Refine
Is part of the Bibliography
- yes (761)
Year of publication
Document Type
- Doctoral Thesis (753)
- Journal article (7)
- Book (1)
Language
- English (761) (remove)
Keywords
- Maus (32)
- Thrombozyt (29)
- Taufliege (19)
- Tissue Engineering (19)
- Genexpression (16)
- Dendritische Zelle (14)
- T-Lymphozyt (14)
- Entzündung (13)
- Angst (12)
- Biene (12)
Institute
- Graduate School of Life Sciences (761) (remove)
Sonstige beteiligte Institutionen
- Helmholtz Institute for RNA-based Infection Research (HIRI) (5)
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg (2)
- Universitätsklinikum Münster (2)
- Zentrum für Infektionsforschung (ZINF) Würzburg (2)
- Bio-Imaging Center Würzburg (1)
- Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München (1)
- CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - the development agency of the Brazilian Federal Government (1)
- Carl-Ludwig-Institut für Physiologie, Universität Leipzig (1)
- Chair of Experimental Biomedicine I (1)
- DAAD - Deutscher Akademischer Austauschdienst (1)
Acceleration is a central aim of clinical and technical research in magnetic resonance imaging (MRI) today, with the potential to increase robustness, accessibility and patient comfort, reduce cost, and enable entirely new kinds of examinations. A key component in this endeavor is image reconstruction, as most modern approaches build on advanced signal and image processing. Here, deep learning (DL)-based methods have recently shown considerable potential, with numerous publications demonstrating benefits for MRI reconstruction. However, these methods often come at the cost of an increased risk for subtle yet critical errors. Therefore, the aim of this thesis is to advance DL-based MRI reconstruction, while ensuring high quality and fidelity with measured data. A network architecture specifically suited for this purpose is the variational network (VN). To investigate the benefits these can bring to non-Cartesian cardiac imaging, the first part presents an application of VNs, which were specifically adapted to the reconstruction of accelerated spiral acquisitions. The proposed method is compared to a segmented exam, a U-Net and a compressed sensing (CS) model using qualitative and quantitative measures. While the U-Net performed poorly, the VN as well as the CS reconstruction showed good output quality. In functional cardiac imaging, the proposed real-time method with VN reconstruction substantially accelerates examinations over the gold-standard, from over 10 to just 1 minute. Clinical parameters agreed on average.
Generally in MRI reconstruction, the assessment of image quality is complex, in particular for modern non-linear methods. Therefore, advanced techniques for precise evaluation of quality were subsequently demonstrated.
With two distinct methods, resolution and amplification or suppression of noise are quantified locally in each pixel of a reconstruction. Using these, local maps of resolution and noise in parallel imaging (GRAPPA), CS, U-Net and VN reconstructions were determined for MR images of the brain. In the tested images, GRAPPA delivers uniform and ideal resolution, but amplifies noise noticeably. The other methods adapt their behavior to image structure, where different levels of local blurring were observed at edges compared to homogeneous areas, and noise was suppressed except at edges. Overall, VNs were found to combine a number of advantageous properties, including a good trade-off between resolution and noise, fast reconstruction times, and high overall image quality and fidelity of the produced output. Therefore, this network architecture seems highly promising for MRI reconstruction.
Articular cartilage defects represent one of the most challenging clinical problem for orthopedic surgeons and cartilage damage after trauma can result in debilitating joint pain, functional impairment and in the long-term development of osteoarthritis. The lateral cartilage-cartilage integration is crucial for the long-term success and to prevent further tissue degeneration. Tissue adhesives and sealants are becoming increasingly more popular and can be a beneficial approach in fostering tissue integration, particularly in tissues like cartilage where alternative techniques, such as suturing, would instead introduce further damage. However, adhesive materials still require optimization regarding the maximization of adhesion strength on the one hand and long-term tissue integration on the other hand. In vitro models can be a valuable support in the investigation of potential candidates and their functional mechanisms. For the conducted experiments within this work, an in vitro disc/ring model obtained from porcine articular cartilage tissue was established. In addition to qualitative evaluation of regeneration, this model facilitates the implementation of biomechanical tests to quantify cartilage integration strength. Construct harvesting for histology and other evaluation methods could be standardized and is ethically less questionable compared to in vivo testing. The opportunity of cell culture technique application for the in vitro model allowed a better understanding of cartilage integration processes.
Tissue bonding requires chemical or physical interaction of the adhesive material and the substrate. Adhesive hydrogels can bind to the defect interface and simultaneously fill the gap of irregularly shaped defect voids. Fibrin gels are derived from the physiological blood-clot formation and are clinically applied for wound closure. Within this work, comparisons of different fibrin glue formulations with the commercial BioGlue® were assessed, which highlighted the need for good biocompatibility when applied on cartilage tissue in order to achieve satisfying long-term integration. Fibrin gel formulations can be adapted with regard to their long-term stability and when applied on cartilage disc/ring constructs improved integrative repair is observable. The kinetic of repairing processes was investigated in fibrin-treated cartilage composites as part of this work. After three days in vitro cultivation, deposited extracellular matrix (ECM) was obvious at the glued interface that increased further over time. Interfacial cell invasion from the surrounding native cartilage was detected from day ten of tissue culture. The ECM formation relies on molecular factors, e.g., as was shown representatively for ascorbic acid, and contributes to increasing integration strengths over time. The experiments performed with fibrin revealed that the treatment with a biocompatible adhesive that allows cartilage neosynthesis favors lateral cartilage integration in the long term. However, fibrin has limited immediate bonding strength, which is disadvantageous for use on articular cartilage that is subject to high mechanical stress. The continuing aim of this thesis was to further develop adhesive mechanisms and new adhesive hydrogels that retain the positive properties of fibrin but have an increased immediate bonding strength.
Two different photochemical approaches with the advantage of on-demand bonding were tested. Such treatment potentially eases the application for the professional user. First, an UV light induced crosslinking mechanism was transferred to fibrin glue to provide additional bonding strength. For this, the cartilage surface was functionalized with highly reactive light-sensitive diazirine groups, which allowed additional covalent bonds to the fibrin matrix and thus increased the adhesive strength. However, the disadvantages of this approach were the multi-step bonding reactions, the need for enzymatic pretreatment of the cartilage, expensive reagents, potential UV-light damage, and potential toxicity hazards. Due to the mentioned disadvantages, no further experiments, including long-term culture, were carried out. A second photosensitive approach focused on blue light induced crosslinking of fibrinogen (RuFib) via a photoinitiator molecule instead of using thrombin as a crosslinking mediator like in normal fibrin glue. The used ruthenium complex allowed inter- and intramolecular dityrosine binding of fibrinogen molecules. The advantage of this method is a one-step curing of fibrinogen via visible light that further achieved higher adhesive strengths than fibrin. In contrast to diazirine functionalization of cartilage, the ruthenium complex is of less toxicological concern. However, after in vitro cultivation of the disc/ring constructs, there was a decrease in integration strength. Compared to fibrin, a reduced cartilage synthesis was observed at the defect. It is also disadvantageous that a direct adjustment of the adhesive can only be made via protein concentration, since fibrinogen is a natural protein that has a fixed number of tyrosine binding sites without chemical modification.
An additional cartilage adhesive was developed that is based on a mussel-inspired adhesive mechanism in which reactivity to a variety of substrates is enabled via free DOPA amino acids. DOPA-based adhesion is known to function in moist environments, a major advantage for application on water-rich cartilage tissue surrounded by synovial liquid. Reactive DOPA groups were synthetically attached to a polymer, here POx, to allow easy chemical modifiability, e.g. insertion of hydrolyzable ester motifs for tunable degradation. The possibility of preparing an adhesive hybrid hydrogel of POx in combination with fibrinogen led to good cell compatibility as was similarly observed with fibrin, but with increased immediate adhesive strength. Degradation could be adjusted by the amount of ester linkages on the POx and a direct influence of degradation rates on the development of integration in the in vitro model could be shown.
Hydrogels are well suited to fill defect gaps and immediate integration can be achieved via adhesive properties. The results obtained show that for the success of long-term integration, a good ability of the adhesive to take up synthesized ECM components and cells to enable regeneration is required. The degradation kinetics of the adhesive must match the remodeling process to avoid intermediate loss of integration power and to allow long-term firm adhesion to the native tissue.
Hydrogels are not only important as adhesives for smaller lesions, but also for filling large defect volumes and populating them with cells to produce tissue engineered cartilage. Many different hydrogel types suitable for cartilage synthesis are reported in the literature. A long-term stable fibrin formulation was tested in this work not only as an adhesive but also as a bulk hydrogel construct. Agarose is also a material widely used in cartilage tissue engineering that has shown good cartilage neosynthesis and was included in integration assessment. In addition, a synthetic hyaluronic acid-based hydrogel (HA SH/P(AGE/G)) was used. The disc/ring construct was adapted for such experiments and the inner lumen of the cartilage ring was filled with the respective hydrogel. In contrast to agarose, fibrin and HA-SH/P(AGE/G) gels have a crosslink mechanism that led to immediate bonding upon contact with cartilage during curing. The enhanced cartilage neosynthesis in agarose compared to the other hydrogel types resulted in improved integration during in vitro culture. This shows that for the long-term success of a treatment, remodeling of the hydrogel into functional cartilage tissue is a very high priority. In order to successfully treat larger cartilage defects with hydrogels, new materials with these properties in combination with chemical modifiability and a direct adhesion mechanism are one of the most promising approaches.
In the initiation phase of acute graft-versus-host disease (aGvHD), CD4+ T cells are activated by hematopoietic antigen presenting cells in secondary lymphoid organs whereas in effector phase by non-hematopoietic cells in the small intestine. We hypothesized that alloreactive CD4+ T cells primarily home to the secondary lymphoid organs subsequent to allogeneic hematopoietic cell transplantation in the initiation phase of aGvHD and are activated by the non-hematopoietic lymph node stromal cells via MHC class II. To test this hypothesis, we employed CD4+ T cell-dependent major mismatch aGvHD mouse model to study this correlation.
Upon analyzing the early events following allo-HCT with bioluminescence imaging, flow cytometry and whole-mount light sheet fluorescence microscopy, we found that allogeneic T cells exclusively home to the spleen, lymph nodes and the Peyer’s patches and not to the intestinal lamina propria in the initiation phase of aGvHD. Utilizing mice devoid of partial or complete hematopoietic antigen presentation we could show allogeneic CD4+ T cells activation in the lymphoid organs of MHCIIΔCD11c and MHCIIΔ BM chimeric mice early after allo-HCT. MHCIIΔ BM chimeras failure of thymic negative selection and developing tissue wasting disease upon syn-HCT deemed them unsuitable to study non-hematopoietic antigen presentation in aGvHD. To overcome this challenge, we generated MHCIIΔVav1 mice that lack MHC class II expression on all hematopoietic cells. MHCIIΔVav1 mice were susceptible to aGvHD and LNSCs from these animals activated allogeneic CD4+ T cells in mixed lymphocyte reaction. Likewise, mesenteric lymph nodes from CD11c.DTR mice surgically transplanted into a MHCIIΔ mouse could activate CD4+ T cells in vivo, clearly demonstrating LNSCs as non-hematopoietic APCs of the lymphoid organs.
We specifically target lymph node stromal cell subsets via the Cre/loxP system, we employed single cell RNA sequencing and selected Ccl19 and VE-Cadherin to specifically target the fibroblastic reticular cells and endothelial cells of the lymph nodes respectively. In MHCIIΔCcl19 mice, alloreactive CD4+ T cells activation was discreetly reduced in the initiation phase of aGvHD whereas absence of MHCII on fibroblastic reticular cells resulted in hyper-activation of allogeneic CD4+ T cells leading to poor survival. This phenotype was modulated by the regulatory T cells that were able to rescue H2-Ab1fl mice but not the MHCIIΔCcl19 subsequent to GvHD.
Knock-out of MHCII on endothelial cells MHCIIΔVE Cadherin, resulted only in modest reduction of CD4+ T cells activation in the initiation phase of GvHD, conversely MHCIIΔVE Cadherin mice showed a protective phenotype compared against littermates H2-Ab1fl mice in long-term survival. Furthermore, to pin-point endothelial cells MHCII antigen presentation we generated MHCIIΔVE Cadherin ΔVav1 animals devoid of antigen presentation in both endothelial and hematopoietic compartments. LNSCs from MHCIIΔVE Cadherin ΔVav1 were unable to activate alloreactive CD4+ T cells in mixed lymphocyte reaction.
Altogether, we demonstrate for the first time that MHC class II on the lymph node stromal cells plays a crucial role in the modulation of allogeneic CD4+ T cells in the initiation and later in the effector phase of graft-versus-host-disease.
In the face of threat, animals react with a defensive reaction to avoid or reduce harm. This defensive reaction encompasses apart from behavioral changes also physiological, analgetic, and endocrine adaptations. Nonetheless, most animal studies on fear and anxiety are based on behavioral observations only, disregarding other aspects of the defensive reaction, or integrating their inter-related dynamics only insufficiently. The first part of this thesis aimed in characterizing patterned associations of behavioral and physiological responses, termed integrated defensive states. Analyzing cardiac and behavioral responses in mice undergoing multiple fear and anxiety paradigms revealed a complex and dynamic interaction of those readouts on both, short and long timescales. Microstates, stereotypical combinations of i.e. freezing and decelerating heart rates, are short-lasting and were, in turn, shown to be influenced by slow acting macrostate changes. One of those higher order macrostates, called `rigidity`, was defined as a latent process that constrains the range of momentary displayed heart rate values. Furthermore, integrated defensive states were found to be highly dependent on the cue and the context the animals are confronted with. Importantly, same behavioral observations, i.e. freezing, were associated with distinct cardiac responses, highlighting the importance of multivariate analysis of integrated defensive states. Defensive states are orchestrated by the brain, which has evolved evolutionary conserved survival circuits. A central brain area of these circuits is the periaqueductal gray (PAG) in the midbrain. It plays a pivotal role in mediating defensive states, as it receives signals about external and internal information from multiple brain regions and sends information to both, higher order brain areas as well as to the brainstem ultimately causing the execution of threat responses. In the second part of this thesis, different neuronal circuit elements in the PAG were optically manipulated in order to gain mechanistic insight into the defense network in the brain underlying the previously delineated cardio-behavioral defensive states. Optical activation of glutamatergic PAG neurons evoked heterogeneous, light-intensity dependent responses. However, a further molecular restriction of the glutamatergic neuronal population targeting only Chx10+ neurons, led to a cardio-behavioral state that resembled spontaneous freezing-bradycardia bouts.
In summary, this thesis presents a multivariate description of defensive states, which includes the complex interaction of cardiac and behavioral responses on different timescales and, furthermore, functionally dissects different excitatory and inhibitory PAG circuit elements mediating these defensive states.
The emergence of human induced pluripotent stem cells (iPSCs) and the rise of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing technology innovated the research platform for scientists based on living human pluripotent cells. The revolutionary combination of both Nobel Prize-honored techniques enables direct disease modeling especially for research focused on genetic diseases. To allow the study on mutation-associated pathomechanisms, we established robust human in vitro systems of three inherited cardiomyopathies: arrhythmogenic cardiomyopathy (ACM), dilated cardiomyopathy with juvenile cataract (DCMJC) and dilated cardiomyopathy with ataxia (DCMA).
Sendai virus vectors encoding OCT3/4, SOX2, KLF4, and c-MYC were used to reprogram human healthy control or mutation-bearing dermal fibroblasts from patients to an embryonic state thereby allowing the robust and efficient generation of in total five transgene-free iPSC lines. The nucleofection-mediated CRISPR/Cas9 plasmid delivery in healthy control iPSCs enabled precise and efficient genome editing by mutating the respective disease genes to create isogenic mutant control iPSCs. Here, a PKP2 knock-out and a DSG2 knock-out iPSC line were established to serve as a model of ACM. Moreover, a DNAJC19 C-terminal truncated variant (DNAJC19tv) was established to mimic a splice acceptor site mutation in DNAJC19 of two patients with the potential of recapitulating DCMA-associated phenotypes. In total eight self-generated iPSC lines were assessed matching internationally defined quality control criteria. The cells retained their ability to differentiate into cells of all three germ layers in vitro and maintained a stable karyotype. All iPSC lines exhibited a typical stem cell-like morphology as well as expression of characteristic pluripotency markers with high population purities, thus validating the further usage of all iPSC lines in in vitro systems of ACM, DCMA and DCMJC.
Furthermore, cardiac-specific disease mechanisms underlying DCMA were investigated using in vitro generated iPSC-derived cardiomyocytes (iPSC-CMs). DCMA is an autosomal recessive disorder characterized by life threatening early onset cardiomyopathy associated with a metabolic syndrome. Causal mutations were identified in the DNAJC19 gene encoding an inner mitochondrial membrane (IMM) protein with a presumed function in mitochondrial biogenesis and cardiolipin (CL) remodeling. In total, two DCMA patient-derived iPSC lines (DCMAP1, DCMAP2) of siblings with discordant cardiac phenotypes, a third isogenic mutant control iPSC line (DNAJC19tv) as well as two control lines (NC6M and NC47F) were directed towards the cardiovascular lineage upon response to extracellular specification cues. The monolayer cardiac differentiation approach was successfully adapted for all five iPSC lines and optimized towards ventricular subtype identity, higher population purities and enhanced maturity states to fulfill all DCMA-specific requirements prior to phenotypic investigations. To provide a solid basis for the study of DCMA, the combination of lactate-based metabolic enrichment, magnetic-activated cell sorting, mattress-based cultivation and prolonged cultivation time was performed in an approach-dependent manner. The application of the designated strategies was sufficient to ensure adult-like characteristics, which included at least 60-day-old iPSC-CMs. Therefore, the novel human DCMA platform was established to enable the study of the pathogenesis underlying DCMA with respect to structural, morphological and functional changes.
The disease-associated protein, DNAJC19, is constituent of the TIM23 import machinery and can directly interact with PHB2, a component of the membrane bound hetero-oligomeric prohibitin ring complexes that are crucial for phospholipid and protein clustering in the IMM. DNAJC19 mutations were predicted to cause a loss of the DnaJ interaction domain, which was confirmed by loss of full-length DNAJC19 protein in all mutant cell lines. The subcellular investigation of DNAJC19 demonstrated a nuclear restriction in mutant iPSC-CMs. The loss of DNAJC19 co-localization with mitochondrial structures was accompanied by enhanced fragmentation, an overall reduction of mitochondrial mass and smaller cardiomyocytes. Ultrastructural analysis yielded decreased mitochondria sizes and abnormal cristae providing a link to defects in mitochondrial biogenesis and CL remodeling. Preliminary data on CL profiles revealed longer acyl chains and a more unsaturated acyl chain composition highlighting abnormities in the phospholipid maturation in DCMA.
However, the assessment of mitochondrial function in iPSCs and dermal fibroblasts revealed an overall higher oxygen consumption that was even more enhanced in iPSC-CMs when comparing all three mutants to healthy controls. Excess oxygen consumption rates indicated a higher electron transport chain (ETC) activity to meet cellular ATP demands that probably result from proton leakage or the decoupling of the ETC complexes provoked by abnormal CL embedding in the IMM.
Moreover, in particular iPSC-CMs presented increased extracellular acidification rates that indicated a shift towards the utilization of other substrates than fatty acids, such as glucose, pyruvate or glutamine. The examination of metabolic features via double radioactive tracer uptakes (18F-FDG, 125I-BMIPP) displayed significantly decreased fatty acid uptake in all mutants that was accompanied by increased glucose uptake in one patient cell line only, underlining a highly dynamic preference of substrates between mutant iPSC-CMs.
To connect molecular changes directly to physiological processes, insights on calcium kinetics, contractility and arrhythmic potential were assessed and unraveled significantly increased beating frequencies, elevated diastolic calcium concentrations and a shared trend towards reduced cell shortenings in all mutant cell lines basally and upon isoproterenol stimulation. Extended speed of recovery was seen in all mutant iPSC-CMs but most striking in one patient-derived iPSC-CM model, that additionally showed significantly prolonged relaxation times. The investigations of calcium transient shapes pointed towards enhanced arrhythmic features in mutant cells comprised by both the occurrence of DADs/EADs and fibrillation-like events with discordant preferences.
Taken together, new insights into a novel in vitro model system of DCMA were gained to study a genetically determined cardiomyopathy in a patient-specific manner upon incorporation of an isogenic mutant control. Based on our results, we suggest that loss of full-length DNAJC19 impedes PHB2-complex stabilization within the IMM, thus hindering PHB-rings from building IMM-specific phospholipid clusters. These clusters are essential to enable normal CL remodeling during cristae morphogenesis. Disturbed cristae and mitochondrial fragmentation were observed and refer to an essential role of DNAJC19 in mitochondrial morphogenesis and biogenesis. Alterations in mitochondrial morphology are generally linked to reduced ATP yields and aberrant reactive oxygen species production thereby having fundamental downstream effects on the cardiomyocytes` functionality. DCMA-associated cellular dysfunctions were in particular manifested in excess oxygen consumption, altered substrate utilization and abnormal calcium kinetics. The summarized data highlight the usage of human iPSC-derived CMs as a powerful tool to recapitulate DCMA-associated phenotypes that offers an unique potential to identify therapeutic strategies in order to reverse the pathological process and to pave the way towards clinical applications for a personalized therapy of DCMA in the future.
Motor neuron diseases (MNDs) encompass a variety of clinically and genetically heterogeneous disorders, which lead to the degeneration of motor neurons (MNs) and impaired motor functions. MNs coordinate and control movement by transmitting their signal to a target muscle cell. The synaptic endings of the MN axon and the contact site of the muscle cell thereby form the presynaptic and postsynaptic structures of the neuromuscular junction (NMJ). In MNDs, synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is an early target in the pathophysiological cascade leading to MN death. In this study, we established new experimental strategies to analyze human MNDs by patient derived induced pluripotent stem cells (iPSCs) and investigated pathophysiological mechanisms in two different MNDs.
To study human MNDs, specialized cell culture systems that enable the connection of MNs to their target muscle cells are required to allow the formation of NMJs. In the first part of this study, we established and validated a human neuromuscular co-culture system consisting of iPSC derived MNs and 3D skeletal muscle tissue derived from myoblasts. We generated 3D muscle tissue by culturing primary myoblasts in a defined extracellular matrix in self-microfabricated silicone dishes that support the 3D tissue formation. Subsequently, iPSCs from healthy donors and iPSCs from patients with the progressive MND Amyotrophic Lateral Sclerosis (ALS) were differentiated into MNs and used for 3D neuromuscular co-cultures. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the functionality of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of ALS and found a decrease in neuromuscular coupling, muscle contraction, and axonal outgrowth in co-cultures with MNs harboring ALS-linked superoxide dismutase 1 (SOD1) mutation. In summary, this co-culture system presents a human model for MNDs that can recapitulate aspects of ALS pathophysiology.
In the second part of this study, we identified an impaired unconventional protein secretion (UPS) of Sod1 as pathological mechanisms in Pleckstrin homology domain-containing family G member 5 (Plekhg5)-associated MND. Sod1 is a leaderless cytosolic protein which is secreted in an autophagy-dependent manner. We found that Plekhg5 depletion in primary MNs and NSC34 cells leads to an impaired secretion of wildtype Sod1, indicating that Plekhg5 drives the UPS of Sod1 in vitro. By interfering with different steps during the biogenesis of autophagosomes, we could show that Plekhg5-regulated Sod1 secretion is determined by autophagy. To analyze our findings in a clinically more relevant model we utilized human iPSC MNs from healthy donors and ALS patients with SOD1 mutations. We observed reduced SOD1 secretion in ALS MNs which coincides with reduced protein expression of PLEKHG5 compared to healthy and isogenic control MNs. To confirm this correlation, we depleted PLEKHG5 in control MNs and found reduced extracellular SOD1 levels, implying that SOD1 secretion depends on PLEKHG5. In summary, we found that Plekh5 regulates the UPS of Sod1 in mouse and human MNs and that Sod1 secretion occurs in an autophagy dependent manner. Our data shows an unreported mechanistic link between two MND-associated proteins.
According to the WHO, foodborne derived enteric infections are a global disease burden and often manifest in diseases that can potentially reach life threatening levels, especially in developing countries. These diseases are caused by a variety of enteric pathogens and affect the gastrointestinal tract, from the gastric to the intestinal to the rectal tissue. Although the complex mucosal structure of these organs is usually well prepared to defend the body against harmful agents, specialised pathogens such as Salmonella enterica can overcome the intestinal defence mechanism. After ingestion, Salmonella are capable of colonising the gut and establishing their proliferative niche, thereby leading to inflammatory processes and tissue damage of the host epithelium. In order to understand these processes, the scientific community in the last decades mostly used cell line based in vitro approaches or in vivo animal studies. Although these approaches provide fundamental insights into the interactions between bacteria and host cells, they have limited applicability to human pathology. Therefore, tissue engineered primary based approaches are important for modern infection research. They exhibit the human complexity better than traditional cell lines and can mimic human-obligate processes in contrast to animal studies.
Therefore, in this study a tissue engineered human primary model of the small intestinal epithelium was established for the application of enteric infection research with the exemplary pathogen Salmonella Typhimurium.
To this purpose, adult stem cell derived intestinal organoids were used as a primary human cell source to generate monolayers on biological or synthetic scaffolds in a Transwell®-like setting. These tissue models of the intestinal epithelium were examined for their comparability to the native tissue in terms of morphology, morphometry and barrier function. Further, the gene expression profiles of organotypical mucins, tight junction-associated proteins and claudins were investigated. Overall, the biological scaffold-based tissue models showed higher similarity to the native tissue - among others in morphometry and polarisation. Therefore, these models were further characterised on cellular and structural level. Ultrastructural analysis demonstrated the establishment of characteristic microvilli and tight-junction connections between individual epithelial cells. Furthermore, the expression pattern of typical intestinal epithelial protein was addressed and showed in vivo-like localisation. Interested in the cell type composition, single cell transcriptomic profiling revealed distinct cell types including proliferative cells and stem cells, progenitors, cellular entities of the absorptive lineage, Enterocytes and Microfold-like cells. Cells of the secretory lineage were also annotated, but without distinct canonical gene expression patterns. With the organotypical polarisation, protein expression, structural features and the heterogeneous cell composition including the rare Microfold-like cells, the biological scaffold-based tissue model of the intestinal epithelium demonstrates key requisites needed for infection studies with Salmonella.
In a second part of this study, a suitable infection protocol of the epithelial tissue model with Salmonella Typhimurium was established, followed by the examination of key features of the infection process. Salmonella adhered to the epithelial microvilli and induced typical membrane ruffling during invasion; interestingly the individual steps of invasion could be observed. After invasion, time course analysis showed that Salmonella resided and proliferated intracellularly, while simultaneously migrating from the apical to the basolateral side of the infected cell. Furthermore, the bacterial morphology changed to a filamentous phenotype; especially when the models have been analysed at late time points after infection. The epithelial cells on the other side released the cytokines Interleukin 8 and Tumour Necrosis Factor α upon bacterial infection in a time-dependent manner. Taken together, Salmonella infection of the intestinal epithelial tissue model recapitulates important steps of the infection process as described in the literature, and hence demonstrates a valid in vitro platform for the investigation of the Salmonella infection process in the human context.
During the infection process, intracellular Salmonella populations varied in their bacterial number, which could be attributed to increased intracellular proliferation and demonstrated thereby a heterogeneous behaviour of Salmonella in individual cells. Furthermore, by the application of single cell transcriptomic profiling, the upregulation of Olfactomedin-4 (OLFM4) gene expression was detected; OLFM4 is a protein involved in various functions including cell immunity as well as proliferating signalling pathways and is often used as intestinal stem cell marker. This OLFM4 upregulation was time-dependent, restricted to Salmonella infected cells and seemed to increase with bacterial mass. Investigating the OLFM4 regulatory mechanism, nuclear factor κB induced upregulation could be excluded, whereas inhibition of the Notch signalling led to a decrease of OLFM4 gene and protein expression. Furthermore, Notch inhibition resulted in decreased filamentous Salmonella formation. Taken together, by the use of the introduced primary epithelial tissue model, a heterogeneous intracellular bacterial behaviour was observed and a so far overlooked host cell response – the expression of OLFM4 by individual infected cells – could be identified; although Salmonella Typhimurium is one of the best-studied enteric pathogenic bacteria. This proves the applicability of the introduced tissue model in enteric infection research as well as the importance of new approaches in order to decipher host-pathogen interactions with higher relevance to the host.
In the scope of climate warming and the increase in frequency and intensity of severe heat waves in Central Europe, identification of temperate tree species that are suited to cope with these environmental changes is gaining increasing importance. A number of tree physiological characteristics are associated with drought-stress resistance and survival following severe heat, but recent studies have shown the importance of plant hydraulic and anatomical traits for predicting drought-induced tree mortality, such as vessel diameter, and their potential to predict species distribution in a changing climate.
A compilation of large global datasets is required to determine traits related to drought-induced embolism and test whether embolism resistance can be determined solely by anatomical traits. However, most measurements of plant hydraulic traits are labour-intense and prone to measurement artefacts. A fast, accurate and widely applicable technique is necessary for estimating xylem embolism resistance (e.g., water potential at 50% loss of conductivity, P50), in order to improve forecasts of future forest changes. These traits and their combination must have evolved following the selective pressure of the environmental conditions in which each species occurs. Describing these environmental-trait relationships can be useful to assess potential responses to environmental change and mitigation strategies for tree species, as future warmer temperatures may be compounded by drier conditions.
RNA viruses rely entirely on the host machinery for their protein synthesis and harbor non-canonical translation mechanisms, such as alternative initiation and programmed –1 ribosomal frameshifting (–1PRF), to suit their specific needs. On the other hand, host cells have developed a variety of defensive strategies to safeguard their translational apparatus and at times transiently shut down global translation. An infection can lead to substantial translational remodeling in cells and translational control is critical during antiviral response. Due to their sheer diversity, this control is likely unique to each RNA virus and the intricacies of post-transcriptional regulation are unclear in certain viral species.
Here, we explored different aspects of translational regulation in virus-infected cells in detail. Using ribosome profiling, we extensively characterized the translational landscape in HIV-1 infected T cells, uncovering novel features of gene regulation in both host and virus. Additionally, we show that substantial pausing occurs prior to the frameshift site indicating complex regulatory mechanisms involving upstream viral RNA elements that can act as cis- regulators of frameshifting.
