle before the onset of genome replication and morphogenesis and after viral entry. The importance of EGFR signaling was additionally investigated by the determination of EGFR half-life in ZIKV-infected cells upon EGF stimulation. While the EGFR half-life was similar in uninfected and Uganda-infected cells, a delay in EGFR degradation was observed in French Polynesia-infected cells. This observation might indicate an extended usurpation of the EGFR signaling since EGFR seems to still be active in the endosomes. Moreover, disruption of lipid rafts by MβCD, a cholesterol-depleting agent, hampered ZIKV entry. In uninfected cells, MβCD treatment led to the activation of EGFR, but at the same time prevented EGFR internalization, indicating that EGFR activation exclusively is not sufficient for an efficient ZIKV entry and further supporting the importance of EGFR internalization during the ZIKV entry process.
Taken together, this study uncovers EGFR as a relevant host factor in the early stages of ZIKV infection, providing novel insights into the ZIKV entry process. Since numerous monoclonal antibodies and substances that target EGFR are licensed, repurposing these compounds might be a helpful tool for the establishment of an antiviral therapy in case of ZIKV re-emergence.
Focused electron and ion beam induced deposition (FEBID/FIBID) methods have gained significant attention in recent years because of their unique ability for the maskless fabrication of arbitrary three-dimensional shapes. Both techniques enable material deposition down to the nanoscale for applications in materials science and condensed matter physics. However, the number of suitable precursor molecules, especially for high purity deposits, is usually still very limited to date. Additionally, both the FEBID and FIBID process are very complex when assessed in detailed and the development of process-optimize, tailored precursor molecules is not yet possible.
In the first part of this work hexacarbonyl vanadium (V(CO)6) and dimanganese decacarbonyl (Mn2(CO)10) are investigated for their use in FEBID in order to complement the already existing data on transition metal carbonyl precursors. In addition, chemical vapor deposition (CVD) has been carried out to compare compositional differences for electron induced and purely thermal processes. FEBID using V(CO)6 resulted in the formation of a vanadium (oxy)carbide material with a V:C ratio of approx. 0.6-0.9. The material shows a temperature-dependent normalized electrical conductance typical for granular metals in agreement with TEM analysis. Additionally, characterization of the crystalline fractions reveals a cubic VC1-xOx phase in agreement with the phase observed in CVD thin films. Thermal decomposition using CVD yielded material of higher purity with V:C ratios of 1.1-1.3. In contrast, an insulating material with approx. 40 at% Mn is obtained for FEBID using Mn2(CO)10 as precursor with very similar compositions being observed for CVD thin films.
The second part of this work deals with the deposition of defined alloy materials by focused charged particle beam deposition. Three silyl substituted transition metal carbonyl complexes have been synthesized and tested for FEBID, FIBID and CVD. The three precursors investigated were: H3SiMn(CO)5, H3SiCo(CO)4, and H2Si(Co(CO)4)2. FEBID experiments with the manganese derivative show the selective loss of silicon, and metal/metalloid contents of up to 49 at%. Contrary, material derived from both cobalt derivatives did retain the 1:1 and 2:1 Co:Si ratios respectively, resulting in metal/metalloid contents of up to 62 at%. Temperature-dependent normalized electrical conductance measurements of as-grown and post-growth electron beam irradiated samples reveal behavior typical for granular metals except for the as-grown CoSi material which is located on the insulating side of the metal-insulator transition. Ga+-FIBID revealed H2Si(Co(CO)4)2 to be a very suitable precursor, retaining the predefined Co:Si ratio in the deposits, while significant loss of silicon was observed for H3SiCo(CO)4 derived deposits. Contrary to FEBID high metal/metalloid contents of up to 90 at% are obtained. Additionally, temperature dependent electrical properties of dicobalt silicide and the expected ferromagnetic behavior have been observed for the Co2Si-FIBID material. Further analysis enables the proposition of different dominating decomposition channels in FEBID and FIBID based on microstructural features such as bubble formation in FIBID materials.