We also characterized the mechanistic details of trans- modulation of frameshifting by host- and virus-encoded proteins. Host antiviral protein ZAP-S binds to the SARS-CoV-2 frameshift site and destabilizes the stimulatory structure, leading to frameshift inhibition. On the other hand, EMCV 2A protein stabilizes the viral frameshift site, thereby, activating EMCV frameshifting. While both proteins were shown to be antagonistic in their mechanism, they interact with the host translational machinery. Furthermore, we showed that frameshifting can be regulated not just by proteins, but also by small molecules. High-throughput screening of natural and synthetic compounds identified two potent frameshift inhibitors that also impeded viral replication, namely trichangion and compound 25. Together, this work largely enhances our understanding of gene regulation mechanisms in virus-infected cells and further validates the druggability of viral –1 PRF site.
Hereditary spastic paraplegias (HSPs) are genetically-determined, neurodegenerative disorders characterized by progressive weakness and spasticity of the lower limbs. Spastic paraplegia type 11 (SPG11) is a complicated form of HSP, which is caused by mutations in the SPG11 gene encoding spatacsin, a protein possibly involved in lysosomal reformation. Based on our previous studies demonstrating that secondary neuroinflammation can be a robust amplifier of various genetically-mediated diseases of both the central and peripheral nervous system, we here test the possibility that neuroinflammation may modify the disease outcome also in a mouse model for SPG11. Spg11-knockout (Spg11-/-) mice develop early walking pattern and behavioral abnormalities, at least partially reflecting motor, and behavioral changes typical for patients. Furthermore, we detected a progressive increase in axonal damage and axonal spheroid formation in the white and grey matter compartments of the central nervous system of Spg11-/- mice. This was accompanied by a concomitant substantial increase of secondary inflammation by cytotoxic CD8+ and CD4+ T-lymphocytes. We here provide evidence that disease-related changes can be ameliorated/delayed by the genetic deletion of the adaptive immune system. Accordingly, we provide evidence that repurposing clinically approved immunomodulators (fingolimod/FTY720 or teriflunomide), that are in use for treatment of multiple sclerosis (MS), also improve disease symptoms in mice, when administered in an early (before neural damage) or late (after/during neural damage) treatment regime.
This work provides strong evidence that immunomodulation can be a therapeutic option for the still untreatable SPG11, including its typical neuropsychological features. This poses the question if inflammation is not only a disease amplifier in SPG11 but can act as a unifying factor also for other genetically mediated disorders of the CNS. If true, this may pave the way to therapeutic options in a wide range of still untreatable, primarily genetic, neurological disorders by repurposing approved immunomodulators.
After myocardial infarction, an inflammatory response is induced characterized by a sterile inflammation, followed by a reparative phase in order to induce cardiac healing. Neutrophils are the first immune cells that enter the ischemic tissue. Neutrophils have various functions in the ischemic heart, such as phagocytosis, production of reactive oxygen species or release of granule components. These functions can not only directly damage cardiac tissue, but are also necessary for initiating reparative effects in post-ischemic healing, indicating a dual role of neutrophils in cardiac healing after infarction.
In recent years, evidence has been growing that neutrophils show phenotypic and functional differences in distinct homeostatic and pathogenic settings.
Preliminary data of my working group using single-cell RNA-sequencing revealed the time- dependent heterogeneity of neutrophils, with different populations showing distinct gene expression profiles in ischemic hearts of mice, including the time-dependent appearance of a SiglecFhigh neutrophil population. To better understand the dynamics of neutrophil heterogeneity in the ischemic heart, my work aimed to validate previous findings at the protein level, as well as to investigate whether the distinct neutrophil populations show functional differences. Furthermore, in vivo depletion experiments were performed in order to modulate circulating neutrophil levels.
Hearts, blood, bone marrow and spleens were processed and analyzed from mice after 1 day and 3 days after the onset of cardiac ischemia and analyzed using flow cytometry.
Results showed that the majority of cardiac neutrophils isolated at day 3 after myocardial infarction were SiglecFhigh, whereas nearly no SiglecFhigh neutrophils could be isolated from ischemic hearts at day 1 after myocardial infarction.
No SiglecFhigh neutrophils could be found in the blood, spleen and bone marrow either after 1 day or 3 days after myocardial infarction, indicating that the SiglecFhigh state of neutrophils is unique to the ischemic cardiac tissue.
When I compared SiglecFhigh and SiglecFlow neutrophils regarding their phagocytosis activity and ROS production, SiglecFhigh neutrophils showed a higher phagocytosis ability than their SiglecFlow counterparts, as well as higher ROS production capacity.
In vivo depletion experiments could not achieve successful and efficient depletion of cardiac neutrophils either 1 day or 3 days after myocardial infarction, but led to a shift of a higher percentage of SiglecFhigh expressing neutrophils in the depletion group. Bone marrow neutrophil levels only showed partial depletion at day 3 after MI. Regarding blood neutrophils, depletion efficiently reduced circulating neutrophils at both time points, 1 and 3 days after MI. To summarize, this work showed the time-dependent presence of different neutrophil states in the ischemic heart. The main population of neutrophils isolated 3 days after MI showed a high expression of SiglecF, a unique state that could not be detected at different time points or other organs. These SiglecFhigh neutrophils showed functional differences regarding their phagocytosis ability and ROS production. Further investigation is needed to reveal what role these SiglecFhigh neutrophils could play within the ischemic heart.
To better target neutrophil depletion in vivo, more efficient or different anti-neutrophil strategies are needed.
Gold nanoparticles of diameter ca. 60 nm have been synthesized based on Turkevich and Frens protocols. We have demonstrated that the carboxyl-modified gold nanoparticles can be coupled covalently with antibodies (Ab) of interest using the EDC/NHS coupling procedure. Binding studies with Ab-grafted AuNPs and GpL fusion proteins proved that conjugation of AuNPs with antibodies enables immobilization of antibodies with preservation of a significant antigen binding capacity. More importantly, our findings showed that the conjugation of types of anti-TNF receptors antibodies such as anti-Fn14 antibodies (PDL192 and 5B6) (Aido et al., 2021), anti-CD40, anti-4-1BB and anti-TNFR2 with gold nanoparticles confers them with potent agonism. Thus, our results suggest that AuNPs can be utilized as a platform to immobilize anti-TNFR antibodies which, on the one hand, helps to enhance their agonistic activity in comparison to “free” inactive antibodies by mimicking the effect of cell-anchored antibodies or membrane-bound TNF ligands and, on the other hand, allows to develop new generations of drug delivery systems. These constructs are characterized with their biocompatibility and their tunable synthesis process.
In a further work part, we combined the benefits of the established system of Ab-AuNPs with materials used widely in the modern biofabrication approaches such as the photo-crosslinked hydrogels, methacrylate-modified gelatin (GelMA), combined with embedded variants of human cell lines. The acquired results demonstrated clearly that the attaching of proteins like antibodies to gold nanoparticles might reduce their release rate from the crosslinked hydrogels upon the very low diffusion of gold nanoparticles from the solid constructs to the surrounding medium yielding long-term local functioning proteins-attached particles. Moreover, our finding suggests that hydrogel-embedded AuNP-immobilized antibodies, e.g. anti-TNFα-AuNPs or anti-IL1-AuNPs enable local inhibitory functions,
To sum up, our results demonstrate that AuNPs can act as a platform to attach anti-TNFR antibodies to enhance their agonistic activity by resembling the output of cell-anchoring or membrane bounding. Gold nanoparticles are considered, thus, as promising tool to develop the next generation of drug delivery systems, which may contribute to cancer therapy. On top of that, the embedding of anti-inflammatory-AuNPs in the biofabricated hydrogel presents new innovative strategy of the treatment of autoinflammatory diseases.
Neisseria meningitidis (the meningococcus) is one of the major causes of bacterial meningitis, a life-threatening inflammation of the meninges. Traversal of the meningeal blood-cerebrospinal fluid barrier (mBCSFB), which is composed of highly specialized brain endothelial cells (BECs), and subsequent interaction with leptomeningeal cells (LMCs) are critical for disease progression. Due to the human-exclusive tropism of N. meningitidis, research on this complex host-pathogen interaction is mostly limited to in vitro studies. Previous studies have primarily used peripheral or immortalized BECs alone, which do not retain relevant barrier phenotypes in culture. To study meningococcal interaction with the mBCSFB in a physiologically more accurate context, BEC-LMC co-culture models were developed in this project using BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in combination with LMCs derived from tumor biopsies.
Distinct BEC and LMC layers as well as characteristic expression of cellular markers were observed using transmission electron microscopy (TEM) and immunofluorescence staining. Clear junctional expression of brain endothelial tight and adherens junction proteins was detected in the iBEC layer. LMC co-culture increased iBEC barrier tightness and stability over a period of seven days, as determined by sodium fluorescein (NaF) permeability and transendothelial electrical resistance (TEER). Infection experiments demonstrated comparable meningococcal adhesion and invasion of the BEC layer in all models tested, consistent with previously published data. While only few bacteria crossed the iBEC-LMC barrier initially, transmigration rates increased substantially over 24 hours, despite constant high TEER. After 24 hours of infection, deterioration of the barrier properties was observed including loss of TEER and altered expression of tight and adherens junction components. Reduced mRNA levels of ZO-1, claudin-5, and VE-cadherin were detected in BECs from all models. qPCR and siRNA knockdown data suggested that transcriptional downregulation of these genes was potentially but not solely mediated by Snail1. Immunofluorescence staining showed reduced junctional coverage of occludin, indicating N. meningitidis-induced post-transcriptional modulation of this protein, as previous studies have suggested. Together, these results suggest a potential combination of transcellular and paracellular meningococcal traversal of the mBCSFB, with the more accessible paracellular route becoming available upon barrier disruption after prolonged N. meningitidis infection. Finally, N. meningitidis induced cellular expression of pro-inflammatory cytokines and chemokines such as IL-8 in all mBCSFB models. Overall, the work described in this thesis highlights the usefulness of advanced in vitro models of the mBCSFB that mimic native physiology and exhibit relevant barrier properties to study infection with meningeal pathogens such as N. meningitidis.
Introduction.
Mobile health (mHealth) integrates mobile devices into healthcare, enabling remote monitoring, data collection, and personalized interventions. Machine Learning (ML), a subfield of Artificial Intelligence (AI), can use mHealth data to confirm or extend domain knowledge by finding associations within the data, i.e., with the goal of improving healthcare decisions. In this work, two data collection techniques were used for mHealth data fed into ML systems: Mobile Crowdsensing (MCS), which is a collaborative data gathering approach, and Ecological Momentary Assessments (EMA), which capture real-time individual experiences within the individual’s common environments using questionnaires and sensors. We collected EMA and MCS data on tinnitus and COVID-19. About 15 % of the world’s population suffers from tinnitus.
Materials & Methods.
This thesis investigates the challenges of ML systems when using MCS and EMA data. It asks: How can ML confirm or broad domain knowledge? Domain knowledge refers to expertise and understanding in a specific field, gained through experience and education. Are ML systems always superior to simple heuristics and if yes, how can one reach explainable AI (XAI) in the presence of mHealth data? An XAI method enables a human to understand why a model makes certain predictions. Finally, which guidelines can be beneficial for the use of ML within the mHealth domain? In tinnitus research, ML discerns gender, temperature, and season-related variations among patients. In the realm of COVID-19, we collaboratively designed a COVID-19 check app for public education, incorporating EMA data to offer informative feedback on COVID-19-related matters. This thesis uses seven EMA datasets with more than 250,000 assessments. Our analyses revealed a set of challenges: App user over-representation, time gaps, identity ambiguity, and operating system specific rounding errors, among others. Our systematic review of 450 medical studies assessed prior utilization of XAI methods.
Results.
ML models predict gender and tinnitus perception, validating gender-linked tinnitus disparities. Using season and temperature to predict tinnitus shows the association of these variables with tinnitus. Multiple assessments of one app user can constitute a group. Neglecting these groups in data sets leads to model overfitting. In select instances, heuristics outperform ML models, highlighting the need for domain expert consultation to unveil hidden groups or find simple heuristics.
Conclusion.
This thesis suggests guidelines for mHealth related data analyses and improves estimates for ML performance. Close communication with medical domain experts to identify latent user subsets and incremental benefits of ML is essential.
Development Of A Human iPSC-Derived Cortical Neuron Model Of Adaptor- Protein-Complex-4-Deficiency
(2024)
Adaptor-protein-4-deficiency (AP-4-deficiency) is an autosomal-recessive childhood- onset form of complicated hereditary spastic paraplegia (HSP) caused by bi-allelic loss- of-function mutations in one of the four subunits of the AP-4-complex. These four conditions are named SPG47 (AP4B1, OMIM #614066), SPG50 (AP4M1, OMIM #612936), SPG51 (AP4E1, OMIM #613744) and SPG52 (AP4S1, OMIM #614067), respectively and all present with global developmental delay, progressive spasticity and seizures. Imaging features include a thinning of the corpus callosum, ventriculomegaly and white matter changes. AP-4 is a highly conserved heterotetrameric complex, which is responsible for polarized sorting of transmembrane cargo including the autophagy- related protein 9 A (ATG9A). Loss of any of the four subunits leads to an instable complex and defective sorting of AP-4-cargo. ATG9A is implicated in autophagosome formation and neurite outgrowth. It is missorted in AP-4-deficient cells and CNS-specific knockout of Atg9a in mice results in a phenotype reminiscent of AP-4-deficiency. However, the AP-4-related cellular phenotypes including ATG9A missorting have not been investigated in human neurons.
Thus, the aim of this study is to provide the first human induced pluripotent stem cell- derived (iPSC) cortical neuron model of AP-4-deficiency to explore AP-4-related phenotypes in preparation for a high-content screening. Under the hypothesis that AP-4- deficiency leads to ATG9A missorting, elevated ATG9A levels, impaired autophagy and neurite outgrowth in human iPSC-derived cortical neurons, in vitro biochemical and imaging assays including automated high-content imaging and analysis were applied. First, these phenotypes were investigated in fibroblasts from three patients with compound heterozygous mutations in the AP4B1 gene and their sex-matched parental controls. The same cell lines were used to generate iPSCs and differentiate them into human excitatory cortical neurons.
This work shows that ATG9A is accumulating in the trans-Golgi-network in AP-4- deficient human fibroblasts and that ATG9A levels are increased compared to parental controls and wild type cells suggesting a compensatory mechanism. Protein levels of the AP4E1-subunit were used as a surrogate marker for the AP-4-complex and were decreased in AP-4-deficient fibroblasts with co-immunoprecipitation confirming the instability of the complex. Lentiviral re-expression of the AP4B1-subunit rescues this corroborating the fact that a stable AP-4-complex is needed for ATG9A trafficking. Surprisingly, autophagic flux was present in AP-4-deficient fibroblasts under nutrient- rich and starvation conditions. These phenotypic markers were evaluated in iPSC-derived cortical neurons and here, a robust accumulation of ATG9A in the juxtanuclear area was seen together with elevated ATG9A protein levels. Strikingly, assessment of autophagy markers under nutrient-rich conditions showed alterations in AP-4-deficient iPSC- derived cortical neurons indicating dysfunctional autophagosome formation. These findings point towards a neuron-specific impairment of autophagy and need further investigation. Adding to the range of AP-4-related phenotypes, neurite outgrowth and branching are impaired in AP-4-deficient iPSC-derived cortical neurons as early as 24h after plating and together with recent studies point towards a distinct role of ATG9A in neurodevelopment independent of autophagy.
Together, this work provides the first patient-derived neuron model of AP-4-deficiency and shows that ATG9A is sorted in an AP-4-dependent manner. It establishes ATG9A- related phenotypes and impaired neurite outgrowth as robust markers for a high-content screening. This disease model holds the promise of providing a platform to further study AP-4-deficiency and to search for novel therapeutic targets.
Different effects of conditional Knock-Out of Stat3 on the sensory epithelium of the Organ of Corti
(2024)
The mammalian cochlea detects sound in response to vibration at frequency-dependent positions along the cochlea duct. The sensory outer hair cells, which are surrounded by supporting cells, act as a signal amplifier by changing their cell length. This is called electromotility. To ensure correct electrical transmission during mechanical forces, a certain resistance of the sensory epithelium is a prerequisite for correct transduction of auditory information. This resistance is managed by microtubules and its posttranslational modification in the supporting cells of the sensory epithelium of the cochlea. Stat3 is a transcription factor, with its different phosphorylation sites, is involved in many cellular processes like differentiation, inflammation, cell survival and microtubule dynamics, depending on cell type and activated pathway. While Stat3 has a wide range of intracellular roles, the question arose, how and if Stat3 is involved in cells of the organ of Corti to ensure a correct hearing.
To test this, Cre/loxp system were used to perform conditional Knock-Out (cKO) of Stat3 in outer hair cells or supporting cells either before hearing onset or after hearing onset. Hearing performances included DPOAE and ABR measurements, while molecular were performed by sequencing. Additionally, morphological examination was used by immunohistochemistry and electron microscopy.
A cKO of Stat3 before and after hearing onset in outer hair cells leads to hearing impairments, whereas synapses, nerve fibers and mitochondria were not affected. Bulk sequencing analyzation of outer hair cells out of cKO mice before hearing onset resulted in a disturbance of cellular homeostasis and extracellular signals. A cKO of Stat3 in the outer hair cells after hearing onset resulted in inflammatory signaling pathway with increased cytokine production and upregulation of NF-kb pathway. In supporting cells, cKO of Stat3 only after hearing onset resulted in a hearing impairment. However, synapses, nerve soma and fibers were not affected of a cKO of Stat3 in supporting cells. Nevertheless, detyronisated modification of microtubules were altered, which can lead to an instability of supporting cells during hearing.
In conclusion, Stat3 likely interact in a cell-specific and function-specific manner in cells of the organ of Corti. While a cKO in outer hair cells resulted in increased cytokine production, supporting cells altered its stability due to decreased detyronisated modification of microtubules. Together the results indicated that Stat3 is an important protein for hearing performances. However, additional investigations of the molecular mechanism are needed to understand the role of Stat3 in the cells of the organ of Corti.
The RNAs of many viruses contain a frameshift stimulatory element (FSE) that grants access to an alternate reading frame via −1 programmed ribosomal frameshifting (PRF). This −1PRF is essential for effective viral replication. The −1PRF efficiency relies on the presence of conserved RNA elements within the FSE, such as a slippery sequence, spacer, and a downstream secondary structure – often a hairpin or a pseudoknot. The PRF efficiency is also affected by trans-acting factors such as proteins, miRNAs and metabolites. The interactions of these factors with the RNA and the translation machinery have not yet been completely understood. Traditional ensemble methods used previously to study these events focus on the whole population of molecular species. This results in innate averaging of the molecular behavior and a loss of heterogeneity information.
Here, we first established the experimental workflow to study the RNA structures and the effect of potential trans-acting factors using single-molecule force spectroscopy technique, optical tweezers. Additionally, to streamline the data analysis, we developed an algorithm for automatized data processing.
Next, we harnessed this knowledge to study viral RNA elements responsible for stimulation of PRF and how the presence of trans-acting factors affects the RNA behavior. We further complemented these single-molecule structural data with ensemble functional assays to gain a complex view on the dynamics behind the programmed ribosomal frameshifting.
Specifically, two different viral RNA elements have been studied in the presented work. First, the dynamics of SARS-CoV-2 FSE and the role of extended sequences have been explored. Then, the mode of action of the host-encoded trans-acting factor ZAP-S inhibition of SARS-CoV-2 PRF has been examined. Finally, the mechanism of the trans-acting viral factor induced PRF in Encephalomyocarditis virus (EMCV) has been uncovered.
Ischemia-reperfusion injury (I/R injury) is a common complication in ischemic stroke (IS) treatment, which is characterized by a paradoxical perpetuation of tissue damage despite the successful re-establishment of vascular perfusion. This phenomenon is known to be facilitated by the detrimental interplay of platelets and inflammatory cells at the vascular interface. However, the spatio-temporal and molecular mechanisms underlying these cellular interactions and their contribution to infarct progression are still incompletely understood. Therefore, this study intended to clarify the temporal mechanisms of infarct growth after cerebral vessel recanalization. The data presented here could show that infarct progression is driven by early blood-brain-barrier perturbation and is independent of secondary thrombus formation. Since previous studies unravelled the secretion of platelet granules as a molecular mechanism of how platelets contribute to I/R injury, special emphasis was placed on the role of platelet granule secretion in the process of barrier dysfunction. By combining an in vitro approach with a murine IS model, it could be shown that platelet α-granules exerted endothelial-damaging properties, whereas their absence (NBEAL2-deficiency) translated into improved microvascular integrity. Hence, targeting platelet α-granules might serve as a novel treatment option to reduce vascular integrity loss and diminish infarct growth despite recanalization.
Recent evidence revealed that pathomechanisms underlying I/R injury are already instrumental during large vessel occlusion. This indicates that penumbral tissue loss under occlusion and I/R injury during reperfusion share an intertwined relationship. In accordance with this notion, human observational data disclosed the presence of a neutrophil dominated immune response and local platelet activation and secretion, by the detection of the main components of platelet α-granules, within the secluded vasculature of IS patients. These initial observations of immune cells and platelets could be further expanded within this thesis by flow cytometric analysis of local ischemic blood samples. Phenotyping of immune cells disclosed a yet unknown shift in the lymphocyte population towards CD4+ T cells and additionally corroborated the concept of an immediate intravascular immune response that is dominated by granulocytes. Furthermore, this thesis provides first-time evidence for the increased appearance of platelet-leukocyte-aggregates within the secluded human vasculature. Thus, interfering with immune cells and/or platelets already under occlusion might serve as a potential strategy to diminish infarct expansion and ameliorate clinical outcome after IS.
Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted:
1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI
2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis
3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics
Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice.
The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition.
In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly – at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe−/− mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD.
Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis.
Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models – two parameters highly influencing plaque development and progression – there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis.
Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development.
Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis.
Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models.
In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.
Drug Discovery based on Oxidative Stress and HDAC6 for Treatment of Neurodegenerative Diseases
(2024)
Most antioxidants reported so far only achieved limited success in AD clinical trials. Growing evidences suggest that merely targeting oxidative stress will not be sufficient to fight AD. While multi-target directed ligands could synergistically modulate different steps in the neurodegenerative process, offering a promising potential for treatment of this complex disease.
Fifteen target compounds have been designed by merging melatonin and ferulic acid into the cap group of a tertiary amide HDAC6 inhibitor. Compound 10b was screened as the best hybrid molecule exhibit potent HDAC6 inhibition and potent antioxidant capacity. Compound 10b also alleviated LPS-induced microglia inflammation and led to a switch from neurotoxic M1 to the neuroprotective M2 microglial phenotype. Moreover, compound 10b show pronounced attenuation of spatial working memory and long-term memory damage in an in vivo AD mouse model. Compound 10b can be a potentially effective drug candidate for treatment of AD and its druggability worth to be further studied.
We have designed ten novel neuroprotectants by hybridizing with several common antioxidants, including ferulic acid, melatonin, lipoic acid, and trolox. The trolox hybrid compound exhibited the most potent neuroprotective effects in multiple neuroprotection assays. Besides, we identified the synergistic effects between trolox and vitamin K derivative, and our trolox hybrid compound showed comparable neuroprotection with the mixture of trolox and vitamin K derivative.
We have designed and synthesized 24 quinone derivatives based on five kinds of different quinones including ubiquinone, 2,3,5-trimethyl-1,4-benzoquinone, memoquin, thymoquinone, and anthraquinone. Trimethylbenzoquinone and thymoquinone derivatives showed more potent neuroprotection than other quinones in oxytosis assay. Therefore, trimethylbenzoquinone and thymoquinone derivatives can be used as lead compounds for further mechanism study and drug discovery for treatment of neurodegenerative disease.
We designed a series of photoswitchable HDAC inhibitors, which could be effective molecular tools due to the high spatial and temporal resolution. In total 23 target compounds were synthesized and photophysicochemically characterized. Azoquinoline-based compounds possess more thermally stable cis-isomers in buffer solution, which were further tested in enzyme-based HDAC inhibition assay. However, none of those tested compounds show significant differences in activities between trans-isomers and corresponding cis-isomers.
Ecophysiological adaptations of the cuticular water permeability within the Solanaceae family
(2024)
The cuticle, a complex lipidic layer synthesized by epidermal cells, covers and protects primary organs of all land plants. Its main function is to avoid plant desiccation by limiting non-stomatal water loss. The cuticular properties vary widely among plant species. So far, most of the cuticle-related studies have focused on a limited number of species, and studies addressing phylogenetically related plant species are rare. Moreover, comparative studies among organs from the same plant species are still scarce.
Thus, this study focus on organ-specificities of the cuticle within and between plant species of the Solanaceae family. Twenty-seven plant species of ten genera, including cultivated and non- cultivated species, were investigated to identify potential cuticular similarities. Structural, chemical and functional traits of fully expanded leaves, inflated fruiting calyces, and ripe fruits were analyzed.
The surface morphology was investigated by scanning electron microscopy. Leaves were mainly amphistomatic and covered by an epicuticular wax film. The diversity and distribution of trichomes varied among species. Only the leaves of S. grandiflora were glabrous. Plant species of the Leptostemonum subgenus had numerous prickles and non-glandular stellate trichomes. Fruits were stomata-free, except for S. muricatum, and a wax film covered their surface. Last, lenticel- like structures and remaining scars of broken trichomes were found on the surface of some Solanum fruits.
Cuticular water permeability was used as indicators of the cuticular transpiration barrier efficiency. The water permeability differed among plant species, organs and fruit types with values ranging up to one hundred-fold. The minimum leaf conductance ranged from 0.35 × 10-5 m s-1 in S. grandiflora to 31.54 × 10-5 m s-1 in S. muricatum. Cuticular permeability of fruits ranged from 0.64 × 10-5 m s-1 in S. dulcamara (fleshy berry) to 34.98 × 10-5 m s-1 in N. tabacum (capsule). Generally, the cuticular water loss of dry fruits was about to 5-fold higher than that of fleshy fruits.
Interestingly, comparisons between cultivated and non-cultivated species showed that wild species have the most efficient cuticular transpiration barrier in leaves and fruits. The average permeability of leaves and fruits of wild plant species was up to three-fold lower in comparison to the cultivated ones. Moreover, ripe fruits of P. ixocarpa and P. peruviana showed two-times lower cuticular transpiration when enclosed by the inflated fruiting calyx.
The cuticular chemical composition was examined using gas chromatography. Very-long-chain aliphatic compounds primarily composed the cuticular waxes, being mostly dominated by n- alkanes (up to 80% of the total wax load). Primary alkanols, alkanoic acids, alkyl esters and branched iso- and anteiso-alkanes were also frequently found. Although in minor amounts, sterols, pentacyclic triterpenoids, phenylmethyl esters, coumaric acid esters, and tocopherols were identified in the cuticular waxes. Cuticular wax coverages highly varied in solanaceous (62- fold variation). The cuticular wax load of fruits ranged from 0.55 μg cm−2 (Nicandra physalodes) to 33.99 μg cm−2 (S. pennellii), whereas the wax amount of leaves varied from 0.90 μg cm−2 (N. physalodes) to 28.42 μg cm−2 (S. burchellii). Finally, the wax load of inflated fruiting calyces ranged from 0.56 μg cm−2 in P. peruviana to 2.00 μg cm−2 in N. physalodes.
For the first time, a comparative study on the efficiency of the cuticular transpiration barrier in different plant organs of closely related plant species was conducted. Altogether, the cuticular chemical variability found in solanaceous species highlight species-, and organ-specific wax biosynthesis. These chemical variabilities might relate to the waterproofing properties of the plant cuticle, thereby influencing leaf and fruit performances. Additionally, the high cuticular water permeabilities of cultivated plant species suggest a potential existence of a trade-off between fruit organoleptic properties and the efficiency of the cuticular transpiration barrier. Last, the high cuticular water loss of the solanaceous dry fruits might be a physiological adaptation favouring seed dispersion.
After priming in Peyer's patches (PPs) and mesenteric lymph nodes (mLN) T- cells infiltrate the intestine through lymphatic draining and homing through the bloodstream. However, we found that in mouse models of acute graft-versus-host disease (GvHD), a subset of alloreactive T-cells directly migrates from PPs to the adjacent intestinal lamina propria (LP), bypassing the normal lymphatic drainage and vascular trafficking routes. Notably, this direct migration occurred in irradiated and unirradiated GvHD models, indicating that irradiation is not a prerequisite for this observed behavior.
Next, we established a method termed serial intravascular staining (SIVS) in mouse models to systematically investigate the trafficking and migration of donor T- cells in the early stages of acute GvHD initiation. We found that the direct migration of T-cells from PPs to LP resulted in faster recruitment of cells after allogeneic hematopoietic cell transplantation (allo-HCT). These directly migrating T-cells were found to be in an activated and proliferative state, exhibiting a TH1/TH17-like phenotype and producing cytokines such as IFN-γ and TNF-α. Furthermore, we observed that the directly migrating alloreactive T-cells expressed specific integrins (α4+, αE+) and chemokine receptors (CxCR3+, CCR5+, and CCR9+). Surprisingly, blocking these integrins and chemokine-coupled receptors did not hinder the direct migration of T- cells from PPs to LP, suggesting the involvement of alternative mechanisms. Previous experiments ruled out the involvement of S1PR1 and topographical features of macrophages, leading us to hypothesize that mediators of cytoskeleton reorganization, such as Coro1a, Dock2, or Cdc42, may play a role in this unique migration process.
Additionally, we observed that directly migrating T-cells created a local inflammatory microenvironment, which attracts circulating T-cells. Histological analysis confirmed that alloreactive PPs-derived T-cells and bloodborne T-cells colocalized. We employed two experimental approaches, including either photoconversion of T-cells in PPs or direct transfer of activated T-cells into the vasculature, to demonstrate this colocalization. We hypothesize that cytokines released by migrating T-cells, such as IFN-γ and TNF-α, may play a role in recruiting T-cells from the vasculature, as inhibiting chemokine-coupled receptors did not impair recruitment.
The unicellular pathogen Trypanosoma brucei is the causative agent of African
trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream
form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF.
In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis.
Biofabrication technologies must address numerous parameters and conditions to reconstruct tissue complexity in vitro. A critical challenge is vascularization, especially for large constructs exceeding diffusion limits. This requires the creation of artificial vascular structures, a task demanding the convergence and integration of multiple engineering approaches. This doctoral dissertation aims to achieve two primary objectives: firstly, to implement and refine engineering methods for creating artificial microvascular structures using Melt Electrowriting (MEW)-assisted sacrificial templating, and secondly, to deepen the understanding of the critical factors influencing the printability of bioink formulations in 3D extrusion bioprinting.
In the first part of this dissertation, two innovative sacrificial templating techniques using MEW are explored. Utilizing a carbohydrate glass as a fugitive material, a pioneering advancement in the processing of sugars with MEW with a resolution under 100 microns was made. Furthermore, by introducing the “print-and-fuse” strategy as a groundbreaking method, biomimetic branching microchannels embedded in hydrogel matrices were fabricated, which can then be endothelialized to mirror in vivo vascular conditions.
The second part of the dissertation explores extrusion bioprinting. By introducing a simple binary bioink formulation, the correlation between physical properties and printability was showcased. In the next step, employing state-of-the-art machine-learning approaches revealed a deeper understanding of the correlations between bioink properties and printability in an extended library of hydrogel formulations.
This dissertation offers in-depth insights into two key biofabrication technologies. Future work could merge these into hybrid methods for the fabrication of vascularized constructs, combining MEW's precision with fine-tuned bioink properties in automated extrusion bioprinting.
Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells is an effective treatment for hematological malignancies that are refractory to conventional chemotherapy. To address a wider variety of cancer entities, there is a need to identify and characterize additional target antigens for CAR-T cell therapy. The two members of the receptor tyrosine kinase-like orphan receptor family, ROR1 and ROR2, have been found to be overexpressed on cancer cells and to correlate with aggressive cancer phenotypes. Recently, ROR1-specific CAR-T cells have entered testing in phase I clinical trials, encouraging us to assess the suitability of ROR2 as a novel target for CAR-T cell therapy. To study the therapeutic potential of targeting ROR2 in solid and hematological malignancies, we selected two representative cancer entities with high unmet medical need: renal cell carcinoma and multiple myeloma.
Our data show that ROR2 is commonly expressed on primary samples and cell lines of clear cell renal cell carcinoma and multiple myeloma. To study the efficacy of ROR2-specific CAR T cell therapy, we designed two CAR constructs with 10-fold binding affinity differences for the same epitope of ROR2. We found both cell products to exhibit antigen-specific anti-tumor reactivity in vitro, including tumor cell lysis, secretion of the effector cytokines interleukin-2 (IL-2) and interferon-gamma (IFNγ), and T cell proliferation. In vivo studies revealed ROR2 specific CAR-T cells to confer durable responses, significant survival benefits and long-term persistence of CAR-expressing T cells. Overall, there was a trend towards more potent anti-tumor efficacy upon treatment with T cells that expressed the CAR with higher affinity for ROR2, both in vitro and in vivo.
We performed a preclinical safety and toxicology assessment comprising analyses of ROR2 expression in healthy human and murine tissues, cross-reactivity, and adoptive T cell transfer in immunodeficient mice. We found ROR2 expression to be conserved in mice, and low-level expression was detectable in the male and female reproductive system as well as parts of the gastrointestinal tract. CAR-T cells targeting human ROR2 were found to elicit similarly potent reactivity upon recognition of murine ROR2. In vivo analyses showed transient tissue-specific enrichment and activation of ROR2-specific CAR-T cells in organs with high blood circulation, such as lung, liver, or spleen, without evidence for clinical toxicity or tissue damage as determined by histological analyses.
Furthermore, we humanized the CAR binding domain of ROR2-specific CAR-T cells to mitigate the risk of adverse immune reactions and concomitant CAR-T cell rejection. Functional analyses confirmed that humanized CARs retained their specificity and functionality against ROR2-positive tumor cells in vitro.
In summary, we show that ROR2 is a prevalent target in RCC and MM, which can be addressed effectively with ROR2-specific CAR-T cells in preclinical models. Our preliminary toxicity studies suggest a favorable safety profile for ROR2-specific CAR-T cells. These findings support the potential to develop ROR2-specific CAR-T cells clinically to obtain cell products with broad utility.
Biological systems are in dynamic interaction. Many responses reside in the core concepts of biological systems interplay (competition and cooperation). In infection situation, the competition between a bacterial system and a host is shaped by many stressors at spatial and temporal determinants. Reactive chemical species are universal stressors against all biological systems since they potentially damage the basic requirements of these systems (nucleic acids, proteins, carbohydrates, and lipids). Either produced endogenously or exogenously, reactive chemical species affect the survival of pathogens including the gram-positive
Staphylococcus aureus (S. aureus). Therefore, bacteria developed strategies to overcome the toxicity of reactive species.
S. aureus is a widely found opportunistic pathogen. In its niche, S. aureus is in permanent contact with surrounding microbes and host factors. Deciphering the deterministic factors
in these interactions could facilitate pinpointing novel bacterial targets. Identifying
the aforementioned targets is crucial to develop new strategies not only to kill the pathogenic organisms but also to enhance the normal flora to minimize the pathogenicity and virulence of potential pathogens. Moreover, targeting S. aureus stress response can be used
to overcome bacterial resistance against host-derived factors. In this study, I identify a novel
S. aureus stress response factor against reactive electrophilic, oxygen, and hypochlorite species to better understand its resilience as a pathogen.
Although bacterial stress response is an active research field, gene function is a current bottleneck in characterizing the understudied bacterial strategies to mediate stress conditions. I aimed at understanding the function of a novel protein family integrated
in many defense systems of several biological systems.
In bacteria, fungi, and plants, old yellow enzymes (OYEs) are widely found. Since the first isolation of the yellow flavoprotein, OYEs are used as biocatalysts for decades to reduce activated C=C bonds in α,β-unsaturated carbonyl compounds. The promiscuity
of the enzymatic catalysis is advantageous for industrial applications.
However, the physiological function of OYEs, especially in bacteria, is still puzzling.
Moreover, the relevance of the OYEs in infection conditions remained enigmatic.
Here, I show that there are two groups of OYEs (OYE flavin oxidoreductase, OfrA and OfrB) that are encoded in staphylococci and some firmicutes. OfrA (SAUSA300_0859) is more conserved than OfrB (SAUSA300_0322) in staphylococci and is a part of the staphylococcal core genome.
A reporter system was established to report for ofrA in S. aureus background.
The results showed that ofrA is induced under electrophilic, oxidative, and hypochlorite stress. OfrA protects S. aureus against quinone, methylglyoxal, hydrogen peroxide,
and hypochlorite stress. Additionally, the results provide evidence that OfrA supports
thiol-dependent redox homeostasis. At the host-pathogen interface, OfrA promotes S. aureus fitness in murine macrophage cell line. In whole human blood, OfrA is involved in S. aureus survival indicating a potential clinical relevance to bacteraemia.
In addition, ofrA mutation affects the production of the virulence factor staphyloxanthin via the upper mevalonate pathway. In summary, decoding OfrA function and its proposed mechanism of action in S. aureus shed the light on a conserved stress response within multiple organisms.
The mold Aspergillus fumigatus (A. fumigatus) is known as human pathogen and can cause life-threatening infections in humans with a weakened immune system. This is a known complication in patients receiving glucocorticoids, e.g. after hematopoietic stem cell transplantation or solid organ transplantation. Although research in the field of immune cell/fungus interaction has discovered key strategies how immune cells fight against infectious fungi, our knowledge is still incomplete. In order to develop effective treatment options against fungal infections, a detailed understanding of their interactions is crucial. Thus, visualization of immune cell and fungus is an excellent approach to gain further knowledge. For a detailed view of such interaction processes, a high optical resolution on nanometer scale is required. There is a variety of super resolution microscopy techniques, enabling fluorescence imaging beyond the diffraction limit. This work combines the use of three complementary super resolution microscopy techniques, in order to study immune cell/fungus interaction from different points of view.
Aim of this work is the introduction of the recently invented imaging technique named expansion microscopy (ExM) for the study of immune cell/fungus interactions. The core aspect of this method is the physical magnification of the specimen, which increases the distance between protein structures that are close to each other and which can therefore be imaged separately.
The simultaneous magnification of primary human natural killer (NK) cells and A. fumigatus hyphae was established in this work using ExM. Reorganization of cytoskeletal components of interacting NK cells was demonstrated here, by expansion of the immunological synapse (IS), formed between NK cells and A. fumigatus. In addition, reorganization of the microtubule-organizing center (MTOC) towards fungal hyphae and an accumulation of actin at the IS has been observed. Furthermore, ExM has been used to visualize lytic granules of NK cells after degranulation. After magnification of the specimen, lysosome associated protein 1 (LAMP1) was shown to surround perforin. In absence of the plasma membrane-exposed degranulation marker LAMP1, a “ring-shaped” structure was often observed for fluorescently labeled perforin. Volume calculation of lytic granules demonstrated the benefit of ExM. Compared to pre-expansion images, analyses of post-expansion images showed two volume distributions for degranulated and non-degranulated NK cells. In addition, this work emphasizes the importance of determining the expansion factor for a structure in each species, as variations of expansion factors have been observed. This factor, as well as possible sample distortions should be considered, when ExM is used in order to analyze the interaction between two species.
A second focus of this work is the visualization of a chimeric antigen receptor (CAR), targeting an epitope on the cell wall of A. fumigatus. Structured illumination microscopy (SIM) revealed that the CAR is part of the immunological synapse of primary human CAR T cells and CAR-NK-92 cells. At the interaction site, an accumulation of the CAR was observed, as well as the presence of perforin. CAR accumulation at fungal hyphae was further demonstrated by automated live cell imaging of interacting CAR-NK-92 cells, expressing a fluorescent fusion protein.
Additionally, the use of direct stochastic optical reconstruction microscopy (dSTORM) gave first insights in CAR expression levels on the basal membrane of CAR-NK-92 cells, with single molecule sensitivity. CAR cluster analyses displayed a heterogeneous CAR density on the basal membrane of transfected NK 92 cells.
In summary, this work provides insights into the application of ExM for studying the interaction of primary human NK cells and A. fumigatus for the first time. Furthermore, this thesis presents first insights regarding the characterization of an A. fumigatus-targeting CAR, by applying super-resolution fluorescence microscopy, like SIM and dSTORM.
mRNA is co- or post-transcriptionally processed from a precursor mRNA to a mature mRNA. In addition to 5'capping and splicing, these modifications also include polyadenylation, the addition of a polyA tail to the 3'end of the mRNA. In recent years, alternative polyadenylation in particular has increasingly been taken into account as a mechanism for regulating gene expression. It is assumed that approximately 70-75 % of human protein coding genes contain alternative polyadenylation signals, which are often located within intronic sequences of protein-coding genes. The use of such polyadenylation signals leads to shortened mRNA transcripts and thus to the generation of C-terminal shortened protein isoforms.
Interestingly, the majority of microRNAs, small non-coding RNAs that play an essential role in post-transcriptional gene regulation, are also encoded in intronic sequences of protein-coding genes and are co-transcriptionally expressed with their host genes. The biogenesis of microRNA has been well studied and is well known, but mechanisms that may influence the expression regulation of mature microRNAs are just poorly understood.
In the presented work, I aimed to investigate the influence of alternative intronic polyadenylation on the biogenesis of microRNAs. The human ion channel TRPM1 could already be associated with melanoma pathogenesis and truncated isoforms of this protein have already been described in literature. In addition, TRPM1 harbors a microRNA, miR211, in its sixth intron, which is assumed to act as a tumor suppressor. Since both, TRPM1 and miR211 have already been associated with melanoma pathogenesis, the shift towards truncated transcripts during the development of various cancers is already known and it has been shown that certain microRNAs play a crucial role in the development and progression of melanoma, melanoma cell lines were used as an in vitro model for these investigations.
Gastroesophageal junction (GEJ), demarcating the region where the distal esophagus meets with the proximal stomach region, is known for developing pathological conditions, including metaplasia and esophageal adenocarcinoma (EAC). It is essential to understand the mechanisms of developmental stages which lead to EAC since the incidence rate of EAC increased over 7-fold during the past four decades, and the overall five years survival rate is 18.4%. In most cases, patients are diagnosed in the advanced stage without prior symptoms. The main precursor for the development of EAC is a pre-malignant condition called Barrett's esophagus (BE). BE is the metaplastic condition where the multilayered squamous epithelium of the native esophagus is replaced by specialized single-layered columnar epithelium, which shows the molecular characteristics of the gastric as well as intestinal epithelium. The main risk factors for BE development include chronic gastro-esophageal acid reflux disease (GERD), altered microbiota, and altered retinoic acid signaling (RA). The cell of origin of BE is under debate due to a lack of clear evidence demonstrating the process of BE initiation. Here, I investigated how GEJ homeostasis is maintained in healthy tissue by stem cell regulatory morphogens, the role of vitamin A (RA signaling), and how its alteration contributes to BE development.
In the first part of my thesis, I showed the presence of two types of epithelial cells, the squamous type in the esophagus and the columnar type in the stomach region in the GEJ, using single-molecule RNA in situ hybridization (smRNA-ISH) and immunohistochemistry. Employing lineage tracing in the mouse model, I have demonstrated that the esophageal epithelial and stomach epithelial cells derived from two distinct epithelial stem cell lineages in the GEJ. The border between squamous and columnar epithelial cells in the Squamo-columnar junction (SCJ) of GEJ is regulated by opposing Wnt microenvironments. The regeneration of stomach columnar epithelial stem cells is maintained by Wnt activating signal from the stromal compartment while squamous epithelial stem cells of the esophagus are maintained by the Wnt inhibitory signals. I recapitulated the in vivo GEJ epithelial stem cell maintenance by using in vitro epithelial 3D organoid culture model. The growth and propagation of stomach columnar epithelial organoids depend on Wnt growth factors, while squamous epithelial organoids' development needs Wnt-deficient culture conditions.
Further, single-cell RNA sequence (scRNA-seq) analysis of organoid-derived epithelial cells revealed the non-canonical Wnt/ planar cell polarity (PCP) pathway involvement in regulating the squamous epithelial cells. In contrast, columnar stomach epithelial cells are regulated by the canonical Wnt/ beta-catenin and non-canonical Wnt/Ca2+ pathways. My data indicate that the SCJ epithelial cells that merge at the GEJ are regulated by opposing stromal Wnt factors and distinct Wnt pathway signaling in the epithelial cells.
In the second part of the thesis, I investigated the role of Vitamin A-derived bioactive compound RA on esophageal and stomach epithelial stem cells. In vitro treatment of esophageal and stomach, epithelial organoids with RA or its pharmacological inhibitor BMS 493 revealed that each cell type was regulated distinctly. I observed that enhanced RA promoted esophageal stem cell differentiation and loss of stratification, while RA inhibition led to enhanced stemness and regeneration of the esophagus stratified epithelium. As opposed to the esophagus, RA signaling is active in the stomach organoids, and inhibition of RA reduces the growth of stomach organoids. Global transcriptomic data and scRNA-seq data revealed that RA signaling induces dormancy phenotype in the esophageal cells. In contrast, the absence of RA in stomach epithelial cells induces the expression of genes associated with BE. Thus, spatially defined regulation of Wnt and RA signaling at GEJ is critical for healthy homeostasis, and its perturbation leads to disease development.
This compilation focuses on adolescent mental disorders and their prevention. It comprises three distinct studies, each contributing to a deeper understanding of this critical topic. This work addresses a critical gap in the understanding of, and approach to, adolescent mental health, and as a result reveals a critically important and urgently needed policy implication for action. The thematic structure of these studies begins with an examination of the epidemiology of child and adolescent mental disorders. Baseline data were collected from N = 877 adolescents with a mean age of 12.43 years (SD = 0.65). Mental health problems, such as depressive symptoms, non-suicidal self-injury, suicidal ideation, symptoms of eating disorders, and gender differences, are thoroughly examined. Results revealed a significant portion of our sample displaying mental health problems as early as the 6th and 7th grades, with girls generally being more affected than boys. The findings underscore the importance of early adolescence in the emergence of mental health problems and thereby emphasize the need for preventive measures. Moving beyond prevalence estimates, the compilation delves into the etiology of these disorders, exploring their potential correlation with a COVID-19 infection. Understanding the early signs and risk factors is crucial for timely support. While numerous studies have investigated potential risk and protective factors during the pandemic, our focus shifts to adolescents’ coping when an infection with the virus was involved (N = 2,154, M = 12.31, SD = 0.67). We hypothesized that students infected or with close family members infected, would exhibit an increased psychopathology and a decreased functioning of protective factors such as self-efficacy or self-esteem. We found no connection between infection and the mental health status within our sample, but protective factors and mental well-being were positively associated. Thus, universal primary prevention appears to be the preferred approach for promoting mental health. Lastly, the compilation introduces LessStress, a noteworthy contribution to more evidence-based prevention programs. This universal approach is designed to reduce stress in schools, accompanied by a cluster-randomized trial to evaluate its effectiveness (estimated sample size N = 1,894). Existing studies have demonstrated the effectiveness of stress prevention, leading us to introduce a short and easy-to-implement prevention program. There is positive evidence for one-lesson interventions in schools for promoting well-being and health behaviors among adolescents. LessStress is designed based on a life skills approach that not only imparts psychoeducational content but also teaches skills relevant to everyday life and directly applicable. Throughout these studies, a common thread emerges: the pressing need to address mental disorders during childhood and adolescence. These formative years play a pivotal role in the development of mental health problems. These formative years play a crucial role in the development of mental health problems. They highlight the importance of epidemiological data collection and analysis based on the latest models to develop prevention interventions that are not only effective but also reach young people on a global level.
Early-onset torsion dystonia (DYT-TOR1A, DYT1) is an inherited hyperkinetic movement disorder caused by a mutation of the TOR1A gene encoding the torsinA protein. DYT-TOR1A is characterized as a network disorder of the central nervous system (CNS), including predominantly the cortico-basal ganglia-thalamo-cortical loop resulting in a severe generalized dystonic phenotype. The pathophysiology of DYTTOR1A is not fully understood. Molecular levels up to large-scale network levels of the CNS are suggested to be affected in the pathophysiology of DYT-TOR1A. The reduced penetrance of 30% - 40% indicates a gene-environmental interaction, hypothesized as “second hit”. The lack of appropriate and phenotypic DYT-TOR1A animal models encouraged us to verify the “second hit” hypothesis through a unilateral peripheral nerve trauma of the sciatic nerve in a transgenic asymptomatic DYT-TOR1A rat model (∆ETorA), overexpressing the human mutated torsinA protein. In a multiscale approach, this animal model was characterized phenotypically and pathophysiologically.
Nerve-injured ∆ETorA rats revealed dystonia-like movements (DLM) with a partially generalized phenotype. A physiomarker of human dystonia, describing increased theta oscillation in the globus pallidus internus (GPi), was found in the entopeduncular nucleus (EP), the rodent equivalent to the human GPi, of nerve-injured ∆ETorA rats. Altered oscillation patterns were also observed in the primary motor cortex. Highfrequency stimulation (HFS) of the EP reduced DLM and modulated altered oscillatory activity in the EP and primary motor cortex in nerve-injured ∆ETorA rats. Moreover, the dopaminergic system in ∆ETorA rats demonstrated a significant increased striatal dopamine release and dopamine turnover. Whole transcriptome analysis revealed differentially expressed genes of the circadian clock and the energy metabolism, thereby pointing towards novel, putative pathways in the pathophysiology of DYTTOR1A dystonia.
In summary, peripheral nerve trauma can trigger DLM in genetically predisposed asymptomatic ΔETorA rats leading to neurobiological alteration in the central motor network on multiple levels and thereby supporting the “second hit” hypothesis. This novel symptomatic DYT-TOR1A rat model, based on a DYT-TOR1A genetic background, may prove as a valuable chance for DYT-TOR1A dystonia, to further investigate the pathomechanism in more detail and to establish new treatment strategies.
The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment.
De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis.
SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis.
SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation.
It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth.
A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation.
In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients.
Barth Syndrome (BTHS) is an inherited X-chromosomal linked disorder, characterized by early development of cardiomyopathy, immune system defects, skeletal muscle myopathy and growth retardation. The disease displays a wide variety of symptoms including heart failure, exercise intolerance and fatigue due to the muscle weakness. The cause of the disease are mutations in the gene encoding for the mitochondrial transacylase Tafazzin (TAZ), which is important for remodeling of the phospholipid cardiolipin (CL). All mutations result in a pronounced decrease of the functional enzyme leading to an increase of monolysocardiolipin (MLCL), the precursor of mature CL, and a decrease in mature CL itself. CL is a hallmark phospholipid of mitochondrial membranes, highly enriched in the inner mitochondrial membrane (IMM). It is not only important for the formation of the cristae structures, but also for the function of different protein complexes associated with the mitochondrial membrane. Reduced levels of mature CL cause remodeling of the respiratory chain supercomplexes, impaired respiration, defects in the Krebs cycle and a loss of mitochondrial calcium uniporter (MCU) protein. The defective Ca2+ handling causes impaired redox homeostasis and energy metabolism resulting in cellular arrhythmias and defective electrical conduction. In an uncompensated situation, blunting mitochondrial Ca2+ uptake provokes increased mitochondrial emission of H2O2 during workload transitions, related to oxidation of NADPH, which is required to regenerate anti-oxidative enzymes. However, in the hearts and cardiac myocytes of mice with a global knock-down of the Taz gene (Taz-KD), no increase in mitochondrial ROS was observed, suggesting that other metabolic pathways may have compensated for reduced Krebs cycle activation.
The healthy heart produces most of its energy by consuming fatty acids. In this study, the fatty acid uptake into mitochondria and their further degradation was investigated, which showed a switch of the metabolism in general in the Taz-KD mouse model. In vivo studies revealed an increase of glucose uptake into the heart and decreased fatty acid uptake and oxidation. Disturbed energy conversion resulted in activation of retrograde signaling pathways, implicating overall changes in the cell metabolism. Upregulated integrated stress response (ISR) was confirmed by increased levels of the downstream target, i.e., the activating transcription factor 4 (ATF4). A Tafazzin knockout mouse embryonal fibroblast cell model (TazKO) was used to inhibit the ISR using siRNA transfection or pharmaceutical inhibition. This verified the central role of
II
the ISR in regulating the metabolism in BTHS. Moreover, an increased metabolic flux into glutathione biosynthesis was observed, which supports redox homeostasis. In vivo PET-CT scans depicted elevated activity of the xCT system in the BTHS mouse heart, which transports essential amino acids for the biosynthesis of glutathione precursors. Furthermore, the stress induced signaling pathway also affected the glutamate metabolism, which fuels into the Krebs cycle via -ketoglutarate and therefore supports energy converting pathways. In summary, this thesis provides novel insights into the energy metabolism and redox homeostasis in Barth syndrome cardiomyopathy and its regulation by the integrated stress response, which plays a central role in the metabolic alterations. The aim of the thesis was to improve the understanding of these metabolic changes and to identify novel targets, which can provide new possibilities for therapeutic intervention in Barth syndrome.
The variant surface glycoprotein (VSG) of African trypanosomes plays an essential role in protecting the parasites from host immune factors. These trypanosomes undergo antigenic variation resulting in the expression of a single VSG isoform out of a repertoire of around 2000 genes. The molecular mechanism central to the expression and regulation of the VSG is however not fully understood.
Gene expression in trypanosomes is unusual due to the absence of typical RNA polymerase II promoters and the polycistronic transcription of genes. The regulation of gene expression is therefore mainly post-transcriptional. Regulatory sequences, mostly present in the 3´ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 100 % conserved 16mer motif within the 3´ UTR has been shown to modulate the stability of VSG transcripts and hence their expression. As a stability-associated sequence element, the absence of nucleotide substitutions in the motif is however unusual. It was therefore hypothesised that the motif is involved in other essential roles/processes besides stability of the VSG transcripts.
In this study, it was demonstrated that the 100 % conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. It was further shown that the intact motif in the active VSG 3´ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Crosstalk between the VSG and procyclin genes during differentiation to the insect vector stage is also unaffected in cells with a mutated 16mer motif. Ectopic overexpression of a second VSG however requires the intact motif to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The 16mer motif therefore plays a dual role in VSG in situ switching and stability of VSG transcripts. The additional role of the 16mer in the essential process of antigenic variation appears to be the driving force for the 100 % conservation of this RNA motif.
A screen aimed at identifying candidate RNA-binding proteins interacting with the 16mer motif, led to the identification of a DExD/H box protein, Hel66. Although the protein did not appear to have a direct link to the 16mer regulation of VSG expression, the DExD/H family of proteins are important players in the process of ribosome biogenesis. This process is relatively understudied in trypanosomes and so this candidate was singled out for detailed characterisation, given that the 16mer story had reached a natural end point. Ribosome biogenesis is a major cellular process in eukaryotes involving ribosomal RNA, ribosomal proteins and several non-ribosomal trans-acting protein factors. The DExD/H box proteins are the most important trans-acting protein factors involved in the biosynthesis of ribosomes. Several DExD/H box proteins have been directly implicated in this process in yeast. In trypanosomes, very few of this family of proteins have been characterised and therefore little is known about the specific roles they play in RNA metabolism. Here, it was shown that Hel66 is involved in rRNA processing during ribosome biogenesis. Hel66 localises to the nucleolus and depleting the protein led to a severe growth defect. Loss of the protein also resulted in a reduced rate of global translation and accumulation of rRNA processing intermediates of both the small and large ribosomal subunits. Hel66 is therefore an essential nucleolar DExD/H protein involved in rRNA processing during ribosome biogenesis. As very few protein factors involved in the processing of rRNAs have been described in trypanosomes, this finding represents an important platform for future investigation of this topic.
Cardiovascular disease is one of the leading causes of death worldwide and, so far, echocardiography, nuclear cardiology, and catheterization are the gold standard techniques used for its detection. Cardiac magnetic resonance (CMR) can replace the invasive imaging modalities and provide a "one-stop shop" characterization of the cardiovascular system by measuring myocardial tissue structure, function and perfusion of the heart, as well as anatomy of and flow in the coronary arteries. In contrast to standard clinical magnetic resonance imaging (MRI) scanners, which are often operated at a field strength of 1.5 or 3 Tesla (T), a higher resolution and subsequent cardiac parameter quantification could potentially be achieved at ultra-high field, i.e., 7 T and above.
Unique insights into the pathophysiology of the heart are expected from ultra-high field MRI, which offers enhanced image quality in combination with novel contrast mechanisms, but suffers from spatio-temporal B0 magnetic field variations. Due to the resulting spatial misregistration and intra-voxel dephasing, these B0-field inhomogeneities generate a variety of undesired image artifacts, e.g., artificial image deformation. The resulting macroscopic field gradients lead to signal loss, because the effective transverse relaxation time T2* is shortened. This affects the accuracy of T2* measurements, which are essential for myocardial tissue characterization. When steady state free precession-based pulse sequences are employed for image acquisition, certain off-resonance frequencies cause signal voids. These banding artifacts complicate the proper marking of the myocardium and, subsequently, systematic errors in cardiac function measurements are inevitable. Clinical MR scanners are equipped with basic shim systems to correct for occurring B0-field inhomogeneities and resulting image artifacts, however, these are not sufficient for the advanced measurement techniques employed for ultra-high field MRI of the heart.
Therefore, this work focused on the development of advanced B0 shimming strategies for CMR imaging applications to correct the spatio-temporal B0 field variations present in the human heart at 7 T. A novel cardiac phase-specific shimming (CPSS) technique was set up, which featured a triggered B0 map acquisition, anatomy-matched selection of the shim-region-of-interest (SROI), and calibration-based B0 field modeling. The influence of technical limitations on the overall spherical harmonics (SH) shim was analyzed. Moreover, benefits as well as pitfalls of dynamic shimming were debated in this study. An advanced B0 shimming strategy was set up and applied in vivo, which was the first implementation of a heart-specific shimming approach in human UHF MRI at the time.
The spatial B0-field patterns which were measured in the heart throughout this study contained localized spots of strong inhomogeneities. They fluctuated over the cardiac cycle in both size and strength, and were ideally addressed using anatomy-matched SROIs. Creating a correcting magnetic field with one shim coil, however, generated eddy currents in the surrounding conducting structures and a resulting additional, unintended magnetic field. Taking these shim-to-shim interactions into account via calibration, it was demonstrated for the first time that the non-standard 3rd-order SH terms enhanced B0-field homogeneity in the human heart. However, they were attended by challenges for the shim system hardware employed in the presented work, which was indicated by the currents required to generate the optimal 3rd-order SH terms exceeding the dynamic range of the corresponding shim coils. To facilitate dynamic shimming updated over the cardiac cycle for cine imaging, the benefit of adjusting the oscillating CPSS currents was found to be vital. The first in vivo application of the novel advanced B0 shimming strategy mostly matched the simulations.
The presented technical developments are a basic requirement to quantitative and functional CMR imaging of the human heart at 7 T. They pave the way for numerous clinical studies about cardiac diseases, and continuative research on dedicated cardiac B0 shimming, e.g., adapted passive shimming and multi-coil technologies.
Platelets play an important role in haemostasis by mediating blood clotting at sites of blood vessel damage. Platelets, also participate in pathological conditions including thrombosis and inflammation. Upon vessel damage, two glycoprotein receptors, the GPIb-IX-V complex and GPVI, play important roles in platelet capture and activation.
GPIb-IX-V binds to von Willebrand factor and GPVI to collagen. This initiates a signalling cascade resulting in platelet shape change and spreading, which is dependent on the actin cytoskeleton. This thesis aimed to develop and implement different super-resolution microscopy techniques to gain a deeper understanding of the conformation and location of these receptors in the platelet plasma membrane, and to provide insights into their signalling pathways. We suggest direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM) as the best candidates for imaging single platelets, whereas expansion microscopy (ExM) is ideal for imaging platelets aggregates.
Furthermore, we highlighted the role of the actin cytoskeleton, through Rac in GPVI signalling pathway. Inhibition of Rac, with EHT1864 in human platelets induced GPVI and GPV, but not GPIbα shedding. Furthermore, EHT1864 treatment did not change GPVI dimerisation or clustering, however, it decreased phospholipase Cγ2 phosphorylation levels, in human, but not murine platelets, highlighting interspecies differences. In summary, this PhD thesis demonstrates that; 1) Rac alters GPVI signalling pathway in human but not mouse platelets; 2) our newly developed ExM protocol can be used to image platelet aggregates labelled with F(ab’) fragments
Electrochemical impedance spectroscopy (EIS) is a valuable technique analyzing electrochemical behavior of biological systems such as electrical characterization of cells and biomolecules, drug screening, and biomaterials in biomedical field. In EIS, an alternating current (AC) power signal is applied to the biological system, and the impedance of the system is measured over a range of frequencies.
In vitro culture models of endothelial or epithelial barrier tissue can be achieved by culturing barrier tissue on scaffolds made with synthetic or biological materials that provide separate compartments (apical and basal sides), allowing for further studies on drug transport. EIS is a great candidate for non-invasive and real-time monitoring of the electrical properties that correlate with barrier integrity during the tissue modeling. Although commercially available transendothelial/transepithelial electrical resistance (TEER) measurement devices are widely used, their use is particularly common in static transwell culture. EIS is considered more suitable than TEER measurement devices in bioreactor cultures that involve dynamic fluid flow to obtain accurate and reliable measurements. Furthermore, while TEER measurement devices can only assess resistance at a single frequency, EIS measurements can capture both resistance and capacitance properties of cells, providing additional information about the cellular barrier's characteristics across various frequencies. Incorporating EIS into a bioreactor system requires the careful optimization of electrode integration within the bioreactor setup and measurement parameters to ensure accurate EIS measurements. Since bioreactors vary in size and design depending on the purpose of the study, most studies have reported using an electrode system specifically designed for a particular bioreactor. The aim of this work was to produce multi-applicable electrodes and established methods for automated non-invasive and real-time monitoring using the EIS technique in bioreactor cultures. Key to the electrode material, titanium nitride (TiN) coating was fabricated on different substrates (materials and shape) using physical vapor deposition (PVD) and housed in a polydimethylsiloxane (PDMS) structure to allow the electrodes to function as independent units. Various electrode designs were evaluated for double-layer capacitance and morphology using EIS and scanning electron microscopy (SEM), respectively. The TiN-coated tube electrode was identified as the optimal choice. Furthermore, EIS measurements were performed to examine the impact of influential parameters related to culture conditions on the TiN-coated electrode system. In order to demonstrate the versatility of the electrodes, these electrodes were then integrated into in different types of perfusion bioreactors for monitoring barrier cells. Blood-brain barrier (BBB) cells were cultured in the newly developed dynamic flow bioreactor, while human umblical vascular endothelial cells (HUVECs) and Caco-2 cells were cultured in the miniature hollow fiber bioreactor (HFBR). As a result, the TiN-coated tube electrode system enabled investigation of BBB barrier integrity in long-term bioreactor culture. While EIS measurement could not detect HUVECs electrical properties in miniature HFBR culture, there was the possibility of measuring the barrier integrity of Caco-2 cells, indicating potential usefulness for evaluating their barrier function. Following the bioreactor cultures, the application of the TiN-coated tube electrode was expanded to hemofiltration, based on the hypothesis that the EIS system may be used to monitor clotting or clogging phenomena in hemofiltration. The findings suggest that the EIS monitoring system can track changes in ion concentration of blood before and after hemofiltration in real-time, which may serve as an indicator of clogging of filter membranes. Overall, our research demonstrates the potential of TiN-coated tube electrodes for sensitive and versatile non-invasive monitoring in bioreactor cultures and medical devices.
Aging is known to be a risk factor for structural abnormalities and functional decline in the nervous system. Characterizing age-related changes is important to identify putative pathways to overcome deleterious effects and improve life quality for the elderly. In this study, the peripheral nervous system of 24-month-old aged C57BL/6 mice has been investigated and compared to 12-month-old adult mice. Aged mice showed pathological alterations in their peripheral nerves similar to nerve biopsies from elderly human individuals, with nerve fibers showing demyelination and axonal damage. Such changes were lacking in nerves of adult 12-month-old mice and adult, non-aged humans. Moreover, neuromuscular junctions of 24-month-old mice showed increased denervation compared to adult mice. These alterations were accompanied by elevated numbers of macrophages in the peripheral nerves of aged mice. The neuroinflammatory conditions were associated with impaired myelin integrity and with a decline of nerve conduction properties and muscle strength in aged mice.
To determine the pathological impact of macrophages in the aging mice, macrophage depletion was performed in mice by oral administration of CSF-1R specific kinase (c-FMS) inhibitor PLX5622 (300 mg/kg body weight), which reduced the number of macrophages in the peripheral nerves by 70%. The treated mice showed attenuated demyelination, less muscle denervation and preserved muscle strength. This indicates that macrophage-driven inflammation in the peripheral nerves is partially responsible for the age-related neuropathy in mice.
Based on previous observations that systemic inflammation can accelerate disease progression in mouse models of neurodegenerative diseases, it was hypothesized that systemic inflammation can exacerbate the peripheral neuropathy found in aged mice. To investigate this hypothesis, aged C57BL/6 mice were intraperitoneally injected with a single dose of lipopolysaccharide (LPS; 500 μg/kg body weight) to induce systemic inflammation by mimicking bacterial infection, mostly via activation of Toll-like receptors (TLRs). Altered endoneurial macrophage activation, highlighted by Trem2 downregulation, was found in LPS injected aged mice one month after injection. This was accompanied by a so far rarely observed form of axonal perturbation, i.e., the occurrence of “dark axons” characterized by a damaged cytoskeleton and an increased overall electron density of the axoplasm. At the same time, however, LPS injection reduced demyelination and muscle denervation in aged mice. Interestingly, TREM2 deficiency in aged mice led to similar changes to LPS injection. This suggests that LPS injection likely mitigates aging-related demyelination and muscle denervation via Trem2 downregulation.
Taken together, this study reveals the role of macrophage-driven inflammation as a pathogenic mediator in age-related peripheral neuropathy, and that targeting macrophages might be an option to mitigate peripheral neuropathies in aging individuals. Furthermore, this study shows that systemic inflammation may be an ambivalent modifier of age-related nerve damage, leading to a distinct type of axonal perturbation, but in addition to functionally counteracting, dampened demyelination and muscle denervation. Translationally, it is plausible to assume that tipping the balance of macrophage polarization to one direction or the other may determine the functional outcome in the aging peripheral nervous system of the elderly.
Emotional-associative learning processes such as fear conditioning and extinction are highly relevant to not only the development and maintenance of anxiety disorders (ADs), but also to their treatment. Extinction, as the laboratory analogue to behavioral exposure, is assumed a core process underlying the treatment of ADs. Although exposure-based treatments are highly effective for the average patient suffering from an AD, there remains a gap in treatment efficacy with over one third of patients failing to achieve clinically significant symptom relief. There is ergo a pressing need for intensified research regarding the underlying neural mechanisms of aberrant emotional-associative learning processes and the neurobiological moderators of treatment (non-)response in ADs.
The current thesis focuses on different applications of the fundamental principles of fear conditioning and extinction by using two example cases of ADs from two different multicenter trials. First, we targeted alterations in fear acquisition, extinction, and its recall as a function of psychopathology in panic disorder (PD) patients compared to healthy subjects using fMRI. Second, exposure-based therapy and pre-treatment patient characteristics exerting a moderating influence on this essential learning process later on (i.e. treatment outcome) were examined using multimodal functional and structural neuroimaging in spider phobia.
We observed aberrations in emotional-associative learning processes in PD patients compared to healthy subjects indicated by an accelerated fear acquisition and an attenuated extinction recall. Furthermore, pre-treatment differences related to defensive, regulatory, attentional, and perceptual processes may exert a moderating influence on treatment outcome to behavioral exposure in spider phobia. Although the current results need further replication, on an integrative meta level, results point to a hyperactive defensive network system and deficient emotion regulation processes (including extinction processes) and top-down control in ADs. This speaks in favor of transdiagnostic deficits in important functional domains in ADs.
Deficits in transdiagnostic domains such as emotion regulation processes could be targeted by enhancing extinction learning or by means of promising tools like neurofeedback. The detection of pre-treatment clinical response moderators, for instance via machine learning frameworks, may help in supporting clinical decision making on individually tailored treatment approaches or, respectively, to avoid ineffective treatment and its related financial costs. In the long run, the identification of neurobiological markers which are capable of detecting non-responders a priori represents an ultimate goal.
This thesis identifies how the printing conditions for a high-resolution additive manufacturing technique, melt electrowriting (MEW), needs to be adjusted to process electroactive polymers (EAPs) into microfibers. Using EAPs based on poly(vinylidene difluoride) (PVDF), their ability to be MEW-processed is studied and expands the list of processable materials for this technology.
Cardiovascular disease and the acute consequence of myocardial infarc- tion remain one of the most important causes of morbidity and mortality in all western societies. While much progress has been made in mitigating the acute, life-threatening ischemia caused by infarction, heart failure of the damaged my- ocardium remains prevalent. There is mounting evidence for the role of T cells in the healing process after myocardial infarction, but relevant autoantigens, which might trigger and regulate adaptive immune involvement have not been discov- ered in patients.
In this work, we discovered an autoantigenic epitope in the adrenergic receptor beta 1, which is highly expressed in the heart. This autoantigenic epitope causes a pro-inflammatory immune reaction in T cells isolated from pa- tients after myocardial infarction (MI) but not in control patients. This immune reaction was only observed in a subset of MI patients, which carry at least one allele of the HLA-DRB1*13 family. Interestingly, HLA-DRB1*13 was more com- monly expressed in patients in the MI group than in the control group.
Taken together, our data suggests antigen-specific priming of T cells in MI patients, which leads to a pro-inflammatory phenotype. The primed T cells react to a cardiac derived autoantigen ex vivo and are likely to exhibit a similar phenotype in vivo. This immune phenotype was only observed in a certain sub- set of patients sharing a common HLA-allele, which was more commonly ex- pressed in MI patients, suggesting a possible role as a risk factor for cardiovas- cular disease.
While our results are observational and do not have enough power to show strong clinical associations, our discoveries provide an essential tool to further our understanding of involvement of the immune system in cardiovascu- lar disease. We describe the first cardiac autoantigen in the clinical context of MI and provide an important basis for further translational and clinical research in cardiac autoimmunity.
Stroke and myocardial infarction are the most prominent and severe consequences of pathological thrombus formation. For prevention and/or treatment of thrombotic events there is a variety of anti-coagulation and antiplatelet medication that all have one side effect in common: the increased risk of bleeding. To design drugs that only intervene in the unwanted aggregation process but do not disturb general hemostasis, it is crucial to decipher the exact clotting pathway which has not been fully understood yet. Platelet membrane receptors play a vital role in the clotting pathway and, thus, the aim of this work is to establish a method to elucidate the interactions, clustering, and reorganization of involved membrane receptors such as GPIIb/IIIa and GPIX as part of the GPIb-IX-V complex. The special challenges regarding visualizing membrane receptor interactions on blood platelets are the high abundancy of the first and the small size of the latter (1—3µm of diameter). The resolution limit of conventional fluorescence microscopy and even super-resolution approaches prevents the successful differentiation of densely packed receptors from one another. Here, this issue is approached with the combination of a recently developed technique called Expansion Microscopy (ExM). The image resolution of a conventional fluorescence microscope is enhanced by simply enlarging the sample physically and thus pulling the receptors apart from each other. This method requires a complex sample preparation and holds lots of obstacles such as variable or anisotropic expansion and low images contrast. To increase ExM accuracy and sensitivity for interrogating blood platelets, it needs optimized sample preparation as well as image analysis pipelines which are the main part of this thesis. The colocalization results show that either fourfold or tenfold expanded, resting platelets allow a clear distinction between dependent, clustered, and independent receptor organizations compared to unexpanded platelets.Combining dual-color Expansion and confocal fluorescence microscopy enables to image in the nanometer range identifying GPIIb/IIIa clustering in resting platelets – a pattern that may play a key role in the clotting pathway
Ibrutinib serves as an efficient second-line therapy in relapsed/refractory mantle cell lymphoma. However, resistance to the BTK inhibitor results in a poor prognosis for patients. Since the mechanisms leading to resistance in initially responding tumor cells are poorly understood, this work aimed to decipher acquired features in ibrutinib-surviving cells of a sensitive mantle cell lymphoma cell line and evaluate these potential therapeutic targets in ibrutinib-treated mantle cell lymphoma.
Time-resolved single-cell RNA sequencing was performed to track the transcriptomic evolution of REC-1 cells across 6 and 48 hours of treatment. Single-cell analysis uncovered a subpopulation of REC-1 with potentially greater aggressiveness and survival advantage by benefiting from interaction with the tumor microenvironment. Upregulation of B-cell receptor genes, elevated surface antigen expression of CD52 and metabolic rewiring to higher dependence on oxidative phosphorylation were identified as further potential resistance features of ibrutinib-surviving cells. RNA sequencing after prolonged incubation corroborated the increase in CD52 and oxidative phosphorylation as dominant characteristics of the cells surviving the 4-day treatment, highlighting their potential as therapeutic targets in combination with ibrutinib treatment. Concomitant use of ibrutinib and the oxidative phosphorylation inhibitor IACS-010759 increased toxicity compared to ibrutinib monotherapy due to higher apoptosis and greater inhibition of proliferation. For anti-CD52 therapy, a consecutive approach with ibrutinib pretreatment followed by incubation of surviving cells with a CD52 monoclonal antibody and human serum yielded a synergistic effect, as ibrutinib-surviving mantle cell lymphoma cells were rapidly depleted by complement-dependent cytotoxicity. Regarding the effects on primary tumor cells from mantle cell lymphoma patients, ibrutinib induced upregulation of CD52 in some cases, and increased toxicity of anti-CD52 therapy was observed in ibrutinib-sensitive patient samples after pretreatment with the BTK inhibitor. The likely favorable in vivo efficacy of an anti-CD52 therapy might therefore be restricted to a subgroup of mantle cell lymphoma patients, also in view of the associated side effects.
Given the need for new therapeutic options in mantle cell lymphoma to overcome resistance to ibrutinib, this work highlights the potentially beneficial use of an oxidative phosphorylation inhibitor as add-on therapy. In addition, the findings suggest to further assess the value of anti-CD52 therapy as consolidation to ibrutinib in ibrutinib-sensitive patients with elevated CD52 surface levels on tumor cells to target resistant clones and minimize risk of minimal residual disease and relapse.
This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.
Pain conditions and chronic pain disorders are among the leading reasons for seeking medical help and immensely burden patients and the healthcare system. Therefore, research on the underlying mechanisms of pain processing and modulation is necessary and warranted. One crucial part of this pain research includes identifying resilience factors that protect from chronic pain development and enhance its treatment. The ability to use emotion regulation strategies has been suggested to serve as a resilience factor, facilitating pain regulation and management. Acceptance has been discussed as a promising pain regulation strategy, but results in this domain have been mixed so far. Moreover, the allocation of acceptance in Gross’s (1998) process model of emotion regulation has been under debate. Thus, comparing acceptance with the already established strategies of distraction and reappraisal could provide insights into underlying mechanisms. This dissertation project consisted of three successive experimental studies which aimed to investigate these strategies by applying different modalities of individually adjusted pain stimuli of varying durations. In the first study (N = 29), we introduced a within-subjects design where participants were asked to either accept (acceptance condition) or react to the short heat pain stimuli (10 s) without using any pain regulation strategies (control condition). In the second study (N = 36), we extended the design of study 1 by additionally applying brief, electrical pain stimuli (20 ms) and including the new experimental condition distraction, where participants should distract themselves from the pain experience by imagining a neutral situation. In the third study (N = 121), all three strategies, acceptance, distraction, and reappraisal were compared with each other and additionally with a neutral control condition in a mixed design. Participants were randomly assigned to one of three strategy groups, including a control condition and a strategy condition. All participants received short heat pain stimuli of 10 s, alternating with tonic heat pain stimuli of 3 minutes. In the reappraisal condition, participants were instructed to imagine the pain having a positive outcome or valence. The self-reported pain intensity, unpleasantness, and regulation ratings were measured in all studies. We further recorded the autonomic measures heart rate and skin conductance continuously and assessed the habitual emotion regulation styles and pain-related trait factors via questionnaires. Results revealed that the strategies acceptance, distraction, and reappraisal significantly reduced the self-reported electrical and heat pain stimulation with both durations compared to a neutral control condition. Additionally, regulatory efforts with acceptance in study 2 and with all strategies in study 3 were reflected by a decreased skin conductance level compared to the control condition. However, there were no significant differences between the strategies for any of the assessed variables. These findings implicate similar mechanisms underlying all three strategies, which led to the proposition of an extended process model of emotion regulation. We identified another sequence in the emotion-generative process and suggest that acceptance can flexibly affect at least four sequences in the process. Correlation analyses further indicated that the emotion regulation style did not affect regulatory success, suggesting that pain regulation strategies can be learned effectively irrespective of habitual tendencies. Moreover, we found indications that trait factors such as optimism and resilience facilitated pain regulation, especially with acceptance. Conclusively, we propose that acceptance could be flexibly used by adapting to different circumstances. The habitual use of acceptance could therefore be considered a resilience factor. Thus, acceptance appears to be a promising and versatile strategy to prevent the development of and improve the treatment of various chronic pain disorders. Future studies should further examine factors and circumstances that support effective pain regulation with acceptance.
This work deals with the acceleration of cardiovascular MRI for the assessment
of functional information in steady-state contrast and for viability assessment
during the inversion recovery of the magnetization. Two approaches
are introduced and discussed in detail. MOCO-MAP uses an exponential
model to recover dynamic image data, IR-CRISPI, with its low-rank plus
sparse reconstruction, is related to compressed sensing.
MOCO-MAP is a successor to model-based acceleration of parametermapping
(MAP) for the application in the myocardial region. To this end, it
was augmented with a motion correction (MOCO) step to allow exponential
fitting the signal of a still object in temporal direction. Iteratively, this
introduction of prior physical knowledge together with the enforcement of
consistency with the measured data can be used to reconstruct an image
series from distinctly shorter sampling time than the standard exam (< 3 s
opposed to about 10 s). Results show feasibility of the method as well as
detectability of delayed enhancement in the myocardium, but also significant
discrepancies when imaging cardiac function and artifacts caused already by
minor inaccuracy of the motion correction.
IR-CRISPI was developed from CRISPI, which is a real-time protocol
specifically designed for functional evaluation of image data in steady-state
contrast. With a reconstruction based on the separate calculation of low-rank
and sparse part, it employs a softer constraint than the strict exponential
model, which was possible due to sufficient temporal sampling density via
spiral acquisition. The low-rank plus sparse reconstruction is fit for the use on
dynamic and on inversion recovery data. Thus, motion correction is rendered
unnecessary with it.
IR-CRISPI was equipped with noise suppression via spatial wavelet filtering.
A study comprising 10 patients with cardiac disease show medical
applicability. A comparison with performed traditional reference exams offer
insight into diagnostic benefits. Especially regarding patients with difficulty
to hold their breath, the real-time manner of the IR-CRISPI acquisition provides
a valuable alternative and an increase in robustness.
In conclusion, especially with IR-CRISPI in free breathing, a major acceleration
of the cardiovascular MR exam could be realized. In an acquisition
of less than 100 s, it not only includes the information of two traditional
protocols (cine and LGE), which take up more than 9.6 min, but also allows
adjustment of TI in retrospect and yields lower artifact level with similar
image quality.
Attention-deficit/hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder described in psychiatry today. ADHD arises during early childhood and is characterized by an age-inappropriate level of inattention, hyperactivity, impulsivity, and partially emotional dysregulation. Besides, substantial psychiatric comorbidity further broadens the symptomatic spectrum. Despite advances in ADHD research by genetic- and imaging studies, the etiopathogenesis of ADHD remains largely unclear. Twin studies suggest a heritability of 70-80 % that, based on genome-wide investigations, is assumed to be polygenic and a mixed composite of small and large, common and rare genetic variants. In recent years the number of genetic risk candidates is continuously increased. However, for most, a biological link to neuropathology and symptomatology of the patient is still missing. Uncovering this link is vital for a better understanding of the disorder, the identification of new treatment targets, and therefore the development of a more targeted and possibly personalized therapy.
The present thesis addresses the issue for the ADHD risk candidates GRM8, FOXP2, and GAD1. By establishing loss of function zebrafish models, using CRISPR/Cas9 derived mutagenesis and antisense oligonucleotides, and studying them for morphological, functional, and behavioral alterations, it provides novel insights into the candidate's contribution to neuropathology and ADHD associated phenotypes. Using locomotor activity as behavioral read-out, the present work identified a genetic and functional implication of Grm8a, Grm8b, Foxp2, and Gad1b in ADHD associated hyperactivity. Further, it provides substantial evidence that the function of Grm8a, Grm8b, Foxp2, and Gad1b in activity regulation involves GABAergic signaling. Preliminary indications suggest that the three candidates interfere with GABAergic signaling in the ventral forebrain/striatum. However, according to present and previous data, via different biological mechanisms such as GABA synthesis, transmitter release regulation, synapse formation and/or transcriptional regulation of synaptic components. Intriguingly, this work further demonstrates that the activity regulating circuit, affected upon Foxp2 and Gad1b loss of function, is involved in the therapeutic effect mechanism of methylphenidate. Altogether, the present thesis identified altered GABAergic signaling in activity regulating circuits in, presumably, the ventral forebrain as neuropathological underpinning of ADHD associated hyperactivity. Further, it demonstrates altered GABAergic signaling as mechanistic link between the genetic disruption of Grm8a, Grm8b, Foxp2, and Gad1b and ADHD symptomatology like hyperactivity. Thus, this thesis highlights GABAergic signaling in activity regulating circuits and, in this context, Grm8a, Grm8b, Foxp2, and Gad1b as exciting targets for future investigations on ADHD etiopathogenesis and the development of novel therapeutic interventions for ADHD related hyperactivity. Additionally, thigmotaxis measurements suggest Grm8a, Grm8b, and Gad1b as interesting candidates for prospective studies on comorbid anxiety in ADHD. Furthermore, expression analysis in foxp2 mutants demonstrates Foxp2 as regulator of ADHD associated gene sets and neurodevelopmental disorder (NDD) overarching genetic and functional networks with possible implications for ADHD polygenicity and comorbidity. Finally, with the characterization of gene expression patterns and the generation and validation of genetic zebrafish models for Grm8a, Grm8b, Foxp2, and Gad1b, the present thesis laid the groundwork for future research efforts, for instance, the identification of the functional circuit(s) and biological mechanism(s) by which Grm8a, Grm8b, Foxp2, and Gad1b loss of function interfere with GABAergic signaling and ultimately induce hyperactivity.
PRC1 serves as a microtubule-bundling protein and is a potential therapeutic target for lung cancer
(2023)
Protein regulator of cytokinesis 1 (PRC1) is a microtubule-associated protein with essential roles in mitosis and cytokinesis. Furthermore, the protein is highly expressed in several cancer types which is correlated with aneuploidy and worse patient outcome. In this study it was investigated, whether PRC1 is a potential target for lung cancer as well as its possible nuclear role.
Elevated PRC1 expression was cell cycle-dependent with increasing levels from S-phase to G2/M-phase of the cell cycle. Thereby, PRC1 localized at the nucleus during interphase and at the central spindle and midbody during mitosis and cytokinesis. Genome-wide expression profiling by RNA sequencing of ectopically expressed PRC1 resulted in activation of the p53 pathway. A mutant version of PRC1, that is unable to enter the nucleus, induced the same gene sets as wildtype PRC1, suggesting that PRC1 has no nuclear-specific functions in lung cancer cells. Finally, PRC1 overexpression leads to proliferation defects, multi-nucleation, and enlargement of cells which was directly linked to microtubule-bundling within the cytoplasm.
For analysis of the requirement of PRC1 in lung cancer, different inducible cell lines were generated to deplete the protein by RNA interference (RNAi) in vitro. PRC1 depletion caused proliferation defects and cytokinesis failures with increased numbers of bi- and multi-nucleated cells compared to non-induced lung cancer cells. Importantly, effects in control cells were less severe as in lung cancer cells. Finally, p53 wildtype lung cancer cells became senescent, whereas p53 mutant cells became apoptotic upon PRC1 depletion. PRC1 is also required for tumorigenesis in vivo, which was shown by using a mouse model for non-small cell lung cancer driven by oncogenic K-RAS and loss of p53. Here, lung tumor area, tumor number, and high-grade tumors were significantly reduced in PRC1 depleted conditions by RNAi.
In this study, it is shown that PRC1 serves as a microtubule-bundling protein with essential roles in mitosis and cytokinesis. Expression of the protein needs to be tightly regulated to allow unperturbed proliferation of lung cancer cells. It is suggested that besides phosphorylation of PRC1, the nuclear localization might be a protective mechanism for the cells to prevent perinuclear microtubule-bundling. In conclusion, PRC1 could be a potential target of lung cancer as mono therapy or in combination with a chemotherapeutic agent, like cisplatin, which enhanced the negative effects on proliferation of lung cancer cells in vitro.
This thesis aimed the development of a correlated device which combines FluidFM® with Fluorescence Microscopy (FL) (FL-FluidFM®) and enables the simultaneous quantification of adhesion forces and fluorescent visualization of mature cells. The implementation of a PIFOC was crucial to achieve a high-resolution as well as a stable but dynamic focus level. The functionality of SCFS after hardware modification was verified by comparing two force-curves, both showing the typical force progression and measured with the optimized and conventional hardware, respectively. Then, the integration of FL was examined by detaching fluorescently labeled REF52 cells. The fluorescence illumination of the cytoskeleton showed the expected characteristic force profile and no evidence of interference effects. Afterwards a corresponding correlative data analysis was addressed including manual force step fitting, the identification of visualized cellular unbinding, and a time-dependent correlation. This procedure revealed a link between the area of cytoskeletal unbinding and force-jumps. This was followed by a comparison of the detachment characteristics of intercellular connected HUVECs and individual REF52 cells. HUVECs showed maximum detachment forces in the same order of magnitude as the ones of single REF52 cells. This contrasted with the expected strong cohesiveness of endothelial cells and indicated a lack of cell-cell contact formation. The latter was confirmed by a comparison of HUVECs, primary HBMVECs, and immortalized EA.hy926 cells fluorescently labeled for two marker proteins of intercellular junctions. This unveiled that both the previous cultivation duration and the cell type have a major impact on the development of intercellular junctions. In summary, the correlative FL FluidFM® represents a powerful novel approach, which enables a truly contemporaneous performance and, thus, has the potential to reveal new insights into the mechanobiological properties of cell adhesion.
In the recent years, translational studies comparing imaging data of animals and humans have gained increasing
scientific interests with crucial findings stemming from both, human and animal work. In order to harmonize
statistical analyses of data from different species and to optimize the transfer of knowledge between them, shared
data acquisition protocols and combined statistical approaches have to be identified. Following this idea, methods
of data analysis, which have until now mainly been used to model neural responses of electrophysiological
recordings from rodent data, were applied on human hemodynamic responses (i.e. Blood-Oxygen-Level-
Dependent BOLD signal) as measured via functional magnetic resonance imaging (fMRI).
At the example of two attention and impulsivity networks, timing dynamics and amplitude of the fMRI signal were
determined (study 1). Study 2 described the same parameters frequency-specifically, and in study 3, the
complexity of neural processing was quantified in terms of fractality. Determined parameters were compared with
regard to the subjects’ task performance / impulsivity to validate findings with regard to reports of the current
scientific debate.
In a general discussion, overlapping as well as additional information of methodological approaches were
discussed with regard to its potential for biomarkers in the context of neuropsychiatric disorders.
The present cumulative dissertation summarizes three clinical studies, which examine
subgroups of patients within the fibromyalgia syndrome (FMS). FMS entails chronic pain and
associated symptoms, and its pathophysiology is incompletely understood (1). Previous studies
show that there is a subgroup of patients with FMS with objective histological pathology of the
small nerve fibers of the peripheral nervous system (PNS). Another subgroup of FMS patients
does not show any signs of pathological changes of the small nerve fibers. The aim of this
dissertation was to compare FMS patients with healthy controls, and these two FMS subgroups
for differences in the central nervous system (CNS) in order to explore possible interactions
between PNS and the CNS. Regarding the CNS, differences of FMS patients with healthy
controls have already been found in studies with small sample sizes, but no subgroups have yet
been identified. Another aim of this thesis was to test whether the subgroups show a different
response to different classes of pain medication. The methods used in this thesis are structural
and functional magnetic resonance imaging (MRI), magnetic resonance diffusion imaging and
magnetic resonance spectroscopy. For the evaluation of clinical symptoms, we used
standardized questionnaires. The subgroups with and without pathologies of the PNS were
determined by skin biopsies of the right thigh and lower leg based on the intraepidermal nerve
fiber density (IENFD) of the small nerve fibers.
1) In the first MRI study, 43 female patients with the diagnosis of FMS and 40 healthy
control subjects, matched in age and body mass index, were examined with different MRI
sequences. Cortical thickness was investigated by structural T1 imaging, white matter integrity
by diffusion tensor imaging and functional connectivity within neuronal networks by functional
resting state MRI. Compared to the controls, FMS patients had a lower cortical volume in
bilateral frontotemporoparietal regions and the left insula, but a higher cortical volume in the
left pericalcarine cortex. Compared to the subgroup without PNS pathology, the subgroup with
PNS pathology had lower cortical volume in both pericalcarine cortices. Diffusion tensor
imaging revealed an increased fractional anisotropy (FA) of FMS patients in corticospinal
pathways such as the corona radiata, but also in regions of the limbic systems such as the fornix
and cingulum. Subgroup comparison again revealed lower mean FA values of the posterior
thalamic radiation and the posterior limb of the left internal capsule in the subgroup with PNS
pathology. In the functional connectivity analysis FMS patients, compared to controls, showed
a hypoconnectivity between the right median frontal gyrus and the posterior cerebellum and
the right crus cerebellum, respectively. In the subgroup comparisons, the subgroup with PNS
pathology showed a hyperconnectivity between both inferior frontal gyri, the right posterior
parietal cortex and the right angular gyrus. In summary, these results show that differences in
brain morphology and functional connectivity exist between FMS patients with and without
PNS pathology. These differences were not associated with symptom duration or severity and,
in some cases, have not yet been described in the context of FMS. The differences in brain
morphology and connectivity between subgroups could also lead to a differential response to
treatment with centrally acting drugs. Further imaging studies with FMS patients should take
into account this heterogeneity of FMS patient cohorts.
2) Following the results from the first MRI study, drug therapies of FMS patients and
their treatment response were compared between PNS subgroups. As there is no licensed drug
for FMS in Europe, the German S3 guideline recommends amitriptyline, duloxetine and
pregabalin for temporary use. In order to examine the current drug use in FMS patients in
Germany on a cross-sectional basis, 156 patients with FMS were systematically interviewed.
The drugs most frequently used to treat pain in FMS were non-steroidal anti-inflammatory
drugs (NSAIDs) (28.9%), metamizole (15.4%) and amitriptyline (8.8%). Pain relief assessed by
patients on a numerical rating scale from 0-10 averaged 2.2 points for NSAIDs, 2.0 for
metamizole and 1.5 for amitriptyline. Drugs that were discontinued for lack of efficacy and not
for side effects were acetaminophen (100%), flupirtine (91.7%), selective serotonin reuptake
inhibitors (81.8%), NSAIDs (83.7%) and weak opioids (74.1%). Patients were divided into
subgroups with and without PNS pathology as determined by skin biopsies. We found no
differences in drug use and effect between the subgroups. Taken together, these results show
that many FMS patients take medication that is not in accordance with the guidelines. The
reduction of symptoms was best achieved with metamizole and NSAIDs. Further longitudinal
studies on medication in FMS are necessary to obtain clearer treatment recommendations.
3) Derived from previous pharmacological and imaging studies (with smaller case
numbers), there is a hypothesis in the FMS literature that hyperreactivity of the insular cortex
may have an impact on FMS. The hyperreactivity seems to be due to an increased concentration
of the excitatory neurotransmitter glutamate in the insular cortex of FMS patients. The
hypothesis is supported by magnetic resonance spectroscopy studies with small number of
cases, as well as results from pharmacological studies with glutamate-inhibiting medication.
Studies from animal models have also shown that an artificially induced increase in glutamate
in the insular cortex can lead to reduced skin innervation. Therefore, the aim of this study was
to compare glutamate and GABA concentrations in the insular cortex of FMS patients with
those of healthy controls using magnetic resonance imaging. There was no significant
difference of both neurotransmitters between the groups. In addition, there was no correlation
between the neurotransmitter concentrations and the severity of clinical symptoms. There
were also no differences in neurotransmitter concentrations between the subgroups with and
without PNS pathology. In conclusion, our study could not show any evidence of a correlation
of glutamate and GABA concentrations with the symptoms of FMS or the pathogenesis of
subgroups with PNS pathologies.
Monarch butterflies are famous for their annual long-distance migration. Decreasing temperatures and reduced daylight induce the migratory state in the autumn generation of monarch butterflies. Not only are they in a reproductive diapause, they also produce fat deposits to be prepared for the upcoming journey: Driven by their instinct to migrate, they depart from their eclosion grounds in the northern regions of the North American continent and start their southern journey to their hibernation spots in Central Mexico. The butterflies cover a distance of up to 4000 km across the United States. In the next spring, the same butterflies invert their preferred heading direction due to seasonal changes and start their northward spring migration. The spring migration is continued by three consecutive butterfly generations, until the animals repopulate the northern regions in North America as non-migratory monarch butterflies. The monarch butterflies’ migratory state is genetically and epigenetically regulated, including the directed flight behavior. Therefore, the insect’s internal compass system does not only have to encode the butterflies preferred, but also its current heading direction. However, the butterfly’s internal heading representation has to be matched to external cues, to avoid departing from its initial flight path and increasing its risk of missing its desired destination. During the migratory flight, visual cues provide the butterflies with reliable orientation information. The butterflies refer to the sun as their main orientation cue. In addition to the sun, the butterflies likely use the polarization pattern of the sky for orientation. The sky compass signals are processed within a region in the brain, termed the central complex (CX). Previous research on the CX neural circuitry of the monarch butterflies demonstrated that tangential central complex neurons (TL) carry the visual input information into the CX and respond to a simulated sun and polarized light. However, whether these cells process additional visual cues like the panoramic skyline is still unknown. Furthermore, little is known about how the migratory state affects visual cue processing. In addition to this, most experiments studying the monarch butterfly CX focused on how neurons process single visual cues. However, how combined visual stimuli are processed in the CX is still unknown.
This thesis is investigating the following questions:
1) How does the migratory state affect visual cue processing in the TL cells within the monarch butterfly brain?
2) How are multiple visual cues integrated in the TL cells?
3) How is compass information modulated in the CX?
To study these questions, TL neurons from both animal groups (migratory and non-migratory) were electrophysiologically characterized using intracellular recordings while presenting different simulated celestial cues and visual sceneries. I showed that the TL neurons of migratory butterflies are more narrowly tuned to the sun, possibly helping them in keeping a directed flight course during migration. Furthermore, I found that TL cells encode a panoramic skyline, suggesting that the CX network combines celestial and terrestrial information. Experiments with combined celestial stimuli revealed that the TL cells combine both cue information linearly. However, if exposing the animals to a simulated visual scenery containing a panoramic skyline and a simulated sun, the single visual cues are weighted differently. These results indicate that the CX’s input region can flexibly adapt to different visual cue conditions. Furthermore, I characterize a previously unknown neuron in the monarch butterfly CX which responds to celestial stimuli and connects the CX with other brain neuropiles. How this cell type affects heading direction encoding has yet to be determined.
The mammalian central clock, located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, controls circadian rhythms in behaviour such as the sleep-wake cycle. It is made up of approximately 20,000 heterogeneous neurons that can be classified by their expression of neuropeptides. There are three major populations: AVP neurons (arginine vasopressin), VIP neurons (vasoactive intestinal peptide), and GRP neurons (gastrin releasing peptide). How these neuronal clusters form functional units to govern various aspects of rhythmic behavior is poorly understood. At a molecular level, biological clocks are represented by transcriptional-posttranslational feedback loops that induce circadian oscillations in the electrical activity of the SCN and hence correlate with behavioral circadian rhythms. In mammals, the sleep wake cycle can be accurately predicted by measuring electrical muscle and brain activity. To investigate the link between the electrical activity of heterogeneous neurons of the SCN and the sleep wake cycle, we optogenetically manipulated AVP neurons in vivo with SSFO (stabilized step function opsin) and simultaneously recorded an electroencephalogram (EEG) and electromyogram (EMG) in freely moving mice. SSFO-mediated stimulation of AVP positive neurons in the anterior hypothalamus increased the total amount of wakefulness during the hour of stimulation. Interestingly, this effect led to a rebound in sleep in the hour after stimulation. Markov chain sleep-stage transition analysis showed that the depolarization of AVP neurons through SSFO promotes the transition from all states to wakefulness. After the end of stimulation, a compensatory increase in transitions to NREM sleep was observed. Ex vivo, SSFO activation in AVP neurons causes depolarization and modifies the activity of AVP neurons. Therefore, the results of this thesis project suggest an essential role of AVP neurons as mediators between circadian rhythmicity and sleep-wake behaviour.
Cancer is one of the leading causes of death worldwide. The underlying tumorigenesis is driven by the accumulation of alterations in the genome, eventually disabling tumor suppressors and activating proto-oncogenes.
The MYC family of proto-oncogenes shows a strong deregulation in the majority of tumor entities. However, the exact mechanisms that contribute to MYC-driven oncogenesis remain largely unknown. Over the past decades, the influence of the MYC protein on transcription became increasingly apparent and was thoroughly investigated. Additionally, in recent years several publications provided evidence for so far unreported functions of MYC that are independent of a mere regulation of target genes. These findings suggest an additional role of MYC in the maintenance of genomic stability and this role is strengthened by key findings presented in this thesis.
In the first part, I present data revealing a pathway that allows MYC to couple transcription elongation and DNA double-strand break repair, preventing genomic instability of MYC-driven tumor cells. This pathway is driven by a rapid transfer of the PAF1 complex from MYC onto RNAPII, a process that is mediated by HUWE1. The transfer controls MYC-dependent transcription elongation and, simultaneously, the remodeling of chromatin structure by ubiquitylation of histone H2B. These regions of open chromatin favor not only elongation but also DNA double-strand break repair.
In the second part, I analyze the ability of MYC proteins to form multimeric structures in response to perturbation of transcription and replication. The process of multimerization is also referred to as phase transition. The observed multimeric structures are located proximal to stalled replication forks and recruit factors of the DNA-damage response and transcription termination machinery. Further, I identified the HUWE1-dependent ubiquitylation of MYC as an essential step in this phase transition. Cells lacking the ability to form multimers display genomic instability and ultimately undergo apoptosis in response to replication stress.
Both mechanisms present MYC as a stress resilience factor under conditions that are characterized by a high level of transcriptional and replicational stress. This increased resilience ensures oncogenic proliferation.
Therefore, targeting MYC’s ability to limit genomic instability by uncoupling transcription elongation and DNA repair or disrupting its ability to multimerize presents a therapeutic window in MYC-dependent tumors.
Since the advent of high-throughput sequencing technologies in the mid-2010s, RNA se-
quencing (RNA-seq) has been established as the method of choice for studying gene
expression. In comparison to microarray-based methods, which have mainly been used to
study gene expression before the rise of RNA-seq, RNA-seq is able to profile the entire
transcriptome of an organism without the need to predefine genes of interest. Today,
a wide variety of RNA-seq methods and protocols exist, including dual RNA sequenc-
ing (dual RNA-seq) and multi RNA sequencing (multi RNA-seq). Dual RNA-seq and
multi RNA-seq simultaneously investigate the transcriptomes of two or more species, re-
spectively. Therefore, the total RNA of all interacting species is sequenced together and
only separated in silico. Compared to conventional RNA-seq, which can only investi-
gate one species at a time, dual RNA-seq and multi RNA-seq analyses can connect the
transcriptome changes of the species being investigated and thus give a clearer picture of
the interspecies interactions. Dual RNA-seq and multi RNA-seq have been applied to a
variety of host-pathogen, mutualistic and commensal interaction systems.
We applied dual RNA-seq to a host-pathogen system of human mast cells and Staphylo-
coccus aureus (S. aureus). S. aureus, a commensal gram-positive bacterium, can become
an opportunistic pathogen and infect skin lesions of atopic dermatitis (AD) patients.
Among the first immune cells S. aureus encounters are mast cells, which have previously
been shown to be able to kill the bacteria by discharging antimicrobial products and re-
leasing extracellular traps made of protein and deoxyribonucleic acid (DNA). However,
S. aureus is known to evade the host’s immune response by internalizing within mast
cells. Our dual RNA-seq analysis of different infection settings revealed that mast cells
and S. aureus need physical contact to influence each other’s gene expression. We could
show that S. aureus cells internalizing within mast cells undergo profound transcriptome
changes to adjust their metabolism to survive in the intracellular niche. On the host side,
we found out that infected mast cells elicit a type-I interferon (IFN-I) response in an
autocrine manner and in a paracrine manner to non-infected bystander-cells. Our study
provides the first evidence that mast cells are capable to produce IFN-I upon infection
with a bacterial pathogen.
Mammalian phoshoglycolate phosphatase (PGP, also known as AUM) belongs to the ubiquitous HAD superfamily of phosphatases. As several other members of HAD phosphatases, the Mg2+-dependent dephosphorylation is conducted via a nucleophilic attack from a conserved aspartate residue in the catalytic cleft. The protein structure of PGP could not yet be solved entirely. Only a hybrid consisting of the PGP cap and the PDXP core (pyridoxal phosphatase, closest enzyme paralog) was crystallizable so far. PGP is able to efficiently dephosphorylate 2-phosphoglycolate, 2-phospho-L-lactate, 4-phospho-D-erythronate, and glycerol-3-phosphate in vitro which makes them likely physiological substrates. The first three substrates can be derived from metabolic side reactions (during glycolysis) and inhibit key enzymes in glycolysis and pentose phosphate pathway, the latter is situated at the intersection between glycolysis and lipogenesis. 2-phosphoglycolate can also be released in the context of repair of oxidative DNA damage. The activity of purified PGP can be reversibly inhibited by oxidation - physiologically likely in association with epidermal growth factor (EGF) signal transduction. In fact, an association between persistently lacking PGP activity (via downregulation) and the presence of hyperphosphorylated proteins after EGF stimulation has been identified. Reversible oxidation and transient inactivation of PGP may be particularly important for short-term and feedback regulatory mechanisms (as part of the EGF signaling). Furthermore, cellular proliferation in PGP downregulated cells is constantly reduced. Whole-body PGP inactivation in mice is embryonically lethal. Despite the many well-known features and functions, the knowledge about PGP is still incomplete.
In the present work the influence of reactive oxygen species (ROS) on PGP activity in cells und a possible connection between oxidative stress and the proliferation deficit of PGP downregulated cells was investigated. For the experiments, a spermatogonial cell line was used (due to the high PGP expression in testis). PGP activity can be reversibly inhibited in cellular lysates by H2O2 (as a ROS representative). Reversible oxidation could thus indeed be physiologically important. More oxidative DNA damage (by bleomycin) showed no PGP-dependent effects here. EGF stimulation (as an inducer of transient and well-controlled ROS production), low concentrations of menadione (as an oxidant) and N-acetylcysteine (as an antioxidant) were able to approximate the proliferation rate in PGP downregulated cells to that of control cells. The redox regulation of PGP could thus have an influence on cellular proliferation as a feedback mechanism - a mechanism that could not take place in PGP downregulated cells. However, the connections are probably even more complex and cannot be elucidated by a sole examination of the proliferation rate. The present results can thus only be regarded as preliminary experiments.
For a better understanding of the features and functions of PGP, this work then focused on specific regulation of enzyme activity by pharmacologically applicable small molecules. Four potent inhibitors had previously been identified in a screening campaign. In this work, three of these four inhibiting compounds could be further characterized in experiments with highly purified, recombinant murine and human PGP. Compounds #2 and #9 showed competitive inhibition properties with a markedly rising KM value with little or no change in vmax. The results were consistent for all tested protein variants: the murine and the human PGP as well as a PGP/PDXP hybrid protein. Compound #1 was the most potent and interesting PGP-inhibitory molecule: less change in KM and a constant decrease in vmax as well as a lower impact on the PGP/PDXP hybrid hint at a mixed mode of inhibition as a combination of competitive and non-competitive inhibition. The characterization of the potential inhibitors can serve as a basis for further structural analysis and studies on the complex physiological role of PGP.
Despite accounting for only a small proportion of all skin cancers, malignant melanoma
displays a serious health risk with increasing incidence and high mortality rate. Fortunately,
advances in the treatment of malignant melanoma now prolong survival and enhance response
and treatment efficacy. Established biomarkers help evaluate disease progression and
facilitate choosing appropriate and individual treatment options. However, the need for easily
accessible and reliable biomarkers is rising to predict patient-specific clinical outcome.
Eosinophil infiltration into the tumor and high peripheral eosinophil counts prior and during
treatment have been associated with better response in patients for various cancer entities,
including melanoma. An analysis of a heterogeneous study cohort reported high serum ECP
levels in non-responders. Hence, eosinophil frequency and serum ECP as a soluble
eosinophil-secreted mediator were suggested as prognostic biomarkers in melanoma. We
examined whether melanoma patients treated with first-line targeted therapy could also benefit
from the effects of eosinophils. In total, 243 blood and serum samples from patients with
advanced melanoma were prospectively and retrospectively collected before and after drug
initiation. To link eosinophil function to improved clinical outcome, soluble serum markers and
peripheral blood counts were used for correlative studies using a homogeneous study cohort.
In addition, functional and phenotypical characterizations provided insights into the expression
profile and activity of freshly isolated eosinophils, including comparisons between patients and
healthy donors.
Our data showed a significant correlation between high pre-treatment blood eosinophil counts
and improved response to targeted therapy and by trend to combinatorial immunotherapy in
patients with metastatic melanoma. In accordance with previous studies our results links
eosinophil blood counts to better response in melanoma patients. High pre-treatment ECP
serum concentration correlated with response to immunotherapy but not to targeted therapy.
Eosinophils from healthy donors and patients showed functional and phenotypical similarities.
Functional assays revealed a strong cytotoxic potential of blood eosinophils towards
melanoma cells in vitro, inducing apoptosis and necrosis. In addition, in vitro cytotoxicity was
an active process of peripheral eosinophils and melanoma cells with bidirectional features and
required close cell-cell interaction. The extent of cytotoxicity was dose-dependent and showed
susceptibility to changes in physical factors like adherence. Importantly, we provide evidence
of an additive tumoricidal function of eosinophils and combinatorial targeted therapy in vitro. In
summary, we give valuable insights into the complex and treatment-dependent role of
eosinophils in melanoma. As a result, our data support the suggestion of eosinophils and their
secreted mediators as potential prognostic biomarkers. It will take additional studies to
examine the molecular mechanisms that underlie our findings.
The nervous system relies on an orchestrated assembly of complex cellular entities called neurons, which are specifically committed to information management and transmission. Inter-neuronal communication takes place via synapses, membrane-membrane junctions which ensure efficient signal transfer. Synaptic neurotransmission involves release of presynaptic neurotransmitters and their reception by cognate receptors at postsynaptic terminals. Inhibitory neurotransmission is primarily mediated by the release of neurotransmitters GABA (γ-Aminobutyric acid) and glycine, which are precisely sensed by GABA type-A receptors (GABAARs) and glycine receptors (GlyRs), respectively. GABAAR assembly and maintenance is coordinated by various postsynaptic neuronal factors including the scaffolding protein gephyrin, the neuronal adaptor collybistin (CB) and cell adhesion proteins of the neuroligin (NL) family, specifically NL2 and NL4.
At inhibitory postsynaptic specializations, gephyrin has been hypothesized to form extended structures underneath the plasma membrane, where its interaction with the receptors leads to their stabilization and impedes their lateral movement. Gephyrin mutations have been associated with various brain disorders, including autism, schizophrenia, Alzheimer’s disease, and epilepsy. Furthermore, gephyrin loss is lethal and causes mice to die within the first post-natal day. Gephyrin recruitment from intracellular deposits to postsynaptic membranes primarily relies on the adaptor protein CB.
As a moonlighting protein, CB, a guanine nucleotide exchange factor (GEF), also catalyzes a nucleotide exchange reaction, thereby regenerating the GTP-bound state of the small GTPase Cdc42 from its GDP-bound form. The CB gene undergoes alternative splicing with the majority of CB splice variants featuring an N-terminal SH3 domain followed by tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains. Previous studies demonstrated that the most widely expressed, SH3-domain containing splice variant (CB2SH3+) preferentially adopts a closed conformation, in which the N-terminally located SH3 domain forms intra-molecular interaction with the DH-PH domain tandem. Previous cell-based studies indicated that SH3 domain-encoding CB variants remain untargeted and colocalize with intracellular gephyrin deposits and hence require additional factors which interact with the SH3 domain, thus inducing an open or active conformation. The SH3 domain-deficient CB isoform (CB2SH3-), on the contrary, adopts an open conformation, which possess enhanced postsynaptic gephyrin-clustering and also effectively replenishes the GTP-bound small GTPase-Cdc42 from its GDP-bound state.
Despite the fundamental role of CB as a neuronal adaptor protein maintaining the proper function of inhibitory GABAergic synapses, its interactions with the neuronal scaffolding protein gephyrin and other post synaptic neuronal factors remain poorly understood. Moreover, CB interaction studies with the small GTPase Cdc42 and TC10, a closely related member of Cdc42 subfamily, remains poorly characterized. Most importantly, the roles of the neuronal factors and small GTPases in CB conformational activation have not been elucidated.
This PhD dissertation primarily focuses on delineating the molecular basis of the interactions between CB and postsynaptic neuronal factors. During the course of my PhD dissertation, I engineered a series of CB FRET (Förster Resonance Energy Transfer) sensors to characterize the CB interaction with its binding partners along with outlining their role in CB conformational activation. Through the aid of these CB FRET sensors, I analyzed the gephyrin-CB interaction, which, due to technical limitations remained unaddressed for more than two decades (refer Chapter 2 for more details). Subsequently, I also unraveled the molecular basis of the interactions between CB and the neuronal cell adhesion factor neuroligin 2 (refer chapter 2) and the small GTPases Cdc42 and TC10 (refer chapter 3) and describe how these binding partners induce a conformational activation of CB.
In summary, this PhD dissertation provides strong evidence of a closely knit CB communication network with gephyrin, neuroligin and the small GTPase TC10, wherein CB activation from closed/inactive to open/active states is effectively triggered by these ligands.
Plexus injury often occurs after motor vehicle accidents and results in lifelong disability with severe neuropathic pain. Surgical treatment can partially restore motor functions, but sensory loss and neuropathic pain persist. Regenerative medicine concepts, such as cell replacement therapies for restoring dorsal root ganglia (DRG) function, set high expectations. However, up to now, it is unclear which DRG cell types are affected by nerve injury and can be targeted in regenerative medicine approaches.
This study followed the hypothesis that satellite glial cells (SGCs) might be a suitable endogenous cell source for regenerative medicine concepts in the DRG. SGCs originate from the same neural crest-derived cell lineage as sensory neurons, making them attractive for neural repair strategies in the peripheral nervous system. Our hypothesis was investigated on three levels of experimentation. First, we asked whether adult SGCs have the potential of sensory neuron precursors and can be reprogrammed into sensory neurons in vitro. We found that adult mouse DRG harbor SGC-like cells that can still dedifferentiate into progenitor-like cells. Surprisingly, expression of the early developmental transcription factors Neurog1 and Neurog2 was sufficient to induce neuronal and glial cell phenotypes. In the presence of nerve growth factor, induced neurons developed a nociceptor-like phenotype expressing functional nociceptor markers, such as the ion channels TrpA1, TrpV1 and NaV1.9. In a second set of experiments, we used a rat model for peripheral nerve injury to look for changes in the DRG cell composition. Using an unbiased deep learning-based approach for cell analysis, we found that cellular plasticity responses after nerve injury activate SGCs in the whole DRG. However, neither injury-induced neuronal death nor gliosis was observed. Finally, we asked whether a severe nerve injury changed the cell composition in the human DRG. For this, a cohort of 13 patients with brachial plexus injury was investigated. Surprisingly, in about half of all patients, the injury-affected DRG showed no characteristic DRG tissue. The complete entity of neurons, satellite cells, and axons was lost and fully replaced by mesodermal/connective tissue. In the other half of the patients, the basic cellular entity of the DRG was well preserved. Objective deep learning-based analysis of large-scale bioimages of the “intact” DRG showed no loss of neurons and no signs of gliosis.
This study suggests that concepts for regenerative medicine for restoring DRG function need at least two translational research directions: reafferentation of existing DRG units or full replacement of the entire multicellular DRG structure. For DRG replacement, SGCs of the adult DRG are an attractive endogenous cell source, as the multicellular DRG units could possibly be rebuilt by transdifferentiating neural crest-derived sensory progenitor cells into peripheral sensory neurons and glial cells using Neurog1 and Neurog2.
Empathy, the act of sharing another person’s affective state, is a ubiquitous driver for helping others and feeling close to them. These experiences are integral parts of human behavior and society. The studies presented in this dissertation aimed to investigate the sustainability and stability of social closeness and prosocial decision-making driven by empathy and other social motives. In this vein, four studies were conducted in which behavioral and neural indicators of empathy sustainability were identified using model-based functional magnetic resonance imaging (fMRI).
Applying reinforcement learning, drift-diffusion modelling (DDM), and fMRI, the first two studies were designed to investigate the formation and sustainability of empathy-related social closeness (study 1) and examined how sustainably empathy led to prosocial behavior (study 2). Using DDM and fMRI, the last two studies investigated how empathy combined with reciprocity, the social norm to return a favor, on the one hand and empathy combined with the motive of outcome maximization on the other hand altered the behavioral and neural social decision process.
The results showed that empathy-related social closeness and prosocial decision tendencies persisted even if empathy was rarely reinforced. The sustainability of these empathy effects was related to recalibration of the empathy-related social closeness learning signal (study 1) and the maintenance of a prosocial decision bias (study 2). The findings of study 3 showed that empathy boosted the processing of reciprocity-based social decisions, but not vice versa. Study 4 revealed that empathy-related decisions were modulated by the motive of outcome maximization, depending on individual differences in state empathy.
Together, the studies strongly support the concept of empathy as a sustainable driver of social closeness and prosocial behavior.
Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes.
The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration.
Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.
Fear and anxiety disorders – interaction of AVP and OXT brain systems with the serotonergic system
(2023)
Anxiety disorders pose a great burden onto society and economy and can have devastating consequences for affected individuals. Treatment options are still limited to psychopharmacotherapy originally developed for the treatment of depression and behavioral therapy. A combination of genetic traits together with aversive events is most likely the cause of these diseases. Gene x environment studies are trying to find a link between genetic traits and specific negative circumstances. In a first study, we focused on social anxiety disorder (SAD), which is the second most-common anxiety disorder after specific phobias. We used a social fear conditioning (SFC) paradigm, which is able to mimic the disease in a mouse model. We wanted to investigate protein levels, as well as mRNA expression of immediate early genes (IEGs), to determine brain areas affected by the paradigm. We also included genes of the vasopressin (AVP)-, oxytocin (OXT)-, neuropeptide Y (NPY)-, and the serotonin system, to investigate the effects of SFC on neurotransmitter gene expression levels in brain regions related to social as well as fear-related behavior. AVP and OXT regulate a lot of different social and anxiety-related behaviors, both positive and negative. Finding a link between different neurotransmitter systems in the development of anxiety disorders could help to identify potential targets for new treatment approaches, which are desperately needed, because the rate of patients not responding to available treatment is very high.
We were able to show altered gene expression of the IEGs cFos and Fosl2, as well as a change in number and density of cFOS-positive cells in the dorsal hippocampus, indicating an influence of SFC on neuronal activity. Our results reveal a possible involvement of anterior dentate gyrus (DG), as well as cornu ammonis area 1 (CA1) and CA3 in the dorsal hippocampus during the expression of social fear. Contrary to our hypothesis, we were not able to see changes in neuronal activity through expression changes of IEGs in the amygdala. Significant higher IEG immunoreactivity and gene expression in the dorsal hippocampus of animals without fear conditioning (SFC-), compared to animals with fear conditioning (SFC+), indicate an involvement of different hippocampal regions in two possible scenarios. Either as elevated gene expression in SFC- animals compared to SFC+ animals or as reduction in SFC+ animals compared to SFC- animals. However, this question cannot be answered without an additional control of basal IEG-activity without social interaction. The NPY system in general and the neuropeptide y receptor type 2 in particular seem to be involved in regulating the response to social fear, mostly through the septum region. In addition to that, a possible role for the induction of social fear response could be identified in the serotonergic system and especially the serotonin receptor 2a of the PVN.
In a second study we focused on changes in the serotonergic system. A polymorphism in the human serotonin transporter (5-HTT) gene is associated with higher risks for the development of anxiety disorders. This makes the 5-HTT a widely used target to study possible causes and the development of anxiety disorders. In mice, a genetically induced knockout of the 5-Htt gene is associated with increased anxiety-like behavior. High amounts of stress during pregnancy, also known as prenatal stress, significantly increase the risk to develop psychiatric disorders for the unborn child. We utilized a prenatal stress paradigm in mice heterozygous for the 5-Htt gene. Some of the animals which had been subjected to prenatal stress showed noticeably “unsocial” interaction behavior towards conspecifics. Again, we were searching for links between the serotonergic system and AVP- and OXT systems. Through quantitative gene expression analysis, we were able to show that both AVP and OXT neuromodulator systems are affected through prenatal stress in female mice, but not in male mice. The 5-Htt genotype seems to be only slightly influential to AVP, OXT or any other neurotransmitter system investigated. Gene expression of AVP and OXT brain systems is highly influenced through the estrous cycle stages of female mice. Additionally, we analyzed the AVP and OXT neuropeptide levels of mice with different 5-Htt genotypes and in both sexes, in order to see whether the production of AVP and OXT is influenced by 5-Htt genotype. On neuropeptide level, we were able to identify a sex difference for vasopressin-immunoreactive (ir) cells in the PVN, with male mice harboring significantly more positive cells than female mice.
Platelets play an important role in the body, since they are part of the hemostasis
system, preventing and stopping blood loss. Nevertheless, when platelet or
coagulation system function are impaired, uncontrolled bleedings but also irreversible
vessel occlusion followed by ischemic tissue damage can occur. Therefore,
understanding platelet function and activation, mechanisms which are controlled by a
variety of platelet membrane receptors and other factors is important to advance out
knowledge of hemostasis and platelet malfunction. For a complete picture of platelet
function and their modulating behavior it is desired to be able to quantify receptor
distributions and interactions of these densely packed molecular ensembles in the
membrane. This challenges scientists for several reasons. Most importantly, platelets
are microscopically small objects, challenging the spatial resolution of conventional
light microscopy. Moreover, platelet receptors are highly abundant on the membrane
so even super-resolution microscopy struggles with quantitative receptor imaging on
platelets.
With Expansion microscopy (ExM), a new super-resolution technique was introduced,
allowing resolutions to achieve super-resolution without using a super-resolution
microscope, but by combining a conventional confocal microscopy with a highly
processed sample that has been expanded physically. In this doctoral thesis, I
evaluated the potential of this technique for super-resolution platelet imaging by
optimizing the sample preparation process and establishing an imaging and image
processing pipeline for dual-color 3D images of different membrane receptors. The
analysis of receptor colocalization using ExM demonstrated a clear superiority
compared to conventional microscopy. Furthermore, I identified a library of
fluorescently labeled antibodies against different platelet receptors compatible with
ExM and showed the possibility of staining membrane receptors and parts of the
cytoskeleton at the same time.
Spinal muscular atrophy (SMA) is a genetic pediatric condition that affects lower motoneurons leading to their degeneration and muscle weakness. It is caused by homozygous loss or mutations in the Survival Motor Neuron 1 (SMN1) gene; however, the pathomechanism leading to motoneuron degeneration is not fully resolved. Cultured embryonic SMA motoneurons display axon elongation and differentiation defects accompanied by collapsed growth cones with a disturbed actin cytoskeleton. Intriguingly, motoneurons cultured from mice deficient for the Tropomyosin-kinase receptor B (TrkB), exhibit similar pathological features. Thus, the question arises whether SMA motoneurons suffer from defective Brain-derived neurotrophic factor (BDNF)/TrkB signaling and whether there is a link to the disturbed actin cytoskeleton. In the recent years, modifier genes such as Plastin 3 (PLS3) were shown to beneficially interfere with SMA pathology. Nevertheless, the mechanism of how the actin-bundler PLS3 counteracts SMN deficiency is not well understood. In this study, we investigated TrkB localization and its activation in cultured SMA motoneurons and neuromuscular junctions (NMJs). While TrkB levels are only mildly affected locally in axon terminals, BDNF-mediated TrkB phosphorylation was massively disturbed. The activity-dependent TrkB translocation to the cell surface and its activation via BDNF were shown to be Pls3-dependent processes, that can be abolished by knockdown of Pls3. In contrast, PLS3 overexpression in SMA motoneurons rescued the defects on morphological and functional level. In particular, the relocation of TrkB to the cell surface after BDNF-induced internalization is disturbed in SMA, which is based on an actin-dependent TrkB translocation defect from intracellular stores. Lastly, AAV9-mediated PLS3 overexpression in vivo in neonatal SMA mice provided further evidence for the capacity of PLS3 to modulate actin dynamics necessary for accurate BDNF/TrkB signaling. In conclusion, we provide a novel role for PLS3 in mediating proper alignment of transmembrane proteins as prerequisite for their appropriate functioning. Hence, PLS3 is required for a key process indispensable for the development and function of motoneurons even beyond the context of SMA.
Dietary fatty acids serve as objective biomarkers for the estimation of habitual diet mainly because biomarkers are free of memory bias or inaccuracies of food databases. The aim of the present work encompassed the implementation of a gas chromatographical method coupled with a mass spectrometrical and flame-ionization detector for analysis of fatty acid biomarkers in human biospecimens, their analytical determination and statistical evaluation in two different study populations and different biospecimens as well as the elaboration of adverse reactions to food ingredients with special focus on food allergies and food intolerances in the context of a possible implementation into an application for consumer health. The first aim was the identification of potential influence of fatty acid biomarkers on desaturase and elongase indexes (Δ9DI, Δ6DI, Δ5DI and ELOVLI5), which are factors in type 2 diabetes risk, in breast adipose tissue from healthy women. Influence of further variables on respective indexes was also investigated. 40 samples were investigated and potential variables were either collected by questionnaire or determined. Principle component analysis was applied for fatty acid biomarkers (PCdiet1, PCdiet2 and PCdiet3 representative for the dietary intake of vegetable oils/nuts, fish and partially hydrogenated vegetable oils), endogenous estrogens (PCE1) and oxysterols (PCOxy1). Multiple linear regression models were applied. Δ9DI and Δ6DI were influenced non-significantly and significantly negatively by PCdiet2 supporting a putative beneficial effect of vegetable oils and nuts on type 2 diabetes risk factors. ELOVLI5 and Δ5DI were influenced significantly and non-significantly positively by PCdiet1 supporting a putative beneficial effect of fish consumption on type 2 diabetes risk factors. On the other hand, PCdiet1 also significantly and non-significantly positively influenced Δ9DI and Δ6DI supporting a putative adverse effect of fish biomarkers on type 2 diabetes risk factors. The opposing influences of PCdiet1 suggesting an ambivalent role of dietary intake of fish on investigated indexes. Δ6DI was significantly positively influenced by PCdiet3 and number of pregnancies supporting a putative adverse effect of partially hydrogenated vegetable oils and pregnancies on type 2 diabetes risk factors. Lifestyle factors like smoking significantly and non-significantly influenced Δ9DI and Δ6DI putatively adversely. Δ5DI was influenced significantly positively by estrogen active drugs suggesting a putative beneficial effect on type 2 diabetes risk factors. It must be considered that a variation coefficient of up to 0.44 only explained 44% of variance of the respective indexes, suggesting other influencing factors might play a role. The second aim was the implementation of a gas chromatographical method coupled with a mass spectrometrical and flame-ionization detector for analysis of fatty acid biomarkers in human biospecimens. The method was optimized for separation and detection of 40 fatty acids. Mean recovery for tridecanoic acid was x(tridecanoic acid) = 90.51% and for nonadecanoic acid x(nonadecanoic acid) = 96.21%. Thus, there was no significant loss of fatty acids with shorter and longer carbon chains over the extraction process to be expected. Limit of detections were calculated in adipose tissue samples and ranged from 0.007 to 0.077% of the proportion of the respective fatty acid to total fatty acids. The third aim was the investigation if differentiation between breast glandular and adipose tissue had a relevant impact on the analysis of dietary fatty acid biomarkers or if contamination of breast glandular with breast adipose tissue and vice versa was neglectable for the analysis of dietary fatty acid biomarkers. No statistical significant differences were observed for all investigated fatty acid biomarkers (pentadecanoic-, heptadecanoic-, trans palmitoleic-, eicosapentaenoic-, docosahexaenoic-, linoleic and α-linolenic acid) between breast glandular and adipose tissue. Thus, differentiation between breast glandular and adipose tissue seems not to be necessary for the analysis of fatty acids serving as biomarkers for the intake of specific food groups. Potential influence of mixed breast tissue on fatty acid biomarkers analysis seems to be neglectable. The fourth aim was the determination of fatty acid biomarkers in adipose tissue in another study population from healthy participants. 27 adipose tissue samples were analyzed. Milk and ruminant fat biomarkers exhibited proportions of 0.47% for pentadecanoic acid, 0.34% for heptadecanoic acid and 0.25% for trans palmitoleic acid. Fish fatty acid biomarkers revealed proportions of 0.034% for eicosapentaenoic acid and 0.061% for docosahexaenoic acid. The mean proportion of vegetable oils and nuts biomarkers were 9.58% for linoleic acid and 0.48% for α-linolenic acid in all adipose tissues. Principle component analysis was applied for the fatty acid biomarkers to provide objective markers of habitual diet for this study population. PCdiet1 was mainly characterized by pentadecanoic acid, heptadecanoic acid and trans palmitoleic acid and therefore served as a principle component for the dietary intake of milk and ruminant fat. PCdiet2 and PCdiet3 only exhibited pattern for ω3 and ω6 fatty acids but not for dietary intake of specific food groups and could therefore not used as objective marker. PCdiet1, 2 and 3 explained 82.76% of variance. The last aim of this thesis was the elaboration of adverse reactions to food ingredients with special focus on food allergies and food intolerances in the context of a possible implementation into an application for consumer health. Scientific information on adverse reactions to food ingredients and trigger substances was provided in this thesis and possible implementation strategies were evaluated. For food allergens, which have regulatory requirements in the context of labelling, a strategy was elaborated, where it is necessary to provide information on the list of ingredients, the nexus ’contain’ and the respective food allergen as well as information on the name of the product. For food intolerances, which do not have regulatory requirements, limits were shown in the context of the application. If the elaborated food intolerances shall be implemented into the application, a professional dietary concept has to be developed for every food intolerance because of the complexity of the implementation.
As a major component of the articular cartilage extracellular matrix, hyaluronic acid is a widely used biomaterial in regenerative medicine and tissue engineering. According to its well-known interaction with multiple chondrocyte surface receptors which positively affects many cellular pathways, some approaches by combining mesenchymal stem cells and hyaluronic acid-based hydrogels are already driven in the field of cartilage regeneration and fat tissue. Nevertheless, a still remaining major problem is the development of the ideal matrix for this purpose. To generate a hydrogel for the use as a matrix, hyaluronic acid must be chemically modified, either derivatized or crosslinked and the resulting hydrogel is mostly shaped by the mold it is casted in whereas the stem cells are embedded during or after the gelation procedure which does not allow for the generation of zonal hierarchies, cell density or material gradients. This thesis focuses on the synthesis of different hyaluronic acid derivatives and poly(ethylene glycol) crosslinkers and the development of different hydrogel and bioink compositions that allow for adjustment of the printability, integration of growth factors, but also for the material and biological hydrogel, respectively bioink properties.
Coxiella burnetii, a Gram negative obligate intracellular bacterium, is the causative
agent of Q fever. It has a world wide distribution and has been documented to
be capable of causing infections in several domestic animals, livestock species,
and human beings. Outbreaks of Q fever are still being observed in livestock
across animal farms in Europe, and primary transmission to humans still oc-
curs especially in animal handlers. Public health authorities in some countries
like Germany are required by law to report human acute cases denoting the
significance of the challenge posed by C. burnetii to public health.
In this thesis, I have developed a platform alongside methods to address the
challenges of genomic analyses of C. burnetii for typing purposes. Identification
of C. burnetii isolates is an important task in the laboratory as well as in the
clinics and genotyping is a reliable method to identify and characterize known
and novel isolates. Therefore, I designed and implemented several methods
to facilitate the genotyping analyses of C. burnetii genomes in silico via a web
platform. As genotyping is a data intensive process, I also included additional
features such as visualization methods and databases for interpretation and
storage of obtained results. I also developed a method to profile the resistome
of C. burnetii isolates using a machine learning approach. Data about antibiotic
resistance in C. burnetii are scarce majorly due to its lifestyle and the difficulty
of cultivation in laboratory media. Alternative methods that rely on homology
identification of resistance genes are also inefficient in C. burnetii, hence, I
opted for a novel approach that has been shown to be promising in other
bacteria species. The applied method relied on an artificial neural network as
well as amino acid composition of position specific scoring matrix profile for
feature extraction. The resulting model achieved an accuracy of ≈ 0.96 on test
data and the overall performance was significantly higher in comparison to
existing models. Finally, I analyzed two new C. burnetii isolates obtained from
an outbreak in Germany, I compared the genome to the RSA 493 reference
isolate and found extensive deletions across the genome landscape.
This work has provided a new digital infrastructure to analyze and character-
ize C. burnetii genomes that was not in existence before and it has also made a
significant contribution to the existing information about antibiotic resistance
genes in C. burnetii.
Mass spectrometry-based quantification of steroids for the diagnostic workup of adrenal tumors
(2023)
Tumors of the adrenal gland belong to the most frequent neoplasms in humans with a prevalence of 3–10 % in adults. The aim of the diagnostic workup is the identification of potentially hormone-secreting and / or malignant tumors, because most of these tumors will require surgical resection. Malignant adrenocortical carcinomas (ACC) are very rare and associated with a poor prognosis in advanced stages, therefore, an early and accurate diagnosis is crucial.
Within this thesis, two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the quantification of steroids in different biomaterials were developed to improve the diagnostic workup of adrenal tumors.
First, an LC-MS/MS method for the simultaneous quantification of cortisol and dexamethasone in serum samples after dexamethasone suppression test (DST) was developed, validated, and applied to 400 clinical samples. Newly established method-specific threshold concentrations for cortisol and dexamethasone increased DST specificity from 67.5 % to 92.4 % while preserving 100 % sensitivity.
Second, an LC-MS/MS method for the quantification of eleven urinary steroids was developed and validated to improve the differentiation between ACC and adrenocortical adenomas (ACA). A decision tree requiring only two steroids was trained for classification and tested based on 24 h urine samples from 268 patients with adrenal tumor. Malignancy was excluded with a negative predictive value of 100 % in an independent validation cohort of 84 samples of 24-h urine. A newly proposed simplified diagnostic workflow with urinary steroid profiling as first tier test could obviate additional adrenal-specific imaging in 42 of 64 patients with ACA.
The new DST method is already in clinical use at the University Hospital Würzburg, whereas the classification model based on urinary steroid profiling will require prospective validation in a larger cohort.
The original habitat of native European honey bees (\(Apis\) \(mellifera\)) is forest, but currently there is a lack of data about the occurrence of wild honey bee populations in Europe. Prior to being kept by humans in hives, honey bees nested as wild species in hollow trees in temperate forests. However, in the 20th century, intensification of silviculture and agriculture with accompanying losses of nesting sites and depletion of food resources caused population declines in Europe. When the varroa mite (Varroa destructor), an invasive ectoparasite from Asia, was introduced in the late 1970s, wild honey bees were thought to be eradicated in Europe. Nevertheless, sporadic, mostly anecdotal, reports from ornithologists or forest ecologists indicated that honey bee colonies still occupy European forest areas. In my thesis I hypothesize that near-natural deciduous forests may provide sufficient large networks of nesting sites representing refugia for wild-living honey bees. Using two special search techniques, i.e. the tracking of flight routes of honey bee foragers (the “beelining” method) and the inspection of known cavity trees, I collected for the first time data on the occurrence and density of wild-living honey bees in forest areas in Germany (CHAPTER 3). I found wild-living honey bee colonies in the Hainich national park at low densities in two succeeding years. In another forest region, I checked known habitat trees containing black woodpecker cavities for occupation by wild-living honey bee colonies. It turned out that honey bees regularly use these cavities and occur in similar densities in both studied forest regions, independent of the applied detection method. Extrapolating these densities to all German forest areas, I estimate several thousand wild-living colonies in Germany that potentially interact in different ways with the forest environment. I conclude that honey bees regularly colonize forest areas in Germany and that networks of mapped woodpecker cavities offer unique possibilities to study the ecology of wild-living honey bees over several years.
While their population status is ambiguous and the density of colonies low, the fact that honey bees can still be found in forests poses questions about food supply in forest environments. Consequently, I investigated the suitability of woodlands as a honey bee foraging habitat (CHAPTER 4). As their native habitat, forests are assumed to provide important pollen and nectar sources for honey bee colonies. However, resource supply might be spatially and temporally restricted and landscape-scale studies in European forest regions are lacking. Therefore, I set up twelve honey bee colonies in observation hives at locations with varying degree of forest cover. Capitalizing on the unique communication behaviour, the waggle dance, I examined the foraging distances and habitat preferences of honey bees over almost an entire foraging season. Moreover, by connecting this decoded dance information with colony weight recordings, I could draw conclusions about the contribution of the different habitat types to honey yield. Foraging distances generally increased with the amount of forest in the surrounding landscape. Yet, forest cover did not have an effect on colony weight. Compared to expectations based on the proportions of different habitats in the surroundings, colonies foraged more frequently in cropland and grasslands than in deciduous and coniferous forests, especially in late summer when pollen foraging in the forest is most difficult. In contrast, colonies used forests for nectar/honeydew foraging in early summer during times of colony weight gain emphasizing forests as a temporarily significant source of carbohydrates. Importantly, my study shows that the ecological and economic value of managed forest as habitat for honey bees and other wild pollinators can be significantly increased by the continuous provision of floral resources, especially for pollen foraging.
The density of these wild-living honey bee colonies and their survival is driven by several factors that vary locally, making it crucial to compare results in different regions. Therefore, I investigated a wild-living honey bee population in Galicia in north-western Spain, where colonies were observed to reside in hollow electric poles (CHAPTER 5). The observed colony density only in these poles was almost twice as high as in German forest areas, suggesting generally more suitable resource conditions for the bees in Galicia. Based on morphometric analyses of their wing venation patterns, I assigned the colonies to the native evolutionary lineage (M-lineage) where the particularly threatened subspecies \(Apis\) \(mellifera\) \(iberiensis\) also belongs to. Averaged over two consecutive years, almost half of the colonies survived winter (23 out of 52). Interestingly, semi-natural areas both increased abundance and subsequent colony survival. Colonies surrounded by more semi-natural habitat (and therefore less intensive cropland) had an elevated overwintering probability, indicating that colonies need a certain amount of semi-natural habitat in the landscape to survive. Due to their ease of access these power poles in Galicia are, ideally suited to assess the population demography of wild-living Galician honey bee colonies through a long-term monitoring.
In a nutshell, my thesis indicates that honey bees in Europe always existed in the wild. I performed the first survey of wild-living bee density yet done in Germany and Spain. My thesis identifies the landscape as a major factor that compromises winter survival and reports the first data on overwintering rates of wild-living honey bees in Europe. Besides, I established methods to efficiently detect wild-living honey bees in different habitat. While colonies can be found all over Europe, their survival and viability depend on unpolluted, flower rich habitats. The protection of near-natural habitat and of nesting sites is of paramount importance for the conservation of wild-living honey bees in Europe.
The monarch butterfly (Danaus plexippus) performs one of the most astonishing behaviors in the animal kingdom: every fall millions of these butterflies leave their breeding grounds in North Amerika and migrate more than 4.000 km southwards until they reach their overwintering habitat in Central Mexico. To maintain their migratory direction over this enormous distance, the butterflies use a time-compensated sun compass. Beside this, skylight polarization, the Earth’s magnetic field and specific mountain ranges seem to guide the butterflies as well the south. In contrast to this fascinating orientation ability, the behavior of the butterflies in their non-migratory state received less attention. Although they do not travel long distances, they still need to orient themselves to find food, mating partners or get away from competitors. The aim of the present doctoral thesis was to investigate use of visual cues for orientation in migrating as well as non-migrating monarch butterflies. For this, field experiments investigating the migration of the butterflies in Texas (USA) were combined with experiments testing the orientation performance of non-migratory butterflies in Germany.
In the first project, I recorded the heading directions of tethered butterflies during their annual fall migration. In an outdoor flight simulator, the butterflies maintained a southwards direction as long as they had a view of the sun’s position. Relocating the position of the sun by 180° using a mirror, revealed that the sun is the animals’ main orientation reference. Furthermore, I demonstrated that when the sun is blocked and a green light stimulus (simulated sun) is introduced, the animals interpreted this stimulus as the ‘real’ sun. However, this cue was not sufficient to set the migratory direction when simulated as the only visual cue in indoor experiments. When I presented the butterflies a linear polarization pattern additionally to the simulated sun, the animals headed in the correct southerly direction showing that multiple skylight cues are required to guide the butterflies during their migration.
In the second project, I, furthermore, demonstrated that non-migrating butterflies are able to maintain a constant direction with respect to a simulated sun. Interestingly, they ignored the spectral component of the stimulus and relied on the intensity instead. When a panoramic skyline was presented as the only orientation reference, the butterflies maintained their direction only for short time windows probably trying to stabilize their flight based on optic-flow information. Next, I investigated whether the butterflies combine celestial with local cues by simulating a sun stimulus together with a panoramic skyline. Under this conditions, the animals’ directedness was increased demonstrating that they combine multiple visual cues for spatial orientation.
Following up on the observation that a sun stimulus resulted in a different behavior than the panoramic skyline, I investigated in my third project which orientation strategies the butterflies use by presenting different simulated cues to them. While a bright stripe on a dark background elicited a strong attraction of the butterflies steering in the direction of the stimulus, the inverted version of the stimulus was used for flight stabilization. In contrast to this, the butterflies maintained arbitrary directions with a high directedness with respect to a simulated sun. In an ambiguous scenery with two identical stimuli (two bright stripes, two dark stripes, or two sun stimuli) set 180° apart, a constant flight course was only achieved when two sun stimuli were displayed suggesting an involvement of the animals’ internal compass. In contrast, the butterflies used two dark stripes for flight stabilization and were alternatingly attracted by two bright stripes. This shows that monarch butterflies use stimulus-dependent orientation strategies and gives the first evidence for different neuronal pathways controlling the output behavior.
To reach their target site, systemic pesticides must enter the plant from a spray droplet applied in the field. The uptake of an active ingredient (AI) takes place via the barrier-forming cuticular membrane, which is the outermost layer of the plant, separating it from the surrounding environment. Formulations are usually used which, in addition to the AI, also contain stabilizers and adjuvants. Adjuvants can either have surface-active properties or they act directly as barrier-modifying agents. The latter are grouped in the class of accelerating adjuvants, whereby individual variants may also have surface-active properties. The uptake of a pesticide from a spray droplet depends essentially on its permeability through the cuticular barrier. Permeability defines a combined parameter, which is the product of AI mobility and AI solubility within the cuticle. In recent decades, several tools have been developed that allowed the determination of individual parameters of organic compound penetration across the cuticular membrane. Nevertheless, earlier studies showed that mainly cuticular waxes are the barrier-determining component of the cuticular membrane and additionally, it was shown that mainly the very-long-chain aliphatic compounds (VLCAs) are responsible for establishing an effective barrier. However, the barrier-determining role of the individual VLCAs, being classified according to their respective functional groups, is still unknown.
Therefore, the following objectives were pursued and achieved in this work: (1) A new ATR-FTIR-based approach was developed to measure the temperature-dependent real-time diffusion kinetics of organic models for active ingredients (AIs) in paraffin wax, exclusively consisting of very-long chain alkanes. (2) The developed ATR-FTIR approach was applied to determine the diffusion kinetics of self-accelerating adjuvants in cuticular model waxes of different VLCA composition. At the same time, wax-specific changes were recorded in the respective IR spectra, which provided information about the respective wax modification. (3) The ATR-FTIR method was used to characterize the diffusion kinetics, as well as to determine the wax-specific sorption capacities for an AI-modeling organic compound and water in cuticular model waxes after adjuvant treatment. Regarding the individual chemical compositions and structures, conclusions were drawn about the adjuvant-specific modes of action (MoA).
In the first chapter, the ATR-FTIR based approach to determine organic compound diffusion kinetics in paraffin wax was successfully established. The diffusion kinetics of the AI modelling organic compounds heptyl parabene (HPB) and 4-cyanophenol (CNP) were recorded, comprising different lipophilicities and molecular volumes typical for AIs used in pesticide formulations. Derived diffusion coefficients ranged within 10-15 m2 s-1, thus being thoroughly higher than those obtained from previous experiments using an approach solely investigating desorption kinetics in reconstituted cuticular waxes. An ln-linear dependence between the diffusion coefficients and the applied diffusion temperature was demonstrated for the first time in cuticular model wax, from which activation energies were derived. The determined activation energies were 66.2 ± 7.4 kJ mol-1 and 56.4 ± 9.8 kJ mol-1, being in the expected range of already well-founded activation energies required for organic compound diffusion across cuticular membranes, which again confirmed the significant contribution of waxes to the cuticular barrier. Deviations from the assumed Fickian diffusion were attributed to co-occurring water diffusion and apparatus-specific properties.
In the second and third chapter, mainly the diffusion kinetics of accelerating adjuvants in the cuticular model waxes candelilla wax and carnauba wax were investigated, and simultaneously recorded changes in the wax-specific portion of the IR spectrum were interpreted as indications of plasticization. For this purpose, the oil derivative methyl oleate, as well as the organophosphate ester TEHP and three non-ionic monodisperse alcohol ethoxylates (AEs) C12E2, C12E4 and C12E6 were selected. Strong dependence of diffusion on the respective principal components of the mainly aliphatic waxes was demonstrated. The diffusion kinetics of the investigated adjuvants were faster in the n-alkane dominated candelilla wax than in the alkyl ester dominated carnauba wax. Furthermore, the equilibrium absorptions, indicating equilibrium concentrations, were also higher in candelilla wax than in carnauba wax. It was concluded that alkyl ester dominated waxes feature higher resistance to diffusion of accelerating adjuvants than alkane dominated waxes with shorter average chain lengths due to their structural integrity. This was also found either concerning candelilla/policosanol (n-alcohol) or candelilla/rice bran wax (alkyl-esters) blends: with increasing alcohol concentration, the barrier function was decreased, whereas it was increased with increasing alkyl ester concentration. However, due to the high variability of the individual diffusion curves, only a trend could be assumed here, but significant differences were not shown. The variability itself was described in terms of fluctuating crystalline arrangements and partial phase separation of the respective wax mixtures, which had inevitable effects on the adjuvant diffusion. However, diffusion kinetics also strongly depended on the studied adjuvants. Significantly slower methyl oleate diffusion accompanied by a less pronounced reduction in orthorhombic crystallinity was found in carnauba wax than in candelilla wax, whereas TEHP diffusion was significantly less dependent on the respective wax structure and therefore induced considerable plasticization in both waxes. Of particular interest was the AE diffusion into both waxes. Differences in diffusion kinetics were also found here between candelilla blends and carnauba wax. However, these depended equally on the degree of ethoxylation of the respective AEs. The lipophilic C12E2 showed approximately Fickian diffusion kinetics in both waxes, accompanied by a drastic reduction in orthorhombic crystallinity, especially in candelilla wax, whereas the more hydrophilic C12E6 showed significantly retarded diffusion kinetics associated with a smaller effect on orthorhombic crystallinity. The individual diffusion kinetics of the investigated adjuvants sometimes showed drastic deviations from the Fickian diffusion model, indicating a self-accelerating effect. Hence, adjuvant diffusion kinetics were accompanied by a distinct initial lag phase, indicating a critical concentration in the wax necessary for effective penetration, leading to sigmoidal rather than to exponential diffusion kinetics.
The last chapter dealt with the adjuvant-affected diffusion of the AI modelling CNP in candelilla and carnauba wax. Using ATR-FTIR, diffusion kinetics were recorded after adjuvant treatment, all of which were fully explicable based on the Fickian model, with high diffusion coefficients ranging from 10-14 to 10-13 m2 s-1. It is obvious that the diffusion coefficients presented in this work consistently demonstrated plasticization induced accelerated CNP mobilities. Furthermore, CNP equilibrium concentrations were derived, from which partition- and permeability coefficients could be determined. Significant differences between diffusion coefficients (mobility) and partition coefficients (solubility) were found on the one hand depending on the respective waxes, and on the other hand depending on treatment with respective adjuvants. Mobility was higher in candelilla wax than in carnauba wax only after methyl oleate treatment. Treatment with TEHP and AEs resulted in higher CNP mobility in the more polar alkyl ester dominated carnauba wax. The partition coefficients, on the other hand, were significantly lower after methyl oleate treatment in both candelilla and carnauba wax as followed by TEHP or AE treatment. Models were designed for the CNP penetration mode considering the respective adjuvants in both investigated waxes. Co-penetrating water, which is the main ingredient of spray formulations applied in the field, was likely the reason for the drastic differences in adjuvant efficacy. Especially the investigated AEs favored an enormous water uptake in both waxes with increasing ethoxylation level. Surprisingly, this effect was also found for the lipophilic TEHP in both waxes. This led to the assumption that the AI permeability is not exclusively determined by adjuvant induced plasticization, but also depends on a “secondary plasticization”, induced by adjuvant-attracted co-penetrating water, consequently leading to swelling and drastic destabilization of the crystalline wax structure.
The successful establishment of the presented ATR-FTIR method represents a milestone for the study of adjuvant and AI diffusion kinetics in cuticular waxes. In particular, the simultaneously detectable wax modification and, moreover, the determinable water uptake form a perfect basis to establish the ATR-FTIR system as a universal screening tool for wax-adjuvants-AI-water interaction in crop protection science.
In highly polarized neurons, endoplasmic reticulum (ER) forms a dynamic and continuous network in axons that plays important roles in lipid synthesis, Ca2+ homeostasis and the maintenance of synapses. However, the mechanisms underlying the regulation of axonal ER dynamics and its function in regulation of local translation still remain elusive. In the course of my thesis, I investigated the fast dynamic movements of ER and ribosomes in the growth cone of wildtype motoneurons as well as motoneurons from a mouse model of Spinal Muscular Atrophy (SMA), in response to Brain-derived neurotrophic factor (BDNF) stimulation. Live cell imaging data show that ER extends into axonal growth cone filopodia along actin filaments and disruption of actin cytoskeleton by cytochalasin D treatment impairs the dynamic movement of ER in the axonal filopodia. In contrast to filopodia, ER movements in the growth cone core seem to depend on coordinated actions of the actin and microtubule cytoskeleton. Myosin VI is especially required for ER movements into filopodia and drebrin A mediates actin/microtubule coordinated ER dynamics. Furthermore, we found that BDNF/TrkB signaling induces assembly of 80S ribosomes in growth cones on a time scale of seconds. Activated ribosomes relocate to the presynaptic ER and undergo local translation. These findings describe the dynamic interaction between ER and ribosomes during local translation and identify a novel potential function for the presynaptic ER in intra-axonal synthesis of transmembrane proteins such as the α-1β subunit of N-type Ca2+ channels in motoneurons. In addition, we demonstrate that in Smn-deficient motoneurons, ER dynamic movements are impaired in axonal growth cones that seems to be due to impaired actin cytoskeleton. Interestingly, ribosomes fail to undergo rapid structural changes in Smn-deficient growth cones and do not associate to ER in response to BDNF. Thus, aberrant ER dynamics and ribosome response to extracellular stimuli could affect axonal growth and presynaptic function and maintenance, thereby contributing to the pathology of SMA.
Social contact is an integral part of daily life. Its health-enhancing effects include reduced negative affective experiences of fear and anxiety, a phenomenon called social buffering. This dissertation studied different forms of social contact and their anxiety-buffering effects with diverse methodologies.
The laboratory-based first study investigated minimal social contact in the context of pain relief learning. Results showed that the observed decreased autonomic and increased subjective fear responses following pain relief learning were independent of social influence. The minimalistic and controlled social setting may have prevented social buffering. Study 2 targeted social buffering in daily life using Ecological Momentary Assessment. We repeatedly assessed individuals’ state anxiety, related cardiovascular responses, and aspects of social interactions with smartphones and portable sensors on five days. Analyses of over 1,500 social contacts revealed gender-specific effects, e.g., heart rate-reducing effects of familiarity in women, but not men. Study 3 examined anxiety, loneliness, and related social factors in the absence of social contact due to social distancing. We constructed and validated a scale measuring state and trait loneliness and isolation, and analysed its link to mental health. Results include a social buffering-like relation of lower anxiety with more trait sociability and sense of belonging.
In sum, the studies showed no fear reduction by minimal social contact, but buffering effects relating to social and personal factors in more complex social situations. Anxiety responses during daily social contacts were lower with more familiar or opposite-gender interaction partners. During limited social contact, lower anxiety related to inter-individual differences in sociability, social belonging, and loneliness. By taking research from lab to life, this dissertation underlined the diverse nature of social contact and its relevance to mental health.
The anaerobe Fusobacterium nucleatum (F. nucleatum) is an important member of the oral microbiome but can also colonize different tissues of the human body. In particular, its association with multiple human cancers has drawn much attention.
This association has prompted growing interest into the interaction of F. nucleatum with cancer, with studies focusing primarily on the host cells. At the same time, F. nucleatum itself remains poorly understood, which includes its transcriptomic architecture but also gene regulation such as global stress responses that typically enable survival of bacteria in new environments. An important aspect of such regulatory networks is the post-transcriptional regulation, which is entirely unknown in F. nucleatum. This paucity extents to any knowledge on small regulatory RNAs (sRNAs), despite their important role as post-transcriptional regulators of the bacterial physiology.
Investigating the above stated aspects is further complicated by the fact that F. nucleatum is phylogenetically distant from all other bacteria, displays very limited genetic tractability and lacks genetic tools for dissecting gene function.
This leaves many open questions on basic gene regulation in F. nucleatum, such as if the bacterium combines transcriptional and post-transcriptional regulation in its adaptation to a changing environment.
To begin answering this question, this works elucidated the transcriptomic landscape of F. nucleatum by performing differential RNA-seq (dRNA-seq). Conducted for five representative strains of all F. nucleatum subspecies and the closely related F. periodonticum, the analysis globally uncovered transcriptional start sites (TSS), 5'untranslated regions (UTRs) and improved the existing annotation. Importantly, the dRNA-seq analysis also identified a conserved suite of sRNAs specific to Fusobacterium.
The development of five genetic tools enabled further investigations of gene functions in F. nucleatum. These include vectors that enable the expression of different fluorescent proteins, inducible gene expression and scarless gene deletion in addition to transcriptional and translational reporter systems.
These tools enabled the dissection of a Sigma E response and uncovered several commonalities with its counterpart in the phylogenetically distant Proteobacteria. The similarities include the upregulation of genes involved in membrane homeostasis but also a Simga E-dependent regulatory sRNA. Surprisingly, oxygen was found to activated Sigma E in F. nucleatum contrasting the typical role of the factor in envelope stress.
The non-coding Sigma E-dependent sRNA, named FoxI, was shown to repress the translation of several envelope proteins which represented yet another parallel to the envelope stress response in Proteobacteria.
Overall, this work sheds light on the RNA landscape of the cancer-associated bacterium leading to the discovery of a conserved global stress response consisting of a coding and a non-coding arm. The development of new genetic tools not only aided the latter discovery but also provides the means for further dissecting the molecular and infection biology of this enigmatic bacterium.
Current therapeutic strategies efficiently improve survival in patients after myocardial infarction (MI). Nevertheless, long-term consequences such as heart failure development, are still one of the leading causes of death worldwide. Inflammation is critically involved in the cardiac healing process after MI and has a dual role, contributing to both tissue healing and tissue damage. In the last decade, a lot of attention was given to targeting inflammation as a potential therapeutic approach in MI, but the poor understanding of inflammatory cell heterogeneity and function is a limit to the development of immune modulatory strategies. The recent development of tools to profile immune cells with high resolution has provided a unique opportunity to better understand immune cell heterogeneity and dynamics in the ischemic heart.
In this thesis, we employed single-cell RNA-sequencing combined with detection of epitopes by sequencing (CITE-seq) to refine our understanding of neutrophils and monocytes/macrophages heterogeneity and dynamic after experimental myocardial infarction.
Neutrophils rapidly invade the infarcted heart shortly after ischemic damage and have previously been proposed to display time-dependent functional heterogeneity. At the single-cell level, we observed dynamic transcriptional heterogeneity in neutrophil populations during the acute post-MI phase and defined previously unknown cardiac neutrophil states. In particular, we identified a locally acquired SiglecFhi neutrophil state that displayed higher ROS production and phagocytic ability compared to newly recruited neutrophils, suggesting the acquisition of specific function in the infarcted heart. These findings highlight the importance of the tissue microenvironment in shaping neutrophil response.
From the macrophage perspective, we characterized MI-associated monocyte-derived macrophage subsets, two with a pro-inflammatory gene signature (MHCIIhiIl1βhi) and three Trem2hi macrophage populations with a lipid associated macrophage (LAM) signature, also expressing pro-fibrotic and tissue repair genes. Combined analysis of blood monocytes and cardiac monocyte/macrophages indicated that the Trem2hi LAM signature is acquired in the infarcted heart.
We furthermore characterized the role of TREM2, a surface protein expressed mainly in macrophages and involved in macrophage survival and function, in the post-MI macrophage response and cardiac repair. Using TREM2 deficient mice, we demonstrate that acquisition of the LAM signature in cardiac macrophages after MI is partially dependent on TREM2. While their cardiac function was not affected, TREM2 deficient mice showed reduced collagen deposition in the heart after MI. Thus, our data in Trem2-deficient mice highlight the role of TREM2 in promoting a macrophage pro-fibrotic phenotype, in line with the pro-fibrotic/tissue repair gene signature of the Trem2hi LAM-signature genes.
Overall, our data provide a high-resolution characterization of neutrophils and macrophage heterogeneity and dynamics in the ischemic heart and can be used as a valuable resource to investigate how these cells modulate the healing processes after MI. Furthermore, our work identified TREM2 as a regulator of macrophage phenotype in the infarcted heart
Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets.
Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin.
In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear.
Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research.
Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition.
Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 %, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 %. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments.
Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation.
As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets.
SUMOylation, as a post-translational modification, plays a crucial role in several biological processes. Small ubiquitin-like modifier (SUMO) proteins can be reversibly linked to the lysine residues located within specific motifs on numerous target proteins, leading to the change of stability, localization, activity of target proteins, mostly by promoting or interfering with the interaction with other molecules. Consequently, it can regulate gene transcription, migration, cell cycle progression, cellular responses to stress, and tumorigenesis.
NFATc1 belongs to the Nuclear Factor of Activated T-cells (NFAT) transcription factor family, which is dephosphorylated and translocates to the nucleus upon cell stimulation, which provokes Ca2+ signalling. NFAT plays a crucial role in the development and function of the immune system. NFATc1 has three SUMOylation sites at the position of aa 349, 702, and 914. In our previous study, we demonstrated that point mutations performed on the SUMOylation sites on all three or only at the lysine residues K702 and K914 lead to enhanced expression of IL-2 in vitro. To evaluate the function of SUMOylation of NFATc1 on T cell-mediated immunity in vivo, we not only generated a transgenic mouse strain (NFATc1/ΔS+ mouse) by point mutations from Lysine to Arginine on the two SUMOylation sites within exon 10 of Nfatc1 to prevent their SUMOylation, but in combination created another mouse strain (NFATc1/ΔBC+ mouse) that is completely Nfatc1 exon 10-ablated by using the LoxP/Cre system. In NFATc1/ΔS+ T cells, we observed enhanced IL-2 production and less IL-17A and IFN-γ expression. In line with exon 10 bearing the relevant SUMO sites, NFATc1/ΔBC+ CD4+ T cells behaved similarly as NFATc1/ΔS+ ones. The mechanism is that elevated IL-2 secretion can counteract the expression of IL-17A and IFN-γ via STAT5 and Blimp-1 induction. Afterwards, Blimp-1 suppressed IL-2 itself as well as Bcl2A1. Next, we performed two disease models with our NFATc1/ΔS+ mice. In a major mismatch model for acute graft-versus-host disease, we found that the mice transplanted with NFATc1/ΔS+ CD3+ T cells developed less severe disease, and T cells proliferated less due to increased Tregs. Moreover, when transferring 2D2.NFATc1/ΔS+ Th1 plus Th17 cells to Rag1-/- mice to induce experimental autoimmune encephalitis, we also observed ameliorated disease compared to animals with transferred WT T cells as well as increased Tregs.
Taking all data together, the deficiency in SUMOylation of NFATc1 leads to an elevated IL-2 secretion in T cells and subsequent activation of STAT5, which competes with STAT3 to inhibit IL-17A production and promotes Treg expansion, as well as to an enforcement of Blimp-1 expression, which suppresses IFN-γ and IL-2 expression. Consequently and despite a short phase of enhanced IL-2 secretion, the deficiency of SUMOylation on NFATc1 can protect from autoreactive and alloreactive diseases.
Moreover, to further understand the function of SUMOylation of NFATc1 in humans, we started by establishing an in vitro 3D culture system for tonsil organoids, which was successful in the presence of feeder cells, along with IL-4 and IL-7 cytokines. To confirm that our 3D tonsil organoids can respond to real antigens, we used CMV peptides and peptides of spike proteins from Covid-19 as real antigens, and co-cultured with tonsil organoids, which indeed can generate memory cells and plasmablasts. In the end, we also compared 3D to 2D cultures. Although the total numbers of all B cell subsets were much less in 3D culture than that in 2D culture, still, it indicates that this in-vitro culture system has its limitation, while being usable to produce the similar results as 2D did. Therefore, this 3D culture system can be used as a platform to investigate NFATc1/ΔS+ or NFATc1/ΔBC+ TFH and TFR cells in the dynamic of human GC responses.
Short functional peptidic probes can maximize the potential of high-end microscopy techniques and multiplex imaging assays and provide new insights into normal and aberrant molecular, cellular and tissue function. Particularly, the visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques and relies either on genetic manipulation or on antibodies that underperform in tissue immunofluorescence. Starting from an endogenous activity-related ligand of gephyrin, a universal marker of the inhibitory post-synapse, I developed a short peptidic multivalent binder with exceptional affinity and selectivity to gephyrin. By tailoring fluorophores to the binder, I have obtained Sylite, a probe for the visualization of inhibitory synapses, with an outstanding signal-to-background ratio, that bests the “gold standard” gephyrin antibodies both in selectivity and in tissue immunofluorescence. In tissue Sylite benefits from simplified handling, provides robust synaptic labeling in record-short time and, unlike antibodies, is not affected by staining artefacts. In super-resolution microscopy Sylite precisely localizes the post-synapse and enables accurate pre- to post-synapse measurements. Combined with complimentary tracing techniques Sylite reveals inhibitory connectivity and profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the periaqueductal gray brain region. Lastly, upon probe optimization for live cell application and with the help of novel thiol-reactive cell penetrating peptide I have visualized inhibitory synapses in living neurons. Taken together, my work provided a versatile probe for conventional and super-resolution microscopy and a workflow for the development and application of similar compact functional synthetic probes.
RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease “Neurodevelopmental Disorder with severe Motor Impairment and Absent Language” (NEDMIAL;OMIM#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3’ → 5’ RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome’s mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5’TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.
Additive manufacturing processes such as 3D printing are booming in the industry due to their high degree of freedom in terms of geometric shapes and available materials. Focusing on patient-specific medicine, 3D printing has also proven useful in the Life Sciences, where it exploits the shape fidelity for individualized tissues in the field of bioprinting. In parallel, the current systems of bioreactor technology have adapted to the new manufacturing technology as well and 3D-printed bioreactors are increasingly being developed. For the first time, this work combines the manufacturing of the tissue and a tailored bioreactor, significantly streamlining the overall process and optimally merging the two processes. This way the production of the tissues can be individualized by customizing the reactor to the tissue and the patient-specific wound geometry. For this reason, a common basis and guideline for the cross-device and cross-material use of 3D printers was created initially. Their applicability was demonstrated by the iterative development of a perfusable bioreactor system, made from polydimethylsiloxane (PDMS) and a lignin-based filament, into which a biological tissue of flexible shape can be bioprinted. Cost-effective bioink-replacements and in silico computational fluid dynamics simulations were used for material sustainability and shape development. Also, nutrient distribution and shear stress could be predicted in this way pre-experimentally.
As a proof of functionality and adaptability of the reactor, tissues made from a nanocellulose-based Cellink® Bioink, as well as an alginate-based ink mixed with Me-PMeOx100-b-PnPrOzi100-EIP (POx) (Alginate-POx bioink) were successfully cultured dynamically in the bioreactor together with C2C12 cell line. Tissue maturation was further demonstrated using hMSC which were successfully induced to adipocyte differentiation. For further standardization, a mobile electrical device for automated media exchange was developed, improving handling in the laboratory and thus reduces the probability of contamination.
In the eusocial insect honeybee (Apis mellifera), many sterile worker bees live together with a reproductive queen in a colony. All tasks of the colony are performed by the workers, undergoing age-dependent division of labor. Beginning as hive bees, they take on tasks inside the hive such as cleaning or the producing of larval food, later developing into foragers. With that, the perception of sweetness plays a crucial role for all honeybees whether they are sitting on the honey stores in the hive or foraging for food. Their ability to sense sweetness is undoubtedly necessary to develop and evaluate food sources. Many of the behavioral decisions in honeybees are based on sugar perception, either on an individual level for ingestion, or for social behavior such as the impulse to collect or process nectar. In this context, honeybees show a complex spectrum of abilities to perceive sweetness on many levels. They are able to perceive at least seven types of sugars and decide to collect them for the colony. Further, they seem to distinguish between these sugars or at least show clear preferences when collecting them. Additionally, the perception of sugar is not rigid in honeybees. For instance, their responsiveness towards sugar changes during the transition from in-hive bees (e.g. nurses) to foraging and is linked to the division of labor. Other direct or immediate factors changing responsiveness to sugars are stress, starvation or underlying factors, such as genotype.
Interestingly, the complexity in their sugar perception is in stark contrast to the fact that honeybees seem to have only three predicted sugar receptors.
In this work, we were able to characterize the three known sugar receptors (AmGr1, AmGr2 and AmGr3) of the honeybee fully and comprehensively in oocytes (Manuscript II, Chapter 3 and Manuscript III, Chapter 4). We could show that AmGr1 is a broad sugar receptor reacting to sucrose, glucose, maltose, melezitose and trehalose (which is the honeybees’ main blood sugar), but not fructose. AmGr2 acts as its co-receptor altering AmGr1’s specificity, AmGr3 is a specific fructose receptor and we proved the heterodimerization of all receptors. With my studies, I was able to reproduce and compare the ligand specificity of the sugar receptors in vivo by generating receptor mutants with CRISPR/Cas9. With this thesis, I was able to define AmGr1 and AmGr3 as the honeybees’ basis receptors already capable to detect all sugars of its known taste spectrum.
In the expression analysis of my doctoral thesis (Manuscript I, Chapter 2) I demonstrated that both basis receptors are expressed in the antennae and the brain of nurse bees and foragers. This thesis assumes that AmGr3 (like the Drosophila homologue) functions as a sensor for fructose, which might be the satiety signal, while AmGr1 can sense trehalose as the main blood sugar in the brain. Both receptors show a reduced expression in the brain of foragers when compared with nurse bees. These results may reflect the higher concentrated diet of nurse bees in the hive. The higher number of receptors in the brain may allow nurse bees to perceive hunger earlier and to consume the food their sitting on. Forager bees have to be more persistent to hunger, when they are foraging, and food is not so accessible. The findings of reduced expression of the fructose receptor AmGr3 in the antennae of nurse bees are congruent with my other result that nurse bees are also less responsive to fructose at the antennae when compared to foragers (Manuscript I, Chapter 2). This is possible, since nurse bees sit more likely on ripe honey which contains not only higher levels of sugars but also monosaccharides (such as fructose), while foragers have to evaluate less-concentrated nectar.
My investigations of the expression of AmGr1 in the antennae of honeybees found no differences between nurse bees and foragers, although foragers are more responsive to the respective sugar sucrose (Manuscript I, Chapter 2). Considering my finding that AmGr2 is the co-receptor of AmGr1, it can be assumed that AmGr1 and the mediated sucrose taste might not be directly controlled by its expression, but indirectly by its co-receptor. My thesis therefore clearly shows that sugar perception is associated with division of labor in honeybees and appears to be directly or indirectly regulated via expression.
The comparison with a characterization study using other bee breeds and thus an alternative protein sequence of AmGr1 shows that co-expression of different AmGr1 versions with AmGr2 alters the sugar response differently. Therefore, this thesis provides first important indications that alternative splicing could also represent an important regulatory mechanism for sugar perception in honeybees.
Further, I found out that the bitter compound quinine lowers the reward quality in learning experiments for honeybees (Manuscript IV, Chapter 5). So far, no bitter receptor has been found in the genome of honeybees and this thesis strongly assumes that bitter substances such as quinine inhibit sugar receptors in honeybees. With this finding, my work includes other molecules as possible regulatory mechanism in the honeybee sugar perception as well. We showed that the inhibitory effect is lower for fructose compared to sucrose. Considering that sugar signals might be processed as differently attractive in honeybees, this thesis concludes that the sugar receptor inhibition via quinine in honeybees might depend on the receptor (or its co-receptor), is concentration-dependent and based on the salience or attractiveness and concentration of the sugar present.
With my thesis, I was able to expand the knowledge on honeybee’s sugar perception and formulate a complex, comprehensive overview. Thereby, I demonstrated the multidimensional mechanism that regulates the sugar receptors and thus the sugar perception of honeybees. With this work, I defined AmGr1 and AmGr3 as the basis of sugar perception and enlarged these components to the co-receptor AmGr2 and the possible splice variants of AmGr1. I further demonstrated how those sugar receptor components function, interact and that they are clearly involved in the division of labor in honeybees. In summary, my thesis describes the mechanisms that enable honeybees to perceive sugar in a complex way, even though they inhere a limited number of sugar receptors. My data strongly suggest that honeybees overall might not only differentiate sugars and their diet by their general sweetness (as expected with only one main sugar receptor). The found sugar receptor mechanisms and their interplay further suggest that honeybees might be able to discriminate directly between monosaccharides and disaccharides or sugar molecules and with that their diet (honey and nectar).
Glioblastoma (GB) is the most aggressive malignant adult brain tumour with a median survival rate of only 15 months. GB tumours are characterized by necrotic and hypoxic core, which leads to nutrient deficient areas contributing to invasive, diffuseinfiltrative and angiogenic nature of these tumours. Cells exposed to nutrient deficient conditions and are known to reprogram their metabolism to produce or procure macro molecules from their environment. This makes cancer cells uniquely dependent on transcriptional regulators and a window of opportunity to target them. Sterol regulatory element binding protein 1 (SREBP1) is a transcriptional regulator of de-novo fatty acid synthesis in cells. The aim of this thesis was to investigate if SREBP1 was involved in restructuring the transcriptional regulation of genes involved in fatty acid biosynthesis upon low serum condition, in mediating interaction with other cell types in the tumour bulk such as endothelial cells, in regulating cancer stem like cells and finally to study its upstream regulation in GB. Global transcriptional analysis on GB cells exposed to low serum conditions revealed that SREBP1 regulated several fatty acid biosynthesis and phospholipid metabolic processes. PLA2G3 was identified as a novel target of SREBP1 in GB that was uniquely regulated in low serum condition. Analysis of total fatty acid and lipid species revealed that loss of SREBP1 in low serum condition changes the proportion of saturated, MUFAs and PUFAs. These changes were not specific to loss of PLA2G3 but as a result of downregulation of many genes regulated by SREBP1 in the fatty acid biosynthetic pathway. Next, treatment of HUVEC’s (endothelial cells) with condition medium from SREBP1-silenced U87 cells inhibited sprouting and tube formation capacity compared to the control condition, emphasizing the role of SREBP1 in angiogenesis and release of signalling mediators. Further, SREBP1 was shown to be important for proliferation of patient derived stem like cells and becomes indispensable for forming neurospheres in long term cultures, indicating its role in maintaining stemness. Also, inhibition of SREBP function by blocking the esterification of cholesterol using inhibitors targeting SOAT1 showed impairment in the viability of GB cells exposed to serum-depleted condition. Overall, SREBP1 plays an important role in maintaining tumour growth in nutrient deficient conditions and help in interaction with tumour microenvironment contributing to the aggressiveness of this tumour and poses itself as an attractive and unique target for GB treatment
Alzheimer´s disease (AD) is a neurodegenerative disease and the most common form of dementia with still no preventive or curative treatment. Besides several risk factors, age is one of the major risks for AD and with an aging society, there is an urgent need for disease modifying agents. The strategy to address only one target within the intertwined network of AD failed so far.
Natural products especially the phytochemical flavonoids, which are poly-phenolic natural products, have shown great potential as disease modifying agents against neurodegenerative disorders like Alzheimer´s disease (AD) with activities even in vivo. Flavonoids are produced by many plants and the native Californian plant Eriodictyon californicum is particularly rich in flavonoids. One of the major flavonoids of E. californicum is sterubin, a very potent agent against oxidative stress and inflammation, two hallmarks and drivers of AD and neurodegeneration. Herein, racemic sterubin was synthesized and separated into its pure (R)- and (S)-enantiomer by chiral HPLC. The pure enantiomers showed comparable neuroprotection in vitro with no significant differences. The stereoisomers were configurationally stable in methanol, but fast racemization was observed in culture medium. Moreover, the activity of sterubin was investigated in vivo, in an AD mouse model. Sterubin showed a significant positive impact on short- and long-term memory at low dosages.
A promising concept for the increase of activity of single flavonoids is hybridization with aromatic acids like cinnamic or ferulic acids. Hybridization of the natural products taxifolin and silibinin with cinnamic acid led to an overadditive effect of these compounds in phenotypic screening assays related to neurodegeneration and AD. Because there are more potent agents as taxifolin or silibinin, the hybrids were further developed, and different flavonoid cinnamic acid hybrids were synthesized. The connection between flavonoids and cinnamic acid was achieved by an amide instead of a labile ester to improve the stability towards hydrolysis to gain better “druggability” of the compounds. To investigate the oxidation state of the C-ring of the flavonoid part, the dehydro analogues of the respective hybrids were also synthesized. The compounds show neuroprotection against oxytosis, ferroptosis and ATP-depletion in the murine hippocampal cell line HT22. While no overall trend within the flavanones compared to the flavones could be assigned, the taxifolin and the quercetin derivative were the most active compounds in course of all assays. The quercetin derivate even shows greater activity than the taxifolin derivate in every assay. As desired no hydrolysis product was found in cellular uptake experiments after 4h, whereas different metabolites were found. The last part of this work focused on synthetic bioisoteres of the natural product curcumin. Due to the drawbacks of curcumin and flavonoids arising from poor pharmacokinetics, rapid metabolism and sometimes instability in aqueous medium, we have examined the biological activity of azobenzene compounds designed as bioisoteres of curcumin, carrying the pharmacophoric catechol group of flavonoids. These bioisosteres exceeded their parent compounds in counteracting intracellular oxidative stress, neuroinflammation and amyloid-beta aggregation. By incorporating an azobenzene moiety and the isosteric behaviour to the natural parent compounds, these compounds may act as molecular tools for further investigation towards the molecular mode of action of natural products.
Due to the global aging society and the enormous global incidence and prevalence rates that will result in the coming years, Alzheimer's Dementia (AD) represents a growing challenge for the health care system. The pathogenesis, which is unclear in parts, the chronic progression of AD, which often lasts for years, as well as insufficient diagnostic and therapeutic options complicate an adequate psychotherapeutic and medical approach to the disease. To date, AD is also considered an incurable disease.
Therefore, it is essential to gain deeper insights into the early detection or even prevention of AD. Consideration of prodromal syndromes such as Mild Cognitive Impairment (MCI) can provide significant evidence about high-risk groups for AD progression and differentiate cognitively "normal" aging individuals from those with pathological cognitive decline. Thus, for example, functional Near-Infrared Spectroscopy (fNIRS) imaging helps identify early neurodegenerative processes. In contrast, potential risk factors and predictors of later-onset clinical symptoms of MCI and AD can most often be revealed and quantified via the use of neuropsychiatric test batteries.
The present thesis consists of four studies and aimed to assess and describe the pathological cognitive decline in a sample of elderly study participants (age: ≥ 70 years; N = 604 at baseline) of the longitudinal, observational, and prospective "Vogel Study" from Würzburg, Germany, who were primarily healthy at baseline, over two measurement time points approximately 3 years apart, to differentiate between healthy and diseased study participants and to define predictors of MCI/AD and longitudinal study dropout.
Studies 1 and 2 differentiated healthy study participants from MCI patients based on the baseline hemodynamic response of the parietal cortex recorded by fNIRS during the processing of a paradigm (here: Angle Discrimination Task [ADT]) for visual-spatial processing performance. Neuronal hypoactivity was found in the MCI patients, with both healthy study participants and MCI patients showing higher superior and right hemispheric activation. MCI patients had more difficulty resolving the paradigm. Thus, no evidence of possible compensatory mechanisms was uncovered in the MCI patients.
Study 3 first defined the four latent factors declarative memory, working memory, attention, and visual-spatial processing based on structural equation model (SEM) calculations of the sample using adequate measurement (in-)variant confirmatory factor models from the baseline assessment to the first of a total of two follow-up assessments after approximately 3 years. This allowed a dimensional assessment of pathological cognitive decline versus classificatory-categorical assignment (healthy/diseased) of the sample. In addition, the superiority of the latent factor approach over a composite approach was demonstrated. Next, using a mixed-model approach, predictive analyses were calculated for the prediction of latent factors at first follow-up by baseline risk factors. The sex of study participants proved to be the best predictor of cognitive change in all the cognitive domains, with females performing better than men in the memory domains. Specifically, for declarative memory, older age predicted lower performance regardless of sex. Additional predictive evidence emerged for low serum levels of Brain-Derived Neurotrophic Factor (BDNF) on lower attention performance and higher depression symptoms on lower visual-spatial processing performance.
Study 4 further reported baseline predictors of study dropout at first follow-up. Cognitive performance, as defined in Study 3 using the four latent cognitive factors, was a predictor of study dropout for cognitive decline in the domains of declarative memory, attention, and visual-spatial processing. Conspicuous dementia screening on the Mini-Mental Status Examination (MMSE) also predicted dropout.
Overall, both the use of fNIRS imaging to detect visual-spatial processing performance in the parietal cortex during applying ADT and the dimensional perspective of the neuropsychiatric test battery in the context of prediction and dropout analyses were found to be suitable for early detection research of MCI and AD. Finally, the results will be interpreted in the overall context and implications, limitations, and perspectives will be discussed.
Ubiquitylation is a protein post translational modification, in which ubiquitin is covalently attached to target protein substrates resulting in diverse cellular outcomes. Besides ubiquitin, various ubiquitin-like proteins including FAT10 exist, which are also conjugated to target proteins. The underlying modification mechanisms are conserved. In the initial step, ubiquitin or a ubiquitin-like protein is thioester-linked to a catalytic cysteine in the E1activating enzyme in an ATP-dependent manner. The respective protein modifier is then transferred to an E2 conjugating enzyme in a transthioesterification reaction. Finally, an E3 ubiquitin ligase E3 catalyzes the covalent attachment of the protein modifier to a substrate. In the case of ubiquitin, multiple ubiquitin molecules can be attached to a substrate in the form of either linear or branched polyubiquitin chains but also as single ubiquitin modifications. Depending on the nature of the ubiquitin chain, the substrates are destined to various cellular processes such as their targeted destruction by the proteasome but also non-degradative outcomes may occur.
As stated above FAT10 is a ubiquitin-like protein modifier which typically targets proteins for proteasomal degradation. It consists of two ubiquitin-like domains and is mainly expressed in cells of the human immune system. The reported involvement of FAT10 modifications in cancers and other diseases has caught the attention of the scientific community as an inhibition of the FAT10ylation process may provide avenues for novel therapeutic approaches. UBA6 is the E1 activating enzyme that resides at the apex of the FAT10 proteasomal degradation pathway. UBA6 not only recognizes FAT10 but can also activate ubiquitin as efficiently as the ubiquitin specific E1 UBA1. The dual specificity of UBA6 may complicate the inhibition FAT10ylation since targeting the active site of UBA6 will also inhibit the UBA6-catalyzed ubiquitin activation. Therefore, it is important to understand the underlying principles for the dual specificity of UBA6 prior to the development of compounds interfering with FAT10ylation.
In this thesis important novel insights into the structure and function of UBA6 were derived by X-ray crystallography and biochemical methods. The first crystal structure of UBA6 reveals the multidomain architecture of this enzyme in atomic detail. The enzyme is composed of a rigid core including its active and inactive adenylation domains as well as a 4 helix bundle. Overall, the molecule adopts a “Y” shape architecture with the core at the base and the first and second catalytic half domains forming one arm of the “Y” and the ubiquitin fold domain constituting the other arm. While UBA6 shares the same domain architecture as UBA1, substantial differences were revealed by the crystal structure. In particular, the first catalytic half domain undergoes a significant shift to a position more distal from the core. This rigid body movement is assumed to generate room to accommodate the second ubiquitin-like domain of FAT10. Differences are also observed in a hydrophobic platform between the core and the first catalytic half domain and the adenylation active site in the core, which together from the binding sites for ubiquitin and FAT10. Site directed mutagenesis of key residues in these areas altered the UBA6-catalyzed activation of ubiquitin and FAT10. UBA6 variants were generated with the goal of trying to block the activation of FAT10 while still maintaining that of ubiquitin activation, in order to fully explain the dual specificity of UBA6. However, none of these mutations could block the activation of FAT10, while some of these UBA6 variants blocked ubiquitin activation. Preliminary inhibition assays with a group of E1 inhibitors belonging to the adenosyl sulfamate family demonstrated potent inhibition of FAT10ylation for two compounds. The dual specificity of UBA6 hence needs to be further examined by biochemical and structural methods. In particular, the structure of a complex between UBA6 and ubiquitin or FAT10 would provide key insights for further biochemical studies, ultimately allowing the targeted inhibition of the FAT10ylation machinery.
MDSCs are suppressive immune cells with a high relevance in various pathologies including cancer, autoimmunity, and chronic infections. Surface marker expression of MDSCs resembles monocytes and neutrophils which have immunostimulatory functions instead of suppressing T cells. Therefore, finding specific surface markers for MDSCs is important for MDSC research and therapeutic MDSC manipulation. In this study, we analyzed if the integrin VLA-1 has the potential as a novel MDSC marker. VLA-1 was expressed by M-MDSCs but not by G-MDSCs as well as by Teff cells. VLA-1 deficiency did not impact iNOS expression, the distribution of M-MDSC and G-MDSC subsets, and the suppressive capacity of MDSCs towards naïve and Teff cells in vitro. In mice, VLA-1 had no effect on the homing capability of MDSCs to the spleen, which is a major reservoir for MDSCs. Since the splenic red pulp contains collagen IV and VLA-1 binds collagen IV with a high affinity, we found MDSCs and Teff cells in this area as expected. We showed that T cell suppression in the spleen, indicated by reduced T cell recovery and proliferation as well as increased apoptosis and cell death, partially depended on VLA-1 expression by the MDSCs. In a mouse model of multiple sclerosis, MDSC injection prior to disease onset led to a decrease of the disease score, and this effect was significantly reduced when MDSCs were VLA-1 deficient. The expression of Sema7A by Teff cells, a ligand for VLA-1 which is implicated in negative T cell regulation, resulted in a slightly stronger Teff cell suppression by MDSCs compared to Sema7A deficient T cells. Live cell imaging and intravital 2-photon microscopy showed that the interaction time of MDSCs and Teff cells was shorter when MDSCs lacked VLA 1 expression, however VLA-1 expression had no impact on MDSC mobility. Therefore, the VLA-1-dependent interaction of MDSC and Teff cells on collagen IV in the splenic red pulp is implicated MDSC-mediated Teff cell suppression.
Mineral biocements are brittle materials, which usually results in catastrophic failure during mechanical loading. Here, previous works demonstrated the feasibility of reducing brittleness by a dual-setting approach, in which a silica sol was simultaneously gelled during the setting of a brushite forming cement. The current thesis aimed at further improving this concept by both using a novel silicate based cement matrix for an enhanced bonding between cement and silica matrix as well as multifunctional silica precursors to increase the network density of the gel. Due to its well-known biocompatibility and osteogenic regeneration capacity, baghdadite was chosen as mineral component of such composites. This required in a first approach the conversion of baghdadite ceramics into self-setting cement formulations. This was investigated initially by using baghdadite as reactive filler in a brushite forming cement (Chapter 4). Here, the ß-TCP component in a equimolar mixture of ß-TCP and acidic monocalcium phosphate anhydrous was subsequently replaced by baghdadite at various concentrations (0, 5, 10, 20, 30, 50, and 100 wt%) to study the influence on physicochemical cement properties such as mechanical performance, radiopacity, phase composition and microstructure. X-ray diffraction profiles demonstrated the dissolution of baghdadite during the cement reaction without affecting the crystal structure of the precipitated brushite phase. In addition, EDX analysis showed that calcium is homogeneously distributed in the cement matrix, while zirconium and silicon form cluster-like aggregates ranging in size from a few micrometers to more than 50 µm. X-ray images and µ-CT analyses indicate improved X-ray visibility with increased incorporation of baghdadite in brushite cement, with an aluminum equivalent thickness nearly doubling at a baghdadite content of 50 wt%. At the same time, the compressive strength of brushite cement increased from 12.9 ± 3.1 MPa to 21.1 ± 4.1 MPa at a baghdadite content of 10 wt%. Cell culture medium conditioned with powdered brushite cement approached physiological pH values when increasing amounts of baghdadite were added to the cement (pH = 6.47 for pure brushite, pH = 7.02 for brushite with 20 wt% baghdadite substitution). Baghdadite substitution also affected the ion content in the culture medium and thus the proliferation activity of primary human osteoblasts in vitro. The results demonstrated for the first time the suitability of baghdadite as a reactive cement additive for improving the radiopacity, mechanical performance, and cytocompatibility of brushite cements.
A second approach (Chapter 5) aimed to produce single component baghdadite cements by an increase of baghdadite solubility to initiate a self-setting cement reaction. For this, the material was mechanically activated by longer grinding times of up to 24h leading to both a decrease in particle and crystallite size as well as a partial amorphization of baghdadite. Baghdadite cements were formed by adding water at a powder to liquid ratio of 2.0 g/ml. Maximum compressive strengths were determined to be ~2 MPa after 3 days of setting for a 24-hour ground material. Inductively coupled plasma mass spectrometry (ICP-MS) measurements showed an incongruent dissolution profile of the set cements, with preferential dissolution of calcium and only minor release of zirconium ions. Cement formation occurs under alkaline conditions, with the unground raw powder resulting in a pH of 11.9 during setting, while prolonged grinding increases the pH to about 12.3.
Finally, mechanically activated baghdadite cements were combined with inorganic silica networks (Chapter 6) to create dual-setting cements with a further improvement of mechanical performance. While a modification of the cement pastes with a TEOS derived sol was already thought to improve strength, it was hypothesized that using multi-arm silica precursors can further enhance their mechanical performance due to a higher network density. In addition, this should also reduce pore size of both gels and cement and hence will be able to adjust the release kinetics of incorporated drugs. For this, multi-armed silica precursors were synthesized by the reaction of various multivalent alcohols (ethylene glycol, glycerine, pentaerythrit) with an isocyanate modified silica precursor. After hydrolysis under acidic conditions, the sols were mixed with baghdadite cement powders in order to allow a simultaneous gel formation and cement setting. Since the silica monomers have a high degree of linkage sites, this resulted in a branched network that interpenetrated with the growing cement crystals. In addition to minor changes in the crystalline phase composition as determined by X-ray diffraction, the novel composites exhibited improved mechanical properties with up to 20 times higher compressive strength and further benefit from an about 50% lower overall porosity than the reference pure baghdadite cement. In addition, the initial burst release of the model drug vancomycin was completely inhibited by the added silica matrix. This observation was verified by testing for the antimicrobial activity with Staphylococcus aureus by measuring the inhibition zones of selected samples after 24 h and 48 h, whereas the antimicrobial effectiveness of a constant vancomycin release could be demonstrated.
The current thesis clearly demonstrated the high potential of baghdadite as a cement formulation for medical application. The initially poor mechanical properties of such cements can be overcome by special processing techniques or by combination with silica networks. The achieved mechanical performance is > 10 MPa and hence suitable for bone replacement under non-load bearing conditions. The high intrinsic radiopacity as well as the alkaline pH during setting may open the way ahead to further dental applications, e.g. as root canal sealers or filler in dental composites. Here, the high pH is thought to lead to antimicrobial properties of such materials similar to commonly applied calcium hydroxide or calcium silicates, however combined with an intrinsic radiopacity for X-ray imaging. This would simplify such formulations to single component materials which are less susceptible to demixing processes during transport, storage or processing.
Cellular growth and proliferation are among the most important processes for cells and
organisms. One of the major determinants of these processes is the amount of proteins
and consequently also the amount of ribosomes. Their synthesis involves several hundred
proteins and four different ribosomal RNA species, is highly coordinated and very
energy-demanding. However, the molecular mechanims of transcriptional regulation of
the protein-coding genes involved, is only poorly understood in mammals.
In this thesis, unbiased genome-wide knockout reporter screens were performed, aiming
to identify previously unknown transcriptional regulators of ribosome biogenesis
factors (RiBis), which are important for the assembly and maturation of ribosomes,
and ribosomal proteins (RPs), which are ribosomal components themself. With that
approach and follow-up (validation) experiments, ALDOA and RBM8A among others,
could be identified as regulators of ribosome biogenesis.
Depletion of the glycolytic enzyme ALDOA led to a downregulation of RiBi- and RPpromoter
driven reporters on protein and transcript level, as well as to a downregulation
of ribosome biogenesis gene transcripts and of mRNAs of other genes important for
proliferation.
Reducing the amount of the exon junction complex protein RBM8A, led to a more prominent
downregulation of one of the fluorescent reporters, but this regulation was independent
of the promoter driving the expression of the reporter. However, acute protein
depletion experiments in combination with nascent RNA sequencing (4sU-Seq)
revealed, that mainly cytosolic ribosomal proteins (CRPs) were downregulated upon
acute RBM8A withdrawal. ChIP experiments showed RBM8A binding to promoters of
RP genes, but also to other chromatin regions. Total POL II or elongating and initiating
POL II levels were not altered upon acute RBM8A depletion.
These data provide a starting point for further research on the mechanisms of transcriptional
regulation of RP and RiBi genes in mammals.
Abstract:
COVID-19 has impressively shown how quickly an emerging pathogen can have a massive impact on our entire lives and show how infectious diseases spread regardless of national borders and economic stability. We find ourselves in a post-antibiotic era and have rested too long on the laurels of past research, so today more and more people are dying from infections with multi-resistant germs.
Infections are highly plastic and heterogeneous processes that are strongly dependent on the individual, whether on the host or pathogen side.
Improving our understanding of the pathogenicity of microorganisms and finding potential targets for a completely new class of drugs is a declared goal of current basic research. To tackle this challenge, single-cell RNA sequencing (scRNA-seq) is our most accurate tool.
In this thesis we implemented different state of the art scRNA-seq technologies to better understand infectious diseases. Furthermore, we developed a new method which is capable to resolve the transcriptome of a single bacterium. Applying a poly(A)-independent scRNA-seq protocol to three different, infection relevant growth conditions we can report the faithful detection of growth-dependent gene expression patterns in individual Salmonella Typhimurium and Pseudomonas aeruginosa bacteria. The data analysis shows that this method not only allows the differentiation of various culture conditions but can also capture transcripts across different RNA species.
Furthermore, using state of the art imaging and single-cell RNA sequencing technologies, we comprehensively characterized a human intestinal tissue model which in further course of the project was used as a Salmonella enterica serovar Typhimurium infection model. While most infection studies are conducted in mice, lacking a human intestinal physiology, the in vitro human tissue model allows us to directly infer in vivo pathogenesis. Combining immunofluorescent imaging, deep single-cell RNA sequencing and HCR-FISH, applied in time course experiments, allows an unseen resolution for studying heterogeneity and the dynamics of Salmonella infection which reveals details of pathogenicity contrary to the general scientific opinion.
The platelet cytoskeleton ensures normal size and discoid shape under resting conditions and undergoes immediate reorganization in response to changes in the extracellular environment through integrin-based adhesion sites, resulting in actomyosin-mediated contractile forces. Mutations in the contractile protein non-muscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild to moderate bleeding tendency in human patients. It is insufficiently understood which factors contribute to the hemostatic defect found in MYH9-related disease patients. Therefore, a better understanding of the underlying biophysical mechanisms in thrombus formation and stabilization is warranted.
This thesis demonstrates that an amino acid exchange at the positions 702, 1424 and 1841 in the heavy chain of the contractile protein non-muscle myosin IIA, caused by heterozygous point mutations in the gene, resulted in macrothrombocytopenia and increased bleeding in mice, reflecting the clinical hallmark of the MYH9-related disease in human patients. Basic characterization of biological functions of Myh9 mutant platelets revealed overall normal surface glycoprotein expression and agonist-induced activation when compared to wildtype platelets. However, myosin light chain phosphorylation after thrombin-activation was reduced in mutant platelets, resulting in less contractile forces and a defect in clot retraction. Altered biophysical characteristics with lower adhesion and interaction forces of Myh9 mutant platelets led to reduced thrombus formation and stability. Platelets from patients with the respective mutations recapitulated the findings obtained with murine platelets, such as impaired thrombus formation and stiffness.
Besides biological and biophysical characterization of mutant platelets from mice and men, treatment options were investigated to prevent increased bleeding caused by reduced platelet forces. The antifibrinolytic agent tranexamic acid was applied to stabilize less compact thrombi, which are presumably more vulnerable to fibrinolysis. The hemostatic function in Myh9 mutant mice was improved by interfering with the fibrinolytic system. These results show the beneficial effect of fibrin stabilization to reduce bleeding in MYH9-related disease.
This work developed during the first funding period of the subproject B05 in the framework of the interdisciplinary research consortium TRR 225 ‘From the Fundamentals of Biofabrication toward functional Tissue Models’ and was part of a cooperation between the Orthopedic Department represented by Prof. Dr. Regina Ebert and the Institute of Organic Chemistry represented by Prof. Dr. Jürgen Seibel.
This project dealed with cellular behavior during the bioprinting process and how to influence it by modifying the cell glycocalyx with functional target molecules. The focus was on the impact of potential shear stress, that cells experience when they get processed in thermoresponsive bioinks, and a way to increase the cell stiffness via metabolic glycoengineering to attenuate shear forces. For the characterization of the metabolic glycoengineering, four different peracetylated and four non-acetylated modified monosaccharides (two mannose and two sialic acid sugars) were tested in primary human mesenchymal stromal cells (hMSC) and telomerase-immortalized hMSC (hMSC-TERT). Viability results demonstrated a dose-dependent correlation for all sugars, at which hMSC-TERT seemed to be more susceptible leading to lower viability rates. The assessment of the incorporation efficiencies was performed by click chemistry using fluorescent dyes and revealed also a dose-dependent correlation for all mannose and sialic acid sugars, while glucose and galactose variants were not detected in the glycocalyx. However, incorporation efficiencies were highest when using mannose sugars in the primary hMSC. A subsequent analysis of the temporal retention of the incorporated monosaccharides showed a constant declining fluorescence signal up to 6 d for azido mannose in hMSC-TERT, whereas no signal could be detected for alkyne mannose after 2 d. Investigation of the differentiation potential and expression of different target genes revealed no impairment after incubation with mannose sugars, indicating a normal phenotype for hMSC-TERT. Following the successful establishment of the method, either a coumarin derivative or an artificial galectin 1 ligand were incorporated into the cell glycocalyx of hMSC-TERT as functional target molecule. The biophysical analysis via shear flow deformation cytometry revealed a slightly increased cell stiffness and lowered fluidity for both molecules. A further part of this project aimed to control lectin-mediated cell adhesion by artificial galectin 1 ligands. As that hypothesis was settled in the work group of Prof. Dr. Jürgen Seibel, this work supported with an initial characterization of galectin 1 as part of the hMSC biology. A stable galectin 1 expression at gene and protein level in both hMSC and hMSC-TERT could be confirmed, at which immunocytochemical stainings could detect the protein only in the glycocalyx. The treatment of hMSC-TERT with a galectin 1 ligand in different concentrations did not show an altered gene expression of galectin 1. However, these first data in addition to the investigation of stiffness confirmed the applicability of specific and artificial
IV
galectin 1 ligands in biofabrication approaches to alter cell properties of hMSC. To conclude, metabolic glycoengineering has been successfully implemented in hMSC and hMSC-TERT to introduce glycocalyx modifications which reside there for several days. A proof of concept was carried out by the increase of cell stiffness and fluidity by the incorporation of a coumarin derivative or an artificial galectin 1 ligand.
For the characterization of shear stress impact on cells after printing in thermoresponsive bioinks, the processing of hMSC-TERT (mixing or additionally printing) with Pluronic F127 or Polyoxazoline-Polyoxazine (POx-POzi) polymer solution was investigated. While there were no changes in viability when using POx-POzi bioink, processing with Pluronic F127 indicated slightly lower viability and increased apoptosis activity. Assessment of cellular responses to potential shear stress showed no reorganization of the cytoskeleton independent of the bioink, but highly increased expression of the mechanoresponsive proto-oncogene c Fos which was more pronounced when using Pluronic F127 and just mixed with the bioinks. Interestingly, processing of the mechanoresponsive reporter cell line hMSC-TERT-AP1 revealed slightly elevated mechanotransduction activity when using POx-POzi polymer and just mixed with the bioinks as well. In conclusion, hMSC-TERT embedded in thermoresponsive bioinks might shortly experience shear stress during the printing process, but that did not lead to remarkable cell damage likely due to the rheological properties of the bioinks. Furthermore, the printing experiments also suggested that cells do not sense more shear stress when additionally printed.
Even though exposure-based cognitive behavioral therapy (CBT) constitutes a first-line treatment for anxiety disorders, a substantial proportion of patients does not respond in a clinically significant manner. The identification of pre-treatment patient characteristics that are associated with treatment outcome might aid in improving response rates. Therefore, the present doctoral thesis aimed at investigating moderators of treatment outcome in anxiety disorders: first, we investigated the neural correlates of comorbidity among primary panic disorder/agoraphobia (PD/AG) and secondary social anxiety disorder (SAD) moderating treatment outcome towards exposure-based CBT. Second, pre-treatment functional resting-state connectivity signatures of treatment response in specific phobia were studied. Within the first study, we compared PD/AG patients with or without secondary SAD regarding their clinical and neurofunctional outcome towards a manualized CBT treatment focusing on PD/AG symptoms. Prior to treatment, PD/AG+SAD compared to PD/AG-SAD patients exhibited a specific neural signature within the temporal lobe, which was attenuated to the level of PD/AG-SAD patients afterwards. CBT was equally effective in both groups. Thus, comorbidity among those two anxiety disorders did not alter treatment outcome substantially. This might be due to the high overlap of shared pathophysiological features within both disorders. In the second study, we assessed pre-treatment functional resting-state connectivity within a sample of spider phobic patients that were treated with massed in virtuo exposure. We found responders already prior to treatment to be characterized by stronger inhibitory frontolimbic connectivity as well as heightened connectivity between the amygdala and regions related to the ventral visual stream. Furthermore, patients demonstrating high within-session extinction exhibited pronounced intrinsic prefrontal connectivity. Our results point to responders exhibiting a brain prepared for the mechanism of action of exposure. Taken together, results highlight the major impact of pre-treatment characteristics on treatment outcome. Both, PD/AG+SAD patients as well as responders within the SpiderVR study exhibited heightened activation or connectivity within the ventral visual pathway and the amygdala. Pronounced visual processing together with enhanced executive control and emotion regulation seem to constitute a fruitful soil for successful exposure. The results provide starting points for personalized treatment approaches in order to improve treatment success in the anxiety disorders. Future studies are needed to investigate the benefit of neuroscientifically informed CBT augmentation strategies such as repetitive transcranial magnetic stimulation.
Honeybees are among the few animals that rely on eusociality to survive. While the
task of queen and drones is only reproduction, all other tasks are accomplished by sterile
female worker bees. Different tasks are mostly divided by worker bees of different ages
(temporal polyethism). Young honeybees perform tasks inside the hive like cleaning and
nursing. Older honeybees work at the periphery of the nest and fulfill tasks like guarding
the hive entrance. The oldest honeybees eventually leave the hive to forage for resources
until they die. However, uncontrollable circumstances might force the colony to adapt or
perish. For example, the introduced Varroa destructor mite or the deformed wing virus
might erase a lot of in-hive bees. On the other hand, environmental events might kill a
lot of foragers, leaving the colony with no new food intake. Therefore, adaptability of
task allocation must be a priority for a honeybee colony.
In my dissertation, I employed a wide range of behavioral, molecular biological and analytical techniques to unravel the underlying molecular and physiological mechanisms of
the honeybee division of labor, especially in conjunction with honeybee malnourishment.
The genes AmOARα1, AmTAR1, Amfor and vitellogenin have long been implied to
be important for the transition from in-hive tasks to foraging. I have studied in detail
expression of all of these genes during the transition from nursing to foraging to understand how their expression patterns change during this important phase of life. My focus
lay on gene expression in the honeybee brain and fat body. I found an increase in the
AmOARα1 and the Amforα mRNA expression with the transition from in-hive tasks to
foraging and a decrease in expression of the other genes in both tissues. Interestingly,
I found the opposite pattern of the AmOARα1 and AmTAR1 mRNA expression in the
honeybee fat body during orientation flights. Furthermore, I closely observed juvenile
hormone titers and triglyceride levels during this crucial time. Juvenile hormone titers
increased with the transition from in-hive tasks to foraging and triglyceride levels decreased.
Furthermore, in-hive bees and foragers also differ on a behavioral and physiological level.
For example, foragers are more responsive towards light and sucrose. I proposed that
modulation via biogenic amines, especially via octopamine and tyramine, can increase
or decrease the responsiveness of honeybees. For that purpose, in-hive bees and foragers were injected with both biogenic amines and the receptor response was quantified
1
using electroretinography. In addition, I studied the behavioral response of the bees to
light using a phototaxis assay. Injecting octopamine increased the receptor response and
tyramine decreased it. Also, both groups of honeybees showed an increased phototactic
response when injected with octopamine and a decreased response when injected with
tyramine, independent of locomotion.
Additionally, nutrition has long been implied to be a driver for division of labor. Undernourished honeybees are known to speed up their transition to foragers, possibly to
cope with the missing resources. Furthermore, larval undernourishment has also been
implied to speed up the transition from in-hive bees to foragers, due to increasing levels
of juvenile hormone titers in adult honeybees after larval starvation. Therefore, I reared
honeybees in-vitro to compare the hatched adult bees of starved and overfed larvae to
bees reared under the standard in-vitro rearing diet. However, first I had to investigate
whether the in-vitro rearing method affects adult honeybees.
I showed effects of in-vitro rearing on behavior, with in-vitro reared honeybees foraging
earlier and for a shorter time than hive reared honeybees. Yet, nursing behavior was
unaffected.
Afterwards, I investigated the effects of different larval diets on adult honeybee workers.
I found no effects of malnourishment on behavioral or physiological factors besides a
difference in weight. Honeybee weight increased with increasing amounts of larval food,
but the effect seemed to vanish after a week.
These results show the complexity and adaptability of the honeybee division of labor.
They show the importance of the biogenic amines octopamine and tyramine and of the
corresponding receptors AmOARα1 and AmTAR1 in modulating the transition from inhive bees to foragers. Furthermore, they show that in-vitro rearing has no effects on
nursing behavior, but that it speeds up the transition from nursing to foraging, showing
strong similarities to effects of larval pollen undernourishment. However, larval malnourishment showed almost no effects on honeybee task allocation or physiology. It seems
that larval malnourishment can be easily compensated during the early lifetime of adult
honeybees.
Systemic and local mechanisms of small fiber pathology in female patients with fibromyalgia syndrome
(2023)
Fibromyalgia syndrome (FMS) is a largely heterogeneous chronic pain syndrome of unclear pathophysiology, which lacks objective diagnostics and specific treatment. An immune-related shift towards a pro-inflammatory profile is discussed at a systemic level. Small fiber pathology (SFP) and local participation of non-neuronal skin cells like keratinocytes in cutaneous nociception are potential peripheral contributors. Small RNAs, particularly microRNAs (miRs) and newly described tRNA fragments (tRFs) act as posttranscriptional key regulators of gene expression and may modulate systemic and peripheral cell pathways. On cellular level, the exact mechanisms of keratinocyte-intraepidermal nerve fiber (IENF) interaction in the skin are insufficiently understood.
Via small RNA sequencing and quantitative real-time PCR, we investigated miR and tRF signatures in whole blood cells and skin biopsy-derived keratinocytes of female FMS patients versus healthy controls. We applied gene target prediction analysis to uncover underlying cellular pathways affected by dysregulated small RNAs. Altered FMS small RNAs from blood were compared with their expression in disease controls, i.e. Parkinson`s patients and patients with major depression and chronic pain. Association of SFP with small RNAs was investigated via correlation with clinical parameter. To explore keratinocyte-nerve fiber interactions with high relevance for SFP and cutaneous nociception, we adapted a super-resolution array tomography (srAT) approach and expansion microscopy (ExM) for human skin samples. Further, we created a fully human 2D co-culture model of primary keratinocytes and induced pluripotent stem cell derived sensory neurons.
Blood miR deregulation indicated systemic modulation of immune processes exerted by CholinomiRs and by miRs targeting the FoxO signaling pathway. Short sized tRFs were associated with mRNA metabolism and splicing. This supports the hypothesis of an inflammatory/autoimmunity component in FMS. Expression of blood small RNAs in FMS were discriminative against disease controls, highlighting their potential as objective biomarker. Blood small RNAs were predominantly upregulated and correlations between miR and clinical parameter reflected rather pain in general than SFP.
In FMS keratinocytes, a downregulation of miRs and tRFs was evident. Pathways for adenosine monophosphate-activated protein kinase (AMPK), adherens junction, and focal adhesion were predicted to be affected by miRs, while tRFs may influence proliferation, migration, and cell growth. Similar to blood miRs, altered miRs in keratinocytes correlated mostly with widespread pain and pain severity parameter. TRFs were partially associated with more severe IENF loss. Small RNAs in FMS keratinocytes may modulate pathways that define how keratinocytes interact with each other and with IENF.
These interactions include nerve fiber ensheathment, a conserved epithelial mechanism, which we visualize in human epidermis and a fully human co-culture model. Additionally, we revealed plaques of connexin 43, a pore forming protein involved in intercellular communication, at keratinocyte- nerve fiber contact sites. Objective quantification of these morphological findings in FMS and other diseases with SFP may inherit diagnostic value similar to IENF density.
We provide evidence for distinct miR and tRF signatures in FMS with implications for systemic immune regulation and local cell-cell interaction pathways. In the periphery we explored novel keratinocyte-nerve fiber interactions relevant for SFP and cutaneous nociception.
The research that is compiled in this thesis can be divided in two parts. The first part, consisting of four chapters, is centered around the role of epigenetic dysregulation in the etiopathophysiology of sporadic alzheimer's disease (sAD). In addition to providing insights into the most recent developments in neuroepigenomic studies of this disease, the first part of the thesis also touches upon remaining challenges, and provides a future outlook on possible developments in the field. The second part, which includes three more chapters, is focused on the application of induced pluripotent stem cell (iPSC)-based disease models for the study of AD, including but not limited to mechanistic studies on epigenetic dysregulation using this platform. Aside from outlining the research that has been conducted using iPSC-based models for sAD to date, the second part of the thesis also provides insights into the acquisition of disease-relevant neural cultures based on directed differentiation of iPSCs, and furthermore includes an experimental approach for the establishment of such a model system.
Autoantibodies against proteins of the node of Ranvier have been identified in a subset of patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Main antigens targeted by autoantibodies are the paranodal proteins contactin 1 (CNTN1), neurofascin (NF) 155 or contactin associated protein (Caspr) as well as the nodal NF186. Several studies investigated the role of anti-paranodal autoantibodies in the pathophysiology of CIDP leading to the current knowledge that immunoglobulin G (IgG)4 deposition leads to detachment of myelin from the axon at the paranodes. However, many questions remain unsolved. Thus, autoantibodies against NF155 have been well studied and their pathogenicity has been proven in an animal model in vivo. However, in some patients, autoantibodies against all isoforms of NF are detectable. These anti-pan-NF autoantibodies occur more rarely and lead to a very severe clinical phenotype. As the pathogenesis of patient-derived autoantibodies against pan-NF has never been investigated in vivo before, we used an animal model to study the effect of acute exposure to anti-pan-NF IgG3 by intraneural injections to the rat sciatic nerve. In addition, we used anti-NF155 IgG4 from a seropositive patient. Behavioral testings as well as nerve conduction studies did not re- veal any deficits after injected neither for anti-NF155 nor for anti-pan-NF autoantibodies. This leads to the suspicion that the disease is more likely induced by a chronic process.
A common symptom in patients with anti-CNTN1 associated neuropathy is sensory ataxia and therefore, an involvement of dorsal root ganglia (DRGs) is hypothesized. We show that sera from anti-CNTN1 positive patients specifically bind to DRG neurons in vitro and reduce surface expression of CNTN1. This is most probably due to internalization mediated by coexisting IgG3 although IgG4 is the predominant subclass of autoantibodies. As it is known that CNTN1 interacts with the β1 subunit of specific sodium channels we analyzed channel expression and sodium currents of DRG neurons after incubation with anti-CNTN1 positive patients’ sera. We identified reduced sodium currents after long-term treatment with patients’ material although surface channel expression remained stable. We therefore concluded that CNTN1 might influence channel properties indirectly through auxiliary β1 subunits. Moreover, we suggest an involvement of DRG neurons in the pathogenesis of anti-CNTN1 associated CIDP as medium-large size neurons are more affected than small neurons. However, the exact mechanism of how anti-CNTN1 autoantibodies influence sodium channels should be subject of further studies.
Furthermore, preliminary results indicate that the epitope for anti-CNTN1 autoantibodies from seropositive patients might be associated with distinct clinical features. We could show that autoantibodies might be either directed against a conformational epitope as binding is prevented after deletion of the first immunoglobulin (Ig) domain of CNTN1 or against the fibronectin type III (FnIII) domains. Strikingly, both patients with FnIII do- main specificity had very high titers of anti-CNTN1 autoantibodies and a chronic disease progression, whereas patients binding to a conformational epitope or to the Ig domains are related to a relapsing-remitting or even monophasic disease course. However, these results need to be further confirmed before a clear statement can be made.
In conclusion, the present study contributes to elucidate the pathogenesis of peripheral neuropathies associated with anti-paranodal autoantibodies. However, further studies are required including a higher number of patients as well as considering effects on structures like DRGs besides the node of Ranvier to fully understand the disease mechanisms.
Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain.
The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis.
Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group’s macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera.
Megakaryocytes (MKs) are the largest cells of the hematopoietic system and the precursor cells of platelets. During proplatelet formation (PPF) bone marrow (BM) MKs extent large cytoplasmic protrusions into the lumen of sinusoidal blood vessels. Under homeostatic conditions PPF occurs exclusively in the direction of the sinusoid, while platelet generation into the marrow cavity is prevented. So far, the mechanisms regulating this process in vivo are still not completely understood, especially when PPF is deregulated during disease. This thesis investigated the mechanisms of PPF in native BM and after myeloablation by total body irradiation (TBI).
First, we have identified a specialized type of BM stromal cells, so called CXCL12-abundant reticular (CAR) cells, as novel possible regulators of PPF. By using complementary high-resolution microscopy techniques, we have studied the morphogenetic events at the MK/vessel wall interface in new detail, demonstrating that PPF formation preferentially occurs at CAR cell-free sites at the endothelium.
In the second part of this thesis, we analyzed the processes leading to BM remodeling in response to myeloablation by TBI. We used confocal laser scanning microscopy (CLSM) to study the kinetic of radiation-triggered vasodilation and mapped extracellular matrix (ECM) proteins after TBI. We could demonstrate that collagen type IV and laminin α5 are specifically degraded at BM sinusoids. At the radiation-injured vessel wall we observed ectopic release of platelet-like particles into the marrow cavity concomitantly to aberrant CAR cell morphology, suggesting that the balance of factors regulating PPF is disturbed after TBI. ECM proteolysis is predominantly mediated by the matrix metalloproteinase MMP9, as revealed by gelatin-zymography and by a newly established BM in situ zymography technique. In transgenic mice lacking MMP9 vascular recovery was delayed, hinting towards a role of MMP9 in vessel reconstitution after myeloablation.
In a third series of experiments, we studied the irradiated BM in the context of hematopoietic stem cell transplantation (HSCT). By using mice as BM donors that ubiquitously express the fluorescent reporter protein dsRed we tracked engraftment of donor cells and especially MKs in the recipient BM. We found a distinct engraftment pattern and cluster formation for MKs, which is different from other blood cell lineages.
Finally, we assessed platelet function after TBI and HSCT and were the first to demonstrate that platelets become massively hyporeactive, particularly upon stimulation of the collagen receptor GPVI.
In summary, our findings shed light on the processes of PPF during health and disease which will help to develop treatments for aberrant thrombopoiesis.